A variant of the KLK4 gene is expressed as a cis sense-antisense chimeric transcript in prostate cancer cells

In humans, more than 30,000 chimeric transcripts originating from 23,686 genes have been identified. The mechanisms and association of chimeric transcripts arising from chromosomal rearrangements with cancer are well established, but much remains unknown regarding the biogenesis and importance of other chimeric transcripts that arise from nongenomic alterations. Recently, a SLC45A3–ELK4 chimera has been shown to be androgen-regulated, and is overexpressed in metastatic or high-grade prostate tumors relative to local prostate cancers. Here, we characterize the expression of a KLK4 cis sense–antisense chimeric transcript, and show other examples in prostate cancer. Using non-protein-coding microarray analyses, we initially identified an androgen-regulated antisense transcript within the 3′ untranslated region of the KLK4 gene in LNCaP cells. The KLK4 cis-NAT was validated by strand-specific linker-mediated RT-PCR and Northern blotting. Characterization of the KLK4 cis-NAT by 5′ and 3′ rapid amplification of cDNA ends (RACE) revealed that this transcript forms multiple fusions with the KLK4 sense transcript. Lack of KLK4 antisense promoter activity using reporter assays suggests that these transcripts are unlikely to arise from a trans-splicing mechanism. 5′ RACE and analyses of deep sequencing data from LNCaP cells treated ±androgens revealed six high-confidence sense–antisense chimeras of which three were supported by the cDNA databases. In this study, we have shown complex gene expression at the KLK4 locus that might be a hallmark of cis sense–antisense chimeric transcription.

Comparison of koala LPCoLN and human strains of Chlamydia pneumoniae highlights extended genetic diversity in the species

Background Chlamydia pneumoniae is a widespread pathogen causing upper and lower respiratory tract infections in addition to a range of other diseases in humans and animals. Previous whole genome analyses have focused on four essentially clonal (> 99% identity) C. pneumoniae human genomes (AR39, CWL029, J138 and TW183), providing relatively little insight into strain diversity and evolution of this species. Results We performed individual gene-by-gene comparisons of the recently sequenced C. pneumoniae koala genome and four C. pneumoniae human genomes to identify species-specific genes, and more importantly, to gain an insight into the genetic diversity and evolution of the species. We selected genes dispersed throughout the chromosome, representing genes that were specific to C. pneumoniae, genes with a demonstrated role in chlamydial biology and/or pathogenicity (n = 49), genes encoding nucleotide salvage or amino acid biosynthesis proteins (n = 6), and extrachromosomal elements (9 plasmid and 2 bacteriophage genes). Conclusions We have identified strain-specific differences and targets for detection of C. pneumoniae isolates from both human and animal origin. Such characterisation is necessary for an improved understanding of disease transmission and intervention.

Replication of the Frank-Starling response in a Mock Circulation loop

Mock circulation loops (MCLs) are used to evaluate cardiovascular devices prior to in-vivo trials; however they lack the vital autoregulatory responses that occur in humans. This study aimed to develop and implement a left and right ventricular Frank-Starling response in a MCL. A proportional controller based on ventricular end diastolic volume was used to control the driving pressure of the MCL’s pneumatically operated ventricles. Ventricular pressure-volume loops and end systolic pressure-volume relationships were produced for a variety of healthy and pathological conditions and compared with human data to validate the simulated Frank-Starling response. The non-linear Frank-Starling response produced in this study successfully altered left and right ventricular contractility with changing preload and was validated with previously reported data. This improvement to an already detailed MCL has resulted in a test rig capable of further refining cardiovascular devices and reducing the number of in-vivo trials.

Fitness and fatness in childhood obesity : implications of physical activity

Overweight and obesity are a significant cause of poor health worldwide, particularly in conjunction with low levels of physical activity (PA). PA is health-protective and essential for the physical growth and development of children, promoting physical and psychological health while simultaneously increasing the probability of remaining active as an adult. However, many obese children and adolescents have a unique set of physiological, biomechanical, and neuromuscular barriers to PA that they must overcome. It is essential to understand the influence of these barriers on an obese child's motivation in order to exercise and tailor exercise programs to the special needs of this population. Chapter Outline • Introduction • Defining Physical Activity, Exercise, and Physical Fitness • Physical Activity, Physical Fitness, And Motor Competence In Obese Children • Physical Activity and Obesity in Children • Physical Fitness in Obese Children • Balance and Gait in Obese Children • Motor Competence in Obese Children • Physical Activity Guidelines for Obese Children • Clinical Assessment of the Obese Child • Physical Activity Characteristics: Mode • Physical Activity Characteristics: Intensity • Physical Activity Characteristics: Frequency • Physical Activity Characteristics: Duration • Conclusion

Partial agonists of the [alpha]3[beta]4[ast] neuronal nicotinic acetylcholine receptor reduce ethanol consumption and seeking in rats

Alcohol use disorders (AUDs) impact millions of individuals and there remain few effective treatment strategies. Despite evidence that neuronal nicotinic acetylcholine receptors (nAChRs) have a role in AUDs, it has not been established which subtypes of the nAChR are involved. Recent human genetic association studies have implicated the gene cluster CHRNA3-CHRNA5-CHRNB4 encoding the α3, α5, and β4 subunits of the nAChR in susceptibility to develop nicotine and alcohol dependence; however, their role in ethanol-mediated behaviors is unknown due to the lack of suitable and selective research tools. To determine the role of the α3, and β4 subunits of the nAChR in ethanol self-administration, we developed and characterized high-affinity partial agonists at α3β4 nAChRs, CP-601932, and PF-4575180. Both CP-601932 and PF-4575180 selectively decrease ethanol but not sucrose consumption and operant self-administration following long-term exposure. We show that the functional potencies of CP-601932 and PF-4575180 at α3β4 nAChRs correlate with their unbound rat brain concentrations, suggesting that the effects on ethanol self-administration are mediated via interaction with α3β4 nAChRs. Also varenicline, an approved smoking cessation aid previously shown to decrease ethanol consumption and seeking in rats and mice, reduces ethanol intake at unbound brain concentrations that allow functional interactions with α3β4 nAChRs. Furthermore, the selective α4β2(*) nAChR antagonist, DHβE, did not reduce ethanol intake. Together, these data provide further support for the human genetic association studies, implicating CHRNA3 and CHRNB4 genes in ethanol-mediated behaviors. CP-601932 has been shown to be safe in humans and may represent a potential novel treatment for AUDs.

Negative affect-induced food intake in non-dieting women is reward driven and associated with restrained-disinhibited eating subtype

In humans the presence of negative affect is thought to promote food intake, but there is widespread variability. Susceptibility to negative affect-induced eating may depend on trait eating behaviours, notably ‘emotional eating’, ‘restrained eating’ and ‘disinhibited eating’, but the evidence is not consistent. In the present study, 30 non-obese, non-dieting women were given access to palatable food whilst in a state of negative or neutral affect, induced by a validated autobiographical recall technique. As predicted, food intake was higher in the presence of negative affect; however, this effect was moderated by the pattern of eating behaviour traits and enhanced wanting for the test food. Specifically, the High Restraint-High Disinhibition subtype in combination with higher scores on emotional eating and food wanting was able to predict negative-affect intake (adjusted R2 = .61). In the absence of stress, individuals who are both restrained and vulnerable to disinhibited eating are particularly susceptible to negative affect food intake via stimulation of food wanting. Identification of traits that predispose individuals to overconsume and a more detailed understanding of the specific behaviours driving such overconsumption may help to optimise strategies to prevent weight gain.

Modulation of the Chlamydia trachomatis In vitro transcriptome response by the sex hormones estradiol and progesterone

BACKGROUND:Chlamydia trachomatis is a major cause of sexually transmitted disease in humans. Previous studies in both humans and animal models of chlamydial genital tract infection have suggested that the hormonal status of the genital tract epithelium at the time of exposure can influence the outcome of the chlamydial infection. We performed a whole genome transcriptional profiling study of C. trachomatis infection in ECC-1 cells under progesterone or estradiol treatment.RESULTS:Both hormone treatments caused a significant shift in the sub-set of genes expressed (25% of the transcriptome altered by more than 2-fold). Overall, estradiol treatment resulted in the down-regulation of 151 genes, including those associated with lipid and nucleotide metabolism. Of particular interest was the up-regulation in estradiol-supplemented cultures of six genes (omcB, trpB, cydA, cydB, pyk and yggV), which suggest a stress response similar to that reported previously in other models of chlamydial persistence. We also observed morphological changes consistent with a persistence response. By comparison, progesterone supplementation resulted in a general up-regulation of an energy utilising response.CONCLUSION:Our data shows for the first time, that the treatment of chlamydial host cells with key reproductive hormones such as progesterone and estradiol, results in significantly altered chlamydial gene expression profiles. It is likely that these chlamydial expression patterns are survival responses, evolved by the pathogen to enable it to overcome the host's innate immune response. The induction of chlamydial persistence is probably a key component of this survival response.

Polycaprolactone-based scaffold plus rhBMP-2 in a sheep thoracic spine fusion model

We report the application of a novel scaffold design in a sheep thoracic spine model for spine deformity correction. The combination of the calcium-phosphate coated polycaprolactone scaffolds with recombinant human bone morphogenic protein-2 (rhBMP-2) are intended as a future bone graft substitute in ensuring the stability of bony intervertebral fusion. A solid free-form fabrication process based on melt extrusion has been utilized in the manufacturing of these scaffolds. To date there are no studies examining the use of such biodegradable implants in a sheep thoracic spine model. The success of anterior scoliosis surgery in humans depends on achieving a solid bony fusion between adjacent vertebrae after the intervertebral discs have been surgically cleared and the disc spaces filled with graft material. Due to limited availability of autograft, there is much current interest in the development of synthetic scaffolds in combination with growth factors such as recombinant human bone morphogenetic protein (rhBMP-2) to achieve a solid bony fusion following scoliosis surgery.

The effect of oral metformin on insulin sensitivity in insulin-resistant ponies

Metformin may be an effective therapeutic option for insulin-resistant (I-R) horses/ponies because, in humans, it reportedly enhances insulin sensitivity (SI) of peripheral tissues without stimulating insulin secretion. To determine the effect of metformin on insulin and glucose dynamics in I-R ponies, six ponies were studied in a cross-over design by Minimal Model analysis of a frequently-sampled intravenous glucose tolerance test (FSIGT). Metformin was administered at 15. mg/kg bodyweight (BW), orally, twice-daily, for 21. days to the metformin-treated group. The control group received a placebo. A FSIGT was conducted before and after treatment. The Minimal Model of glucose and insulin dynamics rendered indices describing SI, glucose effectiveness (Sg), acute insulin response to glucose (AIRg) and the disposition index (DI). The body condition score (BCS), BW and cresty neck score (CNS) were also assessed. There was no significant change in SI, Sg, AIRg, DI, BW, BCS or CNS in response to metformin, or over time in the control group. There were no measurable benefits of metformin on SI, consistent with recent work showing that the bioavailability of metformin in horses is poor, and chronic dosing may not achieve therapeutic blood concentrations. Alternatively, metformin may only be effective in obese ponies losing weight or with hyperglycaemia.

A tissue engineering solution for segmental defect regeneration in load-bearing long bones

The reconstruction of large defects (>10 mm) in humans usually relies on bone graft transplantation. Limiting factors include availability of graft material, comorbidity, and insufficient integration into the damaged bone. We compare the gold standard autograft with biodegradable composite scaffolds consisting of medical-grade polycaprolactone and tricalcium phosphate combined with autologous bone marrow-derived mesenchymal stem cells (MSCs) or recombinant human bone morphogenetic protein 7 (rhBMP-7). Critical-sized defects in sheep - a model closely resembling human bone formation and structure - were treated with autograft, rhBMP-7, or MSCs. Bridging was observed within 3 months for both the autograft and the rhBMP-7 treatment. After 12 months, biomechanical analysis and microcomputed tomography imaging showed significantly greater bone formation and superior strength for the biomaterial scaffolds loaded with rhBMP-7 compared to the autograft. Axial bone distribution was greater at the interfaces. With rhBMP-7, at 3 months, the radial bone distribution within the scaffolds was homogeneous. At 12 months, however, significantly more bone was found in the scaffold architecture, indicating bone remodeling. Scaffolds alone or with MSC inclusion did not induce levels of bone formation comparable to those of the autograft and rhBMP-7 groups. Applied clinically, this approach using rhBMP-7 could overcome autograft-associated limitations.

A spatiotemporal analysis of gait freezing and the impact of pedunculopontine nucleus stimulation

Gait freezing is an episodic arrest of locomotion due to an inability to take normal steps. Pedunculopontine nucleus stimulation is an emerging therapy proposed to improve gait freezing, even where refractory to medication. However, the efficacy and precise effects of pedunculopontine nucleus stimulation on Parkinsonian gait disturbance are not established. The clinical application of this new therapy is controversial and it is unknown if bilateral stimulation is more effective than unilateral. Here, in a double-blinded study using objective spatiotemporal gait analysis, we assessed the impact of unilateral and bilateral pedunculopontine nucleus stimulation on triggered episodes of gait freezing and on background deficits of unconstrained gait in Parkinson’s disease. Under experimental conditions, while OFF medication, Parkinsonian patients with severe gait freezing implanted with pedunculopontine nucleus stimulators below the pontomesencephalic junction were assessed during three conditions; off stimulation, unilateral stimulation and bilateral stimulation. Results were compared to Parkinsonian patients without gait freezing matched for disease severity and healthy controls. Pedunculopontine nucleus stimulation improved objective measures of gait freezing, with bilateral stimulation more effective than unilateral. During unconstrained walking, Parkinsonian patients who experience gait freezing had reduced step length and increased step length variability compared to patients without gait freezing; however, these deficits were unchanged by pedunculopontine nucleus stimulation. Chronic pedunculopontine nucleus stimulation improved Freezing of Gait Questionnaire scores, reflecting a reduction of the freezing encountered in patients’ usual environments and medication states. This study provides objective, double-blinded evidence that in a specific subgroup of Parkinsonian patients, stimulation of a caudal pedunculopontine nucleus region selectively improves gait freezing but not background deficits in step length. Bilateral stimulation was more effective than unilateral.

Plant-made vaccines for humans and animals

Summary: The concept of using plants to produce high-value pharmaceuticals such as vaccines is 20 years old this year and is only now on the brink of realisation as an established technology. The original reliance on transgenic plants has largely given way to transient expression; proofs of concept for human and animal vaccines and of efficacy for animal vaccines have been established; several plant-produced vaccines have been through Phase I clinical trials in humans and more are scheduled; regulatory requirements are more clear than ever, and more facilities exist for manufacture of clinic-grade materials. The original concept of cheap edible vaccines has given way to a realisation that formulated products are required, which may well be injectable. The technology has proven its worth as a means of cheap, easily scalable production of materials: it now needs to find its niche in competition with established technologies. The realised achievements in the field as well as promising new developments will be reviewed, such as rapid-response vaccines for emerging viruses with pandemic potential and bioterror agents. © 2010 The Author. Journal compilation © 2010 Blackwell Publishing Ltd.

Brominated flame retardants in the Australian population : 1993-2009

Brominated flame retardants, including hexabromocyclododecane (HBCD) and polybrominated diphenyl ethers (PBDEs) are used to reduce the flammability of a multitude of electrical and electronic products, textiles and foams. The use of selected PBDEs has ceased, however, use of decaBDE and HBCD continues. While elevated concentrations of PBDEs in humans have been observed in Australia, no data is available on other BFRs such as HBCD. This study aimed to provide background HBCD concentrations from a representative sample of the Australian population and to assess temporal trends of HBCD and compare with PBDE concentrations over a 16 year period. Samples of human milk collected in Australia from 1993 to 2009, primarily from primiparae mothers were combined into 12 pools from 1993 (2 pools); 2001; 2002/2003 (4 pools); 2003/2004; 2006; 2007/2008 (2 pools); and 2009. Concentrations of ∑HBCD ranged from not quantified (nq) to 19 ng g−1 lipid while α-HBCD and γ-HBCD ranged from nq to 10 ng g−1 lipid and nq to 9.2 ng g−1 lipid. β-HBCD was detected in only one sample at 3.6 ng g−1 lipid while ∑4PBDE ranged from 2.5 to 15.8 ng g−1 lipid. No temporal trend was apparent in HBCD concentrations in human milk collected in Australia from 1993 to 2009. In comparison, PBDE concentrations in human milk show a peak around 2002/03 (mean ∑4PBDEs = 9.6 ng g−1 lipid) and 2003/04 (12.4 ng g−1 lipid) followed by a decrease in 2007/08 (2.7 ng g−1 lipid) and 2009 (2.6 ng g−1 lipid). In human blood serum samples collected from the Australian population, PBDE concentrations did not vary greatly (p = 0.441) from 2002/03 to 2008/09. Continued monitoring including both human milk and serum for HBCD and PBDEs is required to observe trends in human body burden of HBCD and PBDEs body burden following changes to usage.

Prenatal Vitamin D deficiency induces an early and more severe experimental autoimmune encephalomyelitis in the second generation

In a previous study, we demonstrated that mouse adult F(1) offspring, exposed to a vitamin d deficiency during pregnancy, developed a less severe and delayed Experimental Autoimmune Encephalomyelitis (EAE), when compared with control offspring. We then wondered whether a similar response was observed in the subsequent generation. To answer this question, we assessed F(2) females whose F(1) parents (males or females) were vitamin d-deprived when developing in the uterus of F(0) females. Unexpectedly, we observed that the vitamin d deficiency affecting the F(0) pregnant mice induced a precocious and more severe EAE in the F(2) generation. This paradoxical finding led us to assess its implications for the epidemiology of Multiple Sclerosis (MS) in humans. Using the REFGENSEP database for MS trios (the patient and his/her parents), we collected the parents' dates of birth and assessed a potential season of birth effect that could potentially be indicative of the vitamin d status of the pregnant grandmothers. A trend for a reduced number of births in the Fall for the parents of MS patients was observed but statistical significance was not reached. Further well powered studies are warranted to validate the latter finding.

Antigenic specificity of a monovalent versus polyvalent MOMP based Chlamydia pecorum vaccine in koalas (Phascolarctos cinereus)

Chlamydia continues to be a major pathogen of koalas. The bacterium is associated with ocular, respiratory and urogenital tract infections and a vaccine is considered the best option to limit the decline of mainland koala populations. Over the last 20 years, efforts to develop a chlamydial vaccine in humans have focussed on the use of the chlamydial major outer membrane protein (MOMP). Potential problems with the use of MOMP-based vaccines relate to the wide range of genetic diversity in its four variable domains. In the present study, we evaluated the immune response of koalas vaccinated with a MOMP-based C. pecorum vaccine formulated with genetically and serologically diverse MOMPs. Animals immunised with individual MOMPs developed strong antibody and lymphocyte proliferation responses to both homologous as well as heterologous MOMP proteins. Importantly, we also showed that vaccine induced antibodies which effectively neutralised various heterologous strains of koala C. pecorum in an in vitro assay. Finally, we also demonstrated that the immune responses in monovalent as well as polyvalent MOMP vaccine groups were able to recognise whole chlamydial elementary bodies, illustrating the feasibility of developing an effective MOMP based C. pecorum vaccine that could protect against a range of strains.