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W Li

Publications and source records attributed to W Li.

At least 577 records · Page 32Linked to original sources
PubMed1999

Construction and analytical application of ion-selective piezoelectric sensor for atropine sulfate.

The method describes the use of a piezoelectric quartz crystal (PQC) as a substitute for ion-selective electrodes. The approach is feasible when the membrane materials are electrically non-conductive and membrane potential measurements are consequently not possible. An ion-selective piezoelectric sensor sensitive to atropine sulfate was constructed by coating a PVC membrane containing activant on one the side of a PQC. On the basis of selective adsorption of atropine ions across the modified film and the sensitive mass response of PQC, the method exhibits a sensitive, rapid response and is easy to operate without pretreatment of the sample. The logarithm of the frequency shift gave a linear relationship with the logarithm of atropine sulfate concentration in the 1.0 x 10(-8)-1.0 x 10(-3) M range with a detection limit of 5.0 x 10(-9) M at pH 7.0. Recoveries were from 98.7-102.2%. Two activants, atropine tetraphenylborate and atropine dipicrylaminate, were synthesized and investigated. Influencing factors were also examined and optimized. The results for real samples obtained by the proposed method agreed with those obtained by conventional methods.

Atropine↗
PubMed1999

Development and characterization of an SV40 immortalized porcine ameloblast-like cell line.

Enamel is secreted as a protein matrix by the ameloblasts. These same cells then control the maturation of the enamel matrix, secreting proteinases that hydrolyze proteins as mineralization progresses, until mature enamel containing less than 1% protein by weight remains. Further understanding of the factors that control ameloblast function and differentiation requires an in vitro cell culture system. In this study, we report immortalization of enamel organ epithelial cells and the selection of a cell line with characteristics of ameloblasts. Porcine enamel organ cells were dissected from unerupted porcine molars, cultured in serum-free medium, and passaged twice. These cells were transfected with an origin-of-replication defective SV40 plasmid by calcium phosphate precipitation, and a cell line with mRNA expression characteristic of ameloblasts was cloned. This cell line (PABSo-E) expressed mRNA for amelogenin, matrix metalloproteinase-20 (enamelysin), and enamel matrix serine proteinase 1 (EMSP1), but not ameloblastin. PABSo-E cells have been passaged more than 55 times, while continuing to maintain characteristics of ameloblasts. These cells will be useful for future studies of ameloblast function.

Ameloblasts↗
PubMed1999

Activation of recombinant bovine matrix metalloproteinase-20 and its hydrolysis of two amelogenin oligopeptides.

Enamelysin is a matrix metalloproteinase (MMP-20) secreted by ameloblasts, previously shown to hydrolyze recombinant amelogenin. The purpose of this study was to use recombinant MMP-20 to further investigate the specific hydrolysis of peptide fragments containing cleavage sites for tyrosine-rich amelogenin peptide (TRAP) and leucine-rich amelogenin peptide (LRAP). MMP-20 cDNA was isolated from a subtracted bovine cDNA library, reconstructed into pRSET A vector, and overexpressed in BL21 Escherichia coli. The recombinant MMP-20 was purified using Mono-S ion exchange and nickel affinity chromatography. The proteinase was renatured by dialysis against buffer containing 50 microM zinc and 5 mM calcium and autolysed to form several active fragments. The varying sizes and activities of the activated enzyme fragments appeared to be due to sequential autolysis at different location of the carboxyl terminus of the intact enzyme. Two synthetic peptides corresponding to amelogenin amino acid sequences 36-49 and 181-188 were hydrolyzed by the activated rMMP-20. Mass spectrometry and amino acid composition analysis showed that the cleavage sites were between the tryptophan and leucine (45 and 46) for TRAP and between proline and alanine (186-187) for LRAP. These results indicate that MMP-20 can be autoactivated, and activated MMP-20 has a functional role in the initial cleavage of amelogenin.

Alanine↗
PubMed1999

Integrin alphavbeta3-RGDS interaction mediates fibrin-induced morphological changes of glomerular endothelial cells.

BACKGROUND: In our previous studies, we found that intraglomerular deposition of fibrin and its metabolites was related to glomerular sclerosis and reduced renal function. It has been reported that both overlying and underlying fibrin may induce specific morphological changes of cultured endothelial cells from large blood vessels. The dependency of these morphological changes on the integrin alphavbeta3-arginyl-glycyl-aspartyl-serine (RGDS) interaction is still controversial. We hypothesized that glomerular endothelial cells (GECs) stimulated by fibrin might undergo morphological changes through an integrin alphavbeta3-RGDS interaction. Methods. In vitro studies were performed to examine the growing status of GECs stimulated by overlying and underlying fibrin gels in the presence or absence of the following: 50 microg/ml anti-alphavbeta3 integrin monoclonal antibody 23C6 or nonimmune mouse IgG, 1 mg/ml synthetic RGDS or arginyl-glycyl-glycyl-serine (RGGS) peptide, 10 mg/ml sodium heparin, 100 microg/ml cycloheximide, and 10 microM actinomycin D. Fast protein liquid chromatography (FPLC)-purified fibrinogen and the third to fifth passages of human GECs were also used in this study. RESULTS: GECs developed capillary tube structure after 60 hours of culturing on fibrin gels, and GECs cultured on gelatin-coated plates displayed a monolayer of cobblestone-like cells in the presence or absence of 23C6 and synthetic RGDS peptide. Fibrin-induced capillary tube formation was promoted by 23C6 and inhibited by RGDS peptide, cycloheximide, and actinomycin D. Disorganization of the GEC monolayer was induced by overlying fibrin, but was not induced by overlying agarose gels and glass cover slips or culturing in fibrinogen, 0.05 NIH U/ml thrombin, fibrin supernatants, as well as in fibrin degradation products. Disorganization of GEC monolayer can be induced by both des-AA-fibrin and des-AABB-fibrin and was unaffected by heparin. Furthermore, both 23C6 and synthetic RGDS peptide prevented disorganization of GECs induced by overlying fibrin, whereas nonimmune mouse IgG, synthetic RGGS peptide, cycloheximide, and actinomycin D had no similar effect. CONCLUSIONS: GECs cultured on fibrin gels may develop capillary structure spontaneously, and GECs covered by fibrin gels may undergo disorganization. Our data suggest that these GEC morphological changes are mediated by an integrin alphavbeta3-RGDS interaction.

Antibodies, Monoclonal↗
PubMed1999

Primary nasal and nasopharyngeal lymphomas: a comparative study of clinical presentation and treatment outcome.

Primary nasal lymphoma (NSL) and nasopharyngeal lymphoma (NPL) are uncommon extranodal lymphomas that have often been grouped together in studies published in the literature. It is unclear whether or not NSL and NPL are biologically different entities. We reviewed the records of 25 NSL and 19 NPL patients who were managed between 1985 and 1995, to evaluate and compare their clinical characteristics and treatment outcome. Clinical variables, including age, sex, stage, tumour bulk and treatment modality, were assessed for their prognostic influence on survival. Nasal obstruction (80%), nasal discharge (64%) and epistaxis (60%) were the predominant symptoms in NSL patients; neck swelling/mass (42%), nasal obstruction (37%) and hearing impairment (32%) were common findings in NPL patients. Limited stage disease (I/II) was present in all NSL and in 80% of the NPL patients. Thirty-six per cent of the NSL and 32% of the NPL patients had bulky disease. The majority of the immunophenotyped NSLs were of T-cell lineage (75%, 12/16) and most immunophenotyped NPLs were of B-cell lineage (69%, 11/16). The patients were treated with radiotherapy alone (14%), chemotherapy alone (23%) or chemotherapy plus radiotherapy (64%). NSL patients showed a lower complete remission rate and higher relapse rate than NPL patients, although the difference did not reach statistical significance. Both the 5-year disease-free survival (36% versus 76%; P = 0.007) and overall survival (33% versus 82%; P = 0.003) were significantly worse in NSL compared with NPL patients. Advanced age (>60 years; P = 0.03) and bulky disease (P = 0.04) were also associated with inferior survival times. The analysis of sex, stage and type of therapy showed no evidence of significant impact on the survival. Despite the close anatomical relationship in origin, NSL and NPL were noted to behave as biologically distinct entities, in which the NSLs demonstrated a higher frequency of T-cell tumours and a much worse prognosis than NPL. In addition to the primary site, advanced age and bulky disease were also associated with reduced survival.

Adult↗
PubMed1999

Pre-enrichment of modified low density lipoproteins with alpha-tocopherol mitigates adverse effects on cultured retinal capillary cells.

PURPOSE: We determined whether pre-enrichment of low density lipoproteins (LDL) with alpha-tocopherol mitigates their adverse effects, following in vitro glycation, oxidation or glycoxidation, towards cultured bovine retinal capillary endothelial cells (RCEC) and pericytes. METHODS: LDL, while still in plasma obtained and pooled from non-diabetic humans, was supplemented in vitro with alpha-tocopherol. It was then isolated and modified in vitro by glycation, minimal oxidation, and glycoxidation. Bovine RCEC and pericytes were exposed to LDL (100mg protein/ ml) for three days. Cell count was determined by cell counting, supernatant levels of plasminogen activator inhibitor-1 (PAI-1) and endothelin-1 (ET-1) by ELISA, and nitrite levels by spectroscopic colorimetric assay. RESULTS: While pre-enrichment of LDL with alpha-tocopherol did not reduce the measured extent of lipoprotein modification, it abolished the reduction in cell count observed with glycated, oxidized and glycoxidized LDL v. normal LDL. Pre-enrichment of LDL with alpha-tocopherol also reduced RCEC supernatant PAI-1 and ET-1 (corrected for cell counts) and increased RCEC and pericyte-associated supernatant nitrite levels: such effects of alpha-tocopherol may inhibit clot formation and favor vasodilatation. CONCLUSIONS: Enrichment of LDL with alpha-tocopherol abolishes adverse effects of glycated, mildly oxidized, and glycoxidized LDL on cultured retinal cell count, and mitigates adverse effects on modulators of fibrinolysis and vascular tone. Direct evidence is required before Vitamin E supplementation is recommended for people with diabetes.

Animals↗
PubMed1999

Cytomegalovirus UL97 and glycoprotein B (gB) sequences in tissues from immunocompromised patients with ganciclovir-resistant virus infection.

The pathogenesis of ganciclovir-resistant cytomegalovirus (CMV) was investigated by analysing UL97 and gB sequences in tissues obtained from 4 immunocompromised patients with infections caused by ganalciclovir-resistant virus. UL97 and gB sequences were obtained by automated sequencing of CMV DNA amplified from lysates prepared from deparaffinized tissue sections. Patient 1 contained wild-type UL97 and gB3 sequences. Patient 2 harboured genetically distinct viruses in his lung: one with a ganciclovir-resistance UL97 mutation and a gB3 genotype, and another without UL97 mutations and a gB1 genotype. In patient 3, a ganciclovir-resistant UL97 mutant virus with a gB1 genotype was cultured from the lung, whereas the CMV in the brain did not contain mutations and its genotype was gB2. In patient 4, ganciclovir-resistance UL97 sequences were found in oesophageal tissue prior to the isolation of a ganciclovir-resistant CMV from the blood. All viruses in this patient had a gB3 genotype. CMV containing ganciclovir-resistance UL97 mutations may cause end-organ disease in immunocompromised individuals. In these subjects, CMV circulating in the blood may have similar or different UL97 and gB genotypes than the virus causing end-organ disease.

Antiviral Agents↗
PubMed1999

Cytokine induction of iNOS and sPLA2 in immortalized astrocytes (DITNC): response to genistein and pyrrolidine dithiocarbamate.

Using an immortalized astrocyte cell line (DITNC), we showed that lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) but not interferon-alpha (IFN-alpha) could individually induce secretory phospholipase A2 (sPLA2) mRNA and enzymatic activity. However, induction of inducible nitric oxide synthase (iNOS) mRNA and NO production by cytokines required the presence of IFN-gamma. Using a three-cytokine mixture (TNF-alpha, IL-1beta, and IFN-gamma) that could maximally induce both iNOS and sPLA2, the increase in these mRNA species reached a maximum by 4-8 h, followed by a decline up to 48 h. L-N6-(1-Iminoethyl)lysine acetate (L-NIL) inhibited cytokine-induced NO production with IC50 of 25 microM, but this compound did not affect iNOS mRNA. Furthermore, L-NIL exerted no effect on sPLA2 mRNA or sPLA2 activity. Pyrrolidine dithiocarbamate (PDTC), an inhibitor for NF-kappaB, was more effective in inhibiting iNOS mRNA and NO production than for sPLA2. Surprisingly, genistein inhibited both NO production and sPLA2 activity with IC50 of 72 microM and 88 microM, respectively. On the other hand, daidzein, a genistein analog lacking tyrosine kinase inhibitor activity, was not effective in inhibition of NO production at 250 microM. These results demonstrate distinct pathways for induction of iNOS and sPLA2 in DITNC cells by cytokines and shed new insight on transcriptional regulation for these two mRNA species.

Animals↗
PubMed1999

Urinary bladder carcinogenicity of dimethylarsinic acid in male F344 rats.

The present study was conducted to determine the carcinogenicity of dimethylarsinic acid (DMA) administered to male F344 rats in a 2 year bioassay. A total of 144 rats (10 weeks old at the start) were divided into four groups of 36 rats each. Groups 1-4 received DMA (purity 100%) at concentrations of 200, 50, 12.5 and 0 p.p.m. in the drinking water, respectively, for 104 weeks. From weeks 97 to 104, urinary bladder tumors were observed in 12 of 31, eight of 31 and none of 33 in groups 1-3, respectively. No bladder tumors were observed in group 4. The present study demonstrated that long-term p. o. administration of DMA induced urinary bladder carcinomas in male F344 rats. Therefore, the results indicate that DMA is carcinogenic for the rat urinary bladder, which may be related to the human carcinogenicity of arsenicals.

Administration, Oral↗
PubMed1999

Evidence for multiple promoter elements orchestrating male-specific regulation of the her-1 gene in Caenorhabditis elegans.

The sex-determining gene her-1 is required for male development in Caenorhabditis elegans. In XO males, two her-1 mRNAs, her-1a and her-1b, are transcribed from two separate promoters: P1, located in the 5'-flanking region, and P2, located in the large second intron. In XX hermaphrodites, accumulation of both her-1 transcripts is repressed by the sdc genes, which in turn are negatively regulated by the xol-1 gene. When introduced into a xol-1(y9) background, transgenic arrays, including 3.4 kb of her-1 intron 2 sequence (P2), result in phenotypes that mimic those of sdc(lf) mutants, including suppression of XO lethality and masculinization of both XX and XO animals. The masculinization, but not the suppression of XO lethality, is dependent on endogenous her-1 activity. These effects could therefore result from sequestration (titration) of sdc gene products by sequences in the arrays, causing derepression of her-1 (masculinizing effect) and disruption of the dosage compensation machinery (allowing survival of XO animals). We used these effects as an assay in a deletion analysis of the two her-1 promoter regions to define potential cis-regulatory sites required for the putative titration. Several regions in P2 contributed to these effects. P1 was effective only in combination with certain P2 sequences and only if a particular P1 site previously implicated in her-1 repression was intact. These results suggest that normal repression of transcription from P1 in XX animals may involve cooperative interaction with sequences in the P2 region. In experiments to test for a possible role of the her-1b transcript in regulation of sdc genes, no significant effects could be demonstrated.

Animals↗
PubMed1999

Homologs of the Caenorhabditis elegans masculinizing gene her-1 in C. briggsae and the filarial parasite Brugia malayi.

The masculinizing gene her-1 in Caenorhabditis elegans (Ce-her-1) encodes a novel protein, HER-1A, which is required for male development. To identify conserved elements in her-1 we have cloned and characterized two homologous nematode genes: one by synteny from the closely related free-living species C. briggsae (Cb-her-1) and the other, starting with a fortuitously identified expressed sequence tag, from the distantly related parasite Brugia malayi (Bm-her-1). The overall sequence identities of the predicted gene products with Ce-HER-1A are only 57% for Cb-HER-1, which is considerably lower than has been found for most homologous briggsae genes, and 35% for Bm-HER-1. However, conserved residues are found throughout both proteins, and like Ce-HER-1A, both have putative N-terminal signal sequences. Ce-her-1 produces a larger masculinizing transcript (her-1a) and a smaller transcript of unknown function (her-1b); both are present essentially only in males. By contrast, Cb-her-1 appears to produce only one transcript, corresponding to her-1a; it is enriched in males but present also in hermaphrodites. Injection of dsRNA transcribed from Cb-her-1 into C. briggsae hermaphrodites (RNA interference) caused XO animals to develop into partially fertile hermaphrodites. Introducing a Cb-her-1 construct as a transgene under control of the C. elegans unc-54 myosin heavy chain promoter caused strong masculinization of both C. briggsae and C. elegans hermaphrodites. Introduction of a similar Bm-her-1 construct into C. elegans caused only very weak, if any, masculinization. We conclude that in spite of considerable divergence the Cb gene is likely to be a functional ortholog of Ce-her-1, while the function of the distantly related Bm gene remains uncertain.

Amino Acid Sequence↗
PubMed1999

Exploring the conformational diversity of loops on conserved frameworks.

Loops are structurally variable regions, but the secondary structural elements bracing loops are often conserved. Motifs with similar secondary structures exist in the same and different protein families. In this study, we made an all-PDB-based analysis and produced 495 motif families accessible from the Internet. Every motif family contains some variable loops spanning a common framework (a pair of secondary structures). The diversity of loops and the convergence of frameworks were examined. In addition, we also identified 119 loops with conformational changes in different PDB files. These materials can give some directions for functional loop design and flexible docking.

Computer Simulation↗
PubMed1999

Determination of indinavir, a HIV-1 protease inhibitor, in human plasma using ion-pair reversed-phase high-performance liquid chromatography.

Indinavir is widely prescribed as a component of potent antiretroviral therapy for the treatment of HIV-1 infection. Because virologic failure of therapy can result from subtherapeutic drug levels, monitoring of indinavir levels may be important in clinical management. We have developed a simple, accurate, and precise high-performance liquid chromatographic (HPLC) assay for measurement of indinavir concentration in human plasma. In our method, indinavir was extracted from plasma with dichloromethane at pH 10.4, which resulted in quantitative recovery of indinavir and the internal standard (IS), methyl-indinavir (86% and 80%-97%, respectively). Chromatographic separation was accomplished using a Luna C18 (2) (Phenomenex) analytic column with a mobile phase composed of acetonitrile:phosphate buffer (25 mM) and 0.2% triethylamine pH 7.0 (34.5:65.5, v/v). Ion-paired reagent triethylamine was necessary to ensure an appropriate retention time for indinavir and differentiate it from other protease inhibitors that were coextracted. Quantification was performed at 210 nm. The standard curves were linear (r2>0.999) over the concentration range 25-5,000 ng/mL, when 1-mL aliquots of plasma were extracted. Inter- and intraday coefficients of variation were acceptable. The assay was used to determine trough and peak levels of in plasma from 12 subjects who received indinavir 1200 mg every 12 hours, 1000 mg every 12 hours, or 800 mg every 8 hours. The concentrations of indinavir found in this study (trough 26-768 ng/mL; peak at 1 hr 3,309-17,568 ng/mL) has a wider range than defined previously (trough 50-300 ng/mL; peak 6,000-12,000 ng/mL). This study illustrates three potential uses of indinavir monitoring: to assess individual dosing regimen, to assess patient compliance, and to monitor unusual indinavir levels caused by changed drug clearance.

Chromatography, High Pressure Liquid↗
PubMed1999

mRNA expression of complement components and regulators in rat arterial smooth muscle cells.

The presence of C5b-9 complexes, some complement regulators, and abundant cytokines in atherosclerotic lesions has been reported. However, it is unclear whether these complement-associated proteins are produced by vascular smooth muscle cells (SMCs) and how they are influenced by the cytokines. In the present study, we demonstrated, by the reverse transcription-polymerase chain reaction method, the mRNA expression of complement components (C3, C4, and C5) and membrane regulators (decay-accelerating factor, membrane cofactor protein, Crry, and CD59) in cultured SMCs derived from the rat carotid artery. The expression of C9 mRNA was also induced upon stimulation by interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and/or lipopolysaccharide (LPS). Northern blot analysis showed that the mRNA expression of C3, C4, DAF and Crry was up-regulated, but that of CD59 was down-regulated by IFN-gamma, TNF-alpha and/or LPS alone or by synergy. The increase of C3 mRNA by TNF-alpha or LPS and that of C4 mRNA by IFN-gamma was induced in a dose-dependent manner. The results indicate that the arterial SMCs of rat have the ability to produce complement components and regulators, which is affected by cytokines and/or LPS. Since atherosclerosis is characterized by the intimal proliferation of SMCs, the complement system including its regulators may be involved in the pathogenesis of the disease.

Animals↗
PubMed1999

Osteopontin (OPN) distribution in premalignant and malignant lesions of oral epithelium and expression in cell lines derived from squamous cell carcinoma of the oral cavity.

The objectives of this study were to assess the immunolocalization of human osteopontin (OPN) in oral lesions and to identify human cell lines of oral squamous cell carcinoma (OSCC) origin that express OPN mRNA. OPN was localized using immunohistochemistry in the following oral specimens: normal epithelium (n=6), epithelial hyperplasia (n=4), epithelial dysplasia (n=28), carcinoma in situ (n=11) and squamous cell carcinoma (n=43). Cell lines UMSCC-1, MDA TU 138, MDA 686LN, SCC4, SCC9, SCC25, CAL 27 and MDA 1483 were characterized for OPN mRNA expression using Northern blotting. OPN was not detected in normal oral epithelium. Intracellular and intercellular immunoreactivity was seen in 75% of hyperplasias, 57% of dysplasias, 54% of carcinoma in situ and 67% of squamous cell carcinomas. UMSCC-1 expressed high levels of OPN mRNA. We conclude that OPN protein is detectable in premalignant and malignant lesions arising from oral epithelium. UMSCC-1 may be a useful cell line in which to conduct in vitro studies designed to clarify the role of OPN in OSCC.

Adolescent↗
PubMed1999

[Efficacy of influenza vaccine among geriatric inpatients: effect of previous vaccination and antibody induction by single and twice injections].

To investigate the effect of previous influenza vaccination and the difference in antibody induction by single and twice injection of influenza vaccine in the elderly, hemagglutination inhibition (HI) antibody titers of the three types of influenza viruses were measured. Influenza vaccination was done for 217 inpatients. For the patients who had influenza vaccination in the year prior to the study, influenza vaccine was administered once to 77 patients and twice to another 70 patients. Influenza vaccine was injected twice to 70 patients who had not received influenza vaccine in the previous years. The influenza vaccine induced an increase in HI titer in almost all patients. The geometric mean of the HI titer and the frequency of patients with HI titers over 128x were similar after vaccination in the groups of patients who were injected twice, irrespective of whether or not influenza vaccination was given in the year prior to the study. The geometric means of the HI titers for influenzas A/H3N2 and B and the frequency of HI titers over 128x for influenza A/H3N2 after vaccination were lower in the patients who received vaccine once than in the patients vaccinated twice. These results suggest that prior vaccination does not diminish antibody response to influenza vaccine significantly in the elderly when influenza vaccine is injected twice. Although single injection is inferior to twice injection in antibody induction with some vaccine virus strains, induction of HI titers over 128x is found in more than 70% of elderly. Single injection of influenza vaccine may be practically effective and useful for protection of influenza infection in the elderly.

Aged↗
PubMed1999

[Evaluation of a rapid enzyme immunoassay for detection of influenza A virus among adult and elderly patients].

Directigen Flu A, an enzyme immunoassay membrane test for the detection of influenza A virus, was compared with serum hemagglutinine inhibition assay in adult and elderly patients. Throat specimens were collected by vigorous swabbing from febrile patients who were suspect for influenza by clinical symptoms and tested with Directigen Flu A. Serum samples were collected from the patients at onset of illness and two to eleven weeks later and tested for antibody titer to influenza A/H1N1, A/H3N2, and B. A total of 57 patients were involved in this study; 32 under 59 years of age and 25 over 60 years of age. Influenza was diagnosed in 42 patients by HI titer elevation (73.7%). Directigen Flu A was positive in 25 patients. Results of Directigen Flu A and HI test agreed in 40 patients. Sensitivity and specificity of Directigen Flu A were 59.5% (25/42) and 100% (15/15), respectively. There was no significant difference in sensitivity between the two age groups. These results indicate the potential usefulness of Directigen Flu A in the surveillance of influenza A infection in the geriatric population. Directigen Flu A gives results within 15 minutes without expensive equipment. Use of Directigen Flu permits the timely administration of antiviral therapy and infection control among hospitalized and institutionalized geriatric patients.

Adult↗