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Broader cross-reactivity after conjugation of V3 based multiple antigen peptides to HBsAg.

Vaccines against highly variable pathogens should elicite antibodies to a huge number of clinical isolates. For this purpose, new strategies to overcome the variability are needed. We have previously reported a useful method to conjugate multiple antigen peptides (MAPs) to carrier proteins. Also, we have suggested that these conjugates might enhance cross-reactivity in comparison to other synthetic structures. In this work, MAPs were synthesized and their respective conjugates to HBsAg were obtained. Two peptides from the V3 loop of HIV-1 were included in the MAPs as B cell epitopes because of their variability. Groups of mice were immunized and the immunogenicity and the level of cross-reaction to a panel of five heterologous V3 peptides were studied. Our results show that sera from mice immunized with MAPs coupled to HBsAg recognize a higher number of heterologous peptides (P < 0.05). This behavior was related neither to the immunogenicity nor the antigenicity of the synthetic structures. These results have important implications for the choice of better immunogens against variable epitopes.

AIDS Vaccines↗

Vascular smooth muscle cell proliferation requires both p38 and BMK1 MAP kinases.

Vascular smooth muscle cell (VSMC) proliferation is a key event in the progression of atherosclerosis. Induction of both c-fos (through the transcription factor Elk-1) and c-jun, both immediate early genes, is important for the stimulation of VSMC proliferation and migration. It was earlier found that p38 mitogen-activated protein (MAP) kinase upregulates c-jun gene transcription through phosphorylation of two myocyte enhancer factor 2 (MEF2) family transcription factors, MEF2A and MEF2C, while big MAP kinase 1 (BMK1) may upregulate c-jun gene transcription through MEF2A, MEF2C, and also MEF2D. Here, we report that inhibition of BMK1 by a dominant negative form of MEK5 or pharmacologic inhibition of p38 by SB 203580 additively suppress serum-induced VSMC proliferation. This additive effect of p38 and BMK1 inhibition implies that these two kinases coordinately regulate MEF2 transcription factors. The exclusive activation of MEF2D by BMK1 appears required for this cooperative upregulation of c-jun in VSMC, and coactivation of p38 and BMK1 also has additive effects on the activation of a reporter gene linked to the c-jun promoter in our experimental system. Thus, coordinate activity of both the p38 and BMK1 pathways appears necessary for optimal transcription of c-jun and, pari pasu, VSMC proliferation. These results may have implications for the future design of pharmacologic agents for inhibition of VSMC growth.

Animals↗

Combined radiofrequency ablation-cooling catheter for reversible cryothermal mapping and ablation.

Reversible cryothermal mapping of cardiac arrhythmias has been performed intraoperatively. However, a steerable cooling catheter for reversible mapping has not yet been developed. We therefore developed and tested a cooling system consisting of a -15 degrees C hypertonic saline reservoir and a 7F steerable catheter also capable of radiofrequency (RF) ablation. Using excised ovine hearts placed in a 37 degrees C circulating saline bath, we measured the temperatures at depths of 0 mm, 1 mm, and 2 mm. The temperature after 90 seconds of cooling was 16.5 +/- 2.1 degrees C at 0 mm compared to 23.9 +/- 4.1 degrees C at 1 mm and 31.1 +/- 3.9 degrees C at 2 mm depth (p < 0.01). These data suggest that a 7F steerable combined RF ablation-cooling catheter may achieve temperatures suitable for mapping arrhythmias such as atrial tachycardias and right ventricular outflow tract tachycardias. Further enhancements to achieve lower temperatures at depth may be needed to reversibly map other arrhythmias such as left ventricular tachycardias.

Animals↗

Mast cell tryptase stimulates DLD-1 carcinoma through prostaglandin- and MAP kinase-dependent manners.

We found that striptease-positive mast cells were abundant in the invasive front of human colon adenocarcinoma by examining 30 cases. Because tryptase has been suggested to be the agonist proteinase for protease-activated receptor-2 (PAR-2), we investigated the effects of stimulation of PAR-2 by tryptase on the cell signaling and proliferation of DLD-1, a human colon carcinoma cell line. PAR-2 stimulation by tryptase induced the increase in [Ca(2+)](i), which was desensitized by the prior application of PAR-2 activating peptide (AP). The proliferative responses of DLD-1 to tryptase and PAR-2 AP were associated with the phosphorylation of MEK and MAP kinase. Inhibition of MEK by PD98059 completely inhibited the proliferation-enhancing effects of tryptase and PAR-2 AP as well as phosphorylation of MAP kinase. Moreover, tryptase and PAR-2 AP stimulated the production of prostaglandin E2 and the inhibition of prostaglandin synthesis by indomethacin or NS398 resulted in the complete inhibition of the proliferative responses to tryptase and PAR-2 AP. Furthermore, the tryptase-stimulated proliferation of DLD-1 was concentration-dependently inhibited by nafamostat mesilate, a specific inhibitor of tryptase. These results as a whole indicated that tryptase has proliferative effects on DLD-1 through cyclooxygenase- and MAP kinase-dependent manners acting on PAR-2 by its proteolytic activity.

Benzamidines↗

An encyclopedia of human enhancer-gene regulatory interactions.

Identifying transcriptional enhancers and their target genes is essential for understanding gene regulation and the effect of human genetic variation on disease1-6. Here we create and evaluate a resource of more than 92&#x2009;million enhancer-gene regulatory interactions across 1,458 biosamples covering 369 cell types and tissues, by integrating predictive models, chromatin states, three-dimensional contacts and large-scale genetic perturbations generated by the ENCODE Consortium7. We first create a systematic benchmarking pipeline to compare predictive models, assembling a dataset of 10,356 element-gene pairs measured in CRISPR perturbation experiments, more than 30,000 fine-mapped expression quantitative trait loci and 569 fine-mapped genome-wide association study&#xa0;(GWAS) variants linked to a probable causal gene. Using this framework, we develop ENCODE-rE2G, a predictive model achieving state-of-the-art performance across several prediction tasks, demonstrating that iterative perturbations and supervised machine learning can build increasingly accurate predictive models of enhancer regulation. Using ENCODE-rE2G, we build an encyclopedia of enhancer-gene regulatory interactions in the human genome, revealing global properties of enhancer networks, identifying differences in regulatory complexity across genes and improving analyses linking noncoding variants to target genes and cell types for common complex diseases. By interpreting the model, we find that beyond enhancer activity and three-dimensional enhancer-promoter contacts, additional features that&#xa0;guide enhancer-promoter communication include promoter class and enhancer-enhancer synergy. These genome-wide maps of enhancer-gene regulatory interactions, benchmarking software, predictive models and insights about enhancer function provide a valuable resource for future studies of gene regulation and human genetics.

Humans↗

Proteins binding to site C2 (muE3) in the immunoglobulin heavy-chain enhancer exist in multiple oligomeric forms.

We describe the purification to near homogeneity of proteins binding to site C2 (muE3) in the immunoglobulin heavy-chain enhancer. Proteins binding to this site produce four protein-DNA complexes which are distinguished by their mobility in gel retardation assays and their elution properties in an anion exchange column. DNA affinity-purified preparations of three chromatographically separated pools, containing different subsets of the four complexes, each contained three polypeptides of 42.5, 44, and 45 kilodaltons (kDa). UV crosslinking of protein to enhancer DNA demonstrated that site C2-binding activities in the three different pools bound DNA through proteins of similar sizes (about 45 kDa), even though the protein-DNA complexes formed by these binding activities were quite distinct. Gel exclusion chromatography and equilibrium binding analyses indicated that the distinct protein-DNA complexes were due to different oligomeric forms of the individual subunits and that a larger multimeric form bound with high affinity to the heavy-chain enhancer site C2, while a smaller species had a much lower affinity for heavy-chain enhancer sequences. Purified protein has been used to map high-affinity binding sites for site C2-binding proteins within an immunoglobulin heavy-chain promoter and at site KE3 in the kappa light-chain enhancer.

Animals↗

Antigenic regions defined by monoclonal antibodies correspond to structural domains of avian lysozyme.

The epitopes recognized by six new BALB/c hybridomas specific for the protein antigen hen egg-white lysozyme (HEL) were mapped in detail. Although fine specificities of all the antibodies were distinct, many of the epitopes overlap in complex patterns. The antibodies could be grouped into three complementation groups, one of which also included the previously characterized HyHEL -5 which is specific for Arg68 . Complex interactions were observed among the antibodies, both among and within complementation groups, including nonreciprocal competition and enhanced binding. Two of the complementation groups mapped near the catalytic site in a new antigenic region. The antibodies HyHEL -8 and HyHEL -10 had very similar and over-lapping specificities, and may recognize very closely related epitopes. The results suggest that the epitopes may form a continuous antigenic surface, and that antigenic regions correspond to structural domains defined by the tertiary structure of HEL.

Animals↗

Mapping 5' termini of JC virus early RNAs.

Within its enhancer promoter region, the MAD-1 strain of JC virus (JCV) has two 98-base-pair tandem repeats, each containing a TATA box-like sequence. In the present study, polyadenylated early JCV mRNAs were isolated 5 or 29 days after infection of primary human fetal glial (PHFG) cells. By using S1 nuclease, the 5' termini of the early mRNAs were mapped to nucleotide position(s) (np) 122 through 125, which lies within an AT rich region (at np 113 through 127). In contrast, when JCV DNA was transcribed in vitro, we observed a single major cluster of 5' start sites at np 94 through 97, which is approximately 25 base pairs downstream from one of the TATA boxes. By day 5, the earliest time at which JCV RNA was detected, viral DNA replication had begun; it continued for at least an additional 20 days. Since more late than early RNA was present at 5 days postinfection, the early RNAs whose synthesis began at np 122 through 125 may be analogous to SV40 late early mRNA (Ghosh and Lebowitz, J. Virol. 40:224-240, 1981). However, we have not detected RNAs with 5' termini 25 to 30 bp downstream from the TATA box at earlier times. While JCV contains two identical TATA boxes, one in each of the 98-bp repeats, only the upstream TATA box functions as an early promoter element.

Cell Line↗

[Hemodynamic changes after application of vasoactive substances in cats with unilateral renal artery constriction in addition to sinoaortal baroreceptor deafferentation].

The relationship between total peripheral resistance (TPR), cardiac output (CO) and venous return (VR) and their role in the response of mean arterial pressure (MAP) on intravenous adrenalin (10 micrograms/kg) and angiotensin II (0.5 micrograms/kg), resp., were investigated in normal cats (A), cats with unilateral renal artery stenosis (B) and cats with sinoaortal baroreceptor deafferentation (C). In the maximum MAP increase in group A there participated both TPR and VR through altered CO. In group B the MAP increased after angiotensin II only in consequence of an enhanced TPR, whereas the VR response was dampened; only after adrenalin the VR contributed a little to MAP increase through CO. In group C, the MAP increase after both substances was due exclusively to increased TPR; at a restricted ejection capacity of the heart the VR cannot act on the level of blood pressure.

Angiotensin II↗

[Possibilities of the use of MR tomography-based cerebral blood volume maps in the diagnosis of brain tumors].

PURPOSE: Today contrast enhanced MR imaging is a reliable method for detecting mostly distinguishing between different histological types of tumours. In this study we use a MR-based method to measure the regional cerebral blood volume (rCBV). Using this technique we try to judge the grading and vitality of the tumours. METHODS: 26 patients with various types of brain tumours were examined. To calculate rCBV-maps of one slice, low-dosed Gd-DTPA was injected as a bolus. Using the relaxation effect the obtained signal intensity-time curves were converted pixel-wise into rCBV images. For the tumours rCBV-ratios were calculated relative to the corresponding area in the contralateral hemisphere. RESULTS: In the investigated group all tumours were detected on the basis of a raised rCBV-ratio. Since only vital parts of the tumour are perfused, the rCBV maps may be used to determine the place of biopsy. CONCLUSIONS: The differential diagnosis of all histological tumour types was not possible on the basis of rCBV values. Distinction between low grade and high grade gliomas was also not significant. However, a low grade glioma can be excluded if the morphological images definitely indicate an astrocytoma and if the rCBV-ratio was higher than 2.

Adult↗

Chromosomal assignment and tissue expression of CBF-C/NFY-C, the third subunit of the mammalian CCAAT-binding factor.

The mammalian CCAAT-binding factor CBF (NFY) consists of three subunits, CBF-A, CBF-B, and CBF-C. All three subunits are evolutionarily conserved and are essential for DNA binding of CBF. In this study we report the identification of human and plant homologs of CBF-C. Northern analysis revealed that, like the other two subunits, CBF-C was produced at equal levels in all rat tissues that were examined. We assigned the mouse CBF-C gene (designated Nfyc) to chromosome 4 with tight linkage to Lmyc. Our mouse linkage data suggest that the human NFYC homolog will map to 1p32.

Amino Acid Sequence↗

Myocardial T1 mapping for detection of left ventricular myocardial fibrosis in chronic aortic regurgitation: pilot study.

OBJECTIVE: The aim of this study was to identify diffuse myocardial fibrosis secondary to chronic aortic regurgitation by comparing the T1 relaxation times of left ventricular myocardium in a pilot patient group with a previously established normal range of times. SUBJECTS AND METHODS: Eight patients with chronic aortic regurgitation and normal coronary arteries awaiting surgical valve replacement underwent a comprehensive MRI examination that included assessment of left ventricular function, severity of valvular regurgitation, and presence of overt myocardial scar evidenced by delayed enhancement. For each patient, myocardial T1 relaxation times determined with a modified Look-Locker technique before and after contrast administration were compared with values previously established for 15 healthy volunteers. RESULTS: There was no statistical difference (p > 0.05) in slice-averaged myocardial T1 relaxation times either before or after gadolinium administration in the patient group compared with the normal range of times. Segmental averaged T1 relaxation times in segments with abnormal wall motion did, however, show statistically significant differences from healthy controls 10, 15, and 20 minutes after administration of gadolinium (510 vs 476 milliseconds, p = 0.001; 532 vs 501 milliseconds, p = 0.002; 560 vs 516 milliseconds, p = 0.001, respectively). Two of the aortic regurgitation patients also had focal areas of myocardial delayed enhancement. CONCLUSION: Segment-based myocardial T1 mapping has the potential for showing differences between relaxation times in aortic regurgitation and in normal hearts, suggesting the existence of a diffuse myocardial fibrotic process.

Adult↗

Angiotensin converting enzyme inhibition improves baroreflex-induced noradrenaline spillover responses in rabbits with heart failure.

Impaired baroreflex function is a characteristic feature of congestive heart failure (CHF), although the mechanism is obscure. This study examined the hypothesis that activation of the renin-angiotensin system contributes to baroreflex dysfunction in CHF. The acute effects of an angiotensin converting enzyme inhibitor, enalaprilat, on baroreflex-mediated changes in heart rate (HR), total and renal noradrenaline (NA) spillover rates were examined in conscious rabbits with doxorubicin-induced cardiomyopathic CHF. Studies were performed under resting conditions and in response to changes in mean arterial pressure (MAP) induced by sodium nitroprusside and phenylephrine infusions. Seven saline-treated (normal group) and 11 doxorubicin-treated rabbits (1 mg/kg administered intravenously twice weekly) were studied after 4 and 6 weeks' treatment. Five CHF rabbits received saline (C group) and 6 enalaprilat infusion (ACEI group) during each study period. After 4 weeks of doxorubicin, baroreflex-HR responses were normal, whereas baroreflex-NA spillover responses were enhanced. Enalaprilat infusion shifted the HR-MAP curve downwards to the left but had no effect on the NA spillover-MAP curves. After 6 weeks of doxorubicin, when CHF was established, baroreflex-HR and NA spillover curves were depressed. At this stage, enalaprilat had little effect on the HR-MAP curve but restored towards normal the NA spillover-MAP curves. The results suggest that the endogenous renin-angiotensin system contributes to attenuated baroreflex responses when CHF is established.

Adrenergic alpha-Agonists↗

Phosphorylation of retinoid X receptor suppresses its ubiquitination in human hepatocellular carcinoma.

Retinoid X receptor alpha (RXRalpha) has emerged as an important nuclear receptor involved in hepatocarcinogenesis, because its ligand suppresses the development of hepatocellular carcinoma (HCC) in both experimental and clinical studies. We have demonstrated that phosphorylation of RXRalpha at serine 260 interferes with its function and delays its degradation in cultured human HCC, leading to enhanced cellular proliferation. Here, we show that in normal liver and in nonproliferating hepatocyte cultures, RXRalpha is unphosphorylated and highly ubiquitinated, rendering it sensitive to proteasome-mediated degradation. On the other hand, phosphoserine 260 RXRalpha is resistant to ubiquitination and proteasome-mediated degradation in both human HCC tissues and a human HCC cell line, HuH7. In these tissues and cells, serine 260 is phosphorylated by mitogen-activated protein (MAP) kinase. In proliferating normal hepatocytes, similar to HCC cells, RXRalpha is also phosphorylated at serine 260 and resistant to ubiquitin-mediated degradation by proteasome, but this ubiquitination of RXRalpha is differentially regulated between HCC cells and normal hepatocytes. In proliferating hepatocytes, 9-cis retinoic acid (9cRA), a ligand to RXRalpha, suppresses MAP kinase-mediated phosphorylation and thereby enhances ubiquitination of RXRalpha, whereas it fails to exert these effects in HCC cells. In conclusion, switching of the ubiquitin/proteasome-dependent degradation of RXRalpha by phosphorylation at serine 260 may be responsible for the aberrant growth of HCC and its suppression by retinoids.

Carcinoma, Hepatocellular↗

The murine SP-C promoter directs type II cell-specific expression in transgenic mice.

Genomic DNA from the mouse pulmonary surfactant protein C (SP-C) gene was analyzed in transgenic mice to identify DNA essential for alveolar type II cell-specific expression. SP-C promoter constructs extending either 13 or 4.8 kb upstream of the transcription start site directed lung-specific expression of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. In situ hybridization analysis demonstrated alveolar cell-specific expression in the lungs of adult transgenic mice, and the pattern of 4.8 SP-C-CAT expression during development paralleled that of the endogenous SP-C gene. With the use of deletion constructs, lung-specific, low-level CAT activity was detected in tissue assays of SP-C-CAT transgenic mice retaining 318 bp of the promoter. In transient and stable cell transfection experiments, the 4.8-kb SP-C promoter was 90-fold more active as a stably integrated gene. These findings indicate that 1) the 4.8-kb SP-C promoter is sufficient to direct cell-specific and developmental expression, 2) an enhancer essential for lung-specific expression maps to the proximal 318-bp promoter, and 3) the activity of the 4.8-kb SP-C promoter construct is highly dependent on its chromatin environment.

Animals↗

Enhancing spatial estimates of metal pollutants in raw wastewater irrigated fields using a topsoil organic carbon map predicted from aerial photography.

Various approaches have been used to estimate metal pollutant element (TE) contents at unsampled locations in a 15-ha contaminated site located in the plain of Pierrelaye-Bessancourt (about 24 km Northwest of Paris). 87 samples of soil plough layer were randomly sampled at each mesh of a regular square grid over the whole study area and the total contents of Cd, Cr, Cu, Ni, Pb, and Zn were measured. A first set of 50 measurements, randomly selected from the 87 samples, was used for the prediction and another set of 37 measurements was kept for the validation. Topsoil organic carbon contents (SOC) were measured at 75 sites with 50 measurements sharing the same locations as TE. An aerial photography of the study area showing bare soils was selected for relating brightness intensities and SOC. Mapping procedures used were ordinary kriging (OK), cokriging (COK), collocated cokriging (CC), and kriging with external drift (KED). SOC maps used as exhaustively sampled information in KED and CC of TE were obtained by KED and CC procedures, respectively, accounting for 75 SOC measurements and the brightness intensities of numerical counts provided by the visible bands of the aerial photograph bare soils. Consequently, for each TE, four maps were generated: two maps resulting from KED and CC procedures (KED-SOC75P, CC-SOC75P), another one provided by standard cokriging (COK-TE50SOC75) accounting for TE prediction set plus 75 SOC measurements, and the last one corresponding to that estimated by ordinary kriging from only prediction set measurements (OK50). Three indices: (1) the mean prediction error (ME) and the mean absolute prediction error (|ME|); (2) the root mean square error (RMSE); and (3) the relative improvement (RI) of accuracy, as well as residuals analysis, were computed from the validation set (observed data) and predicted values. On the 37 test data, the results showed that the more accurate predictions were systematically those obtained by kriging accounting for SOC map predicted by KED from 75 SOC measurements and brightness values of the aerial photo (KED-SOC75P) followed closely by CC-SOC75P procedure, except for Cu and Zn where CC-SOC75P appeared to be slightly more accurate than KED-SOC75P. In regard to the RI of accuracy between prediction methods, the results confirmed once for all the benefit of accounting for SOC data set plus the exhaustively sampled information provided by the aerial photography regardless of the considered TE. Nevertheless, for Cd, Pb, and Zn, the RI of accuracy was less than 20% between the two most accurate methods (KED-SOC75P and CC-SOC75P) and standard cokriging in which the information provided by the aerial photography is ignored when mapping. The sensitivity of KED-SOC75P and CC-SOC75P approaches to the sampling density of the target variables (TE) was assessed using 10 random subsets of different sizes (25 and 33 observations) drawn from a prediction set that includes 50 data. Results have shown that the TE estimates by KED-SOC75P and CC-SOC75P approaches using only 25 TE samples were much more accurate than the estimates performed by OK50 and COK-TE50SOC75 approaches that use the whole samples of the prediction set. Moreover, the RI of accuracy was reduced by less than 15% if the original sampling density was reduced by a third.

Agriculture↗

Body surface Laplacian mapping in patients with left or right ventricular bundle branch block.

Body surface Laplacian maps (BSLMs) have been previously reported to provide enhanced capability in localizing and resolving multiple spatially separate myocardial events. However, only a few studies have been reported on the clinical applications of BSLM. To test the clinical utility of BSLMs, BSLMs and body surface potential maps (BSPMs) during ventricular depolarization for complete right or left ventricular bundle branch block (CRBBB or CLBBB) were studied in ten patients in each group. As a control group, ten healthy subjects were also studied using the same procedure. One hundred and twenty-eight electrodes were placed uniformly over the entire chest and back of the subjects. BSLMs were computed from recorded potentials, using a numerical algorithm. The BSLMs showed multiple and more localized positive and negative activities compared with the BSPMs. In healthy subjects, the BSLMs showed multiple areas of positive activity overlying the RV, LV, and the RV outflow, and negative activity corresponding to RV free-wall breakthrough and LV anterolateral breakthrough sites, whereas the BSPMs could not separate RV and LV activities. In the patients with CRBBB, the BSLMs showed more localized areas of activity corresponding to the LV apex breakthrough and LV lateral breakthrough, and separated LV lateral and posterior activation. In the patients with CLBBB, the BSLMs showed multiple RV activation, and propagating activation of LV from lateral to posterior. The BSLMs appear to provide enhanced capability in detecting multiple ventricular electrical events associated with normal and abnormal conduction and a more detailed activation sequence of both ventricles in healthy subjects and in the patients with CRBBB and CLBBB. BSLM may provide an important alternative to other imaging modalities in localizing cardiac electrical activity noninvasively.

Action Potentials↗

Enhancement of TNF-alpha-induced apoptosis by immobilized arginine-glycine-aspartate: involvement of a tyrosine kinase-dependent, MAP kinase-independent mechanism.

Extracellular matrix facilitates anchorage-dependent cell survival via interaction of its arginine-glycine-aspartate (RGD) motif with integrins. In this report, we describe an unexpected, apoptosis-promoting the effect of immobilized RGD (iRGD) on tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis. Mesangial cells cultured on RGD-coated plates showed enhanced susceptibility to TNF-alpha-induced apoptosis. iRGD alone did not affect cell survival. In contrast, iRGD did not facilitate but inhibited apoptosis induced by H(2)O(2). Mitogen-activated protein (MAP) kinases and tyrosine kinases are important mediators for the RGD-integrin signaling. Pretreatment with MAP kinase kinase inhibitor PD098059, c-Jun N-terminal kinase (JNK)-c-Jun/AP-1 inhibitor curcumin or p38 MAP kinase inhibitor SB203580 did not attenuate the apoptosis-promoting effect of iRGD. Consistently, transfection with dominant-negative mutants of extracellular signal-regulated kinases, JNK or p38 MAP kinase did not inhibit the effect of iRGD. In contrast, protein tyrosine kinase inhibitors, genistein, and herbimycin A, abrogated the apoptosis-promoting effect of iRGD. Of note, TNF-alpha-induced apoptosis on uncoated plates was not attenuated by tyrosine kinase inhibitors. These data provide the first evidence that iRGD accelerates certain apoptosis. We identified that the effect was mediated by the tyrosine kinase-dependent, MAP kinase-independent mechanism.

Animals↗