Knowledge, attitudes and influencers of North American dog-owners surrounding antimicrobials and antimicrobial stewardship.

OBJECTIVES: To quantify the individual influences of antimicrobial cost, method of administration and drug importance in human medicine on dog-owner antimicrobial preference, and determine knowledge, attitudes and influencers of dog-owners surrounding antimicrobials and antimicrobial stewardship. MATERIALS AND METHODS: Data were collected through an online survey targeting three dog-owner participant groups. These consisted of individuals residing in: (1) Canada, (2) USA and (3) any country recruited through an educational social media site. USA and Canadian participants were financially compensated. Conjoint analysis was used to quantify the influence of antimicrobial cost, method of administration and drug importance in human medicine. Descriptive and analytical statistics were used for data evaluation. RESULTS: A total of 809 surveys were completed. Antimicrobial cost accounted for 47% of dog-owner preferences, followed by method of administration (31%) and drug importance in human medicine (22%). All groups preferred lower cost drugs that were administered once by injection. Participants were more likely to prefer drugs considered "very important" in human medicine, except for the social media participants, who preferred drugs that were "not at all important." Most respondents (86%) reported antimicrobial resistance as important in human medicine and 29% believed antimicrobial use in pets posed a risk for antimicrobial resistance in humans. Participants recruited through social media, and those in the highest education category, were significantly more likely to report antimicrobial use in pets as a risk to people. CLINICAL SIGNIFICANCE: Cost was the most important factor in dog-owner antimicrobial preferences. There is a need for dog-owner antimicrobial stewardship education.

Fluorescent tagging for environmental surface cleaning surveillance in a veterinary hospital.

OBJECTIVES: To evaluate the use of fluorescent tagging for environmental surface cleaning surveillance in a small animal veterinary hospital and identify factors associated with tag removal. MATERIALS AND METHODS: Over 5.5 weeks, a commercial fluorescent dye (Glo Germ) was used to tag (mark) surfaces in a small animal veterinary teaching hospital. Twenty-four hours after tagging, cleaning was assessed with a black light (UV-A source). Surfaces were recorded as cleaned based on complete removal of fluorescent tagging at assessment. Proportions cleaned were calculated overall and by predictors (i.e. surface location/type, primary nature of surface contact - animal/human, week of study). RESULTS: A total of 4984 surfaces were tagged and assessed. Overall cleaning was 50%. Cleaning varied by surface/object (range: 2 to 100%) and hospital location (4 to 78%). Surfaces designated as having primarily animal contact were cleaned more frequently than those with primarily human contact (75%, 42%; P<0.001). Cleaning varied over the study period (range by week: 45 to 54%;); a significant trend was not identified. CLINICAL SIGNIFICANCE: Key surfaces in the small animal veterinary practice environment are unlikely to be adequately cleaned, posing a concern for animal and human health. Commercial products can be effectively used to asses environmental cleaning with findings used to target clinic-specific barriers to improve cleaning and reduce hospital-associated infections.

Dogs on livestock farms: A cross-sectional study investigating potential roles in zoonotic pathogen transmission.

Dogs are often present on livestock farms, where they serve important management and companion roles, yet may be involved in zoonotic pathogen transmission. Numerous factors can potentially alter the risk of exposure to zoonotic pathogens, such as the dog's access to livestock, close dog-human contact and an increasing immunocompromised human population. The objective of this study was to quantify and qualify dog ownership among livestock owners, their dog husbandry and biosecurity practices, the dogs' access to livestock and potential risks for zoonotic pathogen transmission. A questionnaire was developed and mailed to 2,000 presumed Ohio livestock owners. Data were collected on demographics, dog husbandry practices, attitudes surrounding zoonotic diseases and attachment to and preventive veterinary care for the dogs. There were 446 responders who met the study inclusion criteria as an Ohio livestock farm owner, with 297 (67%) also owning dogs. Approximately 52% of dog-owning households included at least one individual at higher disease risk (i.e., <5 years, ≥65 years, diagnosed with an immunocompromising condition). Most respondents had little/no concern for disease transmission from livestock to dogs (90%), from dogs to livestock (87%) and from dogs to people (94%). Dogs were allowed access to livestock by 70% of respondents and nearly all (96%; 198) indicated at least one higher risk dog-livestock management practice. In addition, many reported never leashing or fencing their dog (61%) and rarely to never picking up dog faeces (76%). Households with higher risk members reported similar husbandry, biosecurity and concern levels as households without those members (all p > .05). Numerous opportunities for zoonotic pathogen transmission and low level of zoonotic disease concern suggest a need for improved education and outreach for the livestock dog-owning community, particularly for higher risk households.

The 2008 ICOH Workshop on Skin Notation.

BACKGROUND: On 29 March 2008 the International Commission on Occupational Health (ICOH) Scientific Committee on Occupational and Environmental Dermatoses organized a Skin Notation Workshop hosted by the 11th International Percutaneous Penetration Perspectives Conference (La Grande Motte, France). Skin notation (S) was chosen as a topic for discussion because this is the only example of existing regulation in the field of dermal risk assessment. The issue was discussed in a previous workshop held in Siena, Italy in 2006 with the objective of focussing on the problems related to S, the different assignment criteria and the attempts to improve the S system made by various international and governmental agencies. A position paper was subsequently published. OBJECTIVES: The workshop in France was a continuation of this activity with the aim of evaluating how the different strategies can improve S. METHODS AND DISCUSSION: The Workshop was divided into two sessions. The first was dedicated to lectures focused on different aspects of S. In the second session participants discussed key issues with the aim of exploring the actions needed to improve international S. systems.

Outbreak of giardiasis associated with a community drinking-water source.

Giardiasis is a common waterborne gastrointestinal illness. In 2007, a community giardiasis outbreak occurred in New Hampshire, USA. We conducted a cohort study to identify risk factors for giardiasis, and stool and environmental samples were analysed. Consuming tap water was significantly associated with illness (risk ratio 4.7, 95% confidence interval 1.5-14.4). Drinking-water samples were coliform-contaminated and a suspect Giardia cyst was identified in a home water filter. One well was coliform-contaminated, and testing indicated that it was potentially under the influence of surface water. The well was located 12.5 m from a Giardia-contaminated brook, although the genotype differed from clinical specimens. Local water regulations require well placement at least 15 m from surface water. This outbreak, which caused illness in 31 persons, represents the largest community drinking-water-associated giardiasis outbreak in the USA in 10 years. Adherence to well placement regulations might have prevented this outbreak.

Ergonomic comparison of a chem/bio prototype firefighter ensemble and a standard ensemble.

Firefighter turnout gear and equipment protect the wearer against external hazards but, unfortunately, restrict mobility. The aim of this study was to determine the ease of mobility and comfort while wearing a new prototype firefighter ensemble (PE) with additional chemical/biological hazard protection compared to a standard ensemble (SE) by measuring static and dynamic range of motion (ROM), job-related tasks, and comfort. Eight healthy adults (five males, three females), aged 20-40 years, participated in this study. The study consisted of two repeated phases, separated by five uses of the ensembles. Subjects randomly donned either the SE or PE in either dry or wet conditions on separate days. In each phase, five tests were carried out as follows: baseline (non-ensemble), SE-dry, SE-wet, PE-dry, and PE-wet. There was a significant reduction (P < 0.05) of wrist flexion for PE-dry condition compared to the same SE-dry condition. Donning the PE took 80 s longer than the SE in phase 1, this difference disappeared in phase 2. There was a significant decrease (P < 0.05) in post-test comfort wearing the PE compared to the SE. The data collected in this study suggest that, in spite of design features to enhance chemical/biological hazard protection, the PE design does not decrease the wearer's overall functional mobility compared to the SE. However, subjects seem to be more comfortable wearing the SE compared to the PE. These overall findings support the need for a comprehensive ergonomic evaluation of protective clothing systems to ascertain human factors issues.

Functional and molecular adaptations in skeletal muscle of myoglobin-mutant mice.

Myoglobin is a cytoplasmic hemoprotein that is restricted to cardiomyocytes and oxidative skeletal myofibers and facilitates oxygen delivery during periods of high metabolic demand. Myoglobin content in skeletal muscle increases in response to hypoxic conditions. However, we previously reported that myoglobin-null mice are viable and fertile. In the present study, we define important functional, cellular, and molecular compensatory adaptations in the absence of myoglobin. Mice without myoglobin manifest adaptations in skeletal muscle that include a fiber type transition (type I to type II in the soleus muscle), increased expression of the hypoxia-inducible transcription factors hypoxia-inducible factor (HIF)-1alpha and HIF-2 (endothelial PAS domain protein), stress proteins such as heat shock protein 27, and the angiogenic growth factor vascular endothelial growth factor (soleus muscle), as well as increased nitric oxide metabolism (extensor digitorum longus). The resulting changes in angiogenesis, nitric oxide metabolism, and vasomotor regulation are likely to account for preserved exercise capacity of animals lacking myoglobin. These results demonstrate that mammalian organisms are capable of a broad spectrum of adaptive responses that can compensate for a potentially serious defect in cellular oxygen transport.

Myosin light chain kinase binding to a unique site on F-actin revealed by three-dimensional image reconstruction.

Ca2+-calmodulin-dependent phosphorylation of myosin regulatory light chains by the catalytic COOH-terminal half of myosin light chain kinase (MLCK) activates myosin II in smooth and nonmuscle cells. In addition, MLCK binds to thin filaments in situ and F-actin in vitro via a specific repeat motif in its NH2 terminus at a stoichiometry of one MLCK per three actin monomers. We have investigated the structural basis of MLCK-actin interactions by negative staining and helical reconstruction. F-actin was decorated with a peptide containing the NH2-terminal 147 residues of MLCK (MLCK-147) that binds to F-actin with high affinity. MLCK-147 caused formation of F-actin rafts, and single filaments within rafts were used for structural analysis. Three-dimensional reconstructions showed MLCK density on the extreme periphery of subdomain-1 of each actin monomer forming a bridge to the periphery of subdomain-4 of the azimuthally adjacent actin. Fitting the reconstruction to the atomic model of F-actin revealed interaction of MLCK-147 close to the COOH terminus of the first actin and near residues 228-232 of the second. This unique location enables MLCK to bind to actin without interfering with the binding of any other key actin-binding proteins, including myosin, tropomyosin, caldesmon, and calponin.

Myosin light chain kinase- and PKC-dependent contraction of LES and esophageal smooth muscle.

In smooth muscle cells enzymatically isolated from circular muscle of the esophagus (ESO) and lower esophageal sphincter (LES), ACh-induced contraction and myosin light chain (MLC) phosphorylation were similar. Contraction and phosphorylation induced by purified MLC kinase (MLCK) were significantly greater in LES than ESO. ACh-induced contraction and MLC phosphorylation were inhibited by calmodulin and MLCK inhibitors in LES and by protein kinase C (PKC) inhibitors in ESO. Contraction of LES and ESO induced by the PKC agonist 1,2-dioctanoylglycerol (DG) was unaffected by MLCK inhibitors. Caldesmon and calponin concentration-dependently inhibited ACh-induced contraction of ESO and not LES. In ESO, caldesmon antagonist GS17C reversed caldesmon- but not calponin-induced ACh inhibition. GS17C caused contraction of permeabilized ESO but had much less effect on LES. GS17C-induced contraction was not affected by MLCK inhibitors, suggesting that MLCK may not regulate caldesmon-mediated contraction. DG-induced contraction of ESO and LES was inhibited by caldesmon and calponinin, suggesting that these proteins may regulate PKC-dependent contraction. We conclude that calmodulin and MLCK play a role in ACh-induced LES contraction, whereas the classical MLCK may not be the major kinase responsible for contraction and phosphorylation of MLC in ESO. ESO contraction is PKC dependent. Caldesmon and/or calponin may play a role in PKC-dependent contraction.

Ca2+-independent activity of nitric oxide synthase.

Ca2+-independent forms of nitric-oxide synthase have significant activity when the endogenous calmodulin subunit is Ca2+ free. Further activation is seen when Ca2+ is added. We have examined the activation of a Ca2+-independent nitric-oxide synthase variant and its two point mutants that are more dependent on Ca2+ for activation using mutant calmodulins containing non-functional Ca2+-binding sites. These studies provide evidence that the Ca2+-independent activity of these enzymes can be exerted through specific adapted interactions between the enzyme and the Ca2+-binding site 2 of calmodulin. Further, the results suggest that EGTA-sensitive metals other than Ca2+ complexed to calmodulin may be involved in maximal activation of these nitric-oxide synthase variants.

The impact of protective hoods and their water content on the prevention of head burns in New York City firefighters: laboratory tests and field results.

The New York City Fire Department (FDNY) is the largest fire department in the United States. In 1996, FDNY added the thermal protective hood to its modern protective uniform. The purpose of this study is to determine 1) the effectiveness of hoods in reducing head burns and 2) whether hood water content (dry, damp, or saturated) affects the level of thermal protection. Laboratory tests (radiant heat performance, thermal protective performance, and fully dressed manikin) and FDNY field results were used. Laboratory tests evaluated 4 different conditions (no hood, dry, damp, and saturated hoods) exposed to 4 different heat fluxes (0.1, 0.25, 0.5, and 2.0 cal/cm2/sec) equivalent to approximate air temperatures of 200, 400, 600, and 2,250 degrees F. Field results compared FDNY head burns during 3 winters wearing the hood to 3 winters without hood. Wearing a hood dramatically reduced head burns. This was true for all laboratory tests, at all heat flux exposures, and all hood water content conditions. At 0.1 cal/cm2/sec, dry hoods were superior to wet hoods. At all other heat flux exposures, thermal protection was either not significantly different between water content conditions or improved as water content increased. Confirming these laboratory tests, FDNY field results showed significant decreases in neck burns (by 54%), ear burns (by 60%), and head burn totals (by 46%). Based on combined laboratory and field results, we strongly recommend the use of modern thermal protective hoods.

Nitric oxide contributes to vascular smooth muscle relaxation in contracting fast-twitch muscles.

During skeletal muscle contraction, NO derived from neuronal nitric oxide synthase (nNOS) in skeletal muscle fibers or from endothelial cells (eNOS) may relax vascular smooth muscle contributing to functional hyperemia. To examine the relative importance of these pathways, smooth muscle myosin regulatory light chain (smRLC) phosphorylation was assessed as an index of vascular tone in isolated extensor digitorum longus (EDL) muscles from C57, nNOS(-/-), and eNOS(-/-) mice. The smRLC phosphorylation (in mol phosphate per mol smRLC) in C57 resting muscles (0.12 +/- 0.04) was increased 3.7-fold (0.44 +/- 0.03) by phenylephrine (PE). Reversal of this increase with electrical stimulation (to 0.19 +/- 0.03; P < 0.05) was partially blocked by N(omega)-nitro-l-arginine (NLA). In nNOS(-/-) EDL, the PE-induced increase in smRLC phosphorylation (0.10 +/- 0.02 to 0.49 +/- 0.04) was partially decreased by stimulation (0.25 +/- 0.04). In eNOS(-/-) EDL, the control value for smRLC was increased (0.24 +/- 0.04), and PE-induced smRLC phosphorylation (0.36 +/- 0.06) was decreased by stimulation even in the presence of NLA (to 0.20 +/- 0.02; P < 0.05). These results suggest that in addition to NO-independent mechanisms, NO derived from both nNOS and eNOS plays a role in the integrative vascular response of contracting skeletal muscle.

Familial hypertrophic cardiomyopathy mutations in the regulatory light chains of myosin affect their structure, Ca2+ binding, and phosphorylation.

The effect of the familial hypertrophic cardiomyopathy mutations, A13T, F18L, E22K, R58Q, and P95A, found in the regulatory light chains of human cardiac myosin has been investigated. The results demonstrate that E22K and R58Q, located in the immediate extension of the helices flanking the regulatory light chain Ca(2+) binding site, had dramatically altered Ca(2+) binding properties. The K(Ca) value for E22K was decreased by approximately 17-fold compared with the wild-type light chain, and the R58Q mutant did not bind Ca(2+). Interestingly, Ca(2+) binding to the R58Q mutant was restored upon phosphorylation, whereas the E22K mutant could not be phosphorylated. In addition, the alpha-helical content of phosphorylated R58Q greatly increased with Ca(2+) binding. The A13T mutation, located near the phosphorylation site (Ser-15) of the human cardiac regulatory light chain, had 3-fold lower K(Ca) than wild-type light chain, whereas phosphorylation of this mutant increased the Ca(2+) affinity 6-fold. Whereas phosphorylation of wild-type light chain decreased its Ca(2+) affinity, the opposite was true for A13T. The alpha-helical content of the A13T mutant returned to the level of wild-type light chain upon phosphorylation. The phosphorylation and Ca(2+) binding properties of the regulatory light chain of human cardiac myosin are important for physiological function, and alteration any of these could contribute to the development of hypertrophic cardiomyopathy.

Activation of myosin light chain kinase requires translocation of bound calmodulin.

A novel translocation step is inferred from structural studies of the interactions between the intracellular calcium receptor protein calmodulin (CaM) and one of its regulatory targets. A mutant of CaM missing residues 2-8 (DeltaNCaM) binds skeletal muscle myosin light chain kinase with high affinity but fails to activate catalysis. Small angle x-ray scattering data reveal that DeltaNCaM occupies a position near the catalytic cleft in its complex with the kinase, whereas the native protein translocates to a position near the C-terminal end of the catalytic core. Thus, CaM residues 2-8 appear to facilitate movement of bound CaM away from the vicinity of the catalytic cleft.

Localization and activity of myosin light chain kinase isoforms during the cell cycle.

Phosphorylation on Ser 19 of the myosin II regulatory light chain by myosin light chain kinase (MLCK) regulates actomyosin contractility in smooth muscle and vertebrate nonmuscle cells. The smooth/nonmuscle MLCK gene locus produces two kinases, a high molecular weight isoform (long MLCK) and a low molecular weight isoform (short MLCK), that are differentially expressed in smooth and nonmuscle tissues. To study the relative localization of the MLCK isoforms in cultured nonmuscle cells and to determine the spatial and temporal dynamics of MLCK localization during mitosis, we constructed green fluorescent protein fusions of the long and short MLCKs. In interphase cells, localization of the long MLCK to stress fibers is mediated by five DXRXXL motifs, which span the junction of the NH(2)-terminal extension and the short MLCK. In contrast, localization of the long MLCK to the cleavage furrow in dividing cells requires the five DXRXXL motifs as well as additional amino acid sequences present in the NH(2)-terminal extension. Thus, it appears that nonmuscle cells utilize different mechanisms for targeting the long MLCK to actomyosin structures during interphase and mitosis. Further studies have shown that the long MLCK has twofold lower kinase activity in early mitosis than in interphase or in the early stages of postmitotic spreading. These findings suggest a model in which MLCK and the myosin II phosphatase (Totsukawa, G., Y. Yamakita, S. Yamashiro, H. Hosoya, D.J. Hartshorne, and F. Matsumura. 1999. J. Cell Biol. 144:735-744) act cooperatively to regulate the level of Ser 19-phosphorylated myosin II during mitosis and initiate cytokinesis through the activation of myosin II motor activity.

Functional muscle ischemia in neuronal nitric oxide synthase-deficient skeletal muscle of children with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal disease caused by mutation of the gene encoding the cytoskeletal protein dystrophin. Despite a wealth of recent information about the molecular basis of DMD, effective treatment for this disease does not exist because the mechanism by which dystrophin deficiency produces the clinical phenotype is unknown. In both mouse and human skeletal muscle, dystrophin deficiency results in loss of neuronal nitric oxide synthase, which normally is localized to the sarcolemma as part of the dystrophin-glycoprotein complex. Recent studies in mice suggest that skeletal muscle-derived nitric oxide may play a key role in the regulation of blood flow within exercising skeletal muscle by blunting the vasoconstrictor response to alpha-adrenergic receptor activation. Here we report that this protective mechanism is defective in children with DMD, because the vasoconstrictor response (measured as a decrease in muscle oxygenation) to reflex sympathetic activation was not blunted during exercise of the dystrophic muscles. In contrast, this protective mechanism is intact in healthy children and those with polymyositis or limb-girdle muscular dystrophy, muscle diseases that do not result in loss of neuronal nitric oxide synthase. This clinical investigation suggests that unopposed sympathetic vasoconstriction in exercising human skeletal muscle may constitute a heretofore unappreciated vascular mechanism contributing to the pathogenesis of DMD.

Myosin light chain kinase binding to actin filaments.

Smooth muscle myosin light chain kinase (MLCK) plays important roles in contractile-motile processes of a variety of cells. Three DFRxxL motifs at the kinase N-terminus (residues 2-63) are critical for high-affinity binding to actin-containing filaments [Smith et al. (1999) J. Biol. Chem. 274, 29433-29438]. A GST fusion protein containing residues 1-75 of MLCK (GST75-MLCK) bound maximally to both smooth muscle myofilaments and F-actin at 0.28 and 0.31 mol GST75-MLCK/mol actin with respective K(D) values of 0.1 microM and 0.8 microM. High-affinity binding of MLCK to actin-containing filaments may be due to each DFRxxL motif binding to one actin monomer in filaments.