Enzyme-dependent fluorescence recovery of NADH after photobleaching to assess dehydrogenase activity of isolated perfused hearts.

Reduction of NAD(+) by dehydrogenase enzymes to form NADH is a key component of cellular metabolism. In cellular preparations and isolated mitochondria suspensions, enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) of NADH has been shown to be an effective approach for measuring the rate of NADH production to assess dehydrogenase enzyme activity. Our objective was to demonstrate how dehydrogenase activity could be assessed within the myocardium of perfused hearts using NADH ED-FRAP. This was accomplished using a combination of high intensity UV pulses to photobleach epicardial NADH. Replenishment of epicardial NADH fluorescence was then imaged using low intensity UV illumination. NADH ED-FRAP parameters were optimized to deliver 23.8 mJ of photobleaching light energy at a pulse width of 6 msec and a duty cycle of 50%. These parameters provided repeatable measurements of NADH production rate during multiple metabolic perturbations, including changes in perfusate temperature, electromechanical uncoupling, and acute ischemia/reperfusion injury. NADH production rate was significantly higher in every perturbation where the energy demand was either higher or uncompromised. We also found that NADH production rate remained significantly impaired after 10 min of reperfusion after global ischemia. Overall, our results indicate that myocardial NADH ED-FRAP is a useful optical non-destructive approach for assessing dehydrogenase activity

Suppressed dependence of polarization on epitaxial strain in highly polar ferroelectrics

A combined experimental and computational investigation of coupling between polarization and epitaxial strain in highly polar ferroelectric PbZr_0.2Ti_0.8O_3 (PZT) thin films is reported. A comparison of the properties of relaxed (tetragonality c/a = 1.05) and highly-strained (c/a = 1.09) epitaxial films shows that polarization, while being amongst the highest reported for PZT or PbTiO_3 in either film or bulk forms (P_r = 82 microC/cm^2), is almost independent of the epitaxial strain. We attribute this behavior to a suppressed sensitivity of the A-site cations to epitaxial strain in these Pb-based perovskites, where the ferroelectric displacements are already large, contrary to the case of less polar perovskites, such as BaTiO_3. In the latter case, the A-site cation (Ba) and equatorial oxygen displacements can lead to substantial polarization increases.Comment: 4 pages, 3 figure

Stop the beat to see the rhythm: excitation-contraction uncoupling in cardiac research.

Optical mapping is an imaging technique that is extensively used in cardiovascular research, wherein parameter-sensitive fluorescent indicators are used to study the electrophysiology and excitation-contraction coupling of cardiac tissues. Despite many benefits of optical mapping, eliminating motion artifacts within the optical signals is a major challenge, as myocardial contraction interferes with the faithful acquisition of action potentials and intracellular calcium transients. As such, excitation-contraction uncoupling agents are frequently used to reduce signal distortion by suppressing contraction. When compared with other uncoupling agents, blebbistatin is the most frequently used, as it offers increased potency with minimal direct effects on cardiac electrophysiology. Nevertheless, blebbistatin may exert secondary effects on electrical activity, metabolism, and coronary flow, and the incorrect administration of blebbistatin to cardiac tissue can prove detrimental, resulting in erroneous interpretation of optical mapping results. In this “Getting It Right” perspective, we briefly review the literature regarding the use of blebbistatin in cardiac optical mapping experiments, highlight potential secondary effects of blebbistatin on cardiac electrical activity and metabolic demand, and conclude with the consensus of the authors on best practices for effectively using blebbistatin in optical mapping studies of cardiac tissue

Neurobehavioral Abnormalities in Firest-Degree Relative of Individuals With Autism

CONTEXT: Studying sensorimotor and neurocognitive impairments in unaffected family members of individuals with autism may help identify familial pathophysiological mechanisms associated with the disorder. OBJECTIVE: To determine whether atypical sensorimotor or neurocognitive characteristics associated with autism are present in first-degree relatives of individuals with autism. DESIGN: Case-control comparison of neurobehavioral functions. SETTING: University medical center. PARTICIPANTS: Fifty-seven first-degree relatives of individuals with autism and 40 age-, sex-, and IQ-matched healthy control participants (aged 8-54 years). MAIN OUTCOME MEASURES: Oculomotor tests of sensorimotor responses (saccades and smooth pursuit); procedural learning and response inhibition; neuropsychological tests of motor, memory, and executive functions; and psychological measures of social behavior, communication skills, and obsessive-compulsive behaviors. RESULTS: On eye movement testing, family members demonstrated saccadic hypometria, reduced steady-state pursuit gain, and a higher rate of voluntary response inhibition errors relative to controls. They also showed lateralized deficits in procedural learning and open-loop pursuit gain (initial 100 milliseconds of pursuit) and increased variability in the accuracy of large-amplitude saccades that were confined to rightward movements. In neuropsychological studies, only executive functions were impaired relative to those of controls. Family members reported more communication abnormalities and obsessive-compulsive behaviors than controls. Deficits across oculomotor, neuropsychological, and psychological domains were relatively independent from one another. CONCLUSIONS: Family members of individuals with autism demonstrate oculomotor abnormalities implicating pontocerebellar and frontostriatal circuits and left-lateralized alterations of frontotemporal circuitry and striatum. The left-lateralized alterations have not been identified in other neuropsychiatric disorders and are of interest given atypical brain lateralization and language development associated with the disorder. Similar oculomotor deficits have been reported in individuals with autism, suggesting that they may be familial and useful for studies of neurophysiological and genetic mechanisms in autism

RHYTHM: An Open Source Imaging Toolkit for Cardiac Panoramic Optical Mapping

Fluorescence optical imaging techniques have revolutionized the field of cardiac electrophysiology and advanced our understanding of complex electrical activities such as arrhythmias. However, traditional monocular optical mapping systems, despite having high spatial resolution, are restricted to a two-dimensional (2D) field of view. Consequently, tracking complex three-dimensional (3D) electrical waves such as during ventricular fibrillation is challenging as the waves rapidly move in and out of the field of view. This problem has been solved by panoramic imaging which uses multiple cameras to measure the electrical activity from the entire epicardial surface. However, the diverse engineering skill set and substantial resource cost required to design and implement this solution have made it largely inaccessible to the biomedical research community at large. To address this barrier to entry, we present an open source toolkit for building panoramic optical mapping systems which includes the 3D printing of perfusion and imaging hardware, as well as software for data processing and analysis. In this paper, we describe the toolkit and demonstrate it on different mammalian hearts: mouse, rat, and rabbit

Integrating imaging and RNA-seq improves outcome prediction in cervical cancer

Approaches using a single type of data have been applied to classify human tumors. Here we integrate imaging features and transcriptomic data using a prospectively collected tumor bank. We demonstrate that increased maximum standardized uptake value on pretreatment 18F-fluorodeoxyglucose-positron emission tomography correlates with epithelial-to-mesenchymal transition (EMT) gene expression. We derived and validated 3 major molecular groups, namely squamous epithelial, squamous mesenchymal, and adenocarcinoma, using prospectively collected institutional (n = 67) and publicly available (n = 304) data sets. Patients with tumors of the squamous mesenchymal subtype showed inferior survival outcomes compared with the other 2 molecular groups. High mesenchymal gene expression in cervical cancer cells positively correlated with the capacity to form spheroids and with resistance to radiation. CaSki organoids were radiation-resistant but sensitive to the glycolysis inhibitor, 2-DG. These experiments provide a strategy for response prediction by integrating large data sets, and highlight the potential for metabolic therapy to influence EMT phenotypes in cervical cancer