Exceptional overproduction of a functional human membrane protein

Eukaryotic-especially human-membrane protein overproduction remains a major challenge in biochemistry. Heterologously overproduced and purified proteins provide a starting point for further biochemical, biophysical and structural studies, and the lack of sufficient quantities of functional membrane proteins is frequently a bottleneck hindering this. Here, we report exceptionally high production levels of a correctly folded and crystallisable recombinant human integral membrane protein in its active form; human aquaporin 1 (hAQP1) has been heterologously produced in the membranes of the methylotrophic yeast Pichia pastoris. After solubilisation and a two step purification procedure, at least 90 mg hAQP1 per liter of culture is obtained. Water channel activity of this purified hAQP1 was verified by reconstitution into proteoliposomes and performing stopped-flow vesicle shrinkage measurements. Mass spectrometry confirmed the identity of hAQP1 in crude membrane preparations, and also from purified protein reconstituted into proteoliposomes. Furthermore, crystallisation screens yielded diffraction quality crystals of untagged recombinant hAQP1. This study illustrates the power of the yeast P. pastoris as a host to produce exceptionally high yields of a functionally active, human integral membrane protein for subsequent functional and structural characterization. © 2007 Elsevier Inc. All rights reserved.

Stopping the clock on proteomic degradation by heat treatment at the point of tissue excision

The effectiveness of rapid and controlled heating of intact tissue to inactivate native enzymatic activity and prevent proteome degradation has been evaluated. Mouse brains were bisected immediately following excision, with one hemisphere being heat treated followed by snap freezing in liquid nitrogen while the other hemisphere was snap frozen immediately. Sections were cut by cryostatic microtome and analyzed by MALDI-MS imaging and minimal label 2-D DIGE, to monitor time-dependent relative changes in intensities of protein and peptide signals. Analysis by MALDI-MS imaging demonstrated that the relative intensities of markers varied across a time course (0-5 min) when the tissues were not stabilized by heat treatment. However, the same markers were seen to be stabilized when the tissues were heat treated before snap freezing. Intensity profiles for proteins indicative of both degradation and stabilization were generated when samples of treated and nontreated tissues were analyzed by 2-D DIGE, with protein extracted before and after a 10-min warming of samples. Thus, heat treatment of tissues at the time of excision is shown to prevent subsequent uncontrolled degradation of tissues at the proteomic level before any quantitative analysis, and to be compatible with downstream proteomic analysis.

A study of potential serum markers for a diagnosis of gram-positive and gram-negative bacterial sepsis

Sepsis continues to be a major cause of morbidity and mortality as it can readily lead tosevere sepsis, septic shock, multiple organ failure and death. The onset can be rapid and difficult to define clinically. Despite the numerous candidate markers proposed in the literature, to date a serum marker for sepsis has not been found. The aim of this study was to assay the serum of clinically diagnosed patients with eithera Gram-negative or Gram- positive bacterial sepsis for elevated levels of nine potentialmarkers of sepsis, using commercially produced enzyme linked immunosorbent assays(ELISA). The purpose was to find a test marker for sepsis that would be helpful toclinicians in cases of uncertain sepsis and consequently expose false positive BC'scaused by skin or environmental contaminants. Nine test markers were assayed including IL-6, IL-I 0, ILI2, TNF-α, lipopolysaccharide binding protein, procalcitonin, sE-selectin, sICAM -1 and a potential differential marker for Gram-positive sepsis- anti-lipid S antibody. A total of 445 patients were enrolled into this study from the Queen Elizabeth Hospital and Selly Oak Hospital (Birmingham). The results showed that all the markers were elevated in patients with sepsis and that patients with a Gram-negative sepsis consistently produced higher median/range serum levels than those with a Gram-positive sepsis. No single marker was able to identify all the septic patients. Combining two markers caused the sensitivities and specificities for a diagnosis of sepsis to increase to within a 90% to 100% range. By a process of elimination the markers that survived into the last phase were IL-6 with sICAM -1, and anti-lipid S IgG assays Defining cut-off levels for a diagnosis of sepsis became problematic and a semi-blind trial was devised to test the markers in the absence of both clinical details and positive blood cultures. Patients with pyrexia of unknown origin and negative BC were included in this phase (4). The results showed that IL-6 with sICAM-l are authentic markers of sepsis. There was 82% agreement between the test marker diagnosis and the clinical diagnosis for sepsis in patients with a Gram-positive BC and 78% agreement in cases of Gram-negative Be. In the PUO group the test markers identified 12 cases of sepsis and the clinical diagnosis 15. The markers were shown to differentiate between early sepsis and sepsis, inflammatory responses and infection. Anti-lipid S with IL-6 proved be a sensitive marker for Gram-positive infections/sepsis.

Efficacy of Adding 2% (w/v) Chlorhexidine Gluconate to 70% (v/v) Isopropyl Alcohol for Skin Disinfection Prior to Peripheral Venous Cannulation

We undertook a clinical trial to compare the efficacy of 2% (w/v) chlorhexidine gluconate in 70% (v/v) isopropyl alcohol with the efficacy of 70% (v/v) isopropyl alcohol alone for skin disinfection to prevent peripheral venous catheter colonization and contamination. We found that the addition of 2% chlorhexidine gluconate reduced the number of peripheral venous catheters that were colonized or contaminated. © 2008 by The Society for Healthcare Epidemiology of America. All rights reserved.

Tacit collusion, firm asymmetries and numbers:evidence from EC merger cases

This paper estimates the implicit model, especially the roles of size asymmetries and firm numbers, used by the European Commission to identify mergers with coordinated effects. This subset of cases offers an opportunity to shed empirical light on the conditions where a Competition Authority believes tacit collusion is most likely to arise. We find that, for the Commission, tacit collusion is a rare phenomenon, largely confined to markets of two, more or less symmetric, players. This is consistent with recent experimental literature, but contrasts with the facts on ‘hard-core’ collusion in which firm numbers and asymmetries are often much larger.

Tacit collusion, firm asymmetries and numbers:evidence from EC merger cases

The purpose of this paper is to identify empirically the implicit structural model, especially the roles of size asymmetries and concentration, used by the European Commission to identify mergers with coordinated effects (i.e. collective dominance). Apart from its obvious policy-relevance, the paper is designed to shed empirical light on the conditions under which tacit collusion is most likely. We construct a database relating to 62 candidate mergers and find that, in the eyes of the Commission, tacit collusion in this context virtually never involves more than two firms and requires close symmetry in the market shares of the two firms.

Benefits from competition:some illustrative UK cases

This study provides a powerful demonstration of the real world impact of increasedcompetition. By presenting six market case studies drawn from a variety of sectors itgives evidence of the type and magnitude of the benefits following marketinterventions to develop competition and free up the operation of these sectors. In discussing the types and form such interventions take, whether competition policy,deregulation or liberalisation, this report explores market conditions before and afterintervention, paying careful attention to both the envisaged benefits and the potentialfor negative side effects. Overall, the evidence suggests these benefits materialised,and in a number of instances proved more sizeable than anticipated. Concerns aboutharmful side effects have proved unfounded, with market stimuli impacting not only onthe price and range of goods available but also acting as a motivating force to productand process innovation. As Professor Davies points out, although active competition policy proves an importantcomponent in the competitive process, it is not sufficient in its own right. In order todeliver greater productivity, of which competition is a key driver, the UK needs a pool of resourceful entrepreneurs able to exploit changing market conditions. In order togive these people the best chance of success the framework conditions need to becorrect with strength in the complementary capabilities of innovation, investment, skillsand enterprise. Ensuring the competition framework is world class is central to the DTI’s strategy. The most recent peer review of the UK competition regime demonstrates that the UK isa strong performer, ranked third in the 2004 study, with the US first and Germanysecond. This study provides further evidence of the important role played by thatframework in delivering tangible benefits to consumers.

Goal-based modeling of dynamically adaptive system requirements

Self-adaptation is emerging as an increasingly important capability for many applications, particularly those deployed in dynamically changing environments, such as ecosystem monitoring and disaster management. One key challenge posed by Dynamically Adaptive Systems (DASs) is the need to handle changes to the requirements and corresponding behavior of a DAS in response to varying environmental conditions. Berry et al. previously identified four levels of RE that should be performed for a DAS. In this paper, we propose the Levels of RE for Modeling that reify the original levels to describe RE modeling work done by DAS developers. Specifically, we identify four types of developers: the system developer, the adaptation scenario developer, the adaptation infrastructure developer, and the DAS research community. Each level corresponds to the work of a different type of developer to construct goal model(s) specifying their requirements. We then leverage the Levels of RE for Modeling to propose two complementary processes for performing RE for a DAS. We describe our experiences with applying this approach to GridStix, an adaptive flood warning system, deployed to monitor the River Ribble in Yorkshire, England.

Reinforcing the security of corporate information resources:a critical review of the role of the acceptable use policy

Increasingly users are seen as the weak link in the chain, when it comes to the security of corporate information. Should the users of computer systems act in any inappropriate or insecure manner, then they may put their employers in danger of financial losses, information degradation or litigation, and themselves in danger of dismissal or prosecution. This is a particularly important concern for knowledge-intensive organisations, such as universities, as the effective conduct of their core teaching and research activities is becoming ever more reliant on the availability, integrity and accuracy of computer-based information resources. One increasingly important mechanism for reducing the occurrence of inappropriate behaviours, and in so doing, protecting corporate information, is through the formulation and application of a formal ‘acceptable use policy (AUP). Whilst the AUP has attracted some academic interest, it has tended to be prescriptive and overly focussed on the role of the Internet, and there is relatively little empirical material that explicitly addresses the purpose, positioning or content of real acceptable use policies. The broad aim of the study, reported in this paper, is to fill this gap in the literature by critically examining the structure and composition of a sample of authentic policies – taken from the higher education sector – rather than simply making general prescriptions about what they ought to contain. There are two important conclusions to be drawn from this study: (1) the primary role of the AUP appears to be as a mechanism for dealing with unacceptable behaviour, rather than proactively promoting desirable and effective security behaviours, and (2) the wide variation found in the coverage and positioning of the reviewed policies is unlikely to be fostering a coherent approach to security management, across the higher education sector.

The information security policy unpacked:a critical study of the content of university policies

Ensuring the security of corporate information, that is increasingly stored, processed and disseminated using information and communications technologies [ICTs], has become an extremely complex and challenging activity. This is a particularly important concern for knowledge-intensive organisations, such as universities, as the effective conduct of their core teaching and research activities is becoming ever more reliant on the availability, integrity and accuracy of computer-based information resources. One increasingly important mechanism for reducing the occurrence of security breaches, and in so doing, protecting corporate information, is through the formulation and application of a formal information security policy (InSPy). Whilst a great deal has now been written about the importance and role of the information security policy, and approaches to its formulation and dissemination, there is relatively little empirical material that explicitly addresses the structure or content of security policies. The broad aim of the study, reported in this paper, is to fill this gap in the literature by critically examining the structure and content of authentic information security policies, rather than simply making general prescriptions about what they ought to contain. Having established the structure and key features of the reviewed policies, the paper critically explores the underlying conceptualisation of information security embedded in the policies. There are two important conclusions to be drawn from this study: (1) the wide diversity of disparate policies and standards in use is unlikely to foster a coherent approach to security management; and (2) the range of specific issues explicitly covered in university policies is surprisingly low, and reflects a highly techno-centric view of information security management.