Reduced failure rates associated with playing a new online game developed to support learning of core content in human systems physiology.

A digital mobile card-matching game called eFlip was developed to assist second-year undergraduate medical science students to learn core content and understand key associations in physiology. Our team developed customized content of core physiological associations, of increasing difficulty, for upload on a generic card-matching platform. The generic game was extended with add-ons coded to allow identification and access of student usage data for students who consented to have their game usage linked to academic performance such as final course marks and grades. Here, we describe the development of the eFlip game content, the student usage profile, and the game's impact on learning. Students were invited to use eFlip prior to and during the final examination period. Of those who used eFlip, 152 students consented to participate in a study of game use on learning performance outcomes. Within the students who consented, 74 played the game and 78 did not play the game (nonplayers). The mean course mark of the students who played the game [69.57%; 95% confidence level (CI) (67.22, 71.92)] was higher than that of the nonplayers [65.33%; 95% CI (62.67, 67.99)] (P = 0.02). Playing eFlip was also associated with reduced failure rates in students who played the game (1% failure rate) compared with the students who did not play the game (10% failure rate; P = 0.02). The number of games played by students peaked just prior to the course final examination. Overall, students who chose to play eFlip demonstrated improved grades that were associated with a higher probability of passing the physiology course.

Sex Differences in Photoprotective Responses to 1,25-Dihydroxyvitamin D3 in Mice Are Modulated by the Estrogen Receptor-ß.

Susceptibility to photoimmune suppression and photocarcinogenesis is greater in male than in female humans and mice and is exacerbated in female estrogen receptor-beta knockout (ER-ß-/-) mice. We previously reported that the active vitamin D hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D), applied topically protects against the ultraviolet radiation (UV) induction of cutaneous cyclobutane pyrimidine dimers (CPDs) and the suppression of contact hypersensitivity (CHS) in female mice. Here, we compare these responses in female versus male Skh:hr1 mice, in ER-ß-/-/-- versus wild-type C57BL/6 mice, and in female ER-blockaded Skh:hr1 mice. The induction of CPDs was significantly greater in male than female Skh:hr1 mice and was more effectively reduced by 1,25(OH)2D in female Skh:hr1 and C57BL/6 mice than in male Skh:hr1 or ER-ß-/- mice, respectively. This correlated with the reduced sunburn inflammation due to 1,25(OH)2D in female but not male Skh:hr1 mice. Furthermore, although 1,25(OH)2D alone dose-dependently suppressed basal CHS responses in male Skh:hr1 and ER-ß-/- mice, UV-induced immunosuppression was universally observed. In female Skh:hr1 and C57BL/6 mice, the immunosuppression was decreased by 1,25(OH)2D dose-dependently, but not in male Skh:hr1, ER-ß-/-, or ER-blockaded mice. These results reveal a sex bias in genetic, inflammatory, and immune photoprotection by 1,25(OH)2D favoring female mice that is dependent on the presence of ER-ß.

Skeletal Muscle and the Maintenance of Vitamin D Status.

Vitamin D, unlike the micronutrients, vitamins A, E, and K, is largely obtained not from food, but by the action of solar ultraviolet (UV) light on its precursor, 7-dehydrocholesterol, in skin. With the decline in UV light intensity in winter, most skin production of vitamin D occurs in summer. Since no defined storage organ or tissue has been found for vitamin D, it has been assumed that an adequate vitamin D status in winter can only be maintained by oral supplementation. Skeletal muscle cells have now been shown to incorporate the vitamin D-binding protein (DBP) from blood into the cell cytoplasm where it binds to cytoplasmic actin. This intracellular DBP provides an array of specific binding sites for 25-hydroxyvitamin D (25(OH)D), which diffuses into the cell from the extracellular fluid. When intracellular DBP undergoes proteolytic breakdown, the bound 25(OH)D is then released and diffuses back into the blood. This uptake and release of 25(OH)D by muscle accounts for the very long half-life of this metabolite in the circulation. Since 25(OH)D concentration in the blood declines in winter, its cycling in and out of muscle cells appears to be upregulated. Parathyroid hormone is the most likely factor enhancing the repeated cycling of 25(OH)D between skeletal muscle and blood. This mechanism appears to have evolved to maintain an adequate vitamin D status in winter.

Evolution of the sheep coat: the impact of domestication on its structure and development.

Wild sheep and many primitive domesticated breeds have two coats: coarse hairs covering shorter, finer fibres. Both are shed annually. Exploitation of wool for apparel in the Bronze Age encouraged breeding for denser fleeces and continuously growing white fibres. The Merino is regarded as the culmination of this process. Archaeological discoveries, ancient images and parchment records portray this as an evolutionary progression, spanning millennia. However, examination of the fleeces from feral, two-coated and woolled sheep has revealed a ready facility of the follicle population to change from shedding to continuous growth and to revert from domesticated to primitive states. Modifications to coat structure, colour and composition have occurred in timeframes and to sheep population sizes that exclude the likelihood of variations arising from mutations and natural selection. The features are characteristic of the domestication phenotype: an assemblage of developmental, physiological, skeletal and hormonal modifications common to a wide variety of species under human control. The phenotypic similarities appeared to result from an accumulation of cryptic genetic changes early during vertebrate evolution. Because they did not affect fitness in the wild, the mutations were protected from adverse selection, becoming apparent only after exposure to a domestic environment. The neural crest, a transient embryonic cell population unique to vertebrates, has been implicated in the manifestations of the domesticated phenotype. This hypothesis is discussed with reference to the development of the wool follicle population and the particular roles of Notch pathway genes, culminating in the specific cell interactions that typify follicle initiation.

CYP11A1 in skin: an alternative route to photoprotection by vitamin D compounds.

Topical 1,25-dihydroxyvitamin D (1,25D) and other vitamin D compounds have been shown to protect skin from damage by ultraviolet radiation (UVR) in a process that requires the vitamin D receptor. Yet, while mice which do not express the vitamin D receptor are more susceptible to photocarcinogenesis, mice unable to 1α-hydroxylate 25-hydroxyvitamin D to form 1,25D do not show increased susceptibility to UVR-induced skin tumors. A possible explanation is that an alternative pathway, which does not involve 1α-hydroxylation, may produce photoprotective compounds from vitamin D. The cholesterol side chain cleavage enzyme CYP11A1 is expressed in skin and produces 20-hydroxyvitamin D3 (20OHD) as a major product of vitamin D3. We examined whether topical 20OHD would affect UVR-induced DNA damage, inflammatory edema or immune suppression produced in Skh:hr1 mice. Photoprotection by 20OHD at 23 or 46pmol/cm(2) against cyclobutane pyrimidine dimers (DNA lesions) after UVR in mice was highly effective, up to 98±0.8%, (p<0.001) and comparable to that of 1,25D. Sunburn edema measured as skinfold thickness 24h after UVR was also significantly reduced by 20OHD (p<0.001). In studies of contact hypersensitivity (CHS), which is suppressed by UVR, topical application of 20OHD to mice protected against UVR-induced immunosuppression (p<0.05), similar to the effect of 1,25D at similar doses (46±0.6% protection with 20OHD, 44±0.5% with 1,25D). Both UVR-induced DNA damage and immunosuppression contribute to increased susceptibility to UVR-induced skin tumors. This study indicates a potentially anti-photocarcinogenic role of the naturally occurring vitamin D metabolite, 20OHD, which does not depend on 1α-hydroxylation for generation. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

Protection from ultraviolet damage and photocarcinogenesis by vitamin D compounds.

Vitamin D is primarily produced by a photochemical reaction in skin, using the energy of ultraviolet B radiation. Ultraviolet radiation in sunlight is also responsible for several types of DNA damage, immunosuppression and photoaging. A number of adaptive responses are known to occur in skin to increasing UV exposure, including increased pigmentation, increased thickness of the cornified layer of skin and upregulation of DNA repair pathways. In addition to these known responses, there is now sufficient evidence to suggest that the local vitamin D system in skin, which includes local production of the active hormone, 1,25 dihydroxyvitamin D, together with metabolites of over-irradiation products, and vitamin D receptor(s), also provide an adaptive response to UV. The vitamin D system in skin reduces DNA damage, inflammation and photocarcinogenesis. Because vitamin D is made in skin, sun damage is less than it would be otherwise.

Uptake of 25-hydroxyvitamin D by muscle and fat cells.

Vitamin D status, measured as serum 25-hydroxyvitamin D (25OHD) concentration, is determined by rates of input and of degradation. The half-life of 25OHD is surprisingly long for a steroid and much longer than its blood transporter, vitamin D binding protein. There is some evidence to suggest that vitamin D itself is stored in fat, whereas 25OHD concentrations are usually related to muscle-related parameters such as lean body mass and exercise. Both muscle and fat cells come from the mesenchymal cell lineage. We recently published evidence for net uptake of 25OHD into differentiated muscle cells, in a process that was megalin dependent, and speculated that this uptake might contribute to the extended half-life of 25OHD. Whether 25OHD is also taken up into cells of the adipocyte lineage is not clear. In the current study, we used the C2 muscle cell line as a source of myoblasts that were differentiated in culture to myotubes and 3T3-L1 pre-adipocytes that were differentiated into adipocytes in culture. We incubated the cells with trititated 25OHD and measured net uptake 4 and 16h afterwards. Differentiated myotubes took up labeled 25OHD in a time-dependent process to a far greater extent than myoblasts. In contrast, pre-adipocytes, but not differentiated adipocytes, accumulated labeled 25OHD in a time-dependent manner, though to a lesser extent than myotubes. Myotubes, but not myoblasts, showed megalin expression by immunohistochemistry. Pre-adipocytes, but not adipocytes, also showed expression of megalin. Since skeletal muscle consists mainly of differentiated muscle cells, while adipose tissue is mainly differentiated fat cells, it seems likely that muscle, but not fat tissue, provides a large extravascular pool through which 25OHD circulates and that this protects 25OHD from degradation. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Evidence that Notch and Delta expressions have a role in dermal condensate aggregation during wool follicle initiation.

Notch pathway genes have been implicated in the commitment of mesenchymal cells to a wool follicle cell fate. Notch1 and Delta1 transcripts were quantified in fetal skin of fine-woolled (Merino) and strong-woolled (Tukidale) sheep at two time points: either preceding (d56) or during (d70) the first wave of follicle initiation. DIG-labelled probes for both transcripts were localised in the epithelium, some mesenchymal cells, and in the dermal condensates of primordia. The possibility that condensates selectively incorporated Delta1-labelled mesenchymal cells is considered. The involvement of Notch1 in condensate formation was also explored in cultured fetal skin explants and whisker papilla cells using DAPT to block Notch signalling. In its presence, follicle initiation in skin explants was reduced, and the propensity for cultured papilla cells to aggregate was abolished. Results suggest that Notch1 activation is a prerequisite for mesenchymal aggregation. It is speculated that Delta interactions contribute to condensate formation, in vivo.

Vitamin D and death by sunshine.

Exposure to sunlight is the major cause of skin cancer. Ultraviolet radiation (UV) from the sun causes damage to DNA by direct absorption and can cause skin cell death. UV also causes production of reactive oxygen species that may interact with DNA to indirectly cause oxidative DNA damage. UV increases accumulation of p53 in skin cells, which upregulates repair genes but promotes death of irreparably damaged cells. A benefit of sunlight is vitamin D, which is formed following exposure of 7-dehydrocholesterol in skin cells to UV. The relatively inert vitamin D is metabolized to various biologically active compounds, including 1,25-dihydroxyvitamin D3. Therapeutic use of vitamin D compounds has proven beneficial in several cancer types, but more recently these compounds have been shown to prevent UV-induced cell death and DNA damage in human skin cells. Here, we discuss the effects of vitamin D compounds in skin cells that have been exposed to UV. Specifically, we examine the various signaling pathways involved in the vitamin D-induced protection of skin cells from UV.

1α,25-Dihydroxyvitamin D3 reduces several types of UV-induced DNA damage and contributes to photoprotection.

Vitamin D production requires UVB. In turn, we have shown that vitamin D compounds reduce UV-induced damage, including inflammation, sunburn, thymine dimers, the most frequent type of cyclobutane pyrimidine dimer, immunosuppression, and photocarcinogenesis. Our previous studies have shown most of the photoprotective effects by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) occurred through the nongenomic pathway because similar protection was seen with an analog, 1α,25-dihydroxylumistrol3 (JN), which has little ability to alter gene expression and also because a nongenomic antagonist of 1,25(OH)2D3 abolished protection. In the current study, we tested whether this photoprotective effect would extend to other types of DNA damage, and whether this could be demonstrated in human ex vivo skin, as this model would be suited to pre-clinical testing of topical formulations for photoprotection. In particular, using skin explants, we examined a time course for thymine dimers (TDs), the most abundant DNA photolesion, as well as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is a mutagenic DNA base lesion arising from UV-induced oxidative stress, and 8-nitroguanosine (8-NG). Nitric oxide products, known markers for chronic inflammation and carcinogenesis, are also induced by UV. This study showed that 1,25(OH)2D3 significantly reduced TD and 8-NG as early as 30min post UV, and 8-oxodG at 3h post UV, confirming the photoprotective effect of 1,25(OH)2D3 against DNA photoproducts in human skin explants. At least in part, the mechanism of photoprotection by 1,25(OH)2D3 is likely to be through the reduction of reactive nitrogen species and the subsequent reduction in oxidative and nitrosative damage. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Opening of chloride channels by 1α,25-dihydroxyvitamin D3 contributes to photoprotection against UVR-induced thymine dimers in keratinocytes.

UVR produces vitamin D in skin, which is hydroxylated locally to 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). 1,25(OH)(2)D(3) protects skin cells against UVR-induced DNA damage, including thymine dimers, but the mechanism is unknown. As DNA repair is inhibited by nitric oxide (NO) products but facilitated by p53, we examined whether 1,25(OH)(2)D(3) altered the expression of nitrotyrosine, a product of NO, or p53 after UVR in human keratinocytes. 1,25(OH)(2)D(3) and the nongenomic agonist 1α,25-dihydroxylumisterol(3) reduced nitrotyrosine 16 hours after UVR, detected by a sensitive whole-cell ELISA. p53 was enhanced after UVR, and this was further augmented in the presence of 1,25(OH)(2)D(3). DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), a chloride channel blocker previously shown to prevent 1,25(OH)(2)D(3)-induced chloride currents in osteoblasts, had no effect on thymine dimers on its own but prevented the 1,25(OH)(2)D(3)-induced protection against thymine dimers. Independent treatment with DIDS, at concentrations that had no effect on thymine dimers, blocked UVR-induced upregulation of p53. In contrast, reduction of nitrotyrosine remained in keratinocytes treated with 1,25(OH)(2)D(3) and DIDS at concentrations shown to block decreases in post-UVR thymine dimers. These results suggest that 1,25(OH)(2)D(3)-induced chloride currents help protect from UVR-induced thymine dimers, but further increases in p53 or reductions of nitrotyrosine by 1,25(OH)(2)D(3) are unlikely to contribute substantially to this protection.

Novel vitamin D compounds and skin cancer prevention.

As skin cancer is one of the most costly health issues in many countries, particularly in Australia, the possibility that vitamin D compounds might contribute to prevention of this disease is becoming increasingly more attractive to researchers and health communities. In this article, important epidemiologic, mechanistic and experimental data supporting the chemopreventive potential of several vitamin D-related compounds are explored. Evidence of photoprotection by the active hormone, 1α,25dihydroxyvitamin D3, as well as a derivative of an over-irradiation product, lumisterol, a fluorinated analog and bufalin, a potential vitamin D-like compound, are provided. The aim of this article is to understand how vitamin D compounds contribute to UV adaptation and potentially, skin cancer prevention.

1α,25 dihydroxyvitamin D3 enhances cellular defences against UV-induced oxidative and other forms of DNA damage in skin.

DNA damage induced by ultraviolet radiation is the key initiator for skin carcinogenesis since mutations may arise from the photoproducts and it also contributes to photoimmune suppression. The active vitamin D hormone, 1α,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) reduces thymine dimers, the major photoproduct found in human skin after UV exposure, and suppresses the accumulation of nitric oxide derivatives that lead to more toxic reactive nitrogen species (RNS). We examined whether other forms of DNA damage are reduced by 1,25(OH)(2)D(3), and hypothesized that photoprotection by 1,25(OH)(2)D(3) is, in part, due to the suppression of various forms of promutagenic DNA damage, including thymine dimers, through a reduction of genotoxic RNS. Different forms of UV-induced DNA damage were investigated in irradiated skin cells treated with or without 1,25(OH)(2)D(3), or inhibitors of metabolism and inducible nitric oxide synthase. Keratinocytes were also treated with nitric oxide donors in the absence of UV light. DNA damage was assessed by comet assay incorporating site specific DNA repair endonucleases, and by immunohistochemistry using antibodies to thymine dimers or 8-oxo-7,8-dihydro-2'-deoxyguanosine, and quantified by image analysis. Strand breaks in T4 endonuclease V, endonuclease IV and human 8-oxoguanine DNA glycosylase digests increased more than 2-fold in UV irradiated human keratinocytes, and were reduced by 1,25(OH)(2)D(3) treatment after UV exposure, and also by low temperature, sodium azide and an inhibitor of inducible nitric oxide synthase. Conversely, nitric oxide donors induced all three types of DNA damage in the absence of UV. We present data to show that 1,25(OH)(2)D(3) protects skin cells from at least three forms of UV-induced DNA damage, and provide further evidence to support the proposal that a reduction in RNS by 1,25(OH)(2)D(3) is a likely mechanism for its photoprotective effect against oxidative and nitrative DNA damage, as well as cyclobutane pyrimidine dimers.

The role of the vitamin D receptor and ERp57 in photoprotection by 1α,25-dihydroxyvitamin D3.

UV radiation (UVR) is essential for formation of vitamin D(3), which can be hydroxylated locally in the skin to 1α,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)]. Recent studies implicate 1,25-(OH)(2)D(3) in reduction of UVR-induced DNA damage, particularly thymine dimers. There is evidence that photoprotection occurs through the steroid nongenomic pathway for 1,25-(OH)(2)D(3) action. In the current study, we tested the involvement of the classical vitamin D receptor (VDR) and the endoplasmic reticulum stress protein 57 (ERp57), in the mechanisms of photoprotection. The protective effects of 1,25-(OH)(2)D(3) against thymine dimers were abolished in fibroblasts from patients with hereditary vitamin D-resistant rickets that expressed no VDR protein, indicating that the VDR is essential for photoprotection. Photoprotection remained in hereditary vitamin D-resistant rickets fibroblasts expressing a VDR with a defective DNA-binding domain or a mutation in helix H1 of the classical ligand-binding domain, both defects resulting in a failure to mediate genomic responses, implicating nongenomic responses for photoprotection. Ab099, a neutralizing antibody to ERp57, and ERp57 small interfering RNA completely blocked protection against thymine dimers in normal fibroblasts. Co-IP studies showed that the VDR and ERp57 interact in nonnuclear extracts of fibroblasts. 1,25-(OH)(2)D(3) up-regulated expression of the tumor suppressor p53 in normal fibroblasts. This up-regulation of p53, however, was observed in all mutant fibroblasts, including those with no VDR, and with Ab099; therefore, VDR and ERp57 are not essential for p53 regulation. The data implicate the VDR and ERp57 as critical components for actions of 1,25-(OH)(2)D(3) against DNA damage, but the VDR does not require normal DNA binding or classical ligand binding to mediate photoprotection.

A novel model of wound healing in the SCID mouse using a cultured human skin substitute.

Studies of skin graft behaviour in rodent excisional wound models are limited by the dominance of wound contracture and graft sloughing as primary healing responses. To slow skin contraction, polytetrafluoroethylene (Teflon) rings were inserted into dorso-lateral full-thickness wounds in SCID mice. Cultured skin substitutes (OrCel), composed of cultured human keratinocytes and fibroblasts in a bovine collagen sponge, were implanted within the rings. Examination and histology of grafts 14 days later showed graft take in four of six recipients, with 90% epithelialization and wound contraction of 31-47%. Immunohistochemical studies, using human-specific antisera to distinguish graft from host tissues, showed that regenerated tissue was predominantly human. Staining with anticytokeratin, revealed a multilayered, stratified neoepidermis. HBG were identified in keratinocytes in all epidermal layers. Langerhans cells were absent. Antihuman vimentin, used as a fibroblast marker, confirmed that cells of the neodermis were primarily of human origin. Neoepidermal keratinocytes, primarily in the basal and suprabasal layers, were also stained. Results suggest that the poly(tetrafluoroethylene) ring inhibited graft sloughing and provided a more favourable environment for the skin substitute to regenerate a substantially normal human skin.

ErbB receptors mediate both migratory and proliferative activities in human melanocytes and melanoma cells.

Epidermal growth factor receptor (EGFR) activation by transforming growth factor alpha (TGFalpha) has been implicated in autocrine growth in melanoma, but does not alter melanocyte proliferation. This raises the possibility that different signalling pathways are activated via EGFR or ErbB receptors. Here, we demonstrate that ErbB2, ErbB3 and ErbB4 are expressed in cultured human melanocytes. Western analyses with receptor-specific antisera revealed protein bands with Mr values of 185 and 160 kDa, corresponding to ErbB2 and ErbB3, respectively. Blots probed with ErbB4 antibodies showed bands with Mr values of 180, 120 and 80 kDa, corresponding to the receptor and its reported variants. Two malignant melanoma cell lines expressed ErbB2 and ErbB3, but not the full-length ErbB4 receptor. As TGFalpha binds to EGFR and the heregulins (HRG) bind to ErbB3 and ErbB4, these growth factors were examined for effects on receptor activation and on cell growth and motility in a scratch wound closure assay. In normal melanocytes, HRGbeta1 activated the phosphorylation of tyrosine residues of proteins that immunoprecipitated with EGFR and ErbB4 antisera, and significantly enhanced cell migration but not proliferation. Neither TGFalpha nor HRGalpha1 promoted migration or growth in normal melanocytes. By contrast, TGFalpha stimulated migratory activity in the MM96L cell line, but not in the MELJG line, whereas HRGbeta1 significantly enhanced cell growth, but not migration, in both malignant cell lines. The apparent transition of HRGbeta1 from a migratory to a proliferative function after malignant transformation, and the change in TGFalpha from a non-migratory to a migratory activity in one melanoma line, suggests multiple switches in ErbB signalling pathways via EGFR/ErbB heterodimer formation.