Wild-type YopE O8 triggers diminished, proteasome inhibitor-sensitive cytotoxic alterations of infected cells. (A) Differential sensitivity of YopE-conferred cytotoxicity to proteasome inhibition. HEK293 cells were left untreated or treated with the proteasome inhibitor MG-132 prior to infection with virulence plasmid-cured yersiniae (WA-ΔpYV) or yersiniae producing either wild-type (WA-YopEwt) or K62- and K75-mutagenized (WA-YopEK62R/K75Q) YopE O8. Ninety minutes after onset of infection, the yersiniae were killed by addition of gentamicin. The cells were fixed, and cellular morphologies were microscopically analyzed after a total incubation period of 4.5 h. The numbers of cells with a completely rounded phenotype were quantified from three separate experiments, and mean percentages of rounded versus total numbers of cells SD are indicated. Differences in cell rounding were statistically significant for WA-YopEwt versus WA-YopEK62R/K75Q in the absence of MG-132 and for WA-YopEwt with versus without MG-132 (P
Abstract: Simple SummaryUltrastructural studies of cells and tissues are usually performed using transmission electron microscopy (TEM), which enables imaging at the highest possible resolution. The weak point of TEM is the limited ability to analyze the ultrastructure of large areas and volumes of biological samples. This limitation can be overcome by using modern field-emission scanning electron microscopy (FE-SEM) with high-sensitivity detection, which enables the creation of TEM-like images from the flat surfaces of resin-embedded biological specimens. Several FE-SEM-based techniques for two- and three-dimensional ultrastructural studies of cells, tissues, organs, and organisms have been developed in the 21st century. These techniques have created a new era in structural biology and have changed the role of the scanning electron microscope (SEM) in biological and medical laboratories. Since the premiere of the first commercially available SEM in 1965, these instruments were used almost exclusively to obtain topographical information over a large range of magnifications. Currently, FE-SEM offers many attractive possibilities in the studies of cell and tissue ultrastructure, and they are presented in this review. AbstractThe development of field-emission scanning electron microscopes for high-resolution imaging at very low acceleration voltages and equipped with highly sensitive detectors of backscattered electrons (BSE) has enabled transmission electron microscopy (TEM)-like imaging of the cut surfaces of tissue blocks, which are impermeable to the electron beam, or tissue sections mounted on the solid substrates. This has resulted in the development of methods that simplify and accelerate ultrastructural studies of large areas and volumes of biological samples. This article provides an overview of these methods, including their advantages and disadvantages. The imaging of large sample areas can be performed using two methods based on the detection of transmitted electrons or BSE. Effective imaging using BSE requires special fixation and en bloc contrasting of samples. BSE imaging has resulted in the development of volume imaging techniques, including array tomography (AT) and serial block-face imaging (SBF-SEM). In AT, serial ultrathin sections are collected manually on a solid substrate such as a glass and silicon wafer or automatically on a tape using a special ultramicrotome. The imaging of serial sections is used to obtain three-dimensional (3D) information. SBF-SEM is based on removing the top layer of a resin-embedded sample using an ultramicrotome inside the SEM specimen chamber and then imaging the exposed surface with a BSE detector. The steps of cutting and imaging the resin block are repeated hundreds or thousands of times to obtain a z-stack for 3D analyses.Keywords: scanning electron microscope; array tomography; serial block-face imaging; ultrastructure
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Controller Area Network (CAN) is a robust serial bus designed for board to board communication in noisy environments such as automobile and industrial control systems. MultiCAN developed by Infineon improves upon previous CAN implementations by adding features such as additional CAN nodes, more message objects linked list management of message objects and support for TTCAN level 2. The XC85x-Series is a new member of XC800 family dedicated for CAN applications by integrating a MultiCAN controller which support CAN (V2.0B). The on chip CAN module reduces the CPU load by performing most of the functions required by the networking protocol (masking, filtering and buffering of CAN frames). 2ff7e9595c
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