Small Heat-Shock Proteins,IbpAB, Protect Non-Pathogenic Escherichia coli from Killing by Macrophage-Derived Reactive Oxygen Species

Many intracellular bacterial pathogens possess virulence factors that prevent detection and killing by macrophages. However, similar virulence factors in non-pathogenic bacteria are less well-characterized and may contribute to the pathogenesis of chronic inflammatory conditions such as Crohn’s disease. We hypothesize that the small heat shock proteins IbpAB, which have previously been shown to reduce oxidative damage to proteins in vitro and be upregulated in luminal non-pathogenic Escherichia strain NC101 during experimental colitis in vivo, protect commensal E. coli from killing by macrophage-derived reactive oxygen species (ROS). Using real-time PCR, we measured ibpAB expression in commensal E. coli NC101 within wild-type (wt) and ROS-deficient (gp91phox^-/-) macrophages and in NC101 treated with the ROS generator paraquat. We also quantified survival of NC101 and isogenic mutants in wt and gp91phox^-/- macrophages using gentamicin protection assays. Similar assays were performed using a pathogenic E. coli strain O157:H7. We show that non-pathogenic E. coli NC101inside macrophages upregulate ibpAB within 2 hrs of phagocytosis in a ROS-dependent manner and that ibpAB protect E. coli from killing by macrophage-derived ROS. Moreover, we demonstrate that ROS-induced ibpAB expression is mediated by the small E. coli regulatory RNA, oxyS. IbpAB are not upregulated in pathogenic E. coli O157:H7 and do not affect its survival within macrophages. Together, these findings indicate that ibpAB may be novel virulence factors for certain non-pathogenic E. coli strains.     

Anticoagulation for the Acute Management of Ischemic Stroke

Few prospective studies support the use of anticoagulation during the acute phase of ischemic stroke, though observational data suggest a role in certain populations. Depending on the mechanism of stroke, systemic anticoagulation may prevent recurrent cerebral infarction, but concomitantly carries a risk of hemorrhagic transformation. In this article, we describe a case where anticoagulation shows promise for ischemic stroke and review the evidence that has discredited its use in some circumstances while showing its potential in others.     

Structural Maintenance of Chromosome (SMC) Proteins Link Microtubule Stability to Genome Integrity

Structural maintenance of chromosome (SMC) proteins are key organizers of chromosome architecture and are essential for genome integrity. They act by binding to chromatin and connecting distinct parts of chromosomes together. Interestingly, their potential role in providing connections between chromatin and the mitotic spindle has not been explored. Here, we show that yeast SMC proteins bind directly to microtubules and can provide a functional link between microtubules and DNA. We mapped the microtubule-binding region of Smc5 and generated a mutant with impaired microtubule binding activity. This mutant is viable in yeast but exhibited a cold-specific conditional lethality associated with mitotic arrest, aberrant spindle structures, and chromosome segregation defects. In an in vitro reconstitution assay, this Smc5 mutant also showed a compromised ability to protect microtubules from cold-induced depolymerization. Collectively, these findings demonstrate that SMC proteins can bind to and stabilize microtubules and that SMC-microtubule interactions are essential to establish a robust system to maintain genome integrity.     

Flavone Derivatives as Inhibitors of Insulin Amyloid-Like Fibril Formation

Several natural and synthetic flavone derivatives have been reported to inhibit formation of amyloid fibrils or to remodel existing fibrils. These studies suggest that the numbers and positions of hydroxyl groups on the flavone rings determine their effectiveness as amyloid inhibitors. In many studies the primary method for determining the effectiveness of inhibition is measuring Thioflavin T (ThT) fluorescence. This method demonstrably results in a number of false positives for inhibition. We studied the effects of 265 commercially available flavone derivatives on insulin fibril formation. We enhanced the effectiveness of ThT fluorescence measurements by fitting kinetic curves to obtain halftime of aggregation (t ₅₀). Maximal values of ThT fluorescence varied two fold or more in one third of all cases, but this did not correlate with changes in t ₅₀. Changes in t ₅₀ values were more accurate measures of inhibition of amyloid formation. We showed that without a change in an assay, but just by observing complete kinetic curves it is possible to eliminate numbers of false positive and sometimes even false negative results. Examining the data from all 265 flavones we confirmed previous observations that identified the importance of hydroxyl groups for inhibition. Our evidence suggests the importance of hydroxyl groups at locations 5, 6, 7, and 4’, and the absence of a hydroxyl group at location 3, for inhibiting amyloid formation. However, the main conclusion is that the positions are not additive. The structures and their effects must be thought of in the context of the whole molecule.