Evaluation of Expanded 2-Aminobenzothiazole Library for Inhibition of Pseudomonas aeruginosa Virulence Phenotypes.

Bacterial resistance to antibiotics is a rapidly increasing threat to human health. New strategies to combat resistant organisms are desperately needed. One potential avenue is targeting two-component systems, which are the main bacterial signal transduction pathways used to regulate development, metabolism, virulence, and antibiotic resistance. These systems consist of a homodimeric membrane-bound sensor histidine kinase, and a cognate effector, the response regulator. The high sequence conservation in the catalytic and adenosine triphosphate-binding (CA) domain of histidine kinases and their essential role in bacterial signal transduction could enable broad-spectrum antibacterial activity. Through this signal transduction, histidine kinases regulate multiple virulence mechanisms including toxin production, immune evasion, and antibiotic resistance. Targeting virulence, as opposed to development of bactericidal compounds, could reduce evolutionary pressure for acquired resistance. Additionally, compounds targeting the CA domain have the potential to impair multiple two-component systems that regulate virulence in one or more pathogens. We conducted structure-activity relationship studies of 2-aminobenzothiazole-based inhibitors designed to target the CA domain of histidine kinases. We found these compounds have anti-virulence activities in Pseudomonas aeruginosa, reducing motility phenotypes and toxin production associated with the pathogenic functions of this bacterium.

Interactive Tools for Measuring Visual Scanning Performance and Reaction Time.

Occupational therapists are constantly searching for engaging, high-technology interactive tasks that provide immediate feedback to evaluate and train clients with visual scanning deficits. This study examined the relationship between two tools: the VISION COACH™ interactive light board and the Functional Object Detection© (FOD) Advanced driving simulator scenario. Fifty-four healthy drivers, ages 21-66 yr, were divided into three age groups. Participants performed braking response and visual target (E) detection tasks of the FOD Advanced driving scenario, followed by two sets of three trials using the VISION COACH Full Field 60 task. Results showed no significant effect of age on FOD Advanced performance but a significant effect of age on VISION COACH performance. Correlations showed that participants' performance on both braking and E detection tasks were significantly positively correlated with performance on the VISION COACH (.37 < r < .40, p < .01). These tools provide new options for therapists.

The reliability of a VISION COACH task as a measure of psychomotor skills.

The VISION COACH™ interactive light board is designed to test and enhance participants' psychomotor skills. The primary goal of this study was to examine the test-retest reliability of the Full Field 120 VISION COACH task. One hundred eleven male and 131 female adult participants completed six trials where they responded to 120 randomly distributed lights displayed on the VISION COACH interactive light board. The mean time required for a participant to complete a trial was 101 seconds. Intraclass correlation coefficients, ranging from 0.962 to 0.987 suggest the VISION COACH Full Field 120 task was a reliable task. Cohen's d's of adjacent pairs of trials suggest learning effects did not negatively affect reliability after the third trial.

Understanding the needs of hand control users, driver rehabilitation specialists, and dealers/installers.

Understanding unique perspectives from key stakeholder groups involved in the hand control (HC) industry, including driver rehabilitation specialists (DRSs) who train users how to use their HCs, dealers/installers, and users, may become increasingly important in the United States due to increases in elderly, diabetic, and wounded warrior amputee driving populations. In this exploratory study, phone interviews were conducted with 20 DRSs, 20 dealers/installers, and 20 users regarding their perspectives about HC training, maintenance and operation, and design improvements. Results revealed common views and differences in perspectives about whether HC users should receive training and for how long, when and how often users should receive maintenance on their HCs, and what DRSs, dealers/installers, and users would like to see in the future.

L'appropriation par les primo-adoptants de l' Approche École en santé au Quebec.

L' Approche École en santé (AÉS) est une approche globale de promotion de la santé inspirée des principes des « Health Promoting Schools ¼ (écoles promotrices de santé). L'AÉS est associée à un dispositif d'accompagnement des écoles volontaires pour l'implanter qui comporte des ressources professionnelles d'accompagnement et divers outils. Le présent article rend compte des résultats d'une recherche qualitative portant sur l'appropriation locale de cette approche par des écoles ayant participé à la première vague d'implantation au Québec. Après avoir exposé le contexte, la méthodologie et le cadre théorique de l'étude, nous présenterons les appropriations locales observées, et ce qui semble les avoir conditionnées, du point de vue des acteurs.

Schools' absorptive capacity to innovate in health promotion.

PURPOSE: A comprehensive "health promoting schools" (HPS) approach is advocated by the World Health Organization to foster the health of students. To date, few studies have evaluated schools' capacity to implement it in an optimal way. The purpose of this paper is to present a conceptual framework that identifies core features likely to facilitate the incorporation of innovation, such as HPS, into school functioning. DESIGN/METHODOLOGY/APPROACH: The framework was built by combining dimensions derived from two major strands of literature, i.e. management and HPS. It has taken root in Zahra and George's model of organisation absorptive capacity (AC) for new knowledge but has been adapted to better explore AC in a school context. The contrasting cases of two secondary schools that adopted a HPS approach in Quebec, Canada, for at least three years were used to illustrate the value of the framework. FINDINGS: The framework proposed is a multidimensional model that considers components such as modulators, antecedents, integration mechanisms and strategic levers as potential determinants of AC, i.e. acquisition, assimilation, transformation and exploitation. The conceptual framework helped to qualify and compare AC regarding HPS in the two cases and holds promise to appreciate mechanisms having the greatest influence on it. ORIGINALITY/VALUE: The framework can serve as a conceptual guide to facilitate the absorption of innovation in schools and to design future empirical research to better understand the underlying process by which schools strengthen their capacities to become settings conducive to the health of youth.

Molecular basis for the substrate selectivity of bicyclic and monocyclic extradiol dioxygenases.

Extradiol dioxygenases play a key role in determining the specificities of the microbial aromatic catabolic pathways in which they occur. To identify the structural determinants of specificity in this class of enzymes, variants of 2,3-dihydroxybiphenyl (DHB) 1,2-dioxygenase (DHBD) were investigated. Structural data of the DHBD/DHB complex informed the design of seven variants at four positions: V148W, V148L, M175W, A200I, A200W, P280W, and V148L/A200I. All variants had reduced specificity for DHB. In addition, the V148W, V148L, A200I, and V148L/A200I variants had increased specificity for catechol. Indeed, the V148W variant had a higher apparent specificity for 3-Me catechol than for DHB, although the substitution reduced the kcat for all tested substrates as well as the rate constant of suicide inactivation of the enzyme. These results are consistent with available structural data which suggest that the larger residue at position 148 may partially occlude O2 binding. The results further indicate that in addition to defining substrate specificity, the binding pocket orientates the bound catechol to minimize oxidative inactivation of the enzyme during catalysis.

Characterization of extradiol dioxygenases from a polychlorinated biphenyl-degrading strain that possess higher specificities for chlorinated metabolites.

Recent studies demonstrated that 2,3-dihydroxybiphenyl 1,2-dioxygenase from Burkholderia sp. strain LB400 (DHBDLB400; EC 1.13.11.39) cleaves chlorinated 2,3-dihydroxybiphenyls (DHBs) less specifically than unchlorinated DHB and is competitively inhibited by 2',6'-dichloro-2,3-dihydroxybiphenyl (2',6'-diCl DHB). To determine whether these are general characteristics of DHBDs, we characterized DHBDP6-I and DHBDP6-III, two evolutionarily divergent isozymes from Rhodococcus globerulus strain P6, another good polychlorinated biphenyl (PCB) degrader. In contrast to DHBDLB400, both rhodococcal enzymes had higher specificities for some chlorinated DHBs in air-saturated buffer. Thus, DHBDP6-I cleaved the DHBs in the following order of specificity: 6-Cl DHB > 3'-Cl DHB approximately DHB approximately 4'-Cl DHB > 2'-Cl DHB > 4-Cl DHB > 5-Cl DHB. It also cleaved its preferred substrate, 6-Cl DHB, three times more specifically than DHB. Interestingly, some of the worst substrates for DHBDP6-I were among the best for DHBDP6-III (4-Cl DHB > 5-Cl DHB approximately 6-Cl DHB approximately 3'-Cl DHB > DHB > 2'-Cl DHB approximately 4'-Cl DHB; DHBDP6-III cleaved 4-Cl DHB two times more specifically than DHB). Generally, each of the monochlorinated DHBs inactivated the enzymes more rapidly than DHB. The exceptions were 4-Cl DHB for DHBDP6-I and 2'-Cl DHB for DHBDP6-III. As observed in DHBDLB400, chloro substituents influenced the reactivity of the dioxygenases with O2. For example, the apparent specificities of DHBDP6-I and DHBDP6-III for O2 in the presence of 2'-Cl DHB were lower than those in the presence of DHB by factors of >60 and 4, respectively. DHBDP6-I and DHBDP6-III shared the relative inability of DHBDLB400 to cleave 2',6'-diCl DHB (apparent catalytic constants of 0.088 +/- 0.004 and 0.069 +/- 0.002 s(-1), respectively). However, these isozymes had remarkably different apparent K(m) values for this compound (0.007 +/- 0.001, 0.14 +/- 0.01, and 3.9 +/- 0.4 micro M for DHBDLB400, DHBDP6-I, and DHBDP6-III, respectively). The markedly different reactivities of DHBDP6-I and DHBDP6-III with chlorinated DHBs undoubtedly contribute to the PCB-degrading activity of R. globerulus P6.

Identification and analysis of a bottleneck in PCB biodegradation.

The microbial degradation of polychlorinated biphenyls (PCBs) provides the potential to destroy these widespread, toxic and persistent environmental pollutants. For example, the four-step upper bph pathway transforms some of the more than 100 different PCBs found in commercial mixtures and is being engineered for more effective PCB degradation. In the critical third step of this pathway, 2,3-dihydroxybiphenyl (DHB) 1,2-dioxygenase (DHBD; EC 1.13.11.39) catalyzes aromatic ring cleavage. Here we demonstrate that ortho-chlorinated PCB metabolites strongly inhibit DHBD, promote its suicide inactivation and interfere with the degradation of other compounds. For example, k(cat)(app) for 2',6'-diCl DHB was reduced by a factor of approximately 7,000 relative to DHB, and it bound with sufficient affinity to competitively inhibit DHB cleavage at nanomolar concentrations. Crystal structures of two complexes of DHBD with ortho-chlorinated metabolites at 1.7 A resolution reveal an explanation for these phenomena, which have important implications for bioremediation strategies.

The mechanism-based inactivation of 2,3-dihydroxybiphenyl 1,2-dioxygenase by catecholic substrates.

2,3-Dihydroxybiphenyl 1,2-dioxygenase (EC ), the extradiol dioxygenase of the biphenyl biodegradation pathway, is subject to inactivation during the steady-state cleavage of catechols. Detailed analysis revealed that this inactivation was similar to the O(2)-dependent inactivation of the enzyme in the absence of catecholic substrate, resulting in oxidation of the active site Fe(II) to Fe(III). Interestingly, the catecholic substrate not only increased the reactivity of the enzyme with O(2) to promote ring cleavage but also increased the rate of O(2)-dependent inactivation. Thus, in air-saturated buffer, the apparent rate constant of inactivation of the free enzyme was (0.7 +/- 0.1) x 10(-3) s(-1) versus (3.7 +/- 0.4) x 10(-3) s(-1) for 2,3-dihydroxybiphenyl, the preferred catecholic substrate of the enzyme, and (501 +/- 19) x 10(-3) s(-1) for 3-chlorocatechol, a potent inactivator of 2,3-dihydroxybiphenyl 1,2-dioxygenase (partition coefficient = 8 +/- 2, K(m)(app) = 4.8 +/- 0.7 microm). The 2,3-dihydroxybiphenyl 1,2-dioxygenase-catalyzed cleavage of 3-chlorocatechol yielded predominantly 2-pyrone-6-carboxylic acid and 2-hydroxymuconic acid, consistent with the transient formation of an acyl chloride. However, the enzyme was not covalently modified by this acyl chloride in vitro or in vivo. The study suggests a general mechanism for the inactivation of extradiol dioxygenases during catalytic turnover involving the dissociation of superoxide from the enzyme-catecholic-dioxygen ternary complex and is consistent with the catalytic mechanism.