CytA enables CryIV endotoxins of Bacillus thuringiensis to overcome high levels of CryIV resistance in the mosquito, Culex quinquefasciatus

Cry proteins produced by Bacillus thuringiensis are selective biodegradable insecticides used increasingly in bacterial insecticides and transgenic plants as alternatives to synthetic chemical insecticides. However, the potential for development of resistance and cross-resistance in target insect populations to Cry proteins used alone or in combination threatens the more widespread use of this novel pest control technology. Here we show that high levels of resistance to CryIV proteins in larvae of the mosquito, Culex quinquefasciatus, can be suppressed or reduced markedly by combining these proteins with sublethal quantities of CytA, a cytolytic endotoxin of B. thuringiensis. Resistance at the LC95 level of 127-fold for a combination of three CryIV toxins (CryIVA, B, and D), resulting from 60 generations of continuous selection, was completely suppressed by combining sporulated powders of CytA in a 1:3 ratio with sporulated powders of a CryIVA, CryIVB, and CryIVD strain. Combining the CytA strain with a CryIVA and CryIVB strain also completely suppressed mosquito resistance of 217-fold to the latter toxins at the LC95 level, whereas combination of CytA with CryIVD reduced resistance in a CryIVD-selected mosquito strain from greater than 1,000-fold to less than 8-fold. The CytA/CryIV model provides a potential molecular genetic strategy for engineering resistance management for Cry proteins directly into bacterial insecticides and transgenic plants.

Enhanced resistance in STAT6-deficient mice to infection with ectromelia virus

We inoculated BALB/c mice deficient in STAT6 (STAT6−/−) and their wild-type (wt) littermates (STAT6+/+) with the natural mouse pathogen, ectromelia virus (EV). STAT6−/− mice exhibited increased resistance to generalized infection with EV when compared with STAT6+/+ mice. In the spleens and lymph nodes of STAT6−/− mice, T helper 1 (Th1) cytokines were induced at earlier time points and at higher levels postinfection when compared with those in STAT6+/+ mice. Elevated levels of NO were evident in plasma and splenocyte cultures of EV-infected STAT6−/− mice in comparison with STAT6+/+ mice. The induction of high levels of Th1 cytokines in the mutant mice correlated with a strong natural killer cell response. We demonstrate in genetically susceptible BALB/c mice that the STAT6 locus is critical for progression of EV infection. Furthermore, in the absence of this transcription factor, the immune system defaults toward a protective Th1-like response, conferring pronounced resistance to EV infection and disease progression.

Naturally processed peptides from two disease-resistance-associated HLA-DR13 alleles show related sequence motifs and the effects of the dimorphism at position 86 of the HLA-DR beta chain.

HLA-DR13 has been associated with resistance to two major infectious diseases of humans. To investigate the peptide binding specificity of two HLA-DR13 molecules and the effects of the Gly/Val dimorphism at position 86 of the HLA-DR beta chain on natural peptide ligands, these peptides were acid-eluted from immunoaffinity-purified HLA-DRB1*1301 and -DRB1*1302, molecules that differ only at this position. The eluted peptides were subjected to pool sequencing or individual peptide sequencing by tandem MS or Edman microsequencing. Sequences were obtained for 23 peptides from nine source proteins. Three pool sequences for each allele and the sequences of individual peptides were used to define binding motifs for each allele. Binding specificities varied only at the primary hydrophobic anchor residue, the differences being a preference for the aromatic amino acids Tyr and Phe in DRB1*1302 and a preference for Val in DRB1*1301. Synthetic analogues of the eluted peptides showed allele specificity in their binding to purified HLA-DR, and Ala-substituted peptides were used to identify the primary anchor residues for binding. The failure of some peptides eluted from DRB1*1302 (those that use aromatic amino acids as primary anchors) to bind to DRB1*1301 confirmed the different preferences for peptide anchor residues conferred by the Gly-->Val change at position 86. These data suggest a molecular basis for the differential associations of HLA-DRB1*1301 and DRB1*1302 with resistance to severe malaria and clearance of hepatitis B virus infection.

To Promote the Progress: Why Do States Create New Intellectual Property Rights? ACES Working Papers, August 2012

The question I seek to answer is not about explaining support or resistance to the globalization or harmonization of IP rights. Rather, I am interested in new IP rights: what happened to cause a certain intangible to change from something that was not considered intellectual property to something that was considered intellectual property? Explaining the globalization or harmonization of already-existing IP rights is an important task, but fundamentally different than explaining why some things became intellectual property in the first place.

The impact of organizational culture and reshaping capabilities on change implementation success: The mediating role of readiness for change

It was hypothesized that employees' perceptions of an organizational culture strong in human relations values and open systems values would be associated with heightened levels of readiness for change which, in turn, would be predictive of change implementation success. Similarly, it was predicted that reshaping capabilities would lead to change implementation success, via its effects on employees' perceptions of readiness for change. Using a temporal research design, these propositions were tested for 67 employees working in a state government department who were about to undergo the implementation of a new end-user computing system in their workplace. Change implementation success was operationalized as user satisfaction and system usage. There was evidence to suggest that employees who perceived strong human relations values in their division at Time 1 reported higher levels of readiness for change at pre-implementation which, in turn, predicted system usage at Time 2. In addition, readiness for change mediated the relationship between reshaping capabilities and system usage. Analyses also revealed that pre-implementation levels of readiness for change exerted a positive main effect on employees' satisfaction with the system's accuracy, user friendliness, and formatting functions at post-implementation. These findings are discussed in terms of their theoretical contribution to the readiness for change literature, and in relation to the practical importance of developing positive change attitudes among employees if change initiatives are to be successful.

Repressor- and activator-type ethylene response factors functioning in jasmonate signaling and disease resistance identified via a genome-wide screen of Arabidopsis transcription factor gene expression

To identify transcription factors (TFs) involved in jasmonate (JA) signaling and plant defense, we screened 1,534 Arabidopsis (Arabidopsis thaliana) TFs by real-time quantitative reverse transcription-PCR for their altered transcript at 6 h following either methyl JA treatment or inoculation with the incompatible pathogen Alternaria brassicicola. We identified 134 TFs that showed a significant change in expression, including many APETALA2/ethylene response factor (AP2/ERF), MYB, WRKY, and NACTF genes with unknown functions. Twenty TF genes were induced by both the pathogen and methyl JA and these included 10 members of the AP2/ERF TF family, primarily from the B1a and B3 subclusters. Functional analysis of the B1a TF AtERF4 revealed that AtERF4 acts as a novel negative regulator of JA-responsive defense gene expression and resistance to the necrotrophic fungal pathogen Fusarium oxysporum and antagonizes JA inhibition of root elongation. In contrast, functional analysis of the B3 TF AtERF2 showed that AtERF2 is a positive regulator of JA-responsive defense genes and resistance to F. oxysporum and enhances JA inhibition of root elongation. Our results suggest that plants coordinately express multiple repressor-and activator-type AP2/ERFs during pathogen challenge to modulate defense gene expression and disease resistance.

Altered fungal sensitivity to a plant antimicrobial peptide through over-expression of yeast cDNAs

A yeast cDNA expression library was screened to identify genes and cellular processes that influence fungal sensitivity to a plant antimicrobial peptide. A plasmid-based, GAL1 promoter-driven yeast cDNA expression library was introduced into a yeast genotype susceptible to the antimicrobial peptide MiAMP1 purified from Macadamia integrifolia. Following a screen of 20,000 cDNAs, three yeast cDNAs were identified that reproducibly provided transformants with galactose-dependent resistance to MiAMP1. These cDNAs encoded a protein of unknown function, a component (VMA11) of the vacuolar H+-ATPase and a component (cytochrome c oxidase subunit VIa) of the mitochondrial electron transport chain, respectively. To identify genes that increased sensitivity to MiAMP1, the yeast cDNA expression library was introduced into a yeast mutant with increased resistance to MiAMP1. From 11,000 cDNAs screened, two cDNA clones corresponding to a ser/thr kinase and a ser/thr phosphatase reproducibly increased MiAMP1 susceptibility in the mutant in a galactose-dependent manner. Deletion mutants were available for three of the five genes identified but showed no change in their sensitivity to MiAMP1, indicating that these genes could not be detected by screening of yeast deletion mutant libraries. Yeast cDNA expression library screening therefore provides an alternative approach to gene deletion libraries to identify genes that can influence the sensitivity of fungi to plant antimicrobial peptides.

Technological and organisational change in small- to medium-sized manufacturing companies:a learning organisation perspective

This paper examines the extent to which a learning organisation perspective is attainable in small- to medium-sized manufacturing companies. An audit tool is developed from the literature on organisational learning and recognised processes that lead towards becoming a learning organisation. The paper focuses on the application of the audit tool in three UK automotive component suppliers which are all experiencing pressures for change imposed by the major vehicle manufacturers. The main changes are concerned with tiering of the supply chain and substantial delegation of responsibilities to component suppliers including an increasing emphasis on innovation and continuous improvement. The companies presented in the paper are taken from a research project into the impact of changes in supply chain relationships on the operation of small- and medium-sized manufacturing firms in the West Midlands region of the UK. The ways in which the companies are responding to change are presented together with the results of a self-assessment using the developed audit tool. These results suggest that companies of this type tend to focus on change in those areas that involve least challenge to the established power and authority of management.

Sensitivity of staphylococcus epidermidis to chlorhexidine and associated resistance properties

Staphylococcus epidermidis are common Gram-positive bacteria and are responsible for a number of life-threatening nosocomial infections. Treatment of S. epidermidis infection is problematic because the organism is usually resistant to many antibiotics. The high degree of resistance of this organism to a range of antibiotics and disinfectants is widely known. The aims of this thesis were to investigate and evaluate the susceptibility of isolates of S. epidermidis from various infections to chlorhexidine (CHX) and to other disinfectants such as benzalkonium chloride (BKC), triclosan (TLN) and povidone-iodine (PI). In addition, the mechanisms of resistance of S. epidermidis to chlorhexidine (the original isolates and strains adapted to chlorhexidine by serial passage) were examined and co-resistance to clinically relevant antibiotics investigated. In 3 of the 11 S. epidermidis strains passaged in increasing concentrations of chlorhexidine, resistance to the disinfectant arose (16-fold). These strains were examined further, each showing stable chlorhexidine resistance. Co-resistance to other disinfectants such as BKC, TLN and PI and changes in cell surface hydrophobicity were observed. Increases in resistance were accompanied by an increase in the proportion of neutral lipids and phospholipids in the cell membrane. This increase was most marked in diphosphatidylglycerol. These observations suggest that some strains of S. epidermidis can become resistant to chlorhexidine and related disinfectants/antiseptics by continual exposure. The mechanisms of resistance appear to be related to changes in membrane lipid compositions.

From therapeutic to political education: the centrality of affective sensibility in critical pedagogy

While the need for humanising education is pressing in neoliberal societies, the conditions for its possibility in formal institutions have become particularly cramped. A constellation of factors – the strength of neoliberal ideologies, the corporatisation of universities, the conflation of human freedom with consumer satisfaction, and a wider crisis of hope in the possibility or desirability of social change – make it difficult to apply classical theories of subject-transformation to new work in critical pedagogy. In particular, the growth of interest in pedagogies of comfort (as illustrated in certain forms of ‘therapeutic’ education and concerns about student ‘satisfaction’) and resistance to critical pedagogies suggest that subjectivty has become a primary site of political struggle in education. However, it can no longer be assumed that educators can (or should) liberate students’ repressed desires for ‘humanisation’ by politicising curricula, pedagogy or institutions. Rather, we must work to understand the new meanings and affective conditions of critical subjectivity itself. Bringing critical theories of subject transformation together with new work on ‘pedagogies of discomfort’, I suggest we can create new ways of opening up possibilities for critical education that respond to neoliberal subjectivities without corresponding to or affirming them.

Role of GSH in estrone sulfate binding and translocation by the multidrug resistance protein 1 (MRP1/ABCC1)

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent efflux pump that can confer resistance to multiple anticancer drugs and transport conjugated organic anions. Unusually, transport of several MRP1 substrates requires glutathione (GSH). For example, estrone sulfate transport by MRP1 is stimulated by GSH, vincristine is co-transported with GSH, or GSH can be transported alone. In the present study, radioligand binding assays were developed to investigate the mechanistic details of GSH-stimulated transport of estrone sulfate by MRP1. We have established that estrone sulfate binding to MRP1 requires GSH, or its non-reducing analogue S-methyl GSH (S-mGSH), and further that the affinity (Kd) of MRP1 for estrone sulfate is 2.5-fold higher in the presence of S-mGSH than GSH itself. Association kinetics show that GSH binds to MRP1 first, and we propose that GSH binding induces a conformational change, which makes the estrone sulfate binding site accessible. Binding of non-hydrolyzable ATP analogues to MRP1 decreases the affinity for estrone sulfate. However, GSH (or S-mGSH) is still required for estrone sulfate binding, and the affinity for GSH is unchanged. Estrone sulfate affinity remains low following hydrolysis of ATP. The affinity for GSH also appears to decrease in the post-hydrolytic state. Our results indicate ATP binding is sufficient for reconfiguration of the estrone sulfate binding site to lower affinity and argue for the presence of a modulatory GSH binding site not associated with transport of this tripeptide. A model for the mechanism of GSH-stimulated estrone sulfate transport is proposed.

Low level of cross-resistance between triclosan and antibiotics in Escherichia coli K-12 and E. coli O55 compared to E-coli O157

Misuse of biocides has encouraged the emergence of resistance and cross-resistance in certain strains. This study investigated resistance of triclosan-adapted Escherichia coli K-12 and E. coli O55 to antimicrobial agents and compared these to E. coli O157:H7. Cross-resistance in E. coli K-12 and E. coli O55 was observed however to a lesser extent than in E. coli O157:H7. Triclosan-adapted E. coli K-12 demonstrated cross-resistance to chloramphenicol, whereas triclosan-adapted E. coli O55 exhibited resistance to trimethoprim. In comparison, E. coli O157:H7 was resistant to chloramphenicol, tetracycline, amoxicillin, amoxicillin/clavulanic acid, trimethoprim, benzalkonium chloride and chlorohexidine suggesting strain specific rather than general resistance mechanisms. © 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Lys9 for Glu9 substitution in glucagon-like peptide-1(7-36)amide confers dipeptidylpeptidase IV resistance with cellular and metabolic actions similar to those of established antagonists glucagon-like peptide-1(9-36)amide and exendin (9-39)

The incretin hormone glucagon-like peptide-1(7-36)amide (GLP-1) has been deemed of considerable importance in the regulation of blood glucose. Its effects, mediated through the regulation of insulin, glucagon, and somatostatin, are glucose-dependent and contribute to the tight control of glucose levels. Much enthusiasm has been assigned to a possible role of GLP-1 in the treatment of type 2 diabetes. GLIP-l's action unfortunately is limited through enzymatic inactivation caused by dipeptidylpeptidase IV (DPP IV). It is now well established that modifying GLP-1 at the N-terminal amino acids, His7 and Ala8, can greatly improve resistance to this enzyme. Little research has assessed what effect Glu9-substitution has on GLP-1 activity and its degradation by DPP IV. Here, we report that the replacement of Glu9 of GLP-1 with Lys dramatically increased resistance to DPP IV. This analogue (Lys9)GLP-1, exhibited a preserved GLP-1 receptor affinity, but the usual stimulatory effects of GLP-1 were completely eliminated, a trait duplicated by the other established GLP-1-antagonists, exendin (9-39) and GLP-1 (9-36)amide. We investigated the in vivo antagonistic actions of (Lys9)GLP-1 in comparison with GLP-1(9-36)amide and exendin (9-39) and revealed that this novel analogue may serve as a functional antagonist of the GLP-1 receptor.

An examination of attitude toward change as a predictor of policy/program implementation

Policy/program implementation, e.g., the process of fulfilling policy/program directives, is fundamentally tied to change. Implementation studies have examined the process, identifying many critical organizational variables although individuals perform the activities.^ Many of the studies are predicated on the rational, goal oriented model of organizations and examine implementation, presenting only the goal-oriented view. Organizational change and its resistance, however, are not fully explained by the rational model of organizations. There are other schools of thought providing different views of organizations from which explanation may emerge. Bolman and Deal (1984, 1991a, 1994) provide a different perspective for examining organizations Bolman and Deal argue organizations should be viewed through four different frames or lenses. Framing and reframing organizational action captures the complexity of action and provides better understanding of organizational processes. Understanding of implementation of policies/programs also will benefit from the use of the four-frame approach.^ The goal of this research is to provide a better understanding of the implementation process by examining individual attitudes toward change, the dependent variable of this research, and studying the relationship between the dependent variable and frame. The research was conducted in two phases. In Phase One, a survey was sent to 306 school administrators and teachers in magnet programs in Dade County, Florida. The survey instrument was composed of 55 questions including six from Bolman and Deal's Leadership Orientation Survey (1988) and 38 questions about organizational change. In Phase Two, more in-depth analysis of four school was conducted, to further explore the relationship between frame and attitude toward change.^ The results revealed that frame was a factor in explaining differences in personal Attitude Toward Change and Comfort Level with Change. Individuals using the symbolic frame had more positive attitudes toward change and were also more comfortable with change. The results of Phase Two of the research partially supported this finding in that the most fully implemented program was the product of an administrator who had chosen the symbolic frame. ^

"To hold the world in contempt": The British Empire, war, and the Irish and Indian nationalist press, 1899–1914

The era between the close of the nineteenth century and the onset of the First World War witnessed a marked increase in radical agitation among Indian and Irish nationalists. The most outspoken political leaders of the day founded a series of widely circulated newspapers in India and Ireland, placing these editors in the enviable position of both reporting and creating the news. Nationalist journalists were in the vanguard of those pressing vocally for an independent India and Ireland, and together constituted an increasingly problematic contingent for the British Empire. The advanced-nationalist press in Ireland and the nationalist press in India took the lead in facilitating the exchange of provocative ideas--raising awareness of perceived imperial injustices, offering strategic advice, and cementing international solidarity. Irish and Indian press coverage of Britain's imperial wars constituted one of the premier weapons in the nationalists' arsenal, permitting them to build support for their ideology and forward their agenda in a manner both rapid and definitive. Directing their readers' attention to conflicts overseas proved instructive in how the Empire dealt with those who resisted its policies, and also showcased how it conducted its affairs with its allies. As such, critical press coverage of the Boxer Rebellion, Boer War, Russo-Japanese War, and World War I bred disaffection for the Empire, while attempts by the Empire to suppress the critiques further alienated the public. This dissertation offers the first comparative analysis of the major nationalist press organs in India and Ireland, using the prism of war to illustrate the increasingly persuasive role of the press in promoting resistance to the Empire. It focuses on how the leading Indian and Irish editors not only fostered a nationalist agenda within their own countries, but also worked in concert to construct a global anti-imperialist platform. By highlighting the anti-imperial rhetoric of the nationalist press in India and Ireland and illuminating their strategies for attaining self-government, this study deepens understanding of the seeds of nationalism, making a contribution to comparative imperial scholarship, and demonstrating the power of the media to alter imperial dynamics and effect political change.