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.

Weighted fairness with MCR guarantee and a general-purpose explicit rate allocation algorithm for ATM ABR service

In this paper, weighted fair rate allocation for ATM available bit rate (ABR) service is discussed with the concern of the minimum cell rate (MCR). Weighted fairness with MCR guarantee has been discussed recently in the literature. In those studies, each ABR virtual connection (VC) is first allocated its MCR, then the remaining available bandwidth is further shared among ABR VCs according to their weights. For the weighted fairness defined in this paper, the bandwidth is first allocated according to each VC's weight; if a VC's weighted share is less than its MCR, it should be allocated its MCR instead of the weighted share. This weighted fairness with MCR guarantee is referred to as extended weighted (EXW) fairness. Certain theoretical issues related to EXW, such as its global solution and bottleneck structure, are first discussed in the paper. A distributed explicit rate allocation algorithm is then proposed to achieve EXW fairness in ATM networks. The algorithm is a general-purpose explicit rate algorithm in the sense that it can realise almost all the fairness principles proposed for ABR so far whilst only minor modifications may be needed.

An enhanced explicit rate algorithm for ABR traffic control in ATM networks

A high performance, low computational complexity rate-based flow control algorithm which can avoid congestion and achieve fairness is important to ATM available bit rate service. The explicit rate allocation algorithm proposed by Kalampoukas et al. is designed to achieve max–min fairness in ATM networks. It has several attractive features, such as a fixed computational complexity of O(1) and the guaranteed convergence to max–min fairness. In this paper, certain drawbacks of the algorithm, such as the severe overload of an outgoing link during transient period and the non-conforming use of the current cell rate field in a resource management cell, have been identified and analysed; a new algorithm which overcomes these drawbacks is proposed. The proposed algorithm simplifies the rate computation as well. Compared with Kalampoukas's algorithm, it has better performance in terms of congestion avoidance and smoothness of rate allocation.

Protection by methylproamine of irradiated human keratinocytes correlates with reduction of DNA damage

Purpose: The therapeutic ratio for ionising radiation treatment of tumour is a trade-off between normal tissue side-effects and tumour control. Application of a radioprotector to normal tissue can reduce side-effects. Here we study the effects of a new radioprotector on the cellular response to radiation. Methylproamine is a DNA-binding radioprotector which, on the basis of published pulse radiolysis studies, acts by repair of transient radiation-induced oxidative species on DNA. To substantiate this hypothesis, we studied protection by methylproamine at both clonogenic survival and radiation-induced DNA damage, assessed by γH2AX (histone 2AX phosphorylation at serine 139) focus formation endpoints. Materials and methods: The human keratinocyte cell line FEP1811 was used to study clonogenic survival and yield of γH2AX foci following irradiation (137Cs γ-rays) of cells exposed to various concentrations of methylproamine. Uptake of methylproamine into cell nuclei was measured in parallel. Results: The extent of radioprotection at the clonogenic survival endpoint increased with methylproamine concentration up to a maximum dose modification factor (DMF) of 2.0 at 10 μM. At least 0.1 fmole/nucleus of methylproamine is required to achieve a substantial level of radioprotection (DMF of 1.3) with maximum protection (DMF of 2.0) achieved at 0.23 fmole/nucleus. The γH2AX focus yield per cell nucleus 45 min after irradiation decreased with drug concentration with a DMF of 2.5 at 10 μM. Conclusions: These results are consistent with the hypothesis that radioprotection by methylproamine is mediated by attenuation of the extent of initial DNA damage.

Exploring metrics to express energy expenditure of physical activity in youth

Background Several approaches have been used to express energy expenditure in youth, but no consensus exists as to which best normalizes data for the wide range of ages and body sizes across a range of physical activities. This study examined several common metrics for expressing energy expenditure to determine whether one metric can be used for all healthy children. Such a metric could improve our ability to further advance the Compendium of Physical Activities for Youth. Methods A secondary analysis of oxygen uptake (VO2) data obtained from five sites was completed, that included 947 children ages 5 to 18 years, who engaged in 14 different activities. Resting metabolic rate (RMR) was computed based on Schofield Equations [Hum Nutr Clin Nut. 39(Suppl 1), 1985]. Absolute oxygen uptake (ml.min-1), oxygen uptake per kilogram body mass (VO2 in ml.kg-1.min-1), net oxygen uptake (VO2 – resting metabolic rate), allometric scaled oxygen uptake (VO2 in ml.kg-0.75.min-1) and YOUTH-MET (VO2.[resting VO2] -1) were calculated. These metrics were regressed with age, sex, height, and body mass. Results Net and allometric-scaled VO2, and YOUTH-MET were least associated with age, sex and physical characteristics. For moderate-to-vigorous intensity activities, allometric scaling was least related to age and sex. For sedentary and low-intensity activities, YOUTH-MET was least related to age and sex. Conclusions No energy expenditure metric completely eliminated the influence of age, physical characteristics, and sex. The Adult MET consistently overestimated EE. YOUTH-MET was better for expressing energy expenditure for sedentary and light activities, whereas allometric scaling was better for moderate and vigorous intensity activities. From a practical perspective, The YOUTH-MET may be the more feasible metric for improving of the Compendium of Physical Activities for Youth.

Developing guidelines for intervention to reduce risk of low-speed vehicle run-overs of young children

In Australia, research suggests that up to one quarter of child pedestrian hospitalisations result from driveway run-over incidents (Pinkney et al., 2006). In Queensland, these numbers equate to an average of four child fatalities and 81 children presenting at hospital emergency departments every year (The Commission for Children, Young People and Child Guardian). National comparison shows that these numbers represent a slightly higher per capita rate (23.5% of all deaths). To address this issue, the current research was undertaken with the aim to develop an educative intervention based on data collected from parents and caregivers of young children. Thus, the current project did not seek to use available intervention or educational material, but to develop a new evidence-based intervention specifically targeting driveway run-overs involving young children. To this end, general behavioural and environmental changes that caregivers had undertaken in order to reduce the risk of injury to any child in their care were investigated. Broadly, the first part of this report sought to: • develop a conceptual model of established domestic safety behaviours, and to investigate whether this model could be successfully applied to the driveway setting; • explore and compare sources of knowledge regarding domestic and driveway child safety; and • examine the theoretical implications of current domestic and driveway related behaviour and knowledge among caregivers. The aim of the second part of this research was to develop and test the efficacy of an intervention based on the findings in the first part of the research project. Specifically, it sought to: • develop an educational driveway intervention that is based on current safety behaviours in the domestic setting and informed by existing knowledge of driveway safety and behaviour change theory; and • evaluate its efficacy in a sample of parents and caregivers.

An analytical tool that quantifies cellular morphology changes from three-dimensional fluorescence images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology(1) even in complex tissue sections(2). Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells(3), however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgenössische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

Reduction in resting plasma granulysin as a marker of increased training load

Granulysin is a cytolytic granule protein released by natural killer cells and activated cytotoxic T lymphocytes. The influence of exercise training on circulating granulysin concentration is unknown, as is the relationship between granulysin concentration, natural killer cell number and natural killer cell cytotoxicity. We examined changes in plasma granulysin concentration, natural killer cell number and cytotoxicity following acute exercise and different training loads. Fifteen highly trained male cyclists completed a baseline 40-km cycle time trial (TT401) followed by five weeks of normal training and a repeat time trial (TT402). The cyclists then completed four days of high intensity training followed by another time trial (TT403) on day five. Following one final week of normal training cyclists completed another time trial (TT404). Fasting venous blood was collected before and after each time trial to determine granulysin concentration, natural killer cell number and natural killer cell cytotoxicity. Granulysin concentration increased significantly after each time trial (P<0.001). Pre-exercise granulysin concentration for TT403 was significantly lower than pre-exercise concentration for TT401 (-20.3 +/- 7.5%, P<0.026), TT402 (-16.7 +/- 4.3%, P<0.003) and 7T404 (-21 +/- 4.2%, P<0.001). Circulating natural killer cell numbers also increased significantly post-exercise for each time trial (P<0.001), however there was no significant difference across TT40 (P>0.05). Exercise did not significantly alter natural killer cell cytotoxicity on a per cell basis, and there were no significant differences between the four time trials. In conclusion, plasma granulysin concentration increases following moderate duration, strenuous exercise and is decreased in response to a short-term period of intensified training.

Feasibility of an in-patient exercise intervention for children undergoing haematopoietic stem cell transplant

With improving survival rates following HSCT in children, QOL and management of short- and long-term effects need to be considered. Exercise may help mitigate fatigue and declines in fitness and strength. The aims of this study were to assess the feasibility of an inpatient exercise intervention for children undergoing HSCT and observe the changes in physical and psychological health. Fourteen patients were recruited, mean age 10 yr. A 6MWT, isometric upper and lower body strength, balance, fatigue, and QOL were assessed prior to Tx and six wk post-Tx. A supervised exercise program was offered five days per week during the inpatient period and feasibility assessed through uptake rate. The study had 100% program completion and 60% uptake rate of exercise sessions. The mean (±s.d.) weekly activity was 117.5 (±79.3) minutes. Younger children performed significantly more minutes of exercise than adolescents. At reassessment, strength and fatigue were stabilized while aerobic fitness and balance decreased. QOL revealed a non-statistical trend towards improvement. No exercise-related adverse events were reported. A supervised inpatient exercise program is safe and feasible, with potential physiological and psychosocial benefits.

Initiation of embryogenic cell suspensions of taro (Colocasia esculenta var. esculenta) and plant regeneration

Embryogenic callus was initiated by culturing in vitro taro corm slices on agar-solidified half-strength MS medium containing 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) for 20 days followed by transfer to 1.0 mg/L thidiazuron (TDZ). Callus was subsequently proliferated on solid medium containing 1.0 mg/L TDZ, 0.5 mg/L 2,4- D and 800 mg/L glutamine before transfer to liquid medium containing the same components but with reduced glutamine (100 mg/L). After 3 months in liquid culture on an orbital shaker, cytoplasmically dense cell aggregates began to form. Somatic embryogenesis was induced by plating suspension cells onto solid media containing reduced levels of hormones (0.1 mg/L TDZ, 0.05 mg/L 2,4-D), high concentrations of sucrose (40–50 g/L) and biotin (1.0 mg/L). Embryo maturation and germination was then induced on media containing 0.05 mg/L benzyladenine (BA) and 0.1 mg/L indole-3-acetic acid (IAA). Histological studies of the developing embryos revealed the presence of typical shoot and root poles suggesting that these structures were true somatic embryos. The rate of somatic embryos formation was 500–3,000 per mL settledcell volume while approximately 60% of the embryos regenerated into plants.

Fair flow control of ABR service by per-VC virtual queuing

In this paper, a rate-based flow control scheme based upon per-VC virtual queuing is proposed for the Available Bit Rate (ABR) service in ATM. In this scheme, each VC in a shared buffer is assigned a virtual queue, which is a counter. To achieve a specific kind of fairness, an appropriate scheduler is applied to the virtual queues. Each VC's bottleneck rate (fair share) is derived from its virtual cell departure rate. This approach of deriving a VC's fair share is simple and accurate. By controlling each VC with respect to its virtual queue and queue build-up in the shared buffer, network congestion is avoided. The principle of the control scheme is first illustrated by max–min flow control, which is realised by scheduling the virtual queues in round-robin. Further application of the control scheme is demonstrated with the achievement of weighted fairness through weighted round robin scheduling. Simulation results show that with a simple computation, the proposed scheme achieves the desired fairness exactly and controls network congestion effectively.

An analytical solution for diffusion and nonlinear uptake of oxygen in a spherical cell

We develop a new analytical solution for a reactive transport model that describes the steady-state distribution of oxygen subject to diffusive transport and nonlinear uptake in a sphere. This model was originally reported by Lin (Journal of Theoretical Biology, 1976 v60, pp449–457) to represent the distribution of oxygen inside a cell and has since been studied extensively by both the numerical analysis and formal analysis communities. Here we extend these previous studies by deriving an analytical solution to a generalized reaction-diffusion equation that encompasses Lin’s model as a particular case. We evaluate the solution for the parameter combinations presented by Lin and show that the new solutions are identical to a grid-independent numerical approximation.

The beneficial effects of specialist thoracic surgery on the resection rate for non-small-cell lung cancer

We aimed to evaluate the effect of the appointment of a dedicated specialist thoracic surgeon on surgical practice for lung cancer previously served by cardio-thoracic surgeons. Outcomes were compared for the 240 patients undergoing surgical resection for lung cancer in two distinct 3-year periods: Group A: 65 patients, 1994-1996 (pre-specialist); Group B: 175 patients, 1997-1999 (post-specialist). The changes implemented resulted in a significant increase in resection rate (from 12.2 to 23.4%, P<0.001), operations in the elderly (over 75 years) and extended resections. There were no significant differences in stage distribution, in-hospital mortality or stage-specific survival after surgery. Lung cancer surgery provided by specialists within a multidisciplinary team resulted in increased surgical resection rates without compromising outcome. Our results strengthen the case for disease-specific specialists in the treatment of lung cancer. © 2004 Published by Elsevier Ireland Ltd.

Cetuximab plus chemotherapy in patients with advanced non-small-cell lung cancer (FLEX) : an open-label randomised phase III trial

Background: Use of cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor (EGFR), has the potential to increase survival in patients with advanced non-small-cell lung cancer. We therefore compared chemotherapy plus cetuximab with chemotherapy alone in patients with advanced EGFR-positive non-small-cell lung cancer. Methods: In a multinational, multicentre, open-label, phase III trial, chemotherapy-naive patients (≥18 years) with advanced EGFR-expressing histologically or cytologically proven stage wet IIIB or stage IV non-small-cell lung cancer were randomly assigned in a 1:1 ratio to chemotherapy plus cetuximab or just chemotherapy. Chemotherapy was cisplatin 80 mg/m 2 intravenous infusion on day 1, and vinorelbine 25 mg/m 2 intravenous infusion on days 1 and 8 of every 3-week cycle) for up to six cycles. Cetuximab-at a starting dose of 400 mg/m 2 intravenous infusion over 2 h on day 1, and from day 8 onwards at 250 mg/m 2 over 1 h per week-was continued after the end of chemotherapy until disease progression or unacceptable toxicity had occurred. The primary endpoint was overall survival. Analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00148798. Findings: Between October, 2004, and January, 2006, 1125 patients were randomly assigned to chemotherapy plus cetuximab (n=557) or chemotherapy alone (n=568). Patients given chemotherapy plus cetuximab survived longer than those in the chemotherapy-alone group (median 11·3 months vs 10·1 months; hazard ratio for death 0·871 [95% CI 0·762-0·996]; p=0·044). The main cetuximab-related adverse event was acne-like rash (57 [10%] of 548, grade 3). Interpretation: Addition of cetuximab to platinum-based chemotherapy represents a new treatment option for patients with advanced non-small-cell lung cancer. Funding: Merck KGaA. © 2009 Elsevier Ltd. All rights reserved.