Biology subjects

O'Connor, K. M.

Publications and source records attributed to O'Connor, K. M..

6 recordsLinked to original sources

bioRxiv2022

The translating bacterial ribosome at 1.55 A resolution by open access cryo-EM

Our understanding of protein synthesis has been conceptualised around the structure and function of the bacterial ribosome1-4. This complex macromolecular machine is the target of important antimicrobial drugs5, an integral line of defence against infectious diseases. Here, we describe how open access to state-of-the-art cryogenic electron microscopy facilities combined with bespoke user support offered by the newly established EMBL Imaging Centre enabled structural determination of the translating ribosome from Escherichia coli at 1.55 [A] resolution. The obtained structures allow for direct determination of the rRNA sequence to identify ribosome polymorphism sites in the E. coli strain used in this study and enables interpretation of the ribosomal active and peripheral sites at unprecedented resolution. This includes scarcely populated chimeric hybrid states of the ribosome engaged in several tRNA translocation steps resolved at ~2 [A] resolution. The current map not only improves our understanding of protein synthesis but also allows for more precise structure-based drug design of antibiotics to tackle rising bacterial resistance.

molecular biology↗

bioRxiv2020

Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome

Programmed ribosomal frameshifting is the key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream viral proteins. Here we present the cryo-EM structure of the mammalian ribosome in the process of translating viral RNA paused in a conformation primed for frameshifting. We observe that the viral RNA adopts a pseudoknot structure lodged at the mRNA entry channel of the ribosome to generate tension in the mRNA that leads to frameshifting. The nascent viral polyprotein that is being synthesized by the ribosome paused at the frameshifting site forms distinct interactions with the ribosomal polypeptide exit tunnel. We use biochemical experiments to validate our structural observations and to reveal mechanistic and regulatory features that influence the frameshifting efficiency. Finally, a compound previously shown to reduce frameshifting is able to inhibit SARS-CoV-2 replication in infected cells, establishing coronavirus frameshifting as target for antiviral intervention.

molecular biology↗

bioRxiv2020

Instrumented immobilizing boot quantifies reduced Achilles tendon loading during gait

Achilles tendon ruptures are common injuries that lead to functional deficits in two-thirds of patients. Progressively loading the healing tendon has been associated with superior outcomes, but the loading profiles that patients experience throughout rehabilitation have not yet been established. In this study, we developed and calibrated an instrumented immobilizing boot paradigm that is aimed at longitudinally quantifying patient loading biomechanics to develop personalized rehabilitation protocols. We used a 3-part instrumented insole to quantify the ankle loads generated by the Achilles tendon and secured a load cell in-line with the posterior strut of the immobilizing boot to quantify boot loading. We then collected gait data from five healthy young adults to demonstrate the validity of this instrumented immobilizing boot paradigm to assess Achilles tendon loading during ambulation. We developed a simple calibration procedure to improve the measurement fidelity of the instrumented insole needed to quantify Achilles tendon loading while ambulating with an immobilizing boot. By assessing Achilles tendon loading with the ankle constrained to 0 degrees and 30 degrees plantar flexion, we confirmed that walking with the foot supported in plantar flexion decreased Achilles tendon loading by 60% (P < 0.001). This instrumented immobilizing boot paradigm leverages commercially available sensors and logs data using a small microcontroller secured to the boot and a handheld device, making our paradigm capable of continuously monitoring biomechanical loading outside of the lab or clinic.

bioengineering↗

bioRxiv2019

Ultrasound Echogenicity is Associated with Achilles Tendon Fatigue Damage in a Cadaveric Loading Model

Achilles tendon disorders are among the most difficult sports-related injuries to predict with current diagnostic tools. The purpose of this study was to identify a clinically useful marker for early tendon damage. We hypothesized that alterations in mean echogenicity are linked with changes in vitro tendon mechanics. To test our hypothesis, we harvested Achilles tendons from 10 fresh-frozen cadaveric feet and cyclically fatigued them using a universal test frame while we continuously acquired ultrasound images. Throughout this fatigue protocol, we applied 2 stress tests every 500 loading cycles to quantify changes in ultrasound imaging echogenicity. We continued this fatigue protocol until each tendon either failed completely or survived 150,000 cycles. Tendons that failed during the fatigue loading (6/10) underwent greater changes in mean echogenicity compared to tendons that did not fail (P = 0.031). These tendons that failed during fatigue loading demonstrated greater changes in mean echogenicity that surpassed 1.0%; whereas survivor tendons exhibited less than 0.5% changes in mean echogenicity. We found that changes in mean echogenicity measured with ultrasound increased proportionally with increased tendon damage. The magnitude of these changes was relatively small (<1.5% change in mean echogenicity) but may be an effective predictor of tendon failure. Mean echogenicity is a promising marker for quantifying fatigue damage in cadaveric Achilles tendons during a stress test. Although these changes cannot be detected with the naked eye, computer-based predictive models may effectively assess risk of tendon damage in physically active adults. Level of evidenceControlled laboratory experiment

bioengineering↗

bioRxiv2019

Gastrocnemius muscle remodeling explains functional deficits three months following Achilles tendon rupture

Plantarflexor functional deficits are associated with poor outcomes in patients following Achilles tendon rupture. In this longitudinal study, we analyzed the fascicle length and pennation angle of the medial gastrocnemius muscle and the length of the Achilles tendon using ultrasound imaging. To determine the relationship between muscle remodeling and functional deficits measured at 3 months after injury, we correlated the reduction in fascicle length and increase in pennation angle with peak torque measured during isometric plantarflexor contractions and peak power measured during isokinetic plantarflexor contractions. We found that the medial gastrocnemius underwent an immediate change in structure, characterized by decreased length and increased pennation of the muscle fascicles. This decrease in fascicle length was coupled with an increase in tendon length. These changes in muscle-tendon structure persisted throughout the first three months following rupture. Deficits in peak plantarflexor power were moderately correlated with decreased fascicle length at 120 degrees per second (R2 = 0.424, P = 0.057) and strongly correlated with decreased fascicle length at 210 degrees per second (R2 = 0.737, P = 0.003). However, increases in pennation angle did not explain functional deficits. These findings suggest that muscle-tendon structure is detrimentally affected following Achilles tendon rupture. Plantarflexor power deficits are positively correlated with the magnitude of reductions in fascicle length. Preserving muscle structure following Achilles tendon rupture should be a clinical priority to maintain patient function.

physiology↗

bioRxiv2018

Gastrocnemius fascicles are shorter and more pennate immediately following acute Achilles tendon rupture

The purpose of this study was to characterize the short-term effects of Achilles tendon ruptures on medial gastrocnemius. We hypothesized that the fascicles of the medial gastrocnemius muscle of the injured Achilles tendon would be shorter and more pennate immediately following the injury and would persist throughout 4 weeks post-injury. B-mode longitudinal ultrasound images of the medial gastrocnemius were acquired in 10 adults who suffered acute Achilles tendon ruptures and were treated non-operatively. Ultrasound images were acquired during the initial clinical visit following injury as well as two and four weeks following this initial clinical visit. Resting muscle structure was characterized by measuring fascicle length, pennation angle, muscle thickness, and muscle echo intensity in both the injured and contralateral (control) limbs. Fascicle length was 15% shorter (P < 0.001) and pennation angle was 21% greater (P < 0.001) in the injured muscle compared to the uninjured (control) muscle at the presentation of injury (week 0). These differences in fascicle length and pennation angle persisted throughout the 4 weeks after the injury (P < 0.008). Muscle thickness changes were not detected at any of the post-injury visits (difference < 4%, P > 0.04). Echo intensity of the injured limb was 8% lower at the presentation of the injury and 11% lower (P = 0.008) than the contralateral muscle at 2 weeks following injury (P < 0.001) but returned to within 1% by 4 weeks (P = 0.393). Our results suggest that Achilles tendon ruptures elicit rapid changes in the configuration and quality of the medial gastrocnemius, which may explain long-term functional deficits.

bioengineering↗