UNASSIGNED: The purpose of this study was to investigate the role and mechanism of microtubule-associated protein light chain-3 (LC3)-associated phagocytosis (LAP) in the immune response to Aspergillus fumigatus (A. fumigatus) keratitis.
UNASSIGNED: The formation of single-membrane phagosomes was visualized in the corneas of healthy or A. fumigatus-infected humans and C57BL/6 mice using transmission electron microscopy (TEM). Rubicon siRNA (si-Rubicon) was used to block Rubicon expression. RAW 264.7 cells or mice corneas were infected with A. fumigatus with or without pretreatment of si-Rubicon and scrambled siRNA. RAW 264.7 cells were pretreated with Dectin-1 antibody or Dectin-1 overexpressed plasmid and then stimulated with A. fumigatus. Flow cytometry was used to label macrophages in normal and infected corneas of mice. In mice with A. fumigatus keratitis, the severity of the disease was assessed using clinical scores. We used lentiviral technology to transfer GV348-Ubi-GFP-LC3-II-SV40-Puro Lentivirus into the mouse cornea. The GFP-LC3 fusion protein was visualized in corneal slices using a fluorescence microscope. We detected the mRNA and protein expressions of the inflammatory factors IL-6, IL-1β, and IL-10 using real-time PCR (RT-PCR) and ELISA. We detected the expression of LAP-related proteins Rubicon, ATG-7, Beclin-1, and LC3-II using Western blot or immunofluorescence.
UNASSIGNED: Accumulation of single-membrane phagosomes within macrophages was observed in the corneas of patients and mice with A. fumigatus keratitis using TEM. Flow cytometry (FCM) analysis results show that the number of macrophages in the cornea of mice significantly increases after infection with A. fumigatus. LAP-related proteins were significantly elevated in the corneas of mice and RAW 264.7 cells after infection with A. fumigatus. The si-Rubicon treatment elevated the clinical score of mice. In A. fumigatus keratitis mice, the si-Rubicon treated group showed significantly higher expression of IL-6 and IL-1β and lower expression of IL-10 and LC3-II compared to the control group. In RAW 264.7 cells, treatment with the Dectin-1 overexpressed plasmid upregulated the expression of LAP-related proteins, a process that was significantly inhibited by the Dectin-1 antibody.
UNASSIGNED: LAP participates in the anti-inflammatory immune process of fungal keratitis (FK) and exerts an anti-inflammatory effect. LAP is regulated through the Dectin-1 signaling pathway in A. fumigatus keratitis.

Transmission electron microscopy (TEM) is an imaging technique used to visualize and analyze nano-sized structures and objects such as virus particles. Light microscopy can be used to diagnose diseases or characterize e.g. blood cells. Since samples under microscopes exhibit certain symmetries, such as global rotation invariance, equivariant neural networks are presumed to be useful. In this study, a baseline convolutional neural network is constructed in the form of the commonly used VGG16 classifier. Thereafter, it is modified to be equivariant to the p4 symmetry group of rotations of multiples of 90° using group convolutions. This yields a number of benefits on a TEM virus dataset, including higher top validation set accuracy by on average 7.6% and faster convergence during training by on average 23.1% of that of the baseline. Similarly, when training and testing on images of blood cells, the convergence time for the equivariant neural network is 7.9% of that of the baseline. From this it is concluded that augmentation strategies for rotation can be skipped. Furthermore, when modelling the accuracy versus amount of TEM virus training data with a power law, the equivariant network has a slope of - 0.43 compared to - 0.26 of the baseline. Thus the equivariant network learns faster than the baseline when more training data is added. This study extends previous research on equivariant neural networks applied to images which exhibit symmetries to isometric transformations.

Chlorophyll (Chl) plays a crucial role in photosynthesis, functioning as a photosensitizer. As an integral component of this process, energy absorbed by this pigment is partly emitted as red fluorescence. This signal can be readily imaged by fluorescence microscopy and provides a visualization of photosynthetic activity. However, due to limited resolution, signals cannot be assigned to specific subcellular/organellar membrane structures. By correlating fluorescence micrographs with transmission electron microscopy, researchers can identify sub-cellular compartments and membranes, enabling the monitoring of Chl distribution within thylakoid membrane substructures in cyanobacteria, algae, and higher plant single cells. Here, we describe a simple and effective protocol for correlative light-electron microscopy (CLEM) based on the autofluorescence of Chl and demonstrate its application to selected photosynthetic model organisms. Our findings illustrate the potential of this technique to identify areas of high Chl concentration and photochemical activity, such as grana regions in vascular plants, by mapping stacked thylakoids.

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.

Liquid-transmission electron microscopy (liquid-TEM) provides exciting potential for capturing mineralization events at biomaterial interfaces, though it is largely unexplored. To address this, we established a unique approach to visualize calcium phosphate (CaP)-titanium (Ti) interfacial mineralization events by combining the nanofabrication of Ti lamellae by focused ion beam with in situ liquid-TEM. Multiphasic CaP particles were observed to nucleate, adhere, and form different assemblies onto and adjacent to Ti lamellae. Here, we discuss new approaches for exploring the interaction between biomaterials and liquids at the nanoscale. Driving this technology is crucial for understanding and controlling biomineralization to improve implant osseointegration and direct new pathways for mineralized tissue disease treatment in the future.

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem cells (iPSCs) under specific conditions. Synapse development and maturation in ROs have been studied immunocytochemically and functionally. However, the direct evidence of the synaptic contact ultrastructure is limited, containing both special ribbon synapses and conventional chemical synapses. Transmission electron microscopy (TEM) is characterized by high resolution and a respectable history elucidating retinal development and synapse maturation in humans and various species. It is a powerful tool to explore synaptic structure in ROs and is widely used in the research field of ROs. Therefore, to better explore the structure of RO synaptic contacts at the nanoscale and obtain high-quality microscopic evidence, we developed a simple and repeatable method of RO TEM sample preparation. This paper describes the protocol, reagents used, and detailed steps, including RO fixation preparation, post fixation, embedding, and visualization.

UNASSIGNED: Recent studies have shown that the retinal pigment epithelium (RPE) relies on fatty acid oxidation (FAO) for energy, however, its role in overall retinal health is unknown. The only FAO disorder that presents with chorioretinopathy is long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD). Studying the molecular mechanisms can lead to new treatments for patients and elucidate the role of FAO in the RPE. This paper characterizes the chorioretinopathy progression in a recently reported LCHADD mouse model.
UNASSIGNED: Visual assessments, such as optokinetic tracking and fundus imaging, were performed in wildtype (WT) and LCHADD mice at 3, 6, 10, and 12 months of age. Retinal morphology was analyzed in 12-month retinal cross-sections using hematoxylin and eosin (H&E), RPE65, CD68, and TUNEL staining, whereas RPE structure was assessed using transmission electron microscopy (TEM). Acylcarnitine profiles were measured in isolated RPE/sclera samples to determine if FAO was blocked. Bulk RNA-sequencing of 12 month old male WT mice and LCHADD RPE/sclera samples assessed gene expression changes.
UNASSIGNED: LCHADD RPE/sclera samples had a 5- to 7-fold increase in long-chain hydroxyacylcarnitines compared to WT, suggesting an impaired LCHAD step in long-chain FAO. LCHADD mice have progressively decreased visual performance and increased RPE degeneration starting at 6 months. LCHADD RPE have an altered structure and a two-fold increase in macrophages in the subretinal space. Finally, LCHADD RPE/sclera have differentially expressed genes compared to WT, including downregulation of genes important for RPE function and angiogenesis.
UNASSIGNED: Overall, this LCHADD mouse model recapitulates early-stage chorioretinopathy seen in patients with LCHADD and is a useful model for studying LCHADD chorioretinopathy.

CONCLUSIONS: Petal developmental characteristics in Fumarioideae were similar at early stages, and the specialized nectar holder/pollen container formed by the outer/inner petals. The micro-morphology of these two structures, however, shows diversity in seven species. Elaborate petals have been modified to form different types, including petal lobes, ridges, protuberances, and spurs, each with specialized functions. Nectar holder and pollen container presumably have a function in plant-pollinator interactions. In Fumarioideae, four elaborate petals of the disymmetric/zygomorphic flower present architecture forming the \"nectar holder\" and \"pollen container\" structure at the bottom and top separately. In the present study, the petals of seven species in Fumarioideae were investigated by scanning electron microscopy, light microscope, and transmission electron microscopes. The results show that petal development could divided into six stages: initiation, enlargement, adaxial/abaxial differentiation, elaborate specializations (sacs, spurs, and lobes formed), extension, and maturation, while the specialized \"nectar holder\" and \"pollen container\" structures mainly formed in stage 4. \"Nectar holder\" is developed from the shallow sac/spur differentiated at the base of the outer petal, eventually forming a multi-organized complex structure, together with staminal nectaries (1-2) with individual sizes. A semi-closed ellipsoidal \"pollen container\" is developed from the apical part of the 3-lobed inner petals fused by middle lobes and attain different sizes. The adaxial epidermis cells are specialized, with more distinct punctate/dense columnar protrusions or wavy cuticles presented on obviously thickening cell walls. In addition, a large and well-developed cavity appears between the inner and outer epidermis of the petals. As an exception, Hypecoum erectum middle lobes present stamen mimicry. Elaborate petal structure is crucial for comprehending the petal diversity in Fumarioideae and provides more evidence for further exploration of the reproductive study in Papaveraceae.

A comprehensive light and ultrastructural examination of the cornea in Domestic Pigs (Sus scrofa domesticus) revealed four distinct layers: the anterior epithelium, corneal stroma, Descemet\'s membrane and endothelium. Although Bowman\'s layer was not distinctly identified through histology, histochemical analysis indicated the presence of a rudimentary Bowman\'s layer, possibly vestigial from evolution. Scanning electron microscopy of the outer corneal surface unveiled two cell types, characterized by micro-projections, with light cells exhibiting shorter, thicker projections compared to dark cells. Examination of the inner surface via scanning electron microscopy demonstrated an endothelial layer devoid of cilia and microvilli, yet faint round to oval elevations were observed, potentially representing cell nuclei. Transmission electron microscopy unveiled that basal cells of the anterior epithelium closely adhered to the basement membrane, featuring half desmosomes along the basal surface. These basal cells extensively interconnected through interdigitations and a few desmosomes. The superficial cell layer consisted of a few rows of closely attached flat cells, forming a leak-proof layer with zona occludens. The outermost cells of this layer displayed fine projections to enhance the surface area, facilitating tear film distribution. At lower magnification, Transmission electron microscopy of the corneal stroma revealed alternating light and dark bands, with light bands representing transverse sections of collagen fibril lamellae and dark bands corresponding to longitudinal or oblique sections. Spindle-shaped keratocytes (fibroblasts) were identified as the primary stromal cells, intermingled between the lamellae, and featured long processes in close contact with neighbouring keratocytes. Overall, the histomorphology of the pig cornea resembles that of the human cornea except indistinct Bowman\'s membrane. This detailed understanding of the normal corneal structure in pigs hold great significance for biomedical research, providing a valuable reference for studies involving this animal model.

Chaetae are among the most extensively studied structures in polychaetes, serving as a defining morphological trait for annelids. Capitella teleta stands out as one of the few established annelid models for developmental and morphological studies, thus receiving significant scholarly attention. In this study, we unveil a previously unnoticed glandular structure associated with chaetae within the larvae of C. teleta. Our investigations demonstrate the absence of comparable structures in the chaetal follicles of adults and juveniles (older than 1 week), as well as during active chaetogenesis, underscoring the transient nature of these glands. This indicates that larval chaetal follicles transform into a gland that later disappears. Utilizing histology and transmission electron microscopy, we characterized these glands. Our findings underscore the diversity of chaetal ultrastructure in annelids and show that, even in well-studied species, novel morphological details can be found. We emphasize the importance of examining various life-history stages to capture such transient morphological features. This work lays a crucial morphological foundation and deepens our understanding of chaetae and chaetogenesis in C. teleta, paving the way for more accurate interpretations of future experimental studies on chaetogenesis in this species.