Biomedical subjects

Yan Luo

Publications and source records attributed to Yan Luo.

At least 55 records · Page 3Linked to original sources

PubMed2004

TIN2 binds TRF1 and TRF2 simultaneously and stabilizes the TRF2 complex on telomeres.

Human telomeres contain two related telomeric DNA-binding proteins, TRF1 and TRF2. The TRF1 complex contains the TRF1 interacting partner, TIN2, as well as PIP1 and POT1 and regulates telomere-length homeostasis. The TRF2 complex is primarily involved in telomere protection and contains the TRF2 interacting partner human (h)Rap1 as well as several factors involved in the DNA damage response. A prior report showed that conditional deletion of murine TRF1 reduced the presence of TRF2 on telomeres. Here we showed that TRF2 is also lost from human telomeres upon TRF1 depletion with small interfering RNA prompting a search for the connection between the TRF1 and TRF2 complexes. Using mass spectrometry and co-immunoprecipitation, we found that TRF1, TIN2, PIP1, and POT1 are associated with the TRF2-hRap1 complex. Gel filtration identified a TRF2 complex containing TIN2 and POT1 but not TRF1 indicating that TRF1 is not required for this interaction. Co-immunoprecipitation, Far-Western assays, and two-hybrid assays showed that TIN2, but not POT1 or PIP1, interacts directly with TRF2. Furthermore, TIN2 was found to bind TRF1 and TRF2 simultaneously, showing that TIN2 can link these telomeric proteins. This connection appeared to stabilize TRF2 on the telomeres as the treatment of cells with TIN2 small interfering RNA resulted in a decreased presence of TRF2 and hRap1 at chromosome ends. The TIN2-mediated cooperative binding of TRF1 and TRF2 to telomeres has important implications for the mechanism of telomere length regulation and protection.

Antigens, Surface↗

PubMed2004

AIP1/DAB2IP, a novel member of the Ras-GAP family, transduces TRAF2-induced ASK1-JNK activation.

Previously we have shown that ASK-interacting protein 1 (AIP1, also known as DAB2IP), a novel member of the Ras-GAP protein family, mediates TNF-induced activation of ASK1-JNK signaling pathway. However, the mechanism by which TNF signaling is coupled to AIP1 is not known. Here we show that AIP1 is localized on the plasma membrane in resting endothelial cells (EC) in a complex with TNFR1. TNF binding induces release of AIP1 from TNFR1, resulting in cytoplasmic translocation and concomitant formation of an intracellular signaling complex comprised of TRADD, RIP1, TRAF2, and AIPl. A proline-rich region (amino acids 796-807) is critical for maintaining AIP1 in a closed form, which associates with a region of TNFR1 distinct from the death domain, the site of TNFR1 association with TRADD. An AIP1 mutant with deletion of this proline-rich region constitutively binds to TRAF2 and ASK1. A PERIOD-like domain (amino acids 591-719) of AIP1 binds to the intact RING finger of TRAF2, and specifically enhances TRAF2-induced ASK1 activation. At the same time, the binding of AIP1 to TRAF2 inhibits TNF-induced IKK-NF-kappaB signaling. Taken together, our data suggest that AIP1 is a novel transducer in TNF-induced TRAF2-dependent activation of ASK1 that mediates a balance between JNK versus NF-kappaB signaling.

Adaptor Proteins, Signal Transducing↗

PubMed2004

The ground and excited states of polyenyl radicals C2n-1H2n + 1 (n = 2-13): a valence bond study.

The semiempirical valence bond (VB) method, VBDFT(s), is applied to the ground states and the covalent excited states of polyenyl radicals C2n - 1H2n + 1 (n = 2-13). The method uses a single scalable parameter with a value that carries over from the study of the covalent excited states of polyenes (W. Wu, D. Danovich, A. Shurki, S. Shaik, J. Phys. Chem. A, 2000, 104, 8744). Whenever comparison is possible, the VB excitation energies are found to be in good accord with sophisticated molecular orbital (MO)-based methods like CASPT2. The symmetry-adapted Rumer structures are used to discuss the state-symmetry and VB constitution of the ground and excited states, and the expansion to VB determinants is used to gain insight on spin density patterns. The theory helps to understand in a coherent and lucid manner the properties of polyenyl radicals, such as the makeup of the various states, their geometries and energies, and the distribution of the unpaired electrons (the neutral solitons).

Journal Article↗

PubMed2004

Pseudosubstrate peptides inhibit Akt and induce cell growth inhibition.

We have designed peptide inhibitors that potently inhibit Akt both in vitro and inside cells. These peptide inhibitors are selective for Akt versus other closely related kinases. The peptides inhibit the in vitro phosphorylation of a biotinylated Bad peptide by Akt with potency up to 100 nM. We have shown that the binding between Akt1 and these peptide inhibitors requires MgATP. Mutating the two putative Akt phosphorylation sites to Ala (nonsubstrate) in these peptides increases the inhibitory potency while mutating the sites to aspartic acid (phosphorylation mimetic) reduces the potency. When delivered into cells, these peptide inhibitors can inhibit cellular Akt activity and cell growth. Thus, these Akt-specific peptide inhibitors provide prototypes for peptide mimetic drugs as well as very useful tools to dissect cellular functions of Akt.

Adenosine Triphosphate↗

PubMed2004

[Prokaryotic expression, purification and refolding of extracellular ligand binding domains of chick Tie-2 and its immunogenicity].

OBJECTIVE: To study the prokaryotic expression of extracellular ligand binding domains of chick Tie-2, the purification and refolding conditions of the recombinant protein, and to observe its immunogenicity in mouse. METHODS: A DNA fragment encoding extracellular ligand binding domains of chick Tie-2 was obtained by PCR from a previous constructed plasmid as a template. The amplified fragment was then inserted into prokaryotic expression vector PQE30, and was expressed in E. coli XL-1 blue by adding isopropyl-beta-D-thiogalactoside(IPTG). The recombinant protein in inclusion bodies was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography under denatured conditions. Then the refolding of the purified protein was performed with gradient dialysis. The target protein was injected into mouse subcutaneously, and the antiserum of the mouse was analyzed by ELISA and Western blot analysis. RESULTS: The recombinant protein was highly expressed in E. coli XL-1 blue, and in mouse it produced the antibody which could specifically recognize the recombinant protein. CONCLUSION: The protein of extracellular ligand binding domains of chick Tie-2 can be highly expressed in prokaryotic expression system, and the expressed protein can induce immune response in mouse. These findings are very important for the further study of this protein in anti-angiogenesis and immunotherapy research.

Animals↗

PubMed2004

[Prokaryotic expression of extracellular ligand binding domains of chick tie-2 and its anti-angiogenesis effect].

OBJECTIVE: To study the prokaryotic expression of extracellular ligand binding domains of chick tie-2, the purification, refolding conditions of the recombinant protein, and its anti-angiogeneic effect. METHODS: A DNA fragment encoding extracellular ligand binding domains of chick tie-2 was obtained by PCR amplification using a previous constructed plasmid as a template. The amplified fragment was then inserted into prokaryotic expression vector pQE30, and was expressed in E.Coli XL-1 blue by adding isopropyl-beta-D-thiogalactoside(IPTG). The recombinant protein in inclusion bodies was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography under denatured conditions. Then the refolding of the purified protein was performed with gradient dialysis. The target protein was injected s.c. into mouse, and the antibody was detected by ELISA and Western blot analysis. The antibody was purified from the antiserum and then incubated with human umbilical endothelial vein cell (HUEVC) to find its anti-angiogenesis in vitro by using propidium iodide(PI) dying through FACS. Alginate encapsulated tumor cell assays were performed and micro-vessel density was determined by counting per high power field in the sections stained with an antibody reactive to CD31 to test its inhibition of angiogenesis. RESULTS: The recombinant protein was highly expressed in E.Coli XL-1 blue, and the antibody produced in mouse could specifically recognize the recombinant protein. The purified antibody could induce apoptosis of HUEVC in vitro. The anti-angiogenic effect of the antibody could also be found in alginate-encapsulate tumor cell assay and by counting micro-vessel density. CONCLUSION: The protein of extracellular ligand binding domains of chick tie-2 can be expressed at high level in the prokaryotic expression system, and the expressed protein can induce immune response in mouse. Furthermore, the antibody can induce the anti-angiogenic effect.

Angiogenesis Inhibitors↗

PubMed2004

Oral health status of institutionalised elderly in Hong Kong.

OBJECTIVES: To describe the oral health status of institutionalized elderly in Hong Kong. METHODS: Older adults in 56 elderly homes were clinically examined in the institution by one of two calibrated dentists using standard methods recommended by the World Health Organization. Information on their perceived oral health status and behaviour was obtained from an interview. RESULTS: A total of 3153 elderly aged 65 years or above (mean = 79.8) were examined. Around 20% of them were edentulous. The mean DMFT score was 23.0 (DT = 2.6; MT = 20.1; FT = 0.3). The percentage of dentate subjects with healthy gingivae, bleeding on probing, calculus, shallow pockets, and deep pockets (according to the highest CPI score) were 1, 2, 41, 37 and 20 respectively. Two-thirds of the elderly reported having difficulties chewing, and over half had not visited a dentist for over 5 years. CONCLUSION: The oral health status of institutionalised elderly in Hong Kong is poor and needs to be improved.

Aged↗

PubMed2004

[Inhibition of tumor growth and metastasis via local administration of recombinant human endostatin adenovirus].

OBJECTIVE: The growth and metastasis of solid tumors are dependent on angiogenesis. Endostatin, the C-terminal proteolytic fragment of collagen XVIII, is a potent endogenous angiogenesis inhibitor. The authors designed a topical antiangiogenic gene therapy with recombinant human endostatin adenovirus (Ad-hEndo) and assessed its effects on the inhibition of angiogenesis in vitro, and tumor growth and metastasis in vivo. METHODS: Malignant cells (A549) were infected with Ad-hEndo. The expression of recombinant protein and the inhibition of cultured human umbilical vein endothelial were investigated. Immunodeficient A549 nude mice were treated with intratumoral injection of Ad-hEndo, the empty vector Ad-control or saline (NS). The dose-response, side effects, and serum concentration of endostatin were observed. RESULTS: Recombinant endostatin protein was detected in the infected tumor cells with different MOI Ad-hEndo and its inhibitory effect on endothelial cells growth was shown. In animal study, the volume of tumor and the number of pulmonary metastatic lesions in the Ad-hEndo treatment group were significantly smaller than those in the control groups (P<0.05). CONCLUSION: The present findings provide evidence of the anti-tumor effects of the endostatin and may be important for the further use of it in topical antiangiogenic gene therapy of cancer.

Adenoviridae↗

PubMed2003

S phase activation of the histone H2B promoter by OCA-S, a coactivator complex that contains GAPDH as a key component.

We have isolated and functionally characterized a multicomponent Oct-1 coactivator, OCA-S which is essential for S phase-dependent histone H2B transcription. The p38 component of OCA-S binds directly to Oct-1, exhibits potent transactivation potential, is selectively recruited to the H2B promoter in S phase, and is essential for S phase-specific H2B transcription in vivo and in vitro. Surprisingly, p38 represents a nuclear form of glyceraldehyde-3-phosphate dehydrogenase, and binding to Oct-1, as well as OCA-S function, is stimulated by NAD(+) but inhibited by NADH. OCA-S also interacts with NPAT, a cyclin E/cdk2 substrate that is broadly involved in histone gene transcription. These studies thus link the H2B transcriptional machinery to cell cycle regulators, and possibly to cellular metabolic state (redox status), and set the stage for studies of the underlying mechanisms and the basis for coordinated histone gene expression and coupling to DNA replication.

Amino Acid Sequence↗

PubMed2003

Patient-centred outcome measures in oral surgery: validity and sensitivity.

The performances of patient-centred outcome measures after oral surgery were evaluated in a prospective cohort study of 100 patients who had third molar extractions. Participants self-completed a questionnaire incorporating a general health (12-item short form health survey: SF-12) and two specific outcome scales to oral health (oral health impact profile: OHIP-14, and The United Kingdom Oral Health related Quality of Life measure: OHQoL-UK) preoperatively, daily during the immediate postoperative period, and at the review appointment (7-days later). Ninety-seven completed the study. History of 'taking time off' work/study because of pericoronitis during the past year was significantly associated with preoperative OHIP-14 and OHQoL-UK scores. There were significant differences in SF-12, OHIP-14, OHQoL-UK scores during the immediate postoperative period compared with preoperatively, when postoperative symptoms were prevalent. At the review appointment, OHIP-14 and OHQoL-UKscores were associated with clinical findings. The measures were valid and sensate in relation to oral surgery. However, those specific to oral health were more discerning than the general scale.

Adult↗

PubMed2003

Can third molar surgery improve quality of life? A 6-month cohort study.

PURPOSE: In this study, we evaluated patients' perceptions of changes in oral health-related quality of life (OHQOL) over a 6-month period after third molar surgery. PATIENTS AND METHODS: One hundred patients participated in this prospective study. Two specific OHQOL measures, the 14-item Oral Health Impact Profile (OHIP-14) and the 16-item UK Oral Health-Related Quality of Life measure (QHQoL-UK), were administered to the study group before surgery. Standardized surgical and analgesic protocols were used. Patients kept a diary of changes in life quality each postoperative day (POD) for 7 days and were contacted at 1, 3, and 6 months after surgery. RESULTS: Both measures identified a significant deterioration in quality of life in the immediate postoperative period (P <.01). However, there was an improvement in OHQOL compared with preoperative status at 1 (P <.05), 3 (P <.05), and 6 (P <.01) months after surgery. Patients perceived physical, social, and psychologic changes in life quality after surgery. Previous pericoronitis was associated with changes in quality of life (P <.05). CONCLUSION: The study concludes that third molar surgery is associated with changes in OHQOL. This has implications for understanding the value of third molar surgery from patients' perspectives and in assessing health gain.

Activities of Daily Living↗

PubMed2003

Incidence and treatment of hepatic artery complications after orthotopic liver transplantation.

AIM: To investigate the incidence and treatment of hepatic artery complications after orthotopic liver transplantation. METHODS: From February 1999 to May 2002, orthotopic liver transplantations (OLT) were performed in 72 patients with end-stage liver diseases with an average age of 40.2 +/- 13.6 years (ranged from 11 to 68 years), 56 were males and 16 females. The preoperative evaluation for the 72 patients was performed using duplex sonography, abdominal CT scan, and angiography of the hepatic artery. All donor grafts were perfused and preserved in University of Wisconsin solution at 4 degrees. OLT was performed with standard techniques with or without a veno-venous bypass. Reconstructions of hepatic artery were performed between the branch patches of gastroduodenal/hepatic or splenic/common hepatic artery confluence of the donors and recipients, and an end-to-end anastomosis between other arterial vessels of the donors and recipients was done. Arterial anastomosis was performed with interrupted 7-0/8-0 monofilament polypropylene suture under 3.5 x loupe magnification. Diagnosis of the complications of hepatic artery after OLT was based on the clinical presentations, ultrasound findings and arterial angiography. All patients were followed up regularly for duplex ultrasound scan after discharge. RESULTS: The overall incidence of arterial complications in 72 patients after OLTs was 1.4% (1/72). One 3 cm pseudoaneurysm at the side of anastomotic site of hepatic artery was found by urgent arteriogram due to hemoperitoneum secondary to bile leakage after OLT. Subsequently the pseudoaneurysm was successfully embolized and the blood flow toward the donor liver in hepatic artery remained. The overall postoperative 30-day mortality rate was 8.33%. The one-year survival rate was 83.72% in 50 patients with benign diseases and was 71.43% in 22 patients with malignant diseases following OLT. No death associated with complications of hepatic artery occurred. CONCLUSION: Careful preoperative evaluations and intraoperative microsurgical technique for hepatic artery reconstructions are the keys in prevention of hepatic artery complications after OLT.

Adolescent↗

PubMed2003

Adeno-associated virus mediated LacZ gene transfect to cultured human iris pigment epithelium cells.

PURPOSE: To study the feasibility of adeno-associated virus mediated gene transfection to cultured human iris pigment epithelium (IPE) cells in vitro. METHODS: Recombinant replication deficient adeno-associated viruses (AAV) expressing LacZ gene were produced without helper virus. The LacZ gene was transduced into cultured human IPE cells. RESULTS: Cultured human IPE cells stained positively anticytokeratin, The titer of rAAV-LacZ was 2.1 x 10(8) virus particles/ml, 42% cultured human IPE cells expressed beta-galactosidase 7 days after transfection and 67% after 14 days. CONCLUSIONS: Recombined AAV produced without helper virus can transfer a foreign gene into human IPE cells with high efficiency in vitro.

Cells, Cultured↗

PubMed2003

[In vitro differentiation of embryonic stem cells mediated by the supernatant of retinal cells and retinoid acid].

PURPOSE: To analyse the differentiation features of embryonic stem cells which were induced by the supernatant of retinal cells and retinoid acid (RA). METHODS: Embryonic bodies (EB) were gained when embryonic stem cells (ESC) were propagated 1 generation after anabiosis. 3.5d EB without digestion were induced at four conditions sorted as condition A, B, C and D. A contains only retinoid acid (RA) in medium; B contains RA and the supernatant from retinal neurons and glia of mouse; C contains RA and the supernatant from retinal pigment epithelium of human; D contains RA and the supernatant of fetal retinal glia. The changes of embryonic stem cells were observed and the immune cytochemistry was adopted to detect the nestin, glial fibrillary acid protein (GFAP), cytokeratin, microtubule-associated protein 2 (MAP-2) and rhodopsin in induced cells. RESULTS: (1) The changes of differentiation features of EB: The characteristics for early stage were almost same at the four conditions. The cells with various shapes could be seen around EB. EB were dispersed 3 weeks later. Additionally, the cells which were no more abounded and not clear boundary appeared at condition A, the round cells with high transparency at condition B, EB and the cells around EB with apparent pigment at condition C, the large cells with simplex shape and glia-like appearance at condition D. (2) Immune cytochemistry showed that the most induced cells were MAP-2 (+) cells, no nestin (+) cells appeared at the four conditions. There were no GFAP, cytokeratin or rhodopsin positive cells in condition A; However, GFAP (+), cytokeratin (+) and rhodopsin(+) cells could be seen at condition B, cytokeratin positive cells at condition C, GFAP positive cells at condition D. CONCLUSION: RA can induce embryonic stem cells into neurons in second induction. Several kinds of the supernatant from retinal cells have an effect on induction of ESC. ESC can be differentiated into cells associated with the origin of the supernatant.

Animals↗

PubMed2003

[An experimental study of inhibiting effect of decoy oligodeoxynucleotides on the gene expression of collagen in NIH3T3 cell].

OBJECTIVE: To investigate the effect of activator protein-1 (AP-1) decoy-oligodeoxynucleotides (Decoy-ODNs) on the expression of fibroblast alpha2 type I collagen, so as to explore the gene therapy of pathologic scar. METHODS: Decoy-ODNs targeting AP-1 were designed and synthesized. NIH3T3 cells were transfected by cationic liposomes. The distribution of Decoy-ODNs in the cells was investigated. The inhibiting effects of Decoy-ODNs on AP-1 were determined by electrophoretic mobility shift assay (EMSA). And the effects of Decoy-ODNs on the collagen synthesis in the cells were analyzed by RT-PCR. RESULTS: AP-1 Decoy-ODNs could competitively inhibit the AP-1 in vitro activity. Cationic liposomes could play roles by effectively transfecting Decoy-ODNs into the plasma and nucleus. The mRNA expression of fibroblast alpha2 type I collagen decreased evidently after 24 hours of Decoy-ODNs action. CONCLUSION: Decoy-ODNs could inhibit the mRNA expression of fibroblast alpha2 type I collagen by antagonizing AP-1.

Animals↗

PubMed2003

[A study on the effects of rAd-gfp-bcl-X(L) on retinal degeneration in mice].

OBJECTIVE: To observe the therapeutic effect of rAd-gfp-bcl-X(L) on retinal degeneration (RD) in mice. METHODS: rAd-gfp-bcl-X(L) granules were injected into the subretinal space of right eyes of RD mice in Group A (postnatal days 10, p10) and Group B (postnatal days 22, p22) (n = 20). The morphological changes were observed by light microscopy and transmission electron microscopy at 15 - 30 days post-injection. Frozen section was used to observe the expression of GFP. The level of bcl-X(L) mRNA and bcl-X(L) protein were assayed by RT-PCR and immunohistochemistry assay. RESULTS: In frozen section, GFP was observed in the treated eyes under fluorescence microscopy, indicating that rAd-gfp-bcl-X(L) had transfected into the retinal cells successfully and that bcl-X(L) was expressed. In Group A, an increased expression of bcl-X(L) mRNA and Bcl-X(L) protein were observed in the treated eyes. The outer nuclear layer of the treated eyes in Group A was thicker than that of the control eyes at 30 days post-injection. The inner segment of treated eyes in Group A was healthier than that of control eyes, as indicated by transmission electron microscopy. No difference was found in the thickness of retina between treated eyes and control eyes in Group B. CONCLUSION: The bcl-X(L) can be expressed and have an anti-apoptosis effect when the rAd-gfp-bcl-X(L) particles are injected into the subretinal space of RD mice's eyes at an early stage. But the expression and protective effects were not observed in RD mice at a later stage. The therapy of rAd-gfp-bcl-X(L) for retinal degenerative diseases still needs further study.

Adenoviridae↗

PubMed2003

Increased expression of intercellular adhesion molecule-1, vascular cellular adhesion molecule-1 and leukocyte common antigen in diabetic rat retina.

PURPOSE: To understand the expression and distribution of intercellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1) and CD45 (Leukocyte Common Antigen) in the control nondiabetic and various courses of diabetic rats retina. To explore the role of adhesion molecules (AMs) and the adhesion of leukocytes to vascular endothelial cells via AMs in diabetic retinopathy (DR). METHODS: Sixty healthy adult male Wistar rats were randomly divided into diabetic groups (induced by Streptozotocin, STZ) and normal control groups. Rats in these two groups were further randomly divided into 3, 7, 14, 30, 90 and 180 days-group, including 5 rats respectively. The immunohistochemical studies of ICAM-1, VCAM-1 and CD45 were carried out in the retinal digest preparations or retinal paraffin sections, and the results were analyzed qualitatively, semi-quantitatively. RESULTS: No positive reaction of VCAM-1 was found, and weak reactions of ICAM-1, CD45 were found in nondiabetic rats retina. The difference of 6 control groups had no statistical significance (P > 0.05). The increased ICAM-1 and CD45 staining pattern were detectable 3 days after diabetes induction, and a few VCAM-1 positive cells were observed in the retinal blood capillaries. The difference of diabetes and control is significant (P < 0.05). Following the course, the expressions of ICAM-1, VCAM-1 and CD45 were increasingly enhanced, reaching a peak at the 14th day. CONCLUSION: Increased expression of ICAM-1, VCAM-1 and leukocytes adhering and stacking in retinal capillaries are the very early events in DR. Coherence of expression and distribution of the three further accounts for it is the key point for the onset of DR that AMs mediates leukocytes adhesion and endothelial cell injury.

Animals↗

PubMed2003

[Study of bcl-X(L) protection on retinal photoreceptors from apoptosis in vitro].

OBJECTIVE: To assess the effect of bcl-X(L), an anti-apoptotic gene, on glutamate-induced apoptosis in cultured retinal photoreceptors. METHODS: Glutamate-induced apoptosis in cultured retinal photoreceptors was established. The experiment was divided into three groups: control, glutamate treatment and rAd-gfp-bcl-X(L) + glutamate transfection group, the protection of bcl-X(L) on retinal photoreceptors from apoptosis was evaluated. 6.5 x 10(12) pfu/L rAd-gfp-bcl-X(L) were transfected into retinal photoreceptors in the rAd-gfp-bcl-X(L) + glutamate group 48 h before Glutamate-induced apoptosis was established. The positive photoreceptors with green fluorescence were identified under fluorescence microscopy. The expression of Bcl-X(2) protein in rAd-gfp-bcl-X(L) transfected and non-transfected neurons were assessed by immunohistochemistry assay. DNA fragment was detected by agarose gel electrophoresis. Nuclei were revealed by hoechst33258 staining. RESULTS: rAd-mediated gene delivery can transfect retinal photoreceptors effectively. Increased expression of Bcl-X(L) protein was demonstrated in the rAd-transfecting neurons. Transfection of bcl-X(L) significantly decreased the number of apoptotic retinal photoreceptors in vitro. CONCLUSION: Transfection of bcl-X(L) protects cultured retinal photoreceptors from apoptosis induced by glutamate. Transfection the gene of bcl-X(L) may be a potential gene therapy for retinal degenerative diseases.

Animals↗