Avian retroviral cardiomyopathy induced by infectious molecular clones of avian leukosis viruses (fowl glioma-inducing virus variants).

We previously described cardiomyocyte abnormality caused by Km_5666 strain, a variant of fowl glioma-inducing virus (FGV) prototype, which is an avian leukosis virus (ALV). However, the cardiac involvement appeared to be eradicated from the flock after a few years. An epidemiological survey from 2017 to 2020 was performed to elucidate the current prevalence of the cardiopathogenic strains in this flock. Four of the 71 bantams pathologically examined showed both glioma and cardiomyocyte abnormality, from which three ALV strains were detected. DNA sequencing revealed that several different ALV strains coexisted in each bantam and that the conserved Km_5666 virus fluid also contained at least two different ALV strains. We generated three infectious molecular clones from these samples, named KmN_77_clone_A, KmN_77_clone_B, and Km_5666_clone. The envSU of KmN_77_clone_A shared high sequence identity with that of Km_5666 (94.1%). In contrast, the envSU of KmN_77_clone_B showed >99.2% nucleotide similarity with that of an FGV variant without cardiopathogenicity. Furthermore, Km_5666_clone experimentally reproduced both gliomas and cardiomyocyte abnormality in chickens. From these results, it is suggested that the pathogenic determinant of cardiomyocyte abnormality is located in envSU similar to that of Km_5666. The cloning technique described here is beneficial for evaluating the viral pathogenicity in cases where affected birds are coinfected with several different ALV strains.

Objectively-measured out-of-home behavior and physical activity in rural older adults.

This cross-sectional study investigated the association of out-of-home behavior measured by global positioning system (GPS) and amounts of physical activity. Rural older adults aged ≥ 65 years (n = 133) participated in this study. Daily step count and physical activity level were measured using an accelerometer. We monitored out-of-home behavior using a GPS sensor and calculated two indicators: out-of-home time and number of nodes (places) visited per day. In results, only the number of nodes visited was significantly associated with step count (B coefficient = 1,324; 95% CI = 622 to 2,026) and physical activity level (B coefficient = 0.05; 95% CI = 0.02 to 0.09) in the fully-adjusted model, while out-of-home time was not. A greater number of nodes visited, rather than out-of-home time, was associated with higher amounts of physical activity in older adults.

Presence of calpain-5 in mitochondria.

Calpains are Ca2+-dependent cysteine proteases that are widely distributed in animal tissues and modulate a variety of cellular processes. There are 15 members of the calpain family in mammals. In animal cells, there are three types of calpains, viz., calpain-1, calpain-2, and calpain-10 in the mitochondria. The three types of calpains have been shown to play significant roles in pathophysiological conditions, including in apoptosis- and necrosis-like cell death. One of the severe retinal diseases, autosomal dominant neovascular inflammatory vitreoretinopathy, is known to be induced by mutations of the calpain-5 gene. However, the distribution of calpain-5 in the retina has not been elucidated. Therefore, in the present study, we determined the localization of calpain-5 in the porcine retina. We detected calpain-5 in the inner segment of photoreceptor cells using immunohistochemistry. With immunoelectron microscopy, calpain-5 was localized in the mitochondria of photoreceptor cells. Western blot analyses showed that calpain-5 was present in each mitochondrial subfraction. Furthermore, we showed that the molecular weight of mitochondrial calpain-5 was slightly smaller than cytosolic one. Our results demonstrated that a novel mitochondrial calpian, calpain-5, was localized in the mitochondria of retinal photoreceptor cells.

Characterization of mitochondrial calpain-5.

Calpain, a Ca2+-dependent cysteine protease, plays a significant role in gene expression, signal transduction, and apoptosis. Mutations in human calpain-5 cause autosomal dominant neovascular inflammatory vitreoretinopathy and the inhibition of calpain-5 activity may constitute an effective therapeutic strategy for this condition. Although calpain-5 is ubiquitously expressed in mammalian tissues and was recently found to be present in the mitochondria as well as in the cytosol, its physiological function and enzymological properties require further elucidation. The objective of the current study was to determine the characteristics of mitochondrial calpain-5 in porcine retinas, human HeLa cells, and C57BL/6J mice using subcellular fractionation. We found that mitochondrial calpain-5 was proteolyzed/autolyzed at low Ca2+ concentrations in mitochondria isolated from porcine retinas and by thapsigargin-induced endoplasmic reticulum (ER) stress in HeLa cells. Further, mitochondrial calpain-5, as opposed to cytosolic calpain-5, was activated during the early stages of ER stress in C57BL/6J mice. These results showed that mitochondrial calpain-5 was activated at low Ca2+ concentrations in vitro and in response to ER stress in vivo. The present study provides new insights into a novel calpain system in the mitochondria that includes stress responses during the early phases of ER stress. Further, activation of mitochondrial calpain-5 by treatment using low-molecular-weight compounds may have therapeutic potential for diseases related to ER stress, including neurodegenerative diseases, metabolic syndromes, diabetes, and cancer.

Data on mitochondrial ultrastructure of photoreceptors in pig, rabbit, and mouse retinas.

Photoreceptors are one of the most energy-consuming cell types within the human body. To meet their high energy demand, photoreceptors possess a mitochondrial cluster in the inner segment of the cell. Interestingly, in several species, the inner segment of cone photoreceptors contains extremely large mitochondria that exceed 2 µm in diameter, called mega-mitochondria. We previously reported that pig retinas also contain mega-mitochondria, however, there are few reports whether mega-mitochondria are present in mammalian photoreceptors. In the present experiment, we analyzed pig, rabbit, and mouse photoreceptors under a scanning electron microscope (SEM), and compared the mitochondrial morphology. Our data showed that all three species present numerous mitochondrial clusters in the ellipsoid zone of photoreceptors, adjacent to the outer segment. In the pig retina, the inner segments of cone and rod photoreceptors were localized in different layers; consequently, we were able to distinguish them easily. Mega-mitochondria were identified only in the inner segment of cone photoreceptors. Also, mitochondria of cone photoreceptors, including mega-mitochondria, were dense cristae and high electron-densities compared to those of rod photoreceptors. In the rabbit retina, cone photoreceptors were existed within the layer of rod photoreceptor outer segment. The rod photoreceptors had a characteristic long outer segment. Cone photoreceptors had a short outer segment, and also had a thick inner segment compared to rod photoreceptors. Most of the mitochondria present in the rod photoreceptor inner segment were long and narrow, whereas mitochondria of cone photoreceptors were fragmented and short. Mega-mitochondria was not detected in rabbit retina. In the mouse retina, most of the photoreceptor cells were rod photoreceptors. Since the shape of the inner segments were very similar, we distinguished cone and rod photoreceptors based on the shape of the outer segments. Some mitochondria of both rod and cone photoreceptors were long and narrow, and there was no significant difference in mitochondrial morphology. Our data showed that mitochondrial morphology in the inner segment of photoreceptors vary among mammalian species. Although mega-mitochondria were present in pig photoreceptors, we could not observe their presence in rabbit nor mouse retinas. To our knowledge, this is a first experiment that perform the wide field observation of rabbit and mouse retina using electron microscopy, and that compare the mitochondrial morphology of photoreceptor cells in pig, rabbit and mouse.