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What is the central question of this study? Chronotype reflects differences in circadian-mediated metabolic and hormonal profiles. But, does resting and/or exercise fuel use differ in early versus late chronotype as it relates to insulin sensitivity? What are the main finding and its importance? Early chronotypes with metabolic syndrome utilized more fat during rest and exercise independent of aerobic fitness when compared with late chronotypes. Early chronotypes were also more physically active throughout the day. Greater fat use was related to non-oxidative glucose disposal. These findings suggest that early chronotypes have differences in fuel selection that associate with type 2 diabetes risk.Early chronotypes (ECs) are often insulin-sensitive, in part, due to physical activity behaviour. It is unclear, however, if chronotypes differ in resting and/or exercise fuel oxidation in relation to insulin action. Using the Morningness-Eveningness Questionnaire (MEQ), adults with metabolic syndrome (ATP III criteria) were classified as EC (MEQ = 63.7 ± 0.9, n = 24 (19F), 54.2 ± 1.2 years) or late chronotype (LC; MEQ = 47.2 ± 1.4, n = 27 (23F), 55.3 ± 1.5 years). Carbohydrate (CHO) and fat oxidation (FOX, indirect calorimetry) were determined at rest, 55% and 85%

Within regenerative endodontics, exciting opportunities exist for the development of next-generation targeted biomaterials that harness epigenetic machinery, including microRNAs (miRNAs), histone acetylation, and DNA methylation, which are used to control pulpitis and to stimulate repair. Although histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) induce mineralisation in dental pulp cell (DPC) populations, their interaction with miRNAs during DPC mineralisation is not known. Here, small RNA sequencing and bioinformatic analysis were used to establish a miRNA expression profile for mineralising DPCs in culture. Additionally, the effects of a HDACi, suberoylanilide hydroxamic acid (SAHA), and a DNMTi, 5-aza-2′-deoxycytidine (5-AZA-CdR), on miRNA expression, as well as DPC mineralisation and proliferation, were analysed. Both inhibitors increased mineralisation. However, they reduced cell growth. Epigenetically-enhanced mineralisation was accompanied by widespread changes in miRNA expression. Bioinformatic analysis identified many differentially expressed mature miRNAs that were suggested to have roles in mineralisation and stem cell differentiation, including regulation of the Wnt and MAPK pathways. Selected candidate miRNAs were demonstrated by qRT-PCR to be differentially regulated at various time points in mineralising DPC cultures treated with SAHA or 5-AZA-CdR. These data validated the RNA sequencing analysis and highlighted an increased and dynamic interaction between miRNA and epigenetic modifiers during the DPC reparative processes.

Objective To compare proteomics and biological function of human dentin matrix molecules (hDMMs) and bovine dentin matrix molecules (bDMMs). Design Dentin powder from human or bovine teeth (n = 4) was demineralized in 10% (v/v) ethylenediaminetetraacetic acid for 7 days. The extracts were dialyzed, lyophilized and proteins were characterized using liquid chromatography-tandem mass spectrometry and shotgun proteomic analysis. To study biological function, mouse-derived undifferentiated dental pulp cells (OD21) were treated with 0.01, 0.1 or 1 μg/mL of hDMMs or bDMMs and proliferation was measured after 24 hours and 48 hours using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell migration was assessed after 24 hours using a Boyden chamber. Alizarin Red S staining was used to evaluate mineral formation. Results There were 307 proteins identified, of which 93 proteins were common to both species. Gene Ontology functional analysis demonstrated similar pattern of biological process in both species which consisted mainly of tissue development and biomineralization. hDMMs and bDMMs both enhanced cell proliferation. After 24 hours, all concentrations of bDMMs promoted cell proliferation (p ≤ 0.05), while hDMMs did not affect proliferation. After 48 hours, groups with 1μg/mL of bDMMs and 0.01μg/mL of hDMMs had increased cell proliferation compared to control (p ≤ 0.0001). All concentrations of hDMMs and bDMMs enhanced cell migration and mineralization (p ≤ 0.0001). Conclusion bDMMs has similar biological functions as hDMMs. Moreover, bDMMs stimulated cell proliferation, migration and differentiation similar to hDMMs.

Background Junior doctors undertake a significant amount of prescribing; however, they are not well prepared for this, and report they would like more training in their undergraduate courses. To address this we tested a pharmacist-led prescribing program for final-year medical students. Methods Sixteen final-year students took part in the program. The program involved students writing prescriptions and getting feedback from clinical pharmacists, undertaking prescribing and calculation tutorials, and spending time in the pharmacy department. Evaluation included a pre- and post-assessment of their confidence and skills in prescribing, and a feedback session discussing the strengths and weakness of the program, and their perceptions about the role of pharmacists. Changes in the pre- and post-assessment of confidence and skills were examined with permutation and Mann-Whitney U tests. Results There was a significant improvement in students’ confidence in prescribing, and a small but consistent improvement in prescribing skills. Of note, no student prescribed inappropriately and potentially harmfully after the program. Participants were positive about the program, and indicated a better understanding about the pharmacists’ role and their ability to support them as junior doctors. Conclusions This study has shown the potential effect of a pharmacist-led prescribing program on the skills and confidence in prescribing by medical students. It provided an interprofessional teaching opportunity, preparing students for a team-based approach to patient management. Electronic supplementary material The online version of this article (10.1186/s12909-019-1486-1) contains supplementary material, which is available to authorized users.

The effects of cryopreservation on mesenchymal stem cell (MSC) phenotype are not well documented; however this process is of increasing importance for regenerative therapies. This study examined the effect of cryopreservation (10% dimethyl-sulfoxide) on the morphology, viability, gene-expression and relative proportion of MSC surface-markers on cells derived from rat adipose, bone marrow and dental pulp. Cryopreservation significantly reduced the number of viable cells in bone marrow and dental pulp cell populations but had no observable effect on adipose cells. Flow cytometry analysis demonstrated significant increases in the relative expression of MSC surface-markers, CD90 and CD29/CD90 following cryopreservation. sqRT-PCR analysis of MSC gene-expression demonstrated increases in pluripotent markers for adipose and dental pulp, together with significant tissue-specific increases in CD44, CD73–CD105 following cryopreservation. Cells isolated from different tissue sources did not respond equally to cryopreservation with adipose tissue representing a more robust source of MSCs.

Stem-cell-based therapies provide a biological basis for the regeneration of mineralised tissues. Stem cells isolated from adipose tissue (ADSCs), bone marrow (BMSCs) and dental pulp (DPSCs) have the capacity to form mineralised tissue. However, studies comparing the capacity of ADSCs with BMSCs and DPSCs for mineralised tissue engineering are lacking, and their ability to regenerate dental tissues has not been fully explored. Characterisation of the cells using fluorescence-activated cell sorting and semi-quantitative reverse transcription PCR for MSC markers indicated that they were immunophenotypically similar. Alizarin red (AR) staining and micro-computed tomography (µCT) analyses demonstrated that the osteogenic potential of DPSCs was significantly greater than that of BMSCs and ADSCs. Scanning electron microscopy and AR staining showed that the pattern of mineralisation in DPSC cultures differed from ADSCs and BMSCs, with DPSC cultures lacking defined mineralised nodules and instead forming a diffuse layer of low-density mineral. Dentine matrix components (DMCs) were used to promote dentinogenic differentiation. Their addition to cultures resulted in increased amounts of mineral deposited in all three cultures and significantly increased the density of mineral deposited in BMSC cultures, as determined by µCT analysis. Addition of DMCs also increased the relative gene expression levels of the dentinogenic markers dentine sialophosphoprotein and dentine matrix protein 1 in ADSC and BMSC cultures. In conclusion, DPSCs show the greatest potential to produce a comparatively high volume of mineralised matrix; however, both dentinogenesis and mineral volume was enhanced in ADSC and BMSC cultures by DMCs, suggesting that these cells show promise for regenerative dental therapies.

Objective Dentine sialophosphoprotein (DSPP) was initially thought to be unique for dentine formation during tooth development, whilst recent reports have shown a much broader expression pattern such as in bone, periodontium and inner ear. Our goal was to explore its expression and potential impact during early nasal cartilage formation in comparison with tooth development. Study design We investigated DSPP expression in the nasal cartilage by immunohistochemistry and in situ hybridisation. We also cloned a 719 bp partial DSPP cDNA from nasal cartilage and analysed its homology to the published mouse DSPP cDNA sequence. In addition, quantitative RT-PCR was undertaken to compare the expression pattern of DSPP in nasal cartilage and tooth germs during embryonic development. Results The expression of DSPP in mouse nasal chondrocytes was detected using in situ hybridisation and immunohistochemical staining. The quantitative RT-PCR data showed that expression levels of DSPP in nasal cartilage are similar to that of tooth: low at E18, and increased during development with the peak level at P3. Furthermore, DSPP levels in nasal cartilage are lower than tooth but higher than bone. Conclusion DSPP is expressed in nasal cartilage, and a similar temporal expression pattern in cartilage and tooth indicates the potential importance of DSPP during development.

As a mineralised connective tissue, dentine is well adapted to its functional role as a major structural component of the tooth. Although similar in composition to bone, dentine matrix is not remodelled physiologically and traditionally, has been regarded as a rather inert tissue. Nevertheless, dentine–pulp demonstrates strong regenerative potential which allows it to respond to disease and traumatic injury. Such responses are strongly influenced by cell–matrix interactions and modified by disease processes, including infection and inflammation. The identification of many bioactive molecules bound within dentine matrix has allowed their potential involvement in regenerative and other tissue responses to be better understood and new opportunities to be recognised for novel clinical therapies.

Background information. ADM (adrenomedullin) has pleiotropic effects, including regulation of inflammation, infection, angiogenesis, mineralized-tissue formation and development. Recently, we demonstrated up-regulation of the ADM transcript in diseased pulpal tissue while the protein is sequestered within the dentine extracellular matrix during dentinogenesis. The present study aimed to characterize ADM localization during rodent dental tissue development and determine its potential effects on dental cells. Finally, we sought to profile ADM transcript levels in adult organs and tissues to compare its expression in teeth relative to other tissues. Results. Immunohistochemical analysis of developmental rat oral tissues indicated that, at E16 (embryonic day 16), ADM was present in dental epithelium and, by El 8, ADM localized to the dental papilla and inner and outer dental epithelia. By E20, ADM was detected in secretory odontoblasts and ameloblasts and exhibited a similar expression profile to that of the key dentinogenesis signalling molecule, TGF-beta 1 (transforming growth factor-beta 1). Cell growth analysis in the dental MDPC-23, OD-21 and control 3T3 cell lines exposed to ADM (range 10(-15)-10(-7) M) together with EDTA-extracted DMPs (dentine matrix proteins) (range 0.00001-1000 mg/ml) containing comparable concentrations of ADM demonstrated that ADM stimulated a biphasic response in dental cell growth, comparable with that of DMPs, with peak stimulation observed at similar to 10(-11) M. For mineralization analysis, cell lines were exposed to combinations of 50 mu g/ml ascorbic acid, 10 mM beta-G (beta-glycerophosphate), 10(-8) M DEX (dexamethasone) and ADM (range 10-15-10-7 M). The results demonstrated that ADM could substitute for DEX to stimulate mineralization. Postnatally, multiple tissue expression profiling indicated abundant ADM levels in tongue and pulpal tissues. Conclusions. During oral and dental tissue development ADM initially localizes to epithelial tissue, whereas during later stages it is present in mineralized secreting cells, including odontoblasts. ADM may regulate proliferation and mineralization processes during development, whereas, in adulthood, it may be important for maintaining dental tissue homoeostasis.

Objective Vascular endothelial growth factor (VEGF) has been implicated in the regulation of dental pulp and dentine repair. Therapeutic ultrasound was shown to be effective for fracture repair. We investigated whether low frequency ultrasound influences the production of VEGF by odontoblast-like cells. Moreover, we examined the direct effects of VEGF on odontoblast-like cell proliferation. Design MDPC-23, an established odontoblast-like cell line, was exposed to increasing intensities of 30 kHz ultrasound using an ultrasonic tip probe. Results After 24 h cell culture, WST-1 analysis of cell viability and number showed a dose-dependent decrease in the number of viable cells with increasing ultrasound power. However, the relative concentration of VEGF as analysed by ELISA and normalised to cell number was significantly increased in the culture supernatants indicating an ultrasound-induced stimulation of odontoblastic VEGF secretion. Analysis of VEGF gene expression by sqRT-PCR revealed the expression of the main VEGF isoforms in the MDPC-23 cells, i.e. VEGF 120 and VEGF 164 as well as to a minor extent VEGF 188 . Low power ultrasound increased gene expression of all VEGF isoforms. Addition of recombinant VEGF to the cell cultures significantly stimulated cell proliferation. Gene expression of the VEGF receptors Flt1/VEGFR1 and KDR/VEGFR2 was detected in the MDPC-23, suggesting the possibility that VEGF may act on the odontoblast-like cells in an autocrine manner. Conclusions Our results indicate that ultrasound promoted VEGF expression and production by odontoblast-like cells and that VEGF may have autocrine effects on these cells. It is proposed that ultrasound may influence odontoblast activity and dentine repair by modulating production of endogenous growth factors in the dentine-pulp complex.

We have recently reported the induction of dental pulp stem cells (DPSCs) into dentin-secreting odontoblast-like cells after stimulation by isolated dentin matrix components, thus mimicking the nature of tissue regeneration seen after tooth disease and injury. After confluency, the cells were further cultured for 21 d in the 10% fetal bovine serum (FBS) Dulbecco's modified Eagle's medium (DMEM) (control), and in this medium, with the addition of dentin extract (DE) and the mineralization supplement (MS) of ascorbic acid and beta-glycerophosphate (treatment). To identify genes associated with this process, specimens were analyzed with a HG-U133A human gene chip and Arrayassist software. A total of 425 genes, among them 21 matrix and eight TGF-beta-related genes, were either up- or downregulated in the experimental group in which the cells showed odontoblast-like differentiation and mineralization. Expression of selected genes was further confirmed by real-time polymerase chain reaction (PCR) analysis. Of the extracellular matrix (ECM)-related genes, two types of collagen genes were upregulated and seven others downregulated. Other ECM-related genes, for example fibulin-1, tenascin C, and particularly thrombospondin 1, were upregulated, and fibulin-2 was downregulated. Most noticeably, the matrix metalloproteinase 1 was induced by the treatment. In the TGF-beta superfamily, upregulation of the type II receptor, endoglin, and growth/differentiation factor 5 was coordinated with the downregulation of activin A, TGF-beta2, and TGF-beta1 itself. This study identifies the matrix and TGF-beta-related gene profiles during the DPSC cell mineralization in which several genes are reported for the first time to be associated with this process, thus greatly expanding our molecular knowledge of the induced disease repair process.

Isolation of sufficient quantities of pure populations of odontoblasts from healthy and diseased teeth will facilitate our understanding of dentinogenesis during development and repair. Here we describe a novel Piezo-power microdissection (PPMD) technique for the isolation of pure populations of odontoblasts and pulpal tissue from formalin-fixed, paraffin-embedded, mature, healthy and carious human teeth. Odontoblasts and pulpal tissue gene expression were subsequently studied in ribonucleic acid isolated from PPMD preparations using a semi-quantitative reverse transcription polymerase chain reaction approach. Data confirmed that the genes for dentine sialophosphoprotein and Nestin are preferentially expressed in odontoblasts, whilst the genes for both collagen-1alpha and collagen-3alpha were expressed preferentially in pulpal tissue, particularly in carious samples. PPMD provides a novel and powerful approach to isolate pure populations of dental tissues and cells from fixed specimens for subsequent downstream molecular analyses.

The vitality of the dentin-pulp complex, both during tissue homeostasis and after injury, is dependent on pulp cell activity and the signalling processes, which regulate the behavior of these cells. Research, particularly over the last ten to fifteen years, has led to a better understanding of the molecular control of cellular behavior. Growth factors play a pivotal role in signalling the events of tissue formation and repair in the dentin-pulp complex. Sequestration of growth factors in the dentin matrix during tissue formation provides a pool of these molecules, which may be released during injury and contribute to signalling of reparative events. Therapeutic intervention with recombinant growth factors also provides possibilities for control of cell activity during repair. Harnessing both endogenous and exogenous sources of growth factors can provide exciting opportunities for novel biological approaches to dental tissue repair and the blueprint for tissue engineering of the tooth. These approaches offer significant potential for improved clinical management of dental disease and maintenance of tooth vitality.

Trans-dentinal stimulation of tertiary dentinogenesis has long been recognized, and has traditionally been ascribed to diffusion of irritant substances arising during injury and restorative treatment. Identification of bio-active components, especially growth factors including TGF-βs, sequestered within dentin matrix provides a new explanation for cellular signaling during tertiary dentinogenesis. Both isolated dentin matrix components and pure growth factors (TGF-βs) have been shown to signal cellular events leading to reactionary and reparative tertiary dentinogenesis. Release of these bio-active components from dentin matrix may arise during carious attack and other injury to the tissue, and also during subsequent surgical intervention and restoration of the tooth. Both cavity-conditioning agents and leaching from restorative materials may contribute to release of these components. Distance of diffusion, as determined by cavity residual dentin thickness, and other restorative parameters may influence the signaling process after release of these components. Careful consideration of the interplay between tissue injury and surgical and restorative material factors is required for optimum exploitation of the exquisite regenerative capacity of dentin-pulp for more biological approaches to clinical treatment of dental disease.

OBJECTIVES: (1) To determine the extent to which Australian general practitioners (GPs) restrict the numbers of agents they prescribe within a drug class ('personal formularies'); (2) To assess concordance of these drug choices with standards based on established guidelines or recognised good prescribing practices; (3) To assess the potential of these measures as indicators of the quality of prescribing. METHODS: Australian Health Insurance Commission (HIC) prescription data (1994 1997) for around 15,400 GPs providing 1500 or more Medicare services per year were analysed. Measures of an individual GP's use of a personal formulary (determined by number of agents) and concordance with prescribing criteria based on specified drugs for five classes of commonly prescribed drugs were derived. RESULTS: Non-steroidal anti-inflammatory drugs (NSAIDs): GP concordance was higher with a non-specified personal formulary (any five NSAIDs) than with a list of specified drugs (five NSAIDs of 'low' or 'medium' risk of gastrointestinal toxicity), and concordance with both increased over time. In 1997, around 70% of GPs used five or fewer NSAIDs for 90% of their prescribing; 47% of GPs had 90% of prescribing from five selected agents. Angiotensin converting enzyme inhibitors/angiotensin-II receptor antagonists: The introduction of new agents appeared to increase the size of the GPs' personal formularies, and concordance with defined standards decreased over time. Antibacterial agents: Concordance with a specified drug standard (nine drugs listed in the Australian Antibiotic Guidelines) increased substantially over time but was largely due to increased prescribing of two heavily promoted drugs. Beta-blocking agents: Over time, GPs restricted most prescribing to two agents, atenolol and metoprolol. Calcium channel blockers: GPs did not appear to restrict prescribing of these drugs; most GPs prescribed all five agents available. CONCLUSIONS: Australian GPs use 'personal formularies'. Formulary size varies with the drug class, can change over time as new agents become available, and its contents can be influenced by promotional activities. Prescribing standards based on numbers of drugs used may not always reflect rational prescribing choices. Criteria based on specified drugs provide more rigorous prescribing standards, but may give a misleading picture of prescribing quality in the absence of information on patients and the indications for treatment. Personal formulary measures are potentially useful prescribing indicators but need to be carefully defined and interpreted. GPs should be encouraged to identify their personal formularies and review the drugs included in them.

To examine the ability of interns to prescribe appropriately for common clinical conditions at the commencement and completion of the intern year. Interns' perceptions of their ability to prescribe and the perceived influences on their practices were also assessed. The study was conducted at a teaching hospital in urban New South Wales, Australia. A self-complete questionnaire was administered to 56 interns at the beginning and end of internship. At the beginning of the year respondents were asked to identify how equipped they felt they were to perform specific functions related to prescribing practice. Interns were also asked to write hospital prescriptions for four common clinical cases scenarios: post-operative pain, urinary tract infection, asthma, and community-acquired pneumonia. At the end of the year interns were asked to prescribe for the same clinical scenarios and also asked to identify the main influences on their practice. At the beginning of the year 54% of interns felt equipped to choose an appropriate drug for common clinical conditions, however, few felt they were able to determine the appropriate dose (23% of respondents) or dose frequency (25%). A previously validated four-point rating scale was used by two assessors to judge appropriateness of prescribing [Kappa = 0.6]. At the beginning of the year at least two-thirds of interns were prescribing 'inappropriately' for all clinical conditions. By the end of the year 75% were prescribing 'appropriately' for all conditions. The main perceived influences on prescribing practices were registrars, consultants, books and pharmacists.The use of hypothetical clinical cases to explore prescribing ability has shown that doctors are ill-equipped to perform various aspects of prescribing on graduating from medical school. Although our findings may not translate into practice directly they highlight the existence of a potential problem that warrants further study, especially in the areas of actual practice and the influences on it in the early postgraduate years.

Hedgehogs are becoming increasingly popular as pets. This article provides a, broad overview of those factors with which practitioners must be familiar to provide guidance to hedgehog owners. The taxonomy, anatomy, and natural history of the common pet species are presented along with commonly observed behaviors. Information on the reproduction and neonatal management is described. Current recommendations for handling, housing, and feeding these animals are also discussed.

Appropriate selection of medical students is a fundamental prerequisite if medical schools are to produce competent and caring doctors. The selection criteria for entry to the medical degree course at the University of Newcastle, New South Wales, are unique in Australia. The purpose of this study was to identify admission criteria that may predict performance in the first postgraduate (intern) year. Performance ratings were obtained from the clinical supervisors of two graduating classes of Newcastle University medical students during their five terms in internship (first postgraduate year). At least one rating was obtained for 93% of interns. A subset analysis of interns with multiple ratings (57%) showed that combining previous study in both humanities and science before medical school entry was predictive of higher intern performance ratings. These interns were rated more favourably than those who had studied science alone. Moreover, students who had earlier studied both humanities and science were twice as likely to complete their medical degree as those who had studied science alone. Age, gender, admission interview results, written psychometric test scores, academic marks, and whether previous tertiary study had been undertaken prior to medical school entry were not predictive of intern performance ratings. Subject spread, including a background in humanities, is important for effective medical practice, at least in the immediate postgraduate period. Perhaps it is time to evaluate the admission criteria by which medical students are selected.

Objective To assess the odontogenic potential of bone marrow mesenchymal stem cells (BMMSCs) to differentiate into odontoblast-like cells under the morphogenetic influence of dentine matrix as a possible basis for new stem cell-mediated therapeutic approaches to pulp diseases. Design BMMSCs were harvested from the whole bone marrow and cells at passages 3–5 were used for subsequent experiments. For in vitro studies, 1 × 10 4 cells were seeded on the surface of dentine slabs and co-cultured for 2 weeks in 24-well plates, then fixed, decalcified, embedded in paraffin and serial sections were processed for analyses. Haematoxylin-eosin (HE) staining was used for the morphological analysis of BMMSCs on the dentine slabs. The protein expression of dentine sialoprotein (DSP) in co-cultured BMMSCs was detected by immunohistochemical (IHC) staining. For in vivo studies, 5 × 10 6 cells were collected as cell pellets, seeded onto dentine slices and transplanted into renal capsules for 6 weeks. Histological analyses of harvested tissues were performed as described for the in vitro studies. Total RNA and protein were extracted from harvested tissues and Dspp /DSP expression was investigated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. Results After 2 weeks of co-culture with dentine slabs, BMMSCs demonstrated good viability in terms of morphological appearance and some showed polarization and extension of their cytoplasmic processes into dentine tubules with DSP expression. In vivo study demonstrated similar morphological changes and DSP expression in cells adjacent to dentine. RT-PCR and Western blot also demonstrated that the expression of Dspp /DSP in the co-cultured BMMSCs groups was higher than in the control groups. Conclusion Dentine matrix can signal morphogenic induction of differentiation of BMMSCs into odontoblast-like cells in vivo and in vitro .

We compared clinical supervisors’ ratings of intern competence in Newcastle University graduates (whose medical school emphasizes community relevance and interpersonal skills) and in medical graduates of the Universities of Sydney and New South Wales, which have more traditional curricula. A 12‐item anchored supervisor rating scale was used to assess the professional competence of 428 interns on five occasions throughout the intern year. Data analysis was performed using two factors of clinical competence and personal characteristics, as well as an average annual total score, linear regression analysis was employed to estimate the adjusted effects of age, sex, and graduating university on the three scores. All three scores were higher for interns who graduated at younger than 25 years, irrespective of university of graduation or sex. Women scored higher on average than men, and Newcastle graduates were rated significantly better on average than Sydney University graduates for the personal characteristics ...

Dentistry is entering an exciting era in which many of the advances in biotechnology offer opportunities for exploitation in novel and more effective therapies. Pulp healing is complex and dependent on the extent of injury, among many other factors. Many of the molecular and cellular processes involved in these healing events recapitulate developmental processes. The regulation of odontoblast activity is clearly central to pulp healing, and an understanding of the mechanisms involved in these processes is necessary to enable laboratory studies to be translated to clinic application. Transcriptome analysis has identified changes in many odontoblast genes during the life-cycle of this cell and its responses to injurious challenge. The p38 MAPKinase pathway appears to be central to the transcriptional control of odontoblasts and may provide a key target for therapeutic intervention. The many recent advances in knowledge of pulpal stem cells and molecular signaling molecules within the tooth, now provide exciting opportunities for clinical translation to novel therapies. Such translation will require the partnership of researchers and skilled clinicians who can effectively apply advances in knowledge to appropriate clinical cases and develop novel therapies which can be realistically introduced into the clinic.

Characterization of the molecular response under caries lesions requires a robust and reliable transcript isolation system, and analysis of data indicated that collection of extracted teeth in either liquid nitrogen/RNA-stabilizing solution facilitated this. Subsequent transcriptional analysis indicated higher general activity in carious pulps, while characterization of inflammatory mediators, including cytokines and S100 proteins, highlighted increasing expression levels associated with both microbial front progression and elevated cellular immune response. Analysis of the pleiotropic hormone adrenomedullin (ADM) indicated that transcript and protein levels are increased in pulpal tissue during caries, and that protein levels sequestered in dentin due to primary dentinogenesis are comparable with those of TGF-β1. Expression analysis of a leucine-rich-repeat-containing protein (LRRC15/Lib) indicated that this highly conserved molecule was up-regulated during caries, is transcriptionally regulated by pro-inflammatory stimuli, and is relatively abundant in mineralized tissues.

Of twenty-five patients with mild to moderate hypertension, recruited and managed in the community, seventeen responded fully to, and completed a randomised cross-over study of, captopril (ceiling dose 100 mg) compared with lisinopril (ceiling dose 40 mg) both given as a single daily dose. Mean supine and standing blood pressures measured at the end of the dose interval were significantly reduced compared to placebo by both compounds at three and six weeks. However, a consistent, significant increase in blood pressure occurred between three and six weeks in both arms of the study despite good and unchanged compliance with a fixed dose of each medication. Both captopril and lisinopril were well tolerated. Drug-related cough was the principal adverse effect.

The hallmark of HIV-1 and SIV infections is CD4(+) T cell depletion. Both direct cell killing and indirect mechanisms related to immune activation have been suggested to cause the depletion of T cells. We have now identified a mechanism by which immune activation-induced fibrosis of lymphoid tissues leads to depletion of naive T cells in HIV-1 infected patients and SIV-infected rhesus macaques. The T regulatory cell response to immune activation increased procollagen production and subsequent deposition as fibrils via the TGF-β1 signaling pathway and chitinase 3-like-1 activity in fibroblasts in lymphoid tissues from patients infected with HIV-1. Collagen deposition restricted T cell access to the survival factor IL-7 on the fibroblastic reticular cell (FRC) network, resulting in apoptosis and depletion of T cells, which, in turn, removed a major source of lymphotoxin-β, a survival factor for FRCs during SIV infection in rhesus macaques. The resulting loss of FRCs and the loss of IL-7 produced by FRCs may thus perpetuate a vicious cycle of depletion of T cells and the FRC network. Because this process is cumulative, early treatment and antifibrotic therapies may offer approaches to moderate T cell depletion and improve immune reconstitution during HIV-1 infection.

Natural SIV infection of sooty mangabeys (SMs) is nonprogressive despite chronic virus replication. Strikingly, it is characterized by low levels of immune activation, while pathogenic SIV infection of rhesus macaques (RMs) is associated with chronic immune activation. To elucidate the mechanisms underlying this intriguing phenotype, we used high-density oligonucleotide microarrays to longitudinally assess host gene expression in SIV-infected SMs and RMs. We found that acute SIV infection of SMs was consistently associated with a robust innate immune response, including widespread upregulation of IFN-stimulated genes (ISGs) in blood and lymph nodes. While SMs exhibited a rapid resolution of ISG expression and immune activation, both responses were observed chronically in RMs. Systems biology analysis indicated that expression of the lymphocyte inhibitory receptor LAG3, a marker of T cell exhaustion, correlated with immune activation in SIV-infected RMs but not SMs. Our findings suggest that active immune regulatory mechanisms, rather than intrinsically attenuated innate immune responses, underlie the low levels of immune activation characteristic of SMs chronically infected with SIV.

Objective: Primary pulp cell cultures are frequently used to study cellular responses, odontogenic potential and stem cell responses. Their isolation and expansion via a range of technical approaches are widely reported. The purpose of this study was to investigate the influence of isolation approach and extended expansion on cell phenotype and behaviour. Design: To determine viable cell isolation, enzymatic dissociation was performed on rodent incisor pulps using collagenase, trypsin, hyaluronidase and ficin. Extended expansion culture of released cells was performed in DMEM and alpha-MEM media. Cultures were subsequently analysed for gene expression, cell proliferation, cell morphology and differentiation capacity up to passage 20. Results: Data indicated that incubation of extirpated and mechanically minced rodent pulpal tissue with 0.25% Trypsin:EDTA and subsequent culture in alpha-MEM medium provided optimal conditions for maximal cell growth and expansion. Under these conditions, extended culture decreased cellular proliferative capacity up to passage 7, whilst higher passages demonstrated recovered growth rates. In general gene expression analysis of osteogenic and dentinogenic associated markers decreased with increasing passage number. Notably expression of TGF beta s-1, -2 and -3 increased up to passage 10 as did the stem cell and pericyte/myofibroblast markers, CD74, Neuroserpin and alpha-SMA. Analysis of molecular phenotypes indicated little difference in lineage differentiation capacity between earlier and later passages. Conclusions: The present study characterizes conditions for primary pulp cell isolation and expansion and indicates that both earlier and later passages maintain differentiation capacity. Continued passage however may result in selection for cells with a pericyte/myofibroblast phenotype. (C) 2009 Elsevier Ltd. All rights reserved.

Background: Antimicrobials are often used inappropriately in paediatric wards of medical college hospitals in Bangladesh. Most of the antimicrobials are prescribed based on clinical grounds–signs and symptoms. This intervention study assessed the effectiveness of a training intervention on antimicrobials prescribing by physicians in paediatric wards of tertiary care level hospitals. Methodology: This study was conducted at medical college hospitals in Bangladesh during the period from 1998 through 2000. The pre-intervention survey of antimicrobial use was conducted during 1998 in five hospitals. The post-intervention survey was conducted after the interactive training during the succeeding year in three of the original five hospitals, of which one was the intervention hospital and two control hospitals. A total of 3,466 admitted paediatric patients' treatment charts (2,171 in the pre-intervention and 1,295 in the post-intervention surveys) were reviewed. Results: The most commonly used antimicrobials were ampicillin, gentamicin, amoxicillin, cloxacillin and ceftriaxone. Appropriate antimicrobial therapy for the most common infectious diseases, pneumonia and diarrhoea, increased by 16.4% and 56.8% respectively in the intervention hospital compared with the two control hospitals and these improvenmts were significant (p = < 0.001 and p = 0.002, for pneumonia and diarrhoea respectively). Conclusions: An interactive, focussed educational intervention, targeted at physicians, appears to have been effective in improving appropriate antimicrobial prescribing for the most common paediatric infectious diseases in a medical college hospital in Bangladesh.

An adult female Central American banded gecko, Coleonyx mitratus, housed with a male of the same species for two years, became thin over an undetermined period of time. After separation from the male, regurgitation was noted. In spite of supportive care, the animal continued to lose weight over the subsequent ten days. Gastrointestinal contrast radiographs indicated a filling defect in the mid-intestinal region with generalized intestinal distention. Exploratory celiotomy revealed gas and fluid distention of all intestinal loops and a soft tissue mass in the cranial coelomic region. Histopathological examination of the soft tissue mass identified it as a rhabdomyosarcoma, a rare neoplasm in reptiles. Intestinal distention was attributed to ileus.

Background information. Substantial evidence indicates the existence of NCSCs (neural crest-derived stem cells) in embryonic mandibular processes; however, they have not been fully investigated or isolated. The aim of the present study was to isolate stem cells from mandibular process during embryonic development by MACS (magnetic-activated cell sorting). The findings show that the cells are multipotent and self-renewing. Results. LNGFR (low-affinity nerve-growth-factor receptor)+ cells were isolated from rat embryonic mandibular processes by MACS. The cells were grown in clonal culture by limiting dilution to assess their developmental potential. Clone analysis indicated that, first, LNGFR+ cells are multipotent, being able to generate at least neurons and Schwann cells, similar to peripheral neural crest stem cells. Secondly, multipotent LNGFR+ cells generate multipotent progenies, indicating that they are capable of self-renewal and therefore are stem cells. Thirdly, manipulation of the medium supplementation alters the fate of the isolated LNGFR+ cells. Conclusions. These results indicate that LNGFR antibodies label NCSCs with high specificity and purity, and suggest that positive selection using these antibodies may become the method of choice for obtaining multipotent cells from rat embryonic mandibular processes for tissue engineering or regenerative therapeutic use.

In this study, the progenitor cells isolated from the human dental pulp were used to study the effects of ethylenediaminetetraacetic acid-soluble dentin extract (DE) on their differentiation and mineralization to better understand tissue injury and repair in the tooth. Mineralization of the matrix was increasingly evident at 14, 21, and 28 d after treatment with a mineralization supplement (MS) (ascorbic acid [AA], beta-glycerophosphate [beta-GP]) and MS + DE. Real-time polymerase chain reaction results showed type I collagen upregulation after the addition of MS + DE at 7 d. Alkaline phosphatase was downregulated after the mineralization became obvious at 14 d. Bone sialoprotein was shown to be upregulated in the mineralized cell groups at all time points and dentin sialophosphoprotein after 7 d. Core binding factor a 1 was upregulated by the treatment of MS and DE at 7, 14, and 21 d. These results indicated that the MS of AA, beta-GP, and DE synergistically induced cell differentiation of pulp progenitor cells into odontoblast-like cells and induced in vitro mineralization.

BACKGROUND AND AIM The relative roles of self and ambulatory blood pressure monitoring (self BPM, ABPM, respectively) have yet to be clearly defined despite accumulating evidence of self BPM benefits, particularly in hypertension management. In particular, measurements comparable to ABPM have been documented with self-measurement, usually twice daily over one or more days. Nevertheless, ABPM offers a series of recordings performed throughout the day that can be invaluable during drug treatment. Consequently, this study evaluated 18 normotensive and 79 hypertensive patients, including 21 not taking hypotensive agents, and measured their blood pressure at least every 2 h while they were also undergoing ABPM. METHODS AND RESULTS Self-measurement was performed between five and 12 times during the day with 77% of subjects preferring self BPM rather than daytime ABPM. When compared with the closest ABP measurement, mean systolic and diastolic pressures were not significantly different throughout the day in both normotensive and hypertensive patients. However, a comparison of a series of ABPM hourly averages (three readings performed closest to the respective self BPM reading) throughout the day in all hypertensive subjects found ABPM readings to be slightly but significantly lower (3.3 systolic, 2.2 mmHg diastolic; P < 0.05). CONCLUSION It is concluded that multiple self-measurements throughout the day provides comparable information to daytime ABPM, is preferred by most patients, and should be a cost-effective alternative in the diagnosis and management of hypertension.

Summary Objective: The function of apoptosis and its regulation in odontoblasts remain unclear. In this study, we characterize the possible role of transforming growth factor (TGF)-β 1 in the induction of apoptosis and the molecular mechanisms that mediate TGF-β1-induced apoptosis in odontoblasts. Methods: Annexin V/propidium iodide staining, cell Death Detection ELISA and DNA ladder were used to examine the effect of TGF-β1 on apoptosis in a mouse odontoblast-like cell line, MDPC-23. Stable cell clones expressing Smad2 or Smad3 dominant negative mutants, or wild-type Smad7 were constructed to investigate the role of Smad proteins in the mediation of apoptosis by TGF-β1 in MDPC-23 cells. The TGF-β1-induced transcriptional activity in stable cell clones expressing Smad proteins was analyzed by a transient transfected TGF-β-responsive reporter gene, p3TP-Lux. Results: TGF-β1 can induce apoptotic cell death in MDPC-23 cells in a dose-dependent manner. Transfection of dominant negative mutant forms of Smad2 or Smad3 blocked TGF-β1-induced apoptosis; moreover, the Smad3 mutant was more efficient than the Smad2 mutant. Transfection of Smad7, an inhibitory Smad, also significantly inhibited TGF-β1-induced apoptosis of these cells. Over-expression of Smad3 dominant negative mutant or Smad7 significantly inhibited TGF-β1-induced transcriptional activity. Conclusion: These results suggest that Smad proteins are involved in TGF-β1-induced apoptosis of odontoblast cells.

Cranial neural crest-derived ectomesenchymal cells represent a population of pluripotent stem cells giving rise to many of the various oro-facial and dental tissues. The factors determining the terminal fate of these cells are still unclear. The potentiality of human embryonic ectomesenchymal cells from the first branchial arch have been investigated when isolated and grown in a three-dimensional (3D)-collagen gel culture system in the presence of dentin matrix-derived non-collagenous proteins (DNCP) and TGFbeta-1. Functional differentiation of cells showing some characteristics of odontoblast-like cells could be observed when the cells were cultured with DNCP+TGFbeta-1 or DNCP, however, only cytological differentiation was observed during culture with TGFbeta-1 alone. The characteristics of these cells was assessed by morphological appearance, expression of the odontoblast phenotype marker dentin sialophosphoprotein (DSPP), increased alkaline phosphatase levels and formation of mineralised nodules in vitro. The results indicate that these embryonic cells from the first branchial arch are capable of responding to the inductive stimulus of DNCP or DNCP+TGFbeta-1 when isolated and grown in the 3D collagen gel culture system. The capacity of the isolated cells to differentiate into mineralizing cells showing some characteristics of odontoblast-like cells under these growth conditions highlights the potential of such approaches for tissue engineering strategies for hard-tissue regeneration after injury.

Objective : Transforming growth factor-β (TGF-β) regulates odontoblast differentiation and stimulates dentine extracellular matrix synthesis. However, until recently, the molecular mechanisms of action of TGF-β have been unknown. Smad proteins have recently been identified as intracellular signalling mediators of TGF-β. In this study, we characterise the role of Smad proteins as mediators of TGF-β in a mouse odontoblast cell line MDPC-23. Methods : Transcription of Smads was detected by RT-PCR. The change of intracellular location of Smad proteins treated by TGF-β1 was evaluated immunocytochemically. Smad function and its role in transcription of dentin sialophosphoprotein (DSPP) were investigated in cotransfection experiments using promoter-luciferase reporter gene constructs. Results : MDPC-23 cells expressed Smad2, Smad3 and Smad4 mRNA. Endogenous Smad2, Smad3 and Smad4 rapidly translocated from the cytoplasm into the nucleus in response to TGF-β1. The activity of the TGF-β-responsive p3TP-Lux reporter construct was stimulated by 12.7-fold with TGF-β1 treatment. Over-expression of wild-type Smad3 promoted TGF-β1-induced luciferase activity, whereas dominant negative Smad3 inhibited it. TGF-β1 also inhibited the activity of DSPP promoter luciferase reporter construct containing the sequence between −791 bp and +54 bp of the mouse DSPP gene. Over-expression of wild-type Smad3 potentiate the inhibitory effect of TGF-β1 on transcriptional regulation of DSPP, while dominant negative Smad3 decreased the effect. In contrast to Smad3, wild-type Smad2 or its dominant negative mutant had little effect on TGF-β1 regulation of the promoter activity of DSPP. Conclusions : Smad2, Smad3 and Smad4 are present and activated by TGF-β1 in MDPC-23 cells. The Smad pathway is functional in these cells and Smad3 appears to be involved in down-regulation of DSPP by TGF-β1. These findings raise the possibility that Smad signalling plays a role in dentinogenesis.

Knowledge of the molecular events that occur in carious disease has so far been constrained due to difficulties in obtaining sufficient quantities of the dental tissues and cells involved. Our histological findings indicate that a pulp-odontoblast cellular complex can be obtained from carious and healthy human teeth when exposed to low-temperatures prior to pulpal extirpation and from rodent teeth processed at room-temperature. In contrast, pulpal tissue extracted from room-temperature processed human teeth and low-temperature processed rodent teeth resulted in the odontoblast layer remaining attached to the pulp chamber. Semi-quantitative RT-PCR (sq-RT-PCR) analysis confirmed that markers previously shown to be preferentially expressed in odontoblasts, namely dentin sialophosphoprotein (DSPP) and Nestin, amplified more readily from the extracted pulp-odontoblast complex, as compared to pulpal tissue alone, in both human and rodent samples. Subsequent gene expression analysis of collagen-1alpha and collagen-3alpha indicated levels were significantly higher in carious pulpal tissue. In addition, analysis characterising the expression of members of the transforming growth factor and bone morphogenic protein families and their receptors indicated in general, that these genes were expressed by healthy odontoblasts and up-regulated in both pulpal cells and odontoblasts in response to carious injury. Use of this temperature-sensitive dental tissue preparation procedure allows detection of differential gene expression in odontoblasts and other pulpal cells in healthy and carious tissue.

Aim Cavity preparation and restoration variables have an uncertain relationship to pulp injury and repair responses. The purpose of this paper is to determine the importance of cavity preparation and restoration variables, by ranking them according to their effect on pulp injury (odontoblast survival) and pulp repair (reactionary dentine secretion). Method The seven studies reviewed are all based on the protocols described by Murray, Smith and colleagues in 2000 and 2001. Results The studies reviewed provide new perspectives on the importance of cavity preparation and restoration variables in mediating pulp activity. Conclusions The onset of post-operative complications may be most productively minimised by focusing practitioner attention to aspects of treatment highlighted in this review.

Objectives: To derive indicators of quality prescribing by Australian general practitioners based on Health Insurance Commission (HIC) data and assess the influence of incomplete capture of data on under-copayment drugs on the validity of these indicators. Design: Two expert groups proposed prescribing indicators that can be derived from aggregate prescribing data, and which reflect important clinical or costeffectiveness issues. Indicators were examined using HIC data and compared with national prescribing trends over time using Australian Statistics on Medicines. The effect of incomplete data capture on indicator interpretation was examined by stratifying GPs into five strata based on the proportion of concession card holders in their practice. Participants: Approximately 14 000 Australian GPs providing 1500 Medicare services per year. Main outcome measures: Measures of prescribing for individual GPs (based on HIC data 1993–1997). Results: Forty-three potentially useful indicators were identified. These covered a fairly narrow range of prescribing activities and many required additional clinical information for interpretation. Indicators based on prescribing rates gave a misleading picture of prescribing trends where the extent of HIC data capture changed over time. Indicators expressed as ratios that reflected choice of agent within a drug class were less affected by incomplete data capture. Conclusions: Indicators of quality prescribing can be derived from HIC data. However, indicators for under-copayment drugs that represent prescribing rates may unfairly classify doctors practising in areas of socioeconomic disadvantage or high morbidity as “high prescribers”. Ratio indicators are more robust, and may be more valid prescribing measures. If HIC data are to be used to monitor the quality of

The recommended treatment of human chloroquine poisoning is diazepam and adrenaline but neither has been evaluated in controlled clinical trials. We investi gated whether diazepam provided any added benefit over barbiturate anaesthesia and whether the protective effect of catecholamines in chloroquine poisoning was mediated through alpha or beta receptor stimulation. Rats, anaesthetised with thiobutobarbitone had a continuous intravenous infusion of 3 mg/kg/min of chlor oquine. This caused a steady decline in pulse rate and blood pressure. When diazepam (3 mg/kg iv) was admi nistered 5 -10 min later, heart rates decreased at a faster rate (P=0.005), blood pressure was consistently lower (P=0.01) and there was a shorter time to arrhythmias and death (P < 0.05). Adrenergic agents were given by titration to attempt to maintain mean blood pressure > 75 mmHg. Compared with the phenylephrine (selective alpha agonist) group, the group treated with isoprenaline (selective beta agonist) had faster heart rates which decreased more slowly (P < 0.0001), higher blood pressure (P=0.005) and longer time to arrhythmias and death (P=0.005). Adrenaline and noradrenaline had intermedi ate effects. Thus beta agonist effect appears to explain the beneficial effects of adrenaline but alpha agonist activity may be harmful. This animal work suggests that a combination of barbiturate anaesthesia and isoprenaline may be better than the diazepam and adrenaline in combatting the effects of chloroquine.

Irrational prescribing is a habit which is difficult to cure. However, prevention is possible and for this reason the WHO Action Programme on Essential Drugs aims to improve the teaching of pharmacotherapy to medical students. The impact of a short problem-based training course in pharmacotherapy, using a WHO manual on the principles of rational prescribing, was measured in an international multi-centre randomised controlled study of 219 undergraduate medical students in Groningen (Netherlands), Kathmandu (Nepal), Lagos (Nigeria), Newcastle (Australia), New Delhi (India), San Francisco (USA), and Yogyakarta (Japan). The manual and the course presented the students, who were about to enter the clinical phase of their studies, with a normative model for pharmacotherapeutic reasoning in which they were taught to generate a "standard" pharmacotherapeutic approach to common disorders, resulting in a set of first-choice drugs called P(ersonal)-drugs. The students were then taught how to apply this set of P-drugs to specific patient problems on the symptomatic treatment of pain, using a six-step problem-solving routine. The impact of the course was measured by tests before training, immediately after, and six months later. After the course, students from the study group performed significantly better than controls in all patient problems presented (p0.05). The students not only remembered how to solve old problems, but they could also apply their skills to new problems. Both retention and transfer effect were maintained at least six months after the training session in all seven medical schools. In view of the impossibility of teaching students all basic knowledge on the thousands of drugs available, this approach seems to be an efficient way of teaching rational prescribing. However, the method should be accompanied by a change in teaching methods away from the habit of transferring knowledge about the drugs towards problem-based teaching of therapeutic reasoning.

1 A descriptive case study of calcium channel-blocking drug (CCB) overdoses in the Hunter Region of NSW was performed to analyse the in-hospital morbidity and mortality of CCB drug overdoses in an Australian population.2 The patients were admitted to major hospitals within the Hunter Region and treated initially with gastrointestinal decontamination, including the use of oral activated charcoal. Further management was required in most cases and included intravenous calcium, atropine and inotropic support.3 Of the 15 CCB overdoses, four patients died. Non-cardiogenic pulmonary oedema occurred in two other cases. Cardiac conduction defects occurred in 11 cases.4 Atropine was found to be effective only after intravenous calcium had been administered.5 Overdose with slow-release verapamil required prolonged treatment with intravenous calcium salts.6 Overdose with verapamil or diltiazem in doses greater than 300 mg carries a significant risk of death and potentially life threatening arrhythmias occur with lower doses.7 Recommended initial management includes early, effective gastrointestinal decontamination. High dose intravenous calcium salts should be given to reverse hypotension and bradycardia. Atropine and inotropic support are frequently required.