Differential RhoA Dynamics in Migratory and Stationary Cells Measured by FRET and Automated Image Analysis

Genetically-encoded biosensors based on fluorescence resonance energy transfer (FRET) have been widely applied to study the spatiotemporal regulation of molecular activity in live cells with high resolution. The efficient and accurate quantification of the large amount of imaging data from these single-cell FRET measurements demands robust and automated data analysis. However, the nonlinear movement of live cells presents tremendous challenge for this task. Based on image registration of the single-cell movement, we have developed automated image analysis methods to track and quantify the FRET signals within user-defined subcellular regions. In addition, the subcellular pixels were classified according to their associated FRET signals and the dynamics of the clusters analyzed. The results revealed that the EGF-induced reduction of RhoA activity in migratory HeLa cells is significantly less than that in stationary cells. Furthermore, the RhoA activity is polarized in the migratory cells, with the gradient of polarity oriented toward the opposite direction of cell migration. In contrast, there is a lack of consistent preference in RhoA polarity among stationary cells. Therefore, our image analysis methods can provide powerful tools for high-throughput and systematic investigation of the spatiotemporal molecular activities in regulating functions of live cells with their shapes and positions continuously changing in time

Refined Qingkailing Protects MCAO Mice from Endoplasmic Reticulum Stress-Induced Apoptosis with a Broad Time Window

In the current study, we are investigating effect of refined QKL on ischemia-reperfusion-induced brain injury in mice. Methods. Mice were employed to induce ischemia-reperfusion injury of brain by middle cerebral artery occlusion (MCAO). RQKL solution was administered with different doses (0, 1.5, 3, and 6 mL/kg body weight) at the same time of onset of ischemia, and with the dose of 1.5 mL/kg at different time points (0, 1.5, 3, 6, and 9 h after MCAO). Neurological function and brain infarction were examined and cell apoptosis and ROS at prefrontal cortex were evaluated 24 h after MCAO, and western blot and intracellular calcium were also researched, respectively. Results. RQKL of all doses can improve neurological function and decrease brain infarction, and it performed significant effect in 0, 1.5, 3, and 6 h groups. Moreover, RQKL was able to reduce apoptotic process by reduction of caspase-3 expression, or restraint of eIF2a phosphorylation and caspase-12 activation. It was also able to reduce ROS and modulate intracellular calcium in the brain. Conclusion. RQKL can prevent ischemic-induced brain injury with a time window of 6 h, and its mechanism might be related to suppress ER stress-mediated apoptotic signaling

Case report: Successful and effective percutaneous closure of a deep femoral artery pseudoaneurysm using proglide device

A 61-year-old man developed severe swelling in the left lower extremity after interventional embolization of liver tumor. Ultrasound examination showed a pseudoaneurysm and thrombosis in the upper thigh on the left. To recognize the causes and determine the effective therapy, lower extremity arteriography was performed. The results revealed a pseudoaneurysm arised from deep femoral artery. Considering of the size of cavity and symptoms of patient, a new method was tried instead of traditional treatment using PROGLIDE device. Postoperative angiography showed a powerful blocking effect. This case study provide us a specific treatment for pseudoaneurysm, and this method provide us a new therapeutic strategy in clinical practice

Computational Analysis of the Spatiotemporal Coordination of Polarized PI3K and Rac1 Activities in Micro-Patterned Live Cells

Polarized molecular activities play important roles in guiding the cell toward persistent and directional migration. In this study, the polarized distributions of the activities of phosphatidylinositol 3-kinase (PI3K) and the Rac1 small GTPase were monitored using chimeric fluorescent proteins (FPs) in cells constrained on micro-patterned strips, with one end connecting to a neighboring cell (junction end) and the other end free of cell-cell contact (free end). The recorded spatiotemporal dynamics of the fluorescent intensity from different cells was scaled into a uniform coordinate system and applied to compute the molecular activity landscapes in space and time. The results revealed different polarization patterns of PI3K and Rac1 activity induced by the growth factor stimulation. The maximal intensity of different FPs, and the edge position and velocity at the free end were further quantified to analyze their correlation and decipher the underlying signaling sequence. The results suggest that the initiation of the edge extension occurred before the activation of PI3K, which led to a stable extension of the free end followed by the Rac1 activation. Therefore, the results support a concerted coordination of sequential signaling events and edge dynamics, underscoring the important roles played by PI3K activity at the free end in regulating the stable lamellipodia extension and cell migration. Meanwhile, the quantification methods and accompanying software developed can provide a convenient and powerful computational analysis platform for the study of spatiotemporal molecular distribution and hierarchy in live cells based on fluorescence images

Visualization of Src activity at different compartments of the plasma membrane by FRET imaging

SummaryMembrane compartments function as segregated signaling platforms for different cellular functions. It is not clear how Src is regulated at different membrane compartments. To visualize local Src activity in live cells, a FRET-based Src biosensor was targeted in or outside of lipid rafts at the plasma membrane, via acylation or prenylation modifications on targeting tags either directly fused to the biosensor or coupled to the biosensor through an inducible heterodimerization system. In response to growth factors and pervanadate, the induction of Src activity in rafts was slower and weaker, dependent on actin and possibly its mediated transportation of Src from perinuclear regions to the plasma membrane. In contrast, the induction of Src activity in nonrafts was faster and stronger, dependent on microtubules. Hence, Src activity is differentially regulated via cytoskeleton at different membrane compartments

A Measurement of Visual Complexity for Heterogeneity in the Built Environment Based on Fractal Dimension and Its Application in Two Gardens

In this study, a fractal dimension-based method has been developed to compute the visual complexity of the heterogeneity in the built environment. The built environment is a very complex combination, structurally consisting of both natural and artificial elements. Its fractal dimension computation is often disturbed by the homogenous visual redundancy, which is textured but needs less attention to process, so that it leads to a pseudo-evaluation of visual complexity in the built environment. Based on human visual perception, the study developed a method: fractal dimension of heterogeneity in the built environment, which includes Potts segmentation and Canny edge detection as image preprocessing procedure and fractal dimension as computation procedure. This proposed method effectively extracts perceptually meaningful edge structures in the visual image and computes its visual complexity which is consistent with human visual characteristics. In addition, an evaluation system combining the proposed method and the traditional method has been established to classify and assess the visual complexity of the scenario more comprehensively. Two different gardens had been computed and analyzed to demonstrate that the proposed method and the evaluation system provide a robust and accurate way to measure the visual complexity in the built environment

Research and Development of Intelligent Cleaning and Mud Content Detection Technology for Machine-Made Sand

In this paper, an improved method of sand and mud content detection for concrete is proposed, and a set of sand and mud content detection device for concrete is designed. In this paper, a concrete gas content meter and a bubble parameter analysis system were used to study the effect of sand clay content on the gas content of commercial concrete, the loss rate of gas content in warp time, the bubble spacing coefficient and the distribution of hardened appearance pores. The pore structure test shows that the increase of mud content will appropriately reduce the proportion of harmless pores in machine-made sand concrete, and play a role in refining the pore structure in machine-made sand concrete. By using PLC controller and automatic detection device of liquid level and turbidity, the problems of complicated operation, inconvenient carrying and poor repeatability of test data are solved. It reduces the influence of human operation error and improves the accuracy of test data

Research and Development of Intelligent Drying Detection Technology for Machine-Made Sand

This paper aims to develop a high precision variable dielectric constant capacitor detector for mechanical sand water content testing. The non-contact on-line test method can effectively solve the problems of low repeatability of test data, large hysteresis error and poor linearity due to the non-uniform water content. In terms of hardware, CAV444 is a high-precision integrated chip, which matches the corresponding test circuit. The system communicates with PC through serial interface and implements the man-machine interface of PC system based on VS. Secondly, the distribution map method is used to measure the water content of machine-made sand, and the error analysis is carried out. The test shows that the output of the capacitive hygrometer is 0-5 V. The delay time should not exceed 0.15%. The repetition rate was within 0.33%. The measurement accuracy of this method is less than 3.52%