Lack of collagen α6(IV) chain in mice does not cause severe-to-profound hearing loss or cochlear malformation, a distinct phenotype from nonsyndromic hearing loss with COL4A6 missense mutation.

Congenital hearing loss affects 1 in every 1000 births, with genetic mutations contributing to more than 50% of all cases. X-linked nonsyndromic hereditary hearing loss is associated with six loci (DFNX1-6) and five genes. Recently, the missense mutation (c.1771G>A, p.Gly591Ser) in COL4A6, encoding the basement membrane (BM) collagen α6(IV) chain, was shown to be associated with X-linked congenital nonsyndromic hearing loss with cochlear malformation. However, the mechanism by which the COL4A6 mutation impacts hereditary hearing loss has not yet been elucidated. Herein, we investigated Col4a6 knockout (KO) effects on hearing function and cochlear formation in mice. Immunohistochemistry showed that the collagen α6(IV) chain was distributed throughout the mouse cochlea within subepithelial BMs underlying the interdental cells, inner sulcus cells, basilar membrane, outer sulcus cells, root cells, Reissner's membrane, and perivascular BMs in the spiral limbus, spiral ligament, and stria vascularis. However, the click-evoked auditory brainstem response analysis did not show significant changes in the hearing threshold of Col4a6 KO mice compared with wild-type (WT) mice with the same genetic background. In addition, the cochlear structures of Col4a6 KO mice did not exhibit morphological alterations, according to the results of high-resolution micro-computed tomography and histology. Hence, loss of Col4a6 gene expression in mice showed normal click ABR thresholds and normal cochlear formation, which differs from humans with the COL4A6 missense mutation c.1771G>A, p.Gly591Ser. Therefore, the deleterious effects in the auditory system caused by the missense mutation in COL4A6 are likely due to the dominant-negative effects of the α6(IV) chain and/or α5α6α5(IV) heterotrimer with an aberrant structure that would not occur in cases with loss of gene expression.

Prolonged Tachycardia with Higher Heart Rate Is Associated with Higher ICU and In-hospital Mortality.

Tachycardia is common in intensive care units (ICUs). It is unknown whether tachycardia or prolonged tachycardia affects patient outcomes. We investigated the association between tachycardia and mortality in critically ill patients. This retrospective cohort study's primary outcome was patient mortality in the ICU and the hospital. We stratified the patients (n=476) by heart rate (HR) as LowHR, MediumHR, and HighHR groups. We also stratified them by their durations of HR >100 (prolonged HR; tachycardia): MildT, ModerateT, and SevereT groups. We determined the six groups' mortality. The ICU mortality rates of the LowHR, MediumHR, and HighHR groups were 1.0%, 1.5%, and 7.9%, respectively; significantly higher in the HighHR vs. LowHR group. The in-hospital mortality rates of these groups were 1%, 4.5%, and 14.6%, respectively; significantly higher in the HighHR vs. LowHR group. The ICU mortality rates of the MildT, ModerateT, and SevereT groups were 0.9%, 5.6%, and 57.1%, respectively. The mortality of the HRT=0 (i.e., all HR ≤ 100) patients was 0%. The in-hospital mortality rates of the MildT, ModerateT, and SevereT groups were 1.8%, 16.7%, and 85.7%, respectively; that of the HRT=0 patients was 0.5%. Both higher HR and prolonged tachycardia were associated with poor outcomes.

Unripe peach (Prunus persica) extract ameliorates damage from UV irradiation and improved collagen XVIII expression in 3D skin model.

INTRODUCTION: Collagen type XVIII regulates cellular activities of adjacent cells at the dermal-epidermal junction (DEJ). To investigate its possible changes during aging, we compared its mRNA levels and protein localization in skin samples from female participants aged 20-70 years old. In addition, we evaluated the beneficial effects of unripe peach extracts in a 3D skin model. METHODS: Sun-exposed or sun-protected female skin samples were compared by DNA array or by immunohistochemistry for basement membrane components. To evaluate protective effects of fresh unripe peach extract, UV-B irradiated human 3D skin models were incubated in the presence or absence of the extract, followed by measurements of mRNA levels by real-time PCR, or by immunohistochemistry. RESULTS: In aged skin samples, COL18A1 mRNA levels were lower and the protein localization exhibited less intensive signal by anti-collagen type XVIII immunostaining. As observed in the skin tissues, collagen type XVIII exists at the DEJ in the 3D skin model. Fresh unripe peach extract significantly improved mRNA levels and partially localizations of collagen type XVIII, suggesting that fresh unripe peach extract ameliorates DEJ damages caused by UV-B irradiation. CONCLUSION: Collagen type XVIII and fresh unripe peach extract can be promising protective cosmetic strategies against skin aging.

Type IV collagen α6 chain is a regulator of keratin 10 in keratinization of oral mucosal epithelium.

Keratinized mucosa is of fundamental importance to maintain healthy gingival tissue, and understanding the mechanisms of oral mucosa keratinization is crucial to successfully manage healthy gingiva. Previous studies have shown a strong involvement of the basement membrane in the proliferation and differentiation of epithelial cells. Therefore, first, to identify the keratinized mucosa-specific basement membrane components, immunohistochemical analysis for the six alpha chains of type IV collagen was performed in 8-week-old mice. No difference in the expression pattern of type IV collagen α1(IV) and α2(IV) chains was observed in the keratinized and non-keratinized mucosa. Interestingly, however, type IV collagen α5(IV) and α6(IV) chains specifically were strongly detected in the keratinized mucosa. To analyze the functional roles of the type IV collagen isoform α6(IV) in oral mucosa keratinization, we analyzed Col4a6-knockout mice. Epithelial developmental delay and low levels of KRT10 were observed in new-born Col4a6-knockout mice. Additionally, in vitro experiments with loss-of function analysis using human gingival epithelial cells confirmed the important role of α6(IV) chain in epithelial keratinization. These findings indicate that α112:α556 (IV) network, which is the only network that includes the α6(IV) chain, is one regulator of KRT10 expression in keratinization of oral mucosal epithelium.

Network of anatomical texts (NAnaTex), an open-source project for visualizing the interaction between anatomical terms.

Anatomy is the science and art of understanding the structure of the body and its components in relation to the functions of the whole-body system. Medicine is based on a deep understanding of anatomy, but quite a few introductory-level learners are overwhelmed by the sheer amount of anatomical terminology that must be understood, so they regard anatomy as a dull and dense subject. To help them learn anatomical terms in a more contextual way, we started a new open-source project, the Network of Anatomical Texts (NAnaTex), which visualizes relationships of body components by integrating text-based anatomical information using Cytoscape, a network visualization software platform. Here, we present a network of bones and muscles produced from literature descriptions. As this network is primarily text-based and does not require any programming knowledge, it is easy to implement new functions or provide extra information by making changes to the original text files. To facilitate collaborations, we deposited the source code files for the network into the GitHub repository ( https://github.com/ryusukemomota/nanatex ) so that anybody can participate in the evolution of the network and use it for their own non-profit purposes. This project should help not only introductory-level learners but also professional medical practitioners, who could use it as a quick reference.

Drosophila type XV/XVIII collagen mutants manifest integrin mediated mitochondrial dysfunction, which is improved by cyclosporin A and losartan.

Vertebrate collagen types XV and XVIII are broadly distributed basement membrane components, classified into a structurally distinct subgroup called "multiplexin collagens". Mutations in mammalian multiplexins are identified in some degenerative diseases such as Knobloch syndrome 1 (KNO1) or skeletal/cardiac myopathies, however, these progressive properties have not been elucidated. Here we investigated Drosophila mutants of Multiplexin (Mp), the only orthologue of vertebrate collagen types XV and XVIII, to understand the pathogenesis of multiplexin-related diseases. The mp mutants exhibited morphological changes in cardiomyocytes and progressive dysfunction of the skeletal muscles, reminiscent phenotypes observed in Col15a1-null mice. Ultrastructural analysis revealed morphologically altered mitochondria in mutants' indirect flight muscles (IFMs), resulting in severely attenuated ATP production and enhanced reactive oxygen species (ROS) production. In addition, mutants' IFMs exhibited diminished ßPS integrin clustering and abolished focal adhesion kinase (FAK) phosphorylation. Furthermore, mutants' defective IFMs are improved by the administrations of cyclosporin A, an inhibitor against mitochondrial permeability transition pore (mPTP) opening or losartan, an angiotensin II type 1 receptor (AT1R) blocker. Thus, our results suggest that Mp modulates mPTP opening and AT1R activity through its binding to integrin and that lack of Mp causes unregulated mPTP opening and AT1R activity, leading to mitochondrial dysfunctions. Hence, our results provide new insights towards the roles of multiplexin collagens in mitochondrial homeostasis and may serve as pharmacological evidences for the potential use of cyclosporin A or losartan for the therapeutic strategies.

Architecture of the subendothelial elastic fibers of small blood vessels and variations in vascular type and size.

Most blood vessels contain elastin that provides the vessels with the resilience and flexibility necessary to control hemodynamics. Pathophysiological hemodynamic changes affect the remodeling of elastic components, but little is known about their structural properties. The present study was designed to elucidate, in detail, the three-dimensional (3D) architecture of delicate elastic fibers in small vessels, and to reveal their architectural pattern in a rat model. The fine vascular elastic components were observed by a newly developed scanning electron microscopy technique using a formic acid digestion with vascular casts. This method successfully visualized the 3D architecture of elastic fibers in small blood vessels, even arterioles and venules. The subendothelial elastic fibers in such small vessels assemble into a sheet of meshwork running longitudinally, while larger vessels have a higher density of mesh and thicker mesh fibers. The quantitative analysis revealed that arterioles had a wider range of mesh density than venules; the ratio of density to vessel size was higher than that in venules. The new method was useful for evaluating the subendothelial elastic fibers of small vessels and for demonstrating differences in the architecture of different types of vessels.

Abnormalities in the fiber composition and capillary architecture in the soleus muscle of type 2 diabetic Goto-Kakizaki rats.

Type 2 diabetes mellitus is linked to impaired skeletal muscle glucose uptake and storage. This study aimed to investigate the fiber type distributions and the three-dimensional (3D) architecture of the capillary network in the skeletal muscles of type 2 diabetic rats. Muscle fiber type transformation, succinate dehydrogenase (SDH) activity, capillary density, and 3D architecture of the capillary network in the soleus muscle were determined in 36-week-old Goto-Kakizaki (GK) rats as an animal model of nonobese type 2 diabetes and age-matched Wistar (Cont) rats. Although the soleus muscle of Cont rats comprised both type I and type IIA fibers, the soleus muscle of GK rats had only type I fibers. In addition, total SDH activity in the soleus muscle of GK rats was significantly lower than that in Cont rats because GK rats had no high-SDH activity type IIA fiber in the soleus muscle. Furthermore, the capillary diameter, capillary tortuosity, and microvessel volume in GK rats were significantly lower than those in Cont rats. These results indicate that non-obese diabetic GK rats have muscle fiber type transformation, low SDH activity, and reduced skeletal muscle capillary content, which may be related to the impaired glucose metabolism characteristic of type 2 diabetes.

Drosophila type XV/XVIII collagen, Mp, is involved in Wingless distribution.

Multiplexin (Mp) is the Drosophila orthologue of vertebrate collagens XV and XVIII. Like them, Mp is widely distributed in the basement membranes of the developing embryos, including those of neuroblasts in the central and peripheral nervous systems, visceral muscles of the gut, and contractile cardioblasts. Here we report the identification of mutant larvae bearing piggyBac transposon insertions that exhibit decrease Mp production associated with abdominal cuticular and wing margin defects, malformation of sensory organs and impaired sensitivity to physical stimuli. Additional findings include the abnormal ultrastructure of fatbody associated with abnormal collagen IV deposition, and reduced Wingless deposition. Collectively, these findings are consistent with the notion that Mp is required for the proper formation and/or maintenance of basement membrane, and that Mp may be involved in establishing the Wingless signaling gradients in the Drosophila embryo.

Comparison of capillary architecture between slow and fast muscles in rats using a confocal laser scanning microscope.

The skeletal muscle is classified into 2 types, slow oxidative or fast glycolytic muscle. For further characterization, we investigated the capillary architecture in slow and fast muscles. The rat soleus and extensor digitorum longus (EDL) muscles were used as representatives of slow and fast muscles, respectively. To investigate capillary density, sections of both types of muscle were stained with alkaline phosphatase; the soleus muscle showed more intense reactivity, indicating that it had a denser capillary structure than the EDL muscle. We then injected fluorescent contrast medium into samples of both muscle types for light and confocal-laser microscopic evaluation. The capillary density and capillary-to-fiber ratio were significantly higher, and the course of the capillaries was more tortuous, in the soleus muscle than in the EDL muscle. Capillary coursed more tortuously in the soleus than in the EDL muscle. Succinate dehydrogenase (SDH) activity, an indicator of mitochondrial oxidative capacity, and vascular endothelial growth factor (VEGF) expression were also significantly higher in the soleus muscle. Thus, we conclude that slow oxidative muscle possess a rich capillary structure to provide demanded oxygen, and VEGF might be involved in the formation and/or maintenance of this highly capillarized architecture.

A disintegrin and metalloproteinase with thrombospondin motifs 9 (ADAMTS9) expression by chondrocytes during endochondral ossification.

A disintegrin and metalloproteinase with thrombospondin motifs 9 (ADAMTS9) is known to influence aggrecan degradation in endochondral ossification, but its role has not been well understood. In the present study, in vitro gene expression of ADAMTS9 was investigated by RT-PCR in ATDC5 cells in which experimentally chondrogenic differentiation had been induced. We also investigated the protein localization and gene expression pattern of ADAMTS9 in the tibia growth plate cartilage of male mice in a day 1 neonate, 7-week-old young adult, and a 12-week-old adult by immunohistochemistry and in situ hybridization and compared the results with the expression of proliferating cell nuclear antigen (PCNA) and type X collagen for the identification of proliferative and hypertrophic chondrocyte phenotypes, respectively. We found the gene expression of ADAMTS9 by ATDC5 cells as a dual mode, both before the expression of type X collagen and after hypertrophic differentiation. The immunoreactivity of ADAMTS9 was observed in chondrocytes of proliferative and hypertrophic zones in the growth plate. The population of ADAMTS9 positive cells decreased with age. The results of the present study suggest that ADAMTS9 might have a role in aggrecan cleavage around the chondrocytes to allow chondrocyte proliferation and hypertrophy.

Type IV collagen alpha chains of the basement membrane in the rat bronchioalveolar transitional segment.

In the present study, we have analyzed the alpha(IV) chain distribution in the subepithelial basement membrane (BM) of the rat pulmonary airway from the bronchi to alveoli. We have furthermore analyzed the alpha(IV) chain distribution in the subepithelial BM of the bronchioalveolar duct junction (BADJ) using alpha(IV) chain specific monoclonal antibodies. Our results show that the BM of the bronchial and bronchiolar epithelium contains [alpha1(IV)]2alpha2(IV) and [alpha5(IV)]2alpha6(IV) molecules and confirmed that the alveolar BM consists of [alpha1(IV)]2alpha2(IV) and alpha3(IV) alpha4(IV)alpha5(IV) molecules. There are also small regions in BADJ consisting of only [alpha1(IV)]2alpha2(IV) molecules without alpha3(IV)alpha4(IV)alpha5(IV) and [alpha5(IV)]2alpha6(IV) molecules. Moreover, the bronchioalveolar stem cells (BASCs)-primordial cells for bronchiolar Clara cells and alveolar type II (AT2) cells - lie adjacent to such small regions. These findings suggest that [alpha1(IV)]2 alpha2(IV) may be important for the BASCs to self-renew or to self-maintain themselves and that microenvironments produced by alpha(IV) chains may be important for cell differentiation.

The differential distribution of type IV collagen alpha chains in the subepithelial basement membrane of the human alimentary canal.

We studied distribution patterns of type IV collagen alpha chains in the subepithelial basement membrane (SBM) of the human gastrointestinal tract - the esophagus through the anal canal - by immunofluorescent microscopy using alpha(IV) chain-specific monoclonal antibodies. The alpha1(IV), alpha2(IV), alpha5(IV), and alpha6(IV) chains were found in the SBM throughout the tract, indicating the localization of [alpha1(IV)](2)alpha2(IV) and [alpha5(IV)](2)alpha6(IV) heterotrimeric molecules. The [alpha1(IV)](2)alpha2(IV) molecule was continuously stained, while the [alpha5(IV)](2)alpha6(IV) molecule was weakly stained in gastric glands and small intestinal crypts. In addition, the SBM at the luminal surface epithelium of the stomach and large intestine contained small amounts of alpha3(IV) and alpha4(IV) chains which combined to form the alpha3(IV)alpha4(IV)alpha5(IV) heterotrimeric molecule with alpha5(IV) chain. The SBM beneath the villous epithelium of the small intestine was also demonstrated to have an alpha3(IV) chain and alpha4(IV) chain. Considering the specific locations of the type IV collagen trimers throughout the gastrointestinal SBM, the supramolecular network containing the alpha3(IV)alpha4(IV)alpha5(IV) molecule appears to function as a selective permeability barrier and /or as a protection against chemical stress from the luminal digestive enzymes.

The distribution of type IV collagen alpha chains in the mouse ovary and its correlation with follicular development.

The present study aims to identify alpha chains of type IV collagen in the basement membrane of the mouse ovarian follicle and examine their changes during follicular development using immunofluorescence microscopy with specific monoclonal antibodies. The basement membrane of the serous mesothelium enveloping the ovary contained all alpha chains of type IV collagen, alpha1(IV) through alpha6(IV) chains. Primordial follicles showed a distinct immunoreactivity against all six alpha chains in their basement membranes. Immunolabeling for alpha3(IV) and alpha4(IV) chains was almost eliminated in the primary follicles. In basement membranes of secondary and Graafian follicles, the immunofluorescent reaction of alpha3(IV) and alpha4(IV) chains disappeared in Graafian follicles, a partial reduction in fluorescent immunostaining intensity to alpha5(IV) and alpha6(IV) chains was observed; only alpha1(IV) and alpha2(IV) chains were not degraded throughout follicular development. On atretic follicles, in addition to alpha1(IV) and alpha2(IV) chains, alpha3(IV), alpha4(IV), alpha5(IV) and alpha6(IV) chains frequently persisted. A basement membrane-like matrix within the follicular granulosa cell layer, such as the focimatrix (focal intraepithelial matrix) and/or Call-Exner body, was also recognized in mouse secondary and Graafian follicles and contained alpha1(IV), alpha2(IV), alpha5(IV) and alpha6(IV) chains but not alpha3(IV) and alpha4(IV) chains. We expect that the decrease in alpha(IV) chains prompts follicular development and is a prerequisite condition for follicular maturation.

The distributions of type IV collagen alpha chains in basement membranes of human epidermis and skin appendages.

Distributions of type IV collagen alpha chains in the basement membrane (BM) of human skin and its appendages were analyzed by immunofluorescent microscopy using chain-specific monoclonal antibodies. The basement membrane beneath the epidermis contained [alpha1(IV)](2)alpha2(IV) and [alpha5(IV)](2)alpha6(IV) but no alpha3(IV)alpha4(IV)alpha5(IV); this held true for at the eccrine sweat glands and glandular ducts, sebaceous glands, hair follicles, and arrector muscles of hair. The secretary portion of the eccrine sweat glands was rich in [alpha1(IV)](2) alpha2(IV) and had less [alpha5(IV)](2)alpha6(IV), while [alpha5(IV)](2) alpha6(IV) was abundant in the ductal portion. In the subepidermal zone, alpha5(IV)/alpha6(IV) chain negative spots (1.9-15.0 microm) were frequently observed. Triple staining samples (Mel.2, alpha2(IV) and alpha5(IV) chains) showed that about 50% of epidermal melanocytes colocalized with such spots. Results suggest that these alpha5(IV)/alpha6(IV) chain negative spots of the subepidermal basement membrane have a particular relationship with melanocytes and are sites for certain interactions between the two.

In situ preparation of colloidal iron by microwave irradiation for transmission electron microscopy.

We attempted to prepare colloidal iron within tissues by means of microwave irradiation. Mouse tissue blocks were fixed with a mixture of paraformaldehyde and ferric chloride in a cacodylate buffer, immersed in a cacodylate buffered ferric chloride solution, and irradiated in a microwave processor. Colloidal iron was prepared within tissues or cells, and was observed in the form of electron dense fine granules (1-2 nm in diameter) by transmission electron microscopy. Collagen fibrils in the connective tissue showed colloidal iron deposition at regular periodical intervals. Cells in the splenic tissue showed that fine colloidal granules were deposited on the ribosomes but not on the nuclear chromatin. This finding suggests that ferric ions could not diffuse into the nucleus, which was surrounded by the nuclear envelope. The podocyte processes of the renal glomerulus were stained diffusedly. Though this microwave in situ colloidal iron preparation method has some limitations, it is convenient for use in biomedical specimen preparation in transmission electron microscopy.

Human erythrocytes possess a cytoplasmic endoskeleton containing beta-actin and neurofilament protein.

The biconcave disc shape of mammalian erythrocytes has been considered to be maintained only with a membrane underlain by a membranous cytoskeleton. Our improved ion-etching/scanning electron microscopy and saponin-ethanol treatment combined with immunocytochemistry in the human red blood cell revealed the three-dimensional structure of this cytoplasmic endoskeleton apart from the classical membranous cytoskeleton. The endoskeletal meshwork images obtained by the saponin-ethanol treatment corresponded to those by the repeated ion-etching method. The actin-rich endoskeleton was divided into two layers, one superficial and the other deep. The superficial filaments were perpendicularly connected to the membranous cytoskeleton, while the deep filaments formed an irregularly directed complicated meshwork. In the transitional hillside region between the convex periphery and concave center, the endoskeletal filaments containing a neurofilament protein ran parallel to the hillside slope toward the concave center. The endoskeleton of the erythrocyte associating with the membranous cytoskeleton may serve to keep its unique biconcave disc shape deformable, pliable, and restorable against external circumstances.