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BackgroundWe conducted a survey to identify what types of health research could be exempted from research ethics reviews in Australia.MethodsWe surveyed active Australian health researchers and members of Human Research Ethics Committees (HREC). We presented the respondents with eight hypothetical research scenarios, involving: N of 1 trials, no treatment studies, linked data sets, surplus samples, audits, surveys, interviews with patients, and professional opinion. We asked whether these scenarios should or should not be exempt from ethics review, and to provide (optional) explanations. We analysed the reasons thematically, to identify Top 3 reasons underlying the decisions.ResultsMost frequent reasons for requiring ethics reviews, included: the need for independent oversight, privacy/confidentiality issues, review of scientific rigour, and publishing considerations. Most frequent reasons for exempting scenarios from reviews, included: level of risk, study design, privacy/confidentiality issues, and standard clinical practice. Four research scenarios listed the same Top 3 reasons for requiring ethics reviews: need for independent oversight, review of scientific rigour, privacy/confidentiality. Reasons for exempting were less uniform, but low risk was a Top 3 reason for 7 scenarios, and study design for 4 scenarios. Privacy/confidentiality was given as a Top 3 reason for both requiring and exempting from ethics the same two scenarios.ConclusionsThe most frequently offered reasons in support of requiring ethics reviews for research scenarios are more uniform than those for exempting them. However, considerable disagreement exists about when the risks of research are so minimal that the exemption is appropriate.

BackgroundWe conducted a survey to identify what types of health/medical research could be exempt from research ethics reviews in Australia.MethodsWe surveyed Australian health/medical researchers and Human Research Ethics Committee (HREC) members. The survey asked whether respondents had previously changed or abandoned a project anticipating difficulties obtaining ethics approval, and presented eight research scenarios, asking whether these scenarios should or should not be exempt from ethics review, and to provide (optional) comments. Qualitative data were analysed thematically; quantitative data in R.ResultsWe received 514 responses. Forty-three per cent of respondents to whom the question applied, reported changing projects in anticipation of obstacles from the ethics review process; 25% reported abandoning projects for this reason. Research scenarios asking professional staff to provide views in their area of expertise were most commonly exempted from ethics review (to prioritise systematic review topics 84%, on software strengths/weaknesses 85%); scenarios involving surplus samples (82%) and N-of-1 (single case) studies (76%) were most commonly required to undergo ethics review. HREC members were 26% more likely than researchers to require ethics review. Need for independent oversight, and low risk, were most frequently cited in support of decisions to require or exempt from ethics review, respectively.ConclusionsConsiderable differences exist between researchers and HREC members, about when to exempt from review the research that ultimately serves the interests of patients and the public. It is widely accepted that evaluative research should be used to reduce clinical uncertainties—the same principle should apply to ethics reviews.

ObjectivesTo identify what types of health and medical research could be exempted from a requirement to undergo research ethics reviews in Australia.DesignA web-based survey.SettingAustralian-based participants completed the survey online between 10 September and 1 November 2019.ParticipantsActive human health and medical researchers and members of Human Research Ethics Committees (HREC).Main outcome measuresParticipants were asked whether they self-identify as a researcher, a member of an Australian human research ethics committee, or both. They were asked whether they had previously changed or abandoned a research project in anticipation of difficulty in obtaining ethics approval; and were presented with 4 hypothetical research scenarios, asking whether they should or should not be exempt from ethics review in Australia. All but the demographic question allowed the respondent to provide a comment clarifying their response. Qualitative data were analysed thematically; quantitative data were analysed in R.ResultsWe received 514 survey responses: 153 from researchers, 196 from HREC members, and 163 from individuals who identified as both. 27% of researchers reported they had changed their projects because they anticipated obstacles resulting from the ethics review process, and 16% abandoned projects for this reason. The most commonly exempted research scenarios involved professional staff providing their views on their area of expertise (84%, 85%); scenarios involving surplus samples and N-of-1 studies in clinical practice were most commonly required to undergo ethics review (82%, 76% respectively). Probability of answering that ethics review was required was the highest for HREC members and nearly identical for respondents who were both HREC members and researchers; it was the lowest for researchers. HREC members were overall 26% more likely to answer that ethics review was required than researchers.ConclusionsOur survey shows considerable differences between the researchers and members of ethics committees, about how best to serve the interests of patients and the public. Those interests are best served by evaluative research to reduce or resolve the uncertainties in the clinical context – the same general principle should be applied to ethics review.

Objectives: To determine age group‐ and cause‐of‐death‐specific contributions to area socioeconomic status (SES), sex and remoteness life expectancy inequalities. Methods: Mortality and estimated residential population data from New South Wales, Australia, over 2010–2012 was used to calculate life expectancy. Inequalities by sociodemographic groups were partitioned into age group‐ and cause‐of‐death‐specific contributions. Results: The largest contributions to SES differentials in life expectancy were observed at 60–84 years of age; for cancer, cardiovascular, endocrine and respiratory causes of death; and additionally external causes of death for males. Sex inequalities ranged from 3.6 to 5.2 years, with common causes of death such as cardiovascular disease and cancer in late adulthood (60+ years) accounting for the bulk of the differences. Smaller differences in life expectancy were observed by remoteness, with the largest contributions observed in ages 85 years and above, and for cardiovascular, mental, cancer and external causes of death. Conclusions: Common causes of death in late adulthood accounted for the bulk of life expectancy inequalities. Implications for public health: Development of policy and interventions aimed at addressing social determinants, such as proposed by the WHO's Global Plan of Action, are needed to help reduce sociodemographic inequalities in lifespan.

Category: Diabetes Introduction/Purpose: Diabetic ulcers carry with it extensive morbidity. The microbiome of diabetic foot infections have previously been shown to be polymicrobial. We hypothesized that ulcer treatment would normalize the foot microbiome. Methods: Microbiome profiles of the fore, mid and hind diabetic foot. Diabetic foot (with ulcer) were taken pre and post treatment with a combination of surgical debridement, local and systemic antibiotics. 10 subjects were studied. Microbiome graphs and images pre and post treatment of feet are shown. Results: The microbiome profile of the diabetic foot almost completely normalized to that of normal flora. Before treatment, the foot microbiome was associated with polymicrobial pathogenic bacteria mostly away from the ulcer. The ulcer itself only contained minimal species, albeit pathogenic bacteria. The differences in profiles between the pre and post treatment were significantly different p < 0.01. Conclusion: Chicken or egg, the foot microbiome almost completely reverts to normal flora with simple ulcer debridement and local/systemic antibiotics. Our data point possibly suggest ulcers could be prevented by reducing pathogenic microbiome load (chlorhexidine washes) or microbiome transplants. We next move to controls as a proof of concept.

This article describes the evaluation of enhanced surveillance that was conducted on an older age-group of children with pertussis (ages 5-9 years), in addition to the routine follow-up of a younger age-group (ages 0-4 years) in Sydney Local Health District during 2015. We found little incremental benefit in this policy in terms of identifying additional high-risk contacts, despite a greatly increased workload.

ObjectivesLevel of education and genetic risk are key predictors of cardiovascular disease (CVD). While several studies have explored the causal mechanisms of education effects, it remains uncertain to what extent genetic risk is mediated by established CVD risk factors. This study sought to investigate this and explored the mediation of education and genetic effects on CVD by established cardiovascular risk factors in the Framingham Heart Study (FHS).DesignProspective observational cohort study.Participants7017 participants from the FHS.SettingCommunity-based cohort of adults in Framingham, Massachusetts, USA.Primary outcome measureIncident CVD. The total effects of education and genetic predisposition using a 63-variant genetic risk score (GRS) on CVD, as well as those mediated by established CVD risk factors, were assessed via mediation analysis based on the counterfactual framework using Cox proportional hazards regression models.ResultsOver a median follow-up time of 12.0 years, 1091 participants experienced a CVD event. Education and GRS displayed significant associations with CVD after adjustment for age and sex and the established risk factors smoking, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), body mass index, systolic blood pressure (SBP) and diabetes. For education effects, smoking, HDL-C and SBP were estimated to mediate 18.8% (95% CI 9.5% to 43%), 11.5% (95% CI 5.7% to 29.0%) and 4.5% (95% CI 1.6% to 13.3%) of the total effect of graduate degree, respectively, with the collective of all risk factors combined mediating 38.5% (95% 24.1% to 64.9%). A much smaller proportion of the effects of GRS were mediated by established risk factors combined (17.6%, 95% CI 2.4% to 35.7%), with HDL-C and TC mediating 11.5% (95% CI 6.2% to 21.5%) and 3.1% (95% CI 0.2% to 8.3%), respectively.ConclusionsUnlike education inequalities, established risk factors mediated only a fraction of GRS effects on CVD. Further research is required to elucidate the underlying causal mechanisms of genetic contributions to CVD.

Summary We determined brain to liver weight ratio (BLWR) thresholds for fetal growth restriction (FGR) using autopsy information on 395 perinatal deaths comprising stillborn babies who died during labour and neonatal deaths. FGR was defined using two methods: (1) birth weight for gestational age (WGA) less than the 10th percentile; and (2) WGA less than the 10th percentile or discordant birth weight/length. The association between BLWR and FGR was investigated using odds ratios, and classification statistics were calculated for a range of BLWR thresholds. Using WGA, 84 cases (21.3%) were FGR and a further 15 cases (n = 99, 25%) had discordant birth weight/length. The BLWR ranged from 1.02 to 7.30 and was positively associated with FGR. BLWR was not associated with FGR for babies with congenital central nervous system or chromosomal abnormalities. Excluding these, for FGR defined using WGA and discordant birth weight/length, a BLWR threshold of 5.0 was 100% predictive of FGR. A BLWR threshold of 3.0 for babies over 28 weeks gestation and 3.7 for more preterm babies optimised case detection while minimising missed and false positive cases. Additional evidence of FGR should be sought for babies with a BLWR of less than 5.0 to confirm FGR.

Objectives Despite being one of the healthiest countries in the world, Australia displays substantial mortality differentials by socioeconomic disadvantage, remoteness and sex. In this study, we examined how these mortality differentials translated to differences in life expectancy between 2001 and 2012. Design and setting Population-based study using mortality and estimated residential population data from Australia9s largest state, New South Wales (NSW), between 2001 and 2012. Age-group-specific death rates by socioeconomic disadvantage quintile, remoteness (major cities vs regional and remote areas), sex and year were estimated via Poisson regression, and inputted into life table calculations to estimate life expectancy. Results Life expectancy decreased with increasing socioeconomic disadvantage in males and females. The disparity between the most and least socioeconomically deprived quintiles was 3.77 years in males and 2.39 years in females in 2012. Differences in life expectancy by socioeconomic disadvantage were mostly stable over time. Gender gaps in life expectancy ranged from 3.50 to 4.93 years (in 2012), increased with increasing socioeconomic disadvantage and decreased by ∼1 year for all quintiles between 2001 and 2012. Overall, life expectancy varied little by remoteness, but was 1.8 years higher in major cities compared to regional/remote areas in the most socioeconomically deprived regions in 2012. Conclusions Socioeconomic disadvantage and sex were strongly associated with life expectancy. The disparity in life expectancy across the socioeconomic spectrum was larger in males and was stable over time. In contrast, gender gaps reduced for all quintiles between 2001 and 2012, and a remoteness effect was evident in 2012, but only for those living in the most deprived areas.

A new N-acetyl-D-glucosamine (GlcNAc) specific lectin was identified and purified from the fruiting body of the Australian indigenous mushroom Psathyrella asperospora. The functional lectin, named PAL, showed hemagglutination activity against neuraminidase treated rabbit and human blood types A, B and O, and exhibited high binding specificity towards GlcNAc, as well as mucin and fetuin, but not against asialofetuin. PAL purified to homogeneity by a combination of ammonium sulfate precipitation, chitin affinity chromatography and size exclusion chromatography, was monomeric with a molecular mass of 41.8 kDa, was stable at temperatures up to 55 °C and between pH 6–10, and did not require divalent cations for optimal activity. De novo sequencing of PAL using LC-MS/MS, identified 10 tryptic peptides that revealed substantial sequence similarity to the GlcNAc recognizing lectins from Psathyrella velutina (PVL) and Agrocybe aegerita (AAL-II) in both the carbohydrate binding and calcium binding sites. Significantly, PAL was also found to exert a potent anti-proliferative effect on HT29 cells (IC50 0.48 μM) that was approximately 3-fold greater than that observed on VERO cells; a difference found to be due to the differential expression of cell surface GlcNAc on HT29 and VERO cells. Further characterization of this activity using propidium iodine staining revealed that PAL induced cell cycle arrest at G2/M phase in a manner dependent on its ability to bind GlcNAc.

Cell-based assays represent a major end point of high throughput screening (HTS) but a key limitation of such assays is the potentially poor membrane permeability of test compounds. In this study, we optimized the conditions for the delivery of the membrane impermeable compound 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) into human cells using hypotonic shift; a method that can promote the uptake of molecules from the extracellular fluid into cell cytoplasm via endocytosis. We showed that uptake of HPTS by cells was a function of hypotonic buffer osmolarity and that delivery was highly efficient with almost 100% of cells displaying uptake. Delivery of HPTS was equally effective at 25°C and 37°C, with delivery of compound proportional to incubation time and concentration of HPTS within the loading medium. The experimental conditions identified in this study could be applied to HTS drug discovery studies providing an effective method of delivering small membrane impermeable compounds into cells.

The RUNX2 transcription factor is essential for osteoblast differentiation and chondrocyte maturation. Rare mutations within RUNXZ cause the dominantly inherited skeletal disorder cleidocranial dysplasia which is charaterized by gross dysgenesis of the skeleton. A unique feature of RUNX2 is the polyglutamine and polyalanine (poly Q/A)domain encoded by a trinucleotide repeat sequence. Such repeat sequences are prone to strand slippage resulting in high mutation rates. We hypothesized that mutations within the RUNX2 poly Q/A domain would exist and these variants would be associated with altered bone density and protein function. 4361 DNA samples were obtained from multiple epidemiological studies of bone density. Bone density was measured by DEXA and ultrasound and expressed as Z-scores around the appropriate age-adjusted mean. A total of 21 subjects were identified as being heterozygous for a wild type 23Q117A allele and a p l y Q/A repeat variant allele. Deletions (ISQ and 164) and insertions (304 and 23A) were identified. Collectively Q/A repeat variants presented with significantly lower Femoral Neck BMD (DEXA) displaying a 0.65 SD decrease (n = 21, p = 0.0004) and lower bone density as measured by quantitative ultrasound of similar magnitude (-0.79 SD, n = 8, p = 0.031). To understand the mechanism via which the rare mutations were decreasing bone density, functional analyses were carried out on 23Q (WT), 16Q and 304 RUNX2. The ability of the proteins to bind to the RUNX2 DNA binding site was assessed by EMSA. The analysis did not reveal any obvious changes in the DNA binding capacities of the mutant proteins. However, reporter gene analysis using the mouse osteocalcin promoter revealed significant decreases in the transactivation function of 169 and 30Q. Finally, the ability of the proteins to localise to the nucleus was assessed. Subcellular analysis revealed the 164 and 30Q proteins displayed defective nuclear localisation compared to WT. We have identified a new class of functionally relevant RUNX2 variants that occur at collective frequency of -0.5%. These mutations are associated with significantly lower bone density and altered protein function.

At present, there are no vaccines approved for the prevention or treatment of malignant melanoma, despite the amount of time and resources that has been invested. In this study, we aimed to develop a self-contained vaccine capable of directly stimulating anticancer CD8(+) T-cell immune responses. To achieve this, three whole-cell melanoma vaccines were developed expressing 4-1BBL or B7.1 T-cell co-stimulatory molecules individually or in combination. The ability of engineered vaccine cell lines to stimulate potent anticancer immune responses in C57BL/6 mice was assessed. Mice vaccinated with cells overexpressing both 4-1BBL and B7.1 (B16-F10-4-1BBL-B7.1-IFNγ/β anticancer vaccine) displayed the greatest increases in CD8(+) T-cell populations (1.9-fold increase versus control within spleens), which were efficiently activated following antigenic stimulation, resulting in a 10.7-fold increase in cancer cell cytotoxicity relative to control. The enhanced immune responses in B16-F10-4-1BBL-B7.1-IFNγ/β-vaccinated mice translated into highly efficient rejection of live tumour burdens and conferred long-term protection against repeated tumour challenges, which were likely due to enhanced effector memory T-cell populations. Similar results were observed when dendritic cell (DC)-deficient LTα(-/-) mice were treated with the B16-F10-4-1BBL-B7.1-IFNγ/β anticancer vaccine, suggesting that the vaccine can directly stimulate CD8(+) T-cell responses in the context of severely reduced DCs. This study shows that the B16-F10-4-1BBL-B7.1-IFNγ/β anticancer vaccine acted as a highly effective antigen-presenting cell and is likely to be able to directly stimulate CD8(+) T-cells, without requiring co-stimulatory signals from either CD4(+) T-cells or DCs, and warrants translation of this technology into the clinical setting.

RUNX2 gene SNPs were genotyped in subjects from the upper and lower deciles of age- and weight-adjusted femoral neck BMD. Of 16 SNPs in RUNX2 and its two promoters (P1 and P2), only SNPs in the P2 promoter were significantly associated with BMD. These P2 promoter SNPs were functionally different in gel-shift and promoter activity assays. Introduction: Specific osteoblast genes are induced by Runx2, a cell-specific transcription factor that is a candidate gene for controlling BMD. We tested the hypothesis that RUNX2 genetic variation is associated with BMD. Materials and Methods: From a population repository of normal subjects, the age- and weight-adjusted femoral neck BMD was ranked, and the upper and lower deciles (n = 132 each) were taken to represent the adjusted extremes of the population distribution. In these 264 subjects, we identified 16 allelic variations within the RUNX2 gene and promoters (P1 and P2) through DNA sequencing and denaturing high-performance liquid chromatography. Characterization of these alleles was performed through allele-specific cloning, transfection into ROS 17/2.8 cells, luciferase reporter analysis, and electrophoretic mobility shift assays. Results: Within the P2 promoter were three polymorphic nucleotides for which the minor alleles were over-represented in the upper decile of BMD (0.117 and 0.064 in the upper and lower deciles, respectively). These alleles are in near complete linkage disequilibrium with each other and represent a haplotype block that is significantly associated with increased BMD. The common and rare P2 promoter alleles were cloned upstream of luciferase, and when transfected into osteoblast-like cells, the construct representing the rare haplotype showed significantly greater P2 promoter activity than the common haplotype. Conclusions: Because the high BMD allele had higher P2 promoter activity, the data suggest that greater RUNX2 P2 promoter activity is associated with higher BMD.

We hypothesized that by taking subjects from the extremes of a population, we would specifically find alleles related to the bone mineral density trait, if such alleles exist for any particular target gene. We tested this idea using RUNX2, the well known osteoblast transcription factor. From a population of 1300 subjects the age-weight adjusted femoral neck bone mineral density (BMD) was ranked and the upper and lower deciles taken to represent the adjusted extremes. After adjusting and ranking, the two groups (n=130 each) were not significantly different for age or weight. In these 260 subjects, we identified 19 allelic variations within the RUNX2 gene and promoter (P1 and P2), and characterized these novel variations with respect to BMD strata by genotype using dHPLC. A general linkage disequilibrium (LD) analysis of RUNX2 showed that LD drops off with physical distance, as expected. Within the P2 promoter were three polymorphic nucleotides for which the minor alleles were over represented in the upper decile of BMD. These alleles are in complete LD with each other and represent a haplotype block that is significantly associated with increased BMD. Constructs made from the "normal" and the "high BMD" alleles were cloned upstream of otherwise identical luciferase constructs. Despite there being only 3 nucleotide differences between the two constructs, when transfected into osteoblast-like cells the high BMD allele construct showed significantly greater P2 promoter activity than the normal allele. Comparative homology studies showed that the high BMD alleles were the major forms in other animals (dog, rat, mouse, chimp), suggesting that the human normal BMD alleles represent the evolutionary change. The rat sequence was identical to the human high BMD allele, permitting an analysis of DNAprotein interactions in the well characterized ROS17/2.8 rat osteoblast cell line. These data showed abundant nuclear factor binding to one the three polymorphic sites in the human normal BMD allele that does not occur in the high BMD allele (and rat sequence). The data suggest that the high BMD allele does not interact with a factor that otherwise binds strongly to the normal human allele. Since the high BMD allele had higher P2 promoter activity, the binding activity may be a repressor that limits the expression of the normal allele. In any event, the nuclear factor is a candidate for the allele-specific regulation of the RUNX2 promoter.

Runt related transcription factor 2 (RUNX2) is a key regulator of osteoblast differentiation. Several variations within the RUNX2 gene have been found to be associated with significant changes in BMD, which is a major risk factor for fracture. In this study we report that an 18 bp deletion within the polyalanine tract (17A>11A) of RUNX2 is significantly associated with fracture. Carriers of the 11A allele were found to be nearly twice as likely to have sustained fracture. Within the fracture category, there was a significant tendency of 11A carriers to present with fractures of distal radius and bones of intramembranous origin compared to bones of endochondral origin (p = 0.0001). In a population of random subjects, the 11A allele was associated with decreased levels of serum collagen cross links (CTx, p = 0.01), suggesting decreased bone turnover. The transactivation function of the 11A allele showed a minor quantitative decrease. Interestingly, we found no effect of the 11A allele on BMD at multiple skeletal sites. These findings suggest that the 11A allele is a biologically relevant polymorphism that influences serum CTx and confers enhanced fracture risk in a site-selective manner related to intramembranous bone ossification.

RUNX2 is an essential transcription factor required for skeletal development and cartilage formation. Haploinsufficiency of RUNX2 leads to cleidocranial displaysia (CCD) a skeletal disorder characterised by gross dysgenesis of bones particularly those derived from intramembranous bone formation. A notable feature of the RUNX2 protein is the polyglutamine and polyalanine (23Q/17A) domain coded by a repeat sequence. Since none of the known mutations causing CCD characterised to date map in the glutamine repeat region, we hypothesised that Q-repeat mutations may be related to a more subtle bone phenotype. We screened subjects derived from four normal populations for Q-repeat variants. A total of 22 subjects were identified who were heterozygous for a wild type allele and a Q-repeat variant allele: (15Q, 16Q, 18Q and 30Q). Although not every subject had data for all measures, Q-repeat variants had a significant deficit in BMD with an average decrease of 0.7SD measured over 12 BMD-related parameters (p = 0.005). Femoral neck BMD was measured in all subjects (-0.6SD, p = 0.0007). The transactivation function of RUNX2 was determined for 16Q and 30Q alleles using a reporter gene assay. 16Q and 30Q alleles displayed significantly lower transactivation function compared to wild type (23Q). Our analysis has identified novel Q-repeat mutations that occur at a collective frequency of about 0.4%. These mutations significantly alter BMD and display impaired transactivation function, introducing a new class of functionally relevant RUNX2 mutants.

Background Real-time quantitative RT-PCR (qPCR) is a powerful technique capable of accurately quantitating mRNA expression levels over a large dynamic range. This makes qPCR the most widely used method for studying quantitative gene expression. An important aspect of qPCR is selecting appropriate controls or normalization factors to account for any differences in starting cDNA quantities between samples during expression studies. Here, we report on the selection of a concise set of housekeeper genes for the accurate normalization of quantitative gene expression data in differentiating osteoblasts, osteoclasts and macrophages. We implemented the use of geNorm, an algorithm that determines the suitability of genes to function as housekeepers by assessing expression stabilities. We evaluated the expression stabilities of 18S, ACTB, B2M, GAPDH, HMBS and HPRT1 genes. Findings Our analyses revealed that 18S and GAPDH were regulated during osteoblast differentiation and are not suitable for use as reference genes. The most stably expressed genes in osteoblasts were ACTB, HMBS and HPRT1 and their geometric average constitutes a suitable normalization factor upon which gene expression data can be normalized. In macrophages, 18S and GAPDH were the most variable genes while HMBS and B2M were the most stably expressed genes. The geometric average of HMBS and B2M expression levels forms a suitable normalization factor to account for potential differences in starting cDNA quantities during gene expression analysis in macrophages. The expression stabilities of the six candidate reference genes in osteoclasts were, on average, more variable than that observed in macrophages but slightly less variable than those seen in osteoblasts. The two most stably expressed genes in osteoclasts were HMBS and B2M and the genes displaying the greatest levels of variability were 18S and GAPDH. Notably, 18S and GAPDH were the two most variably expressed control genes in all three cell types. The geometric average of HMBS, B2M and ACTB creates an appropriate normalization factor for gene expression studies in osteoclasts. Conclusion We have identified concise sets of genes suitable to use as normalization factors for quantitative real-time RT-PCR gene expression studies in osteoblasts, osteoclasts and macrophages.

Rapid mineralization of cultured osteoblasts could be a useful characteristic in stem cell-mediated therapies for fracture and other orthopedic problems. Dimethyl sulfoxide (DMSO) is a small amphipathic solvent molecule capable of stimulating cell differentiation. We report that, in primary human osteoblasts, DMSO dose-dependently enhanced the expression of osteoblast differentiation markers alkaline phosphatase activity and extracellular matrix mineralization. Furthermore, similar DMSO-mediated mineralization enhancement was observed in primary osteoblast-like cells differentiated from mouse mesenchymal cells derived from fat, a promising source of starter cells for cell-based therapy. Using a convenient mouse pre-osteoblast model cell line MC3T3-E1, we further investigated this phenomenon showing that numerous osteoblast-expressed genes were elevated in response to DMSO treatment and correlated with enhanced mineralization. Myocyte enhancer factor 2c (Mef2c) was identified as the transcription factor most induced by DMSO, among the numerous DMSO-induced genes, suggesting a role for Mef2c in osteoblast gene regulation. Immunohistochemistry confirmed expression of Mef2c in osteoblast-like cells in mouse mandible, cortical, and trabecular bone. shRNAi-mediated Mef2c gene silencing resulted in defective osteoblast differentiation, decreased alkaline phosphatase activity, and matrix mineralization and knockdown of osteoblast specific gene expression, including osteocalcin and bone sialoprotein. A flow on knockdown of bone-specific transcription factors, Runx2 and osterix by shRNAi knockdown of Mef2c, suggests that Mef2c lies upstream of these two important factors in the cascade of gene expression in osteoblasts.

Loading of the rat ulna is an ideal model to examine stress fracture healing. The aim of this study was to undertake a detailed examination of the histology, histomorphometry and gene expression of the healing and remodelling process initiated by fatigue loading of the rat ulna. Ulnae were harvested 1, 2, 4, 6, 8, and 10 weeks following creation of a stress fracture. Stress fracture healing involved direct remodelling that progressed along the fracture line as well as woven bone proliferation at the site of the fracture. Histomorphometry demonstrated rapid progression of basic multicellular units from 1 to 4 weeks with significant slowing down of healing by 10 weeks after loading. Quantitative PCR was performed at 4 hours, 24 hours, 4 days, 7 days, and 14 days after loading. Gene expression was compared to an unloaded control group. At 4 hours after fracture, there was a marked 220-fold increase (P

Objective To determine a practical approach for deriving life expectancy estimates in Australian New South Wales local government areas which display a large diversity in population sizes. Design Population-based study utilising mortality and estimated residential population data. Setting 153 local government areas in New South Wales, Australia. Outcome measures Key performance measures of Chiang II, Silcocks, adjusted Chiang II and Bayesian random effects model methodologies of life expectancy estimation including agreement analysis of life expectancy estimates and comparison of estimate SEs. Results Chiang II and Silcocks methods produced almost identical life expectancy estimates across a large range of population sizes but calculation failures and excessively large SEs limited their use in small populations. A population of 25 000 or greater was required to estimate life expectancy with SE of 1 year or less using adjusted Chiang II (a composite of Chiang II and Silcocks methods). Data aggregation offered some remedy for extending the use of adjusted Chiang II in small populations but reduced estimate currency. A recently developed Bayesian random effects model utilising the correlation in mortality rates between genders, age groups and geographical areas markedly improved the precision of life expectancy estimates in small populations. Conclusions We propose a hybrid approach for the calculation of life expectancy using the Bayesian random effects model in populations of 25 000 or lower permitting the precise derivation of life expectancy in small populations. In populations above 25 000, we propose the use of adjusted Chiang II to guard against violations of spatial correlation, to benefit from a widely accepted method that is simpler to communicate to local health authorities and where its slight inferior performance compared with the Bayesian approach is of minor practical significance.