Dataset Information

Gold rotor bead tracking for high-speed measurements of DNA twist, torque and extension.

ABSTRACT: Single-molecule measurements of DNA twist and extension have been used to reveal physical properties of the double helix and to characterize structural dynamics and mechanochemistry in nucleoprotein complexes. However, the spatiotemporal resolution of twist measurements has been limited by the use of angular probes with high rotational drag, which prevents detection of short-lived intermediates or small angular steps. We introduce gold rotor bead tracking (AuRBT), which yields >100× improvement in time resolution over previous techniques. AuRBT employs gold nanoparticles as bright low-drag rotational and extensional probes, which are monitored by instrumentation that combines magnetic tweezers with objective-side evanescent darkfield microscopy. Our analysis of high-speed structural dynamics of DNA gyrase using AuRBT revealed an unanticipated transient intermediate. AuRBT also enables direct measurements of DNA torque with >50× shorter integration times than previous techniques; we demonstrated high-resolution torque spectroscopy by mapping the conformational landscape of a Z-forming DNA sequence.

SUBMITTER: Lebel P

PROVIDER: S-EPMC4211898 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Gold rotor bead tracking for high-speed measurements of DNA twist, torque and extension.

Lebel Paul P Basu Aakash A Oberstrass Florian C FC Tretter Elsa M EM Bryant Zev Z

Nature methods 20140223 4

Single-molecule measurements of DNA twist and extension have been used to reveal physical properties of the double helix and to characterize structural dynamics and mechanochemistry in nucleoprotein complexes. However, the spatiotemporal resolution of twist measurements has been limited by the use of angular probes with high rotational drag, which prevents detection of short-lived intermediates or small angular steps. We introduce gold rotor bead tracking (AuRBT), which yields >100× improvement ...[more]

PMID: 24562422

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Project description:BackgroundSlow gait speed is a powerful predictor of disability in activities of daily living and mortality. Muscle strength and body composition change over time, but their changes differ by sex. How these parameters jointly affect gait speed decline is unknown. Understanding this association could help develop and evaluate the sex-specific effects of lifestyle interventions to delay gait speed decline in older adults. We assessed whether changes in strength (Δstrength), appendicular lean mass (ΔALM), and fat mass (Δfat) jointly relate to change in gait speed and whether the association differs by sex.MethodsThe analytic sample comprised 575 women and 539 men aged 22-95 years enrolled in the Baltimore Longitudinal Study of Aging. Mean follow-up was 4.0 years. Measures included isometric knee extension strength, dual-energy X-ray absorptiometry-assessed ALM and fat mass, and gait speed from the 400 m fast pace walk. Sex-specific linear mixed models were adjusted for follow-up time and baseline age, race, height, ALM, fat mass, peak torque, and gait speed. We also included second-order interaction terms of the key predictive variables (e.g. Δstrength × ΔALM). To interpret the interactions, we estimated average gait declines using the 25th or 75th percentile of the two significant predictive variables and then assessed which condition relates to larger decline in gait speed.ResultsIn both sexes, independent of ΔALM and Δfat, larger decline in strength significantly related to larger decline in gait speed (P = 0.01 for both sexes). In men, interactions between Δstrength × ΔALM and Δfat by ΔALM were associated with change in gait speed; men with greater declines in both muscle strength and ALM or greater declines in both ALM and fat have steeper gait speed decline. In contrast, in women, the interaction between Δfat and ΔALM was associated with change in gait speed; women with an increase in fat mass combined with less decline in ALM have steeper gait speed decline.ConclusionsWhile change in strength affects change in gait speed in both sexes, the effects of body composition change differ by sex. Dual-energy X-ray absorptiometry-based estimates of lean mass may be confounded by intramuscular fat. Future studies should examine sex-specific combined effects of change in strength and body composition on mobility using multiple techniques to measure body composition. Intervention studies should consider testing sex-specific interventions on body composition.

Project description:Proteins lack the duplication mechanism like nucleic acids, so the connection of immunoassays with effective nucleic acid amplification techniques has become a powerful way for the detection of trace protein biomarkers in biological fluids. However, such immunoassays generally suffer from rather stringent DNA sequence design and complicated operations. Herein, we propose a simple but highly sensitive flow cytometric immunoassay (FCI) by employing on-bead terminal deoxynucleotidyl transferase (TdT)-initiated template-free DNA extension as an effective signal amplification pathway (TdT-FCI), and gold nanoparticles (AuNPs) co-functionalized with both the detection antibody and a 3'-OH oligonucleotide (ODN) as the transducer to bridge the immunoassay and subsequent TdT-mediated DNA amplification. The target antigen can sandwich with the capture antibody immobilized on the magnetic beads (MBs) and the detection antibody on the AuNPs to bring a lot of ODNs onto the surface of MBs. Each ODN on the MBs can be effectively elongated by TdT in a template-free manner to produce a long poly(T) tail, which will then bind to many 6-carboxyfluorescein (FAM)-labeled poly(A)25. Since each AuNP can carry multiple ODNs and each extended ODN can ultimately capture numerous FAM-poly(A)25, efficiently amplified fluorophore accumulation on the MBs can be achieved. The fluorescent MBs can be individually interrogated with a flow cytometer and thus quantitative analysis of the target antigen can be realized. Coupled with the powerful flow cytometry analysis, the simple but efficient TdT-based signal amplification mechanism has pushed the detection limit of prostate specific antigen (PSA) down to a low level of 0.5 pg mL-1. Furthermore, based on an elegant bead size-encoding principle, we have further advanced the TdT-FCI for multiplexed antigen detection in a single reaction. Sharing the unique merits of simple design and operation, efficient signal amplification, powerful signal readout and the capability for multiplexed analysis, this TdT-FCI provides a versatile tool for detecting trace antigen biomarkers towards clinical diagnosis as well as prognosis.

Project description:BackgroundWith aging, the ability to generate muscle force decreases, contributing to declines in physical functions such as walking. While most studies assess muscle force by peak torque, the rate of torque development (RTD) reflects a dynamic component of muscle performance that is important for physical function. Using data from the Baltimore Longitudinal Study of Aging, we assessed whether RTD adds significantly to peak torque in associations with lower extremity performance. If so, RTD may help identify weak older adults for screening and intervention.MethodsWe assessed associations of RTD and peak torque with physical performance independent of demographics, BMI, body composition, and each other in 1089 Baltimore Longitudinal Study of Aging participants (49.7% women; aged 26 to 96 years; women, 64.0 ± 13.8 years; men, 68.4 ± 14.4 years). Peak torque was assessed by isometric and 30 deg/s isokinetic knee extension tests. Peak RTD was operationalized as the maximum torque-time slope among successive 50 ms epochs over the first 3 s of a test of knee extension isometric strength, with the knee joint positioned at 120 deg of flexion. A battery of lower extremity performance tests included gait speed during a 6 m walk at usual and fast pace (6 m usual and fast), time to complete a 400 m walk at fast pace (400 m), distance covered in a 2.5 min walk at normal pace (2.5 min), time to complete 5 and 10 chair stands, and two summary tests of lower extremity performance. Sex-stratified generalized linear regression models were adjusted for age, race, BMI, appendicular lean mass, and whole body fat mass.ResultsIn men, independent of either measure of peak torque and cofactors, RTD was a significant (P < 0.05) predictor of all lower extremity performance tests except the 400 m and 2.5 min walks. In women, independent of peak torque, RTD was only a significant independent correlate of the 6 m fast walk (P < 0.001).ConclusionsRTD independently contributes to physical functions in men but less in women. The mechanisms underlying the sex difference are unclear and require further study.

Dataset Information

Gold rotor bead tracking for high-speed measurements of DNA twist, torque and extension.

Publications

Gold rotor bead tracking for high-speed measurements of DNA twist, torque and extension.

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