Selection-Free Zinc-Finger Nuclease Engineering by Context-Dependent Assembly (CoDA)

Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe Context-Dependent Assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA ZFNs, we rapidly altered 20 genes in zebrafish, Arabidopsis, and soybean. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multi-gene pathways or genome-wide alterations

Pedestrian Injury and Human Behaviour: Observing Road-Rule Violations at High-Incident Intersections

Background Human behaviour is an obvious, yet under-studied factor in pedestrian injury. Behavioural interventions that address rule violations by pedestrians and motorists could potentially reduce the frequency of pedestrian injury. In this study, a method was developed to examine road-rule non-compliance by pedestrians and motorists. The purpose of the study was to examine the potential association between violations made by pedestrians and motorists at signalized intersections, and collisions between pedestrians and motor-vehicles. The underlying hypothesis is that high-incident pedestrian intersections are likely to vary with respect to their aetiology, and thus are likely to require individualized interventions – based on the type and rate of pedestrian and motorist violation. Methods High-incident pedestrian injury intersections in Vancouver, Canada were identified using geographic information systems. Road-rule violations by pedestrians and motorists were documented at each incident hotspot by a team of observers at several different time periods during the day. Results Approximately 9,000 pedestrians and 18,000 vehicles were observed in total. In total for all observed intersections, over 2000 (21%) pedestrians committed one of the observed pedestrian road-crossing violations, while approximately 1000 (5.9%) drivers committed one of the observed motorist violations. Great variability in road-rule violations was observed between intersections, and also within intersections at different observation periods. Conclusions Both motorists and pedestrians were frequently observed committing road-rule violations at signalized intersections, suggesting a potential human behavioural contribution to pedestrian injury at the study sites. These results suggest that each intersection may have unique mechanisms that contribute to pedestrian injury, and may require targeted behavioural interventions. The method described in this study provides the basis for understanding the relationship between violations and pedestrian injury risk at urban intersections. Findings could be applied to targeted prevention campaigns designed to reduce the number of pedestrian injuries at signalized intersections

N-AS-triggered SPMs are direct regulators of microglia in a model of Alzheimer???s disease

Sphingosine kinase1 (SphK1) is an acetyl-CoA dependent acetyltransferase which acts on cyclooxygenase2 (COX2) in neurons in a model of Alzheimer's disease (AD). However, the mechanism underlying this activity was unexplored. Here we show that N-acetyl sphingosine (N-AS) is first generated by acetyl-CoA and sphingosine through SphK1. N-AS then acetylates serine 565 (S565) of COX2, and the N-AS-acetylated COX2 induces the production of specialized pro-resolving mediators (SPMs). In a mouse model of AD, microglia show a reduction in N-AS generation, leading to decreased acetyl-S565 COX2 and SPM production. Treatment with N-AS increases acetylated COX2 and N-AS-triggered SPMs in microglia of AD mice, leading to resolution of neuroinflammation, an increase in microglial phagocytosis, and improved memory. Taken together, these results identify a role of N-AS in the dysfunction of microglia in AD. Neuronal sphingosine kinase 1 (SphK1) acetylates COX2 which is needed for microglial phagocytosis activity, and release of pro-resolving mediators (SPMs) from neurons. Here the authors examine how SphK1-mediates COX2 acetylation, and how this leads to increased secretion of SPMs from neurons in the context of Alzheimer's disease models

NLRP3 is activated in Alzheimer's disease and contributes to pathology in APP/PS1 mice.

Alzheimer's disease is the world's most common dementing illness. Deposition of amyloid-β peptide drives cerebral neuroinflammation by activating microglia. Indeed, amyloid-β activation of the NLRP3 inflammasome in microglia is fundamental for interleukin-1β maturation and subsequent inflammatory events. However, it remains unknown whether NLRP3 activation contributes to Alzheimer's disease in vivo. Here we demonstrate strongly enhanced active caspase-1 expression in human mild cognitive impairment and brains with Alzheimer's disease, suggesting a role for the inflammasome in this neurodegenerative disease. Nlrp3(-/-) or Casp1(-/-) mice carrying mutations associated with familial Alzheimer's disease were largely protected from loss of spatial memory and other sequelae associated with Alzheimer's disease, and demonstrated reduced brain caspase-1 and interleukin-1β activation as well as enhanced amyloid-β clearance. Furthermore, NLRP3 inflammasome deficiency skewed microglial cells to an M2 phenotype and resulted in the decreased deposition of amyloid-β in the APP/PS1 model of Alzheimer's disease. These results show an important role for the NLRP3/caspase-1 axis in the pathogenesis of Alzheimer's disease, and suggest that NLRP3 inflammasome inhibition represents a new therapeutic intervention for the disease