Combined expression of KLK4, KLK5, KLK6, and KLK7 by ovarian cancer cells leads to decreased adhesion and paclitaxel-induced chemoresistance.

OBJECTIVE: Chemoresistance is a critical feature of advanced ovarian cancer with only 30% of patients surviving longer than 5 years. We have previously shown that four kallikrein-related (KLK) peptidases, KLK4, KLK5, KLK6 and KLK7 (KLK4-7), are implicated in peritoneal invasion and tumour growth, but underlying mechanisms were not identified. We also reported that KLK7 overexpression confers chemoresistance to paclitaxel, and cell survival via integrins. In this study, we further explored the functional consequenses of overexpression of all four KLKs (KLK4-7) simultaneously in the ovarian cancer cell line, OV-MZ-6, and its impact on integrin expression and signalling, cell adhesion and survival as contributors to chemoresistance and metastatic progression. METHODS: Quantitative gene and protein expression analyses, confocal microscopy, cell adhesion and chemosensitivity assays were performed. RESULTS: Expression of α5β1/αvβ3 integrins was downregulated upon combined stable KLK4-7 overexpression in OV-MZ-6 cells. Accordingly, the adhesion of these cells to vitronectin and fibronectin, the extracellular matrix binding proteins of α5β1/αvβ3 integrins and two predominant proteins of the peritoneal matrix, was decreased. KLK4-7-transfected cells were more resistant to paclitaxel (10-100 nmol/L: 38-54%), but not to carboplatin, which was associated with decreased apoptotic stimuli. However, the KLK4-7-induced paclitaxel resistance was not blocked by the MEK1/2 inhibitor, U0126. CONCLUSIONS: This study demonstrates that combined KLK4-7 expression by ovarian cancer cells promotes reduced integrin expression with consequently less cell-matrix attachment, and insensitivity to paclitaxel mediated by complex integrin and MAPK independent interactions, indicative of a malignant phenotype and disease progression suggesting a role for these KLKs in this process.

G-protein β3 subunit gene (GNB3) variant in causation of essential hypertension

Essential hypertensives display enhanced signal transduction through pertussis toxin-sensitive G proteins. The T allele of a C825T variant in exon 10 of the G protein β3 subunit gene (GNB3) induces formation of a splice variant (Gβ3-s) with enhanced activity. The T allele of GNB3 was shown recently to be associated with hypertension in unselected German patients (frequency=0.31 versus 0.25 in control). To confirm and extend this finding in a different setting, we performed an association study in Australian white hypertensives. This involved an extensively examined cohort of 110 hypertensives, each of whom were the offspring of 2 hypertensive parents, and 189 normotensives whose parents were both normotensive beyond age 50 years. Genotyping was performed by polymerase chain reaction and digestion with BseDI, which either cut (C allele) or did not cut (T allele) the 268-bp polymerase chain reaction product. T allele frequency in the hypertensive group was 0.43 compared with 0.25 in the normotensive group (χ2=22; P=0.00002; odds ratio=2.3; 95% CI=1.7 to 3.3). The T allele tracked with higher pretreatment blood pressure: diastolic=105±7, 109±16, and 128±28 mm Hg (mean±SD) for CC, CT, and 7T, respectively (P=0.001 by 1-way ANOVA). Blood pressures were higher in female hypertensives with a T allele (P=0.006 for systolic and 0.0003 for diastolic by ANOVA) than they were in male hypertensives. In conclusion, the present study of a group with strong family history supports a role for a genetically determined, physiologically active splice variant of the G protein β3 subunit gene in the causation of essential hypertension.

Natural, engineered and synthetic inhibitors of kallikrein-related peptidases

There is a rapidly growing appreciation of the important physiological roles played by kallikreins and kallikrein-related peptidases (KLKs). Recent studies have revealed that these enzymes control key events in processes as diverse as inflammation and skin desquamation. Accordingly, there is considerable interest in developing tools to further dissect kallikrein activity, and a burgeoning effort aimed at producing lead inhibitors for therapeutic development. Indeed, several candidate inhibitors are already in clinical trials. This chapter surveys the naturally occurring kallikrein inhibitors, together with strategies for employing these molecules as bioscaffolds, as well as current progress in the development of small-molecule kallikrein inhibitors.

High pressure in situ diffraction studies of metal–hydrogen systems

“Hybrid” hydrogen storage, where hydrogen is stored in both the solid material and as a high pressure gas in the void volume of the tank can improve overall system efficiency by up to 50% compared to either compressed hydrogen or solid materials alone. Thermodynamically, high equilibrium hydrogen pressures in metal–hydrogen systems correspond to low enthalpies of hydrogen absorption–desorption. This decreases the calorimetric effects of the hydride formation–decomposition processes which can assist in achieving high rates of heat exchange during hydrogen loading—removing the bottleneck in achieving low charging times and improving overall hydrogen storage efficiency of large hydrogen stores. Two systems with hydrogenation enthalpies close to −20 kJ/mol H2 were studied to investigate the hydrogenation mechanism and kinetics: CeNi5–D2 and ZrFe2−xAlx (x = 0.02; 0.04; 0.20)–D2. The structure of the intermetallics and their hydrides were studied by in situ neutron powder diffraction at pressures up to 1000 bar and complementary X-ray diffraction. The deuteration of the hexagonal CeNi5 intermetallic resulted in CeNi5D6.3 with a volume expansion of 30.1%. Deuterium absorption filled three different types of interstices, Ce2Ni2 and Ni4 tetrahedra, and Ce2Ni3 half-octahedra and was accompanied by a valence change for Ce. Significant hysteresis was observed between deuterium absorption and desorption which profoundly decreased on a second absorption cycle. For the Al-modified Laves-type C15 ZrFe2−xAlx intermetallics, deuteration showed very fast kinetics of H/D exchange and resulted in a volume increase of the FCC unit cells of 23.5% for ZrFe1.98Al0.02D2.9(1). Deuterium content, hysteresis of H/D uptake and release, unit cell expansion and stability of the hydrides systematically change with the amount of Al content. In the deuteride D atoms exclusively occupy the Zr2(Fe,Al)2 tetrahedra. Observed interatomic distances are Zr–D = 1.98–2.11; (Fe, Al)–D = 1.70–1.75A˚ . Hydrogenation slightly increases the magnetic moment of the Fe atoms in ZrFe1.98Al0.02 and ZrFe1.96Al0.04 from 1.9 �B at room temperature for the alloy to 2.2 �B for its deuteride.

Design, characterisation and in vivo testing of a new, adjustable stiffness, external fixator for the rat femur

Very little is known about the infl uence of the mechanical environment on the healing of large segmental defects. This partly reflects the lack of standardised, well characterised technologies to enable such studies. Here we report the design, construction and characterisation of a novel external fixator for use in conjunction with rat femoral defects. This device not only imposes a predetermined axial stiffness on the lesion, but also enables the stiffness to be changed during the healing process. The main frame of the fi xator consists of polyethylethylketone with titanium alloy mounting pins. The stiffness of the fi xator is determined by interchangeable connection elements of different thicknesses. Fixators were shown to stabilise 5 mm femoral defects in rats in vivo for at least 8 weeks during unrestricted cage activity. No distortion or infections, including pin infections, were noted. The healing process was simulated in vitro by inserting into a 5 mm femoral defect, materials whose Young’s moduli approximated those of the different tissues present in regenerating bone. These studies confirmed that, although the external fixator is the major determinant of axial stiffness during the early phase of healing, the regenerate within the lesion subsequently dominates this property. There is much clinical interest in altering the mechanics of the defect to enhance bone healing. Our data suggest that, if alteration of the mechanical environment is to be used to modulate the healing of large segmental defects, this needs to be performed before the tissue properties become dominant.

Dietary flavonoids from modified apple reduce inflammation markers and modulate gut microbiota in mice

Apples are rich in polyphenols, which provide antioxidant properties, mediation of cellular processes such as inflammation, and modulation of gut microbiota. In this study we compared genetically engineered apples with increased flavonoids [myeloblastis transcription factor 10 (MYB10)] with nontransformed apples from the same genotype, "Royal Gala" (RG), and a control diet with no apple. Compared with the RG diet, the MYB10 diet contained elevated concentrations of the flavonoid subclasses anthocyanins, flavanol monomers (epicatechin) and oligomers (procyanidin B2), and flavonols (quercetin glycosides), but other plant secondary metabolites were largely unaltered. We used these apples to investigate the effects of dietary flavonoids on inflammation and gut microbiota in 2 mouse feeding trials. In trial 1, male mice were fed a control diet or diets supplemented with 20% MYB10 apple flesh and peel (MYB-FP) or RG apple flesh and peel (RG-FP) for 7 d. In trial 2, male mice were fed MYB-FP or RG-FP diets or diets supplemented with 20% MYB10 apple flesh or RG apple flesh for 7 or 21 d. In trial 1, the transcription levels of inflammation-linked genes in mice showed decreases of >2-fold for interleukin-2 receptor (Il2rb), chemokine receptor 2 (Ccr2), chemokine ligand 10 (Cxcl10), and chemokine receptor 10 (Ccr10) at 7 d for the MYB-FP diet compared with the RG-FP diet (P <0.05). In trial 2, the inflammation marker prostaglandin E2 (PGE2) in the plasma of mice fed the MYB-FP diet at 21 d was reduced by 10-fold (P < 0.01) compared with the RG-FP diet. In colonic microbiota, the number of total bacteria for mice fed the MYB-FP diet was 6% higher than for mice fed the control diet at 21 d (P = 0.01). In summary, high-flavonoid apple was associated with decreases in some inflammation markers and changes in gut microbiota when fed to healthy mice.

Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples

Mutations in the genes encoding for either the biosynthetic or transcriptional regulation of the anthocyanin pathway have been linked to color phenotypes. Generally, this is a loss of function resulting in a reduction or a change in the distribution of anthocyanin. Here, we describe a rearrangement in the upstream regulatory region of the gene encoding an apple (Malus x domestica) anthocyanin-regulating transcription factor, MYB10. We show that this modification is responsible for increasing the level of anthocyanin throughout the plant to produce a striking phenotype that includes red foliage and red fruit flesh. This rearrangement is a series of multiple repeats, forming a minisatellite-like structure that comprises five direct tandem repeats of a 23-bp sequence. This MYB10 rearrangement is present in all the red foliage apple varieties and species tested but in none of the white fleshed varieties. Transient assays demonstrated that the 23-bp sequence motif is a target of the MYB10 protein itself, and the number of repeat units correlates with an increase in transactivation by MYB10 protein. We show that the repeat motif is capable of binding MYB10 protein in electrophoretic mobility shift assays. Taken together, these results indicate that an allelic rearrangement in the promoter of MYB10 has generated an autoregulatory locus, and this autoregulation is sufficient to account for the increase in MYB10 transcript levels and subsequent ectopic accumulation of anthocyanins throughout the plant.

The genome of the domesticated apple (Malus × domestica Borkh.)

We report a high-quality draft genome sequence of the domesticated apple (Malus × domestica). We show that a relatively recent (>50 million years ago) genome-wide duplication (GWD) has resulted in the transition from nine ancestral chromosomes to 17 chromosomes in the Pyreae. Traces of older GWDs partly support the monophyly of the ancestral paleohexaploidy of eudicots. Phylogenetic reconstruction of Pyreae and the genus Malus, relative to major Rosaceae taxa, identified the progenitor of the cultivated apple as M. sieversii. Expansion of gene families reported to be involved in fruit development may explain formation of the pome, a Pyreae-specific false fruit that develops by proliferation of the basal part of the sepals, the receptacle. In apple, a subclade of MADS-box genes, normally involved in flower and fruit development, is expanded to include 15 members, as are other gene families involved in Rosaceae-specific metabolism, such as transport and assimilation of sorbitol.

The LCC15-MB human breast cancer cell line expresses osteopontin and exhibits an invasive and metastatic phenotype

We have characterized the LCC15-MB cell line which was recently derived from a breast carcinoma metastasis resected from the femur of a 29-year-old woman. LCC15-MB cells are vimentin (VIM) positive, exhibit a stellate morphology in routine cell culture, and form penetrating colonies when embedded in three-dimensional gels of Matrigel or fibrillar collagen. They show high levels of activity in the Boyden chamber chemomigration and chemoinvasion assays, and like other invasive human breast cancer (HBC) cell lines, LCC15-MB cells activate matrix-metalloproteinase-2 in response to treatment with concanavalin A. In addition, these cells are tumorigenic when implanted subcutaneously in nude mice and recolonize bone after arterial injection. Interestingly, both the primary lesion and the bone metastasis from which LCC15-MB were derived, as well as the resultant cell line, abundantly express the bone matrix protein osteopontin (OPN). OPN is also expressed by the highly metastatic MDA-MB-435 cells, but not other invasive or noninvasive HBC cell lines. Expression of OPN is retained in the subcutaneous xenograft and intraosseous metastases of LCC15-MB as detected by immunohistochemistry. Both VIM and OPN expression have been associated with breast cancer invasion and metastasis, and their expression by the LCC15-MB cell line is consistent with its derivation from a highly aggressive breast cancer. These cells provide a useful model for studying molecular mechanisms important for breast cancer metastasis to bone and, in particular, the implication(s) of OPN and VIM expression in this process.

On cheating immune secret sharing

The paper addresses the cheating prevention in secret sharing. We consider secret sharing with binary shares. The secret also is binary. This model allows us to use results and constructions from the well developed theory of cryptographically strong boolean functions. In particular, we prove that for given secret sharing, the average cheating probability over all cheating vectors and all original vectors, i.e., 1/n 2n ∑c=1...n ∑α∈V n ρc,α , denoted by ρ, satisfies ρ ≥ ½, and the equality holds if and only if ρc,α satisfies ρc,α= ½ for every cheating vector δc and every original vector α. In this case the secret sharing is said to be cheating immune. We further establish a relationship between cheating-immune secret sharing and cryptographic criteria of boolean functions.This enables us to construct cheating-immune secret sharing.

Environmental, personal, and genetic determinants of response to vitamin D supplementation in older adults

CONTEXT AND OBJECTIVE: Suboptimal vitamin D status can be corrected by vitamin D supplementation, but individual responses to supplementation vary. We aimed to examine genetic and nongenetic determinants of change in serum 25-hydroxyvitamin D (25(OH)D) after supplementation. DESIGN AND PARTICIPANTS: We used data from a pilot randomized controlled trial in which 644 adults aged 60 to 84 years were randomly assigned to monthly doses of placebo, 30 000 IU, or 60 000 IU vitamin D3 for 12 months. Baseline characteristics were obtained from a self-administered questionnaire. Eighty-eight single-nucleotide polymorphisms (SNPs) in 41 candidate genes were genotyped using Sequenom MassArray technology. Serum 25(OH)D levels before and after the intervention were measured using the Diasorin Liaison platform immunoassay. We used linear regression models to examine associations between genetic and nongenetic factors and change in serum 25(OH)D levels. RESULTS: Supplement dose and baseline 25(OH)D level explained 24% of the variability in response to supplementation. Body mass index, self-reported health status, and ambient UV radiation made a small additional contribution. SNPs in CYP2R1, IRF4, MC1R, CYP27B1, VDR, TYRP1, MCM6, and HERC2 were associated with change in 25(OH)D level, although only CYP2R1 was significant after adjustment for multiple testing. Models including SNPs explained a similar proportion of variability in response to supplementation as models that included personal and environmental factors. CONCLUSION: Stepwise regression analyses suggest that genetic variability may be associated with response to supplementation, perhaps suggesting that some people might need higher doses to reach optimal 25(OH)D levels or that there is variability in the physiologically normal level of 25(OH)D.

Insurance and transport law : controlling insurance contract terms : Section 54 of the Insurance Contracts Act – compliance, recovery and accountability

Commonwealth legislation covering insurance contracts contains numerous provisions designed to control the operation and effect of terms in life and general insurance contracts. For example, the Life Insurance Act 1995 (Cth) contains provisions regulating the consequences attendant upon incorrect statements in proposals [1] and non-payment of premiums, [2] provides that an insurer may only exclude liability in the case of suicide if it has made express provision for such contingency in its policy, [3] and severely restricts the efficacy of conditions as to war risks. [4] The Insurance Contracts Act 1984 (Cth) is even more intrusive and has a major impact upon contractual provisions in the general insurance field. It is beyond the scope of this note to explore all of these provisions in any detail but examples of controls and constraints imposed upon the operation and effect of contractual provisions include the following. A party is precluded from relying upon a provision in a contract of insurance if such reliance would amount to a failure to act with the utmost good faith. [5] Similarly, a policy provision which requires differences or disputes arising out of the insurance to be submitted to arbitration is void, [6] unless the insurance is a genuine cover for excess of loss over and above another specified insurance. [7] Similarly clause such as conciliation clauses, [8] average clauses, [9] and unusual terms [10] are given qualified operation. [11] However the provision in the Insurance Contracts Act that has the greatest impact upon, and application to, a wide range of insurance clauses and claims is s 54. This section has already generated a significant volume of case law and is the focus of this note. In particular this note examines two recent cases. The first, Johnson v Triple C Furniture and Electrical Pty Ltd [2012] 2 Qd R 337, (hereafter the Triple C case), is a decision of the Queensland Court of Appeal; and the second, Matthew Maxwell v Highway Hauliers Pty Ltd [2013] WASCA 115, (hereafter the Highway Hauliers case), is a decision of the Court of Appeal in Western Australia. This latter decision is on appeal to the High Court of Australia. The note considers too the decision of the New South Wales Court of Appeal in Prepaid Services Pty Ltd v Atradius Credit Insurance NV [2013] NSWCA 252 (hereafter the Prepaid Services case).These cases serve to highlight the complex nature of s 54 and its application, as well as the difficulty in achieving a balance between an insurer and an insured's reasonable expectations.

Common values in assessing health outcomes from disease and injury: Disability weights measurement study for the Global Burden of Disease Study 2010

BACKGROUND Measurement of the global burden of disease with disability-adjusted life-years (DALYs) requires disability weights that quantify health losses for all non-fatal consequences of disease and injury. There has been extensive debate about a range of conceptual and methodological issues concerning the definition and measurement of these weights. Our primary objective was a comprehensive re-estimation of disability weights for the Global Burden of Disease Study 2010 through a large-scale empirical investigation in which judgments about health losses associated with many causes of disease and injury were elicited from the general public in diverse communities through a new, standardised approach. METHODS We surveyed respondents in two ways: household surveys of adults aged 18 years or older (face-to-face interviews in Bangladesh, Indonesia, Peru, and Tanzania; telephone interviews in the USA) between Oct 28, 2009, and June 23, 2010; and an open-access web-based survey between July 26, 2010, and May 16, 2011. The surveys used paired comparison questions, in which respondents considered two hypothetical individuals with different, randomly selected health states and indicated which person they regarded as healthier. The web survey added questions about population health equivalence, which compared the overall health benefits of different life-saving or disease-prevention programmes. We analysed paired comparison responses with probit regression analysis on all 220 unique states in the study. We used results from the population health equivalence responses to anchor the results from the paired comparisons on the disability weight scale from 0 (implying no loss of health) to 1 (implying a health loss equivalent to death). Additionally, we compared new disability weights with those used in WHO's most recent update of the Global Burden of Disease Study for 2004. FINDINGS 13,902 individuals participated in household surveys and 16,328 in the web survey. Analysis of paired comparison responses indicated a high degree of consistency across surveys: correlations between individual survey results and results from analysis of the pooled dataset were 0·9 or higher in all surveys except in Bangladesh (r=0·75). Most of the 220 disability weights were located on the mild end of the severity scale, with 58 (26%) having weights below 0·05. Five (11%) states had weights below 0·01, such as mild anaemia, mild hearing or vision loss, and secondary infertility. The health states with the highest disability weights were acute schizophrenia (0·76) and severe multiple sclerosis (0·71). We identified a broad pattern of agreement between the old and new weights (r=0·70), particularly in the moderate-to-severe range. However, in the mild range below 0·2, many states had significantly lower weights in our study than previously. INTERPRETATION This study represents the most extensive empirical effort as yet to measure disability weights. By contrast with the popular hypothesis that disability assessments vary widely across samples with different cultural environments, we have reported strong evidence of highly consistent results.

Identification of common variants associated with human hippocampal and intracranial volumes

Identifying genetic variants influencing human brain structures may reveal new biological mechanisms underlying cognition and neuropsychiatric illness. The volume of the hippocampus is a biomarker of incipient Alzheimer's disease and is reduced in schizophrenia, major depression and mesial temporal lobe epilepsy. Whereas many brain imaging phenotypes are highly heritable, identifying and replicating genetic influences has been difficult, as small effects and the high costs of magnetic resonance imaging (MRI) have led to underpowered studies. Here we report genome-wide association meta-analyses and replication for mean bilateral hippocampal, total brain and intracranial volumes from a large multinational consortium. The intergenic variant rs7294919 was associated with hippocampal volume (12q24.22; N = 21,151; P = 6.70 × 10 -16) and the expression levels of the positional candidate gene TESC in brain tissue. Additionally, rs10784502, located within HMGA2, was associated with intracranial volume (12q14.3; N = 15,782; P = 1.12 × 10 -12). We also identified a suggestive association with total brain volume at rs10494373 within DDR2 (1q23.3; N = 6,500; P = 5.81 × 10 -7).

Teaching engineering tribology: Elements of assessment design for different learning styles

This study involves teaching engineering students concepts in lubrication engineering that are heavily dependent on mathematics. Excellent learning outcomes have been observed when assessment tasks are devised for a diversity of learning styles. Providing different pathways to knowledge reduces the probability that a single barrier halts progress towards the ultimate learning objective. The interdisciplinary nature of tribology can be used advantageously to tie together multiple elements of engineering to solve real physical problems—an approach that seems to benefit a majority of engineering students. To put this into practice, various assessment items were devised on the study of hydrodynamics, culminating in a project to provide a summative evaluation of the students’ learning achievement. A survey was also conducted to assess other aspects of students’ learning experiences under the headings: ‘perception of learning’ and ‘overall satisfaction’. High degrees of achievement and satisfaction were observed. An attempt has been made to identify the elements contributing to success so that they may be applied to other challenging concepts in engineering education.