Biomedical subjects

J H Elder

Publications and source records attributed to J H Elder.

At least 19 recordsLinked to original sources

PubMed2001

Inhibition of the ERK pathway prevents HIVgp120-induced REM sleep increase.

Approximately 35% of HIV-infected subjects, both children and adults, exhibit alterations in the sleep-waking cycle. HIV surface glycoprotein gp120 has been postulated to contribute to this abnormality. For example, it has been reported that HIVgp120 modifies sleep in freely-moving rats and that it also activates the ERK pathway in brain slices. The goal of this work was to determine if sleep changes induced by HIVgp120 in normal rats are mediated by the MAPK pathway. Our results show that a single intraventricular administration of HIVgp120 selectively increases REMS and that such an increase can be prevented by U0126, an inhibitor of ERK activating enzyme, MEK. In contrast, SB202190, a MAPK-p38 inhibitor, had no effect on HIVgp120-induced increase in REMS. These results suggest that HIVgp120 increases REMS in the rat by specifically affecting the ERK signal transduction pathway.

AIDS Dementia Complex↗

PubMed2001

Design, synthesis, and biological evaluation of HIV/FIV protease inhibitors incorporating a conformationally constrained macrocycle with a small P3' residue.

A series of norstatine-based HIV/FIV protease inhibitors incorporating a 15-membered macrocycle as a mimic of the tripeptide (Ala-Val-Phe), a motif with a small P3' residue elective against the FIV protease and the drug-resistant HIV proteases, has been synthesized. It was found that the macrocycle is important to the overall activity of the inhibitors. Certain inhibitors were developed expressing low nanomolar inhibitory activity against the HIV/FIV proteases and they are also effective against some drug-resistant as well as TL3-resistant HIV proteases.

Amino Acid Motifs↗

PubMed2001

Structure-activity studies of FIV and HIV protease inhibitors containing allophenylnorstatine.

The interaction of P1 and P3 side chains with the combining S1 and S3 hydrophobic subsites of HIV and FIV proteases has been explored using asymmetric competitive inhibitors. The inhibitors evaluated contained (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid (allophenylnorstatine) as the hydroxymethylcarbonyl isostere, (R)-5,5-dimethyl-1, 3-thiazolidine-4-carbonyl as P1', Val as P2 and P2' residues, and a variety of amino acids at the P3 and P3' positions. All inhibitors showed competitive inhibition of both enzymes with higher potency against the HIV protease in vitro. Within this series, 31 (VLE776) is the most effective inhibitor against FIV protease, and it contains Phe at P3, but no P3' residue. VLE776 also exhibited potent antiviral activities against the drug-resistant HIV mutants (G48V and V82F) and the TL3-resistant HIV mutants. Explanation of the inhibition activities was described. In addition, a new strategy was described for development of bifunctional inhibitors, which combine the protease inhibitor and another enzyme inhibitor in one molecule.

Aspartic Acid Endopeptidases↗

PubMed2001

Binding of recombinant feline immunodeficiency virus surface glycoprotein to feline cells: role of CXCR4, cell-surface heparans, and an unidentified non-CXCR4 receptor.

To address the role of CXCR4 in the cell-surface attachment of the feline immunodeficency virus (FIV), a soluble fusion protein, gp95-Fc, consisting of the surface glycoprotein (SU, gp95) of either a primary (PPR) or cell line-adapted (34TF10) FIV strain was fused in frame with the Fc domain of human immunoglobulin G1. The recombinant SU-immunoadhesins were used as probes to investigate the cellular binding of FIV SU. In agreement with the host cell range properties of both viruses, binding of 34TF10 gp95-Fc was observed for all cell lines tested, whereas PPR gp95-Fc bound only to primary feline T cells. 34TF10 gp95-Fc also bound to Jurkat and HeLa cells, consistent with the ability of FIV to use human CXCR4 as a fusion receptor. As expected, 34TF10 gp95-Fc binding to Jurkat cells was blocked by addition of stromal cell-derived factor 1alpha (SDF-1alpha), as was binding to the 3201 feline lymphoma cell line. However, SDF-1alpha, RANTES, macrophage inflammatory protein 1beta, and heparin all failed to inhibit the binding of either gp95-Fc to primary T cells, suggesting that a non-CXCR4 receptor is involved in the binding of FIV SU. In this regard, an unidentified 40-kDa protein species from the surface of primary T cells but not Jurkat and 3201 cells specifically coprecipitated with both gp95-Fc. Yet another type of binding of 34TF10 gp95-Fc to adherent kidney cells was noted. SDF-1alpha failed to block the binding of 34TF10 gp95-Fc to either HeLa, Crandel feline leukemia, or G355-5 cells. However, binding was severely impaired in the presence of soluble heparin, as well as after enzymatic removal of surface heparans or on cells deficient in heparan expression. These overall findings suggest that in addition to CXCR4, a non-CXCR4 receptor and cell-surface heparans also play an important role in FIV gp95 cell surface interactions on specific target cells.

Animals↗

PubMed2001

Molecular basis for the relative substrate specificity of human immunodeficiency virus type 1 and feline immunodeficiency virus proteases.

We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3' region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF/VVNGLVK-NH(2) (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2' position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1', FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2' subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1' subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF/VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVF Psi(CH(2)NH)VVNGL-NH(2.) This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors.

Amino Acid Substitution↗

PubMed2001

Viral evolution in response to the broad-based retroviral protease inhibitor TL-3.

TL-3 is a protease inhibitor developed using the feline immunodeficiency virus protease as a model. It has been shown to efficiently inhibit replication of human, simian, and feline immunodeficiency viruses and therefore has broad-based activity. We now demonstrate that TL-3 efficiently inhibits the replication of 6 of 12 isolates with confirmed resistance mutations to known protease inhibitors. To dissect the spectrum of molecular changes in protease and viral properties associated with resistance to TL-3, a panel of chronological in vitro escape variants was generated. We have virologically and biochemically characterized mutants with one (V82A), three (M46I/F53L/V82A), or six (L24I/M46I/F53L/L63P/V77I/V82A) changes in the protease and structurally modeled the protease mutant containing six changes. Virus containing six changes was found to be 17-fold more resistant to TL-3 in cell culture than was wild-type virus but maintained similar in vitro replication kinetics compared to the wild-type virus. Analyses of enzyme activity of protease variants with one, three, and six changes indicated that these enzymes, compared to wild-type protease, retained 40, 47, and 61% activity, respectively. These results suggest that deficient protease enzymatic activity is sufficient for function, and the observed protease restoration might imply a selective advantage, at least in vitro, for increased protease activity.

Amino Acid Sequence↗

PubMed2000

Identification of efficiently cleaved substrates for HIV-1 protease using a phage display library and use in inhibitor development.

The recognition sequences for substrate cleavage by aspartic protease of HIV-1 are diverse and cleavage specificities are controlled by complex interactions between at least six amino acids around the cleavage site. We have identified 45 efficiently cleaved peptide substrates of HIV-1 protease (PR) using substrate phage display, an approach that can elucidate both context-dependent and context-independent preferences at individual subsites of a protease substrate. Many of the selected peptides were cleaved more efficiently and had lower K(m) values than physiologically relevant substrates of HIV-1 PR. Therefore, mutations occurring in the cleavage sites of the Gag and Gag-pol polyproteins of HIV-1 could significantly lower the K(m) values to better compete against drugs for protease binding while maintaining cleavage rates necessary for viral replication. The most efficiently cleaved peptide substrate derived from these phage, Ac-GSGIF*LETSL-NH(2), was cleaved 60 times more efficiently and had a K(m) approximately 260 times lower than a nine-amino-acid peptide based on the natural reverse transcriptase/integrase cleavage site when assayed at pH 5.6, 0.2 M NaCl. The peptide substrates selected served as frameworks for synthesis of tight binding reduced amide inhibitors of HIV-1 PR. The results show that the most efficiently cleaved substrates serve as the best templates for synthesis of the tightest binding inhibitors. Thus, defining changes in substrate preferences for drug-resistant proteases may aid in the development of more efficacious inhibitors.

Amides↗

PubMed2000

HIV-derived protein gp120 suppresses P3 potential in rats: potential implications in HIV-associated dementia.

Between 20 and 30% of AIDS patients have neurological symptoms characterized by motor impairment, memory loss and progressive dementia. Previous studies have implicated the HIV derived gp120, which produces behavioral deficits and electrophysiological alterations in rats. The goal of the present study was to describe the effect of this protein on the P3 event-related potential (ERP), evoked by a passive discrimination task in rats. We used II rats divided into two groups: HIV gp120 (n = 6) and control (n = 5). We recorded the P3 wave before any treatment (baseline), during the i.c.v. administration of either HIVgp 120 (700 ng/5 days) or saline (pH 7.2), and 24 h, 7, 14 and 21 days after the last injection. There were no changes between groups in the amplitude or latencies of the observed components (N1, P2, N2 and P3) evoked by target stimuli, during baseline or during the injection period. However, the HIV gp120 group showed a significant amplitude reduction in P3 wave 24 h after the last injection, while the N1, P2 and N2 waves remained unchanged. However, from the 7th day through the 21st day, P2 and N2 components also disappeared and only the N1 component could be observed in the HIV gp 20-treated group. These changes in the N2, P2 and P3 potentials, suggesting an alteration in cognitive processes, further support the neurotoxic activity of HIV gp120 and its role in AIDS dementia.

AIDS Dementia Complex↗

PubMed2000

Structural studies of FIV and HIV-1 proteases complexed with an efficient inhibitor of FIV protease.

Three forms of feline immunodeficiency virus protease (FIV PR), the wild type (wt) and two single point mutants, V59I and Q99V, as well as human immunodeficiency virus type 1 protease (HIV-1 PR), were cocrystallized with the C2-symmetric inhibitor, TL-3. The mutants of FIV PR were designed to replace residues involved in enzyme-ligand interactions by the corresponding HIV-1 PR residues at the structurally equivalent position. TL-3 shows decreased (improved) inhibition constants with these FIV PR mutants relative to wt FIV PR. Despite similar modes of binding of the inhibitor to all PRs (from P3 to P3'), small differences are evident in the conformation of the Phe side chains of TL-3 at the P1 and P1' positions in the complexes with the mutated FIV PRs. The differences mimick the observed binding of TL-3 in HIV-1 PR and correlate with a significant improvement in the inhibition constants of TL-3 with the two mutant FIV PRs. Large differences between the HIV-1 and FIV PR complexes are evident in the binding modes of the carboxybenzyl groups of TL-3 at P4 and P4'. In HIV-1 PR:TL-3, these groups bind over the flap region, whereas in the FIV PR complexes, the rings are located along the major axis of the active site. A significant difference in the location of the flaps in this region of the HIV-1 and FIV PRs correlates with the observed conformational changes in the binding mode of the peptidomimetic inhibitor at the P4 and P4' positions. These findings provide a structural explanation of the observed Ki values for TL-3 with the different PRs and will further assist in the development of improved inhibitors.

Animals↗

PubMed2000

HIV- and FIV-derived gp120 alter spatial memory, LTP, and sleep in rats.

Human immunodeficiency virus (HIV)-associated dementia (HAD) has been detected in 20-30% of patients suffering AIDS. The envelope glycoprotein 120 (gp120) derived from HIV seems to play a critical role in the pathophysiology of this dementia. Likewise, the feline immunodeficiency virus (FIV)-derived gp120 causes neurological and electrophysiological abnormalitites in cats. We have studied the effects of gp120 derived from HIV or FIV on learning and memory processing, hippocampal long-term potentiation (LTP), hippocampal neuronal cAMP production, the sleep-waking cycle, and locomotor activity and equilibrium in rats. Results showed that while both HIV- and FIV-gp120 impaired the rat's performance in the Barnes maze task, only HIVgp120 impaired the induction and maintenance of LTP. However, both glycoproteins induced a significant decrease in the posttetanic potentiation. HIVgp120 also caused a significant reduction in cAMP production in the hippocampus. Regarding the sleep-waking cycle, HIV- and FIV-gp120 increased the waking state and slow-wave sleep 1 (SWS1), while decreasing both SWS2 and REM sleep. Locomotor activity and equilibrium were significantly altered by these glycoproteins. These results suggest that HIVgp120 causes neurophysiological abnormalities and therefore may facilitate HAD development in AIDS patients.

AIDS Dementia Complex↗

PubMed2000

The effects of blur and size on monocular and stereoscopic localization.

Monocular localization of non-abutting stimuli and stereoscopic localization of the same second-order targets are performed with the same precision (Wilcox, L.M. & Hess, R.F. (1996) Is the site of non-linear filtering in stereopsis before or after binocular combination? Vision Research, 36, 391-399). Further, both tasks show a similar dependence on the scale of the stimulus. Since prior studies used Gaussian-enveloped stimuli, modifications of stimulus scale produced concurrent changes in edge blur. The experiments reported here assess the relative contributions of size and blur to the observed dependence on envelope scale for both monocular localization and stereoacuity. Stereoacuity for first-order targets was found to be an order of magnitude better than stereoacuity for second-order targets and monocular acuity for both first- and second-order targets. Further, while first-order stereopsis was found to depend solely on blur, second-order stereoacuity and monocular acuity were affected by both size and blur. These results suggest that while stereoacuity for first-order stimuli may be determined by a correlative process limited by early additive noise, stereoacuity for second-order stimuli and monocular acuity for non-abutting targets are more likely limited by stimulus-dependent spatial subsampling.

Depth Perception↗

PubMed2000

Structures of feline immunodeficiency virus dUTP pyrophosphatase and its nucleotide complexes in three crystal forms.

dUTP pyrophosphatase (dUTPase) cleaves the alpha-beta phosphodiester of dUTP to form pyrophosphate and dUMP, preventing incorporation of uracil into DNA and providing the substrate for thymine synthesis. Seven crystal structures of feline immunodeficiency virus (FIV) dUTPase in three crystal forms have been determined, including complexes with substrate (dUTP), product (dUMP) or inhibitor (dUDP) bound. The native enzyme has been refined at 1.40 A resolution in a hexagonal crystal form and at 2.3 A resolution in an orthorhombic crystal form. In the dUDP complex in a cubic crystal form refined at 2.5 A resolution, the C-terminal conserved P-loop motif is fully ordered. The analysis defines the roles of five sequence motifs in interaction with uracil, deoxyribose and the alpha-, beta- and gamma-phosphates. The enzyme utilizes adaptive recognition to bind the alpha- and beta-phosphates. In particular, the alpha-beta phosphodiester adopts an unfavorable eclipsed conformation in the presence of the P-loop. This conformation may be relevant to the mechanism of alpha-beta phosphodiester bond cleavage.

Animals↗

PubMed2000

Multiple antiviral activities of cyanovirin-N: blocking of human immunodeficiency virus type 1 gp120 interaction with CD4 and coreceptor and inhibition of diverse enveloped viruses.

Cyanovirin-N (CV-N) is a cyanobacterial protein with potent neutralizing activity against human immunodeficiency virus (HIV). CV-N has been shown to bind HIV type 1 (HIV-1) gp120 with high affinity; moreover, it blocks the envelope glycoprotein-mediated membrane fusion reaction associated with HIV-1 entry. However, the inhibitory mechanism(s) remains unclear. In this study, we show that CV-N blocked binding of gp120 to cell-associated CD4. Consistent with this, pretreatment of gp120 with CV-N inhibited soluble CD4 (sCD4)-dependent binding of gp120 to cell-associated CCR5. To investigate possible effects of CV-N at post-CD4 binding steps, we used an assay that measures sCD4 activation of the HIV-1 envelope glycoprotein for fusion with CCR5-expressing cells. CV-N displayed equivalently potent inhibitory effects when added before or after sCD4 activation, suggesting that CV-N also has blocking action at the level of gp120 interaction with coreceptor. This effect was shown not to be due to CV-N-induced coreceptor down-modulation after the CD4 binding step. The multiple activities against the HIV-1 envelope glycoprotein prompted us to examine other enveloped viruses. CV-N potently blocked infection by feline immunodeficiency virus, which utilizes the chemokine receptor CXCR4 as an entry receptor but is CD4 independent. CV-N also inhibited fusion and/or infection by human herpesvirus 6 and measles virus but not by vaccinia virus. Thus, CV-N has broad-spectrum antiviral activity, both for multiple steps in the HIV entry mechanism and for diverse enveloped viruses. This broad specificity has implications for potential clinical utility of CV-N.

Animals↗

PubMed2000

Alteration of substrate and inhibitor specificity of feline immunodeficiency virus protease.

Feline immunodeficiency virus (FIV) protease is structurally very similar to human immunodeficiency virus (HIV) protease but exhibits distinct substrate and inhibitor specificities. We performed mutagenesis of subsite residues of FIV protease in order to define interactions that dictate this specificity. The I37V, N55M, M56I, V59I, and Q99V mutants yielded full activity. The I37V, N55M, V59I, and Q99V mutants showed a significant increase in activity against the HIV-1 reverse transcriptase/integrase and P2/nucleocapsid junction peptides compared with wild-type (wt) FIV protease. The I37V, V59I, and Q99V mutants also showed an increase in activity against two rapidly cleaved peptides selected by cleavage of a phage display library with HIV-1 protease. Mutations at Q54K, I98P, and L101I dramatically reduced activity. Mutants containing a I35D or I57G substitution showed no activity against either FIV or HIV substrates. FIV proteases all failed to cut HIV-1 matrix/capsid, P1/P6, P6/protease, and protease/reverse transcriptase junctions, indicating that none of the substitutions were sufficient to change the specificity completely. The I37V, N55M, M56I, V59I, and Q99V mutants, compared with wt FIV protease, all showed inhibitor specificity more similar to that of HIV-1 protease. The data also suggest that FIV protease prefers a hydrophobic P2/P2' residue like Val over Asn or Glu, which are utilized by HIV-1 protease, and that S2/S2' might play a critical role in distinguishing FIV and HIV-1 protease by specificity. The findings extend our observations regarding the interactions involved in substrate binding and aid in the development of broad-based inhibitors.

Amino Acid Sequence↗

PubMed2000

Expanded host cell tropism and cytopathic properties of feline immunodeficiency virus strain PPR subsequent to passage through interleukin-2-independent T cells.

A cytopathic variant of feline immunodeficiency virus (FIV) strain PPR emerged after passage of wild-type virus on an interleukin-2-independent cell line. The virus, termed FIV-PPRglial, displayed a phenotype markedly different from the parental virus, including the ability to productively infect previously refractory cell lines, induction of large syncytia, and accelerated kinetic properties. A chimeric molecular clone, FIV-PPRchim42, containing the FIV-PPRglial envelope within the backbone of FIV-PPR, exhibited all the characteristics of the FIV-PPRglial phenotype, demonstrating that the viral envelope was responsible for the acquired traits. Subsequent molecular characterization revealed that the FIV-PPRglial envelope contained five amino acid substitutions relative to wild-type FIV-PPR. Mutagenic analyses further demonstrated that the acquired phenotype was minimally attributable to a combination of three mutations, specifically, a glutamine-to-proline change within the second constant domain of the surface protein (SU); a threonine-to-proline change within the V4 loop, also in the SU; and a premature stop codon in the cytoplasmic tail of the transmembrane protein. All three changes were required to produce the FIV-PPRglial phenotype. Cotransfection studies with mutant viruses in combination with each other and with FIV-PPR indicated that the truncated cytoplasmic tail was responsible for the induction of syncytium formation. Receptor usage analyses were pursued, and distinctions were observed between FIV-PPR and FIV-PPRglial. In vitro infections with FIV-PPR, FIV-PPRglial, and FIV-34TF10 on two adherent cell lines were ablated in the presence of SDF1alpha, the natural ligand for CXCR4. In contrast, viral infection of T cells was not limited to CXCR4 usage, and inhibition studies indicate the potential involvement of a CC chemokine receptor.

Amino Acid Sequence↗

PubMed2000

Feline immunodeficiency virus Vif localizes to the nucleus.

Monoclonal antibodies prepared against recombinant Vif derived from the 34TF10 strain of feline immunodeficiency virus (FIV) were used to assess the expression and localization of Vif in virus-infected cells. Analyses by Western blotting and by immunoprecipitation from cells infected with FIV-34TF10 revealed the presence of a single 29-kDa species specific for virus-infected cells. Confirmation of antibody specificity was also performed by specific immunoprecipitation of in vitro-transcribed and -translated recombinant Vif. Localization experiments were also performed on virus-infected cells, using different fixation procedures. Results for methanol fixation protocols similar to those reported for localization of human immunodeficiency virus (HIV) Vif showed a predominant cytoplasmic localization for FIV Vif, very similar to localization of HIV type 1 Vif and virtually identical to the localization observed for the Gag antigens of the virus. However, with milder fixation procedures that used 2% formaldehyde at 4 degrees C, FIV Vif was strongly evident in the nucleus. The localization was distinct from the nuclear localization noted with Rev and did not involve the nucleolus. Attempts to show colocalization or coprecipitation of Vif with Gag antigens were unsuccessful. In addition, Vif was not detected in purified FIV virions. The results are consistent with the notion that the primary role of Vif in virus infection initiates in the nucleus.

Amino Acid Sequence↗

PubMed1999

Feline immunodeficiency virus envelope protein (FIVgp120) causes electrophysiological alterations in rats.

Close to 20% of the patients infected with the AIDS virus develops neurological deficit; eventhough HIV does not invade neurons. Consistently with the neurological deficit, HIV(+) subjects show abnormalities in brainstem auditory and visual evoked potentials (BSAEP and VEP) and in sleep patterns. The HIV-derived glycoprotein 120 has been postulated as a neurotoxic; therefore, it may be playing a crucial role in the generation of BSAEP and VEP, as well as in sleep disturbances. To study the role of the virus-derived proteins on the development of these electrophysiological signals' alterations, we have used the feline immunodeficiency virus (FIV)-derived gp120 and evaluated the changes in these electrophysiological signals. We employed 15 adult male Sprague-Dawley rats (250-350 g), chronically implanted for evoked potential and sleep recordings. Results showed that the i.c.v. administration of FIVgp120 (5 ng/10 microliter) produces changes in the latency of both cortical auditory evoked potentials (CAEPs) and VEPs and a decrease in both REM sleep and SWS. These data support the notion that FIVgp120 is neurotoxic to the central nervous system of cats and rats and that this protein suffices to cause electrophysiological alterations. In addition, it suggests that a similar effect may be occurring in humans as a result of HIVgp120's neurotoxic effects.

Analysis of Variance↗

PubMed1999

Videotaped behavioral observations: enhancing validity and reliability.

Observing and quantifying behaviors in natural or clinical settings has long produced challenges for researchers and clinicians. Although nurses are uniquely positioned to conduct behavioral research, they may not be well acquainted with behavioral observation strategies. The author identifies several problems commonly encountered while studying behavior in natural settings and suggests methods for enhancing validity and reliability through videotaped observations. Results of a newly developed calibration procedure are discussed with implications for rater training. Nurse clinicians and researchers should find this information useful for evaluating interventions in clinics, homes, and other applied settings.

Adult↗