Two stacked heme molecules in the binding pocket of the periplasmic heme-binding protein HmuT from Yersinia pestis

The periplasmic binding protein HmuT from Yersinia pestis (YpHmuT) is a component of the heme uptake locus hmu and delivers bound hemin to the inner-membrane-localized, ATP-binding cassette (ABC) transporter HmuUV for translocation into the cytoplasm. The mechanism of this process, heme transport across the inner membrane of pathogenic bacteria, is currently insufficiently understood at the molecular level. Here we describe the crystal structures of the substrate-free and heme-bound states of YpHmuT, revealing two lobes with a central binding cleft. Superposition of the apo and holo states reveals a minor tilting motion of the lobes surrounding concomitant with heme binding. Unexpectedly, YpHmuT binds two stacked hemes in a central binding cleft that is larger than those of the homologous periplasmic heme-binding proteins ShuT and PhuT, both of which bind only one heme. The hemes bound to YpHmuT are coordinated via a tyrosine side chain that contacts the Fe atom of one heme and a histidine that contacts the Fe atom of the other heme. The coordinating histidine is only conserved in a subset of periplasmic heme binding proteins suggesting that its presence predicts the ability to bind two heme molecules simultaneously. The structural data are supported by spectroscopic binding studies performed in solution, where up to two hemes can bind to YpHmuT. Isothermal titration calorimetry suggests that the two hemes are bound in discrete, sequential steps and with dissociation constants (K_D) of ∼ 0.29  and ∼ 29 nM, which is similar to the affinities observed in other bacterial substrate binding proteins. Our findings suggest that the cognate ABC transporter HmuUV may simultaneously translocate two hemes per reaction cycle.     

Immediate Outcome Indicators in Perioperative Care: A Controlled Intervention Study on Quality Improvement in Hospitals in Tanzania

Introduction

Outcome assessment is the standard for evaluating the quality of health services worldwide. In this study, outcome has been divided into immediate and final outcome. Aim was to compare an intervention hospital with a Continuous Quality Improvement approach to a control group using benchmark assessments of immediate outcome indicators in surgical care. Results were compared to final outcome indicators.

Method

Surgical care quality in six hospitals in Tanzania was assessed from 2006–2011, using the Hospital Performance Assessment Tool. Independent observers assessed structural, process and outcome quality using checklists based on evidence-based guidelines. The number of surgical key procedures over the benchmark of 80% was compared between the intervention hospital and the control group. Results were compared to Case Fatality Rates.

Results

In the intervention hospital, in 2006, two of nine key procedures reached the benchmark, one in 2009, and four in 2011. In the control group, one of nine key procedures reached the benchmark in 2006, one in 2009, and none in 2011. Case Fatality Rate for all in-patients in the intervention hospital was 5.5% (n = 12,530) in 2006, 3.5% (n = 21,114) in 2009 and 4.6% (n = 18,840) in 2011. In the control group it was 3.1% (n = 17,827) in 2006, 4.2% (n = 13,632) in 2009 and 3.8% (n = 17,059) in 2011.

Discussion

Results demonstrated that quality assurance improved performance levels in both groups. After the introduction of Continuous Quality Improvement, performance levels improved further in the intervention hospital while quality in the district hospital did not. Immediate outcome indicators appeared to be a better steering tool for quality improvement compared to final outcome indicators. Immediate outcome indicators revealed a need for improvement in pre- and postoperative care.

Conclusion

Quality assurance programs based on immediate outcome indicators can be effective if embedded in Continuous Quality Improvement. Nevertheless, final outcome indicators cannot be neglected.     

Localised invertebrate grazing moderates the effect of warming on competitive fungal interactions

Outcomes of competitive mycelial interactions determine saprotrophic fungal community composition and are regulated by biotic (e.g. invertebrate grazing) and abiotic (e.g. climate) factors. Selective grazing can moderate the effects of elevated temperature on fungal interactions. In natural systems, however, patchy and aggregative distributions of invertebrates exert unequal grazing pressures on competing fungi. We explored whether restricting grazing to the territory of one fungal competitor affected the potential of Oniscus asellus (Isopoda) to control the outcomes of interactions and mediate responses to elevated temperature. Restricted grazing prevented the dominance of any one fungal species in the majority of interactions and, indirectly, altered the influence of warming. The location of grazer restriction was, however, only important during certain interactions. Selective pressures reflected feeding preferences, but grazer location determined the extent of selective grazing pressure exerted. Aggregation of macro-invertebrate grazers appears important in maintaining multi-species assemblages of wood-decomposer fungi in a changing climate.     

Arterial Klotho Expression and FGF23 Effects on Vascular Calcification and Function

Recent studies support a role for FGF23 and its co-receptor Klotho in cardiovascular pathology, yet the underlying mechanisms remain largely elusive. Herein, we analyzed the expression of Klotho in mouse arteries and generated a novel mouse model harboring a vascular smooth muscle cell specific deletion of Klotho (Sm22-KL^−/−). Arterial Klotho expression was detected at very low levels with quantitative real-time PCR; Klotho protein levels were undetectable by immunohistochemistry and Western blot. There was no difference in arterial Klotho between Sm22-KL^−/− and wild-type mice, as well as no changes in serum markers of mineral metabolism. Intravenous delivery of FGF23 elicited a rise in renal (0.005; p<0.01) but not arterial Egr-1 expression, a marker of Klotho-dependent FGF23 signaling. Further, the impact of FGF23 on vascular calcification and endothelial response was evaluated in bovine vascular smooth muscle cells (bVSMC) and in a murine ex vivo model of endothelial function, respectively. FGF23 treatment (0.125–2 ng/mL) did not modify calcification in bVSMCs or dilatory, contractile and structural properties in mice arterial specimen ex vivo. Collectively, these results demonstrate that FGF23-Klotho signaling is absent in mouse arteries and that the vascular response was unaffected by FGF23 treatment. Thus, our data do not support Klotho-mediated FGF23 effects in the vasculature although confirmative studies in humans are warranted.     

Force Control Deficits in Individuals with Parkinson’s Disease,Multiple Systems Atrophy,and Progressive Supranuclear Palsy

Objective

This study examined grip force and cognition in Parkinson’s disease (PD), Parkinsonian variant of multiple system atrophy (MSAp), progressive supranuclear palsy (PSP), and healthy controls. PD is characterized by a slower rate of force increase and decrease and the production of abnormally large grip forces. Early-stage PD has difficulty with the rapid contraction and relaxation of hand muscles required for precision gripping. The first goal was to determine which features of grip force are abnormal in MSAp and PSP. The second goal was to determine whether a single variable or a combination of motor and cognitive measures would distinguish patient groups. Since PSP is more cognitively impaired relative to PD and MSAp, we expected that combining motor and cognitive measures would further distinguish PSP from PD and MSAp.

Methods

We studied 44 participants: 12 PD, 12 MSAp, 8 PSP, and 12 controls. Patients were diagnosed by a movement disorders neurologist and were tested off anti-Parkinsonian medication. Participants completed a visually guided grip force task wherein force pulses were produced for 2 s, followed by 1 s of rest. We also conducted four cognitive tests.

Results

PD, MSAp, and PSP were slower at contracting and relaxing force and produced longer pulse durations compared to controls. PSP produced additional force pulses during the task and were more cognitively impaired relative to other groups. A receiver operator characteristic analysis revealed that the combination of number of pulses and Brief Test of Attention (BTA) discriminated PSP from PD, MSAp, and controls with a high degree of sensitivity and specificity.

Conclusions

Slowness in contracting and relaxing force represent general features of PD, MSAp, and PSP, whereas producing additional force pulses was specific to PSP. Combining motor and cognitive measures provides a robust method for characterizing behavioral features of PSP compared to MSAp and PD.     

A Winter Distribution Model for Bicknell’s Thrush (Catharus bicknelli), a Conservation Tool for a Threatened Migratory Songbird

Conservation planning and implementation require identifying pertinent habitats and locations where protection and management may improve viability of targeted species. The winter range of Bicknell’s Thrush (Catharus bicknelli), a threatened Nearctic-Neotropical migratory songbird, is restricted to the Greater Antilles. We analyzed winter records from the mid-1970s to 2009 to quantitatively evaluate winter distribution and habitat selection. Additionally, we conducted targeted surveys in Jamaica (n = 433), Cuba (n = 363), Dominican Republic (n = 1,000), Haiti (n = 131) and Puerto Rico (n = 242) yielding 179 sites with thrush presence. We modeled Bicknell’s Thrush winter habitat selection and distribution in the Greater Antilles in Maxent version 3.3.1. using environmental predictors represented in 30 arc second study area rasters. These included nine landform, land cover and climatic variables that were thought a priori to have potentially high predictive power. We used the average training gain from ten model runs to select the best subset of predictors. Total winter precipitation, aspect and land cover, particularly broadleaf forests, emerged as important variables. A five-variable model that contained land cover, winter precipitation, aspect, slope, and elevation was the most parsimonious and not significantly different than the models with more variables. We used the best fitting model to depict potential winter habitat. Using the 10 percentile threshold (>0.25), we estimated winter habitat to cover 33,170 km², nearly 10% of the study area. The Dominican Republic contained half of all potential habitat (51%), followed by Cuba (15.1%), Jamaica (13.5%), Haiti (10.6%), and Puerto Rico (9.9%). Nearly one-third of the range was found to be in protected areas. By providing the first detailed predictive map of Bicknell’s Thrush winter distribution, our study provides a useful tool to prioritize and direct conservation planning for this and other wet, broadleaf forest specialists in the Greater Antilles.     

High sodium chloride intake exacerbates immobilization-induced bone resorption and protein losses

We examined, in immobilization, the effect of a diet high in sodium chloride (NaCl) on bone markers, nitrogen balance, and acid-base status. Eight healthy male test subjects participated in a 14-day head-down-tilt bed rest (HDBR) study. During the bed rest period they received, in a randomized crossover design, a high (7.7 meq Na(+)/kg body wt per day) and a low (0.7 meq Na(+)/kg body wt per day) NaCl diet. As expected, 24-h excretion of urinary calcium was significantly greater in the high-NaCl-intake HDBR phase than in the low-NaCl-intake HDBR phase (P < 0.001). High NaCl intake caused a 43-50% greater excretion of the bone resorption markers COOH- (CTX) and NH(2)- (NTX) terminal telopeptide of type I collagen in HDBR than low NaCl in HDBR (CTX/NTX: P < 0.001). Serum concentrations of the bone formation markers bone-specific alkaline phosphatase (bAP) and NH(2)-terminal propeptide of type I procollagen (PINP) were identical in both NaCl intake phases. High NaCl intake led to a more negative nitrogen balance in HDBR (P < 0.001). Changes were accompanied by increased serum chloride concentration (P = 0.008), reduced blood bicarbonate (P = 0.017), and base excess (P = 0.009) whereas net acid excretion was lower during high than during low NaCl intake in immobilization (P < 0.001). High NaCl intake during immobilization exacerbates disuse-induced bone and muscle loss by causing further protein wasting and an increase in bone resorption. Changes in the acid-base status, mainly caused by disturbances in electrolyte metabolism, seem to determine NaCl-induced degradation processes.     

Apparent transverse compressive material properties of the digital flexor tendons and the median nerve in the carpal tunnel

Carpal tunnel syndrome is a frequently encountered peripheral nerve disorder caused by mechanical insult to the median nerve, which may in part be a result of impingement by the adjacent digital flexor tendons. Realistic finite element (FE) analysis to determine contact stresses between the flexor tendons and median nerve depends upon the use of physiologically accurate material properties. To assess the transverse compressive properties of the digital flexor tendons and median nerve, these tissues from ten cadaveric forearm specimens were compressed transversely while under axial load. The experimental compression data were used in conjunction with an FE-based optimization routine to determine apparent hyperelastic coefficients (μ and α) for a first-order Ogden material property definition. The mean coefficient pairs were μ=35.3 kPa, α=8.5 for the superficial tendons, μ=39.4 kPa, α=9.2 for the deep tendons, μ=24.9 kPa, α=10.9 for the flexor pollicis longus (FPL) tendon, and μ=12.9 kPa, α=6.5 for the median nerve. These mean Ogden coefficients indicate that the FPL tendon was more compliant at low strains than either the deep or superficial flexor tendons, and that there was no significant difference between superficial and deep flexor tendon compressive behavior. The median nerve was significantly more compliant than any of the flexor tendons. The material properties determined in this study can be used to better understand the functional mechanics of the carpal tunnel soft tissues and possible mechanisms of median nerve compressive insult, which may lead to the onset of carpal tunnel syndrome.     

Immunological stimuli change expression of genes and neutrophil function in fathead minnow Pimephales promelas Rafinesque

Fathead minnows Pimephales promelas were exposed to lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid [poly(I:C)] to observe immunological responses during simulated bacterial and viral challenge at the level of gene expression and granulocyte function. Complementary DNA libraries were created from LPS- and poly(I:C)-treated fish and c. 5000 expressed sequence tags (ESTs) were sequenced. The ESTs were subjected to BLASTx analysis and 1500 genes were annotated, grouped by function and 20 immune genes were selected for expression studies by real-time PCR. Lipopolysaccharide treatment significantly downregulated expression of interferon regulatory factor 2 binding protein 1 (nine-fold), Chemokine (C-X-C motif) ligand 12a (three-fold) and TNF-related apoptosis-inducing ligand, TRAIL (two-fold). In poly(I:C)-treated fish, a significant upregulation was observed for IFN-inducible and antiviral proteins belonging to the family of Mx proteins (73-fold) and chemokine CCL-C5a (28-fold). Blood neutrophil count was significantly increased in poly(I:C)-treated fish at 24 and 48 h post-injection. Neutrophil extracellular trap release and respiratory burst of kidney granulocytes were suppressed in poly(I:C)-treated fish, while degranulation of primary granules was not affected significantly by the treatment. The changes in gene expression and neutrophil function in P. promelas exposed to LPS and poly(I:C) support the use of this species as an alternative model for studies of pathogen effects on the innate immune system of fishes.     

Effectiveness of a quality-improvement program in improving management of primary care practices

Background:

The European Practice Assessment program provides feedback and outreach visits to primary care practices to facilitate quality improvement in five domains (infrastructure, people, information, finance, and quality and safety). We examined the effectiveness of this program in improving management in primary care practices in Germany, with a focus on the domain of quality and safety.

Methods:

In a before–after study, 102 primary care practices completed a practice assessment using the European Practice Assessment instrument at baseline and three years later (intervention group). A comparative group of 102 practices was included that completed their first assessment using this instrument at the time of the intervention group’s second assessment. Mean scores were based on the proportion of indicators for which a positive response was achieved by all of the practices, on a scale of 0 to 100.

Results:

We found significant improvements in all domains between the first and second assessments in the intervention group. In the domain of quality and safety, improvements in scores (mean scores were based on the proportion of indicators for which a positive response was achieved by all of the practices, on a scale of 0 to 100) were observed in the following dimensions: complaint management (from a mean score of 51.2 at first assessment to 80.7 at second assessment); analysis of critical incidents (from 79.1 to 89.6); and quality development, quality policy (from 40.7 to 55.6). Overall scores at the time of the second assessment were significantly higher in the intervention group than in the comparative group.

Interpretation:

Primary care practices that completed the European Practice Assessment instrument twice over a three-year period showed improvements in practice management. Our findings show the value of the quality-improvement cycle in the context of practice assessment and the use of established organizational standards for practice management with the Europeaen Practice Assessment.A variety of quality-improvement initiatives in health care management have been implemented in most health care systems.^1,2 Countries such as Australia, New Zealand, the United Kingdom and the United States have a long tradition in, and established standards for, quality management in primary care. In Canada, such initiatives in primary care are in their infancy, despite support by the federal Primary Health Care Transition Fund since 2000.³ In Ontario, a comprehensive book was recently issued that provides recommendations on practice management and clinical indicators for improving quality in primary care settings in the province.⁴ The indicators were adopted and refined from the Royal New Zealand College of General Practitioners, the Royal Australian College of General Practitioners’ National Expert Committee on Standards for General Practices, the TOPAS–Europe Association European Practice Assessment, the United Kingdom’s Quality and Outcomes Framework and the Canadian Institute for Health Information.In Germany, similar developments took place. In 2005, the German government stipulated that health care providers implement a system of annual assessment of quality management.⁵ One of the systems available to practices is the European Practice Assessment (www.epa-qm.de), a validated instrument based on quality indicators for assessing practice management.⁶ The five key domains of the European Practice Assessment instrument and their respective dimensions are described in Figure 1.Open in a separate windowFigure 1:The domains and dimensions (and number of indicators) of the European Practice Assessment instrument used to measure the quality of practice management in primary care practices. For an example of how the pentagon shape is used to provide feedback to individual practices, see Appendix 1 (available at www.cmaj.ca/lookup/suppl/doi:10.1503/cmaj.110412/-/DC1). GP = general practitioner.The European Practice Assessment is used to help general practices both assess and improve their quality of practice management set against predefined criteria with embedded quality indicators. The improvement process is ongoing (e.g., through plan–do–study–act cycles⁷): each step is reviewed and redesigned with a view to improving the quality of the end product, thereby fostering continuous improvement.⁸ The multifaceted strategy has three essential components: assessment and feedback using validated instruments based on quality indicators; external support through an outreach educational visit by a trained visitor (physician or nurse) to support the practice in improving areas of management identified by the practice;⁹ and formal accreditation by an external organization.Three requirements have to be fulfilled by practices to receive accreditation: achieve a positive response for more than 50% of the indicators; meet predefined safety indicators (e.g., the vaccination status of staff regarding hepatitis B vaccination is recorded and medical equipment is checked regularly according to national regulations); and highlight areas for continuous quality improvement.Accreditation is one method for assessing and benchmarking the performance of general practice care across a broad range of clinical and organizational domains. It describes a formal process of self-assessment and external and independent peer review to encourage best management practice and can result in recommendations for continuous improvement of safety and quality in the practice.⁸ Practice accreditation can be used for different purposes: quality control, regulation, quality improvement, providing data on performance, and marketing.⁸ In Germany, it is used for quality improvement, leading to a certificate.We conducted a study to determine whether improvements in practice management occurred in general practices that completed the European Practice Assessment twice over three years, compared with general practices that completed the European Practice Assessment once. We focused our analysis on the domain of quality and safety, expecting an association between practice organization and quality improvement.^10,11 We hypothesized that the initial use of the European Practice Assessment and reassessment with it three years later would result in improved scores in the dimensions and indicators within the domain of quality and safety.