Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “ULTRACENTRIFUGATION”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 289 records · Page 16Linked to original sources
PubMed2000

Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling.

A new method for the size-distribution analysis of polymers by sedimentation velocity analytical ultracentrifugation is described. It exploits the ability of Lamm equation modeling to discriminate between the spreading of the sedimentation boundary arising from sample heterogeneity and from diffusion. Finite element solutions of the Lamm equation for a large number of discrete noninteracting species are combined with maximum entropy regularization to represent a continuous size-distribution. As in the program CONTIN, the parameter governing the regularization constraint is adjusted by variance analysis to a predefined confidence level. Estimates of the partial specific volume and the frictional ratio of the macromolecules are used to calculate the diffusion coefficients, resulting in relatively high-resolution sedimentation coefficient distributions c(s) or molar mass distributions c(M). It can be applied to interference optical data that exhibit systematic noise components, and it does not require solution or solvent plateaus to be established. More details on the size-distribution can be obtained than from van Holde-Weischet analysis. The sensitivity to the values of the regularization parameter and to the shape parameters is explored with the help of simulated sedimentation data of discrete and continuous model size distributions, and by applications to experimental data of continuous and discrete protein mixtures.

Confidence Intervals↗
PubMed1980

Graphical analysis of nonideal monomer N-mer, isodesmic, and type II indefinite self-associating systems by equilibrium ultracentrifugation.

A graphical procedure is described by which one can obtain in principle the monomer molecular weight, stoichiometry, equilibrium constant, and second virial coefficient of nonideal monomer N-mer, isodesmic, and type II indefinite self-associating systems. In addition, a method is presented for obtaining both the equilibrium constant and the second virial coefficient from the maximum in a plot of apparent molecular weight vs. concentration if the monomer molecular weight and stoichiometry are known. The usefulness and limitations of the methods are discussed, as well as the quality and range of data required for determination of the relevant parameters. The techniques described are applicable to analysis of self-associating systems by osmotic pressure and light scattering, as well as equilibrium ultracentrifugation measurements.

Chemical Phenomena↗
PubMed1981

Analysis of data from the analytical ultracentrifuge by nonlinear least-squares techniques.

Least-squares analysis of experimental data from the analytical ultracentrifuge is discussed in detail, with particular attention to the use of interference optics in studying nonideal self-associating macromolecular systems. Several samples are given that describe the application of the technique, the expected precision of the results, and some of its limitations. A FORTRAN IV computer program is available from the authors.

Computers↗
PubMed2000

Analytical performance and clinical efficacy of three routine procedures for LDL cholesterol measurement compared with the ultracentrifugation-dextran sulfate-Mg(2+) method.

The diagnosis and management of adults with hypercholesterolemia in the US are largely based on low-density lipoprotein cholesterol (LDL-C) concentration. In order to classify someone correctly into the National Cholesterol Education Program cut-points, LDL-C must be measured with a total error of </=12%. We examined simultaneously the analytical and clinical performance of two homogeneous LDL-C assays (LDL-C(RD), Roche Diagnostics and LDL-C(GZ), Genzyme) and the Friedewald calculation (LDL-C(Fried)). These assays correlated highly with the ultracentrifugation-dextran sulfate-Mg(2+) method (LDL-C(RD): r=0.962, y=1.029x-0.48 mmol/l, n=134; LDL-C(GZ): r=0.961, y=0.986x-0.12 mmol/l, n=134; LDL-C(Fried): r=0.960, y=1.017x-0.18 mmol/l, n=115). The total error requirement was met by the LDL-C(GZ) assay at all clinical decision cut-points, whereas the LDL-C(RD) assay met this requirement only at LDL-C concentrations of 4.92 mmol/l. The LDL-C(Fried) failed to meet the total error requirement, because the compounded imprecision of the three independent tests required for this calculation was high. Both, the LDL-C(GZ) and the LDL-C(RD) assays appeared to be only slightly affected by increasing triglycerides. At the medical decision cut-point range, the LDL-C(RD), LDL-C(GZ) and LDL-C(Fried) assays showed positive predictive values of 89-100, 85-100 and 83-99%, respectively, and negative predictive values of 52-98, 77-98 and 68-98%, respectively. The homogeneous assays provide clinical laboratories with the means to measure LDL-C in hypertriglyceridemic samples and could have a role in the diagnosis and management of hyperlipidemic patients.

Adult↗
PubMed1976

The molecular weight of aspermatogenic antigen as determined by equilibrium ultracentrifugation.

The extraction of antigen from testis and sperm is described. This antigen, when administered in microgram amounts in a single injection, induces aspermatogenesis in 100% of guinea pigs so treated. The deletion of the seminiferous cell line is specific (no cross-reactions) and provides a tool for investigating inhibition of cell replication as well as reproductive control. The antigen is a polypeptide-polysaccharide complex and has been determined to have a molecular weight of 10,520, using the equilibrium ultracentrifugation method.

Animals↗
PubMed1978

Comparative studies of the ethynyl estrogens used in oral contraceptives. VII. Effects with and without progestational agents on ultracentrifugally fractionated plasma lipoproteins in humans, baboons, and beagles.

Ethynyestradiol and mestranol, in doses ranging from 50 to 100 microgram/day, were given to women in 21-day cycles; baboons and beagle dogs received 1 and 4 microgram/kg/day in a similar regimen. After a number of such cycles, megestrol acetate, norethindrone acetate, or dl-norgestrel was given concomitantly. Protein, cholesterol, triglyceride, and phospholipid levels were determined in total plasma and in ultracentrifugally separated lipoprotein fractions. Over the dosage range studied, the effects of the two kinds of estrogen were indistinguishable. Except for human total plasma triglyceride, no dose-related differences were observed. The lowering of serum protein and the increase in cholesterol induced by estrogen were more pronounced in baboons and beagles than in human subjects. The cholesterol-depressing effect of progestational compounds observed in humans was very pronounced in baboons but absent in beagles. In all three species, estrogen increased the lipoprotein fraction cholesterol, except for human low-density lipoprotein cholesterol, which was decreased. Human plasma triglyceride and phospholipid increased on estrogen administration and were decreased by the progestins; in the two animal species, triglyceride is normally very low and the estrogen-induced changes were negligible; the phospholipid rose with estrogen but was unaffected by progestins. In sum, the two animal species show many similarities to, as well as important differences from, the human response of plasma lipids to various contraceptive steroids.

Animals↗
PubMed1990

Particle characterization in centrifugal fields. Comparison between ultracentrifugation and sedimentation field-flow fractionation.

A ten-component mixture of polystyrene latex particles in the 67-1220 nm size range was subjected to analysis by analytical ultracentrifugation (AUC) and sedimentation field-flow fractionation (SdFFF) using programmed and constant fields. The AUC analysis of the mixture yielded diameter values in good agreement with data determined on the separate components; the relative amounts of each component in the mixture were likewise closely reproducing the sample's known composition. Diameters determined by SdFFF, either in a constant- or programmed-field mode, were in good agreement with the AUC for particles smaller than about 500 nm. For the sample's larger components, however, particularly the programmed mode showed diameter values smaller than expected. In addition, field programming resulted in incomplete recoveries of the larger particles, leading to more or less distorted mass distributions for the complex sample. The observed discrepancies, which are thought to result from events at the analytical wall in the FFF channel, suggested a protocol for accurate sizing, as opposed to fingerprinting, of samples with broad size distribution. By tracking sizes and amounts of the different components at different but constant field strengths, and retaining as analytically valid only those data recorded in a retention range from five to about thirty column volumes, it was possible to determine sizes and amounts in good agreement with known parameters for the sample. Unlike the AUC procedure, SdFFF produces fractions of a high degree of uniformity, which lend themselves to a secondary analysis, e.g. by electron microscopy, as shown in the study.

Centrifugation↗
PubMed2002

Reversible dimer formation and stability of the anti-tumour single-chain Fv antibody MFE-23 by neutron scattering, analytical ultracentrifugation, and NMR and FT-IR spectroscopy.

MFE-23 is a single chain Fv (scFv) antibody molecule used to target colorectal cancer through its high affinity for the tumour marker carcinoembryonic antigen (CEA). ScFv molecules are formed from peptide-linked antibody V(H) and V(L) domains, and many of these form dimers. Our recent crystal structure for MFE-23 showed that this formed an unusual symmetric back-to-back association of two monomers that is consistent with a domain-swapped diabody structure. Neutron scattering and modelling fits showed that MFE-23 existed as compact V(H)-V(L)-linked monomers at therapeutically relevant concentrations below 1 mg/ml. Size-exclusion gel chromatography showed that the monomeric and dimeric forms of MFE-23 could be separated, and that the proportions of these two forms depended on the starting MFE-23 concentration. Sedimentation equilibrium experiments by analytical ultracentrifugation at nine concentrations of MFE-23 indicated a reversible monomer-dimer self-association equilibrium with an association constant of 1.9x10(3)-2.2x10(3) M(-1). Sedimentation velocity experiments using the time derivative g(s(*)) method showed that MFE-23-His has a concentration-dependent weight average sedimentation coefficient that increased from 1.8 S for the monomer to about 3-6 S for the dimer. Both values agreed with those calculated from the MFE-23 crystal structure. In relation to the thermal stability of MFE-23, denaturation experiments by (1)H NMR and FT-IR spectroscopy showed that the molecule is stable up to 47 degrees C, after which denaturation was irreversible. MFE-23 dimerisation is discussed in terms of a new model for diabody structures, in which the V(H) and V(L) domains in the monomer are able to dissociate and reassociate to form a dimer, or diabody, but in which symmetric back-to-back contacts between the two monomers are formed. This dimerisation in solution is attributed to the complementary nature of the C-terminal surface of the MFE-23 monomer. Crystal structures for seven other scFv molecules have shown that, while the contact residues for symmetric back-to-back dimer formation in MFE-23 are not fully conserved, in principle, back-to-back contacts can be formed in these too. This offers possibilities for the creation of other forms of scFv molecules.

Amino Acid Sequence↗
PubMed2003

The extended multidomain solution structures of the complement protein Crry and its chimeric conjugate Crry-Ig by scattering, analytical ultracentrifugation and constrained modelling: implications for function and therapy.

Complement receptor-related gene/protein y (Crry) is a cell membrane-bound regulator of complement activation found in mouse and rat. Crry contains only short complement/consensus repeat (SCR) domains. X-ray and neutron scattering was performed on recombinant rat Crry containing the first five SCR domains (rCrry) and mouse Crry with five SCR domains conjugated to the Fc fragment of mouse IgG1 (mCrry-Ig) in order to determine their solution structures at medium resolution. The radius of gyration R(G) of rCrry was determined to be 4.9-5.0 nm, and the R(G) of the cross-section was 1.2-1.5 nm as determined by X-ray and neutron scattering. The R(G) of mCrry-Ig was 6.6-6.7 nm, and the R(G) of the cross-section were 2.3-2.4 nm and 1.3 nm. The maximum dimension of rCrry was 18 nm and that for mCrry-Ig was 26 nm. The neutron data indicated that rCrry and mCrry-Ig have molecular mass values of 45,000 Da and 140,000 Da, respectively, in agreement with their sequences, and sedimentation equilibrium data supported these determinations. Time-derivative velocity experiments gave sedimentation coefficients of 2.4S for rCrry and 5.4S for mCrry-Ig. A medium-resolution model of rCrry was determined using homology models that were constructed for the first five SCR domains of Crry from known crystal and NMR structures, and linked by randomly generated linker peptide conformations. These trial-and-error calculations revealed a small family of extended rCrry structures that best accounted for the scattering and ultracentrifugation data. These were shorter than the most extended rCrry models as the result of minor bends in the inter-SCR orientations. The mCrry-Ig solution data were modelled starting from a fixed structure for rCrry and the crystal structure of mouse IgG1, and was based on conformational searches of the hinge peptide joining the mCrry and Fc fragments. The best-fit models showed that the two mCrry antennae in mCrry-Ig were extended from the Fc fragment. No preferred orientation of the antennae was identified, and this indicated that the accessibility of the antennae for the molecular targets C4b and C3b was not affected by the covalent link to Fc. A structural comparison between Crry and complement receptor type 1 indicated that the domain arrangement of Crry SCR 1-3 is as extended as that of the CR1 SCR 15-17 NMR structure.

Amino Acid Sequence↗
PubMed1990

RecA protein self-assembly. II. Analytical equilibrium ultracentrifugation studies of the entropy-driven self-association of RecA.

We have investigated the self-association of RecA protein from Escherichia coli by equilibrium ultracentrifugation. Monomeric RecA (Mr = 37,842) was observed in reversible equilibrium with trimers, hexamers and dodecamers in the presence of 1.5 M-KCl, 5 mM-Hepes, 1 mM-EDTA, 2 mM-ATP (pH 7.0) at 1 degrees C. The equilibrium was strongly temperature-dependent, with polymerization being favored as the temperature was raised from 1 degrees C 21 degrees C, and was reversible with respect to temperature. The values of both the standard enthalpy and entropy of self-association were positive, indicating that it is an entropy-driven process under these conditions. In the absence of KCl, in 50 mM-citrate, 5 mM-ATP, 5% (v/v) glycerol (pH 6.0) at 4 degrees C, only small amounts of RecA monomer could be detected, while in 10 mM-Tris-acetate, 10% glycerol (pH 7.5) at 4 degrees C, the smallest species present in significant concentration appeared to be the trimer. The majority of the species observed had molecular weights between 228,000 and 456,000, suggesting dominant stoichiometries of six to 12 monomers per oligomer. At pH 6.0, in the absence of ATP, much larger oligomers containing at least 24 monomers also appeared to be present. The data are consistent with an equilibrium mixture of monomers, trimers, hexamers, dodecamers, 24-mers and higher oligomers, with the distribution of oligomers being dependent on solution conditions. Thermodynamic analysis indicates that these oligomeric species are in reversible equilibrium with each other. It is not certain whether trimers assemble directly into hexamers, or whether disassembly into monomers is a prerequisite for the formation of higher oligomers. The possible role of higher-order RecA oligomers in the formation of RecA nucleoprotein filaments is discussed.

Adenosine Triphosphate↗
PubMed1994

Determining confidence intervals for parameters derived from analysis of equilibrium analytical ultracentrifugation data.

In the above discussion, we have introduced the profiling approach of Bates and Watts. It is an easy to implement, empirical approach to the determination of confidence intervals for parameters in nonlinear models. We have applied the approach to the analysis of equilibrium sedimentation data and have demonstrated that, although models for analyzing such data are formally nonlinear they are functionally linear. As such, linear approximation confidence intervals for the parameters are adequate for these models and data sets. Further, we have been able to examine the effect of implementing a multiple independent variable approach (in this case, using multiple rotor speeds) on the precision of the analysis. We found that the standard errors of the parameters were reduced and that this is accounted for by either the increase in the number of data points or the decreases in parameter correlation. In this case, profiling helped to visualize the effect on the sum of squares surface of reducing parameter correlation, making the effect of the small decreases in the correlation of some parameters more evident. Using profiling, it should be easy to explore other methods for the improvement of the analysis of ultracentrifugation data and to be able to quantitate the improvement. With the above discussion as an example, it is likely that the profiling approach should be quite useful and broadly applicable in the analysis of data in terms of nonlinear models.

Computer Simulation↗
PubMed2004

Comparison of scanning electron microscopy, dynamic light scattering and analytical ultracentrifugation for the sizing of poly(butyl cyanoacrylate) nanoparticles.

Nanoparticles represent promising carriers for controlled drug delivery. This work focuses on the size and molecular mass characterization of polyalkylcyanoacrylate nanoparticles formed by anionic emulsion polymerization of butylcyanoacrylate in the presence of poloxamer 188 as a stabilizer. Three different methods were used to determine the size and size distribution of the particle populations: scanning electron microscopy (SEM), dynamic light scattering (DLS), and analytical ultracentrifugation (ANUC). SEM on freeze-dried and Au-shadowed samples showed a relatively narrow distribution of virtually spherical particles with a mean diameter of 167 nm. DLS yielded a monomodal distribution with hydrodynamic diameters around 199 nm (in the absence of additional stabilizer) or 184 nm (in the presence of 1% poloxamer 188). The size distribution determined by ANUC using sedimentation velocity analysis was somewhat more complex, the size of the most abundant particles being around 184 nm. Molar particle mass distributions centered around 2.3x10(9) g/mol. The advantages and disadvantages of the three sizing techniques are discussed.

Enbucrilate↗
PubMed2003

Ultracentrifugation-based approaches to study regulation of Sec6/8 (exocyst) complex function during development of epithelial cell polarity.

The Sec6/8 (exocyst) complex is an essential component of the exocytic apparatus and plays an evolutionarily conserved role in polarized membrane growth. During development of epithelial cell polarity, this cytosolic protein complex is recruited to plasma membrane sites of cell-cell contact, where it facilitates exocytosis to the lateral membrane domain. However, the identity of membrane binding sites for Sec6/8 complex, mechanisms regulating association of Sec6/8 complex with these sites, and the precise function of the complex in polarized trafficking are not known. Biochemical strategies involving differential, rate-zonal, and isopycnic density gradient ultracentrifugation are providing clues to these questions.

Animals↗
PubMed1993

Human factor VIIa and its complex with soluble tissue factor: evaluation of asymmetry and conformational dynamics by ultracentrifugation and fluorescence anisotropy decay methods.

Ultracentrifugation and fluorescence anisotropy decay measurements were used to evaluate the asymmetry and conformational dynamics of human blood clotting enzyme VIIa (VIIa) and the complex it forms with a soluble truncation mutant of human tissue factor (sTF) which acts as an essential cofactor for VIIa. Sedimentation velocity experiments showed that both VIIa and the sTF.VIIa complex are highly asymmetric. In each case, the friction ratio f/fsphere, is consistent with a family of general elliposids ranging from prolate to oblate. Fluorescence anisotropy decay experiments were used to limit the family of elliposids which can describe the hydrodynamic behavior of VIIa and sTF.VIIa. For both VIIa and the sTF.VIIa complex, the oblate ellipsoid of revolution was eliminated. In addition, the fluorescence anisotropy decay data clearly show that upon binding sTF.VIIa loses a segmental motion involving a domain containing the active site of the enzyme. This suggests that sTF causes a stabilization of a limited range of VIIa conformations. This stabilization may be important for proper recognition of the TF.VIIa substrate, factor X.

Chemical Phenomena↗
PubMed1991

Thermodynamics of antiparallel hairpin-double helix equilibria in DNA oligonucleotides from equilibrium ultracentrifugation.

Five highly palindromic DNA dodecamers, four of which may form G-A or I-A purine-purine mispairs at either the 5.8 or 6.7 positions, have been studied at sedimentation equilibrium in the analytical ultracentrifuge. Each DNA oligonucleotide forms an equilibrium mixture of ordered antiparallel hairpin and double-stranded helical structures in solutions of 0.1 or 0.5 M NaCl between 5 and 40 degrees C. The dimeric duplex is favored by conditions of high salt and low temperature. The monomer-dimer equilibrium constants vary from 5 x 10(6) to 5 x 10(3) and are unique for each DNA dodecamer. Analysis of the temperature dependence of the equilibrium constants shows that the double helix to hairpin conversion is driven by a positive entropy change and is associated with an endothermic enthalpy change. The mispair substitutions at the 5.8 positions and the IA(6.7) mispair have the greatest tendency toward hairpin formation and exhibit significantly larger entropy changes than the nonmispaired dGGTACGCGTACC parent sequence and the thermodynamically similar GA(6.7) DNA. The consequences of such hairpin-double helix equilibria must be considered in the interpretation of other kinds of experiments carried out on oligonucleotides at different concentrations.

Base Sequence↗
PubMed1982

Covalently activated glycogen phosphorylase: a phosphorus-31 nuclear magnetic resonance and ultracentrifugation analysis.

Glycogen phosphorylase b reconstituted with pyridoxal pyrophosphate in place of the natural coenzyme, pyridoxal phosphate, is shown to exist in a more activated (R) conformation than does native phosphorylase. Addition of nucleotide activator to the reconstituted enzyme traps it totally in this activated conformation. These conclusions were arrived at on the basis of tertiary structural information obtained from 31P nuclear magnetic resonance studies, which allowed measurement of the nucleotide binding constant, and on the basis of quaternary structural information obtained via ultracentrifugal analysis of the enzyme in the presence of various effectors. Control experiments were performed with another modified form of the enzyme, pyridoxal phosphorylase. It is suggested that the transition-state analogue pyridoxal pyrophosphate, bound at the active site, mimics the actual configuration of enzyme plus substrate achieved during the normal catalytic reaction and therefore traps the enzyme in an activated conformation. These findings agree well with recent results obtained with the alternate transition-state analogue pyridoxal pyrophosphate glucose [Withers, S. G., Madsen, N. B., Sykes, B. D., Takagi, M., Shimomura, S., & Fukui, T. (1981) J. Biol. Chem. 256, 10759] and therefore provide further evidence for the "interacting phosphates" hypothesis presented in the latter paper.

Enzyme Activation↗
PubMed1984

Structure of bovine blood coagulation factor Va. Determination of the subunit associations, molecular weights, and asymmetries by analytical ultracentrifugation.

Thrombin-activated bovine factor V (factor Va), an essential component of the blood clotting cascade pro thrombinase complex, is composed of two nonidentical subunits (Vl and Vh) and Ca2+ in tight association. We have examined Vl, Vh, and factor Va using analytical ultracentrifugation. At pH 7.65 in 50 mM tris(hydroxymethyl)aminomethane, 0.1 M NaCl, 1 mM benzamidine, and 10 mM Ca2+, the Vl subunit has a molecular weight (Mr) of 82 500, an S0(20) ,w = 5. 0(2)S , and, assuming a model of a prolate ellipsoid with 0.3 g of H2O/g of protein, an axial ratio of 5:1. The corresponding values for the Vh subunit are an Mr of 92 300, an S0(20) ,w = 5.2(9) S, and an axial ratio of 5:1. We found these same values for Vl and for Vh in a buffer that contained 2 mM ethylenediaminetetraacetate (EDTA) rather than the 10 mM Ca2+. The Vl subunit undergoes a weak, reversible self-association at 9 degrees C with an apparent monomer-dimer association constant of 5.6 X 10(3) M-1 in the presence of 2 mM EDTA and 2.3 X 10(3) M-1 in the presence of 10 mM Ca2+. Our data indicate that the Vl self-association includes dimer and higher oligomers. Factor Va, examined in the presence of 10 mM Ca2+ and at 20 degrees C, has an Mr of 174 000, and S0(20) ,w = 8.1(8)S, an axial ratio of 5:1, and an apparent Vl-Vh association constant of at least 2.7 X 10(8) M-1. Our results suggest that factor Va self-associates to form higher multimers. When solutions of Va are dialyzed against a buffer that contains no Ca2+ and 2 mM EDTA, the apparent Vl-Vh subunit association constant is reduced to 9.4 X 10(3) M-1. Our hydrodynamic data indicate that there is a substantial decrease in molecular asymmetry when factor V is proteolytically activated by thrombin to form factor Va and that Vl and Vh are arranged "side by side" rather than "end to end" in factor Va.

Animals↗
PubMed1989

Interaction of clotting factor V heavy chain with prothrombin and prethrombin 1 and role of activated protein C in regulating this interaction: analysis by analytical ultracentrifugation.

Changes in the affinity of the heavy subunit of blood coagulation factor Va (Vh) for prothrombin are thought to be important in regulating the rate of thrombin production. Using analytical ultracentrifugation, we have measured the affinity of bovine Vh for prothrombin and for the prethrombin 1 fragment of prothrombin at 23.3 degrees C, pH 7.65, in 50 mM tris(hydroxymethyl)aminomethane, 0.1 M NaCl, 0.1 mM benzamidine, and either 2 mM Ca2+ or 2 mM ethylenediaminetetraacetate (EDTA). Under these conditions a 1:1 complex of Vh with prothrombin is formed that is governed by a dissociation constant (Kd) of 10 microM, regardless of whether the buffer contains Ca2+ or EDTA. An identical Kd is observed when prethrombin 1 is substituted for prothrombin. This indicates that the fragment 1 portion of prothrombin, containing the gamma-carboxyglutamic acid residues, does not influence the association. Substitution of human prethrombin 1 for the bovine molecule also results in a 1:1 Vh-prethrombin 1 complex governed by a slightly weaker Kd (27 microM). Discrete proteolysis of bovine Vh by the anticoagulant activated protein C converts the Vh to a form with little or no affinity for prethrombin 1 (Kd greater than 1 mM), without detectable change in the mass of the Vh.

Animals↗