Discovery and characterization of IGFBP-mediated endocytosis in the human retinal pigment epithelial cell line ARPE-19

Insulin-like growth factor binding proteins (IGFBPs) are prime regulators of IGF-action in numerous cell types including the retinal pigment epithelium (RPE). The RPE performs several functions essential for vision, including growth factor secretion and waste removal via a phagocytic process mediated in part by vitronectin (Vn). In the course of studying the effects of IGFBPs on IGF-mediated VEGF secretion and Vn-mediated phagocytosis in the RPE cell line ARPE-19, we have discovered that these cells avidly ingest synthetic microspheres (2.0 μm diameter) coated with IGFBPs. Given the novelty of this finding and the established role for endocytosis in mediating IGFBP actions in other cell types, we have explored the potential role of candidate cell surface receptors. Moreover, we have examined the role of key IGFBP structural motifs, by comparing responses to three members of the IGFBP family (IGFBP-3, IGFBP-4 and IGFBP-5) which display overlapping variations in primary structure and glycosylation status. Coating of microspheres (FluoSpheres®, sulfate modified polystyrene filled with a fluorophore) was conducted at 37 °C for 1 h using 20 μg/mL of test protein, followed by extensive washing. Binding of proteins was confirmed using a microBCA assay. The negative control consisted of microspheres treated with 0.1% bovine serum albumin (BSA), and all test samples were post-treated with BSA in an effort to coat any remaining free protein binding sites, which might otherwise encourage non-specific interactions with the cell surface. Serum-starved cultures of ARPE-19 cells were incubated with microspheres for 24 h, using a ratio of approximately 100 microspheres per cell. Uptake of microspheres was quantified using a fluorometer and was confirmed visually by confocal fluorescence microscopy. The ARPE-19 cells displayed little affinity for BSA-treated microspheres, but avidly ingested large quantities of those pre-treated with Vn (ANOVA; p < 0.001). Strong responses were also observed towards recombinant formulations of non-glycosylated IGFBP-3, glycosylated IGFBP-3 and glycosylated IGFBP-5 (all p < 0.001), while glycosylated IGFBP-4 induced a relatively minor response (p < 0.05). The response to IGFBP-3 was unaffected in the presence of excess soluble IGFBP-3, IGF-I or Vn. Likewise, soluble IGFBP-3 did not induce uptake of BSA-treated microspheres. Antibodies to either the transferrin receptor or type 1 IGF-receptor displayed slight inhibitory effects on responses to IGFBPs and Vn. Heparin abolished responses to Vn, IGFBP-5 and non-glycosylated IGFBP-3, but only partially inhibited the response to glycosylated IGFBP-3. Our results demonstrate for the first time IGFBP-mediated endocytosis in ARPE-19 cells and suggest roles for the IGFBP-heparin-binding domain and glycosylation status. These findings have important implications for understanding the mechanisms of IGFBP actions on the RPE, and in particular suggest a role for IGFBP-endocytosis.

Identifying the learning development of students who are refugees

Students who are refugees need understanding and support to settle successfully into mainstream Australian classrooms. Teachers not aware of students’ prior learning and the process of second language acquisition may have difficulty providing the most appropriate learning environments to meet these students’ needs. This study found that, with no coordination of information on students’ learning backgrounds nor of their learning needs and development, students were in danger of being identified as at-risk of having a learning disability, with little support to substantiate such claims.

Reading the environment : narrative constructions of ecological subjectivities in Australian children's literature

Ways in which humans engage with the environment have always provided a rich source of material for writers and illustrators of Australian children's literature. Currently, readers are confronted with a multiplicity of complex, competing and/or complementing networks of ideas, theories and emotions that provide narratives about human engagement with the environment at a particular historical moment. This study, entitled Reading the Environment: Narrative Constructions of Ecological Subjectivities in Australian Children's Literature, examines how a representative sample of Australian texts (19 picture books and 4 novels for children and young adults published between 1995 and 2006) constructs fictional ecological subjects in the texts, and offers readers ecological subject positions inscribed with contemporary environmental ideologies. The conceptual framework developed in this study identifies three ideologically grounded positions that humans may assume when engaging with the environment. None of these positions clearly exists independently of any other, nor are they internally homogeneous. Nevertheless they can be categorised as: (i) human dominion over the environment with little regard for environmental degradation (unrestrained anthropocentrism); (ii) human consideration for the environment driven by understandings that humans need the environment to survive (restrained anthropocentrism); and (iii) human deference towards the environment guided by understandings that humans are no more important than the environment (ecocentrism). iv The transdisciplinary methodological approach to textual analysis used in this thesis draws on ecocriticism, narrative theories, visual semiotics, ecofeminism and postcolonialism to discuss the difficulties and contradictions in the construction of the positions offered. Each chapter of textual analysis focuses on the construction of subjectivities in relation to one of the positions identified in the conceptual framework. Chapter 5 is concerned with how texts highlight the negative consequences of human dominion over the environment, or, in the words of this study, living with ecocatastrophe. Chapter 6 examines representations of restrained anthropocentrism in its contemporary form, that is, sustainability. Chapter 7 examines representations of ecocentrism, a radical position with inherent difficulties of representation. According to the analysis undertaken, the focus texts convey the subtleties and complexities of human engagement with the environment and advocate ways of viewing and responding to contemporary unease about the environment. The study concludes that these ways of viewing and responding conform to and/or challenge dominant socio-cultural and political-economic opinions regarding the environment. This study, the first extended work of its kind, makes an original contribution to ecocritical study of Australian children's literature. By undertaking a comprehensive analysis of how texts for children represent human engagement with the environment at a time when important environmental concerns pose significant threats to human existence, I hope to contribute new knowledge to an area of children's literature research that to date has been significantly under-represented.

The impact of changes to the graduated driver licensing program in Queensland, Australia on the experiences of learner drivers

Purpose: Graduated driver licensing (GDL) has been introduced in numerous jurisdictions in Australia and internationally in an attempt to ameliorate the significantly greater risk of death and injury for young novice drivers arising from road crashes. The GDL program in Queensland, Australia, was extensively modified in July 2007. This paper reports the driving and licensing experiences of Learner drivers progressing through the current-GDL program, and compares them to the experiences of Learners who progressed through the former-GDL program. ----- ----- Method: Young drivers (n = 1032, 609 females, 423 males) aged 17 to 19 years (M = 17.43, SD = 0.67) were recruited as they progressed from a Learner to a Provisional driver’s licence. They completed a survey exploring their sociodemographic characteristics, driving and licensing experiences as a Learner. Key measures for a subsample (n = 183) of the current-GDL drivers were compared with the former-GDL drivers (n = 149) via t-tests and chi-square analyses. ----- ----- Results: As expected, Learner drivers progressing through the current-GDL program gained significantly more driving practice than those in the former program, which was more likely to be provided by mothers than in the past. Female learners in the current-GDL program reported less difficulty obtaining supervision than those in the former program. The number of attempts needed to pass the practical driving assessment did not change, nor did the amount of professional supervision. The current-GDL Learners held their licence for a significantly longer duration than those in the former program, with the majority reporting that their Logbook entries were accurate on the whole. Compared to those in the former program, a significantly smaller proportion of male current-GDL Learners reported being detected for a driving offence while the females reported significantly lower crash involvement. Most current-GDL drivers reported undertaking their supervised practice at the end of the Learner period. ----- ----- Conclusions: The enhancements to the GDL program in Queensland appear to have achieved many of their intended results. The current-GDL learners participating in the study reported obtaining a significantly greater amount of supervised driving experience compared to former-GDL learners. Encouragingly, the current-GDL Learners did not report any greater difficulty in obtaining supervised driving practice, and there was a decline in the proportion of current-GDL Learners engaging in unsupervised driving. In addition, the majority of Learners do not appear to be attempting to subvert logbook recording requirements, as evidenced by high rates of self-reported logbook accuracy. The results have implications for the development and the evaluation of GDL programs in Australia and around the world.

Enabling access to and re-use of publicly funded research data as Open Educational Resources : a strategy for overcoming the legal barriers to data access and re-use

Open Educational Resources (OER) are teaching, learning and research materials that have been released under an open licence that permits online access and re-use by others. The 2012 Paris OER Declaration encourages the open licensing of educational materials produced with public funds. Digital data and data sets produced as a result of scientific and non-scientific research are an increasingly important category of educational materials. This paper discusses the legal challenges presented when publicly funded research data is made available as OER, arising from intellectual property rights, confidentiality and information privacy laws, and the lack of a legal duty to ensure data quality. If these legal challenges are not understood, addressed and effectively managed, they may impede and restrict access to and re-use of research data. This paper identifies some of the legal challenges that need to be addressed and describes 10 proposed best practices which are recommended for adoption to so that publicly funded research data can be made available for access and re-use as OER.

Gated and Guarded Community (GACOS) in Malaysia : worth or not?

A 'Gated and Guarded Community' has become a popular trend in the recent years, particularly for housing areas. The increasing in population and income has lead to the increase in housing demand. The 1991 Population Census Report showed that Malaysian population has increased with an average yearly rate of 2.7% per year, that is, from 13.74 million people in 1980 to 19.35 million in 1991, followed by 20.69 million in 1995 and increase to 23.27 in year 2000. This is followed by consistent increase in the average population monthly income. Started from 1995 to 1999, the average annual growth rate of mean monthly income in Malaysia is about 5.2 %, from RM2,020.00 in 1995 to RM2,472.00 in 1999 and increasing constantly. This shows that the human growth usually have correlation between demand, income and housing. This paper presents the factors that involved in determined the Gated and Guarded Community Investment in Malaysia either it is worth to invest or otherwise. Hopefully, the results will also indicate that there may be other factors affecting their investment decision besides security and privacy. This paper is actually to draw attention to some practitioner and collect more information in establishing my research analysis.

Open Access and Scholarly Books: Workshop Report, 19 June 2013

On 19 June 2013 Knowledge Unlatched and the Berkman Center for Internet and Society at Harvard Law School jointly convened a one-day workshop titled Open Access and Scholarly Books in Cambridge, MA. The workshop brought together a group of 21 invited publishers, librarians, academics and Open Access innovators to discuss the challenge of making scholarly books Open Access. This report captures discussions that took place on the day.

Association between a 19 bp deletion polymorphism at the dopamine beta-hydroxylase (DBH) locus and migraine with aura

Migraine is a debilitating neurological disorder, affecting 12% of Caucasian populations. It is well known that migraine has a strong genetic component, although the type and number of genes involved is unclear. Our previous work has investigated dopamine related migraine candidate genes and has reported a significant allelic association with migraine of a microsatellite localised to the promoter region of the dopamine beta-hydroxylase (DBH) gene. The present study performed an association analysis in a larger population of case-controls (275 unrelated Caucasian migraineurs versus 275 controls) examining two different genetic DBH polymorphisms (a functional insertion/deletion promoter and a coding SNP A444G polymorphism). Although no significant association was found for the SNP polymorphism, the results showed a significant association between the insertion/deletion variant and disease (chi(2)=8.92, P=0.011), in particular in migraine with aura (chi(2)=11.53, P=0.003) compared to the control group. Furthermore, the analysis of this polymorphism stratified by gender, revealed that male individuals with the homozygote deletion genotype had three times the risk of developing migraine, compared to females. The DBH insertion/deletion polymorphism is in linkage disequilibrium with the previously reported migraine associated DBH microsatellite and this insertion/deletion polymorphism is functional, which may explain a potential role in susceptibility to migraine.

Association analysis of a highly polymorphic CAG Repeat in the human potassium channel gene KCNN3 and migraine susceptibility

Background Migraine is a polygenic multifactorial disease, possessing environmental and genetic causative factors with multiple involved genes. Mutations in various ion channel genes are responsible for a number of neurological disorders. KCNN3 is a neuronal small conductance calcium-activated potassium channel gene that contains two polyglutamine tracts, encoded by polymorphic CAG repeats in the gene. This gene plays a critical role in determining the firing pattern of neurons and acts to regulate intracellular calcium channels. Methods The present association study tested whether length variations in the second (more 3') polymorphic CAG repeat in exon 1 of the KCNN3 gene, are involved in susceptibility to migraine with and without aura (MA and MO). In total 423 DNA samples from unrelated individuals, of which 202 consisted of migraine patients and 221 non-migraine controls, were genotyped and analysed using a fluorescence labelled primer set on an ABI310 Genetic Analyzer. Allele frequencies were calculated from observed genotype counts for the KCNN3 polymorphism. Analysis was performed using standard contingency table analysis, incorporating the chi-squared test of independence and CLUMP analysis. Results Overall, there was no convincing evidence that KCNN3 CAG lengths differ between Caucasian migraineurs and controls, with no significant difference in the allelic length distribution of CAG repeats between the population groups (P = 0.090). Also the MA and MO subtypes did not differ significantly between control allelic distributions (P > 0.05). The prevalence of the long CAG repeat (>19 repeats) did not reach statistical significance in migraineurs (P = 0.15), nor was there a significant difference between the MA and MO subgroups observed compared to controls (P = 0.46 and P = 0.09, respectively), or between MA vs MO (P = 0.40). Conclusion This association study provides no evidence that length variations of the second polyglutamine array in the N-terminus of the KCNN3 channel exert an effect in the pathogenesis of migraine.

Familial typical migraine: Linkage to chromosome 19p13 and evidence for genetic heterogeneity

Migraine is a frequent familial disorder that, in common with most multifactorial disorders, has an unknown etiology. The authors identified several families with multiple individuals affected by typical migraine using a single set of diagnostic criteria and studied these families for cosegregation between the disorder and markers on chromosome 19, the location of a mutation that causes a rare form of familial hemiplegic migraine (FHM). One large tested family showed both cosegregation and significant allele sharing for markers situated within or adjacent to the FHM locus. Multipoint GENEHUNTER results indicated significant excess allele sharing across a 12.6- cM region containing the FHM Ca2+ channel gene, CACNL1A4 (maximum nonparametric linkage Z score = 6.64, p = 0.0026), with a maximum parametric lod score of 1.92 obtained for a (CAG)(n) triplet repeat polymorphism situated in exon 47 of this gene. The CAG expansion did not, however, appear to be the cause of migraine in this pedigree. Other tested families showed neither cosegregation nor excess allele sharing to chromosome 19 markers. HOMOG analysis indicated heterogeneity, generating a maximum HLOD score of 3.6. It was concluded that Chr19 mutations either in the CACNL1A4 gene or a closely linked gene are implicated in some pedigrees with familial typical migraine, and that the disorder is genetically heterogeneous.

Cryotherapy for docetaxel-induced hand and nail toxicity: Randomised control trial

Purpose This study investigated the efficacy and safety of cryotherapy, in the form of frozen gel gloves, in relation to docetaxel-induced hand and fingernail toxicities. Patients and methods After piloting with 21 patients, a consecutive series sample of patients (n=53) prescribed docetaxel every three weeks, for a minimum of three cycles, was enrolled in this randomised control trial. Participants acted as their own control, with the frozen gel glove worn on one randomised hand for 15 minutes prior to infusion, for the duration of the infusion, and for 15 minutes of after completion of treatment. Hand and nail toxicities were evaluated by two blinded assessors according to CTCAE.v4 criteria. To assess the potential for cross-infection of multi-use gloves, microbial culture and sensitivity swabs were taken of each glove at every tenth use. Results Of the 53 participants enrolled in the main study, 21 provided evaluable data. There was a 60% withdrawal rate due to patient discomfort with the intervention. The mean incidence and severity of toxicities in all evaluable cycles in control and intervention hands respectively were erythroderma Grade 1 (5%/5%); nail discolouration Grade 1 (81%/67%); nail loss Grade 1 (19%/19%) and nail ridging Grade 1 (57%/57%). No significant differences were determined between hand conditions in terms of time to event, nor in terms of toxicity in gloved and non-gloved hands. Conclusion While cryotherapy in the form of frozen gloves for the cutaneous toxicities associated with docetaxel is safe, its limited efficacy, patient discomfort and some logistical issues preclude its use in our clinical setting.

Risks for HIV infection among male street laborers in urban Vietnam

Risks for HIV infection remain unknown in male street laborers. This research investigates patterns of self-reported risk behaviors among these men in urban Vietnam. In a cross-sectional survey using a social mapping technique, 450 men, mostly low-skilled and unregistered migrant laborers across 13 districts in Hanoi were approached for interviews. The study revealed that male street laborers were at high risk of acquiring and transmitting HIV. One in every 12 men reported homosexual or bisexual behavior. These men on average had three sexual partners within the preceding year, and condom use was inconsistent. Close to 95 % of the men had reported sexual encounters with regular partners. One-third with commercial sex workers (CSW) and 24.2 % with casual partners, but just under one-third had ever used condoms with regular partners and CSWs and very few (17.6 %) with casual partners at their last sexual encounter. 17.11 % used illicit drugs sometimes, with 66.7 % of them frequently sharing injecting equipment with peers. These men had limited HIV knowledge; 51.4 % incorrectly believed that, once you trust your partner, you no longer need to use condoms and 42.4 % believed that you can tell by looking at someone if they have HIV. Access to HIV prevention was also limited; only 19.8 % of men had been tested for HIV during the previous 12 months, almost 10 % of whom neither returned for the result nor knew their HIV status. The study provides interesting directions for future research and suggests ways to effectively design prevention strategies for these men.

Desorption electrospray ionisation mass spectrometry reveals in situ modification of a hindered amine light stabiliser resulting from direct N–OR bond cleavage

Detection and characterisation of structural modifications of a hindered amine light stabiliser (HALS) directly from a polyester-based coil coating have been achieved by desorption electrospray ionisation mass spectrometry (DESI-MS) for the first time. In situ detection is made possible by exposing the coating to an acetone vapour atmosphere prior to analysis. This is a gentle and non-destructive treatment that allows diffusion of analyte to the surface without promoting lateral migration. Using this approach a major structural modification of the HALS TINUVIN®123 (bis(1-octyloxy-2,2,6,6-tetramethyl-4-piperidyl) sebacate) was discovered where one N-ether piperidine moiety (N-OC8H17) is converted to a secondary piperidine (N–H). With the use of 2-dimensional DESI-MS imaging the modification was observed to arise during high curing temperatures (ca. 260 °C) and under simulated physiological conditions (80 °C, full solar spectrum). It is proposed that the secondary piperidine derivative is a result of a highly reactive aminyl radical intermediate produced by N–O homolytic bond cleavage. The nature of the bond cleavage is also suggested by ESR spin-trapping experiments employing α-phenyl-N-tert-butyl nitrone (PBN) in toluene at 80 °C. The presence of a secondary piperidine derivative in situ and the implication of N–OR competing with NO–R bond cleavage suggest an alternative pathway for generation of the nitroxyl radical—an essential requirement in anti-oxidant activity that has not previously been described for the N-ether sub-class of HALS.