Extracellular vesicles (EVs) released by endothelial cells support vascular homeostasis. To better understand endothelial cell EV biogenesis, we examined cultured human umbilical vein endothelial cells (HUVECs) prepared by rapid freezing, freeze-substitution, and serial thin section electron microscopy (EM). Thin sections of HUVECs revealed clusters of membrane protrusions on the otherwise smooth cell surface. The protrusions contained membrane-bound organelles, including multivesicular bodies (MVBs), and appeared to be on the verge of pinching off to form microvesicles. Beyond cell peripheries, membrane-bound vesicles with internal MVBs were observed, and serial sections confirmed that they were not connected to cells. These observations are consistent with the notion that these multi-compartmented microvesicles (MCMVs) pinch-off from protrusions. Remarkably, omega figures formed by fusion of vesicles with the MCMV limiting membrane were directly observed, apparently releasing exosomes from the MCMV. In summary, MCMVs are a novel form of EV that bud from membrane protrusions on the HUVEC surface, contain MVBs and release exosomes. These observations suggest that exosomes can be harboured within and released from transiting microvesicles after departure from the parent cell, constituting a new site of exosome biogenesis occurring from endothelial and potentially additional cell types.

Reptile white blood cell (WBC) morphological features are strikingly variable across species. In the Argentine black and white tegu (Salvator merianae), red tegu (Salvator rufescens), and Savannah monitor (Varanus exanthematicus), previous reports described a WBC type with a single distinct, clear, linear- to ovoid- to crescent-shaped inclusion of presumptive monocytic origin. The objective of this study was to further investigate the origin of this unique WBC type with crescent-shaped inclusions. Blood samples from two Argentine black and white tegus, tegu 1, a 4-year-old female, and tegu 2, a 2-year-old presumed male, were submitted for routine hematological evaluation. Additional blood films were prepared and stained with these cytochemical stains: alkaline phosphatase (ALP; naphthol AS-MX phosphate substrate), alpha-naphthyl butyrate esterase, alpha-chloroacetate esterase, myeloperoxidase, Periodic acid-Schiff, and Sudan black B. Blood films from tegu 1 were also stained with a second ALP stain (5-bromo-4-chloro-3-indoxyl-phosphate and nitroblue tetrazolium substrate), Luna, luxol fast blue, and toluidine blue. The blood from tegu 1 was cytocentrifuged to isolate and fix the buffy coat in glutaraldehyde 2.5% aqueous solution for transmission electron microscopy. Six morphologically distinct WBC types were identified from tegu 1, including heterophils, basophils, monocytes, azurophils, lymphocytes, and the unique WBC type, which were identified as eosinophils with inclusions. WBC types in tegu 2 were similar; however, eosinophils lacked a discernable inclusion. Proper WBC identification will be useful in obtaining accurate hemogram data for this species.

UNASSIGNED: Beta-glucan (β-glucan) is a polysaccharide containing β-glycosidic bonds that is an important structure part of different yeast cells.
UNASSIGNED: The purpose of the study is to characterize β-glucan obtained from Candida albicans (C. albicans) isolated from caprine mastitis.
UNASSIGNED: The β-glucan was extracted by using utilizing an Alkaline-acidic extraction technique. The dry weight of extracted β-glucan was 7.47/150 g with 4.98%.
UNASSIGNED: The findings demonstrated that the extracted β-glucan had similarity in the primary peak 5.78 of liquid samples using the method of high-performance liquid chromatography when compared to the standard form of β-glucan. However, scanning electron microscopy studies revealed that the standard of β-glucan was distinct in morphology but similar to β-glucan isolated from C. albicans in terms of particle sizes in the range of 1.60-2.65 m and the lack of cell wall traces. The findings of an investigation using energy-dispersive X-ray fluorescence spectroscopy (EDS/EDX) of extracted and standard β-glucan, showed the principal elements discovered were carbon (C), oxygen (O), and nitrogen (N). Aluminum (Al), silicon (Si), nickel (Ni), and gold (Au) were also present, but in less amounts.
UNASSIGNED: The extracted β-glucan displayed a high degree of similarity and purity to the standard β-glucan, according to the findings of Fourier transform infrared spectroscopy (FT-IR) research.

The determination of the atomic resolution structure of biomacromolecules is essential for understanding details of their function. Traditionally, such a structure determination has been performed with crystallographic or nuclear resonance methods, but during the last decade, cryogenic transmission electron microscopy (cryo-TEM) has become an equally important tool. As the blotting and flash-freezing of the samples can induce conformational changes, external validation tools are required to ensure that the vitrified samples are representative of the solution. Although many validation tools have already been developed, most of them rely on fully resolved atomic models, which prevents early screening of the cryo-TEM maps. Here, a novel and automated method for performing such a validation utilizing small-angle X-ray scattering measurements, publicly available through the new software package AUSAXS, is introduced and implemented. The method has been tested on both simulated and experimental data, where it was shown to work remarkably well as a validation tool. The method provides a dummy atomic model derived from the EM map which best represents the solution structure.

UNASSIGNED: Hashimoto thyroiditis (HT) is an autoimmune disorder associated with hypothyroidism. Lymphocyte infiltration leading to thyroid follicular cell destruction is counteracted by increased collagen production, deposition and scarring. However, only recently a specific subpopulation of modified fibroblasts with contractile properties, namely \"myofibroblasts\" (MFBs) have been linked to HT.
UNASSIGNED: Our ultrastructural study aims to delineate the presence and contribution of MFBs to the fibrotic milieu of HT.
UNASSIGNED: Tissue biopsies were obtained from 5 HT-diagnosed patients and specimens were examined using a Transmission Electron Microscope (TEM).
UNASSIGNED: Histopathological examination indicated extensive microvilli atrophy and atypical vacuolations of the thyroid follicular cells in the HT samples. In addition to interstitial extravasated lymphocytes, capillaries were encircled by MFBs (mean distance from lumen 1.248± 0.43µm) with the characteristic electron-dense α-smooth muscle actin (α-SMA), confirmable in higher magnifications. Myofibroblastic projections were found to have significantly higher representation near the capillary lumen compared to the impaired endothelial lining (P < 0.01).
UNASSIGNED: Our TEM findings suggest that the intrusion of endothelia by myofibroblastic projections can be a significant factor towards the malfunction of follicular cells in HT patients and offer a paradigmal understanding of the ultrastructural interactions that may underlie the HT pathology.

A 23-month-old neutered male dog of unknown ancestry presented with a history of progressive neurological signs that included anxiety, cognitive impairment, tremors, seizure activity, ataxia, and pronounced visual impairment. The clinical signs were accompanied by global brain atrophy. Due to progression in the severity of disease signs, the dog was euthanized at 26 months of age. An examination of the tissues collected at necropsy revealed dramatic intracellular accumulations of autofluorescent inclusions in the brain, retina, and cardiac muscle. The inclusions were immunopositive for subunit c of mitochondrial ATP synthase, and their ultrastructural appearances were similar to those of lysosomal storage bodies that accumulate in some neuronal ceroid lipofuscinosis (NCL) diseases. The dog also exhibited widespread neuroinflammation. Based on these findings, the dog was deemed likely to have suffered from a form of NCL. A whole genome sequence analysis of the proband\'s DNA revealed a homozygous C to T substitution that altered the intron 3-exon 4 splice site of CLN6. Other mutations in CLN6 cause NCL diseases in humans and animals, including dogs. The CLN6 protein was undetectable with immunolabeling in the tissues of the proband. Based on the clinical history, fluorescence and electron-microscopy, immunohistochemistry, and molecular genetic findings, the disorder in this dog was classified as an NCL resulting from the absence of the CLN6 protein. Screening the dog\'s genome for a panel of breed-specific polymorphisms indicated that its ancestry included numerous breeds, with no single breed predominating. This suggests that the CLN6 disease variant is likely to be present in other mixed-breed dogs and at least some ancestral breeds, although it is likely to be rare since other cases have not been reported to date.

We revisited the formula related to the overall tilt angle of a specimen using a side-entry double-tilt sample holder in a transmission electron microscope. Initially, we examined existing formulas in the literature for calculating the overall tilt angle. Subsequently, a new formula was derived, proven to better account for the actions of the double-tilt holder, thereby providing improved accuracy in the calculation. This newly derived formula has been implemented in the Landyne software suite. Furthermore, we demonstrated the accuracy of the new formula through examples. RESEARCH HIGHLIGHTS: A new formula has been derived to calculate overall tilt angles for side-entry double-tilt holders in TEM.

Toxoplasma gondii and Neospora caninum are major worldwide morbidity-causing pathogens. Bumped kinase inhibitors (BKIs) are a compound class that has been optimized to target the apicomplexan calcium-dependent protein kinase 1 (CDPK1) - and several members of this class have proven to be safe and highly active in vitro and in vivo. BKI-1708 is based on a 5-aminopyrazole-4-carboxamide scaffold, and exhibited in vitro IC50 values of 120 nM for T. gondii and 480 nM for N. caninum β-galactosidase expressing strains, and did not affect human foreskin fibroblast (HFF) viability at concentrations up to 25 μM. Electron microscopy established that exposure of tachyzoite-infected fibroblasts to 2.5 μM BKI-1708 in vitro induced the formation of multinucleated schizont-like complexes (MNCs), characterized by continued nuclear division and harboring newly formed intracellular zoites that lack the outer plasma membrane. These zoites were unable to finalize cytokinesis to form infective tachyzoites. BKI-1708 did not affect zebrafish (Danio rerio) embryo development during the first 96 h following egg hatching at concentrations up to 2 μM. Treatments of mice with BKI-1708 at 20 mg/kg/day during five consecutive days resulted in drug plasma levels ranging from 0.14 to 4.95 μM. In vivo efficacy of BKI-1708 was evaluated by oral application of 20 mg/kg/day from day 9-13 of pregnancy in mice experimentally infected with N. caninum (NcSpain-7) tachyzoites or T. gondii (TgShSp1) oocysts. This resulted in significantly decreased cerebral parasite loads and reduced vertical transmission in both models without drug-induced pregnancy interference.

Connectomics provides essential nanometer-resolution, synapse-level maps of neural circuits to understand brain activity and behavior. However, few researchers have access to the high-throughput electron microscopes necessary to generate enough data for whole circuit or brain reconstruction. To date, machine-learning methods have been used after the collection of images by electron microscopy (EM) to accelerate and improve neuronal segmentation, synapse reconstruction and other data analysis. With the computational improvements in processing EM images, acquiring EM images has now become the rate-limiting step. Here, in order to speed up EM imaging, we integrate machine-learning into real-time image acquisition in a singlebeam scanning electron microscope. This SmartEM approach allows an electron microscope to perform intelligent, data-aware imaging of specimens. SmartEM allocates the proper imaging time for each region of interest - scanning all pixels equally rapidly, then re-scanning small subareas more slowly where a higher quality signal is required to achieve accurate segmentability, in significantly less time. We demonstrate that this pipeline achieves a 7-fold acceleration of image acquisition time for connectomics using a commercial single-beam SEM. We apply SmartEM to reconstruct a portion of mouse cortex with the same accuracy as traditional microscopy but in less time.

Large-scale electron microscopy (EM) has enabled the reconstruction of brain connectomes at the synaptic level by serially scanning over massive areas of sample sections. The acquired big EM data sets raise the great challenge of image mosaicking at high accuracy. Currently, it simply follows the conventional algorithms designed for natural images, which are usually composed of only a few tiles, using a single type of keypoint feature that would sacrifice speed for stronger performance. Even so, in the process of stitching hundreds of thousands of tiles for large EM data, errors are still inevitable and diverse. Moreover, there has not yet been an appropriate metric to quantitatively evaluate the stitching of biomedical EM images. Here we propose a two-stage error detection method to improve the EM image mosaicking. It firstly uses point-based error detection in combination with a hybrid feature framework to expedite the stitching computation while maintaining high accuracy. Following is the second detection of unresolved errors with a newly designed metric of EM stitched image quality assessment (EMSIQA). The novel detection-based mosaicking pipeline is tested on large EM data sets and proven to be more effective and as accurate when compared with existing methods.