The interplay between chondrocyte redifferentiation pellet size and oxygen concentration

Chondrocytes dedifferentiate during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their redifferentiation and restoration of the chondrogenic phenotype. Typically 1��105���5��105 chondrocytes are aggregated, resulting in ���macro��� pellets having diameters ranging from 1���2 mm. These macropellets are commonly used to study redifferentiation, and recently macropellets of autologous chondrocytes have been implanted directly into articular cartilage defects to facilitate their repair. However, diffusion of metabolites over the 1���2 mm pellet length-scales is inefficient, resulting in radial tissue heterogeneity. Herein we demonstrate that the aggregation of 2��105 human chondrocytes into micropellets of 166 cells each, rather than into larger single macropellets, enhances chondrogenic redifferentiation. In this study, we describe the development of a cost effective fabrication strategy to manufacture a microwell surface for the large-scale production of micropellets. The thousands of micropellets were manufactured using the microwell platform, which is an array of 360��360 ��m microwells cast into polydimethylsiloxane (PDMS), that has been surface modified with an electrostatic multilayer of hyaluronic acid and chitosan to enhance micropellet formation. Such surface modification was essential to prevent chondrocyte spreading on the PDMS. Sulfated glycosaminoglycan (sGAG) production and collagen II gene expression in chondrocyte micropellets increased significantly relative to macropellet controls, and redifferentiation was enhanced in both macro and micropellets with the provision of a hypoxic atmosphere (2% O2). Once micropellet formation had been optimized, we demonstrated that micropellets could be assembled into larger cartilage tissues. Our results indicate that micropellet amalgamation efficiency is inversely related to the time cultured as discreet microtissues. In summary, we describe a micropellet production platform that represents an efficient tool for studying chondrocyte redifferentiation and demonstrate that the micropellets could be assembled into larger tissues, potentially useful in cartilage defect repair.

Female concentration and the gender wage differential in the United Kingdom

The paper utilizes the methodology proposed by Johnson and Solon (American Economic Review, 76 (5), 1117-1125, 1986) to examine the impact of job segregation on the gender wage gap in the UK in 1991. The results suggest that despite implementation of the UK 1983 Equal Pay Amendment there remains clear evidence that male/female workers in female dominated jobs continue to earn less for work of ���similar worth��� than their counterparts in male dominated jobs within the same firm. This conclusion is insensitive to whether one adopts an occupation or firm based measure of gender concentration.

Serum 25-hydroxyvitamin D concentration in relation to melanoma progress

Dr Wyatt���s study investigated the complex relationship between vitamin D and melanoma, specifically if vitamin D status is associated with more aggressive melanomas. Exposure to solar ultraviolet radiation is the principal risk factor for melanoma and also the main source of vitamin D. This research found that insufficient vitamin D at time of melanoma diagnosis is significantly associated with poorer prognosis (as defined by tumour thickness). These results will contribute to a more refined public health message concerning melanoma and vitamin D, particularly in Queensland, which has the highest global incidence of melanoma, but vitamin D deficiency is not uncommon.

Investigation of hormone receptor genes in migraine

Migraine is a common neurological condition with a complex mode of inheritance. Steroid hormones have long been implicated in migraine, although their role remains unclear. Our investigation considered that genes involved in hormonal pathways may play a role in migraine susceptibility. We therefore investigated the androgen receptor (AR) CAG repeat, and the progesterone receptor (PR) PROGINS insert by cross-sectional association analysis. The results showed no association with the AR CAG repeat in our study group of 275 migraineurs and 275 unrelated controls. Results of the PR PROGINS analysis showed a significant difference in the same cohort, and in an independent follow-up study population of 300 migraineurs and 300 unrelated controls. Analysis of the genotypic risk groups of both populations together indicated that individuals who carried the PROGINS insert were 1.8 times more likely to suffer migraine. Interaction analysis of the PROGINS variant with our previously reported associated ESR1 594A variant showed that individuals who possessed at least one copy of both risk alleles were 3.2 times more likely to suffer migraine. Hence, variants of these steroid hormone receptor genes appear to act synergistically to increase the risk of migraine by a factor of three.

Molecular genetic analyses of RFLPs for PCR-amplified growth hormone gene, renal kallikrein gene and atrial natriuretic factor gene in essential hypertension

The present study examined polymorphisms of genes that might be involved in the onset of essential hypertension (HT). These included the (i) growth hormone gene (GH1), whose locus has recently been linked to elevated blood pressure (BP) in the stroke-prone SHR, although recent sib-pair analysis of a polymorphism near the human chorionic somatomammotropin gene (a member of the GH cluster) was unable to show linkage with HT; (ii) renal kallikrein gene (KLK1); and (iii) atrial natriuretic factor gene (ANF), where a primary defect in production or activity of kallikrein or ANF could cause NaCl retention and vasoconstriction. Association analyses were conducted to compare restriction fragment length polymorphisms (RFLPs) of each gene in 85 HT and 95 normotensive (NT) Caucasian subjects whose parents had a similar BP status at age ���50 years. The frequency of the minor allele of (i) a RsaI RFLP in the promoter of GH1, amplified from leukocyte DNA by the polymerase chain reaction, was 0.15 in the HT group and 0.14 in the NT group (��1=0.34, P=0.55); (ii) a TaqI RFLP for KLK1 was 0.035 in the HT group and 0.015 in the NT group (��2=1.5, P=0.21); and (iii) a XhoI RFLP for ANF was 0.50 in HTs and 0.46 in NTs (��2=0.20, P=0.65). Studies of HT pedigrees found one family in which the ANF locus and HT were not linked, owing to an obligate recombinant. The present data thus provide no evidence for involvement of the growth hormone, renal kallikrein, nor ANF gene in the causation of essential hypertension.

Diurnal rhythm and effects of feeding, exercise and recombinant equine growth hormone on serum insulin concentrations in the horse

Reasons for performing the study As growth hormone increases lean body mass, it could be a therapy for obese horses. However, growth hormone use induces hyperinsulinaemia in some species, so further investigation is warranted. Objectives To investigate the effects of feeding, exercise and growth hormone therapy on basal insulin concentrations in healthy horses. Study design In vivo experimental study. Methods Blood samples were obtained every 30���min from 12 geldings over 24���h, to establish basal serum insulin concentrations, before they underwent a 3-week exercise programme. Horses were allocated into 2 groups and exercised for another 4 weeks. Group���A received daily i.m. injections of recombinant equine growth hormone; 5���mg/day for 5 days, then 12.5���mg/day for 16 days. Blood samples were taken daily before feeding. Insulin vs. time area under curve of Groups���A and B were compared using a Student's unpaired t test. Results Horses demonstrated insulin peaks within 2���h of feeding of 577 �� 108.3���pmol/l at 09.30���h and 342.4 �� 75.7���pmol/l at 17.30���h, despite receiving the same meal. The nadir was between midnight and 07.30���h. Exercise had no effect on basal insulin concentrations prior to equine growth hormone administrations. The equine growth hormone injections increased serum insulin concentrations (P = 0.01) within Group���A, from 44.4 �� 15.3���pmol/l initially to 320.9 �� 238.2���pmol/l by Day 12. Exogenous growth hormone caused variable hyperinsulinaemia, which was alleviated once equine growth hormone administration ceased. Conclusions Single serum samples taken prior to the morning meal provide basal insulin concentrations. Exercise did not change basal insulin concentrations. However, equine growth hormone injections increased basal insulin concentrations, which were not ameliorated by exercise. Potential relevance This therapy is not recommended to address obesity in insulin-resistant equids.

Spatio-temporal modelling of ultrafine particle number concentration

This thesis developed semi-parametric regression models for estimating the spatio-temporal distribution of outdoor airborne ultrafine particle number concentration (PNC). The models developed incorporate multivariate penalised splines and random walks and autoregressive errors in order to estimate non-linear functions of space, time and other covariates. The models were applied to data from the "Ultrafine Particles from Traffic Emissions and Child" project in Brisbane, Australia, and to longitudinal measurements of air quality in Helsinki, Finland. The spline and random walk aspects of the models reveal how the daily trend in PNC changes over the year in Helsinki and the similarities and differences in the daily and weekly trends across multiple primary schools in Brisbane. Midday peaks in PNC in Brisbane locations are attributed to new particle formation events at the Port of Brisbane and Brisbane Airport.

Low muscle glycogen concentration does not suppress the anabolic response to resistance exercise

We determined the effect of muscle glycogen concentration and postexercise nutrition on anabolic signaling and rates of myofibrillar protein synthesis after resistance exercise (REX). Sixteen young, healthy men matched for age, body mass, peak oxygen uptake (VO2peak) and strength (one repetition maximum; 1RM) were randomly assigned to either a nutrient or placebo group. After 48 h diet and exercise control, subjects undertook a glycogen-depletion protocol consisting of one-leg cycling to fatigue (LOW), whereas the other leg rested (NORM). The next morning following an overnight fast, a primed, constant infusion of L-[ring-13C6] phenylalanine was commenced and subjects completed 8 sets of 5 unilateral leg press repetitions at 80% 1RM. Immediately after REX and 2 h later, subjects consumed a 500 ml bolus of a protein/CHO (20 g whey + 40 g maltodextrin) or placebo beverage. Muscle biopsies from the vastus lateralis of both legs were taken at rest and 1 and 4 h after REX. Muscle glycogen concentration was higher in the NORM than LOW at all time points in both nutrient and placebo groups (P < 0.05). Postexercise Akt-p70S6K-rpS6 phosphorylation increased in both groups with no differences between legs (P < 0.05). mTORSer2448 phosphorylation in placebo increased 1 h after exercise in NORM (P < 0.05), whereas mTOR increased ?4-fold in LOW (P < 0.01) and ?11 fold in NORM with nutrient (P < 0.01; different between legs P < 0.05). Post-exercise rates of MPS were not different between NORM and LOW in nutrient (0.070 �� 0.022 vs. 0.068 �� 0.018 %/h) or placebo (0.045 �� 0.021 vs. 0.049 �� 0.017 %/h). We conclude that commencing high-intensity REX with low muscle glycogen availability does not compromise the anabolic signal and subsequent rates of MPS, at least during the early (4 h) postexercise recovery period.

A randomised, concentration-controlled, comparison of standard (5-day) vs. prolonged (15-day) infusions of etoposide phosphate in small-cell lung cancer

Purpose: This randomised trial was designed to investigate the activity and toxicity of continuous infusion etoposide phosphate (EP), targeting a plasma etoposide concentration of either 3 ��g/ml for five days (5d) or 1 ��g/ml for 15 days (15d), in previously untreated SCLC patients with extensive disease. Patients and methods: EP was used as a single agent. Plasma etoposide concentration was monitored on days 2 and 4 in patients receiving 5d EP and on days 2, 5, 8 and 11 in patients receiving 15d EP, with infusion modification to ensure target concentrations were achieved. Treatment was repeated every 21 days for up to six cycles, with a 25% reduction in target concentration in patients with toxicity. Results: The study has closed early after entry of 29 patients (14 with 5d EP, 15 with 15d EP). Objective responses were seen in seven of 12 (58%, confidence interval (CI): 27%-85%) evaluable patients after 5d EP, and two of 14 (14%, CI: 4%42%) evaluable patients after 15d EP (P = 0.038). Grade 3 or 4 neutropenia or leucopenia during the first cycle of treatment was observed in six of 12 patients after 5d EP and 0/14 patients after 15d EP (P = 0.004), with median nadir WBC count of 2.6 x 109/1 after 5d and 5.0 x 109/1 after 15d EP (P = 0.017). Only one of 49 cycles of 15d EP was associated with grade 3 or worse haematological toxicity, compared to 14 of 61 cycles of 5d EP. Conclusions: Although the number of patients entered into this trial was small, the low activity seen at 1 ��g/ml in the 15d arm suggests that this concentration is below the therapeutic window in this setting. Further concentration- controlled studies with prolonged EP infusions are required.

Sex hormone regulation of innate immunity in the female reproductive tract : the role of epithelial cells in balancing reproductive potential with protection against sexually transmitted pathogens

The immune system in the female reproductive tract (FRT) does not mount an attack against HIV or other sexually transmitted infections (STI) with a single endogenously produced microbicide or with a single arm of the immune system. Instead, the body deploys dozens of innate antimicrobials to the secretions of the female reproductive tract. Working together, these antimicrobials along with mucosal antibodies attack many different viral, bacterial and fungal targets. Within the FRT, the unique challenges of protection against sexually transmitted pathogens coupled with the need to sustain the development of an allogeneic fetus have evolved in such a way that sex hormones precisely regulate immune function to accomplish both tasks. The studies presented in this review demonstrate that estradiol and progesterone secreted during the menstrual cycle act both directly and indirectly on epithelial cells and other immune cells in the reproductive tract to modify immune function in a way that is unique to specific sites throughout the FRT. As presented in this review, studies from our laboratory and others demonstrate that the innate immune response is under hormonal control, varies with the stage of the menstrual cycle, and as such is suppressed at mid-cycle to optimize conditions for successful fertilization and pregnancy. In doing so, a window of STI vulnerability is created during which potential pathogens including HIV enter the reproductive tract to infect host targets.

Interleukin-1beta induces human proximal tubule cell injury, alpha-smooth muscle actin expression and fibronectin production

BACKGROUND Tubulointerstitial lesions, characterized by tubular injury, interstitial fibrosis and the appearance of myofibroblasts, are the strongest predictors of the degree and progression of chronic renal failure. These lesions are typically preceded by macrophage infiltration of the tubulointerstitium, raising the possibility that these inflammatory cells promote progressive renal disease through fibrogenic actions on resident tubulointerstitial cells. The aim of the present study, therefore, was to investigate the potentially fibrogenic mechanisms of interleukin-1beta (IL-1beta), a macrophage-derived pro-inflammatory cytokine, on human proximal tubule cells (PTC). METHODS Confluent, quiescent, passage 2 PTC were established in primary culture from histologically normal segments of human renal cortex (N = 11) and then incubated in serum- and hormone-free media supplemented with either IL-1beta (0 to 4 ng/mL) or vehicle (control). RESULTS IL-1beta significantly enhanced fibronectin secretion by up to fourfold in a time- and concentration-dependent fashion. This was accompanied by significant (2.5- to 6-fold) increases in alpha-smooth muscle actin (alpha-SMA) expression, transforming growth factor beta (TGF-beta1) secretion, nitric oxide (NO) production, NO synthase 2 (NOS2) mRNA and lactate dehydrogenase (LDH) release. Cell proliferation was dose-dependently suppressed by IL-1beta. NG-methyl-l-arginine (L-NMMA; 1 mmol/L), a specific inhibitor of NOS, blocked NO production but did not alter basal or IL-1beta-stimulated fibronectin secretion. In contrast, a pan-specific TGF-beta neutralizing antibody significantly blocked the effects of IL-1beta on PTC fibronectin secretion (IL-1beta, 268.1 +/- 30.6 vs. IL-1beta+alphaTGF-beta 157.9 +/- 14.4%, of control values, P < 0.001) and DNA synthesis (IL-1beta 81.0 +/- 6.7% vs. IL-1beta+alphaTGF-beta 93.4 +/- 2.1%, of control values, P < 0.01). CONCLUSION IL-1beta acts on human PTC to suppress cell proliferation, enhance fibronectin production and promote alpha-smooth muscle actin expression. These actions appear to be mediated by a TGF-beta1 dependent mechanism and are independent of nitric oxide release.

Identification of a long non-coding RNA gene, GHSROS (growth hormone secretagogue receptor opposite strand), which stimulates cell migration in non-small cell lung cancer cell lines

The molecular mechanisms involved in non���small cell lung cancer tumourigenesis are largely unknown; however, recent studies have suggested that long non-coding RNAs (lncRNAs) are likely to play a role. In this study, we used public databases to identify an mRNA-like, candidate long non-coding RNA, GHSROS (GHSR opposite strand), transcribed from the antisense strand of the ghrelin receptor gene, growth hormone secretagogue receptor (GHSR). Quantitative real-time RT-PCR revealed higher expression of GHSROS in lung cancer tissue compared to adjacent, non-tumour lung tissue. In common with many long non-coding RNAs, GHSROS is 5' capped and 3' polyadenylated (mRNA-like), lacks an extensive open reading frame and harbours a transposable element. Engineered overexpression of GHSROS stimulated cell migration in the A549 and NCI-H1299 non-small cell lung cancer cell lines, but suppressed cell migration in the Beas-2B normal lung-derived bronchoepithelial cell line. This suggests that GHSROS function may be dependent on the oncogenic context. The identification of GHSROS, which is expressed in lung cancer and stimulates cell migration in lung cancer cell lines, contributes to the growing number of non-coding RNAs that play a role in the regulation of tumourigenesis and metastatic cancer progression.

Hybrid genetic algorithm for elimination of severe stress concentration in railhead ends

This paper presents a computational method for eliminating severe stress concentration at the unsupported railhead ends in rail joints through innovative shape optimization of the contact zone, which is complex due to near field nonlinear contact. With a view to minimizing the computational efforts, hybrid genetic algorithm method coupled with parametric finite element has been developed and compared with the traditional genetic algorithm (GA). The shape of railhead top surface where the wheel contacts nonlinearly was optimized using the hybridized GA method. Comparative study of the optimal result and the search efficiency between the traditional and hybrid GA methods has shown that the hybridized GA provides the optimal shape in fewer computational cycles without losing accuracy. The method will be beneficial to solving complex engineering problems involving contact nonlinearity.

Intermittent Fugu parathyroid hormone 1 (1���34) is an anabolic bone agent in young male rats and osteopenic ovariectomized rats

Human parathyroid hormone (hPTH) is currently the only treatment for osteoporosis that forms new bone. Previously we described a fish equivalent, Fugu parathyroid hormone 1 (fPth1) which has hPTH-like biological activity in vitro despite fPth1(1���34) sharing only 53% identity with hPTH(1���34). Here we demonstrate the in vivo actions of fPth1(1���34) on bone. In study 1, young male rats were injected intermittently for 30 days with fPth1 [30 ��g���1000 ��g/kg body weight (b.w.), (30fPth1���1000fPth1)] or hPTH [30 ��g���100 ��g/kg b.w. (30hPTH���100hPTH)]. In proximal tibiae at low doses, the fPth1 was positively correlated with trabecular bone volume/total volume (TbBV/TV) while hPTH increased TbBV/TV, trabecular thickness (TbTh) and trabecular number (TbN). 500fPth1 and 1000fPth1 increased TbBV/TV, TbTh, TbN, mineral apposition rate (MAR) and bone formation rate/bone surface (BFR/BS) with a concomitant decrease in osteoclast surface and number. In study 2 ovariectomized (OVX), osteopenic rats and sham operated (SHAM) rats were injected intermittently with 500 ��g/kg b.w. of fPth1 (500fPth1) for 11 weeks. 500fPth1 treatment resulted in increased TbBV/TV (151%) and TbTh (96%) in the proximal tibiae due to increased bone formation as assessed by BFR/BS (490%) and MAR (131%). The effect was restoration of TbBV/TV to SHAM levels without any effect on bone resorption. 500fPth1 also increased TbBV/TV and TbTh in the vertebrae (L6) and cortical thickness in the mid-femora increasing bone strength at these sites. fPth1 was similarly effective in SHAM rats. Notwithstanding the low amino acid sequence homology with hPTH (1���34), we have clearly established the efficacy of fPth1 (1���34) as an anabolic bone agent.

Hormone resistance, invasiveness, and metastatic potential in breast cancer

Critical phenotypic changes that occur during the progression of breast cancer include the loss of hormone-dependence, acquired resistance to systemic therapies, and increased metastatic potential. We have isolated a series of MCF-7 human breast cancer variants which exhibit hormone-independent growth, antiestrogen resistance, and increased metastatic potential. Analysis of the phenotypes of these variants strongly suggests that changes in the expression of specific genes may be critical to the generation of phenotypic diversity in the process of malignant progression in breast cancer. Epigenetic changes may contribute significantly to the generation of these phenotypic changes observed during breast cancer progression. Many of the characteristics of the progressed phenotypes appear to have arisen in response to appropriate selective pressures (growth in ovariectomized nude mice; growth in the presence of antiestrogens). These observations are consistent with the concept of clonal selection and expansion in the process of malignant progression.