"In vivo cryotechnique" for paradigm shift to "living morphology" of animal organs

The morphological study has been one of the major approaches in medical and biological fields. For the last century, the conventional chemical fixation and alcohol dehydration were commonly used as an easy preparation method, but it was frequently pointed out that they usually yield many structural artifacts during their preparation processes. Although both conventional quick-freezing and high-pressure freezing methods, by which animal tissues are resected and frozen for physical fixation,can reduce such structural artifacts, the tissues have to be removed from living animal organs for the freezing. Therefore, such specimens are inevitably exposed to noxious stresses of anoxia and ischemia, exhibiting only dead morphological states of animal tissues without blood circulation. To the contrary, our "in vivo cryotechnique", by which all cells and tissues in animal bodies are cryofixed in vivo, can prevent such artifacts of resected specimens. By means of the cryotechnique, it is now possible to reveal the in vivo morphology of cells and tissues in living animal organs. Actually, it has been already applied to several animal organs, such as kidney, liver, intestine, cerebellum, eye ball, blood vessel, and joint cartilage, and brought new morphological findings, reflecting their physiological significance, which had been difficult to demonstrate by the conventional preparation methods. Moreover, its application to immunohistochemistry has also revealed more precise immunolocalizations of dynamically changing molecules in living animal organs, easily translocated by ischemic stresses and anoxia caused during the tissue resection. The "in vivo cryotechnique" allows us to perform novel morphological investigations of "living" morphological states, and develops new medical and biological fields with "living morphology" during this 21st century.Biomedical Reviews 2004; 15: 1-19

Histological Study and LYVE-1 Immunolocalization of Mouse Mesenteric Lymph Nodes with “In Vivo Cryotechnique”

The “in vivo cryotechnique” (IVCT) is a powerful tool to directly freeze living animal organs in order to maintain biological components in frozen tissues, reflecting their native states. In this study, mesenteric lymph nodes of living mice were directly frozen with IVCT, and we did morphological studies and immunohistochemical analyses on a hyaluronic acid receptor, LYVE-1. In lymph nodes, widely open lymphatic sinuses were observed, and many lymphocytes adhered to inner endothelial cells along subcapsular sinuses. The LYVE-1 was clearly immunolocalized at inner endothelial cells of subcapsular sinuses, as well as those of medullary sinuses. Conventional pre-embedding electron microscopy also showed LYVE-1 immunolocalization along both the apical and basal sides of cell membranes of inner endothelial cells. By triple-immunostaining for LYVE-1, smooth muscle actin, and type IV collagen, the LYVE-1 was immunolocalized only in the inner endothelial cells, but not in outer ones which were surrounded by collagen matrix and smooth muscle cells. Thus, the functional morphology of lymph nodes in vivo was demonstrated and LYVE-1 immunolocalization in inner endothelial cells of subcapsular sinuses suggests hyaluronic acid incorporation into lymph node parenchyma

Immunohistochemical analysis of an ectopic endometriosis in the uterine round ligament

A rare case of the inguinal endometriosis was reported with immunohistochemical analysis. A 28-year-old woman had a thumb-sized tumor in the right groin for two years with a gradual increase in size and pain. An operation revealed an elastic hard tumor with an unclear margin and adhesion to the uterine round ligament. The histology showed irregular proliferation of the endometrial glands and stroma. The glandular epithelium stained weakly positive against CD125 antibody and the stromal matrix stained strongly positive against CD10 antibody. The nucleus in both the epithelial and stromal cells stained strongly positive against progesterone and estrogen receptor antibodies, and the cytoplasm in both types of cells stained moderately positive against COX-2 (cyclooxygenase-2) antibody. In conclusion, the combination of estrogen or progesterone receptor antibody for the nucleus and CD10 or COX-2 antibody for the cytoplasm could enhance the accuracy of diagnosis for ectopic endometriosis

Immunohistochemical Distribution of Serum Proteins in Living Mouse Heart with In Vivo Cryotechnique

In vivo cryotechnique (IVCT), which immediately cryofixes target organs in situ, was used to clarify the morphological features of beating heart tissue of living mice. IVCT was performed for diastolic heart tissue under the condition of monitoring with electrocardiogram (ECG). Other mouse hearts were prepared with conventional perfusion-fixation (PF-DH) or immersion-fixation followed by dehydration (IM-DH), and quick-freezing of resected heart tissues (FQF). Immunolocalizations of albumin, immunoglobulin G1 (IgG1), intravenously injected bovine serum albumin (BSA), and connexin 43 were examined after different intervals of BSA injection. In the case of IVCT, the exact stop time of beating mouse hearts was recorded by ECG, and open blood vessels with flowing erythrocytes were observed with less artificial tissue shrinkage than with conventional preparation methods. Both albumin and BSA were well preserved in intercalated discs and t-tubules of cardiomyocytes in addition to blood vessels and interstitial matrices. IgG1 was immunolocalized in interstitial matrices of heart tissues in addition to their blood vessels. At 4 hr after BSA injection, it was immunolocalized in the intercalated discs of cardiomyocytes and lost later at 8 hr. IVCT should prove to be more useful for the morphofunctional examination of dynamically changing heart tissue than conventional preparation methods

The C-Terminal Fragment of Prostate-Specific Antigen, a 2331 Da Peptide, as a New Urinary Pathognomonic Biomarker Candidate for Diagnosing Prostate Cancer

Background and Objectives: Prostate cancer (PCa) is one of the most common cancers and leading cause of cancer-related deaths in men. Mass screening has been carried out since the 1990s using prostate-specific antigen (PSA) levels in the serum as a PCa biomarker. However, although PSA is an excellent organ-specific marker, it is not a cancer-specific marker. Therefore, the aim of this study was to discover new biomarkers for the diagnosis of PCa. Materials and Methods: We focused on urine samples voided following prostate massage (digital rectal examination [DRE]) and conducted a peptidomic analysis of these samples using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS_n). Urinary biomaterials were concentrated and desalted using CM-Sepharose prior to the following analyses being performed by MALDI-TOF/MS_n: 1) differential analyses of mass spectra; 2) determination of amino acid sequences; and 3) quantitative analyses using a stable isotope-labeled internal standard. Results: Multivariate analysis of the MALDI-TOF/MS mass spectra of urinary extracts revealed a 2331 Da peptide in urine samples following DRE. This peptide was identified as a C-terminal PSA fragment composed of 19 amino acid residues. Moreover, quantitative analysis of the relationship between isotope-labeled synthetic and intact peptides using MALDI-TOF/MS revealed that this peptide may be a new pathognomonic biomarker candidate that can differentiate PCa patients from non-cancer subjects. Conclusion: The results of the present study indicate that the 2331 Da peptide fragment of PSA may become a new pathognomonic biomarker for the diagnosis of PCa. A further large-scale investigation is currently underway to assess the possibility of using this peptide in the early detection of PCa

Blastocyst complementation using Prdm14-deficient rats enables efficient germline transmission and generation of functional mouse spermatids in rats.

Murine animal models from genetically modified pluripotent stem cells (PSCs) are essential for functional genomics and biomedical research, which require germline transmission for the establishment of colonies. However, the quality of PSCs, and donor-host cell competition in chimeras often present strong barriers for germline transmission. Here, we report efficient germline transmission of recalcitrant PSCs via blastocyst complementation, a method to compensate for missing tissues or organs in genetically modified animals via blastocyst injection of PSCs. We show that blastocysts from germline-deficient Prdm14 knockout rats provide a niche for the development of gametes originating entirely from the donor PSCs without any detriment to somatic development. We demonstrate the potential of this approach by creating PSC-derived Pax2/Pax8 double mutant anephric rats, and rescuing germline transmission of a PSC carrying a mouse artificial chromosome. Furthermore, we generate mouse PSC-derived functional spermatids in rats, which provides a proof-of-principle for the generation of xenogenic gametes in vivo. We believe this approach will become a useful system for generating PSC-derived germ cells in the future

Population pharmacokinetic modeling of GS‐441524, the active metabolite of remdesivir, in Japanese COVID‐19 patients with renal dysfunction

腎障害患者におけるレムデシビルの薬物動態モデルを構築 --新型コロナウイルス感染症治療薬の適正使用に向けて--. 京都大学プレスリリース. 2021-11-25.Remdesivir, a prodrug of the nucleoside analog GS-441524, plays a key role in the treatment of coronavirus disease 2019 (COVID-19). However, owing to limited information on clinical trials and inexperienced clinical use, there is a lack of pharmacokinetic (PK) data in patients with COVID-19 with special characteristics. In this study, we aimed to measure serum GS-441524 concentrations and develop a population PK (PopPK) model. Remdesivir was administered at a 200 mg loading dose on the first day followed by 100 mg from day 2, based on the package insert, in patients with an estimated glomerular filtration rate (eGFR) greater than or equal to 30 ml/min. In total, 190 concentrations from 37 Japanese patients were used in the analysis. The GS-441524 trough concentrations were significantly higher in the eGFR less than 60 ml/min group than in the eGFR greater than or equal to 60 ml/min group. Extracorporeal membrane oxygenation in four patients hardly affected the total body clearance (CL) and volume of distribution (Vd) of GS-441524. A one-compartment model described serum GS-441524 concentration data. The CL and Vd of GS-441524 were significantly affected by eGFR readjusted by individual body surface area and age, respectively. Simulations proposed a dose regimen of 200 mg on day 1 followed by 100 mg once every 2 days from day 2 in patients with an eGFR of 30 ml/min or less. In conclusion, we successfully established a PopPK model of GS-441524 using retrospectively obtained serum GS-441524 concentrations in Japanese patients with COVID-19, which would be helpful for optimal individualized therapy of remdesivir

Formation of an Ultracarbonaceous Antarctic Micrometeorite through Minimum Aqueous Alteration in a Small Porous Icy Body

A comprehensive study of the organic chemistry and mineralogy of an ultracarbonaceous micrometeorite (UCAMM D05IB80) collected from near the Dome Fuji Station, Antarctica, was carried out to understand the genetic relationship among organic materials, silicates, and water. The micrometeorite is composed of a dense aggregate of ∼5 µm-sized hollow ellipsoidal organic material containing submicrometer-sized phases such as glass with embedded metal and sulfides (GEMS) and mineral grains. There is a wide area of organic material (∼15 × 15 μm) in its interior. Low-Ca pyroxene is much more abundant than olivine and shows various Mg/(Mg + Fe) ratios ranging from ∼1.0 to 0.78, which is common to previous works on UCAMMs. By contrast, GEMS grains in this UCAMM have unusual chemical compositions. They are depleted in both Mg and S, which suggests that these elements were leached out from the GEMS grains during very weak aqueous alteration, without the formation of phyllosilicates. The organic materials have two textures—smooth and globular with an irregular outline—and these are composed of imine, nitrile and/or aromatic nitrogen heterocycles, and amide. The ratio of nitrogen to carbon (N/C) in the smooth region of the organics is ∼0.15, which is five times higher than that of insoluble organic macromolecules in types 1 and 2 carbonaceous chondritic meteorites. In addition, the UCAMM organic materials are soluble in epoxy and are thus hydrophilic; this polar nature indicates that they are very primitive. The surface of the material is coated with an inorganic layer, a few nanometers thick, that consists of C, O, Si, S, and Fe. Sulfur is also contained in the interior, implying the presence of organosulfur moieties. There are no isotopic anomalies of D, 13C, or 15N in the organic material. Interstellar photochemistry alone would not be sufficient to explain the N/C ratio of the UCAMM organics; therefore, we suggest that a very small amount of fluid on a comet must have been necessary for the formation of the UCAMM. The GEMS grains depleted in Mg and S in the UCAMM prove a very weak degree of aqueous alteration; weaker than that of carbonaceous chondrites. Short-duration weak alteration probably caused by planetesimal shock locally melted cometary ice grains and released water that dissolved the organics; the fluid would likely have not mobilized because of the very low thermal conductivity of the porous icy body. This event allowed the formation of the large organic puddle of the UCAMM, as well as organic matter sulfurization, formation of thin membrane-like layers of minerals, and deformation of organic nanoglobules.アクセプト後にタイトル・アブストラクト等変更あり、著者最終稿は変更前のタイトル"Formation of an Ultracarbonaceous Antarctic Micrometeorite through Minimum Aqueous Alteration in a Small Porous Icy Body"This work was supported by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (No. 22224010, PI: H. Nagahara). The STXM facility at the beamline 5.3.2.2, ALS, is supported by the Department of Energy, Basic Energy Sciences Program

Current Performance and On-Going Improvements of the 8.2 m Subaru Telescope

An overview of the current status of the 8.2 m Subaru Telescope constructed and operated at Mauna Kea, Hawaii, by the National Astronomical Observatory of Japan is presented. The basic design concept and the verified performance of the telescope system are described. Also given are the status of the instrument package offered to the astronomical community, the status of operation, and some of the future plans. The status of the telescope reported in a number of SPIE papers as of the summer of 2002 are incorporated with some updates included as of 2004 February. However, readers are encouraged to check the most updated status of the telescope through the home page, http://subarutelescope.org/index.html, and/or the direct contact with the observatory staff.Comment: 18 pages (17 pages in published version), 29 figures (GIF format), This is the version before the galley proo