Transverse magnetic tweezers allowing coincident epi-fluorescence microscopy on horizontally extended DNA

Longitudinal magnetic tweezers (L-MT) have seen wide-scale adoption as the tool-of-choice for stretching and twisting a single DNA molecule. They are also used to probe topological changes in DNA as a result of protein binding and enzymatic activity. However, in the longitudinal configuration, the DNA molecule is extended perpendicular to the imaging plane. As a result, it is only possible to infer biological activity from the motion of the tethered superparamagnetic microsphere. Described here is a “transverse” magnetic tweezers (T-MT) geometry featuring simultaneous control of DNA extension and spatially coincident video-rate epi-fluorescence imaging. Unlike in L-MT, DNA tethers in T-MT are extended parallel to the imaging plane between two micron-sized spheres, and importantly protein targets on the DNA can be localised using fluorescent nanoparticles. The T-MT can manipulate a long DNA construct at molecular extensions approaching the contour length defined by B-DNA helical geometry, and the measured entropic elasticity agrees with the worm-like chain model (force < 35 pN). By incorporating a torsionally constrained DNA tether, the T-MT would allow both the relative extension and twist of the tether to be manipulated, while viewing far-red emitting fluorophore-labelled targets. This T-MT design has the potential to enable the study of DNA binding and remodelling processes under conditions of constant force and defined torsional stress

Trends in match injury risk in professional male rugby union: a 16-season review of 10851 match injuries in the English Premiership (2002-2019):The Professional Rugby Injury Surveillance Project

Objectives The Professional Rugby Injury Surveillance Project is the largest and longest running rugby union injury surveillance project globally and focuses on the highest level of rugby in England.Methods We examined match injuries in professional men’s rugby over the period 2002/2003 to 2018/2019 and described trends in injuries over this time.Results Over the period 2002/2003–2018/2019, 10 851 injuries occurred in 1 24 952 hours of match play, equating to a mean of 57 injuries per club per season and one injury per team per match. The mean incidence, severity (days absence) and burden (days absence/1000 hours) of injury were 87/1000 hours (95% CI 82 to 92), 25 days (95% CI 22 to 28) and 2178 days/1000 hours (95% CI 1872 to 2484), respectively. The tackle accounted for 43% injuries with running the second most common activity during injury (12%). The most common injury location was the head/face with an incidence of 11.3/1000 hours, while the location with the highest overall burden was the knee (11.1 days/1000 hours). Long-term trends demonstrated stable injury incidence and proportion of injured players, but an increase in the mean and median severity of injuries. Concussion incidence, severity and burden increased from the 2009/2010 season onwards and from 2011 to 2019 concussion was the most common injury.Conclusion The rise in overall injury severity and concussion incidence are the most significant findings from this work and demonstrate the need for continued efforts to reduce concussion risk as well as a greater understanding of changes in injury severity over time

Apollo Lightcraft project

The detailed design of a beam-powered transatmospheric vehicle, the Apollo Lightcraft, was selected as the project for the design course. The principal goal is to reduce the LEO payload delivery cost by at least three orders of magnitude below the Space Shuttle Orbiter in the post 2020 era. The completely reusable, single-stage-to-orbit shuttlecraft will take off and land vertically, and have a reentry heat shield integrated with its lower surface. At appropriate points along the launch trajectory, the combined cycle propulsion system will transition through three or four airbreathing modes, and finally use a pure rocket mode for orbital insertion. The objective for the Spring semester propulsion source was to design and perform a detailed theoretical analysis on an advanced combined-cycle engine suitable for the Apollo Lightcraft. The preliminary theoretical analysis of this combined-cycle engine is now completed, and the acceleration performance along representative orbital trajectories was simulated. The total round trip cost is $3430 or$686 per person. This represents a payload delivery cost of $3.11/lb, which is a factor of 1000 below the STS. The Apollo Lightcraft concept is now ready for a more detailed investigation during the Fall semester Transatmosphere Vehicle Design course

Patterns of training volume and injury risk in elite rugby union: an analysis of 1.5 million hours of training exposure over eleven seasons

Rugby union is a popular team sport that demands high levels of physical fitness and skill. The study aim was to examine trends in training volume and its impact on injury incidence, severity and burden over an 11-season period in English professional rugby. Data were recorded from 2007/08 through 2017/18, capturing 1,501,606 h of training exposure and 3,782 training injuries. Players completed, on average, 6 h 48 minutes of weekly training (95% CI: 6 h 30 mins to 7 h 6 mins): this value remained stable over the 11 seasons. The mean incidence of training-related injuries was 2.6/1000 player-hours (95% CI: 2.4 to 2.8) with a mean severity rising from 17 days in 2007/08 to 37 days in 2017/18 (Change/season = 1.773, P &lt;0.01). Rate of change in severity was dependent on training type, with conditioning (non-gym-based) responsible for the greatest increase (2.4 days/injury/season). As a result of increasing severity, injury burden rose from 51 days absence/1000 player-hours in 2007/08 to 106 days’ absence/1000 player-hours in 2017/18. Despite the low incidence of injury in training compared to match-play, training accounted for 34% of all injuries. Future assessments of training intensity may lead to a greater understanding of the rise in injury severity.</p

Live imaging the foreign body response in zebrafish reveals how dampening inflammation reduces fibrosis

Implanting biomaterials in tissues leads to inflammation and a foreign body response (FBR), which can result in rejection. Here, we live image the FBR triggered by surgical suture implantation in a translucent zebrafish model and compare with an acute wound response. We observe inflammation extending from the suture margins, correlating with subsequent avascular and fibrotic encapsulation zones: sutures that induce more inflammation result in increased zones of avascularity and fibrosis. Moreover, we capture macrophages as they fuse to become multinucleate foreign body giant cells (FBGCs) adjacent to the most pro-inflammatory sutures. Genetic and pharmacological dampening of the inflammatory response minimises the FBR (including FBGC generation) and normalises the status of the tissue surrounding these sutures. This model of FBR in adult zebrafish allows us to live image the process and to modulate it in ways that may lead us towards new strategies to ameliorate and circumvent FBR in humans. This article has an associated First Person interview with the first author of the paper

Inhibition of the classical pathway of the complement cascade prevents early dendritic and synaptic degeneration in glaucoma

BACKGROUND: Glaucoma is a complex, multifactorial disease characterised by the loss of retinal ganglion cells and their axons leading to a decrease in visual function. The earliest events that damage retinal ganglion cells in glaucoma are currently unknown. Retinal ganglion cell death appears to be compartmentalised, with soma, dendrite and axon changes potentially occurring through different mechanisms. There is mounting evidence from other neurodegenerative diseases suggesting that neuronal dendrites undergo a prolonged period of atrophy, including the pruning of synapses, prior to cell loss. In addition, recent evidence has shown the role of the complement cascade in synaptic pruning in glaucoma and other diseases. RESULTS: Using a genetic (DBA/2J mouse) and an inducible (rat microbead) model of glaucoma we first demonstrate that there is loss of retinal ganglion cell synapses and dendrites at time points that precede axon or soma loss. We next determine the role of complement component 1 (C1) in early synaptic loss and dendritic atrophy during glaucoma. Using a genetic knockout of C1qa (D2.C1qa (-/-) mouse) or pharmacological inhibition of C1 (in the rat bead model) we show that inhibition of C1 is sufficient to preserve dendritic and synaptic architecture. CONCLUSIONS: This study further supports assessing the potential for complement-modulating therapeutics for the prevention of retinal ganglion cell degeneration in glaucoma. Mol Neurodegener 2016 Apr 6; 11(2):2

Electroconvection in a Suspended Fluid Film: A Linear Stability Analysis

A suspended fluid film with two free surfaces convects when a sufficiently large voltage is applied across it. We present a linear stability analysis for this system. The forces driving convection are due to the interaction of the applied electric field with space charge which develops near the free surfaces. Our analysis is similar to that for the two-dimensional B\'enard problem, but with important differences due to coupling between the charge distribution and the field. We find the neutral stability boundary of a dimensionless control parameter ${\cal R}$ as a function of the dimensionless wave number ${\kappa}$. ${\cal R}$, which is proportional to the square of the applied voltage, is analogous to the Rayleigh number. The critical values ${{\cal R}_c}$ and ${\kappa_c}$ are found from the minimum of the stability boundary, and its curvature at the minimum gives the correlation length ${\xi_0}$. The characteristic time scale ${\tau_0}$, which depends on a second dimensionless parameter ${\cal P}$, analogous to the Prandtl number, is determined from the linear growth rate near onset. ${\xi_0}$ and ${\tau_0}$ are coefficients in the Ginzburg-Landau amplitude equation which describes the flow pattern near onset in this system. We compare our results to recent experiments.Comment: 36 pages, 7 included eps figures, submitted to Phys Rev E. For more info, see http://mobydick.physics.utoronto.ca

Annular electroconvection with shear

We report experiments on convection driven by a radial electrical force in suspended annular smectic A liquid crystal films. In the absence of an externally imposed azimuthal shear, a stationary one-dimensional (1D) pattern consisting of symmetric vortex pairs is formed via a supercritical transition at the onset of convection. Shearing reduces the symmetries of the base state and produces a traveling 1D pattern whose basic periodic unit is a pair of asymmetric vortices. For a sufficiently large shear, the primary bifurcation changes from supercritical to subcritical. We describe measurements of the resulting hysteresis as a function of the shear at radius ratio $\eta \sim 0.8$. This simple pattern forming system has an unusual combination of symmetries and control parameters and should be amenable to quantitative theoretical analysis.Comment: 12 preprint pages, 3 figures in 2 parts each. For more info, see http://mobydick.physics.utoronto.c

Nitrolinoleate inhibits superoxide generation, degranulation, and integrin expression by human neutrophils: novel antiinflammatory properties of nitric oxide-derived reactive species in vascular cells

Nitration of unsaturated fatty acids such as linoleate by NO-derived reactive species forms novel derivatives (including nitrolinoleate [LNO2]) that can stimulate smooth muscle relaxation and block platelet activation by either NO/cGMP or cAMP-dependent mechanisms. Here, LNO2 was observed to inhibit human neutrophil function. LNO2, but not linoleic acid or the nitrated amino acid 3-nitrotyrosine, dose-dependently (0.2 to 1 µmol/L) inhibited superoxide (O2·-) generation, Ca2+ influx, elastase release, and CD11b expression in response to either phorbol 12-myristate 13-acetate or N-formyl-Met-Leu-Phe. LNO2 did not elevate cGMP, and inhibition of guanylate cyclase by 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one did not restore neutrophil responses, ruling out a role for NO. In contrast, LNO2 caused elevations in intracellular cAMP in the presence and absence of phosphodiesterase inhibition, suggesting activation of adenylate cyclase. Compared with phorbol 12-myristate 13-acetate–activated neutrophils, N-formyl-Met-Leu-Phe–activated neutrophils were more susceptible to the inhibitory effects of LNO2, indicating that LNO2 may inhibit signaling both upstream and downstream of protein kinase C. These data suggest novel signaling actions for LNO2 in mediating its potent inhibitory actions. Thus, nitration of lipids by NO-derived reactive species yields products with antiinflammatory properties, revealing a novel mechanism by which NO-derived nitrated biomolecules can influence the progression of vascular disease