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

The Impact of Mutation of Myelodysplasia-Related Genes in De Novo Acute Myeloid Leukemia Carrying NPM1 Mutation

Background: The impact of gene mutations typically associated with myelodysplastic syndrome (MDS) in acute myeloid leukemia (AML) with NPM1 mutation is unclear. Methods: Using a cohort of 107 patients with NPM1-mutated AML treated with risk-adapted therapy, we compared survival outcomes of patients without MDS-related gene mutations (group A) with those carrying concurrent FLT3-ITD (group B) or with MDS-related gene mutations (group C). Minimal measurable disease (MMD) status assessed by multiparameter flow cytometry (MFC), polymerase chain reaction (PCR), and/or next-generation sequencing (NGS) were reviewed. Results: Among the 69 patients treated intensively, group C showed significantly inferior progression-free survival (PFS, p \u3c 0.0001) but not overall survival (OS, p = 0.055) compared to group A. Though groups A and C had a similar MMD rate, group C patients had a higher relapse rate (p = 0.016). Relapse correlated with MMD status at the end of cycle 2 induction (p = 0.023). Survival of group C patients was similar to that of group B. Conclusion: MDS-related gene mutations are associated with an inferior survival in NPM1-mutated AML

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

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