Nanoscale imaging using differential expansion microscopy.

Expensive and time-consuming approaches of immunoelectron microscopy of biopsy tissues continues to serve as the gold-standard for diagnostic pathology. The recent development of the new approach of expansion microscopy (ExM) capable of fourfold lateral expansion of biological specimens for their morphological examination at approximately 70 nm lateral resolution using ordinary diffraction limited optical microscopy, is a major advancement in cellular imaging. Here we report (1) an optimized fixation protocol for retention of cellular morphology while obtaining optimal expansion, (2) an ExM procedure for up to eightfold lateral and over 500-fold volumetric expansion, (3) demonstrate that ExM is anisotropic or differential between tissues, cellular organelles and domains within organelles themselves, and (4) apply image analysis and machine learning (ML) approaches to precisely assess differentially expanded cellular structures. We refer to this enhanced ExM approach combined with ML as differential expansion microscopy (DiExM), applicable to profiling biological specimens at the nanometer scale. DiExM holds great promise for the precise, rapid and inexpensive diagnosis of disease from pathological specimen slides.

Self-Assembly and Biogenesis of the Cellular Membrane are Dictated by Membrane Stretch and Composition.

The cell plasma membrane is a highly dynamic organelle governing a wide range of cellular activities including ion transport, secretion, cell division, growth, and development. The fundamental process involved in the addition of new membranes to pre-existing plasma membranes, however, is unclear. Here, we report, using biophysical, morphological, biochemical, and molecular dynamic simulations, the selective incorporation of proteins and lipids from the cytosol into the cell plasma membrane dictated by membrane stretch and composition. Stretching of the cell membrane as a consequence of volume increase following incubation in a hypotonic solution and results in the incorporation of cytosolic proteins and lipids into the existing plasma membrane. Molecular dynamic simulations further confirm that increased membrane stretch results in the rapid insertion of lipids into the existing plasma membrane. Similarly, depletion of cholesterol from the cell plasma membrane selectively alters the incorporation of lipids into the membrane.

Nanothermometry Reveals Calcium-Induced Remodeling of Myosin.

Ions greatly influence protein structure-function and are critical to health and disease. A 10, 000-fold higher calcium in the sarcoplasmic reticulum (SR) of muscle suggests elevated calcium levels near active calcium channels at the SR membrane and the impact of localized high calcium on the structure-function of the motor protein myosin. In the current study, combined quantum dot (QD)-based nanothermometry and circular dichroism (CD) spectroscopy enabled detection of previously unknown enthalpy changes and associated structural remodeling of myosin, impacting its function following exposure to elevated calcium. Cadmium telluride QDs adhere to myosin, function as thermal sensors, and reveal that exposure of myosin to calcium is exothermic, resulting in lowering of enthalpy, a decrease in alpha helical content measured using CD spectroscopy, and the consequent increase in motor efficiency. Isolated muscle fibers subjected to elevated levels of calcium further demonstrate fiber lengthening and decreased motility of actin filaments on myosin-functionalized substrates. Our results, in addition to providing new insights into our understanding of muscle structure-function, establish a novel approach to understand the enthalpy of protein-ion interactions and the accompanying structural changes that may occur within the protein molecule.

Nanothermometry Measure of Muscle Efficiency.

Despite recent advances in thermometry, determination of temperature at the nanometer scale in single molecules to live cells remains a challenge that holds great promise in disease detection among others. In the present study, we use a new approach to nanometer scale thermometry with a spatial and thermal resolution of 80 nm and 1 mK respectively, by directly associating 2 nm cadmium telluride quantum dots (CdTe QDs) to the subject under study. The 2 nm CdTe QDs physically adhered to bovine cardiac and rabbit skeletal muscle myosin, enabling the determination of heat released when ATP is hydrolyzed by both myosin motors. Greater heat loss reflects less work performed by the motor, hence decreased efficiency. Surprisingly, we found rabbit skeletal myosin to be more efficient than bovine cardiac. We have further extended this approach to demonstrate the gain in efficiency of Drosophila melanogaster skeletal muscle overexpressing the PGC-1α homologue spargel, a known mediator of improved exercise performance in humans. Our results establish a novel approach to determine muscle efficiency with promise for early diagnosis and treatment of various metabolic disorders including cancer.

A Preliminary Investigation of a Relapse Prevention Mobile Application to Maintain Smoking Abstinence Among Individuals With Posttraumatic Stress Disorder.

OBJECTIVES: Smokers with posttraumatic stress disorder (PTSD) have increased difficulty achieving and maintaining abstinence. Contingency management approaches to smoking cessation interventions have demonstrated short-term efficacy but are limited by high rates of relapse. The goal of this pilot study was to evaluate the usability and feasibility of a smartphone-based smoking cessation application (Stay Quit Coach) designed to prevent relapse among individuals with PTSD. METHODS: Smokers (N = 11) were randomized to (1) QUIT4EVER, an intervention combining mobile contingency management smoking cessation counseling and medications, and Stay Quit Coach or (2) a contact control condition that was identical to QUIT4EVER except Stay Quit Coach was not included. The primary outcome was prolonged smoking abstinence. RESULTS: Among those queried during the follow-up periods, average Stay Quit Coach helpfulness ratings were high and ranged from 7.25 to 10 on a 10-point Likert scale (with higher scores corresponding to greater helpfulness). The Stay Quit Coach was rated by participants as being most effective at helping to quit smoking, helping to remain quit, and providing support and relevant information about quitting. Among the three quitters in the QUIT4EVER group, all reported abstinence at 3 and 6 months; however, abstinence was only bioverified for one quitter at 6 months. Among the four quitters in the contact control condition group, three reported abstinence at 3 and 6 months, but abstinence was not confirmed by bioverification. CONCLUSIONS: Smokers with PTSD express interest in and helpfulness of Stay Quit Coach for remaining abstinent after a quit attempt. Combined use of mobile contingency management and Stay Quit Coach is a feasible and acceptable adjunctive smoking cessation treatment for reducing smoking among smokers with PTSD. Adequately powered clinical trials are needed to demonstrate the long-term efficacy of this combined approach to smoking cessation. This study [Use of Technological Advances to Prevent Smoking Relapse among Smokers with PTSD (QUIT4EVER)] was registered on www.clinicaltrials.gov . clinicaltrials.gov identifier: NCT01990079.