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M Osumi

Publications and source records attributed to M Osumi.

At least 37 records · Page 2Linked to original sources
PubMed1999

[Comparison of the newly developed MB redox system with mycobacteria growth indicator tube (MGIT) and 2% Ogawa egg media for recovery of mycobacteria in clinical specimens].

The rate of recovery and the mean time to detection of mycobacteria in clinical specimens were determined in a newly-developed MB Redox system based on liquid medium, and the results were compared with those of MGIT and 2% Ogawa egg media. From 587 sputum specimens processed, totally 203 mycobacterial isolates were detected, of which 177 (87.2%) with MB Redox, 185 (91.1%) with MGIT and 133 (65.6%) with 2% Ogawa medium. The difference in the percentages of positive cultures between either of the two liquid media and 2% Ogawa medium was significant (p < 0.0001). The mean time to detection of the Mycobacterium tuberculosis complex was 17.5 days with MB Redox, 18.7 days with MGIT, and 26.2 days with 2% Ogawa medium. The contamination rates were 1.5, 1.7, and 4.1% for MB Redox, MGIT, and 2% Ogawa medium, respectively. In conclusion, both MB Redox and MGIT systems, based on liquid medium, are more efficient than 2% Ogawa medium for the recovery of mycobacteria in clinical specimens.

Bacteriological Techniques↗
PubMed1998

Ultrastructure of human scalp hair shafts as revealed by freeze-substitution fixation.

Human scalp hair is important as a diagnostic clue to many diseases, in medical jurisprudential investigations, and also as a subject of cosmetic treatments. While many ultrastructural studies of the human hair root including the hair follicle have been reported, few studies have been done on the human hair shaft. We report here the ultrastructure of human scalp hair shafts prepared by a rapid-freezing technique followed by freeze-substitution fixation that allows the observation of fine cell structures. Healthy scalp hair shafts from Japanese females 12-13 years of age were rapid-frozen and then freeze-substituted in OsO4-acetone. In addition, this technique was applied to the study of some changes of the hair shafts (i.e., hair damaged by thioglycolic acid cold permanent waving and white hair). By this method, the hair shaft was rapid-frozen throughout without appreciable ice damage although the hair shaft was nearly 100 microm in diameter. The rapid-freezing technique resulted in excellent preservation of the ultrastructure of the hair shafts: lamellar structures in the cuticle and fine fibrous ultrastructures in the cortex were observed without chemical treatments. Thioglycolic acid treatment affected the ultrastructure of both the cuticle and the cortex. Except for the absence of melanin granules, no significant differences in the ultrastructure were observed between white hair and black hair. The rapid-freezing technique followed by freeze-substitution fixation appears to be the most reliable approach for the morphological evaluation of fully keratinized cells and tissues.

Adolescent↗
PubMed1998

Morphological effects of itraconazole on murine macrophage.

Morphological effects of itraconazole (ITCZ) on murine macrophages were examined by light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and compared with the effects of other antifungal agents. Thioglycolate-induced peritoneal macrophages were prepared from C3H/He J mice and cultured for 20 h in the presence of the antifungal azoles econazole, fluconazole, miconazole, ketoconazole, ITCZ, hydroxy-itraconazole (ITCZ-OH), and a polyene antibiotic amphotericin B (AMPH). Among these reagents, only ITCZ and its derivative ITCZ-OH were effective in causing morphological changes of murine macrophage as determined by LM and SEM. Macrophages treated with 2 microg/ml ITCZ or ITCZ-OH were stretched out bidirectionally, their surface was smooth and their 'ruffles' decreased. TEM observation showed that the bundles of the filamentous structure existed along the cell shape in the cytoplasm. These findings suggest that ITCZ and ITCZ-OH affect the morphology of macrophages.

Animals↗
PubMed1998

[Inhibition of mycelial growth of Candida albicans by ascites fluid of tumor bearing mice].

The effects of ascites fluids and sera of tumor-bearing mice on the mycelial growth of Candida albicans were examined. When the ascites fluids or the sera obtained from mice inoculated with MM46 mammary carcinoma were added to the culture medium, mycelial growth of C. albicans was strongly inhibited. The molecular size of the growth inhibitory factor in the ascites fluids was estimated to be approximately 80 K dalton by gel-filtration chromatography. Ferric chloride (6 microM) neutralized the anti-Candida activity. On the basis of these results including morphological observation, a possible role of a transferrin-like molecule was discussed.

Animals↗
PubMed1998

[Dynamics of cell wall formation from Trichophyton mentagrophytes protoplast].

There is no reference to the cell wall formation during the regeneration process in dermatophyte. Having succeeded in identifying the cell wall formation system of protoplast from Trichophyton mentagrophytes I introduce here the dynamics of the regeneration of this protoplast and the formation of cell wall using ultrahigh resolution low-voltage scanning electron microscopy and transmission electron microscopy (TEM), compare to the formation of yeast cell wall. Protoplasts were formed from mycelia of T. mentagrophytes; they regenerated the cell wall substances and were followed by a network of fibrils after 4-6 hr, which developed into mature mycelia after 21 hr. A network of twisted microfibrils and rope-shaped thick fiber was seen; it was covered with a sheath-shaped structure and was created on the surface of the developing mycelium. Intrafibrillar space and the surface of thick fibers were gradually filled with amorphous particles and a mature cell wall surface was formed. This protoplast regeneration was completely inhibited by the antifungal agent, lanoconazole (2 ng/ml ). The sectioned TEM image revealed that normal mycelium having a thin cell wall developed from the abortive bead-shaped mycelium which was visible in the lose fibrous structure. Based on this result and our previos work on the ultrastructure of the mycelial surface of T. rubrum with lanoconazole, a model of the cell wall composition of Trichophyton is proposed.

Cell Wall↗
PubMed1998

[Augmentation of anti-Candida activity of neutrophils by macrophages treated with itraconazole].

Effects of itraconazole (ITCZ) on anti-Candida activity of a macrophage (MP)-neutrophil axis were examined in vitro. Murine peritoneal MP, stimulated by an enterococcal preparation FK-23, released tumor necrosis factor a (TNF) into the culture medium in 20 hr-culture period. This level of TNF production by the stimulated MP, which were measured by a bioassay or immunological assay, was heightened by co-stimulation with 0.5 mg/ml of ITCZ. Supernatants obtained from the culture medium of FK-23 and ITCZ-treated MP not only contained about 10 ng/ml of TNF but also had the capacity to enhance anti-Candida activity of murine neutrophils. This enhancement was blocked by pretreatment of the MP-supernatant with anti-TNF antibody. Observation by scanning electron microscope suggested that cell walls of the candida mycelia, the growth of which was inhibited by the neutrophils in the presence of either the MP-supernatant or TNF, sustained similar structural damage. These results suggest that ITCZ augments anti-Candida activity of neutrophils through enhanced production of TNF by MP in vitro.

Animals↗
PubMed1997

Two distinct pathways for targeting proteins from the cytoplasm to the vacuole/lysosome.

Stress conditions lead to a variety of physiological responses at the cellular level. Autophagy is an essential process used by animal, plant, and fungal cells that allows for both recycling of macromolecular constituents under conditions of nutrient limitation and remodeling the intracellular structure for cell differentiation. To elucidate the molecular basis of autophagic protein transport to the vacuole/lysosome, we have undertaken a morphological and biochemical analysis of this pathway in yeast. Using the vacuolar hydrolase aminopeptidase I (API) as a marker, we provide evidence that the autophagic pathway overlaps with the biosynthetic pathway, cytoplasm to vacuole targeting (Cvt), used for API import. Before targeting, the precursor form of API is localized mostly in restricted regions of the cytosol as a complex with spherical particles (termed Cvt complex). During vegetative growth, the Cvt complex is selectively wrapped by a membrane sac forming a double membrane-bound structure of approximately 150 nm diam, which then fuses with the vacuolar membrane. This process is topologically the same as macroautophagy induced under starvation conditions in yeast (Baba, M., K. Takeshige, N. Baba, and Y. Ohsumi. 1994. J. Cell Biol. 124:903-913). However, in contrast with autophagy, API import proceeds constitutively in growing conditions. This is the first demonstration of the use of an autophagy-like mechanism for biosynthetic delivery of a vacuolar hydrolase. Another important finding is that when cells are subjected to starvation conditions, the Cvt complex is now taken up by an autophagosome that is much larger and contains other cytosolic components; depending on environmental conditions, the cell uses an alternate pathway to sequester the Cvt complex and selectively deliver API to the vacuole. Together these results indicate that two related but distinct autophagy-like processes are involved in both biogenesis of vacuolar resident proteins and sequestration of substrates to be degraded.

Aminopeptidases↗
PubMed1997

Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae.

We tried genetically to immobilize cellulase protein on the cell surface of the yeast Saccharomyces cerevisiae in its active form. A cDNA encoding FI-carboxymethylcellulase (CMCase) of the fungus Aspergillus aculeatus, with its secretion signal peptide, was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast alpha-agglutinin a protein involved in mating and covalently anchored to the cell wall. The plasmid constructed containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase activity was detected in the cell pellet fraction. The CMCase protein was solubilized from the cell wall fraction by glucanase treatment but not by sodium dodecyl sulphate treatment, indicating the covalent binding of the fusion protein to the cell wall. The appearance of the fused protein on the cell surface was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. These results proved that the CMCase was anchored on the cell wall in its active form.

Aspergillus↗
PubMed1997

[Clinical evaluation of a reagent for detection of DNA of Mycobacterium tuberculosis complex using the ligase chain reaction (LCR) method].

We evaluated the clinical efficacy of LCR MTB, a reagent developed by Abbott in the USA, in the full automatic ligase chain reaction (LCR) for detection of DNA of M. tuberculosis complex using a thermostable ligase. Using 458 samples isolated from patients with tuberculosis, LCR was compared with a smear method and with a culture method, and was also compared with two other methods of gene amplification, MTD and Amplicor, using 340 and 200 of the 458 samples, respectively. The LCR method detected M. tuberculosis in 49.8% (228/458) of the samples, and was superior to the smear method (31.9%, 146/458) and the culture method (39.1%, 179/458) in sensitivity. The LCR method was also superior to the MTD and Amplicor methods; sensitivity were 37.9% (129/340) for MTD vs. 47.6% (162/340) for LCR, and 56.5% (113/200) for Amplicor vs. 59.5% (119/200) for LCR. These favorable results and the convenience of the LCR method, which enables rapid detection of target genes with a high degree of sensitivity, strongly suggest that LCR MTB is useful as a reagent for detection of M. tuberculosis using nucleic acid amplification.

Adolescent↗
PubMed1997

Construction of a starch-utilizing yeast by cell surface engineering.

We have engineered the cell surface of the yeast Saccharomyces cerevisiae by anchoring active glucoamylase protein on the cell wall, and we have endowed the yeast cells with the ability to utilize starch directly as the sole carbon source. The gene encoding Rhizopus oryzae glucoamylase with its secretion signal peptide was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast alpha-agglutinin, a protein involved in mating and covalently anchored to the cell wall. The constructed plasmid containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The glucoamylase activity as not detected in the culture medium, but it was detected in the cell pellet fraction. The glucoamylase protein transferred to the soluble fraction from the cell wall fraction after glucanase treatment but not after sodium dodecyl sulfate treatment, indicating the covalent binding of the fusion protein to the cell wall. Display of the fused protein was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. The transformant cells could surely grow on starch as the sole carbon source. These results showed that the glucoamylase was anchored on the cell wall and displayed as its active form. This is the first example of an application of cell surface engineering to utilize and improve the metabolic ability of cells.

Cell Membrane↗
PubMed1996

Individual expression of Candida tropicalis peroxisomal and mitochondrial carnitine acetyltransferase-encoding genes and subcellular localization of the products in Saccharomyces cerevisiae.

In an n-alkane assimilating yeast, Candida tropicalis, carnitine acetyltransferase (CAT; EC 2.3.1.7) was localized in both peroxisomes and mitochondria. Both CATs were encoded by one gene, CT-CAT, although the initiation sites of translation were suggested to be different. In the present study, the genes corresponding to the supposed C. tropicalis peroxisomal and mitochondrial CATs, which were truncated from the CT-CAT gene, were individually expressed in Saccharomyces cerevisiae, using the C. tropicalis isocitrate lyase promoter (UPR-ICL), which is inducible by oleic acid in concert with proliferation of peroxisomes in S. cerevisiae [Umemura, K., Atomi, H., Kanai, T., Teranishi, Y., Ueda, M., and Tanaka, A. (1995) Appl. Microbiol. Biotechnol. 43, 489-492]. The 71 kDa precursor of mitochondrial CAT, initiating at the first Met, was found to be processed to the mature size (66 kDa) in S. cerevisiae and immunoelectronmicroscopical observation revealed that this enzyme was localized in mitochondria. On the other hand, 68 kDa CAT, initiating at the second Met (residue No. 19), had no cleavable signal and was translocated into peroxisomes and cytosol, but not into mitochondria. The amino-terminal amino acid sequences of individually expressed CATs were identical to those of CATs isolated from alkane-grown C. tropicalis cells, respectively. These results demonstrated that only the 71 kDa protein yielded the 66 kDa protein and that peroxisomal and mitochondrial CATs arose from the difference in the initiation sites of translation.

Amino Acid Sequence↗
PubMed1996

Immunoelectron microscopic observation of the behaviors of peroxisomal enzymes inducibly synthesized in an n-alkane-utilizable yeast cell, Candida tropicalis.

We reported that immunoelectron microscopy was an excellent tool for determining the subcellular localization of thiolase isozymes, acetoacetyl-CoA thiolase (T-I) and 3-ketoacyl-CoA thiolase (T-III) in n-alkane-grown Candida tropicalis cells (KAMASAWA, N. et al., (1992). Cell Struct. Funct., 17: 203-207). Current investigation on the visualization of other peroxisomal enzymes, acyl-CoA oxidase (ACO), catalase (KAT), carnitine acetyltransferase (CAT), isocitrate lyase (ICL) and malate synthase (MS), showed that ACO localized in peroxisomes, KAT in peroxisomes and cytoplasm, and CAT in peroxisomes, mitochondria and cytoplasm. Most of ICL and MS were found in peroxisomes. These results agreed with previous biochemical studies and supported the presumed roles of these enzymes. The same technique was applied to study the process of synthesis and localization of these enzymes early in the cultivation period in n-alkane medium when peroxisomes began to proliferate. ACO and T-III were rapidly induced after transfer of cells from glucose- to n-alkane-media. There was a drastic change of their location from cytoplasm to peroxisomes between 1 h and 2 h after the transfer, while T-I, KAT and CAT were moderately induced in cytoplasm and their location was gradually changed to each organelle. ICL and MS, the key enzymes in the glyoxylate cycle, were already localized in peroxisomes in the glucose-grown cells and respective inducible enzymes also were gradually localized there. This visual analysis is useful for the vivid elucidation of the process of peroxisome proliferation and enzyme transport within a cell.

Alkanes↗
PubMed1996

Schizosaccharomyces pombe is more sensitive to pressure stress than Saccharomyces cerevisiae.

The effects of hydrostatic pressure on ultrastructure, microtubules and microfilaments of Schizosaccharomyces pombe were investigated by fluorescence microscopy, conventional electron microscopy and immunoelectron microscopy. Cells were treated with hydrostatic pressure from 0.1 to 400 MPa for 10 min at room temperature. The nuclear membrane was disrupted at above 100 MPa. At 150 MPa the matrixes of mitochondria had an electron dense area. At 250 MPa the cytoplasmic substances changed dramatically, the cellular organelles could hardly be detected and the fragmented nuclear membrane was barely visible. The fluorescence in alpha-tubulin was lost in most of the cells at 100 MPa. The gold particles for anti alpha-tubulin were not visible in the cells at the same level. Cell cycle specific actin distribution was lost even at 50 MPa, although actin dots localized at the central region remained unchanged. Thick actin cables appeared at 100 MPa. Complete depolymerization of F-actin was observed at 150 MPa. These results suggest that S. pombe cells were more sensitive than Saccharomyces cerevisiae cells. The damage to microtubules and nuclear membrane caused by hydrostatic pressure was though to be followed by breakdown of nuclear division apparatus and the inhibition of nuclear division. This damage might contribute to the frequent formation of polyploidy in S. pombe.

Actin Cytoskeleton↗
PubMed1996

Clinical presentation of pulmonary tuberculosis associated with acquired immunodeficiency syndrome in metropolitan Tokyo.

The clinical features of pulmonary tuberculosis associated with acquired immunodeficiency syndrome (AIDS) in Japan were surveyed utilizing questionnaires completed by 48 institutes around the Tokyo metropolitan area. We found 11 Japanese and six foreign patients. The average number of patients per institute was 0.37. The Japanese patients had advanced human immunodeficiency virus (HIV) infection. A middle aged man, with fever and cough, nonspecific chest X-ray infiltrates, decreased lymphocyte counts, and a negative tuberculin skin test was the typical presentation of the Japanese patients. The clinical diagnosis was often difficult. The smear positive rate was high among those from whom smears were obtained, suggesting high communicability. None of the isolates indicated multidrug-resistant tuberculosis at the time of diagnosis. In conclusion, sputum smear and culture remain the keys to diagnosing tuberculosis in patients with AIDS, although the clinical picture may be atypical for pulmonary tuberculosis.

Acquired Immunodeficiency Syndrome↗
PubMed1996

[A case of AIDS with bronchial tuberculosis].

A case is 48 years-old Japanese man who had a history of frequent sexual contact with prostitutes in Thailand and the Philippines. He presented with chief complaint of chest discomfort in April 1995. His chest X-ray film showed right mediastinal lymph node swelling in other hospital and the sputum smear was strongly positive for acid fast bacilli. In May 1995, he was admitted to our hospital and serological tests for HIV were positive both by EIA and Western blot methods. The CD4 lymphocyte count was 167/microliters. He was diagnosed as a case of AIDS according to the criteria proposed by the AIDS surveillance committee of the Japanese Ministry of Health and Welfare. Although numerous tubercule bacilli were detected in sputum, the chest X-ray did not show abnormal shadow in lung fields. So the diagnosis of bronchial tuberculosis was suspected by these apparently contradictory findings and the bronchoscopy was performed. Biopsy specimen of the bronchial mucous membrane obtained by bronchoscopy confirmed the presence of acid fast bacilli by Ziehl-Neelsen's staining method, however, histological findings were atypical of tuberculosis. A month after the initiation of treatment with isoniazid, rifampicin and ethambutol and AZT, his clinical symptoms improved and the sputum smear and the culture tests for tubercule bacilli converted to negative. Complications of AIDS, (Pneumocystis carinii infection, Cytomegalo virus infection, Kaposi's sarcoma, etc) other than tuberculosis have not developed to date. In the past reports, we could not find reports of bronchial tuberculosis with AIDS. Tuberculous granuloma formation was scarce in this case, and it was suspected that bronchial tuberculosis with AIDS would show characteristic sign as same as pulmonary tuberculosis with AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1996

[Detection of Mycobacterium tuberculosis in clinical specimens other than sputum by the Mycobacterium Tuberculosis Direct Test (MTD)--assessment of sample preparation methods and clinical evaluation].

The Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (MTD) has been widely used as a rapid test for the identification of Mycobacterium tuberculosis complex in clinical samples, and several research groups have verified its clinical usefulness. However, most of the specimens they tested were sputum, and there have been few reports on other specimens. In particular, there have been no reports on assessments of methods of preparing samples other than sputum for the MTD. We assessed methods of preparing samples other than sputum and the influence of a local anesthetic and an anticoagulant that may be present in samples, and also evaluated the MTD as a means of detecting M. tuberculosis in pleural fluid, bronchial lavage cerebrospinal fluid, urine and ascitic fluid. 1. Assessment of three sample preparation methods, i.e., the NALC-NaOH method GuSCN-Diatom nucleic acid extraction method, and the ultrasonication method, revealed that the combination of the NALC-NaOH method and the ultrasonication method, widely used to prepare sputum samples, is also a valid method of preparing other samples. 2. The local anesthetic and the anticoagulant used clinically and remained in specimens did not affect the results of the MTD. 3. Seven (36.8%) of 19 pleural fluid samples from patients diagnosed as tuberculous pleurisy were positive of M. tuberculosis by the MTD, while five (27.8%) of 18 pleural fluid samples cultured for bacteria were positive for M. tuberculosis complex. None of the 20 pleural fluid samples from patients diagnosed as non-tuberculous pleurisy were positive for M. tuberculosis complex either by MTD or culture. 4. Eight (32.0%) of 25 bronchial lavage samples from patients diagnosed as pulmonary tuberculosis were positive for M. tuberculosis complex by the MTD, while 3 (12.0%) were positive by culture. None of the 18 bronchial lavage samples from patients diagnosed as non-tuberculous disease were positive for M. tuberculosis complex either by the MTD or culture. Based on these results, it is concluded that the MTD is a very useful method of detecting M. tuberculosis in clinical samples other than sputum because it is more sensitive than culture on Ogawa's egg medium in detecting M. tuberculosis complex in pleural fluid samples, bronchial lavage samples, and so on, with the same preparation method as used for sputum.

Bacteriological Techniques↗
PubMed1996

[Evaluation of a new detection method of Mycobacterium tuberculosis in clinical specimens by amplification of ribosomal RNA and its clinical application].

A new rapid M. tuberculosis detection method, the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (MTD), which allows direct detection of M.tuberculosis in clinical specimens by amplification of M.tuberculosis ribosomal RNA (rRNA), was evaluated. In the present study, MTD and conventional smear and culture examinations were performed on 225 sputum, 36 pleural fluid (PF), nine cerebrospinal fluid (CSF) and 41 bronchoalveolar lavage fluid (BALF) specimens. Among 225 sputum specimens from 132 patients, 20 culture-negative specimens from 14 patients and one specimen which was culture-positive for M.fortuitum on Ogawa's egg medium were MTD-positive. On the other hand, there were 6 M.tuberculosis culture-positive but MTD-negative specimens from 6 patients. The amount of bacilli in all six cases, however, was very few. The sensitivity of M.tuberculosis detection by MTD, as compared with Ogawa's egg medium and MB-Check, was 93 and 92%, respectively, in sputum specimens, and the specificity of MTD was 80 and 85%, respectively. Taking into account not only culture-positive specimens but also MTD-positive specimens from patients clinically diagnosed as active tuberculosis, the sensitivity and specificity of MTD were 93% and 99%, respectively. Among 36 PF specimens from 31 patients, of which 20 specimens from 16 patients were clinically diagnosed as tuberculous, only 4 from 4 patients were culture-positive for M.tuberculosis while 7 from 5 patients were MTD-positive. The results of MTD in 9 CSF specimens coincided well with those of MB-Check, but one MTD-positive specimen yielded a false-negative result with Ogawa's egg medium. Among 41 BALF specimens from 39 patients, only one was culture-positive while one culture-positive and 7 culture-negative specimens were positive on MTD. All these MTD-positive patients were later verified as having pulmonary tuberculosis either by transbronchial lung biopsy and other examinations, or by the clinical course of the disease, particularly the response to anti-tuberculosis drugs. Inconsistent results were seen in 21 sputum specimens from 15 patients, 4 PF specimens from 3 patients and 7 BALF specimens from 7 patients, all of which were culture-negative for M.tuberculosis but MTD-positive. None of these patients had any clinical findings inconsistent with diagnosis as tuberculosis. It was noted that MTD was very useful for rapid detection of M.tuberculosis in sputum as well as other clinical specimens. We observed the clinical course of 20 pulmonary tuberculosis patients by monthly examinations of sputum, using smears, Ogawa's egg medium, MB-Check and MTD. MTD-positivity rates fell down in parallel with decreased pulmonary tuberculous activity. During the clinical course, out of 61 MTD-negative specimens only 4 (6.6%) were culture-positive for M.tuberculosis, while out of 63 smear-and-culture-negative specimens 6 (9.5%) were MTD-positive. These data suggest that MTD is useful to assess the clinical activity and course of tuberculosis.

Adult↗