Matchout deuterium labelling of proteins for small-angle neutron scattering studies using prokaryotic and eukaryotic expression systems and high cell-density cultures.

Small-angle neutron scattering (SANS) is a powerful technique for the characterisation of macromolecular structures and interactions. Its main advantage over other solution state approaches is the ability to use D2O/H2O solvent contrast variation to selectively match out specific parts of a multi-component system. While proteins, nucleic acids, and lipids are readily distinguished in this way, it is not possible to locate different parts of a protein-protein system without the introduction of additional contrast by selective deuteration. Here, we describe new methods by which 'matchout labelled' proteins can be produced using Escherichia coli and Pichia pastoris expression systems in high cell-density cultures. The method is designed to produce protein that has a scattering length density that is very close to that of 100% D2O, providing clear contrast when used with hydrogenated partner proteins in a complex. This allows the production of a single sample system for which SANS measurements at different solvent contrasts can be used to distinguish and model the hydrogenated component, the deuterated component, and the whole complex. The approach, which has significant cost advantages, has been extensively tested for both types of expression system

A Fisher-Rao Metric for curves using the information in edges

Two curves which are close together in an image are indistinguishable given a measurement, in that there is no compelling reason to associate the measurement with one curve rather than the other. This observation is made quantitative using the parametric version of the Fisher-Rao metric. A probability density function for a measurement conditional on a curve is constructed. The distance between two curves is then defined to be the Fisher-Rao distance between the two conditional pdfs. A tractable approximation to the Fisher-Rao metric is obtained for the case in which the measurements are compound in that they consist of a point x and an angle α which specifies the direction of an edge at x. If the curves are circles or straight lines, then the approximating metric is generalized to take account of inlying and outlying measurements. An estimate is made of the number of measurements required for the accurate location of a circle in the presence of outliers. A Bayesian algorithm for circle detection is defined. The prior density for the algorithm is obtained from the Fisher-Rao metric. The algorithm is tested on images from the CASIA Iris Interval database

A Dimerization Site at SCR-17/18 in Factor H Clarifies a New Mechanism for Complement Regulatory Control

Complement Factor H (CFH), with 20 short complement regulator (SCR) domains, regulates the alternative pathway of complement in part through the interaction of its C-terminal SCR-19 and SCR-20 domains with host cell-bound C3b and anionic oligosaccharides. In solution, CFH forms small amounts of oligomers, with one of its self-association sites being in the SCR-16/20 domains. In order to correlate CFH function with dimer formation and the occurrence of rare disease-associated variants in SCR-16/20, we identified the dimerization site in SCR-16/20. For this, we expressed, in Pichia pastoris, the five domains in SCR-16/20 and six fragments of this with one-three domains (SCR-19/20, SCR-18/20, SCR-17/18, SCR-16/18, SCR-17 and SCR-18). Size-exclusion chromatography suggested that SCR dimer formation occurred in several fragments. Dimer formation was clarified using analytical ultracentrifugation, where quantitative c(s) size distribution analyses showed that SCR-19/20 was monomeric, SCR-18/20 was slightly dimeric, SCR-16/20, SCR-16/18 and SCR-18 showed more dimer formation, and SCR-17 and SCR-17/18 were primarily dimeric with dissociation constants of ~5 µM. The combination of these results located the SCR-16/20 dimerization site at SCR-17 and SCR-18. X-ray solution scattering experiments and molecular modelling fits confirmed the dimer site to be at SCR-17/18, this dimer being a side-by-side association of the two domains. We propose that the self-association of CFH at SCR-17/18 enables higher concentrations of CFH to be achieved when SCR-19/20 are bound to host cell surfaces in order to protect these better during inflammation. Dimer formation at SCR-17/18 clarified the association of genetic variants throughout SCR-16/20 with renal disease

Physiological Oxygen Causes the Release of Volatile Organic Compounds from Human Pluripotent Stem Cells with Possible Roles in Maintaining Self-Renewal and Pluripotency

Human pluripotent stem cells (hPSCs) have widespread potential biomedical applications. There is a need for large-scale in vitro production of hPSCs, and optimal culture methods are vital in achieving this. Physiological oxygen (2% O2) improves key hPSCs attributes, including genomic integrity, viability, and clonogenicity, however, its impact on hPSC metabolism remains un-clear. Here, Selected Ion Flow Tube-Mass Spectrometry (SIFT-MS) was used to detect and quantify metabolic Volatile Organic Compounds (VOCs) in the headspace of hPSCs and their differentiated progeny. hPSCs were cultured in either 2% O2 or 21% O2. Media was collected from cell cultures and transferred into glass bottles for SIFT-MS measurement. The VOCs acetaldehyde and dimethyl sulfide (DMS)/ethanethiol were significantly increased in undifferentiated hPSCs compared to their differentiating counterparts, and these observations were more apparent in 2% O2. Pluripotent marker expression was consistent across both O2 concentrations tested. Transcript levels of ADH4, ADH5, and CYP2E1, encoding enzymes involved in converting ethanol to acetaldehyde, were upregulated in 2% O2, and chemical inhibition of ADH and CYP2E1 decreased acetaldehyde levels in hPSCs. Acetaldehyde and DMS/ethanethiol may be indicators of altered metabolism pathways in physiological oxygen culture conditions. The identification of non-destructive biomarkers for hPSC characterization has the potential to facilitate large-scale in vitro manufacture for future biomedical application.</jats:p

Exploring Norms in Agile Software Teams

The majority of software developers work in teams and are thus influenced by team norms. Norms are shared expectations of how to behave and regulate the interaction between team members. Our aim of this study is to gain more knowledge about team norms in software teams and to increase the understanding of how norms influence teamwork in agile software development projects. We conducted a study of norms in four agile teams located in Norway and Malaysia. The analysis of 22 interviews revealed that we could extract a varied set of both injunctive and descriptive norms. Our results suggest that team norms have an important role in enabling team performance.acceptedVersio

The Role of Individual Variables, Organizational Variables and Moral Intensity Dimensions in Libyan Management Accountants’ Ethical Decision Making

This study investigates the association of a broad set of variables with the ethical decision making of management accountants in Libya. Adopting a cross-sectional methodology, a questionnaire including four different ethical scenarios was used to gather data from 229 participants. For each scenario, ethical decision making was examined in terms of the recognition, judgment and intention stages of Rest’s model. A significant relationship was found between ethical recognition and ethical judgment and also between ethical judgment and ethical intention, but ethical recognition did not significantly predict ethical intention—thus providing support for Rest’s model. Organizational variables, age and educational level yielded few significant results. The lack of significance for codes of ethics might reflect their relative lack of development in Libya, in which case Libyan companies should pay attention to their content and how they are supported, especially in the light of the under-development of the accounting profession in Libya. Few significant results were also found for gender, but where they were found, males showed more ethical characteristics than females. This unusual result reinforces the dangers of gender stereotyping in business. Personal moral philosophy and moral intensity dimensions were generally found to be significant predictors of the three stages of ethical decision making studied. One implication of this is to give more attention to ethics in accounting education, making the connections between accounting practice and (in Libya) Islam. Overall, this study not only adds to the available empirical evidence on factors affecting ethical decision making, notably examining three stages of Rest’s model, but also offers rare insights into the ethical views of practising management accountants and provides a benchmark for future studies of ethical decision making in Muslim majority countries and other parts of the developing world

Demonstration of the Effect of Generic Anatomical Divisions versus Clinical Protocols on Computed Tomography Dose Estimates and Risk Burden

Objective: Choosing to undertake a CT scan relies on balancing risk versus benefit, however risks associated with CT scanning have generally been limited to broad anatomical locations, which do not provided adequate information to evaluate risk against benefit. Our study aimed to determine differences in radiation dose and risk estimates associated with modern CT scanning examinations when computed for clinical protocols compared with those using anatomical area. Methods: Technical data were extracted from a tertiary hospital Picture Archiving Communication System for random samples of 20–40 CT examinations per adult clinical CT protocol. Organ and whole body radiation dose were calculated using ImPACT Monte Carlo simulation software and cancer incidence and mortality estimated using BEIR VII age and gender specific lifetime attributable risk weights. Results: Thirty four unique CT protocols were identified by our study. When grouped according to anatomic area the radiation dose varied substantially, particularly for abdominal protocols. The total estimated number of incident cancers and cancer related deaths using the mean dose of anatomical area were 86 and 69 respectively. Using more specific protocol doses the estimates rose to 214 and 138 incident cancers and cancer related deaths, at least doubling the burden estimated. Conclusions: Modern CT scanning produces a greater diversity of effective doses than much of the literature describes; where a lack of focus on actual scanning protocols has produced estimates that do not reflect the range and complexity of modern CT practice. To allow clinicians, patients and policy makers to make informed risk versus benefit decisions the individual and population level risks associated with modern CT practices are essential

Reovirus exerts potent oncolytic effects in head and neck cancer cell lines that are independent of signalling in the EGFR pathway

Background: reovirus exploits aberrant signalling downstream of Ras to mediate tumor-specific oncolysis. Since ~90% squamous cell carcinomas of the head and neck (SCCHN) over-express EGFR and SCCHN cell lines are sensitive to oncolytic reovirus, we conducted a detailed analysis of the effects of reovirus in 15 head and neck cancer cell lines. Both pre- and post-entry events were studied in an attempt to define biomarkers predictive of sensitivity/resistance to reovirus. In particular, we analysed the role of EGFR/Ras signalling in determining virus-mediated cytotoxicity in SCCHN. Methods: to test whether EGFR pathway activity was predictive of increased sensitivity to reovirus, correlative analyses between reoviral IC50 by MTT assay and EGFR levels by western blot and FACS were conducted. Inhibition or stimulation of EGFR signalling were analysed for their effect on reoviral oncolysis by MTT assay, and viral growth by TCID50 assay. We next analysed the effects of inhibiting signalling downstream of Ras, by specific inhibitors of p38MAPK, PI3-K or MEK, on reoviral killing examined by MTT assay. The role of PKR in reoviral killing was also determined by blockade of PKR using 2-aminopurine and assaying for cell survival by MTT assay. The apoptotic response of SCCHN to reovirus was examined by western blot analysis of caspase 3 cleavage. Results: correlative analyses between reoviral sensitivity and EGFR levels revealed no association. Intermediate sub-viral and core particles showed the same infectivity/cytotoxicity as intact reovirus. Therefore, sensitivity was not determined by cell entry. In 4 cell lines, oncolysis and viral growth were both unaffected by inhibition or stimulation of EGFR signalling. Inhibition of signalling downstream of Ras did not abrogate reoviral oncolysis and, in addition, modulation of PKR using 2-aminopurine did not alter reovirus sensitivity in resistant cell lines. Caspase 3 cleavage was not detected in infected cells and oncolysis was observed in pan-caspase inhibited cells. Conclusions: in summary, reovirus is potently oncolytic in a broad panel of SCCHN cell lines. Attempts to define sensitivity/resistance by analysis of the EGFR/Ras/MAPK pathway have failed to provide a clear predictive biomarker of response. Further analysis of material from in vitro and clinical studies is ongoing in an attempt to shed further light on this issue

A prospective, cross-sectional study of anaemia and peripheral iron status in antiretroviral naïve, HIV-1 infected children in Cape Town, South Africa

BACKGROUND: Anaemia is a common manifestation of paediatric HIV infection. Although there are many causes, anaemia of chronic diseases is the most frequent type. In poor countries iron deficiency is widespread. It is probable that many HIV-infected children in these countries are also iron deficient. This study describes the relationship between paediatric HIV infection and anaemia, and documents the peripheral iron status of antiretroviral naive, HIV-infected children. METHODS: Sixty children were evaluated prospectively. Investigations included CD4+ count, haemoglobin concentration (Hb), red blood cell (RBC) morphology, and iron studies. RESULTS: Anaemia was present in 73% of children. Compared to mild HIV infection, median Hb was lower in children with moderate clinical infection (104 g/L v 112 g/L, p = 0.04) and severe clinical infection (96 g/L v 112 g/L, p = 0.006), and more children with severe infection were anaemic (92% v 58%, 0.04). There was a significant relationship between immunological status and Hb. 68% had abnormal RBC morphology. Significantly more children with moderate and severe disease, and severe immunosuppression had abnormal RBC morphology. 52% were iron-depleted, 20% had iron-deficient erythropoiesis and 18% iron deficiency anaemia (IDA). 16% (7/44) of anaemic children had microcytosis and hypochromia. Median soluble transferrin receptor concentration was significantly higher in those with microcytic hypochromic anaemia (42.0 nmol/L v 30.0 nmol/L, p = 0.008). CONCLUSIONS: Both the proportion of anaemic children and the median Hb were associated with disease status. Iron depletion and IDA are major problems in HIV-infected children in South Africa