Large is fast, small is tight: determinants of speed and affinity in subunit capture by a periplasmic chaperone

The chaperone/usher pathway assembles surface virulence organelles of Gram-negative bacteria, consisting of fibers of linearly polymerized protein subunits. Fiber subunits are connected through 'donor strand complementation': each subunit completes the immunoglobulin (Ig)-like fold of the neighboring subunit by donating the seventh β-strand in trans. Whereas the folding of Ig domains is a fast first-order process, folding of Ig modules into the fiber conformation is a slow second-order process. Periplasmic chaperones separate this process in two parts by forming transient complexes with subunits. Interactions between chaperones and subunits are also based on the principle of donor strand complementation. In this study, we have performed mutagenesis of the binding motifs of the Caf1M chaperone and Caf1 capsular subunit from Yersinia pestis and analyzed the effect of the mutations on the structure, stability, and kinetics of Caf1M-Caf1 and Caf1-Caf1 interactions. The results suggest that a large hydrophobic effect combined with extensive main-chain hydrogen bonding enables Caf1M to rapidly bind an early folding intermediate of Caf1 and direct its partial folding. The switch from the Caf1M-Caf1 contact to the less hydrophobic, but considerably tighter and less dynamic Caf1-Caf1 contact occurs via the zip-out-zip-in donor strand exchange pathway with pocket 5 acting as the initiation site. Based on these findings, Caf1M was engineered to bind Caf1 faster, tighter, or both faster and tighter. To our knowledge, this is the first successful attempt to rationally design an assembly chaperone with improved chaperone function.

Distribution, gene sequence and expression in vivo of the plasmid encoded fimbrial antigen of Salmonella serotype Enteritidis

The pefA gene which encoded the serotype associated plasmid (SAP) mediated fimbrial major subunit antigen of Salmonella enterica serotype Typhimurium shared genetic identity with 128 of 706 salmonella isolates as demonstrated by dot (colony) hybridization. Seventy-seven of 113 isolates of Typhimurium and individual isolates of serotypes Bovis-morbificans, Cholerae-suis and Enteritidis phage type 9b hybridized pefA strongly, whereas 48 isolates of Enteritidis hybridized pefA weakly and one Enteritidis isolate of phage type 14b failed to hybridize. Individual isolates of 294 serotypes and 247 individual isolates of serotype Dublin did not hybridize pefA. Southern hybridization of plasmids extracted from Enteritidis demonstrated that the pefA gene probe hybridized strongly an atypical SAP of 80 kb in size harboured by one Enteritidis isolate of phage-type 9b, whereas the typical SAP of 58 kb in size harboured by 48 Enteritidis isolates hybridized weakly. One Enteritidis isolate of phage type 14b which failed to hybridize pefA in dot (colony) hybridization experiments was demonstrated to be plasmid free. A cosmid library of Enteritidis phage type 4 expressed in Escherichia coli K12 was screened by hybridization for the presence of pef sequences. Recombinant clones which were deduced to harbour the entire pef operon elaborated a PEF-like fimbrial structure at the cell surface. The PEF-like fimbrial antigen was purified from one cosmid clone and used in western blot experiments with sera from chickens infected with Enteritidis phage-type 4. Seroconversion to the fimbrial antigen was observed which indicated that the Enteritidis PEF-like fimbrial structure was expressed at some stage during infection. Nucleotide sequence analysis demonstrated that the pefA alleles of Typhimurium and Enteritidis phage-type 4 shared 76% DNA nucleotide and 82% deduced amino acid sequence identity.

Striving for inclusive design in the built environment: learning strategy adopted by policy implementers at local authorities in England

The research will explore views on inclusive design policy implementation and learning strategy used in practice by Local Authorities’ planning, building control and policy departments in England. It reports emerging research findings. The research aim was developed from an extensive literature review, and informed by a pilot study with relevant Local Authority departments. The pilot study highlighted gaps within the process of policy implementation, a lack of awareness of the process and flaws in the design guidance policy. This has helped inform the development of a robust research design using both a survey and semi-structured interviews. The questionnaire targeted key employees within Local Authorities designed to establish how employees learn about inclusive design policy and to determine their views on current approaches of inclusive design policy implementation adopted by their Local Authorities. The questionnaire produces 117 responses. Interestingly approximately 9 out of 129 Local Authorities approached claimed that they were unable to participate either because an inclusive design policy was not adopted or they were faced with a high workload and thus unable to take part. An emerging finding is a lack of understanding of inclusive design problems, which may lead to problem with inclusive design policy implementation, and thus adversely affect how the built environment can be experienced. There is a strong indication from the survey respondents indicating that they are most likely to learn about inclusive design from policy guides produced by their Local Authorities and from their colleagues.

The quality of referendum debate: the UK's electoral system referendum in the print media

This paper analyses the quality of debate surrounding the UK’s 2011 electoral reform referendum as represented in the print media. It first considers how debate quality in the context of a referendum campaign may best be conceptualized. It then uses content analysis of media coverage to investigate three aspects of that debate: its quantity; the balance between Yes and No arguments, and the quality of reason-giving. It finds that the quantity of debate was comparable to other recent electoral reform referendums. Coverage was predominantly, but not overwhelmingly, hostile to change. The different indicators of the quality of reason-giving present a mixed picture. The paper concludes by considering how the analysis could be extended through further comparison with other cases.

Where theory meets practice: a critical comparison of research into identifying letters and craft knowledge of type design

This paper introduces scientific research findings and accounts of skilled design judgement to: (i) develop an interdisciplinary account of what affects our identification of letters when reading; (ii) analyse the relationship between the approaches of psychologists and designers to explaining how we identify letters; (iii) propose ways in which collaboration may work to make psychological research more relevant to typographic practice. The topics reviewed are addressed within each discipline and cover the contribution of letters and words to reading; letter features; essential or structural forms; uniformity within font design; and letter spacing. Analysis of the literature identifies possible means of reconciling different perspectives, points out some anomalies in interpretation of findings, and proposes how designers may contribute to research planning and dissemination.

Late Annibale and his workshop: invention, imitation and patronage

Discussion of the organisation of Annibale Carracci's workshop in his later years

Regional performance and characteristics of Indian manufacturing industry

This paper investigates the regional characteristics of Indian manufacturing industry. Its aim is to assess whether geography plays any major role in determining the performance or characteristics of Indian manufacturing firms, and in order to do this, it presents the results of cross-section regressions estimated on the basis of a balanced sample of 1607 firms across the 30 Indian states. The results suggest that firm performance and characteristics are related to many of the expected industrial organization variables. However, there is also evidence of significant region–state influences on both the performance and characteristics of Indian manufacturing industry. As such, the results demonstrate that analyses which focus solely on standard non-spatial industrial organization variables will fail to explain much of the cross-sectional variation in firm performance and characteristics. In particular, while there are no systematic simple centre–periphery variations in the Indian regional economic system, there is evidence to suggest that industrial spatial concentration, regional specialization, and regional market size play a key role in determining the performance and characteristics of Indian manufacturing industry.

Claviceps purpurea expressing polygalacturonases escaping PGIP inhibition fully infects PvPGIP2 wheat transgenic plants but its infection is delayed in wheat transgenic plants with increased level of pectin methyl esterification

Claviceps purpurea is a biotrophic fungal pathogen of grasses causing the ergot disease. The infection process of C. purpurea on rye flowers is accompanied by pectin degradation and polygalacturonase (PG) activity represents a pathogenicity factor. Wheat is also infected by C. purpurea and we tested whether the presence of polygalacturonase inhibiting protein (PGIP) can affect pathogen infection and ergot disease development. Wheat transgenic plants expressing the bean PvPGIP2 did not show a clear reduction of disease symptoms when infected with C. purpurea. To ascertain the possible cause underlying this lack of improved resistance of PvPGIP2 plants, we expressed both polygalacturonases present in the C. purpurea genome, cppg1 and cppg2 in Pichia pastoris. In vitro assays using the heterologous expressed PGs and PvPGIP2 showed that neither PG is inhibited by this inhibitor. To further investigate the role of PG in the C. purpurea/wheat system, we demonstrated that the activity of both PGs of C. purpurea is reduced on highly methyl esterified pectin. Finally, we showed that this reduction in PG activity is relevant in planta, by inoculating with C. purpurea transgenic wheat plants overexpressing a pectin methyl esterase inhibitor (PMEI) and showing a high degree of pectin methyl esterification. We observed reduced disease symptoms in the transgenic line compared with null controls. Together, these results highlight the importance of pectin degradation for ergot disease development in wheat and sustain the notion that inhibition of pectin degradation may represent a possible route to control of ergot in cereals.

The roles and competencies of HR managers in Slovenian multinational companies

Purpose – The HRM literature provides various typologies of the HR managers’ roles in organizations. The purpose of this paper is to examine how the roles and required competencies of HR managers in Slovenian multinational companies change when these companies enter the international arena. Design/methodology/approach – The authors explored the total population of 25 Slovenian multinational companies (MNCs) operating in Serbia. In these companies the authors conducted interviews with 16 expatriates working in branches in Serbia, sent questionnaires to the CEOs, and conducted a survey of 50 HR managers and interviews with 15 of them. The authors used a triangulation approach and analyzed the results by multivariate methods and content analysis. Findings – The authors found that the complexity of HR managers’ roles, and expectations of their competencies, increases with an increasing level of internationalization of companies. Orientation to people and conflict resolution are seen as elementary competencies needed in all stages of internationalization. The key competence is seen to be strategic thinking that, according to CEOs and expatriates, goes hand in hand with cultural sensitivity, openness to change and a comprehensive understanding of the international environment and business processes. Practical implications – These results can potentially be used for assessing the HRM roles and competencies in different stages of company internationalization, especially MNCs operating in the ex-communist states of Europe, and will help HR managers to support expatriates, CEOs and other employees working in branches abroad more efficiently. Originality/value – This study contributes to the review and evaluation of the quite limited research on HR managers’ roles and competencies in MNCs. It focuses on MNCs and outward internationalization in the Central and Eastern European region. It contributes to studies of the HR managers’ roles and competencies and is the first study to establish a set of roles and competencies for HR managers in Slovenian MNCs.

Assessing the benefits of domestic hot fill washing appliances

Washing machine and dishwasher appliance use accounts for approximately 10% of electricity demand in EU households. The majority of this demand is due to the operation of electric heating elements inside appliances. This paper investigates the potential benefits that can be realised by adding a hot fill connection to washing appliances, with respect to carbon emissions, demand side management and renewable energy integration. Initial laboratory testing of new hot and cold fill appliances has resulted in modifications to optimise hot fill intake, and a novel numerical model presents a method of characterising appliance electricity use in different configurations. In order to validate model findings and test the use of new hot fill appliances in situ, a pilot study has recorded appliances’ resource consumption at one-minute resolution in fourteen households. The addition of hot fill reduced the total dishwasher and washing machine electricity consumption by 38% and 67% respectively. Depending on how hot water is supplied to appliances it is estimated that hot fill use results in an annual household carbon saving of up to 147 kgCO2. Further to direct electricity reduction, hot fill appliances can offer a method of time shifting demand away from peak periods without inconveniencing occupants’ lifestyles.