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Results for “second generation sequencing”

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PubMed1992

Single-stranded DNA as a recombination substrate in plants as assessed by stable and transient recombination assays.

Two separate assays, one that requires stable integration of recombination products and one that does not, were employed to elucidate the role of single-stranded DNA in extrachromosomal homologous recombination in Nicotiana tabacum. Both assays revealed that single-stranded DNA in linear and in circular forms was an efficient substrate for recombination, provided that the cotransformed recombination substrates were of complementary sequence, so that direct annealing was possible. Recombination was inefficient when both single-stranded recombination partners contained homologous regions of identical sequence and generation of a double-stranded DNA was required prior to heteroduplex formation. These results indicate that direct annealing of single strands is an important initial step for intermolecular recombination in tobacco cells. Annealed cotransformed single-stranded molecules yielded intermediates that could be further processed by either continuous or discontinuous second-strand synthesis. The type of intermediate had no influence on the recombination efficiency. Double-stranded circles were unable to recombine efficiently either with each other or with single-stranded DNA. Our results suggest that a helicase activity is involved in the initial steps of double-stranded DNA recombination which unwinds duplex molecules at the site of double-strand breaks.

Base Sequence↗

PubMedDate not supplied

Time-resolved fluorometry in end-point and real-time PCR quantification of nucleic acids.

Two time-resolved fluorescence-based methods for nucleic acid quantification are described and their results are compared. Both methods use an exogenous internal standard to eliminate errors arising from different steps of the assay. The first method is a competitive end-point assay, where the standard competes for the same primers with the actual target sequence, prostate-specific antigen (PSA) cDNA. The standard and target are quantified in a dual-label plate hybridization with lanthanide-labelled probes after a fixed number of PCR cycles. The second method is based on real-time monitoring of PCR and on the use of a novel homogeneous signal generation principle that relies on the use of a 5'-->3' exonucleolytic DNA polymerase and a probe labelled with an environment sensitive, stable and fluorescent lanthanide chelate. In this assay, a non-competitive, exogenous internal standard is used. Both assays have a wide linear range (50-5 x 10(6) and 10-5 x 10(7) input PSA cDNA molecules for the end-point and real-time assays, respectively) and there is a strong correlation between the results obtained with the two assays (r = 1.0). Being somewhat faster to perform, the real-time format is better suited for assays that require high throughput.

Base Sequence↗

PubMed2005

Digital single-nucleotide polymorphism analysis for allelic imbalance.

Digital single-nucleotide polymorphism (SNP) analysis is developed to amplify a single template from a pool of DNA samples, thereby generating the amplicons that are homogeneous in sequence. Different fluorophores are then applied as probes to detect and discriminate different alleles (paternal vs maternal alleles or wild-type vs mutant), which can be readily counted. In this way, digital SNP analysis transforms the exponential and analog signals from conventional polymerase chain reaction (PCR) to linear and digital ones. Digital SNP analysis has the following advantages. First, statistical analysis of the PCR products becomes available as the alleles can be directly counted. Second, this technology is designed to generate PCR products of the same size; therefore, DNA degradation would not be a problem as it commonly occurs when microsatellite markers are used to assess allelic status in clinical samples. Last, digital SNP analysis is designed to amplify a relatively small amount of DNA samples, which is available in some clinical samples. Digital SNP analysis has been applied in quantification of mutant alleles and detection of allelic imbalance in clinical specimens and it represents another example of the power of PCR and provides unprecedented opportunities for molecular genetic analysis.

Allelic Imbalance↗

PubMed1991

Amplification of polyomavirus DNA sequences stably integrated in rat cells.

To investigate the mechanism by which the polyomavirus large T antigen (T-Ag) promotes amplification of integrated viral sequences, we constructed a rat cell line, Hy2-ts5, carrying two different inserts of polyomavirus DNA. The first insert, designated the middle T (pmt) locus, was devised to analyze homologous recombination between two defective copies of pmt lying 3.3 kb apart on the same chromosome. Reconstitution of a functional pmt by spontaneous recombination occurred at a rate of about 2 x 10(-7) per cell generation. The second locus contained the polyomavirus large T (plt) gene carrying a temperature-sensitive mutation and producing a nonfunctional large T-Ag at 39 degrees C. A shift to the permissive temperature for as little as 24 h induced the production of a functional large T-Ag which, in turn, promoted homologous recombination in the pmt locus at a rate close to 1.0 per cell generation. The particularity of this system is that it allowed recombination products to be analyzed as early as a single cell doubling following the initial recombinational event. Amplification occurred by successive duplications of a discrete sequence in the viral insert. Unequal sister chromatid exchange was ruled out as the recombination mechanism promoted by large T-Ag. Instead, we proposed a model of nonconservative recombination involving mispairing between homologous sequences.

Animals↗

PubMed1983

Extensive polymorphism surrounding the murine Ia A beta chain gene.

A segment of the murine immune response-associated (Ia) antigen A beta chain mRNA has been cloned, and this cDNA clone has been used to examine the organization of the genes encoding these highly polymorphic cell surface proteins. To isolate this clone, pIA beta-1, we screened 5000 cDNA clones by a filter hybridization-selection assay using a rabbit heteroantiserum to identify the A beta chain produced in in vitro translation reactions. This clone corresponds to a segment of the mRNA encoding the beta chain of the murine I-A antigen. The 464 bp nucleotide sequence of the insert is consistent with a cDNA clone encoding the carboxyl end of the murine A beta chain. Comparison of this nucleotide sequence to a human Ia beta chain-derived cDNA clone suggests that the two clones could encode proteins that are about 90% homologous in this region. Southern blot analysis using pIA beta-1 as a probe to investigate the structure of the A beta chain gene in inbred strains of mice allows two conclusions. First, there is only one A beta chain gene homologous to this cDNA clone in each haploid mouse genome. Second, the restriction fragments encoding this gene are highly polymorphic. We postulate that the mechanism which generates polymorphic variation of the A beta chain may be involved in generating the restriction site polymorphism within or near the A beta chain gene.

Animals↗

PubMed2005

Mutations conferring resistance to SCH6, a novel hepatitis C virus NS3/4A protease inhibitor. Reduced RNA replication fitness and partial rescue by second-site mutations.

Drug resistance is a major issue in the development and use of specific antiviral therapies. Here we report the isolation and characterization of hepatitis C virus RNA replicons resistant to a novel ketoamide inhibitor of the NS3/4A protease, SCH6 (originally SCH446211). Resistant replicon RNAs were generated by G418 selection in the presence of SCH6 in a dose-dependent fashion, with the emergence of resistance reduced at higher SCH6 concentrations. Sequencing demonstrated remarkable consistency in the mutations conferring SCH6 resistance in genotype 1b replicons derived from two different strains of hepatitis C virus, A156T/A156V and R109K. R109K, a novel mutation not reported previously to cause resistance to NS3/4A inhibitors, conferred moderate resistance only to SCH6. Structural analysis indicated that this reflects unique interactions of SCH6 with P'-side residues in the protease active site. In contrast, A156T conferred high level resistance to SCH6 and a related ketoamide, SCH503034, as well as BILN 2061 and VX-950. Unlike R109K, which had minimal impact on NS3/4A enzymatic function, A156T significantly reduced NS3/4A catalytic efficiency, polyprotein processing, and replicon fitness. However, three separate second-site mutations, P89L, Q86R, and G162R, were capable of partially reversing A156T-associated defects in polyprotein processing and/or replicon fitness, without significantly reducing resistance to the protease inhibitor.

Binding Sites↗

PubMed1980

The mechanism of action of penicillin. Penicillin acylates the active site of Bacillus stearothermophilus D-alanine carboxypeptidase.

Penicillin kills susceptible bacteria by specifically inhibiting the transpeptidase that catalyzes the final step in cell wall biosynthesis, the cross-linking of peptidoglycan. It was hypothesized (Tipper, D., and Strominger, J. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 1133-1141) that 1) penicillin is a structural analog of the acyl-D-alanyl-D-alanine terminus of the pentapeptide side chains of nascent peptidoglycan, and that 2) penicillin, by virtue of its highly reactive beta-lactam structure, irreversibly acylates the active site of the cell wall transpeptidase. Although the cell wall transpeptidase has proven elusive, a closely related penicillin-sensitive cell wall enzyme, D-alanine carboxypeptidase, has been purified from membranes of Bacillus stearothermophilus by penicillin affinity chromatography. By amino acid sequence analysis of 14C-labeled cyanogen bromide peptides generated and purified from this carboxypeptidase covalently labeled with either [14C]penicillin G or the substrate, [14C]diacetyl-L-lysyl-D-alanyl-D-lactate, it was shown that the penicillin and substrate were both bound as esters to a serine at residue 36. Therefore, the second hypothesis stated above was proven to be correct for D-alanine carboxypeptidase. Several new methods were developed in the course of this work, including 1) a rapid penicillin-binding assay, 2) use of hydroxylamine to protect peptides against carbamylation during ion exchange chromatography in concentrated urea solutions, and 3) gel filtration chromatography in 70% formic acid, a universal solvent for peptides.

Amino Acid Sequence↗

PubMed1993

Unequal signal and coding joint formation in human V(D)J recombination.

Substrates for studying V(D)J recombination in human cells and two human pre-B-cell lines that have active V(D)J recombination activity are described. Using these substrates, we have been able to analyze the relative efficiency of signal joint and coding joint formation. Coding joint formation was five- to sixfold less efficient than signal joint formation in both cell lines. This imbalance between the two halves of the reaction was demonstrated on deletional substrates, where each joint is assayed individually. In both cell lines, the inversional reaction (which requires formation of both a signal and a coding joint) was more than 20-fold less efficient than signal joint formation alone. The signal and coding sequences are identical in all of these substrates. Hence, the basis for these differential reaction ratios appears to be that coding joint and signal joint formation are both inefficient and their combined effects are such that inversions (two-joint reactions) reflect the product of these inefficiencies. Physiologically, these results have two implications. First, they show how signal and coding joint formation efficiencies can affect the ratio of deletional to inversional products at endogenous loci. Second, the fact that not all signal and coding joints go to completion implies that the recombinase is generating numerous broken ends. Such unresolved ends may participate in pathologic chromosomal rearrangements even when the other half of the same reaction may have proceeded to resolution.

B-Lymphocytes↗

PubMed2001

Strategies to generate antibodies capable of distinguishing between proteins with >90% amino acid identity.

Protein engineering is a common strategy for the generation of protein variants with new properties. The engineered variants often have a high degree of similarity with the wild-type progenitor protein, necessitating a tool (e.g., antibody) to distinguish the wild-type and variant protein forms. As part of an overall effort to understand the process of incorporation of amino acids into storage proteins during seed fill in soybean, we have engineered a variant of soybean vegetative storage protein beta (VSPbeta) that is 91.8% identical in amino acid sequence to the wild-type protein, but contains 10% methionine (VSPbeta-Met, unpublished results). Thus, it would be desirable to have antibodies that specifically recognize VSPbeta-Met over the endogenously expressed wild-type protein in transgenic plants. To this end, we compared three strategies for the isolation of VSPbeta-Met-specific antibodies: (1) hybridoma production using VSPbeta-Met protein as the antigen, (2) polyclonal antibody production in rabbits using a peptide antigen corresponding to a methionine-rich region of VSPbeta-Met, and (3) subtractive immunization in mice using VSPbeta-WT as the tolerogen, cyclophosphamide for immunosuppression and VSPbeta-Met as the immunogen. While the first strategy generated antibodies cross-reactive to both antigens, the second strategy generated polyclonal antibodies that preferentially recognized the variant protein in immunoblots. However, using subtractive immunization, we were able to generate mouse polyclonal antibodies that exhibited 10-fold greater reactivity with VSPbeta-Met than VSPbeta-WT in an ELISA.

Amino Acid Sequence↗

PubMed2001

Generation of recombinant human C3dg tetramers for the analysis of CD21 binding and function.

CD21 (complement receptor 2, CR2) binds the terminal proteolytic fragments of the third component of complement (C3) that have been covalently attached to immune complexes or other targets during the activation of complement. We used the technique of in vivo biotinylation to create a recombinant multivalent ligand for CD21. A sequence coding for a biotinylation signal peptide was added to the 3' end of the human C3dg cDNA. The modified C3dg was expressed in Escherichia coli and biotinylated intracellularly by the bacterial biotin holoenzyme synthetase (BirA) enzyme. Monomeric C3dg was unable to bind to CD21 as determined by flow cytometry, while biotinylated recombinant C3dg (rC3dg) complexed with fluorochrome-conjugated streptavidin bound tightly. Binding was observed using CD21 positive B cells but not seen on pre-B cells that do not express this complement receptor. Two assays were used to assess the functional capacity of the recombinant C3dg. First, multimeric C3dg caused the phosphorylation of the mitogen-activated kinase, p38, in mature B lymphoma cells. Second, C3dg greatly enhanced the activation of primary B cells in combination with a sub-stimulatory concentration of anti-IgM monoclonal antibody. These results illustrate the utility of the technique of in vivo biotinylation to generate ligands for cell surface receptors that require multimerization for high avidity binding and function.

Amino Acid Sequence↗

PubMed1990

Transduction of cellular neo mRNA by retrovirus-mediated recombination.

Transduction of cellular oncogenes by retroviruses is thought to be a multistep process, involving transcriptional activation of a cellular gene by upstream proviral integration and joining of cellular DNA to retroviral transcriptional signals, followed by copackaging and recombination with a helper virus genome during reverse transcription. To examine the molecular mechanism of the reverse transcriptase-mediated recombination, we introduced into mouse fibroblast cells a variety of constructs in which the neo selectable marker was joined to flanking retroviruslike or cell-like sequences. After superinfection and copackaging with a replication-competent Mo-MuLVsupF virus, the formation of recombinant neo transducing viruses was assessed in a second round of virus infection by the ability to confer G418 resistance to infected cells. Our results showed that recombinant neo proviruses were generated from neo RNA containing either a 5' or 3' retroviral end, implying that one recombination event with helper virus RNA was sufficient to incorporate the neo gene into proviral DNA. Recombination occurred with an apparent frequency of 10(-4) to 10(-5) per replication cycle in the absence of homology between the two recombining partners. This frequency, however, increased at least 100-fold if homology was provided at the site of recombination. Our results support the hypothesis that neo-transducing viruses arise via reverse transcriptase-mediated recombination of RNA rather than by recombination proceeding through DNA intermediates. Unexpectedly, removal of the retroviral packaging site psi reduced the number of neo recombinants only slightly. Our data indicated that although RNAs lacking the psi site are poorly packaged into virions, those RNAs that are included in the virions undergo frequent recombination, even if there is no selection for recombination. Many of the neo recombinants formed with the psi- constructs had undergone additional recombinations and often incorporated the psi site from the helper RNA.

DNA, Viral↗

PubMed1998

An alternative domain determines nuclear localization in multifunctional protein 4.1.

Multiple protein 4.1 isoforms are originated by alternative pre-mRNA splicing, differential use of two translation initiation sites, and posttranslational modifications. The complexity of alternative splicing events suffered by the 4.1 pre-mRNA makes necessary the direct cloning of 4.1 full-coding cDNA sequences to ensure that the encoded 4.1 proteins are naturally occurring isoforms. We have approached this point by reverse transcription-polymerase chain reaction techniques using RNA from the nucleated human Molt-4 T-cell line as a starting template. Molecular cloning of 4.1 cDNAs using the second translation initiation codon has allowed us to identify two 4.1 isoforms, designated 4.1H and 4.1I, which are differentially targeted to the nucleus (4.1H) and the cytoplasm (4.1I). These two isoforms differ only in the inclusion (4.1H) or exclusion (4.1I) of 21 amino acids encoded by exon 16. A cluster of basic amino acids, KKKR, generated by joining of the sequences encoded by the constitutive exon 13 and the alternative exon 16, is necessary for the nuclear targeting of 4.1H, as demonstrated by site-directed mutagenesis analysis. Immunofluorescence microscopy and biochemical studies indicate that 4.1H belongs to the group of nuclear 4.1 proteins that are distributed diffusely throughout the nucleoplasm and that are extractable in 0.5% Triton X-100. This is the first demonstration of differential nuclear targeting by the presence of an alternative domain, among naturally occurring protein 4.1 isoforms.

Alternative Splicing↗

PubMed1977

Anatomy of herpes simplex virus DNA. IX. Apparent exclusion of some parental DNA arrangements in the generation of intertypic (HSV-1 X HSV-2) recombinants.

We are reporting the physical location of parental DNA sequences in 28 recombinants produced by crossing herpes simplex viruses (HSV) 1 and 2. The parental crosses were of two kinds. In the first, temperature-sensitive mutants of HSV-1 and HSV-2 were crossed to produce wild-type recombinants. In the second, temperature-sensitive mutants of HSV-1 rendered resistant to phosphonoacetic acid were crossed with wild-type HSV-2, and recombinants that multiplied at nonpermissive temperature and were resistant to the drug were selected. The DNAs of the recombinants were mapped with XbaI, EcoRI, HpaI, HsuI, BglII, and, in some instances, KpnI restriction endonucleases. The results were as follows. (i) We established the colinear arrangements of HSV-1 and HSV-2 DNAs. (ii) There was extensive interchange of genomic regions, ranging from the exchange or the entire L of S component of HSV DNA to substitutions of regions within the same component. In some recombinants, the reiterated sequences ab and ac bracketing the L and S components of HSV DNA were heterotypic. Most recombinants grew well and showed no obvious defects. (iii) The number of crossover events ranged from one to as many as six. Although crossover events occurred throughout the DNA, some clustering of crossover events was observed. (iv) Analysis of recombinants permitted localization of several markers used in this study and appears to be a useful technique for marker mapping. (v) As previously reported, HSV DNA consists of four populations, differing in relative orientation of the L and S components. All recombinants could be displayed in one arrangement of L and S such that the number of crossover events was minimized. The data are consistent with the hypothesis that only one arrangement of the parental DNA participates in the generation of recombinants.

Base Sequence↗

PubMed1985

MR technology: effect of even-echo rephasing on calculated T2 values and T2 images.

In multiple spin-echo image sequences of blood flow, the "even-echo" phenomenon produces an absolute increase in signal magnitude from first- to second-echo images of normal vessels harboring slow flow. Distinguishing this from the apparent relatively high signal intensity seen on second-echo images in pathologic foci of stationary tissue is important to the diagnostician. Selected case material containing two tissue types was reviewed retrospectively: tissues known to harbor slow flow, such as normal veins and venous sinuses and vascular malformations, and tissues that have long transverse (T2) relaxation times and appear as intense structures on second-echo images, such as neoplasms, infarcts, and regions of demyelination. Calculations of T2 parameters were made by computer for defined regions of interest. T2 images were also generated. Visual inspection of the acquired images did not reliably distinguish increased intensity due to even-echo rephasing from the relative changes between adjacent tissues seen on second-echo images. More definitive differentiation of the even-echo phenomenon was provided by calculated values of T2 and computer-synthesized T2 images representing acquired intensity data of two-echo sequences. The synthesized images were especially useful when stationary tissue with lengthened T2 values was adjacent to or in proximity to vessels or vascular lesions. A five spin-echo image sequence was valuable for separating slow flow from stationary tissue by a technique of synthesizing T2-difference images using three consecutive echoes.

Brain Diseases↗

PubMed2004

Analysis and synthesis of textured motion: particles and waves.

Natural scenes contain a wide range of textured motion phenomena which are characterized by the movement of a large amount of particle and wave elements, such as falling snow, wavy water, and dancing grass. In this paper, we present a generative model for representing these motion patterns and study a Markov chain Monte Carlo algorithm for inferring the generative representation from observed video sequences. Our generative model consists of three components. The first is a photometric model which represents an image as a linear superposition of image bases selected from a generic and overcomplete dictionary. The dictionary contains Gabor and LoG bases for point/particle elements and Fourier bases for wave elements. These bases compete to explain the input images and transfer them to a token (base) representation with an O(10(2))-fold dimension reduction. The second component is a geometric model which groups spatially adjacent tokens (bases) and their motion trajectories into a number of moving elements--called "motons." A moton is a deformable template in time-space representing a moving element, such as a falling snowflake or a flying bird. The third component is a dynamic model which characterizes the motion of particles, waves, and their interactions. For example, the motion of particle objects floating in a river, such as leaves and balls, should be coupled with the motion of waves. The trajectories of these moving elements are represented by coupled Markov chains. The dynamic model also includes probabilistic representations for the birth/death (source/sink) of the motons. We adopt a stochastic gradient algorithm for learning and inference. Given an input video sequence, the algorithm iterates two steps: 1) computing the motons and their trajectories by a number of reversible Markov chain jumps, and 2) learning the parameters that govern the geometric deformations and motion dynamics. Novel video sequences are synthesized from the learned models and, by editing the model parameters, we demonstrate the controllability of the generative model.

Algorithms↗

PubMed2000

Identification of oxidant-sensitive proteins: TNF-alpha induces protein glutathiolation.

Reactive oxygen species are thought to play a role in a variety of physiologic and pathophysiological processes. One possible mediator of oxidant effects at the molecular level is a subset of proteins containing reactive cysteine thiols that can be readily oxidized. The transient incorporation of glutathione into cellular proteins is an established response to oxidant stress and could provide a mechanism for reversible covalent modification in response to reactive oxygen species. To better understand the function of protein S-glutathiolation in vivo, a biotinylated membrane-permeant analogue of glutathione, biotinylated glutathione ethyl ester, was developed and used to detect proteins into which glutathione is incorporated under oxidant stress. Oxidant stress from exogenous hydrogen peroxide or generated in response to TNF-alpha was found to increase incorporation of biotinylated glutathione ethyl ester into several HeLa cell proteins. The identity of two of these proteins was determined by peptide sequencing and mass spectrometric peptide mapping. A 23 kDa S-glutathiolated protein was identified as thioredoxin peroxidase II, a member of the peroxiredoxin family of peroxidases known to play a role in redox-dependent growth factor and cytokine signal transduction. A second, 36 kDa, protein was identified as annexin II. Further investigation revealed a single reactive cysteine in the annexin II tail domain. Deletion of the identified cysteine was found to abolish S-glutathiolation of annexin II. These findings demonstrate a specific posttranslational modification associated with an endogenously generated oxidant stress and suggest a mechanism by which TNF-alpha might selectively regulate protein function in a redox-dependent fashion.

Animals↗

PubMed1996

Characterization of mitochondrial DNA using low-stringency single specific primer amplification analyzed by laser induced fluorescence--capillary electrophoresis.

Polymerase chain reaction (PCR)-based DNA typing is routinely used in forensics for identity testing. Those assays that distinguish single nucleotide polymorphisms (SNPs) require other biochemical reactions in addition to PCR to identify the sequence polymorphisms. Low-stringency sequence-specific PCR (LSSP-PCR) is an example of a recent method that does not require additional biochemical treatments. The analysis of LSSP-PCR by capillary electrophoresis (CE) to discriminate the highly polymorphic mitochondrial DNA (mtDNA) D-loop region is described. The DNA from five individuals were amplified (first step) using sequence-specific primers to produce 1021 bp fragments containing the D-loop region. Each fragment was isolated by electroelution using CE and UV detection, and subjected to a second amplification (second step) using a single primer annealed under low stringency conditions. This generated a range or profile of PCR products for each sample, which were resolved and analyzed by CE with the intercalator TOTO-1 and laser-induced fluorescence (LIF) detection. The LSSP-PCR profiles were unique for each individual, indicating that this technique may be applicable for forensic identity testing.

DNA Primers↗

PubMed2006

Matrix-assisted laser desorption/ionization in-source decay combined with tandem time-of-flight mass spectrometry of permethylated oligosaccharides: targeted characterization of specific parts of the glycan structure.

Matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) has been introduced in recent years as a valuable tool for the structural characterization of permethylated oligosaccharides. In this report, we describe the combination of MALDI in-source decay (ISD) with the subsequent TOF/TOF-MS analyses of specific fragments, allowing the detailed characterization of the selected part of the oligosaccharide molecule. Part of the second-generation fragment ions were different from those observed in conventional MALDI-TOF/TOF-MS experiments. Other fragments, which had already been observed in conventional MALDI-TOF/TOF-MS and again showed up in second-generation fragment analysis, could be assigned to specific parts of the molecule. Our approach disclosed different structural features of the oligosaccharides: due to permethylation, the glycosidic linkage fragments allowed the distinction between terminal, monosubstituted and disubstituted monosaccharides and indicated the oligosaccharide sequence. Moreover, substitution positions were deduced based on characteristic cross-ring fragmentation by high-energy collision-induced fragmentation. In conclusion, combination of MALDI-ISD with TOF/TOF-MS allows the detailed characterization of specific moieties of permethylated oligosaccharides and is, therefore, a powerful technique for structural glycomics.

Animals↗