A method to increase reproducibility in adult ventricular myocyte sizing and flow cytometry: Avoiding cell size bias in single cell preparations.

RATIONALE:Flow cytometry (FCM) of ventricular myocytes (VMs) is an emerging technology in adult cardiac research that is challenged by the wide variety of VM shapes and sizes. Cellular variability and cytometer flow cell size can affect cytometer performance. These two factors of variance limit assay validity and reproducibility across laboratories. Washing and filtering of ventricular cells in suspension are routinely done to prevent cell clumping and minimize data variability without the appropriate standardization. We hypothesize that washing and filtering arbitrarily biases towards sampling smaller VMs than what actually exist in the adult heart. OBJECTIVE:To determine the impact of washing and filtering on adult ventricular cells for cell sizing and FCM. METHODS AND RESULTS:Left ventricular cardiac cells in single-cell suspension were harvested from New Zealand White rabbits and fixed prior to analysis. Each ventricular sample was aliquoted before washing or filtering through a 40, 70, 100 or 200μm mesh. The outcomes of the study are VM volume by Coulter Multisizer and light-scatter signatures by FCM. Data are presented as mean±SD. Myocyte volumes without washing or filtering (NF) served as the "gold standard" within the sample and ranged from 11,017 to 46,926μm3. Filtering each animal sample through a 200μm mesh caused no variation in the post-filtration volume (1.01+0.01 fold vs. NF, n = 4 rabbits, p = 0.999) with an intra-assay coefficient of variation (%CV) of <5% for all 4 samples. Filtering each sample through a 40, 70 or 100μm mesh invariably reduced the post-filtration volume by 41±10%, 9.0±0.8% and 8.8±0.8% respectively (n = 4 rabbits, p<0.0001), and increased the %CV (18% to 1.3%). The high light-scatter signature by FCM, a simple parameter for the identification of ventricular myocytes, was measured after washing and filtering. Washing discarded VMs and filtering cells through a 40 or 100μm mesh reduced larger VM by 46% or 11% respectively (n = 6 from 2 rabbits, p<0.001). CONCLUSION:Washing and filtering VM suspensions through meshes 100μm or less biases myocyte volumes to smaller sizes, excludes larger cells, and increases VM variability. These findings indicate that validity and reproducibility across laboratories can be compromised unless cell preparation is standardized. We propose no wash prior to fixation and a 200μm mesh for filtrations to provide a reproducible standard for VM studies using FCM

Scanning nano-spin ensemble microscope for nanoscale magnetic and thermal imaging

Quantum sensors based on solid-state spins provide tremendous opportunities in a wide range of fields from basic physics and chemistry to biomedical imaging. However, integrating them into a scanning probe microscope to enable practical, nanoscale quantum imaging is a highly challenging task. Recently, the use of single spins in diamond in conjunction with atomic force microscopy techniques has allowed significant progress towards this goal, but generalisation of this approach has so far been impeded by long acquisition times or by the absence of simultaneous topographic information. Here we report on a scanning quantum probe microscope which solves both issues, by employing a nano-spin ensemble hosted in a nanodiamond. This approach provides up to an order of magnitude gain in acquisition time, whilst preserving sub-100 nm spatial resolution both for the quantum sensor and topographic images. We demonstrate two applications of this microscope. We first image nanoscale clusters of maghemite particles through both spin resonance spectroscopy and spin relaxometry, under ambient conditions. Our images reveal fast magnetic field fluctuations in addition to a static component, indicating the presence of both superparamagnetic and ferromagnetic particles. We next demonstrate a new imaging modality where the nano-spin ensemble is used as a thermometer. We use this technique to map the photo-induced heating generated by laser irradiation of a single gold nanoparticle in a fluid environment. This work paves the way towards new applications of quantum probe microscopy such as thermal/magnetic imaging of operating microelectronic devices and magnetic detection of ion channels in cell membranes.Comment: 22 pages including Supporting Information. Changes to v1: affiliations and funding information updated, plus minor revisions to the main tex

The role of cosmic ray pressure in accelerating galactic outflows

We study the formation of galactic outflows from supernova explosions (SNe) with the moving-mesh code AREPO in a stratified column of gas with a surface density similar to the Milky Way disk at the solar circle. We compare different simulation models for SNe placement and energy feedback, including cosmic rays (CR), and find that models that place SNe in dense gas and account for CR diffusion are able to drive outflows with similar mass loading as obtained from a random placement of SNe with no CRs. Despite this similarity, CR-driven outflows differ in several other key properties including their overall clumpiness and velocity. Moreover, the forces driving these outflows originate in different sources of pressure, with the CR diffusion model relying on non-thermal pressure gradients to create an outflow driven by internal pressure and the random-placement model depending on kinetic pressure gradients to propel a ballistic outflow. CRs therefore appear to be non-negligible physics in the formation of outflows from the interstellar medium.Comment: 8 pages, 4 figures, accepted for publication in ApJL; movie of simulated gas densities can be found here: http://www.h-its.org/tap-images/galactic-outflows

The alpha-1A adrenergic receptor agonist A61603 reduces cardiac polyunsaturated fatty acid and endocannabinoid metabolites associated with inflammation in vivo

Alpha-1-adrenergic receptors (α1-ARs) are G-protein coupled receptors (GPCRs) with three highly homologous subtypes (α1A, α1B, and α1D). Of these three subtypes, only the α1A and α1B are expressed in the heart. Multiple pre-clinical models of heart injury demonstrate cardioprotective roles for the α1A. Non-selective α1-AR activation promotes glycolysis in the heart, but the functional α1-AR subtype and broader metabolic effects have not been studied

Evidence for the Evolution of Young Early-Type Galaxies in the GOODS/CDF-S Field

We have developed an efficient photometric technique for identifying young early-type galaxy candidates using a combination of photometric redshifts, spectral-type classification, and optical/near-infrared colors. Applying our technique to the GOODS HST/ACS and VLT/ISAAC data we have selected a complete and homogeneous sample of young elliptical candidates among early-type field galaxies. The distribution of structural parameters for these candidates shows that their selection, which is based on early spectral types, is fully consistent with early morphological types. We investigate the evolution of their luminosities and colors as a function of redshift and galaxy mass and find evidence for an increasing starburst mass fraction in these young early-type galaxy candidates at higher redshifts, which we interpret in terms of massive field galaxies experiencing more massive/intense starbursts at higher redshifts. Moreover, we find indications for a systematically larger young elliptical fraction among sub-L*/2 early-type galaxies compared to their brighter counterparts. The total fraction among the field early-type galaxies increases with redshift, irrespective of galaxy luminosity. Our results are most consistent with galaxy formation scenarios in which stars in massive early-type field galaxies are assembled earlier than in their low-mass counterparts.Comment: 11 pages, 10 figures, accepted for publication in A

Seasonal Change in Countermovement Jump Performance in NCAA Women’s Golfers

Background of Study: The countermovement vertical jump (CMJ) task has been reported to have positive associations with golf-specific performance variables. Additionally, the CMJ is commonly used to assess neuromuscular fatigue in athletic populations. Objective: Thus, this investigation sought to examine the changes in CMJ performance throughout a competitive season in NCAA collegiate women’s golfers. Methods: Using a longitudinal study design, six collegiate women golfers completed three sessions (pre, mid, and post) of CMJ testing during the spring competition period. During each testing session, two successful jump trials were collected using a portable force platform sampling at 1000 Hz. During each trial, an arm swing was restricted by the use of a dowel placed across the upper back. A one-way repeated measures analysis of variance was used to determine if differences were present between testing sessions. Results: Propulsive net impulse significantly increased from pre to mid (p 0.05) and pre to post (p 0.05). No other variables showed a statistically significant change over the duration of the study, though moderate effect size increases were in countermovement depth from pre to mid-testing (0.73) and jump height from pre to post-testing (0.72). Conclusions: These findings support previous findings of an increase in vertical jump performance over the course of a season in collegiate golfers, though strategies for maximal performance may shift

Alpha-1–Adrenergic Receptors in Heart Failure: The Adaptive Arm of the Cardiac Response to Chronic Catecholamine Stimulation

Alpha-1-adrenergic receptors are G-protein coupled receptors (GPCRs) activated by catecholamines. The alpha-1A and alpha-1B subtypes are expressed in mouse and human myocardium, whereas the alpha-1D protein is found only in coronary arteries. There are far fewer alpha-1-ARs than beta-ARs in the non-failing heart, but their abundance is maintained or increased in the setting of heart failure, which is characterized by pronounced chronic elevation of catecholamines and b□eta-AR dysfunction. Decades of evidence from gain- and loss-of-function studies in isolated cardiac myocytes and numerous animal models demonstrate important adaptive functions for cardiac alpha-1-ARs, to include physiological hypertrophy, positive inotropy, ischemic preconditioning, and protection from cell death. Clinical trial data indicate that blocking alpha-1-ARs is associated with incident heart failure in patients with hypertension. Collectively, these findings suggest that alpha-1-AR activation might mitigate the well-recognized toxic effects of beta-ARs in the hyperadrenergic setting of chronic heart failure. Thus, exogenous cardioselective activation of alpha-1-ARs might represent a novel and viable approach to the treatment of heart failure

Alpha-1-adrenergic receptors: Targets for agonist drugs to treat heart failure

Evidence from cell, animal, and human studies demonstrates that α1-adrenergic receptors mediate adaptive and protective effects in the heart. These effects may be particularly important in chronic heart failure, when catecholamine levels are elevated and β-adrenergic receptors are down regulated and dysfunctional. This review summarizes these data and proposes that selectively activating α1-adrenergic receptors in the heart may represent a novel and effective way to treat heart failure

Neutrophil-derived microvesicle induced dysfunction of brain microvascular endothelial cells in vitro

The blood-brain barrier (BBB), composed of brain microvascular endothelial cells (BMEC) that are tightly linked by tight junction (TJ) proteins, restricts the movement of molecules between the periphery and the central nervous system. Elevated systemic levels of neutrophils have been detected in patients with altered BBB function, but the role of neutrophils in BMEC dysfunction is unknown. Neutrophils are key players of the immune response and, when activated, produce neutrophil-derived microvesicles (NMV). NMV have been shown to impact the integrity of endothelial cells throughout the body and we hypothesize that NMV released from circulating neutrophils interact with BMEC and induce endothelial cell dysfunction. Therefore, the current study investigated the interaction of NMV with human BMEC and determined whether they altered gene expression and function in vitro. Using flow cytometry and confocal imaging, NMV were shown to be internalized by the human cerebral microvascular endothelial cell line hCMEC/D3 via a variety of energy-dependent mechanisms, including endocytosis and macropinocytosis. The internalization of NMV significantly altered the transcriptomic profile of hCMEC/D3, specifically inducing the dysregulation of genes associated with TJ, ubiquitin-mediated proteolysis and vesicular transport. Functional studies confirmed NMV significantly increased permeability and decreased the transendothelial electrical resistance (TEER) of a confluent monolayer of hCMEC/D3. These findings indicate that NMV interact with and affect gene expression of BMEC as well as impacting their integrity. We conclude that NMV may play an important role in modulating the permeability of BBB during an infection