Investigation of the low-density lipoprotein receptor gene and cholesterol as a risk factor for migraine

The Low-Density Lipoprotein Receptor (LDLR) gene is a cell surface receptor that plays an important role in cholesterol homeostasis. We investigated the (TA)n polymorphism in exon 18 of the LDLR gene on chromosome 19p13.2 performing an association analysis in 244 typical migraine-affected patients, 151 suffering from migraine with aura (MA), 96 with migraine without aura (MO) and 244 unaffected controls. The populations consisted of Caucasians only, and controls were age- and sex-matched. The results showed no significant difference between groups for allele frequency distributions of the (TA)n polymorphism even after separation of the migraine-affected individuals into subgroups of MA and MO affected patients. This is in contradiction to Mochi et al. who found a positive association of this variant with MO. Our study discusses possible differences between the two studies and extends this research by investigating circulating cholesterol levels in a migraine-affected population.

The methylentetrahydrofolate reductase gene variant (C677T) as a risk factor for essential hypertension in Caucasians

Essential hypertension (EH) is a common, multifactorial disorder likely to be influenced by multiple genes of modest effect. The methylenetetrahydrofolate reductase (MTHFR) gene C677T mutation is functionally important, being strongly associated with reduced enzyme activity and increased plasma levels of homocysteine. Mild hyperhomocysteinemia is a known risk factor for cardiovascular disease (CVD) and hypothesised also to be involved in hypertension pathophysiology. The present study was performed to determine the prevalence of the 677T mutation in Australian Caucasian patients diagnosed with EH and to test whether the C677T variant is associated with the disorder. A case-control cohort, consisting of 250 EH patients and 250 age, sex and racially matched normotensive controls, were used for the association study. Comparison of C677T allele frequencies revealed a higher proportion of the mutant allele (T) in the EH group (40%) compared to unaffected controls (34%) (p=0.07). Furthermore, genotypic results indicated that the prevalence of the homozygous mutant genotype (T/T) in the affected group was higher than that of controls (14%:10%) (p=0.17). Interestingly, conditional logistic regression showed that the MTHFR C677T mutation conferred a mild, yet significant increase in risk of essential hypertension after adjusting for body mass index (odds ratio=1.57, 95% confidence interval: 1.04-2.37, p=0.03). These findings require further investigation in large independent samples, but suggest that essential hypertension, like CVD, may be mildly influenced by the MTHFR C677T variant.

Electrospraying of polymers with therapeutic molecules : state of the art

The encapsulation and release of bioactive molecules from polymeric vehicles represents the holy grail of drug and growth factor delivery therapies, whereby sustained and controlled release is crucial in eliciting a positive therapeutic effect. To this end, electrospraying is rapidly emerging as a popular technology for the production of polymeric particles containing bioactive molecules. Compared with traditional emulsion fabrication techniques, electrospraying has the potential to reduce denaturation of protein drugs and affords tighter regulation over particle size distribution and morphology. In this article, we review the importance of the electrospraying parameters that enable reproducible tailoring of the particles' physical and in vitro drug release characteristics, along with discussion of existing in vivo data. Controlled morphology and monodispersity of particles can be achieved with electrospraying, with high encapsulation efficiencies and without unfavorable denaturation of bioactive molecules throughout the process. Finally, the combination of electrospraying with electrospun scaffolds, with an emphasis on tissue regeneration is reviewed, depicting a technique in its relative infancy but holding great promise for the future of regenerative medicine.

Composites for delivery of therapeutics : combining melt electrospun scaffolds with loaded electrosprayed microparticles

A novel strategy is reported to produce biodegradable microfiber-scaffolds layered with high densities of microparticles encapsulating a model protein. Direct electrospraying on highly porous melt electrospun scaffolds provides a reproducible scaffold coating throughout the entire architecture. The burst release of protein is significantly reduced due to the immobilization of microparticles on the surface of the scaffold and release mechanisms are dependent on protein-polymer interactions. The composite scaffolds have a positive biological effect in contact with precursor osteoblast cells up to 18 days in culture. The scaffold design achieved with the techniques presented here endorses these new composite scaffolds as promising templates for growth factor delivery.

Melanocortin-1 receptor genotype is a risk factor for basal and squamous cell carcinoma

MC1R gene variants have previously been associated with red hair and fair skin color, moreover skin ultraviolet sensitivity and a strong association with melanoma has been demonstrated for three variant alleles that are active in influencing pigmentation: Arg151Cys, Arg160Trp, and Asp294His. This study has confirmed these pigmentary associations with MC1R genotype in a collection of 220 individuals drawn from the Nambour community in Queensland, Australia, 111 of whom were at high risk and 109 at low risk of basal cell carcinoma and squamous cell carcinoma. Comparative allele frequencies for nine MC1R variants that have been reported in the Caucasian population were determined for these two groups, and an association between prevalence of basal cell carcinoma, squamous cell carcinoma, solar keratosis and the same three active MC1R variant alleles was demonstrated [odds ratio = 3.15 95% CI (1.7, 5.82)]. Three other commonly occurring variant alleles: Val60Leu, Val92Met, and Arg163Gln were identified as having a minimal impact on pigmentation phenotype as well as basal cell carcinoma and squamous cell carcinoma risk. A significant heterozygote effect was demonstrated where individuals carrying a single MC1R variant allele were more likely to have fair and sun sensitive skin as well as carriage of a solar lesion when compared with those individuals with a consensus MC1R genotype. After adjusting for the effects of pigmentation on the association between MC1R variant alleles and basal cell carcinoma and squamous cell carcinoma risk, the association persisted, confirming that presence of at least one variant allele remains informative in terms of predicting risk for developing a solar-induced skin lesion beyond that information wained through observation of pigmentation phenotype.

Phosphorus as a limiting factor on sustainable greywater irrigation

Water reuse through greywater irrigation has been adopted worldwide and has been proposed as a potential sustainable solution to increased water demands. Despite widespread adoption there is limited domestic knowledge of greywater reuse, there is no pressure to produce lowlevel phosphorus products and current guidelines and legislation, such as those in Australia, may be inadequate due to the lack of long-term data to provide a sound scientific basis. Research has clearly identified phosphorus as a potential environmental risk to waterways from many forms of irrigation. To assess the sustainability of greywater irrigation, this study compared four residential lots that had been irrigated with greywater for four years and adjacent non-irrigated lots that acted as controls. Each lot was monitored for the volume of greywater applied and selected physic-chemical water quality parameters and soil chemistry profiles were analysed. The non-irrigated soil profiles showed low levels of phosphorus and were used as controls. The Mechlich3 Phosphorus ratio (M3PSR) and Phosphate Environmental Risk Index (PERI) were used to determine the environmental risk of phosphorus leaching from the irrigated soils. Soil phosphorus concentrations were compared to theoretical greywater irrigation loadings. The measured phosphorus soil concentrations and the estimated greywater loadings were of similar magnitude. Sustainable greywater reuse is possible; however incorrect use and/or a lack of understanding of how household products affect greywater can result in phosphorus posing a significant risk to the environment.

The B subunit of coagulation factor XIII is linked to renin and the Duffy blood group to α-spectrin on human chromosome 1

Family linkage studies were used to detect two linkage relationships on human chromosome 1. The B subunit of coagulation factor XIII showed significant linkage to renin with a maximum lod score of 5.071 at a distance of 10 cM. Significant linkage was also shown between the Duffy blood group and α-spectrin with linkage results giving a combined lod score of 3.194 at 5 cM.

Pten and Ndufb8 aberrations in cervical cancer tissue

Cervical cancer is one of the world's major health issues. Despite many studies in this field, the carcinogenetic events of malignant conversion in cervical tumours have not been significantly characterised. The first aim of this project was to investigate the mutation status of the tumour suppressor gene- Phosphatase and Tension Homolog (PTEN)- in cervical cancer tissue. The second aim of this study was the analysis in the same cervical cancer tissue for aberrations in the mitochondrial electron transport chain subunit gene NDUFB8, which is localised to the same chromosomal contig as PTEN. The third aim was the evaluation of the potential therapeutic anti-cancer drug 2,4-Thiazolidinediones (TZDs) and its affect in regulating the PTEN protein in a cervical cancer cell line (HeLa). To approach the aims, paraffin-embedded cancerous cervical tissue and non-cancerous cervical tissue were obtained. DNA recovered from those tissues was then used to investigate the putative genomic changes regarding the NDUFB8 gene utilising SYBR Green I Real-Time PCR. The PTEN gene was studied via Dual-Labelled probe Real-Time PCR. To investigate the protein expression change of the PTEN protein, HeLa cells were firstly treated with different concentrations of 2,4-Thiazolidinediones and the level of PTEN protein expression was then observed utilising standard protein assays. Results indicated that there were putative copy-number changes between the cancerous cervical tissue and non-cancerous cervical tissue, with regard to the PTEN locus. This implies a potential gain of the PTEN gene in cancerous cervical tissue. With regards to normal cervical tissue versus cancerous cervical tissue no significant melting temperature differences were observed with the SYBR Green I Real-Time PCR in respect to the NDUFB8 gene. A putative up-regulation of PTEN protein was observed in TZD treated HeLa cells. © 2008 Springer Science+Business Media, LLC.

Common PPARγ variants C161T and Pro12Ala are not associated with inflammatory bowel disease in an Australian cohort

Background & Aims: Peroxisome proliferator-activated receptor (PPAR) γ is a transcription factor, highly expressed in colonic epithelial cells, adipose tissue and macrophages, with an important role in the regulation of inflammatory pathways. The common PPARγ variants C161T and Pro12Ala have recently been associated with Ulcerative Colitis (UC) and an extensive UC phenotype respectively, in a Chinese population. PPARγ Pro12Ala variant homozygotes appear to be protected from the development of Crohn's disease (CD) in European Caucasians. Methods: A case-control study was performed for both variants (CD n=575, UC n=306, Controls n=360) using a polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis in an Australian IBD cohort. A transmission disequilibrium test was also performed using CD trios for the PPARγ C161T variant. Genotype-phenotype analyses were also undertaken. Results: There was no significant difference in genotype distribution data or allele frequency between CD and UC patients and controls. There was no difference in allele transmission for the C161T variant. No significant relationship between the variants and disease location was observed. Conclusions: We were unable to replicate in a Caucasian cohort the recent association between PPARγ C161T and UC or between PPARγ Pro12Ala and an extensive UC phenotype in a Chinese population. There are significant ethnic differences in genetic susceptibility to IBD and its phenotypic expression.

Detection of mRNA for nuclear receptor co-activators 1 and 3 in archival breast cancer tissue and surrounding stroma : a tissue expression study

Before the age of 75 years, approximately 10% of women will be diagnosed with breast cancer, one of the most common malignancies and a leading cause of death among women. The objective of this study was to determine if expression of the nuclear receptor coactivators 1 and 3 (NCoA1 and NCoA3) varied in breast cancer grades. RNA was extracted from 25 breast tumours and transcribed into cDNA which underwent semi-quantitative polymerase chain reaction, normalised using 18S. Analysis indicated that an expression change for NCoA1 in cancer grades and estrogen receptor alpha negative tissue (P= 0.028 and 0.001 respectively). NCoA1 expression increased in grade 3 and estrogen receptor alpha negative tumours, compared to controls. NCoA3 showed a similar, but not significant, trend in grade and a non-significant decrease in estrogen receptor alpha negative tissues. Expression of NCoA1 in late stage and estrogen receptor alpha negative breast tumours may have implications to breast cancer treatment, particularly in the area of manipulation of hormone signalling systems in advanced tumours.

Expression of glucocorticoid and progesterone nuclear receptor genes in archival breast cancer tissue.

BACKGROUND: Previous studies in our laboratory have shown associations of specific nuclear receptor gene variants with sporadic breast cancer. In order to investigate these findings further, we conducted the present study to determine whether expression levels of the progesterone and glucocorticoid nuclear receptor genes vary in different breast cancer grades. METHODS: RNA was extracted from paraffin-embedded archival breast tumour tissue and converted into cDNA. Sample cDNA underwent PCR using labelled primers to enable quantitation of mRNA expression. Expression data were normalized against the 18S ribosomal gene multiplex and analyzed using analysis of variance. RESULTS: Analysis of variance indicated a variable level of expression of both genes with regard to breast cancer grade (P = 0.00033 for glucocorticoid receptor and P = 0.023 for progesterone receptor). CONCLUSION: Statistical analysis indicated that expression of the progesterone nuclear receptor is elevated in late grade breast cancer tissue.

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 (χ1=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 (χ2=1.5, P=0.21); and (iii) a XhoI RFLP for ANF was 0.50 in HTs and 0.46 in NTs (χ2=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.

Perspectives in multiphasic osteochondral tissue engineering

Critical-sized osteochondral defects are clinically challenging, with limited treatment options available. By engineering osteochondral grafts using a patient's own cells and osteochondral scaffolds designed to facilitate cartilage and bone regeneration, osteochondral defects may be treated with less complications and better long-term clinical outcomes. Scaffolds can influence the development and structure of the engineered tissue, and there is an increased awareness that osteochondral tissue engineering concepts need to take the in vivo complexities into account in order to increase the likelihood of successful osteochondral tissue repair. The developing trend in osteochondral tissue engineering is the utilization of multiphasic scaffolds to recapitulate the multiphasic nature of the native tissue. Cartilage and bone have different structural, mechanical, and biochemical microenvironments. By designing osteochondral scaffolds with tissue-specific architecture, it may be possible to enhance osteochondral repair within shorter timeframe. While there are promising in vivo outcomes using multiphasic approaches, functional regeneration of osteochondral constructs still remains a challenge. In this review, we provide an overview of in vivo osteochondral repair studies that have taken place in the past three years, and define areas which needs improvement in future studies

Mechanical and biological characterization of a porous poly-L-lactic acid-co-ε-caprolactone scaffold for tissue engineering

This article presents a method for making highly porous biodegradable scaffold that may ultimately be used for tissue engineering. Poly(L-lactic-co-1-caprolactone) acid (70:30) (PLCL) scaffold was produced using the solvent casting/leaching out method, which entails dissolving the polymer and adding a porogen that is then leached out by immersing the scaffold in distillated water. Tensile tests were performed for three types of scaffolds, namely pre-wetted, dried, and UV-irradiated scaffolds and their mechanical properties were measured. The prewetted PLCL scaffold possessed a modulus of elasticity 0.92+0.09 MPa, a tensile strength of 0.12+0.03 MPa and an ultimate strain of 23+5.3%. No significant differences in the modulus elasticity, tensile strength, nor ultimate strain were found between the pre-wetted, dried, and UV irradiated scaffolds. The PLCL scaffold was seeded by human fibroblasts in order to evaluate its biocompatibility by Alamar bluew assays. After 10 days of culture, the scaffolds showed good biocompatibility and allowed cell proliferation. However, the fibroblasts stayed essentially at the surface. This study shows the possibility to use the PLCL scaffold in dynamic mechanical conditions for tissue engineering

The effect of polystyrene sodium sulfonate grafting on polyethylene terephthalate artificial ligaments on in vitro mineralisation and in vivo bone tissue integration

This study investigates the impact of polystyrene sodium sulfonate (PolyNaSS) grafting onto the osseo-integration of a polyethylene terephthalate artificial ligament (Ligament Advanced Reinforcement System, LARS™) used for Anterior Cruciate Ligament (ACL). The performance of grafted and non-grafted ligaments was assessed in vitro by culturing human osteoblasts under osteogenic induction and this demonstrated that the surface modification was capable of up-regulating the secretion of ALP and induced higher level of mineralisation as measured 6 weeks post-seeding by Micro-Computed Tomography. Grafted and non-grafted LARS™ were subsequently implanted in an ovine model for ACL reconstruction and the ligament-to-bone interface was evaluated by histology and biomechanical testings 3 and 12 months post-implantation. The grafted ligaments exhibited more frequent direct ligament-to-bone contact and bone formation in the core of the ligament at the later time point than the non-grafted specimens, the grafting also significantly reduced the fibrous encapsulation of the ligament 12 months post-implantation. However, this improved osseo-integration was not translated into a significant increase in the biomechanical pull-out loads. These results provide evidences that PolyNaSS grafting improved the osseo-integration of the artificial ligament within the bone tunnels. This might positively influence the outcome of the surgical reconstructions, as higher ligament stability is believed to limit micro-movement and therefore permits earlier and enhanced healing.