Biomedical subjects

L Chen

Publications and source records attributed to L Chen.

At least 685 records · Page 38Linked to original sources

PubMed1998

Analysis of homogeneity of treatment effect in adaptive multicenter clinical trials.

In this paper, an exact test for analyzing the homogeneity of treatment effect in adaptive multicenter clinical trials is proposed. Extensive simulation studies are performed to investigate the large sample behavior of a commonly used test statistic for testing homogeneity of treatment effect. When the sample size in each center is large relative to the number of centers, the asymptotic null distribution of the test statistic is reasonable. On the other hand, when the data are relatively sparse, the proposed exact test should be used to incorporate the adaptive nature of the design.

Algorithms↗

PubMed1998

Comparison of cardiac troponin I, creatine kinase-MB, and myoglobin for detection of acute ischemic myocardial injury in a swine model.

To study the comparative value of the levels of cardiac troponin I (cTnI), creatine kinase-MB isoenzyme (CK-MB), and myoglobin in the detection of acute ischemic myocardial injury, we serially measured plasma concentrations of these cardiac proteins in 12 pigs with myocardial ischemia subtending severe coronary artery stenoses and in 5 pigs with a sham operation performed, but without coronary artery stenosis. In the stenosis group, flow in the left anterior descending (LAD) artery was reduced by 36% and maintained for 24 hours (n = 3), 7 days (n = 6), or 4 weeks (n = 3). Flow in the coronary artery was measured by a flowmeter, and regional left ventricular dysfunction was monitored by echocardiography. Myocardial infarction was identified with triphenyltetrazolium chloride staining. All pigs with stenosis of the LAD had significant ultrastructural abnormalities consisting of loss of myofibrils and an increase in mitochondria and glycogen deposition. Cardiac proteins were released in all pigs with stenosis of the LAD artery during the development of myocardial ischemia; the levels of cTnI, CK-MB, and myoglobin increased significantly relative to the baseline. The sensitivity and specificity for cTnI were higher than for CK-MB or myoglobin. Results of this study show that cTnI is the better marker for the detection of acute ischemic myocardial injury. Increased levels of cTnI can be found in reversible and irreversible myocardial ischemic injury in this model.

Acute Disease↗

PubMed1998

Comparative investigation on the mutagenicities of organophosphate, phthalimide, pyrethroid and carbamate insecticides by the Ames and lactam tests.

The Salmonella lactam test is a newly developed method for detecting genotoxins. This technique is based on the ability of DNA damaging agents to reverse expression of the beta-lactamase gene, an important gene that enables microbes to resist beta-lactam antibiotics. A construct p-SELECT Control DNA plasmid containing a beta-lactamase gene site was constructed in many mutant forms, including point and frameshift mutants. These mutant constructs were introduced into Salmonella tester strains whose mutagenicity is based on their ability to reverse expression of the beta-lactamase gene. Fourteen pesticides were evaluated for genotoxicity using our newly developed Salmonella typhimurium strains JK947 and JK3, which are useful for detecting base substitution mutations. Six pesticides, namely allethrin, captan, folpet, monocrotophos, acephate and carbofuran, proved highly mutagenic in strain JK947, while the first four pesticides were more weakly mutagenic in strain JK3. In comparison, results from the Ames test show strain JK947 to be more sensitive to these pesticides than strains TA100 and TA1535. Strains TA98 and JK1 proved insensitive to allethrin, captan, folpet, acephate, carbofuran and monocrotophos. Among the many advantages of the lactam test are: large numbers of cells can treated and the test is operationally simple and inexpensive; revertant colonies form faster in the lactam test (16 h) than in the Ames test (48 h); the lactam test can detect mutagens present in biological specimens contaminated by histidine and biotin, samples that may give false positive results in the Ames test.

Ampicillin Resistance↗

PubMed1998

High level expression and secretion of Fc-X fusion proteins in mammalian cells.

We have developed a general expression system that enhances the production and secretion of proteins in mammalian cells. The protein of interest is expressed as a fusion to a signal peptide and the Fc fragment of immunoglobulin as the N-terminal fusion partner, which can direct the cellular processes into expressing and secreting high levels of many different types of proteins. These include secretory proteins, enzymes and soluble domains of membrane proteins, as well as nuclear and regulatory proteins. Typical expression levels of these proteins from stable cell lines ranged from several to 100 microg/ml in conditioned media. The Fc domain helps to solubilize hydrophobic proteins and provides a handle for easy detection and purification of the fusion proteins; and it can be cleaved off by treatment with protease if desired.

Animals↗

PubMed1998

The ovarian blood follicle barrier is both charge- and size-selective in mice.

This report characterizes the permeability and selectivity properties of the ovarian blood-follicle barrier. Proteins of similar size but opposite net charge possess strikingly different permeabilities with respect to this barrier. Inter-alpha-inhibitor (I alpha I, 220 kDa, pI approximately 6.2) is excluded from the follicle until an ovulatory stimulus, whereas immunoglobulin G (IgG, 155 kDa, pI approximately 6.5-7.0) passes into the follicle without an ovulatory stimulus. However, cationization of I alpha I results in its influx into the follicle in the absence of an ovulatory signal. Conversely, anionization of IgG results in its exclusion from the follicle unless an ovulatory stimulus (hCG administration) is provided. Molecular size also plays a role in blood-follicle barrier selectivity. For example, cationization of alpha 2-macroglobulin (pI approximately 8.5; 700 kDa) fails to facilitate its entry into unstimulated follicles. Conversely, negatively charged BSA (pl approximately 4.5; 66 kDa) passes freely into unstimulated follicles. These studies support the hypothesis that the blood-follicle barrier is size-selective but that charge sign and density play a role in the permeability of this barrier to proteins within an intermediate size range.

Alpha-Globulins↗

PubMed1998

Longitudinal assessment of valvular heart disease by echocardiography.

Over the past few years, important publications have addressed the issue of longitudinal assessment of valvular heart disease. The American College of Cardiology and the American Heart Association jointly published guidelines on echocardiography. Aortic stenosis continues to be well studied, with papers characterizing the rates of progression of mean and peak gradients, as well as aortic valve area changes. Doppler assessment of the progression of aortic regurgitation has been shown in larger studies than have been performed in the past. Based on an important study in patients with chronic aortic regurgitation, the optimal timing of surgery in asymptomatic mild to moderate aortic regurgitation may be predicted by indices of left ventricular function and left ventricular stress. Finally, mitral stenosis and mitral regurgitation studies evaluated rates of progression and also optimal timing for surgery.

Cardiac Surgical Procedures↗

PubMed1998

Right ventricular overload causes the decrease in cardiac output after nitric oxide synthesis inhibition in endotoxemia.

OBJECTIVE: To determine whether the decrease in cardiac output after nitric oxide synthase inhibition in endotoxemia is due to increased left ventricular afterload or right ventricular afterload. DESIGN: Prospective, randomized, unblinded study. SETTING: Research laboratory at an academic, university medical center. SUBJECTS: Nonanesthetized, sedated, mechanically ventilated pigs. INTERVENTIONS: Pigs were infused with 250 microg/kg of endotoxin over 30 mins. Normal saline was infused to maintain pulmonary artery occlusion pressure (PAOP) at a value not exceeding 1.5 times the baseline value. Left ventricular dimensions and function were studied using echocardiography. Right ventricular volumes and ejection fraction were determined via a rapid thermistor pulmonary artery catheter. We also measured mean arterial pressure (MAP), cardiac output, pulmonary arterial pressure, and calculated pulmonary and systemic resistances. Gastric tonometry was used as an index of gastric mucosal oxygenation and peripheral oxygenation. When MAP had decreased to < or =60 mm Hg or had decreased 30 mm Hg from baseline, nine animals received NG-nitro-L-arginine methyl ester (L-NAME) at 15 mg/kg to restore MAP to baseline. A second group of animals (n = 6) continued to receive normal saline, ensuring that PAOP did not exceed 1.5 times its baseline value. A third group of pigs (n = 5) did not receive endotoxin and served as the time control. In this group, a balloon was used to occlude the descending thoracic aorta and to increase MAP by approximately the same amount as in the L-NAME group. MEASUREMENTS AND MAIN RESULTS: Endotoxin caused an increase in pulmonary arterial pressure and right ventricular volumes, and a decrease in gastric mucosal pH. Cardiac output was maintained in the animals receiving the saline infusion. By 2 hrs, pulmonary arterial pressure had decreased but was still notably higher than baseline. However, by this time, MAP had decreased to < or =60 mm Hg. L-NAME administration restored MAP to its baseline value but resulted in worsening pulmonary hypertension, increased right ventricular volumes, and decreased cardiac output, compared with the saline group. Three animals that received L-NAME died of right ventricular failure. We did not observe any evidence of left ventricular dysfunction with increased left ventricular afterload. Moreover, the restoration of MAP with L-NAME infusion did not correct gastric mucosal acidosis. No changes were noted in the time-control group. Occlusion of the thoracic aorta increased MAP but did not change cardiac output. This finding demonstrates that increases in left ventricular afterload of the magnitude seen with the infusion of L-NAME do not lead to decreases in cardiac output. CONCLUSION: The decrease in cardiac output after nitric oxide synthase inhibition in endotoxemia is due to increased right ventricular afterload and not to left ventricular afterload.

Animals↗

PubMed1998

Melagatran, an oral active-site inhibitor of thrombin, prevents or delays formation of electrically induced occlusive thrombus in the canine coronary artery.

Intravenous administration of thrombin inhibitors, such as hirudin, has been shown to decrease the frequency of coronary artery reocclusion after thrombolysis. However, recent findings in large clinical trials in patients with unstable angina and myocardial infarction have failed to demonstrate a sustained antithrombotic effect after cessation of drug treatment. These findings indicate a need for a prolonged antithrombotic regimen, preferably an orally active thrombin inhibitor. To test the hypothesis that a regimen consisting of oral thrombin inhibitor will delay or prevent the formation of occlusive clot, anesthetized dogs were given saline (n = 9) or a single dose of a novel active site low-molecular-weight thrombin inhibitor melagatran by nasogastric tube (1.5 mg/kg, n = 6; 2.5 mg/kg, n = 6), and 15 min later, a potent thrombogenic stimulus in the form of anodal current (100 microA) was applied to the intimal surface of the narrowed left anterior descending coronary artery (LAD). All saline-treated dogs developed stable thrombus, indicated by zero flow at 34 +/- 7 min after initiation of direct current. On the other hand, one of the six dogs given high-dose melagatran did not develop thrombotic occlusion of the LAD during the entire 4 h of observation. Mean time to occlusive thrombus formation in 11 other dogs was prolonged 4-5 times as compared with that in the saline-treated dogs (p < 0.001). Spontaneous thrombolysis was observed in three of 11 dogs after initial clot formation. Overall, the coronary artery was patent for 68% (low dose) and 75% (high dose) of the observation period in melagatran-treated dogs (vs. 14% of observation period in saline-treated dogs). Peak plasma concentration was 0.87 +/- 0.22 microM in dogs given low-dose and 1.38 +/- 0.30 microM in dogs given high-dose melagatran. The activated partial thromboplastin time (aPTT) increased 1.5-fold at peak plasma concentration of melagatran. These observations imply (a) thrombin generation plays a critical role in thrombus formation in narrowed coronary arteries, (b) oral melagatran prevents or delays thrombus formation, whereas the aPTT is only modestly prolonged, and (c) the thrombus formed in the presence of melagatran is prone to spontaneous lysis in this canine model of coronary thrombosis.

Administration, Oral↗

PubMed1998

Femoral nerve transfer for treatment of brachial plexus root avulsion.

Femoral nerve transfer to the muscular branches of the thenar and hypothenar muscles was performed to determine its protective effect on the hand intrinsic muscles. Seven cases of brachial plexus root avulsion treated from May of 1989 to October of 1991 were involved. The femoral nerve transfer to the muscular branches of the thenar and hypothenar muscles was done at the same stage of multiple neurotization. The muscular branches derived from the femoral nerve were isolated and coapted with the thenar muscle branch of the median nerve and the deep branch of the ulnar nerve. A groin flap was harvested simultaneously to form a skin-tube pedicle that covered the nerve bridge. At the second stage, when regeneration of the median and ulnar nerves was found to reach as far as the level of the wrist, the femoral nerve was divided and the muscular branches of the thenar and hypothenar muscles were anastomosed with the regenerated median and ulnar nerves. All the cases were followed up for more than 6 years. Six months after femoral nerve transfer, muscle power of the interosseous muscles and adductor pollicis recovered to MRC3, whereas that of the abductor pollicis brevis recovered to MRC1 to 2. Five cases underwent second-stage transfer. Four to five years of follow-up revealed that the muscle power of the interosseous muscles and adductor pollicis was MRC2 in one case, MRC1 in three cases, and MRC0 in one case. As for the donor area, muscle power of the quadriceps femoris reduced to M3 to 4 within 1 month after femoral nerve transfer and recovered to normal at 3 months. In conclusion, femoral nerve transfer to the muscular branches of the thenar and hypothenar muscles has some protective effect on the hand intrinsic muscles. The outcome of the second stage, however, is not satisfactory.

Adolescent↗

PubMed1998

Effect of Helicobacter pylori eradication on antral somatostatin cell density in humans.

OBJECTIVE: As Helicobacter pylori infection is associated with an elevation in plasma gastrin with normal antral gastrin cell counts, an abnormality in antral somatostatin cells may be associated with the infection. We evaluated the effect of eradication of H. pylori on antral somatostatin cell density in the light of antral gastrin cell density and plasma gastrin levels. DESIGN: Prospective study. METHODS: Of 25 dyspeptic patients with H. pylori infection, nine had H. pylori successfully eradicated and the rest remained infected. Antral biopsies were immunostained for somatostatin cells and plasma gastrin measured before and 4 weeks after H. pylori eradication therapy. Ten other dyspeptic patients without H. pylori infection had their somatostatin cell density evaluated as controls. RESULTS: Somatostatin cell density in the patients without H. pylori infection at the outset was significantly higher than that in the patients with H. pylori infection at the outset (median 57 [18-83] vs. 37 [6-80] cells/mm) respectively (P <0.05). Somatostatin cell density increased after H. pylori eradication (before treatment, median 50 [15-72]; after treatment 71 [39-107] cells/mm) (P < 0.05) but was unchanged with persistent H. pylori infection. Plasma gastrin decreased after H. pylori eradication (before treatment, median 70 [45-100]; after treatment 30 [10-100] ng/l) (P < 0.05) but was unchanged with persistent H. pylori infection. CONCLUSIONS: Following eradication of H. pylori, there is an increase in somatostatin cell density with a fall in plasma gastrin. This supports the theory that H. pylori infection results in a decrease in somatostatin cell density and, as the latter is an inhibitor of gastrin cells, this results in an increased plasma gastrin.

Adult↗

PubMed1998

Complete genomic sequence and analysis of the prion protein gene region from three mammalian species.

The prion protein (PrP), first identified in scrapie-infected rodents, is encoded by a single exon of a single-copy chromosomal gene. In addition to the protein-coding exon, PrP genes in mammals contain one or two 5'-noncoding exons. To learn more about the genomic organization of regions surrounding the PrP exons, we sequenced 10(5) bp of DNA from clones containing human, sheep, and mouse PrP genes isolated in cosmids or lambda phage. Our findings are as follows: (1) Although the human PrP transcript does not include the untranslated exon 2 found in its mouse and sheep counterparts, the large intron of the human PrP gene contains an exon 2-like sequence flanked by consensus splice acceptor and donor sites. (2) The mouse Prnpa but not the Prnpb allele found in 44 inbred lines contains a 6593 nucleotide retroviral genome inserted into the anticoding strand of intron 2. This intracisternal A-particle element is flanked by duplications of an AAGGCT nucleotide motif. (3) We found that the PrP gene regions contain from 40% to 57% genome-wide repetitive elements that independently increased the size of the locus in all three species by numerous mutations. The unusually long sheep PrP 3'-untranslated region contains a "fossil" 1.2-kb mariner transposable element. (4) We identified sequences in noncoding DNA that are conserved between the three species and may represent biologically functional sites.

Amino Acid Sequence↗

PubMed1998

Xa21D encodes a receptor-like molecule with a leucine-rich repeat domain that determines race-specific recognition and is subject to adaptive evolution.

The rice Xa21 gene confers resistance to Xanthomonas oryzae pv oryzae in a race-specific manner. Analysis of the inheritance patterns and resistance spectra of transgenic plants carrying six Xa21 gene family members indicated that one member, designated Xa21D, displayed a resistance spectrum identical to that observed for Xa21 but conferred only partial resistance. Xa21D encodes a receptor-like protein carrying leucine-rich repeat (LRR) motifs in the presumed extracellular domain. The Xa21D transcript terminates shortly after the stop codon introduced by the retrotransposon Retrofit. Comparison of nucleotide substitutions in the LRR coding regions of Xa21 and Xa21D provided evidence of adaptive selection. Both functional and evolutionary evidence indicates that the Xa21D LRR domain controls race-specific pathogen recognition.

Adaptation, Physiological↗

PubMed1998

Ex vivo expansion of human hematopoietic stem cells: implications for the modern blood bank.

Pluripotent hematopoietic stem cells (PHSC) are rare cells within the marrow that are capable of self-renewal and differentiation into multiple hematopoietic lineages. Following myeloablative chemotherapy and radiation therapy and marrow transplantation, hematological reconstitution occurs after a period of 2-3 weeks. Recently, a number of laboratories have shown that both early and delayed phases of engraftment are mediated by PHSC within a graft and that engraftment can be accelerated by transplanting greater numbers of PHSC. Increasing efforts have been directed, therefore, towards developing methods to expand PHSC ex vivo. In this report, we describe an endothelial cell-based culture system to which exogenous cytokines are added which appears to permit the ex vivo expansion of PHSC. Refinement of these technologies will potentially have a major impact on the ability of blood banks to improve the quality of hematopoietic stem cell grafts.

Adult↗

PubMed1998

Cloning and expression of the inositol monophosphatase gene from Methanococcus jannaschii and characterization of the enzyme.

Inositol monophosphatase (EC 3.1.3.25) plays a pivotal role in the biosynthesis of di-myo-inositol-1,1'-phosphate, an osmolyte found in hyperthermophilic archaeal. Given the sequence homology between the MJ109 gene product of Methanococcus jannaschii and human inositol monophosphatase, the MJ109 gene was cloned and expressed in Escherichia coli and examined for inositol monophosphatase activity. The purified MJ109 gene product showed inositol monophosphatase activity with kinetic parameters (K(m) = 0.091 +/- 0.016 mM; Vmax = 9.3 +/- 0.45 mumol of Pi min-1 mg of protein-1) comparable to those of mammalian and E. coli enzymes. Its substrate specificity, Mg2+ requirement, Li+ inhibition, subunit association (dimerization), and heat stability were studied and compared to those of other inositol monophosphatases. The lack of inhibition by low concentrations of Li+ and high concentrations of Mg2+ and the high rates of hydrolysis of glucose-1-phosphate and p-nitrophenylphosphate are the most pronounced differences between the archaeal inositol monophosphatase and those from other sources. The possible causes of these kinetic differences are discussed, based on the active site sequence alignment between M. jannaschii and human inositol monophosphatase and the crystal structure of the mammalian enzyme.

Amino Acid Sequence↗

PubMed1998

Biosynthesis of Di-myo-inositol-1,1'-phosphate, a novel osmolyte in hyperthermophilic archaea.

Biosynthesis of di-myo-inositol-1,1'-phosphate (DIP) is proposed to occur with myo-inositol and myo-inositol 1-phosphate (I-1-P) used as precursors. Activation of the I-1-P with CTP and condensation of the resultant CDP-inositol (CDP-I) with myo-inositol then generates DIP. The sole known biosynthetic pathway of inositol in all organisms is the conversion of D-glucose-6-phosphate to myo-inositol. This conversion requires two key enzymes: L-I-1-P synthase and I-1-P phosphatase. Enzymatic assays using 31P nuclear magnetic resonance spectroscopy as well as a colorimetric assay for inorganic phosphate have confirmed the occurrence of L-I-1-P synthase and a moderately specific I-1-P phosphatase. The enzymatic reaction that couples CDP-I with myo-inositol to generate DIP has also been detected in Methanococcus igneus. 13C labeling studies with [2,3-13C]pyruvate and [3-13C]pyruvate were used to examine this pathway in M. igneus. Label distribution in DIP was consistent with inositol units formed from glucose-6-phosphate, but the label in the glucose moiety was scrambled via transketolase and transaldolase activities of the pentose phosphate pathway.

Cell-Free System↗

PubMed1998

Mta has properties of an RNA export protein and increases cytoplasmic accumulation of Epstein-Barr virus replication gene mRNA.

The Epstein-Barr virus (EBV) Zta and Mta regulatory proteins were previously found to be required for efficient replication of oriLyt in cotransfection-replication assays, but the contribution of Mta to the replication process was unknown. We now demonstrate that Mta regulates replication gene expression. Using the polymerase processivity factor BMRF1 as an example, we found that in transfected cells, total BMRF1 mRNA levels were unaffected by Mta but that the amounts of cytoplasmic BMRF1 RNA and protein were greatly increased in the presence of Mta. Mta also increased cytoplasmic accumulation of the BALF2, BALF5, BSLF1, and BBLF4 replication gene mRNAs but did not affect cytoplasmic levels of BBLF2/3 mRNA. Thus, five of the six core replication genes require Mta for efficient accumulation of cytoplasmic RNA. The contribution of Mta to posttranscriptional RNA processing was examined. Examination of Mta localization in transfected cells by indirect immunofluorescence revealed that Mta colocalized with the splicing factor SC35. We also found that Mta has RNA binding activity. Glutathione S-transferase-Mta bound to BMRF1 and BMLF1 transcripts but not to a control cellular gene RNA. Mta contains a consensus leucine-rich nuclear export signal. Such signal sequences are characteristic of proteins that undergo nuclear export. Examination of Mta localization in a heterokaryon assay provided evidence that Mta shuttles between the nucleus and the cytoplasm. Our experiments indicate that Mta functions in RNA processing and transport and mediates cytoplasmic accumulation of a number of EBV early mRNAs.

Amino Acid Sequence↗

PubMed1998

Role of laminin-1, collagen IV, and an autocrine factor(s) in regulated secretion by lacrimal acinar cells.

Adhesion to novel basement membrane component BM180 in the presence of laminin-1 promotes stimulus-secretion coupling in lacrimal acinar cells [G. W. Laurie, J. D. Glass, R. A. Ogle, C. M. Stone, J. R. Sluss, and L. Chen. Am. J. Physiol. 270 (Cell Physiol. 39): C1743-C1750, 1996]. The identity of the active laminin-1 site and the possibility that other promoters of coupling are present in the acinar cell microenvironment were probed by use of different substrates, media, neutralizing antibodies and cell numbers. Regulated peroxidase secretion was unaffected by basement membrane coat concentration and was detectable at reduced levels in serum-free medium. Anti-laminin-1 antibodies, particularly against sites in the beta1 and gamma1 chains, but not alpha1 chains, partially suppressed regulated secretion, as did an anti-collagen IV antibody. Without effect were RGD peptide and antibodies against entactin, the beta1-integrin subunit, and several growth factors. Increasing cell number in serum-free medium revealed an unknown, serum-maskable, secretion-enhancing activity with a remarkable specificity for regulated secretion. Stimulus-secretion coupling, therefore, appears to be modulated by several extracellular factors whose relative contributions remain to be determined.

Animals↗

PubMed1998

Evidence suggesting that nitric oxide mediates iron-induced toxicity in cultured proximal tubule cells.

The potential role of nitric oxide (NO) in iron-induced toxicity was studied in proximal tubule cells in primary culture. NO production (NO2-/NO3-) was significantly increased in iron-treated compared with control cells (3.43 +/- 0.08 vs. 1.56 +/- 0.08 nmol/dish, P < 0.01). NO synthase (NOS) activity was also induced by iron treatment (16.2 +/- 2.0 vs. 0.4 +/- 0.2 nmol of [3H]citrulline/mg protein, P < 0.01). L-Arginine, a substrate for NOS, augmented iron-induced NO production and cell damage [lactate dehydrogenase (LDH) leakage], whereas aminoguanidine, an inhibitor of NOS, reduced iron-induced NO production and LDH leakage. Sodium nitroprusside, an exogenous NO donor, induced LDH leakage in a dose-dependent manner, but no effect on lipid peroxidation (malo-ndialdehyde bis[dimethyl acetal] (MDA) production) was observed. Superoxide dismutase and catalase decreased iron-induced MDA production but did not affect LDH leakage or NO production. Dimethylpyrroline N-oxide and desferal prevented MDA production, LDH leakage, and NO production. We concluded that NO is one of the mediators of iron-induced toxicity in proximal tubule cells. NO-induced toxicity is not dependent on lipid peroxidation. This may explain the variable effect of different antioxidants on cell damage and lipid peroxidation in iron-induced cytotoxicity.

Animals↗