An Efficient Method for Isolating and Purifying Nuclei from Mice Brain for Single-Molecule Imaging Using High-Speed Atomic Force Microscopy.

Nuclear pore complexes (NPCs) on the nuclear membrane surface have a crucial function in controlling the movement of small molecules and macromolecules between the cell nucleus and cytoplasm through their intricate core channel resembling a spiderweb with several layers. Currently, there are few methods available to accurately measure the dynamics of nuclear pores on the nuclear membranes at the nanoscale. The limitation of traditional optical imaging is due to diffraction, which prevents achieving the required resolution for observing a diverse array of organelles and proteins within cells. Super-resolution techniques have effectively addressed this constraint by enabling the observation of subcellular components on the nanoscale. Nevertheless, it is crucial to acknowledge that these methods often need the use of fixed samples. This also raises the question of how closely a static image represents the real intracellular dynamic system. High-speed atomic force microscopy (HS-AFM) is a unique technique used in the field of dynamic structural biology, enabling the study of individual molecules in motion close to their native states. Establishing a reliable and repeatable technique for imaging mammalian tissue at the nanoscale using HS-AFM remains challenging due to inadequate sample preparation. This study presents the rapid strainer microfiltration (RSM) protocol for directly preparing high-quality nuclei from the mouse brain. Subsequently, we promptly utilize HS-AFM real-time imaging and cinematography approaches to record the spatiotemporal of nuclear pore nano-dynamics from the mouse brain.

The rise of circulatory endothelin (ET)-1 and endothelin receptors (ETA, ETB) expression in kidney of obese wistar rat.

BACKGROUND: Endothelin (ET)-1, a circulatory protein, and its receptors (ETA and ETB) in various organs were reported to play a pivotal role in many diseases, including obesity. However, the changes of ETA and ETB expression in ventricle and kidney in obesity was less reported. The study is designed to observe the level of circulatory ET-1 and expression of ETA/ETB in ventricle and kidney of obese, as compared to non-obese, Wistar rats. METHODS: Groups of obese 14 and 34 weeks Wistar rats were compared to non-obese controls at similar ages. The obesity status was achieved by feeding the with high calories protein diet CP 551 + milk powder, while the control group was fed with a standard calorie protein AD II diet. The concentration of circulatory ET-1, ETA and ETB of ventricle and kidney were measured by Enzyme Linked Immunosorbent Assay (ELISA) technique after the termination of both groups at 14th and 24th weeks. RESULTS: The level of circulatory ET-1, expression of ETA and ETB in kidney, and LDL of obese rats were significantly higher than control rats (T-Test, P<0.05) in the elder groups, while no differences of the ETA and ETB were found in the ventricle. No differences of the levels of circulatory ET-1, ETA and ETB expression were found between obese and control groups of younger rats (P>0.05). HDL levels were under normal value for both groups. CONCLUSION: Obesity in elder obese rats leads to dysregulation of kidney vessels through activity of ET-1 and ETA/ETB.