The superficial layers of the mammalian superior colliculus (SC) contain neurons that are generally responsive to visual stimuli but can differ considerably in morphology and response properties. To elucidate the structure and function of these neurons, we combined extracellular recording and juxtacellular labeling, detailed anatomical reconstruction, and ultrastructural analysis of the synaptic contacts of labeled neurons, using transmission electron microscopy. Our labeled neurons project to different brainstem nuclei. Of particular importance are neurons that fit the morphological criteria of the wide field (WF) neurons and whose dendrites are horizontally oriented. They display a rather characteristic axonal projection pattern to the nucleus of optic tract (NOT); thus, we call them superior collicular WF projecting to the NOT (SCWFNOT) neurons. We corroborated the morphological characterization of this neuronal type as a distinct neuronal class with the help of unsupervised hierarchical cluster analysis. Our ultrastructural data demonstrate that SCWFNOT neurons establish excitatory connections with their targets in the NOT. Although, in rodents, the literature about the WF neurons has focused on their extensive projection to the lateral posterior nucleus of the thalamus, as a conduit for information to reach the visual association areas of the cortex, our data suggest that this subclass of WF neurons may participate in the optokinetic nystagmus.

AQP4 is expressed in the endfeet membranes of subpial and perivascular astrocytes and in the ependymal cells that line the ventricular system. The sporadic appearance of obstructive congenital hydrocephalus (OCHC) has been observed in the offspring of AQP4-/- mice (KO) due to stenosis of Silvio\'s aqueduct. Here, we explore whether the lack of AQP4 expression leads to abnormal development of ependymal cells in the aqueduct of mice. We compared periaqueductal samples from wild-type and KO mice. The microarray-based transcriptome analysis reflected a large number of genes with differential expression (809). Gene sets (GS) associated with ependymal development, ciliary function and the immune system were specially modified qPCR confirmed reduced expression in the KO mice genes: (i) coding for transcription factors for ependymal differentiation (Rfx4 and FoxJ1), (ii) involved in the constitution of the central apparatus of the axoneme (Spag16 and Hydin), (iii) associated with ciliary assembly (Cfap43, Cfap69 and Ccdc170), and (iv) involved in intercellular junction complexes of the ependyma (Cdhr4). By contrast, genes such as Spp1, Gpnmb, Itgax, and Cd68, associated with a Cd11c-positive microglial population, were overexpressed in the KO mice. Electron microscopy and Immunofluorescence of vimentin and γ-tubulin revealed a disorganized ependyma in the KO mice, with changes in the intercellular complex union, unevenly orientated cilia, and variations in the planar cell polarity of the apical membrane. These structural alterations translate into reduced cilia beat frequency, which might alter cerebrospinal fluid movement. The presence of CD11c + microglia cells in the periaqueductal zone of mice during the first postnatal week is a novel finding. In AQP4-/- mice, these cells remain present around the aqueduct for an extended period, showing peak expression at P11. We propose that these cells play an important role in the normal development of the ependyma and that their overexpression in KO mice is crucial to reduce ependyma abnormalities that could otherwise contribute to the development of obstructive hydrocephalus.

The evolution of a multilayer sample surface during focused ion beam processing was simulated using the level set method and experimentally studied by milling a silicon dioxide layer covering a crystalline silicon substrate. The simulation took into account the redeposition of atoms simultaneously sputtered from both layers of the sample as well as the influence of backscattered ions on the milling process. Monte Carlo simulations were applied to produce tabulated data on the angular distributions of sputtered atoms and backscattered ions. Two sets of test structures including narrow trenches and rectangular boxes with different aspect ratios were experimentally prepared, and their cross sections were visualized in scanning transmission electron microscopy images. The superimposition of the calculated structure profiles onto the images showed a satisfactory agreement between simulation and experimental results. In the case of boxes that were prepared with an asymmetric cross section, the simulation can accurately predict the depth and shape of the structures, but there is some inaccuracy in reproducing the form of the left sidewall of the structure with a large amount of the redeposited material. To further validate the developed simulation approach and gain a better understanding of the sputtering process, the distribution of oxygen atoms in the redeposited layer derived from the numerical data was compared with the corresponding elemental map acquired by energy-dispersive X-ray microanalysis.

Layered lithiated oxides are promising materials for next generation Li-ion battery cathode materials; however, instability during cycling results in poor performance over time compared to the high capacities theoretically possible with these materials. Here we report the characterizations of a Li1.47Mn0.57Al0.13Fe0.095Co0.105Ni0.095O2.49 high-entropy layered oxide (HELO) with the Li2MO3 structure where M = Mn, Al, Fe, Co, and Ni. Using electron microscopy and X-ray spectroscopy, we identify a homogeneous Li2MO3 structure stabilized by the entropic contribution of oxygen vacancies. This defect-driven entropy would not be attainable in the LiMO2 structure sometimes observed in similar materials as a secondary phase owing to the presence of fewer O sites and a 3+ oxidation state for the metal site; instead, a Li2-γMO3-δ is produced. Beyond Li2MO3, this defect-driven entropy approach to stabilizing novel compositions and phases can be applied to a wide array of future cathode materials including spinel and rock salt structures.

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.