Automatic Hotspots Detection for Intracellular Calcium Analysis in Fluorescence Microscopic Videos

In recent years, life-cell imaging techniques and their software applications have become powerful tools to investigate complex biological mechanisms such as calcium signalling. In this paper, we propose an automated framework to detect areas inside cells that show changes in their calcium concentration i.e. the regions of interests or hotspots, based on videos taken after loading living mouse cardiomyocytes with fluorescent calcium reporter dyes. The proposed system allows an objective and efficient analysis through the following four key stages: (1) Pre-processing to enhance video quality, (2) First level segmentation to detect candidate hotspots based on adaptive thresholding on the frame level, (3) Second-level segmentation to fuse and identify the best hotspots from the entire video by proposing the concept of calcium fluorescence hit-ratio, and (4) Extraction of the changes of calcium fluorescence over time per hotspot. From the extracted signals, different measurements are calculated such as maximum peak amplitude, area under the curve, peak frequency, and inter-spike interval of calcium changes. The system was tested using calcium imaging data collected from Heart muscle cells. The paper argues that the automated proposal offers biologists a tool to speed up the processing time and mitigate the consequences of inter-intra observer variability

Self-organized Vortex State in Two-dimensional Dictyostelium Dynamics

We present results of experiments on the dynamics of Dictyostelium discoideum in a novel set-up which constraints cell motion to a plane. After aggregation, the amoebae collect into round ''pancake" structures in which the cells rotate around the center of the pancake. This vortex state persists for many hours and we have explicitly verified that the motion is not due to rotating waves of cAMP. To provide an alternative mechanism for the self-organization of the Dictyostelium cells, we have developed a new model of the dynamics of self-propelled deformable objects. In this model, we show that cohesive energy between the cells, together with a coupling between the self-generated propulsive force and the cell's configuration produces a self-organized vortex state. The angular velocity profiles of the experiment and of the model are qualitatively similar. The mechanism for self-organization reported here can possibly explain similar vortex states in other biological systems.Comment: submitted to PRL; revised version dated 3/8/9

Calcium-sensing receptor antagonists abrogate airway hyperresponsiveness and inflammation in allergic asthma

Airway hyperresponsiveness and inflammation are fundamental hallmarks of allergic asthma that are accompanied by increases in certain polycations, such as eosinophil cationic protein. Levels of these cations in body fluids correlate with asthma severity. We show that polycations and elevated extracellular calcium activate the human recombinant and native calcium-sensing receptor (CaSR), leading to intracellular calcium mobilization, cyclic adenosine monophosphate breakdown, and p38 mitogen-activated protein kinase phosphorylation in airway smooth muscle (ASM) cells. These effects can be prevented by CaSR antagonists, termed calcilytics. Moreover, asthmatic patients and allergen-sensitized mice expressed more CaSR in ASMs than did their healthy counterparts. Indeed, polycations induced hyperreactivity in mouse bronchi, and this effect was prevented by calcilytics and absent in mice with CaSR ablation from ASM. Calcilytics also reduced airway hyperresponsiveness and inflammation in allergen-sensitized mice in vivo. These data show that a functional CaSR is up-regulated in asthmatic ASM and targeted by locally produced polycations to induce hyperresponsiveness and inflammation. Thus, calcilytics may represent effective asthma therapeutics

Retention of Memory through Metamorphosis: Can a Moth Remember What It Learned As a Caterpillar?

Insects that undergo complete metamorphosis experience enormous changes in both morphology and lifestyle. The current study examines whether larval experience can persist through pupation into adulthood in Lepidoptera, and assesses two possible mechanisms that could underlie such behavior: exposure of emerging adults to chemicals from the larval environment, or associative learning transferred to adulthood via maintenance of intact synaptic connections. Fifth instar Manduca sexta caterpillars received an electrical shock associatively paired with a specific odor in order to create a conditioned odor aversion, and were assayed for learning in a Y choice apparatus as larvae and again as adult moths. We show that larvae learned to avoid the training odor, and that this aversion was still present in the adults. The adult aversion did not result from carryover of chemicals from the larval environment, as neither applying odorants to naïve pupae nor washing the pupae of trained caterpillars resulted in a change in behavior. In addition, we report that larvae trained at third instar still showed odor aversion after two molts, as fifth instars, but did not avoid the odor as adults, consistent with the idea that post-metamorphic recall involves regions of the brain that are not produced until later in larval development. The present study, the first to demonstrate conclusively that associative memory survives metamorphosis in Lepidoptera, provokes intriguing new questions about the organization and persistence of the central nervous system during metamorphosis. Our results have both ecological and evolutionary implications, as retention of memory through metamorphosis could influence host choice by polyphagous insects, shape habitat selection, and lead to eventual sympatric speciation

Spontaneous, pro-arrhythmic calcium signals disrupt electrical pacing in mouse pulmonary vein sleeve cells

The pulmonary vein, which returns oxygenated blood to the left atrium, is ensheathed by a population of unique, myocyte- like cells called pulmonary vein sleeve cells (PVCs). These cells autonomously generate action potentials that propagate into the left atrial chamber and cause arrhythmias resulting in atrial fibrillation; the most common, often sustained, form of cardiac arrhythmia. In mice, PVCs extend along the pulmonary vein into the lungs, and are accessible in a lung slice preparation. We exploited this model to study how aberrant Ca2+ signaling alters the ability of PVC networks to follow electrical pacing. Cellular responses were investigated using real-time 2-photon imaging of lung slices loaded with a Ca2+- sensitive fluorescent indicator (Ca2+ measurements) and phase contrast microscopy (contraction measurements). PVCs displayed global Ca2+ signals and coordinated contraction in response to electrical field stimulation (EFS). The effects of EFS relied on both Ca2+ influx and Ca2+ release, and could be inhibited by nifedipine, ryanodine or caffeine. Moreover, PVCs had a high propensity to show spontaneous Ca2+ signals that arose via stochastic activation of ryanodine receptors (RyRs). The ability of electrical pacing to entrain Ca2+ signals and contractile responses was dramatically influenced by inherent spontaneous Ca2+ activity. In PVCs with relatively low spontaneous Ca2+ activity (2+ activity (>1.5 Hz), electrical pacing was less effective; PVCs became unpaced, only partially-paced or displayed alternans. Because spontaneous Ca2+ activity varied between cells, neighboring PVCs often had different responses to electrical pacing. Our data indicate that the ability of PVCs to respond to electrical stimulation depends on their intrinsic Ca2+ cycling properties. Heterogeneous spontaneous Ca2+ activity arising from stochastic RyR opening can disengage them from sinus rhythm and lead to autonomous, pro-arrhythmic activity

Apical Transport of Influenza A Virus Ribonucleoprotein Requires Rab11-positive Recycling Endosome

Influenza A virus RNA genome exists as eight-segmented ribonucleoprotein complexes containing viral RNA polymerase and nucleoprotein (vRNPs). Packaging of vRNPs and virus budding take place at the apical plasma membrane (APM). However, little is known about the molecular mechanisms of apical transport of newly synthesized vRNP. Transfection of fluorescent-labeled antibody and subsequent live cell imaging revealed that punctate vRNP signals moved along microtubules rapidly but intermittently in both directions, suggestive of vesicle trafficking. Using a series of Rab family protein, we demonstrated that progeny vRNP localized to recycling endosome (RE) in an active/GTP-bound Rab11-dependent manner. The vRNP interacted with Rab11 through viral RNA polymerase. The localization of vRNP to RE and subsequent accumulation to the APM were impaired by overexpression of Rab binding domains (RBD) of Rab11 family interacting proteins (Rab11-FIPs). Similarly, no APM accumulation was observed by overexpression of class II Rab11-FIP mutants lacking RBD. These results suggest that the progeny vRNP makes use of Rab11-dependent RE machinery for APM trafficking