Special care dentistry: how prepared are we?

AIM: To assess and evaluate the confidence of the undergraduate dental team in Irish dental schools regarding their training in the field of special care dentistry (SCD). METHOD: A questionnaire was distributed amongst the undergraduate dental teams within Cork University Dental School and Hospital (CUDSH) and Dublin Dental University Hospital (DDUH). The data were analysed to assess for differences between the median responses of the various groups. RESULTS: A total of 139 students participated in the study; 82 from DDUH (59 dental science students, seven dental hygiene students and 16 dental nursing students) and 57 from CUDSH (43 dental science students and 14 dental hygiene students). Twenty-two per cent (n = 30) of respondents agreed that their training had been sufficient in providing treatment or assistance for patients requiring SCD. Seventy-five per cent agreed that more emphasis should be placed on clinical training in SCD (n = 76). Thirty-one per cent of students anticipated a high level of confidence in treating patients requiring SCD upon graduation (n = 43), with dental science students feeling the least prepared amongst the dental team. CONCLUSION: Confidence levels of the undergraduate dental team were low with regard to SCD. The existing hands-on clinical training in SCD was deemed insufficient by the respondents. Students believed more emphasis should be placed on SCD training in the undergraduate curriculum.

A RA-dependent, tumour-growth suppressive transcription complex is the target of the PML-RARalpha and T18 oncoproteins.

PML and Tif1a are fused to RARA and Braf, respectively, resulting in the production of PML-RARalpha and Tif1alpha-B-Raf (T18) oncoproteins. Here we show that PML, Tif1alpha and RXRalpha/RARalpha function together in a transcription complex that is dependent on retinoic acid (RA). We found that PML acts as a ligand-dependent coactivator of RXRalpha/RARalpha. PML interacts with Tif1alpha and CBP. In Pml-/- cells, the RA-dependent induction of genes such as RARB2 and the ability of Tif1alpha and CBP to act as transcriptional coactivators on RA are impaired. We show that both PML and Tif1alpha are growth suppressors required for the growth-inhibitory activity of RA. T18, similar to PML-RARalpha, disrupts the RA-dependent activity of this complex in a dominant-negative manner resulting in a growth advantage. Our data define a new pathway for the control of cell growth and tumorigenesis, and provide a new model for the pathogenesis of acute promyelocytic leukaemia (APL).

The value of education in special care dentistry as a means of reducing inequalities in oral health.

People with disability are subject to inequality in oral health both in terms of prevalence of disease and unmet healthcare needs. Over 18% of the global population is living with moderate to severe functional problems related to disability, and a large proportion of these persons will require Special Care Dentistry at some point in their lifetime. It is estimated that 90% of people requiring Special Care Dentistry should be able to access treatment in a local, primary care setting. Provision of such primary care is only possible through the education and training of dentists. The literature suggests that it is vital for the dental team to develop the necessary skills and gain experience treating people with special needs in order to ensure access to the provision of oral health care. Education in Special Care Dentistry worldwide might be improved by the development of a recognised academic and clinical discipline and by providing international curricula guidelines based on the International Classification of Functioning, Disability and Health (ICF, WHO). This article aims to discuss the role and value of promoting and harmonising education in Special Care Dentistry as a means of reducing inequalities in oral health.

Mediator complexes and transcription.

Over the past decade, various components of the transcription machinery have been identified as potential targets for activators. Recently, metazoan versions of yeast Mediator have been isolated and found to act as key coactivators to many transcription factors. Recent work has defined the composition, function and biology of metazoan mediator complexes, which has led us to propose a new nomenclature for the variously named versions of the mediator complex.

Prediction of nonSVR to therapy with pegylated interferon-alpha2a and ribavirin in chronic hepatitis C genotype 1 patients after 4, 8 and 12 weeks of treatment.

In patients with chronic hepatitis C genotype 1, the current algorithm for treatment discontinuation is based on no early virological response (<2 log decline in hepatitis C virus (HCV)-RNA) at 12 weeks. It is important to determine whether prediction of nonsustained virological response (NR) before 12 weeks can be robustly obtained by statistical methods. We used longitudinal discriminant analysis (LDA) to build and cross-validate models including baseline patient characteristics and measurements of serum HCV-RNA in the first 4, 8 or 12 weeks of treatment. The performance of each model was evaluated by the partial AUC (PA) index, exploring the accuracy of prediction in the range of high negative predictive values. Models were compared by computing 95% confidence intervals for the difference in PA indices. NR was best predicted before week 12 by a single HCV-RNA measurement at week 8 taken together with gender, BMI and age (W8 model, PA index = 0.857). This model was not inferior to models that included a measurement at week 12 (PA index = 0.831). The best model obtained with LDA within the first 4 weeks, which included measurements at days 4, 8 and at week 4, was found to be inferior to the week 8 model (PA index = 0.796). These results indicate that lack of sustained viral response is best predicted after 8 weeks of treatment and that waiting until 12 weeks does not improve the prediction.

A modified approach to estimating sample size for simple logistic regression with one continuous covariate.

Different methods for the calculation of sample size for simple logistic regression (LR) with one normally distributed continuous covariate give different results. Sometimes the difference can be large. Furthermore, some methods require the user to specify the prevalence of cases when the covariate equals its population mean, rather than the more natural population prevalence. We focus on two commonly used methods and show through simulations that the power for a given sample size may differ substantially from the nominal value for one method, especially when the covariate effect is large, while the other method performs poorly if the user provides the population prevalence instead of the required parameter. We propose a modification of the method of Hsieh et al. that requires specification of the population prevalence and that employs Schouten's sample size formula for a t-test with unequal variances and group sizes. This approach appears to increase the accuracy of the sample size estimates for LR with one continuous covariate.

In vitro transcription enhancement by purified derivatives of the glucocorticoid receptor.

Mammalian glucocorticoid receptors enhance transcription from linked promoters by binding to glucocorticoid response element (GRE) DNA sequences. Understanding the mechanism of receptor action will require biochemical studies with purified components. Enhancement was observed in vitro with derivatives of the receptor that were expressed in Escherichia coli, purified, and added to a cell-free extract from Drosophila embryo nuclei. Transcription from promoters linked to one or multiple GREs was selectively enhanced by as much as six times. The effect was weaker with only one GRE, and enhancement was abolished by a point mutation that inactivates the GRE in vivo.

Sparing of the brain in neonatal undernutrition: amino acid transport and incorporation into brain and muscle.

Rates of tyrosine and lysine transport and incorporation into protein were measured in control and undernourished weanling rats. Undernutrition was induced by feeding lactating dams a low protein diet (12 percent casein) from birth to day 21. At weaning, body and brain weights of undernourished rats were 50 percent and 88 percent, respectively, of control values. Lysine and tyrosine transport rates into skeletal muscle were reduced by over 75 percent, more than twice the reduction seen in brain. Rates of amino acid incorporation into muscle protein were reduced by approximately 50 percent; the change in rate of incorporation into brain protein was not statistically significant. These data indicate that, in spite of marked retardation of amino acid transport into brain, the brain seems fully capable of maintaining normal rates of protein synthesis.

Solution structure of the glucocorticoid receptor DNA-binding domain.

The three-dimensional structure of the DNA-binding domain (DBD) of the glucocorticoid receptor has been determined by nuclear magnetic resonance spectroscopy and distance geometry. The structure of a 71-residue protein fragment containing two "zinc finger" domains is based on a large set of proton-proton distances derived from nuclear Overhauser enhancement spectra, hydrogen bonds in previously identified secondary structure elements, and coordination of two zinc atoms by conserved cysteine residues. The DBD is found to consist of a globular body from which the finger regions extend. A model of the dimeric complex between the DBD and the glucocorticoid response element is proposed. The model is consistent with previous results indicating that specific amino acid residues of the DBD are involved in protein-DNA and protein-protein interactions.

Nuclear receptor cofactors as chromatin remodelers.

Nuclear receptors regulate transcription in direct response to their cognate hormonal ligands. Ligand binding leads to the dissociation of corepressors and the recruitment of coactivators. Many of these factors, acting in large complexes, have emerged as chromatin remodelers through intrinsic histone modifying activities or through other novel functions. In addition, other ligand-recruited complexes appear to act more directly on the transcriptional apparatus, suggesting that transcriptional regulation by nuclear receptors may involve a process of both chromatin alterations and direct recruitment of key initiation components at regulated promoters.

A historical cohort study on glycemic-control and cancer-risk among patients with diabetes.

AIMS: This population-based historical cohort study examined whether poor glycemic-control (i.e., high glucose and HbA1c blood levels) in patients with diabetes is associated with cancer-risk. METHODS: From a large healthcare database, patients aged 21-89 years, diagnosed with diabetes before January 2002 (prevalent) or during 2002-2010 (incident), were followed for cancer during 2004-2012 (excluding cancers diagnosed within the first 2 years since diabetes diagnosis). Risks of selected cancers (all-sites, colon, breast, lung, prostate, pancreas and liver) were estimated according to glycemic-control in a Cox regression model with time-dependent covariates, adjusted for age, sex, ethnic origin, socioeconomic status, smoking and parity. Missing glucose or HbA1c values were imputed. RESULTS: Among 440,000 patients included in our analysis, cancer was detected more than 2 years after diabetes diagnosis in 26,887 patients (6%) during the follow-up period. Associations of poor glycemic-control with all-sites cancer and most specific cancers were either null or only weak (hazard ratios (HRs) for a 1% HbA1c or a 30 mg/dl glucose increase between 0.94 and 1.09). Exceptions were pancreatic cancer, for which there was a strong positive association (HRs: 1.26-1.51), and prostate cancer, for which there was a moderate negative association (HRs: 0.85-0.96). CONCLUSION: Overall, poor glycemic-control appears to be only weakly associated with cancer-risk, if at all. A substantial part of the positive association with pancreatic cancer is attributable to reverse causation, with the cancer causing poorer glycemic-control prior to its diagnosis. The negative association with prostate cancer may be related to lower PSA levels in those with poor control.

Tyrosine hydroxylase in human adrenal and pheochromocytoma: localization, kinetics, and catecholamine inhibition.

The properties of partially purified tyrosine hydroxylase from six pheochromocytomas were compared with partially purified normal human and bovine adrenal medulla enzyme. Substrate and inhibition kinetics, cofactor requirements, and intracellular localization of the enzyme from normal and tumor chromaffin tissue of humans were similar, as was the amount of enzyme activity per gram of tissue. Contrary to previous reports, the sensitivity to catecholamine inhibition of the pheochromocytoma enzyme from the six tumors studied was similar to that of both human and bovine adrenal medulla tyrosine hydroxylase. These results suggest that the excessive synthesis and secretion of catecholamines in some pheochromocytomas is not the result of a reduced sensitivity of tyrosine hydroxylase to catecholamine inhibition.

Selective effects of ligands on vitamin D3 receptor- and retinoid X receptor-mediated gene activation in vivo.

Steroid/nuclear hormone receptors are ligand-regulated transcription f factors that play key roles in cell regulation, differentiation, and oncogenesis. Many nuclear receptors, including the human 1,25-dihydroxyvitamin D3 receptor (VDR), bind cooperatively to DNA either as homodimers or as heterodimers with the 9-cis retinoic acid (RA) receptor (retinoid X-receptor [RXR]). We have previously reported that the ligands for VDR and RXR can differentially modulate the affinity of the receptors' interaction with DNA in vitro, primarily by modulating the dimerization status of these receptors. These experiments suggested a complex interaction between VDR and RXR and their respective ligands on inducible target genes in vivo. To examine these effects in cells, we used a transient-transfection strategy whereby we simultaneously introduced two different reporter plasmids that are selectively inducible by each ligand. Although VDR can bind as a homodimer to the osteopontin gene vitamin D response element, we find that a RXR-VDR heterodimer must be the transactivating species from the element in vivo, since RXR enhances and 9-cis RA and other RXR-specific ligands attenuate this induction. Conversely, when VDR is overexpressed, vitamin D3 attenuates 9-cis RA induction from an RXR-responsive element. These effects, however, appear to be very sensitive to both the relative ratios of the two receptors and their respective target elements. Functional RXR-VDR complexes are strictly dependent on the DNA-binding polarity. Chimeric versions of VDR and RXR were also constructed to examine the putative activities of homodimeric receptors; a VDR chimera can transactivate in the absence of RXR, demonstrating that VDR has intrinsic transactivation properties. Taken together, these results establish a complex, sensitive cross talk in vivo between two ligands and their receptors that signal through two distinct endocrine pathways.

Ligand modulates the conversion of DNA-bound vitamin D3 receptor (VDR) homodimers into VDR-retinoid X receptor heterodimers.

Protein dimerization facilitates cooperative, high-affinity interactions with DNA. Nuclear hormone receptors, for example, bind either as homodimers or as heterodimers with retinoid X receptors (RXR) to half-site repeats that are stabilized by protein-protein interactions mediated by residues within both the DNA- and ligand-binding domains. In vivo, ligand binding among the subfamily of steroid receptors unmasks the nuclear localization and DNA-binding domains from a complex with auxiliary factors such as the heat shock proteins. However, the role of ligand is less clear among nuclear receptors, since they are constitutively localized to the nucleus and are presumably associated with DNA in the absence of ligand. In this study, we have begun to explore the role of the ligand in vitamin D3 receptor (VDR) function by examining its effect on receptor homodimer and heterodimer formation. Our results demonstrate that VDR is a monomer in solution; VDR binding to a specific DNA element leads to the formation of a homodimeric complex through a monomeric intermediate. We find that 1,25-dihydroxyvitamin D3, the ligand for VDR, decreases the amount of the DNA-bound VDR homodimer complex. It does so by significantly decreasing the rate of conversion of DNA-bound monomer to homodimer and at the same time enhancing the dissociation of the dimeric complex. This effectively stabilizes the bound monomeric species, which in turn serves to favor the formation of a VDR-RXR heterodimer. The ligand for RXR, 9-cis retinoic acid, has the opposite effect of destabilizing the heterodimeric-DNA complex. These results may explain how a nuclear receptor can bind DNA constitutively but still act to regulate transcription in a fully hormone-dependent manner.

Discrete roles for peroxisome proliferator-activated receptor gamma and retinoid X receptor in recruiting nuclear receptor coactivators.

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a major role in adipogenesis. PPARgamma binds to DNA as a heterodimer with retinoid X receptor (RXR), and PPARgamma-RXR can be activated by ligands specific for either receptor; the presence of both ligands can result in a cooperative effect on the transactivation of target genes. How these ligands mediate transactivation, however, remains unclear. PPARgamma is known to interact with both the p160/SRC-1 family of coactivators and the distinct, multisubunit coactivator complex called DRIP. A single DRIP subunit, DRIP205 (TRAP220, PBP), binds directly to PPARgamma. Here we report that PPARgamma and RXR selectively interacted with DRIP205 and p160 proteins in a ligand-dependent manner. At physiological concentrations, RXR-specific ligands only induced p160 binding to RXR, and PPARgamma-specific ligands exclusively recruited DRIP205 but not p160 coactivators to PPARgamma. This selectivity was not observed in interaction assays off DNA, implying that the specificity of coactivator binding in response to ligand is strongly influenced by the allosteric effects of DNA-bound heterodimers. These coactivator-selective effects were also observed in transient-transfection assays in the presence of overexpressed p160 or DRIP coactivators. The results suggest that the cooperative effects of PPARgamma- and RXR-specific ligands may occur at the level of selective coactivator recruitment.

Retinoid X receptor:vitamin D3 receptor heterodimers promote stable preinitiation complex formation and direct 1,25-dihydroxyvitamin D3-dependent cell-free transcription.

The numerous members of the steroid/nuclear hormone receptor superfamily act as direct transducers of circulating signals, such as steroids, thyroid hormone, and vitamin or lipid metabolites, and modulate the transcription of specific target genes, primarily as dimeric complexes. The receptors for 9-cis retinoic acid and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], RXR and VDR, respectively, as members of this superfamily, form a heterodimeric complex and bind cooperatively to vitamin D responsive elements (VDREs) to activate or repress the transcription of a multitude of genes which regulate a variety of physiological functions. To directly investigate RXR- and VDR-mediated transactivation, we developed a cell-free transcription system for 1,25(OH)2D3 signaling by utilizing crude nuclear extracts and a G-free cassette-based assay. Transcriptional enhancement in vitro was dependent on purified, exogenous RXR and VDR and was responsive to physiological concentrations of 1,25(OH)2D3. We found that RXR and VDR transactivated selectively from VDRE-linked templates exclusively as a heterodimeric complex, since neither receptor alone enhanced transcription in vitro. By the addition of low concentrations of the anionic detergent Sarkosyl to limit cell-free transcription to a single round and the use of agarose gel mobility shift experiments to assay factor complex assembly, we observed that 1,25(OH)2D3 enhanced RXR:VDR-mediated stabilization or assembly of preinitiation complexes to effect transcriptional enhancement from VDRE-linked promoter-containing DNA.

A two-hit mechanism for vitamin D3-mediated transcriptional repression of the granulocyte-macrophage colony-stimulating factor gene: vitamin D receptor competes for DNA binding with NFAT1 and stabilizes c-Jun.

We previously described a control element in the granulocyte-macrophage colony-stimulating factor (GM-CSF) enhancer that is necessary and sufficient to mediate both transcriptional activation in response to T-cell stimuli and transcriptional repression by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] through the vitamin D3 receptor (VDR). This DNA element is a composite site that is recognized by both Fos-Jun and NFAT1; it is directly bound by VDR in the absence of a retinoid X receptor as an apparent monomer, and it is bound in a unique tertiary conformation. We describe here the mechanism by which VDR elicits its transcriptional inhibitory effect. Firstly, VDR outcompetes NFAT1 for binding to the composite site. Overexpression of NFAT1 in vivo by transient transfection is able to relieve the 1,25(OH)2D3-dependent repression. Secondly, VDR stabilizes the binding of a Jun-Fos heterodimer to the adjacent AP-1 portion of the element. This appears to occur through a direct interaction between VDR and c-Jun, as demonstrated in vitro by direct glutathione S-transferase coprecipitation assays. In vivo, overexpression of c-Jun, but not c-Fos, leads to a rescue of the 1, 25(OH)2D3-mediated repression. Transfected FLAG-VDR bound to the NFAT1-AP-1 DNA binding element can be selectively precipitated from nuclear extracts that are made from cells treated with activating agents in the presence of 1,25(OH)2D3. VDR is not detected in the complex in the absence of the ligand. Thus, VDR acts selectively on the two components required for activation of this promoter/enhancer: it competes with NFAT1 for binding to the composite site, positioning itself adjacent to Jun-Fos on the DNA. Co-occupancy apparently leads to an inhibitory effect on c-Jun's transactivation function. These two events mediated by VDR effectively block the NFAT1-AP-1 activation complex, resulting in an attenuation of activated GM-CSF transcription.

Transcriptional repression of the interleukin-2 gene by vitamin D3: direct inhibition of NFATp/AP-1 complex formation by a nuclear hormone receptor.

T-lymphocyte proliferation is suppressed by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D3, and is associated with a decrease in interleukin 2 (IL-2), gamma interferon, and granulocyte-macrophage colony-stimulating factor mRNA levels. We report here that 1,25(OH)2D3-mediated repression in Jurkat cells is cycloheximide resistant, suggesting that it is a direct transcriptional repressive effect on IL-2 expression by the vitamin D3 receptor (VDR). We therefore examined vitamin D3-mediated repression of activated IL-2 expression by cotransfecting Jurkat cells with IL-2 promoter/reporter constructs and a VDR overexpression vector and by DNA binding. We delineated an element conferring both DNA binding by the receptor in vitro and 1,25(OH)2D3-mediated repression in vivo to a short 40-bp region encompassing an important positive regulatory element, NF-AT-1, which is bound by a T-cell-specific transcription factor, NFATp, as well as by AP-1. VDR DNA-binding mutants were unable to either bind to this element in vitro or repress in vivo; the VDR DNA-binding domain alone, however, bound the element but also could not repress IL-2 expression. These results indicate that DNA binding by VDR is necessary but not sufficient to mediate IL-2 repression. By combining partially purified proteins in vitro, we observed the loss of the bound NFATp/AP-1-DNA complex upon inclusion of VDR or VDR-retinoid X receptor. Order of addition and off-rate experiments indicate that the VDR-retinoid X receptor heterodimer blocks NFATp/AP-1 complex formation and then stably associates with the NF-AT-1 element. This direct inhibition by a nuclear hormone receptor of transcriptional activators of the IL-2 gene may provide a mechanistic explanation of how vitamin derivatives can act as potent immunosuppressive agents.

A differential screen for ligand-regulated genes: identification of HoxA10 as a target of vitamin D3 induction in myeloid leukemic cells.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the hormonal ligand for vitamin D3, is a potent inducer of myeloid-leukemic-cell differentiation. Such cells differentiate exclusively into monocytes/macrophages in response to this ligand. Since 1,25(OH)2D3 transduces its hormone signal through the vitamin D3 receptor (VDR), a ligand-modulated transcription factor and member of the nuclear hormone receptor superfamily, we sought to identify direct VDR target genes induced during this differentiation process. To do so, we applied a modified differential screen with a nascent-RNA purification strategy using biases for immediate-early-response genes induced by 1,25(OH)2D3 in the myelomonocytic cell line U937. Using this screen, we had previously identified p21Waf1/Cip1 as a gene transcriptionally induced by 1,25(OH)2D3 and demonstrated that this induction facilitates the differentiation of U937 cells into monocytes/macrophages (24). Here, we describe in detail our differential screen strategy and the identification and isolation of 20 1,25(OH)2D3-inducible genes or unknown cDNAs by means of this screen. One gene newly identified as a target of VDR regulation in myeloid cells is the homeobox HoxA10 gene. HoxA10 protein may act as a general regulator of cell growth, since overexpression of HoxA10 facilitated the differentiation of U937 cells into monocytes/macrophages independent of 1,25(OH)2D3 and acted to strongly inhibit the growth of the breast cancer cell line MCF-7 by arresting these cells in G1.

The DRIP complex and SRC-1/p160 coactivators share similar nuclear receptor binding determinants but constitute functionally distinct complexes.

Transcriptional activation requires both access to DNA assembled as chromatin and functional contact with components of the basal transcription machinery. Using the hormone-bound vitamin D(3) receptor (VDR) ligand binding domain (LBD) as an affinity matrix, we previously identified a novel multisubunit coactivator complex, DRIP (VDR-interacting proteins), required for transcriptional activation by nuclear receptors and several other transcription factors. In this report, we characterize the nuclear receptor binding features of DRIP205, a key subunit of the DRIP complex, that interacts directly with VDR and thyroid hormone receptor in response to ligand and anchors the other DRIP subunits to the nuclear receptor LBD. In common with other nuclear receptor coactivators, DRIP205 interaction occurs through one of two LXXLL motifs and requires the receptor's AF-2 subdomain. Although the second motif of DRIP205 is required only for VDR binding in vitro, both motifs are used in the context of an retinoid X receptor-VDR heterodimer on DNA and in transactivation in vivo. We demonstrate that both endogenous p160 coactivators and DRIP complexes bind to the VDR LBD from nuclear extracts through similar sequence requirements, but they do so as distinct complexes. Moreover, in contrast to the p160 family of coactivators, the DRIP complex is devoid of any histone acetyltransferase activity. The results demonstrate that different coactivator complexes with distinct functions bind to the same transactivation region of nuclear receptors, suggesting that they are both required for transcription activation by nuclear receptors.