Construction and evaluation of plasmid vectors optimized for constitutive and regulated gene expression in Burkholderia cepacia complex isolates

Genetic studies with Burkholderia cepacia complex isolates are hampered by the limited availability of cloning vectors and by the inherent resistance of these isolates to the most common antibiotics used for genetic selection. Also, some of the promoters widely employed for gene expression in Escherichia coli are inefficient in B. cepacia. In this study, we have utilized the backbone of the vector pME6000, a derivative of the pBBR1 plasmid that was originally isolated from Bordetella bronchiseptica, to construct a set of vectors useful for gene expression in B. cepacia. These vectors contain either the constitutive promoter of the S7 ribosomal protein gene from Burkholderia sp. strain LB400 or the arabinose-inducible P(BAD) promoter from E. coli. Promoter sequences were placed immediately upstream of multiple cloning sites in combination with the minimal sequence of pME6000 required for plasmid maintenance and mobilization. The functionality of both vectors was assessed by cloning the enhanced green fluorescent protein gene (e-gfp) and determining the levels of enhanced green fluorescent protein expression and fluorescence emission for a variety of clinical and environmental isolates of the B. cepacia complex. We also demonstrate that B. cepacia carrying these constructs can readily be detected intracellularly by fluorescence microscopy following the infection of Acanthamoeba polyphaga.

Regulation of Epithelial Differentiation and Proliferation by the Stroma: A Role for the Retinoblastoma Protein.

Signaling between the epithelium and stromal cells is crucial for growth, differentiation, and repair of the epithelium. Although the retinoblastoma protein (Rb) is known to regulate the growth of keratinocytes in a cell-autonomous manner, here we describe a function of Rb in the stromal compartment. We find that Rb depletion in fibroblasts leads to inhibition of differentiation and enhanced proliferation of the epithelium. Analysis of conditioned medium identified that keratinocyte growth factor (KGF) levels were elevated following Rb depletion. These findings were also observed with organotypic co-cultures. Treatment of keratinocytes with KGF inhibited differentiation and enhanced keratinocyte proliferation, whereas reduction of KGF levels in Rb-depleted fibroblasts was able to restore expression of differentiation markers. Our findings suggest a crucial role for dermal fibroblasts in regulating the differentiation and proliferation of keratinocytes, and we demonstrate a role for stromal Rb in this cross-talk.Journal of Investigative Dermatology advance online publication, 14 June 2012;doi:10.1038/jid.2012.201.

HOXA/PBX3 knockdown impairs growth and sensitizes cytogenetically normal acute myeloid leukemia cells to chemotherapy

The cytogenetically normal subtype of acute myeloid leukemia (CN-AML) is associated with Intermediate risk which complicates therapeutic options. Lower overall HOX/TALE expression appears to correlate with more favorable prognosis/better response to treatment in some leukemias and solid cancer. The functional significance of the associated gene expression and response to chemotherapy is not known. Three independent microarray datasets obtained from large patient cohorts along with quantitative PCR validation was used to identify a four gene HOXA/TALE signature capable of prognostic stratification. Biochemical analysis was used to identify interactions between the four encoded proteins and targeted knockdown used to examine the functional importance of sustained expression of the signature in leukemia maintenance and response to chemotherapy. An eleven HOXA/TALE code identified in an Intermediate risk (n=315) compared to a Favourable group of patients (n=105) was reduced to a four gene signature of HOXA6, HOXA9, PBX3 and MEIS1 by iterative analysis of independent platforms. This signature maintained the Favorable/Intermediate risk partition and where applicable, correlated with overall survival in CN-AML. We further show that cell growth and function is dependent on maintained levels of these core genes and that direct targeting of HOXA/PBX3 sensitizes CN-AML cells to standard chemotherapy. Together the data support a key role for HOXA/TALE in CN-AML and demonstrate that targeting of clinically significant HOXA/PBX3 elements may provide therapeutic benefit to these patients.

Entinostat prevents leukemia maintenance in a collaborating oncogene-dependent model of CN-AML.

The incidence of refractory acute myeloid leukemia (AML) is on the increase due in part to an aging population that fails to respond to traditional therapies. High throughput genomic analysis promises better diagnosis, prognosis and therapeutic intervention based on improved patient stratification. Relevant pre-clinical models are urgently required to advance drug development in this area. The collaborating oncogenes, HOXA9 and MEIS1, are frequently co-overexpressed in cytogenetically normal AML (CN-AML) and a conditional transplantation mouse model was developed that demonstrated oncogene-dependency and expression levels comparable to CN-AML patients. Integration of gene signatures obtained from the mouse model and a cohort of CN-AML patients using statistically significant connectivity Map (sscMap) analysis identified Entinostat as a drug with the potential to alter the leukemic condition towards the normal state. Ex vivo treatment of leukemic cells, but not age-matched normal bone marrow controls, with Entinostat validated the gene signature and resulted in reduced viability in liquid culture, impaired colony formation and loss of the leukemia initiating cell. Furthermore, in vivo treatment with Entinostat resulted in prolonged survival of leukemic mice. This study demonstrates that the HDAC inhibitor Entinostat inhibits disease maintenance and prolongs survival in a clinically relevant murine model of cytogenetically normal AML. © 2013 AlphaMed Press

Combination of CFD and DOE to analyse solid particle erosion in elbows

Solid particle erosion is a major concern in the engineering industry, particularly where transport of slurry flow is involved. Such flow regimes are characteristic of those in alumina refinement plants. The entrainment of particulate matter, for example sand, in the Bayer liquor can cause severe erosion in pipe fittings, especially in those which redirect the flow. The considerable costs involved in the maintenance and replacement of these eroded components led to an interest in research into erosion prediction by numerical methods at Rusal Aughinish alumina refinery, Limerick, Ireland, and the University of Limerick. The first stage of this study focused on the use of computational fluid dynamics (CFD) to simulate solid particle erosion in elbows. Subsequently an analysis of the factors that affect erosion of elbows was performed using design of experiments (DOE) techniques. Combining CFD with DOE harnesses the computational power of CFD in the most efficient manner for prediction of elbow erosion. An analysis of the factors that affect the erosion of elbows was undertaken with the intention of producing an erosion prediction model. © 2009 Taylor & Francis.

A light microscopic and autoradiographic study of non-irradiated and irradiated ocular wounds

Focal gamma irradiation was used to limit the intraocular extension of scar tissue which typically occurs after posterior perforating injury to the eye. Standard posterior perforating injuries were created in the right eye of forty-eight rabbits, half of which had the site of perforation focally irradiated using a Cobalt 60 ophthalmic plaque. Non-irradiated wounds healed with profuse formation of highly cellular and vascularised granulation tissue which invaded the vitreous to form contractile vitreo-retinal membranes. In irradiated eyes vitreo-retinal membrane formation was infrequent; the wounds showing only sparse granulation tissue with little or no extension into the vitreous cavity. Autoradiographic studies carried out in a second group of 40 animals showed that the episclera was the main source of the proliferating fibroblasts, and cell counts confirmed that the inflammatory and repair responses in irradiated wounds were both delayed and attenuated.

Keratinocyte Growth-Factor Promotes Epithelial Survival and Resolution in a Human Model of Lung Injury

Rationale: Increasing epithelial repair and regeneration may hasten resolution of lung injury in patients with the Acute Respiratory Distress Syndrome (ARDS). In animal models of ARDS, Keratinocyte Growth Factor (KGF) reduces injury and increases epithelial proliferation and repair. The effect of KGF in the human alveolus is unknown.

Objectives: To test whether KGF can attenuate alveolar injury in a human model of ARDS.

Methods: Volunteers were randomized to intravenous KGF (60 μg/kg) or placebo for 3 days, before inhaling 50μg lipopolysaccharide. Six hours later, subjects underwent bronchoalveolar lavage (BAL) to quantify markers of alveolar inflammation and cell-specific injury.

Measurements and Main Results: KGF did not alter leukocyte infiltration or markers of permeability in response to LPS. KGF increased BAL concentrations of Surfactant Protein D (SP-D), MMP-9, IL-1Ra, GM-CSF and CRP. In vitro, BAL fluid from KGF-treated subjects (KGF BAL) inhibited pulmonary fibroblast proliferation, but increased alveolar epithelial proliferation. Active MMP-9 increased alveolar epithelial wound repair. Finally, BAL from the KGF pre-treated group enhanced macrophage phagocytic uptake of apoptotic epithelial cells and bacteria compared with BAL from the placebo-treated group. This effect was blocked by inhibiting activation of the GM-CSF receptor.

Conclusions: KGF treatment increases BAL SP-D, a marker of type II alveolar epithelial cell proliferation in a human model of ALI. Additionally KGF increases alveolar concentrations of the anti-inflammatory cytokine IL-1Ra, and mediators that drive epithelial repair (MMP-9) and enhance macrophage clearance of dead cells and bacteria (GM-CSF).

English Language Education at University: Trends and Challenges in Teaching and Learning Academic Discourse in the UK

This chapter discusses English Language Education at university and highlights a number of trends and their associated challenges in teaching and learning academic discourse. Academic discourse refers to the ways in which language is used by participants in academia. It encompasses written discourse, from article and book publishing, PhD theses to course assignments; spoken discourse, from study groups, tutorials, conference presentations to inaugural lectures; and more recently, computer-mediated discourse, from asynchronous text-based conferencing to academic blogs. The role of English language educators in preparing students and academics for successful participation in these academic events, or the academy, in English is not to be underestimated. Academic communication is not only vital to an individual’s success at university, but to the maintenance and creation of academic communities and to scientific progress itself (Hyland, 2009). This chapter presents an overview of academic discourse and discusses recent issues which have an impact on teaching and learning English at university and discusses their associated challenges: first, the increasing internationalisation of universities. Second, the emergence of a mobile academe in its broadest sense, in which students and academics move across traditional geopolitical, institutional and disciplinary boundaries, is discussed. Third, the growth of UK transnational higher education is examined as a trend which sees academics and students vicariously or otherwise involved in English language teaching and learning. Fourth, the chapter delves into the rapid and ongoing development in technology assisted and online learning. While responding to trends can be difficult, they can also inspire ingenuity. Furthermore, such trends and challenges will not emerge in the same manner in different contexts. The discussion in this chapter is illustrated with examples from a UK context but the implications of the trends and challenges are such that they reach beyond borders.

Extreme learning machines for virtual metrology and etch rate prediction

Virtual metrology (VM) aims to predict metrology values using sensor data from production equipment and physical metrology values of preceding samples. VM is a promising technology for the semiconductor manufacturing industry as it can reduce the frequency of in-line metrology operations and provide supportive information for other operations such as fault detection, predictive maintenance and run-to-run control. Methods with minimal user intervention are required to perform VM in a real-time industrial process. In this paper we propose extreme learning machines (ELM) as a competitive alternative to popular methods like lasso and ridge regression for developing VM models. In addition, we propose a new way to choose the hidden layer weights of ELMs that leads to an improvement in its prediction performance.

Design of a Virtual Reality Framework for Maintainability and assemblability test of complex systems

This paper presents a unique environment whose features are able to satisfy requirements for both virtual maintenance and virtual manufacturing through the conception of original virtual reality (VR) architecture. Virtual Reality for the Maintainability and Assemblability Tests (VR_MATE) encompasses VR hardware and software and a simulation manager which allows customisation of the architecture itself as well as interfacing with a wide range of devices employed in the simulations. Two case studies are presented to illustrate VR_MATE's unique ability to allow for both maintainability tests and assembly analysis of an aircraft carriage and a railway coach cooling system respectively. The key impact of this research is the demonstration of the potentialities of using VR techniques in industry and its multiple applications despite the subjective character within the simulation. VR_MATE has been presented as a framework to support the strategic and operative objectives of companies to reduce product development time and costs whilst maintaining product quality for applications which would be too expensive to simulate and evaluate in the real world.

Uncovering Dependence Clusters and Linchpin Functions

Dependence clusters are (maximal) collections of mutually dependent source code entities according to some dependence relation. Their presence in software complicates many maintenance activities including testing, refactoring, and feature extraction. Despite several studies finding them common in production code, their formation, identification, and overall structure are not well understood, partly because of challenges in approximating true dependences between program entities. Previous research has considered two approximate dependence relations: a fine-grained statement-level relation using control and data dependences from a program’s System Dependence Graph and a coarser relation based on function-level controlflow reachability. In principal, the first is more expensive and more precise than the second. Using a collection of twenty programs, we present an empirical investigation of the clusters identified by these two approaches. In support of the analysis, we consider hybrid cluster types that works at the coarser function-level but is based on the higher-precision statement-level dependences. The three types of clusters are compared based on their slice sets using two clustering metrics. We also perform extensive analysis of the programs to identify linchpin functions – functions primarily responsible for holding a cluster together. Results include evidence that the less expensive, coarser approaches can often be used as e�ective proxies for the more expensive, finer-grained approaches. Finally, the linchpin analysis shows that linchpin functions can be e�ectively and automatically identified.