Protection of eukaryotic cells against pore-forming toxins from pathogenic bacteria

Bacterial infections are a leading cause of mortality and morbidity. Many species of pathogenic bacteria secrete pore-forming toxins to damage eukaryotic cells and facilitate pathogen invasion. Although cells can repair this damage, little is known about the intrinsic protection of cells against these toxins. Side-chain oxysterols and steroids can reduce the severity of bacterial diseases by suppressing immunity. Here we tested the hypothesis that oxysterols and steroids might also enhance the intrinsic protection of eukaryotic cells against pore-forming toxins. We first used the cholesterol-dependent cytolysin pyolysin, which forms pores in bovine endometrial cells. We found that 25-hydroxycholesterol or 27-hydroxycholesterol treatment protected bovine endometrial cells against pyolysin, and that these oxysterols are present in the reproductive tract. The oxysterols reduced pyolysin-induced leakage of potassium and lactate dehydrogenase by > 65%, limited changes to the actin cytoskeleton, and prevented cytolysis. The oxysterols also protected human cervical, lung, and liver epithelial cells against pyolysin damage, and protected cells against Staphylococcus aureus α-hemolysin. Mechanistically, oxysterol cytoprotection was partially dependent on activating acetyl-coenzyme A acetyltransferase and liver X receptors. The steroids, progesterone, oestradiol, or hydrocortisone were not protective in bovine endometrial cells. However, hydrocortisone and dexamethasone protected several types of human cells against pyolysin, reducing cytolysis from > 75% to < 25%, via a glucocorticoid receptor dependent mechanism. Treatment with these glucocorticoids also protected human cells against α-hemolysin and another cholesterol-dependent cytolysin, streptolysin O. However, glucocorticoid cytoprotection was reversibly blocked by the presence of ≥ 4% serum, which led to the discovery that glucocorticoid cytoprotection depended on the rate limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase. In conclusion, side-chain oxysterols and glucocorticoids enhance the intrinsic protection of eukaryotic cells against pore-forming toxins. These findings imply that oxysterols and glucocorticoids could help limit the severity of disease caused by pathogens that secrete pore-forming toxins

Manipulating bovine granulosa cell energy metabolism limits inflammation

Bovine granulosa cells are often exposed to energy stress, due to the energy demands of lactation, and exposed to lipopolysaccharide from postpartum bacterial infections. Granulosa cells mount innate immune responses to lipopolysaccharide, including the phosphorylation of mitogen-activated protein kinases and production of pro-inflammatory interleukins. Cellular energy depends on glycolysis, and energy stress activates intracellular AMPK (AMP-activated protein kinase), which in turn inhibits mTOR (mechanistic target of rapamycin). Here, we tested the hypothesis that manipulating glycolysis, AMPK or mTOR to mimic energy stress in bovine granulosa cells limits the inflammatory responses to lipopolysaccharide. We inhibited glycolysis, activated AMPK or inhibited mTOR in granulosa cells isolated from 4–8 mm and > 8.5 mm diameter ovarian follicles, and then challenged the cells with lipopolysaccharide and measured the production of interleukins IL-1α, IL-1β, and IL-8. We found that inhibiting glycolysis with 2-deoxy-D-glucose reduced lipopolysaccharide-stimulated IL-1α > 80%, IL-1β > 90%, and IL-8 > 65% in granulosa cells from 4–8 mm and > 8.5 mm diameter ovarian follicles. Activating AMPK with AICAR also reduced lipopolysaccharide-stimulated IL-1α > 60%, IL-1β > 75%, and IL-8 > 20%, and shortened the duration of lipopolysaccharide-stimulated phosphorylation of the mitogen-activated protein kinase ERK1/2 and JNK. However, only the mTOR inhibitor Torin 1, and not rapamycin, reduced lipopolysaccharide-stimulated IL-1α and IL-1β. In conclusion, manipulating granulosa cell energy metabolism with a glycolysis inhibitor, an AMPK activator, or an mTOR inhibitor, limited inflammatory responses to lipopolysaccharide. Our findings imply that energy stress compromises ovarian follicle immune defences

Attributional perceptions of dating outcomes

Attributional responses made by undergraduate college students to interpersonal relationship outcomes were investigated to determine if the actor-observer effect exists in a similar manner for successful and unsuccessful dating situations, or if the effect is mediated by an egoserving bias tendency. Participants rated the influence of the Weinerian Factors of ability, effort, luck and task difficulty on successful and unsuccessful dating outcomes in terms of Self, an Other Male, or an Other Female. Participants attributed a significantly greater amount of internal responsibility to the others as compared to self for unsuccessful dating outcomes but not for successful outcomes. Additionally, participants, when responding to Self outcomes, were shown to take more responsibility for successful situations as compared to unsuccessful situations. This general pattern was extended to Other Female, but not to Other Male

Yersinia ruckeri isolates recovered from diseased Atlantic Salmon (Salmo salar) in Scotland are more diverse than those from Rainbow Trout (Oncorhynchus mykiss) and represent distinct subpopulations

Yersinia ruckeri is the etiological agent of enteric redmouth (ERM) disease of farmed salmonids. Enteric redmouth disease is traditionally associated with rainbow trout (Oncorhynchus mykiss, Walbaum), but its incidence in Atlantic salmon (Salmo salar) is increasing. Yersinia ruckeri isolates recovered from diseased Atlantic salmon have been poorly characterized, and very little is known about the relationship of the isolates associated with these two species. Phenotypic approaches were used to characterize 109 Y. ruckeri isolates recovered over a 14-year period from infected Atlantic salmon in Scotland; 26 isolates from infected rainbow trout were also characterized. Biotyping, serotyping, and comparison of outer membrane protein profiles identified 19 Y. ruckeri clones associated with Atlantic salmon but only five associated with rainbow trout; none of the Atlantic salmon clones occurred in rainbow trout and vice versa. These findings suggest that distinct subpopulations of Y. ruckeri are associated with each species. A new O serotype (designated O8) was identified in 56 biotype 1 Atlantic salmon isolates and was the most common serotype identified from 2006 to 2011 and in 2014, suggesting an increased prevalence during the time period sampled. Rainbow trout isolates were represented almost exclusively by the same biotype 2, serotype O1 clone that has been responsible for the majority of ERM outbreaks in this species within the United Kingdom since the 1980s. However, the identification of two biotype 2, serotype O8 isolates in rainbow trout suggests that vaccines containing serotypes O1 and O8 should be evaluated in both rainbow trout and Atlantic salmon for application in Scotland

Oxysterols protect bovine endometrial cells against pore‐forming toxins from pathogenic bacteria

Many species of pathogenic bacteria secrete toxins that form pores in mammalian cell membranes. These membrane pores enable the delivery of virulence factors into cells, result in the leakage of molecules that bacteria can use as nutrients, and facilitate pathogen invasion. Inflammatory responses to bacteria are regulated by the side-chain-hydroxycholesterols 27-hydroxycholesterol and 25-hydroxycholesterol, but their effect on the intrinsic protection of cells against pore-forming toxins is unclear. Here, we tested the hypothesis that 27-hydroxycholesterol and 25-hydroxycholesterol help protect cells against pore-forming toxins. We treated bovine endometrial epithelial and stromal cells with 27-hydroxycholesterol or 25-hydroxycholesterol, and then challenged the cells with pyolysin, which is a cholesterol-dependent cytolysin from Trueperella pyogenes that targets these endometrial cells. We found that treatment with 27-hydroxycholesterol or 25-hydroxycholesterol protected both epithelial and stomal cells against pore formation and the damage caused by pyolysin. The oxysterols limited pyolysin-induced leakage of potassium and lactate dehydrogenase from cells, and reduced cytoskeletal changes and cytolysis. This oxysterol cytoprotection against pyolysin was partially dependent on reducing cytolysin-accessible cholesterol in the cell membrane and on activating liver X receptors. Treatment with 27-hydroxycholesterol also protected the endometrial cells against Staphylococcus aureus α hemolysin. Using mass spectrometry, we found 27-hydroxycholesterol and 25-hydroxycholesterol in uterine and follicular fluid. Furthermore, epithelial cells released additional 25-hydroxycholesterol in response to pyolysin. In conclusion, both 27-hydroxycholesterol and 25-hydroxycholesterol increased the intrinsic protection of bovine endometrial cells against pore-forming toxins. Our findings imply that side-chain-hydroxycholesterols may help defend the endometrium against pathogenic bacteria

Apparatus and method for centrifugation and robotic manipulation of samples

A device for centrifugation and robotic manipulation of specimen samples, including incubating eggs, and uses thereof are provided. The device may advantageously be used for the incubation of avian, reptilian or any type of vertebrate eggs. The apparatus comprises a mechanism for holding samples individually, rotating them individually, rotating them on a centrifuge collectively, injecting them individually with a fixative or other chemical reagent, and maintaining them at controlled temperature, relative humidity and atmospheric composition. The device is applicable to experiments involving entities other than eggs, such as invertebrate specimens, plants, microorganisms and molecular systems

Cytomegalovirus distribution and evolution in hominines

Herpesviruses are thought to have evolved in very close association with their hosts. This is notably the case for cytomegaloviruses (CMVs; genus Cytomegalovirus) infecting primates, which exhibit a strong signal of co-divergence with their hosts. Some herpesviruses are however known to have crossed species barriers. Based on a limited sampling of CMV diversity in the hominine (African great ape and human) lineage, we hypothesized that chimpanzees and gorillas might have mutually exchanged CMVs in the past. Here, we performed a comprehensive molecular screening of all 9 African great ape species/subspecies, using 675 fecal samples collected from wild animals. We identified CMVs in eight species/subspecies, notably generating the first CMV sequences from bonobos. We used this extended dataset to test competing hypotheses with various degrees of co-divergence/number of host switches while simultaneously estimating the dates of these events in a Bayesian framework. The model best supported by the data involved the transmission of a gorilla CMV to the panine (chimpanzee and bonobo) lineage and the transmission of a panine CMV to the gorilla lineage prior to the divergence of chimpanzees and bonobos, more than 800,000 years ago. Panine CMVs then co-diverged with their hosts. These results add to a growing body of evidence suggesting that viruses with a double-stranded DNA genome (including other herpesviruses, adenoviruses, and papillomaviruses) often jumped between hominine lineages over the last few million years.Peer Reviewe

2015 Research & Innovation Day Program

A one day showcase of applied research, social innovation, scholarship projects and activities.https://first.fanshawec.ca/cri_cripublications/1002/thumbnail.jp