Biomedical subjects

Makoto Ito

Publications and source records attributed to Makoto Ito.

At least 19 recordsLinked to original sources

PubMed2026

Cooperation and the evolution of bacterial niche breadth.

Bacteria exhibit varying niche breadths, with generalists thriving in diverse environments and specialists confined to specific habitats. Although genes for cooperative traits have been suggested to influence niche breadth evolution, their precise role remains unclear. We used a combination of phylogeny-based comparative methods to test causal hypotheses about the directionality of the relationship between genes for cooperation and bacterial niche breadth evolution across 25,785 species. Our results revealed 1) a positive correlation between the proportion of genes for cooperation and niche breadth; 2) genes for cooperation influenced niche breadth evolution, with a decreased proportion of such genes promoting niche contraction as the predominant evolutionary direction; and 3) genes for cooperation experience more frequent gain and loss within species rather than across species. These findings suggest a role of bacterial cooperation in influencing niche breadth evolution and maintaining the ecological versatility of bacteria. While our results are consistent with a simple relationship under the hypotheses tested, more complex causal scenarios are possible, including the role of factors that influence both cooperation and niche breadth.

Phylogeny↗

PubMed2007

Ceramidase enhances phospholipase C-induced hemolysis by Pseudomonas aeruginosa.

We previously reported the purification, molecular cloning, and characterization of a neutral ceramidase from Pseudomonas aeruginosa strain AN17 (Okino, N., Tani, M., Imayama, S., and Ito, M. (1998) J. Biol. Chem. 273, 14368-14373; Okino, N., Ichinose, S., Omori, A., Imayama, S., Nakamura, T., and Ito, M. (1999) J. Biol. Chem. 274, 36616-36622). Interestingly, the gene encoding the enzyme is adjacent to that encoding hemolytic phospholipase C (plcH) in the genome of Pseudomonas aeruginosa, which is a well known pathogen for opportunistic infections. We report here that simultaneous production of PlcH and ceramidase was induced by several lipids and PlcH-induced hemolysis was significantly enhanced by the action of the ceramidase. When the strain was cultured with sphingomyelin or phosphatidylcholine, production of both enzymes drastically increased, causing the increase of hemolytic activity in the cell-free culture supernatant. Ceramide and sphingosine were also effective in promoting the production of ceramidase but not that of PlcH. Furthermore, we found that the hemolytic activity of a Bacillus cereus sphingomyelinase was significantly enhanced by addition of a recombinant Pseudomonas ceramidase. TLC analysis of the erythrocytes showed that ceramide produced from sphingomyelin by the sphingomyelinase was partly converted to sphingosine by the ceramidase. A ceramidase-null mutant strain caused much less hemolysis of sheep erythrocytes than did the wild-type strain. Sphingosine was detected in the erythrocytes co-cultured with the wild-type strain but not the mutant strain. Finally, we found that the enhancement of PlcH-induced hemolysis by the ceramidase occurred in not only sheep but also human erythrocytes. These results may indicate that the ceramidase enhances the PlcH-induced cytotoxicity and provide new insights into the role of sphingolipid-degrading enzymes in the pathogenicity of P. aeruginosa.

Amidohydrolases↗

PubMed2006

Fucosyl-GM1a, an endoglycoceramidase-resistant ganglioside of porcine brain.

The use of bovine brain has been prohibited in many countries because of the world-wide prevalence of mad cow disease, and thus porcine brain is expected to be a new source for the preparation of gangliosides. Here, we report the presence of a ganglioside in porcine brain which is strongly resistant to hydrolysis by endoglycoceramidase, an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. Five major gangliosides (designated PBG-1, 2, 3, 4, 5) were extracted from porcine brain by Folch's partition, followed by mild alkaline hydrolysis and PBA column chromatography. We found that PBG-2, but not the others, was strongly resistant to hydrolysis by the enzyme. After the purification of PBG-2 with Q-Sepharose, Silica gel 60 and Prosep-PB chromatographies, the structure of PBG-2 was determined by GC, GC-MS, FAB-MS and NMR spectroscopy as Fucalpha1-2Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-4Glcbeta1-1'Cer (fucosyl-GM1a). The ceramide was mainly composed of C18:0 and C20:0 fatty acids and d18:1 and d20:1 sphingoid bases. The apparent kcat/Km for fucosyl-GM1a was found to be 30 times lower than that for GM1a, indicating that terminal fucosylation makes GM1a resistant to hydrolysis by the enzyme. This report indicates the usefulness of endoglycoceramidase to prepare fucosyl-GM1a from porcine brain.

Animals↗

PubMed2006

Knockdown of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebrafish.

Nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N) is an enzyme that dephosphorylates and concomitantly downregulates multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) in vitro. However, the functional roles of this enzyme in vivo are not well understood. To investigate the biological significance of CaMKP-N during zebrafish embryogenesis, we cloned and characterized zebrafish CaMKP-N (zCaMKP-N). Based on the nucleotide sequences in the zebrafish whole genome shotgun database, we isolated a cDNA clone for zCaMKP-N, which encoded a protein of 633 amino acid residues. Transiently expressed full-length zCaMKP-N in mouse neuroblastoma, Neuro2a cells, was found to be localized in the nucleus. In contrast, the C-terminal truncated mutant lacking RKKRRLDVLPLRR (residues 575-587) had cytoplasmic staining, suggesting that the nuclear localization signal of zCaMKP-N exists in the C-terminal region. Ionomycin treatment of CaMKIV-transfected Neuro2a cells resulted in a marked increase in the phosphorylated form of CaMKIV. However, cotransfection with zCaMKP-N significantly decreased phospho-CaMKIV in ionomycin-stimulated cells. Whole mount in situ hybridization analysis of zebrafish embryos showed that zCaMKP-N is exclusively expressed in the head and neural tube regions. Gene knockdown of zCaMKP-N using morpholino-based antisense oligonucleotides induced significant morphological abnormalities in zebrafish embryos. A number of apoptotic cells were observed in brain and spinal cord of the abnormal embryos. These results suggest that zCaMKP-N plays a crucial role in the early development of zebrafish.

Amino Acid Sequence↗

PubMed2006

Ceramide/sphingosine/sphingosine 1-phosphate metabolism on the cell surface and in the extracellular space.

Sphingolipid metabolites, ceramide, sphingosine, and sphingosine 1-phosphate, have emerged as a new class of lipid biomodulators of various cell functions. These metabolites are known to function not only as intracellular second messengers, but also in the extracellular space. Sphingosine 1-phosphate especially has numerous functions as an important extracellular mediator that binds to cell surface S1P receptors. Recent studies have also shown that sphingolipid-metabolizing enzymes function not only in intracellular organelles but also in the extracellular spaces, including the outer leaflet of the plasma membrane. This review focuses on the metabolic enzymes (acid and alkaline sphingomyelinases, neutral ceramidase, and sphingosine kinase) that are involved in the production of the sphingolipid metabolites in these extracellular spaces, and on the metabolic pathway itself.

Amidohydrolases↗

PubMed2006

Structure of beta-1,3-xylooligosaccharides generated from Caulerpa racemosa var. laete-virens beta-1,3-xylan by the action of beta-1,3-xylanase.

Recently we reported the molecular cloning and characterization of a novel beta-1,3-xylanase from the marine bacterium Vibrio sp. AX-4 [Kiyohara et al. (2005) Biochem. J. 388, 949-957]. We report here the structural analysis of oligosaccharides generated from beta-1,3-xylan of a siphonous green alga, Caulerpa racemosa var. laete-virens, by the action of beta-1,3-xylanase. The enzyme degraded the polysaccharide producing oligosaccharides with different R(f)s on TLC (EX2-EX5). Sugar component, linkage, and MALDI-TOF-MS analyses revealed that EX2 and EX3 were Xyl-1,3-Xyl and Xyl-1,3-Xyl-1,3-Xyl, respectively. On the other hand, EX4 was a mixture of Glc-1,3-Xyl-1,3-Xyl, Xyl-1,4-Xyl-1,3-Xyl and Xyl-1,3-Xyl-1,4-Xyl, while EX5 was a mixture of tetra-saccharides containing 3-substitued Glc in addition to the same components of EX4. Branching was not likely present in EXOs prepared from the polysaccharide by the enzyme. These results strongly suggest that the C. racemosa beta-1,3-xylan is a linear heteropolysaccharide containing 1,3-Glc and 1,4-Xyl both of which are thought to be located within a beta-1,3-Xyl chain and linked via covalent bonds. This report indicates the usefulness of the enzyme for the structural analysis of beta-1,3-xylan.

Caulerpa↗

PubMed2006

Neuronal organization of the rat inferior colliculus participating in four major auditory pathways.

The central nucleus of the inferior colliculus (CNIC) contains different types of neurons and is a source of ascending projection to the medial geniculate body (MGB), commissural projection to the contralateral IC, direct descending projection to the cochlea nucleus (CN) and indirect projection to the CN via the superior olivary complex (SOC). Using a retrograde tracing technique, we examined what kind of neurons and what percentage of neurons of each type recognized in the CNIC participated in the above-mentioned four projection pathways. We also examined whether the individual CNIC neurons send the collateral to the MGB, the contralateral IC, the CN and the SOC. In the present study, we demonstrated that the neurons participating in the four projections could be morphologically classified into two types of neurons with soma size variation. The percentages of neurons of each type differed among the four projection pathways. Using a double-labeling technique, we found very few double-labeled neurons, indicating the collateral projections to the ipsilateral MGB and the contralateral IC. There were no double-labeled neurons in the collateral projections between the other combinations of targets. Therefore, we conclude that the ascending projection, the commissural projection and the descending projection to these targets arise from separate populations of neurons.

Animals↗

PubMed2006

A novel phosphatidylcholine which contains pentadecanoic acid at sn-1 and docosahexaenoic acid at sn-2 in Schizochytrium sp. F26-b.

Docosahexaenoic acid (DHA, 22:6n-3)-containing phospholipids are a ubiquitous component of the central nervous system and retina, however their physiological and pharmacological functions have not been fully elucidated. Here, we report a novel DHA-containing phosphatidylcholine (PC) in a marine single cell eukaryote, Schizochytrium sp. F26-b. Interestingly, 31.8% of all the fatty acid in F26-b is DHA, which is incorporated into triacylglycerols and various phospholipids. In phospholipids, DHA was found to make up about 50% of total fatty acid. To identify phospholipid species containing DHA, the fraction of phospholipids from strain F26-b was subjected to normal phase high-performance liquid chromatography (HPLC). It was found that DHA was incorporated into PC, lyso-PC, phosphatidylethanolamine, and phosphatidylinositol. The major DHA-containing phospholipid was PC in which 32.5% of the fatty acid was DHA. The structure of PC was analyzed further by phospholipase A2 treatment, fast atom bombardment mass spectrometry, and 1H- and 13C-NMR after purification of the PC with reverse phase HPLC. Collectively, it was clarified that the major PC contains pentadecanoic acid (C15:0) at sn-1 and DHA at sn-2; the systematic name of this novel PC is therefore "1-pentadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine."

Animals↗

PubMed2006

RFamide peptide QRFP43 causes obesity with hyperphagia and reduced thermogenesis in mice.

QRFP, an RFamide peptide, was recently identified as an endogenous ligand of an orphan G protein-coupled receptor, GPR103. Recent investigation revealed that acute intracerebroventricular (ICV) administration of QRFP26/P518/26RFa, a constitutive part of QRFP43 (43-amino acid-residue form of QRFP), increases appetite in mice, but its role in long-term energy homeostasis remains unknown. In the present study, we examined the effects of chronic administration of QRFP43 on feeding behavior, body weight regulation, and energy expenditure in mice. Intracerebroventricular infusion of QRFP43 for 13 d resulted in a significant increase in body weight and fat mass with hyperphagia. Weight gain and hyperphagia were more evident when mice were fed a moderately high-fat diet. Pair feeding of QRFP43-infused mice did not increase body weight but significantly increased fat mass and plasma concentrations of insulin, leptin, and cholesterol when compared with controls. Moreover, significant decreases in rectal temperature and expression of brown adipose tissue uncoupling protein-1 mRNA were observed in QRFP43-infused ad libitum- and pair-fed mice. The present results suggest that QRFP plays an important role in energy homeostasis by regulating appetite and energy expenditure.

Animals↗

PubMed2006

Development of nonalcoholic steatohepatitis model through combination of high-fat diet and tetracycline with morbid obesity in mice.

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease closely associated with obesity, type 2 diabetes and hyperlipidemia. For further understanding of NASH, development and characterization of appropriate animal models with metabolic abnormalities is important. Based on the "two hit theory", we tried to develop a new murine model of NASH with metabolic abnormalities. For the first hit to achieve metabolic abnormalities, a high-fat diet (HFD: 60 cal% fat) was fed to C57BL/6 mice for 10 weeks. For the second hit, 30mg/kg tetracycline was injected intraperitoneally once daily for 10 days. The HFD-fed mice treated with tetracycline showed robust increases in triglyceride content and expression levels of proinflammatory cytokine mRNAs in the liver. In addition, plasma ALT levels were significantly elevated by this combinational treatment. Furthermore, histological examination demonstrated that combinational treatment induced multifocal inflammatory cellular infiltration in the livers of all mice, and thus caused mild steatohepatitis. The HFD-tetracycline model could be useful for further understanding NASH.

Journal Article↗

PubMed2006

Targeted chemotherapy against intraperitoneally disseminated colon carcinoma using a cationized gelatin-conjugated HVJ envelope vector.

The hemagglutinating virus of Japan envelope (HVJ-E; Sendai virus) vector derived from inactivated HVJ particles can be used to deliver DNA, proteins, and drugs into cells both in vitro and in vivo. HVJ-E is capable of delivering bleomycin, an anticancer drug, to various cancer cell lines, thereby producing 300-fold greater cytotoxicity than administration of bleomycin alone. In a mouse model of peritoneally disseminated colon cancer, we injected HVJ-E containing the luciferase gene into the peritoneum. Unexpectedly, luciferase gene expression was not observed within the tumor deposits or any organs. However, when combined with cationized gelatin (CG), CG-HVJ-E produced a high level of luciferase gene expression primarily within the tumor deposits. Forty-eight hours after introducing colon cancer cells into the peritoneum of experimental mice, CG-HVJ-E with or without bleomycin was injected into the abdominal cavity. Following six injections of bleomycin-incorporated CG-HVJ-E, complete responses were observed in 40% of the mice examined. All of the mice that received either empty CG-HVJ-E or bleomycin alone died within 40 days of having cancer cells introduced into the peritoneum. When the mice with complete responses were rechallenged with colon cancer cells from the same cell line, no tumors developed. Thus, CG-HVJ-E may suppress peritoneal dissemination of cancer.

Animals↗

PubMed2006

Arrhythmogenesis in the short-QT syndrome associated with combined HERG channel gating defects: a simulation study.

BACKGROUND: This study aimed to show the mechanism how the HERG channel gating defects causes life-threatening arrhythmia in the short-QT syndrome, using a simulation model of ventricular action potentials (APs). METHODS AND RESULTS: To evaluate the electrophysiological consequences of the short-QT syndrome at the level of the cardiac AP, the Markov model of wild-type (WT) KCNH2 channel was modified to obtain a model of the KCNH2 channel with the N588K mutation associated with the short-QT syndrome. Two parameters (betai and betabeta) were changed to reconstruct the N588K mutant Markov model, which successfully reproduced the experimental results of voltage-clamp recordings. The WT and mutant models were then integrated into the Luo-Rudy theoretical model of the cardiac ventricular AP. Unexpectedly, 1 parameter change alone, which caused gain of function, could shorten the AP duration (APD) but failed to induce early after-depolarizations (EADs). Only the condition with the combined gating defects could lead to EAD. CONCLUSIONS: Although the gain of function for KCNH2 shortened APD in the short-QT syndrome, this simulation study suggested that arrhythmogenesis was associated not only with gain of function, but also with accelerated deactivation of KCNH2.

Action Potentials↗

PubMed2006

Gender and age effects on ventricular repolarization abnormality in Japanese general carriers of a G643S common single nucleotide polymorphism for the KCNQ1 gene.

BACKGROUND: The KCNQ1 single nucleotide polymorphism (SNP), G643S, is known to be associated with secondary long QT syndrome (LQTS) and to cause a mild reduction in KCNQ1 current. However, the precise incidence and its association with QT intervals remain unknown in the greater cohort of the population in Japan. METHODS AND RESULTS: The genotype was screened at codon 643 of KCNQ1 in 992 residents of a farming community. Eighty-eight individuals (female/male =52/36, 8.9%) were found to have a heterozygous G643S SNP. Matching both gender and age, we randomly selected 243 control (G643G) cases and compared the electrocardiogram parameters in both groups; QT, QTf (QT corrected by Fridericia's formula) intervals, the peak and the end of the T wave (Tpe) interval, and the Tpe/QT ratio. The latter 2 reflect the transmural dispersion of ventricular repolarization (TDR). In G643S carriers, both Tpe and Tpe/QT were significantly longer than in non-carriers, without significant QT prolongation. Both genders showed a tendency for an increase in QTf with aging. In females, both Tpe and Tpe/QT showed a similar significant increase with age, which was not observed in males. CONCLUSIONS: In elderly females, G643S might be an independent risk factor for secondary LQTS by causing a greater TDR.

Adult↗

PubMed2006

[Clinical characteristics of perioperative pulmonary thromboembolism in Japan--Results of the perioperative thromboembolism research in the Japanese Society of Anesthesiologists].

BACKGROUND: Although we reported the perioperative mortality to be 4.41 (per 10000 cases) and morbidity to be 18.0% with the pulmonary thromboembolism (PTE) in Japan based on the research conducted by the Japanese Society of Anesthesiologists (JSA), the characteristics of these patients have not yet been assessed. METHODS: Results of 809 registry cases of perioperative PTE patients were analyzed regarding the differences in gender, characteristics of obesity, cases that resulted in death, feature of PTE cases with abdominal surgery and hip/lower extremity surgery, and changes in the results of two researchs. RESULTS: Considerably higher number of obesity cases were observed in women than in men, and the obesity rate was approximately 1.5-3-times higher in men than that observed in the general female population. The predictors of mortality were old age, long-term rest and gender (male). More than 80% of the patients who had undergone abdominal surgery contracted PTE in the postoperative period; but, more than 60% of the patients who had undergone hip/lower limb surgery along with a bone fracture contracted PTE in the preoperative or intra-operative periods. When compared with the results of the research carried out in 2002 and 2003, the incidence around caesarean section appeared to have decreased significantly (P = 0.016), and use of elastic stoking and intra-pneumonic compression appeared to have increased significantly (P<0.01). However, the rate of using anticoagulant drugs appeared to remain unchanged. CONCLUSIONS: The results of this study indicate the clinical characteristics of patients with perioperative PTE in Japan, the predictors of perioperative mortality, and the changes observed between the results of the researches carried out in 2002 and 2003.

Age Factors↗

PubMed2006

[Incidence and characteristics of perioperative pulmonary thromboembolism in Japan in 2004].

BACKGROUND: The incidence of perioperative pulmonary thromboembolism (PTE) is reported not to be low in Japan. The aim of this study is to investigate the incidence and characteristics of perioperative PTE in Japan in 2004. METHODS: A questionnaire was mailed to 960 institutions registered as the teaching hospitals of the Japanese Society of Anesthesiologists (JSA). The survey details included age, sex, type of surgery, and risk factors of patients operated in 2004. RESULTS: Effective responses were received from 642 institutions (66.9%) of 960 institutions. A total of 409 cases of perioperative PTE were reported from 245 institutions (25.5% of the institutions responded). The incidence of perioperative PTE was 3.62 per 10,000 cases. Cardiac arrest occurred at the onset in 65 cases (15.9%). Of the patients, 150 (36.7%) had malignancy; 142 (34.7%) were obese; and 121 (29.6%) were bed-ridden (> 4 days). In 273 cases (66.7%), the patients were over 60 years of age. PTE was found to be more frequent in females than in males (males, 147 cases; females, 259 cases). The types of surgery resulted in perioperative PTE with high frequency were "spinal surgery" (6.95 per 10,000 cases) and "limbs and/or hip joint surgery" (9.79 per 10,000 cases). Eighty-nine patients (21.8%) died from perioperative PTE. CONCLUSIONS: The incidence of perioperative PTE was not low and its mortality was high in Japan in 2004. In particular, the incidence of perioperative PTE was found to be high in females, the elderly, patients with malignancy, and patients who had undergone orthopedic surgery.

Adolescent↗

PubMed2005

Substitution of the N-glycan function in glycosyltransferases by specific amino acids: ST3Gal-V as a model enzyme.

The sialyltranferase ST3Gal-V transfers a sialic acid to lactosylceramide. We investigated the role of each of the N-glycans modifying mouse ST3Gal-V (mST3Gal-V) by measuring the in vitro enzyme activity of Chinese hamster ovary (CHO) cells transfected with ST3Gal-V cDNA or its mutants. By examining mutants of mST3Gal-V, in which each asparagine was replaced with glutamine (N180Q, N224Q, N334Q), we determined that all three sites are N-glycosylated and that each N-glycan is required for enzyme activity. Despite their importance, N-glycosylation sites in ST3Gal-V are not conserved among species. Therefore, we considered whether the function in the activity that is performed in mST3Gal-V by the N-glycan could be substituted for by specific amino acid residues selected from the ST3Gal-V of other species or from related sialyltransferases (ST3Gal-I, -II, -III, and -IV), placed at or near the glycosylation sites. To this end, we constructed a series of interspecies mutants for mST3Gal-V, specifically, mST3Gal-V-H177D-N180S (medaka or tetraodon type), mST3Gal-V-N224K (human type), and mST3Gal-V-T336Q (zebrafish type). The ST3Gal-V activity of these mutants was quite similar to that of the wild-type enzyme. Thus, we have demonstrated here that the N-glycans on mST3Gal-V are required for activity but can be substituted for specific amino acid residues placed at or near the glycosylation sites. We named this method SUNGA (substitution of N-glycan functions in glycosyltransferases by specific amino acids). Furthermore, we verified that the ST3Gal-V mutant created using the SUNGA method maintains its high activity when expressed in Escherichia coli thereby establishing the usefulness of the SUNGA method in exploring the function of N-glycans in vivo.

Amino Acid Sequence↗

PubMed2005

Crystallization and preliminary X-ray analysis of the Pax6 paired domain bound to the Pax6 gene enhancer.

Pax6 is a member of the Pax family of transcription factors and is essential for eye development. Pax6 has two DNA-binding domains: the paired domain and the homeodomain. The Pax6 paired domain is involved in Pax6 gene autoregulation by binding to its enhancer. In this study, crystallization and preliminary X-ray diffraction analysis of the mammalian Pax6 paired domain in complex with the Pax6 gene enhancer was attempted. The Pax6 paired domain complexed with an optimized 25 bp DNA fragment was crystallized by the hanging-drop vapour-diffusion method. The crystal diffracted synchrotron radiation to 3.0/3.7 A resolution and belongs to the monoclinic space group P2(1), with unit-cell parameters a = 62.21, b = 70.69, c = 176.03 A, beta = 90.54 degrees. Diffraction data were collected to 3.7 A resolution.

Binding Sites↗

PubMed2005

Frontal lobe function in bipolar disorder: a multichannel near-infrared spectroscopy study.

Frontal lobe dysfunction has been implicated as one of the pathophysiological bases of bipolar disorder. Detailed time courses of brain activation in the bipolar disorder group were investigated using multichannel near-infrared spectroscopy (NIRS), a recently developed functional neuroimaging technology with a high time resolution, and were compared with those in the major depression and healthy control groups. Seventeen patients with bipolar disorder, 11 equally depressed patients with major depression, and 17 healthy controls participated in the study. Changes in oxy hemoglobin concentration ([oxy-Hb]) during cognitive and motor tasks were monitored using frontal and temporal probes of two sets of 24-channel NIRS machines. [oxy-Hb] increases in the bipolar disorder group were smaller than those in the healthy control group during the early period of a verbal fluency task, larger than those in the major depression and healthy control groups during the late period of this task, and were smaller than those in the major depression group during a finger-tapping task. Depressive symptoms and antidepressant dosages did not correlate with [oxy-Hb] changes in the two patient groups. Bipolar disorder and major depression were characterized by preserved but delayed and reduced frontal lobe activations, respectively, in the present high-time-resolution study by multichannel NIRS.

Adult↗