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Background: Poultry products are the largest food category linked to salmonellosis in Canada. Bacteriophages (phages) have been proposed as a novel antimicrobial in the poultry industry due to their documented ubiquity, efficacy, and safety benefits. Materials and Methods: A library of 78 lytic phages was rapidly screened against 50 prominent poultry-associated Salmonella enterica isolates procured from British Columbia, Canada. Results: A phage cocktail was successfully formulated using only three sewage-isolated phages (SE4, SE13, and SE20) to achieve broad-spectrum antimicrobial efficacy across all S. enterica serovars. Highly promising phages were also characterized using one-step growth curves and transmission electron microscopy. Conclusion: Relative host efficiency is a new agar-based semiquantitative metric developed here for the rapid comparison of different phages against a panel of known bacterial targets., Competing Interests: No competing financial interests exist., (Copyright 2020, Mary Ann Liebert, Inc., publishers.)

Bacteriophages have shown promise as therapeutic alternatives to antibiotics for the control of infectious bacteria, including the human pathogen Salmonella. However, the development of effective phage-based applications requires the elucidation of key interactions between phages and target hosts, particularly since host resistance to phage is inevitable. Little is known about the alteration of host phenotypes following the development of resistance to phage. The aim of this study is to evaluate the antibiotic susceptibility and virulence of a Salmonella isolate following the development of resistance to bacteriophage SI1. We observed enhanced susceptibility to tetracycline and decreased invasion capacity in a differentiated Caco-2 intestinal cell line. Whole genome sequence analysis revealed an array of mutations, most notably, truncations in vgrG1_2 , a core gene involved in Type VI secretion and mutations in the lipopolysaccharide, thereby indicating the plausible attachment site of phage SI1. These findings shed light on understanding the underlying mechanism for phage immunity within the host. Importantly, we reveal an associated genetic cost to the bacterial host with developing resistance to phages. Taken together, these results will aid in advancing strategies to delay or eliminate the development of host resistance when designing informed phage-based antimicrobials.

Background: People after stroke benefit from comprehensive secondary prevention programs including cardiac rehabilitation (CR), yet there is little understanding of eligibility for exercise and barriers to use., Objective: The aim of this study was to examine eligibility for CR; enrollment, adherence, and completion; and factors affecting use., Design: This was a prospective study of 116 consecutive people enrolled in a single outpatient stroke rehabilitation (OSR) program located in Toronto, Ontario, Canada., Methods: Questionnaires were completed by treating physical therapists for consecutive participants receiving OSR and included reasons for CR ineligibility, reasons for declining participation, demographics, and functional level. CR eligibility criteria included the ability to walk ≥100 m (no time restriction) and the ability to exercise at home independently or with assistance. People with or without hemiplegic gait were eligible for adapted or traditional CR, respectively. Logistic regression analyses were used to examine factors associated with use indicators., Results: Of 116 participants receiving OSR, 82 (70.7%) were eligible for CR; 2 became eligible later. Sixty (71.4%) enrolled in CR and 49 (81.7%) completed CR, attending 87.1% (SD = 16.6%) of prescribed sessions. The primary reasons for ineligibility included being nonambulatory or having poor ambulation (52.9%; 18/34 patients) and having severe cognitive deficits and no home exercise support (20.6%; 7/34). Frequently cited reasons for declining CR were moving or travel out of country (17.2%; 5/29 reasons), lack of interest (13.8%; 4/29), transportation issues (10.3%; 3/29), and desiring a break from therapy (10.3%; 3/29). In a multivariate analysis, people who declined CR were more likely to be women, have poorer attendance at OSR, and not diabetic. Compared with traditional CR, stroke-adapted CR resulted in superior attendance (66.1% [SD = 22.9%] vs 87.1% [SD = 16.6%], respectively) and completion (66.7% vs 89.7%, respectively). The primary reasons for dropping out were medical (45%) and moving (27%)., Limitations: Generalizability to other programs is limited, and other, unmeasured factors may have affected outcomes., Conclusions: An OSR-CR partnership provided an effective continuum of care, with approximately 75% of eligible people participating and more than 80% completing. However, just over 1 of 4 eligible people declined participation; therefore, strategies should target lack of interest, transportation, women, and people without diabetes. An alternative program model is needed for people who have severe ambulatory or cognitive deficits and no home exercise support., (© 2019 American Physical Therapy Association.)

Salmonella enterica ( S. enterica ) is a causative agent of multiple outbreaks of foodborne illness associated with fresh produce, including pre-cut melon and leafy vegetables. Current industrial antimicrobial interventions have been shown to reduce microbial populations by <90%. Consequently, bacteriophages have been suggested as an alternative to chemical sanitizers. Seven S. enterica strains from four serovars (10 5  CFU/mL) were separately inoculated onto excised pieces of Romaine lettuce leaf and cantaloupe flesh treated with a five-strain bacteriophage cocktail 24 h before S. enterica inoculation. S. enterica , total aerobic populations and water activity were measured immediately after inoculation and after 1 and 2 days of incubation at 8 °C. The efficacy of the bacteriophage cocktail varied between strains. Populations of S. enterica Enteritidis strain S3, S. Javiana S203, S. Javiana S200 were reduced by > 3 log CFU/g and S. Newport S2 by 1 log CFU/g on both lettuce and cantaloupe tissues at all sampling times. In contrast, populations of strains S. Thompson S193 and S194 were reduced by 2 log CFU/g on day 0 on lettuce, but were not significantly different (P > 0.05) from the controls thereafter, S. Newport S195 populations were reduced on lettuce by 1 log CFU/g on day 0 and no reductions were found on cantaloupe tissue. Both aerobic populations and water activity were higher on cantaloupe than on lettuce. The water activity of lettuce decreased significantly (P < 0.05) from 0.845 ± 0.027 on day 0-0.494 ± 0.022 on day 1, but that of cantaloupe remained between 0.977 and 0.993 from day 0-2. The results of this study showed that bacteriophages can reduce S. enterica populations on lettuce and cantaloupe tissues but that the magnitude of the effect was strain-dependent., (© 2019 The Authors.)

Phages infecting members of the opportunistic human pathogen, Salmonella enterica , are widespread in natural environments and offer a potential source of agents that could be used for controlling populations of this bacterium; yet, relatively little is known about these phages. Here we describe the isolation and characterization of 45 phages of Salmonella enterica from disparate geographic locations within British Columbia, Canada. Host-range profiling revealed host-specific patterns of susceptibility and resistance, with several phages identified that have a broad-host range (i.e., able to lyse >40% of bacterial hosts tested). One phage in particular, SE13, is able to lyse 51 out of the 61 Salmonella strains tested. Comparative genomic analyses also revealed an abundance of sequence diversity in the sequenced phages. Alignment of the genomes grouped the phages into 12 clusters with three singletons. Phages within certain clusters exhibited extraordinarily high genome homology (>98% nucleotide identity), yet between clusters, genomes exhibited a span of diversity (<50% nucleotide identity). Alignment of the major capsid protein also supported the clustering pattern observed with alignment of the whole genomes. We further observed associations between genomic relatedness and the site of isolation, as well as genetic elements related to DNA metabolism and host virulence. Our data support the knowledge framework for phage diversity and phage-host interactions that are required for developing phage-based applications for various sectors, including biocontrol, detection and typing., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Integrin αIIbβ3, a transmembrane heterodimer, mediates platelet aggregation when it switches from an inactive to an active ligand-binding conformation following platelet stimulation. Central to regulating αIIbβ3 activity is the interaction between the αIIb and β3 extracellular stalks, which form a tight heterodimer in the inactive state and dissociate in the active state. Here, we demonstrate that alanine replacements of sensitive positions in the heterodimer stalk interface destabilize the inactive conformation sufficiently to cause constitutive αIIbβ3 activation. To determine the structural basis for this effect, we performed a structural bioinformatics analysis and found that perturbing intersubunit contacts with favorable interaction geometry through substitutions to alanine quantitatively accounted for the degree of constitutive αIIbβ3 activation. This mutational study directly assesses the relationship between favorable interaction geometry at mutation-sensitive positions and the functional activity of those mutants, giving rise to a simple model that highlights the importance of interaction geometry in contributing to the stability between protein-protein interactions.

The Salmonella Syst-OMICS consortium is sequencing 4,500 Salmonella genomes and building an analysis pipeline for the study of Salmonella genome evolution, antibiotic resistance and virulence genes. Metadata, including phenotypic as well as genomic data, for isolates of the collection are provided through the Salmonella Foodborne Syst-OMICS database (SalFoS), at https://salfos.ibis.ulaval.ca/. Here, we present our strategy and the analysis of the first 3,377 genomes. Our data will be used to draw potential links between strains found in fresh produce, humans, animals and the environment. The ultimate goals are to understand how Salmonella evolves over time, improve the accuracy of diagnostic methods, develop control methods in the field, and identify prognostic markers for evidence-based decisions in epidemiology and surveillance.

αIIbβ3 activation in platelets is followed by activation of the tyrosine kinase c-Src associated with the carboxyl terminus of the β3 cytosolic tail. Exogenous peptides designed to interact with the αIIb transmembrane (TM) domain activate single αIIbβ3 molecules in platelets by binding to the αIIb TM domain and causing separation of the αIIbβ3 TM domain heterodimer. Here we asked whether directly activating single αIIbβ3 molecules in platelets using the designed peptide anti-αIIb TM also initiates αIIbβ3-mediated outside-in signaling by causing activation of β3-associated c-Src. Anti-αIIb TM caused activation of β3-associated c-Src and the kinase Syk, but not the kinase FAK, under conditions that precluded extracellular ligand binding to αIIbβ3. c-Src and Syk are activated by trans-autophosphorylation, suggesting that activation of individual αIIbβ3 molecules can initiate αIIbβ3 clustering in the absence of ligand binding. Consistent with this possibility, incubating platelets with anti-αIIb TM resulted in the redistribution of αIIbβ3 from a homogenous ring located at the periphery of discoid platelets into nodular densities consistent with clustered αIIbβ3. Thus, these studies indicate that not only is resting αIIbβ3 poised to undergo a conformational change that exposes its ligand-binding site, but it is poised to rapidly assemble into intracellular signal-generating complexes as well., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The Inventory of Reading Occupations (IRO) is an assessment tool of children's reading participation. In this study, we used Rasch methods to determine the internal validity of the IRO. Participants included 192 typical and struggling readers from kindergarten to third grade from five different states in the United States. We analyzed the fit of each of the items in the 17 reading categories, the test items in the three dimensions of reading participation, and the contexts of reading in the IRO. Analysis indicated that the IRO items support the Rasch model of unidimensionality. Analysis also indicated that 1 of the 30 test items can be revised to strengthen test validity. Moreover, the analysis also suggested that the IRO is more useful for first- to third-grade students. This study provides evidence of internal validity of a useful tool to assess children's reading participation., (Copyright © 2016 by the American Occupational Therapy Association, Inc.)

Platelet spreading is critical for hemostatic plug formation and thrombosis. However, the detailed dynamics of platelet spreading as a function of receptor-ligand adhesive interactions has not been thoroughly investigated. Using reflection interference contrast microscopy, we found that both adhesive interactions and PAR4 activation affect the dynamics of platelet membrane contact formation during spreading. The initial growth of close contact area during spreading was controlled by the combination of different immobilized ligands or PAR4 activation on fibrinogen, whereas the growth of the total area of spreading was independent of adhesion type and PAR4 signaling. We found that filopodia extend to their maximal length and then contract over time; and that filopodial protrusion and expansion were affected by PAR4 signaling. Upon PAR4 activation, the integrin α(IIb)β(3) mediated close contact to fibrinogen substrata and led to the formation of ringlike patterns in the platelet contact zone. A systematic study of platelet spreading of GPVI-, α(2)-, or β(3)-deficient platelets on collagen or fibrinogen suggests the integrin α(2) is indispensable for spreading on collagen. The platelet collagen receptors GPVI and α(2) regulate integrin α(IIb)β(3)-mediated platelet spreading on fibrinogen. This work elucidates quantitatively how receptor-ligand adhesion and biochemical signals synergistically control platelet spreading., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Activated platelets shed surface proteins, potentially modifying platelet function as well as providing a source of bioactive fragments. Previous studies have identified several constituents of the platelet sheddome, but the full extent of shedding is unknown. Here we have taken a global approach, analyzing protein fragments in the supernate of activated platelets using mass spectroscopy and looking for proteins originating from platelet membranes. After removing plasma proteins and microparticles, 1048 proteins were identified, including 69 membrane proteins. Nearly all of the membrane proteins had been detected previously, but only 10 had been shown to be shed in platelets. The remaining 59 are candidates subject to confirmation. Based on spectral counts, protein representation in the sheddome varies considerably. As proof of principle, we validated one of the less frequently detected proteins, semaphorin 7A, which had not previously been identified in platelets. Surface expression, cleavage, and shedding of semaphorin 7A were demonstrated, as was its association with α-granules. Finally, cleavage of semaphorin 7A and 12 other proteins was substantially reduced by an inhibitor of ADAM17, a known sheddase. These results define a subset of membrane proteins as sheddome candidates, forming the basis for further studies examining the impact of ectodomain shedding on platelet function.

Although much is known about extrinsic regulators of platelet function such as nitric oxide and prostaglandin I(2) (PGI(2)), considerably less is known about intrinsic mechanisms that prevent overly robust platelet activation after vascular injury. Here we provide the first evidence that regulators of G-protein signaling (RGS) proteins serve this role in platelets, using mice with a G184S substitution in G(i2α) that blocks RGS/G(i2) interactions to examine the consequences of lifting constraints on G(i2)-dependent signaling without altering receptor:effector coupling. The results show that the G(i2α)(G184S) allele enhances platelet aggregation in vitro and increases platelet accumulation after vascular injury when expressed either as a global knock-in or limited to hematopoietic cells. Biochemical studies show that these changes occur in concert with an attenuated rise in cyclic adenosine monophosphate levels in response to prostacyclin and a substantial increase in basal Akt activation. In contrast, basal cyclic adenosine monophosphate (cAMP) levels, agonist-stimulated increases in [Ca(++)](i), Rap1 activation, and α-granule secretion were unaffected. Collectively, these observations (1) demonstrate an active role for RGS proteins in regulating platelet responsiveness, (2) show that this occurs in a pathway-selective manner, and (3) suggest that RGS proteins help to prevent unwarranted platelet activation as well as limiting the magnitude of the normal hemostatic response.

We recently reported that Semaphorin 4D (Sema4D) and its receptors are expressed on the platelet surface and showed that Sema4D((-/-)) mice have a selective defect in collagen-induced platelet aggregation and an impaired vascular injury response. Here we investigated the mechanisms involved, tested the role of platelet-platelet contacts in Sema4D-mediated events, and examined the relationship between Sema4D-dependent signaling and integrin α(IIb)β(3) outside-in signaling. The results show that spleen tyrosine kinase (Syk) activation, an early step in collagen signaling via the glycoprotein VI (GPVI)/FcRγ complex, is greatly reduced in Sema4D((-/-)) platelets and can be restored by adding soluble Sema4D. Earlier events, including FcRγ phosphorylation, occur normally; later events are impaired. In contrast, when engagement of α(IIb)β(3) was blocked, Sema4D((-/-)) and control platelets were indistinguishable in assays of Syk activation, adhesion, spreading on collagen, and activation of α(IIb)β(3). Finally, we found that, unlike the Sema4D knockout, α(IIb)β(3) blockade inhibited FcRγ phosphorylation and that stimulating aggregation with Mn(2+) failed to normalize Syk activation in the absence of Sema4D. Collectively, these results show that α(IIb)β(3) and Sema4D jointly promote collagen responses by amplifying Syk activation, partly by forming integrin-mediated contacts that enable the binding of Sema4D to its receptors and partly through integrin outside-in signaling. These 2 processes are interdependent, but distinguishable.

Objectives: 1) To measure the proportion of patients with chronic rhinosinusitis (CRS) who experience clinically significant improvement after endoscopic sinus surgery (ESS) in a prospective, multi-institutional fashion. 2) To identify preoperative characteristics that predict clinically significant improvement in quality of life (QOL) after ESS., Study Design: Prospective, multi-institutional cohort study., Setting: Academic tertiary care centers., Subjects and Methods: A total of 302 patients with CRS from three centers were enrolled between July 2004 and December 2008 and followed for an average of 17.4 months postoperatively. Preoperative patient characteristics, CT scan, endoscopy score, and pre- and postoperative quality of life (QOL) data were collected. Univariate and multivariate analyses were performed., Results: Patients improved an average of 15.8 percent (18.9 points) on the Rhinosinusitis Disability Index and 21.2 percent (21.2 points) on the Chronic Sinusitis Survey (both P < 0.001). Patients significantly improved on all eight Medical Outcomes Study Short Form-36 (SF-36) subscales (all P < 0.001). Among patients with poor baseline QOL, 71.7 percent of patients experienced clinically significant improvement on the RSDI and 76.1 percent on the CSS. Patients undergoing primary surgery were 2.1 times more likely to improve on the RSDI (95% confidence interval [CI], 1.2, 3.4; P = 0.006) and 1.8 times more likely to improve on the CSS (95% CI, 1.1, 3.1; P = 0.020) compared with patients undergoing revision surgery., Conclusion: In this prospective, multi-institutional study, most patients experienced clinically significant improvement across multiple QOL outcomes after ESS. Specific patient characteristics provided prognostic value with regard to outcomes.

Objectives: Peripherally located frontal sinus pathology may be unreachable with standard endoscopic techniques. Patients with superiorly or laterally based lesions often undergo osteoplastic flap with or without obliteration. Image-guided frontal trephination (IGFT) can localize pathology and provide excellent exposure. We present 13 patients in whom this technique was applied., Study Design: Medical records of 13 patients undergoing IGFT were retrospectively reviewed., Results: The patients' mean age was 49.2 years, (range 14-79); follow-up time was 29.9 months (range 12-39). Indications for IGFT were superiorly or laterally based mucoceles (3), fibrous dysplasia or osteoma (3), type 4 frontal cells (3), and frontal recess stenosis or ossification (4). In five patients, IGFT was combined with endoscopic transethmoid frontal sinusotomy; eight patients were treated through a trephination approach, and three patients underwent trephination with unilateral frontal sinus obliteration. One patient required revision; all others remain symptom free., Conclusions/significance: IGFT offers an attractive alternative to osteoplastic flap.

Objective: The surgical treatment of epistaxis associated with hereditary hemorrhagic telangiectasia (HHT) is varied. Laser therapy is often inadequate for larger complex lesions. This study sought to determine if bipolar cautery can be effectively and safely used in treating HHT-associated epistaxis., Study Design and Setting: Records from all patients with HHT treated surgically over 8 years were reviewed retrospectively. Outcomes or complications were noted in the clinic on follow-up evaluation., Results: Twenty-seven patients with HHT who underwent surgical treatment of epistaxis were evaluated; 18 were treated with bipolar cautery. Forty-two separate bipolar treatments were performed. No new septal perforations or synechiae were noted. Twenty-two of 42 treatments were coupled with ancillary laser treatments. The bipolar was also used as the sole technique in 20 procedures., Conclusion: Bipolar electrocautery is a safe and effective tool for the intraoperative control of HHT-related epistaxis., Significance: Bipolar electrocautery may be used as an adjunct to laser techniques or as a stand-alone technique., Ebm Rating: C-4.