The impact of pneumolysin on the macrophage response to Streptococcus pneumoniae is strain-dependent

Streptococcus pneumoniae is the world's leading cause of pneumonia, bacteremia, meningitis and otitis media. A major pneumococcal virulence factor is the cholesterol-dependent cytolysin, which has the defining property of forming pores in cholesterol-containing membranes. In recent times a clinically significant and internationally successful serotype 1 ST306 clone has been found to express a non-cytolytic variant of Ply (Ply306). However, while the pneumococcus is a naturally transformable organism, strains of the ST306 clonal group have to date been virtually impossible to transform, severely restricting efforts to understand the role of non-cytolytic Ply in the success of this clone. In this study isogenic Ply mutants were constructed in the D39 background and for the first time in the ST306 background (A0229467) to enable direct comparisons between Ply variants for their impact on the immune response in a macrophage-like cell line. Strains that expressed cytolytic Ply were found to induce a significant increase in IL-1β release from macrophage-like cells compared to the non-cytolytic and Ply-deficient strains in a background-independent manner, confirming the requirement for pore formation in the Ply-dependent activation of the NLRP3 inflammasome. However, cytolytic activity in the D39 background was found to induce increased expression of the genes encoding GM-CSF (CSF2), p19 subunit of IL-23 (IL23A) and IFNβ (IFNB1) compared to non-cytolytic and Ply-deficient D39 mutants, but had no effect in the A0229467 background. The impact of Ply on the immune response to the pneumococcus is highly dependent on the strain background, thus emphasising the importance of the interaction between specific virulence factors and other components of the genetic background of this organism

Intraindividual personality change: Situational influences, patterns of change, and frequency-based measurement

A large amount of research supports the idea that little change in personality occurs over time (e.g., Caspi & Roberts, 2001). However, changes in an individual’s personality across different situations, known as intraindividual, or within-person, variability, has not received nearly as much empirical attention, in part due to the difficulties inherent in measuring personality across situations. The present study evaluated a measure of within-person variability called frequency-based measurement by comparing it with situational personality expression. The study involved two phases: a one-time measure of trait personality using frequency-based measurement and a five day experience sampling (ESM) study involving repeated measures of state personality. Mean levels of the Big Five traits (i.e., extraversion, agreeableness, conscientiousness, neuroticism, and openness to experience), as measured by the frequency-based format, were significantly positively correlated with ESM measures of the traits, providing initial construct validity for this novel format. A major prediction in the study, that frequency-based variability in personality would be related to the state variability found in the ESM study, was not supported. However, variability in frequency-based measures of conscientiousness and neuroticism were related to both self-monitoring and self-concept clarity, and frequency-based variability in agreeableness was related to self-concept clarity. All of these relationships were small, indicating some divergent validity for frequency-based measurement. Finally, variability in any one of the Big Five traits was significantly positively correlated with variability in all of the other traits, in both measures (i.e., frequency-based measurement and ESM ratings). This provides support for traitedness (Baumeister & Tice, 1988), a theoretical approach to intraindividual variability that could help to explain why some individuals vary more than others depending on the situation. Overall, the present study found some support for the validity of frequency-based measurement as a method of evaluating intraindividual personality variability without the need for cost and time-intensive longitudinal studies

Testing noninvasive methods for extracting DNA from degraded wildlife scat

Genetic sampling is used in many wildlife fields to gather data on populations or individuals. In noninvasive genetic sampling, animals do not need to be captured. DNA can be gathered from hair, scat, or other residue shed into the environment. However, this can result in degraded DNA, so it is necessary to find a method of DNA extraction that balances costs and maximizes DNA quality. Common noninvasive DNA extraction methods include spin column and magnetic bead technology. Magnetic bead extraction has the potential to be automated, saving materials and labor costs. In this study, I compared the DNA concentration, purity, and genotyping success of the DNeasy Blood & Tissue (BT) kit, QIAamp Stool Mini kit (ST), QIAamp PowerFecal kit (PF), and Norgen Biotek Magnetic Bead (MB) kit using 25 coyote scat samples that had been stored at room temperature for 3 years to see which methods are optimal under conditions of highly degraded DNA. The BT and PF kits resulted in the highest DNA concentration, and the BT and ST samples had the best purity. The PF kit had the best genotyping success, followed by the BT kit. Overall, the PF kit had the best results. The BT kit, while resulting in slightly less DNA and worse amplification than the PF kit, is much cheaper, so the trade-off may be worth it if funding is limited. The MB kit resulted in very little DNA of low purity, so using magnetic beads to automate extraction will likely result in reduced success.Keywords: Canis latrans, DNA extraction, scat, noninvasive genetic sampling, magnetic bead

Numerical and Experimental Investigation of Multistable Systems

<p>The focus of this dissertation is on phenomena exhibited by multistable systems. Two phenomena of particular importance are chaos control and stochastic resonance. In this work, both models that can predict ordered responses and experiments in which ordered responses occur are explored. In addition, parameter identification methods are presented and improved. </p><p>Chaos control, when implemented with delays, can be an effective way to stabilize unstable periodic orbits within a multistable system experiencing a chaotic response. Delayed control is easy to implement physically but greatly increases the complexity of analyzing such systems. In this work, the spectral element method was adapted to evaluate unstable periodic orbits stabilized by feedback control implemented with delays. Examples are presented for Duffing systems in which the delay is equal to the forcing period. The spectral approach is also extended to analyze the control of chaos with arbitrary delays. Control with arbitrary delays can also be used to stabilize equilibria within the chaotic response. These methods for arbitrary delays are explored in self-excited, chaotic systems.</p><p>Stochastic resonance occurs in multistable systems when an increase in noise results in an ordered response. It is well known that noise excitation of multistable systems results in the system escaping from potential wells or switching between wells. In stochastic resonance, a small external signal is amplified due to these switching events. Methods for modeling stochastic resonance in both underdamped and overdamped systems are presented. In addition, stochastic resonance in a bistable, composite beam excited by colored noise is investigated experimentally. The experimental results are compared with analytical models, and the effect of modal masses on the analytical expressions is explored. Finally, an alternative approach for calculating the effect of colored noise excitation is proposed.</p><p>In order to implement analysis methods related to delay differential equations or stochastic resonance, the parameters of the system must be known in advance or determined experimentally. Parameter identification methods provide a natural connection between experiment and theory. In this work, the harmonic balance parameter identification method was applied to beam energy harvesters and is improved using weighting matrices. The method has been applied to a nonlinear, bistable, piezoelectric beam with a tip mass. Then, an experimental method of determining the number of restoring force coefficients necessary to accurately model the systems was demonstrated. The harmonic balance method was also applied to a bistable, beam system undergoing stochastic resonance. Finally, a new weighting strategy is presented based on the signal to noise ratio of each harmonic.</p>Dissertatio

Pore-forming activity of S. pneumoniae pneumolysin disrupts the paracellular localization of the epithelial adherens junction protein E-cadherin

Streptococcus pneumoniae, a common cause of community-acquired bacterial pneumonia, can cross the respiratory epithelial barrier to cause lethal septicemia and meningitis. S. pneumoniae pore-forming toxin pneumolysin (PLY) triggers robust neutrophil (PMN) infiltration that promotes bacterial transepithelial migration in vitro and disseminated disease in mice. Apical infection of polarized respiratory epithelial monolayers by S. pneumoniae at a multiplicity of infection (MOI) of 20 resulted in recruitment of PMNs, loss of 50% of the monolayer, and PMN-dependent bacterial translocation. Reducing the MOI to 2 decreased PMN recruitment two-fold and preserved the monolayer, but apical-to-basolateral translocation of S. pneumoniae remained relatively efficient. At both MOI of 2 and 20, PLY was required for maximal PMN recruitment and bacterial translocation. Co-infection by wild-type S. pneumoniae restored translocation by a PLY-deficient mutant, indicating that PLY can act in trans. Investigating the contribution of S. pneumoniae infection on apical junction complexes in the absence of PMN transmigration, we found that S. pneumoniae infection triggered the cleavage and mislocalization of the adherens junction (AJ) protein E-cadherin. This disruption was PLY-dependent at MOI of 2 and was recapitulated by purified PLY, requiring its pore-forming activity. In contrast, at MOI of 20, E-cadherin disruption was independent of PLY, indicating that S. pneumoniae encodes multiple means to disrupt epithelial integrity. This disruption was insufficient to promote bacterial translocation in the absence of PMNs. Thus, S. pneumoniae triggers cleavage and mislocalization of E-cadherin through PLY-dependent and -independent mechanisms, but maximal bacterial translocation across epithelial monolayers requires PLY-dependent neutrophil transmigration

The effect of selected rest break activities on reaction time, balance, and perceived discomfort after one hour of simulated occupational whole-body vibration exposure in healthy adults

Copyright statement: This is an Open Access article distributed under the terms of the creative commons Attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.This work was supported by Agrivita Canada, Mitacs Accelerate, University of Saskatchewan College of Medicine Research Award, University of Saskatchewan College of Medicine MPT Research Project Funding, and WorkSafeBC Ralph McGinn Postdoctoral Fellowship. The funding sources had no involvement in study design, data collection, analysis and interpretation of data, writing the manuscript, and decision to submit the article for publication.Peer ReviewedBackground & Objective Negative health effects from occupational whole-body vibration (WBV) exposure during machinery operation include alterations in proprioception, vestibular function, reaction time, stress, motor response, and decrements in musculoskeletal health. To reduce WBV exposure during machinery operation, it may be possible to incorporate short rest break activities throughout the day. This study aims to determine if there are intervention activities that can minimize decrements in cognitive, proprioceptive, and musculoskeletal effects related to WBV exposure during machine operation. Materials & Methods Eleven healthy adults participated in four 1-hour sessions of ecologically valid WBV exposure followed by one of four 5-minute activities: sitting, walking, 2 minutes of gaze stabilization exercise (GSE) coupled with 3 minutes of trunk mobility exercise (GSE+MOBIL), or 2 minutes of GSE coupled with a 3-minute walk (GSE+WALK). Baseline and post-activity measurements (rating of perceived discomfort, balance and postural sway measurements, 5-minute psychomotor vigilance task test) were submitted to a paired t-test to determine the effect of WBV exposure and activities on physical, cognitive, and sensorimotor systems and to a repeated measures ANOVA to determine any differences across activities. Results We observed degradation of the slowest 10% reaction speed outcomes between baseline and post-activity after walking (7.3%, p<0.05) and sitting (8.6%, p<0.05) but not after GSE+MOBIL or GSE+WALK activities. Slowest 10% reaction speeds after GSE+MOBIL activity was faster than all other activities. Rating of perceived discomfort was higher after SIT and WALK activities. There were no notable differences in balance outcomes. Conclusion When compared to sitting for 5 minutes, an activity including GSE and an active component, such as walking or trunk mobility exercises, resulted in maintenance of reaction time after WBV exposure. If confirmed in occupational environments, GSE may provide a simple, rapid, effective, and inexpensive means to protect against decrements in reaction time after WBV exposure

Author response

Many pathogenic bacteria produce pore-forming toxins to attack and kill human cells. We have determined the 4.5 Å structure of the ~2.2 MDa pore complex of pneumolysin, the main virulence factor of Streptococcus pneumoniae, by cryoEM. The pneumolysin pore is a 400 Å ring of 42 membrane-inserted monomers. Domain 3 of the soluble toxin refolds into two ~85 Å β-hairpins that traverse the lipid bilayer and assemble into a 168-strand β-barrel. The pore complex is stabilized by salt bridges between β-hairpins of adjacent subunits and an internal α-barrel. The apolar outer barrel surface with large sidechains is immersed in the lipid bilayer, while the inner barrel surface is highly charged. Comparison of the cryoEM pore complex to the prepore structure obtained by electron cryo-tomography and the x-ray structure of the soluble form reveals the detailed mechanisms by which the toxin monomers insert into the lipid bilayer to perforate the target membrane

Structural insights into Clostridium perfringens delta toxin pore formation

Clostridium perfringens Delta toxin is one of the three hemolysin-like proteins produced by C. perfringens type C and possibly type B strains. One of the others, NetB, has been shown to be the major cause of Avian Nectrotic Enteritis, which following the reduction in use of antibiotics as growth promoters, has become an emerging disease of industrial poultry. Delta toxin itself is cytotoxic to the wide range of human and animal macrophages and platelets that present GM2 ganglioside on their membranes. It has sequence similarity with Staphylococcus aureus β-pore forming toxins and is expected to heptamerize and form pores in the lipid bilayer of host cell membranes. Nevertheless, its exact mode of action remains undetermined. Here we report the 2.4 Å crystal structure of monomeric Delta toxin. The superposition of this structure with the structure of the phospholipid-bound F component of S. aureus leucocidin (LukF) revealed that the glycerol molecules bound to Delta toxin and the phospholipids in LukF are accommodated in the same hydrophobic clefts, corresponding to where the toxin is expected to latch onto the membrane, though the binding sites show significant differences. From structure-based sequence alignment with the known structure of staphylococcal α-hemolysin, a model of the Delta toxin pore form has been built. Using electron microscopy, we have validated our model and characterized the Delta toxin pore on liposomes. These results highlight both similarities and differences in the mechanism of Delta toxin (and by extension NetB) cytotoxicity from that of the staphylococcal pore-forming toxins

Isoprenoids increase bovine endometrial stromal cell tolerance to the cholesterol-dependent cytolysin from Trueperella pyogenes†

Preventing postpartum uterine disease depends on the ability of endometrial cells to tolerate the presence of the bacteria that invade the uterus after parturition. Postpartum uterine disease and endometrial pathology in cattle is most associated with the pathogen Trueperella pyogenes. Trueperella pyogenes secretes a cholesterol-dependent cytolysin, pyolysin, which causes cytolysis by forming pores in the plasma membrane of endometrial stromal cells. The aim of the present study was to identify cell-intrinsic pathways that increase bovine endometrial stromal cell tolerance to pyolysin. Pyolysin caused dose-dependent cytolysis of bovine endometrial stromal cells and leakage of lactate dehydrogenase into supernatants. Cell tolerance to pyolysin was increased by inhibitors that target the mevalonate and cholesterol synthesis pathway, but not the mitogen-activated protein kinase, cell cycle, or metabolic pathways. Cellular cholesterol was reduced and cell tolerance to pyolysin was increased by supplying the mevalonate-derived isoprenoid farnesyl pyrophosphate, or by inhibiting farnesyl-diphosphate farnesyltransferase 1 or geranylgeranyl diphosphate synthase 1 to increase the abundance of farnesyl pyrophosphate. Supplying the mevalonate-derived isoprenoid geranylgeranyl pyrophosphate also increased cell tolerance to pyolysin, but independent of changes in cellular cholesterol. However, geranylgeranyl pyrophosphate inhibits nuclear receptor subfamily 1 group H receptors (NR1H, also known as Liver X receptors), and reducing the expression of the genes encoding NR1H3 or NR1H2 increased stromal cell tolerance to pyolysin. In conclusion, mevalonate-derived isoprenoids increased bovine endometrial stromal cell tolerance to pyolysin, which was associated with reducing cellular cholesterol and inhibiting NR1H receptors