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PubMed1997

Activation of phospholipase D by growth factors and oncogenes in murine fibroblasts follow alternative but cross-talking pathways.

Phospholipase D (PLD) is activated by a variety of stimuli, including mitogenic stimulation by growth factors and oncogene transformation. Activation of PLD by growth factors requires protein kinase C (PKC) since depletion of the enzyme by down-regulation or direct inhibition by specific drugs completely abrogates this effect. Transformation by the ras and src oncogenes is also associated with an increase in basal PLD activity. However, this effect is not dependent on PKC, suggesting that growth factors and oncogenes may activate PLD by two independent mechanisms. Here we demonstrate that activation of PLD by phorbol esters is greatly enhanced in ras-transformed cells, suggesting synergistic activation of PLD by ras oncogenes and PKC. Also, ras-transformed cells showed a dramatic attenuation of the PLD activation induced by growth factors, although receptor function was still detectable. This attenuation paralleled the specific uncoupling of the phosphatidylinositol-specific phospholipase C (PI-PLC) pathway, indicating that activation of PLD by growth factors may be mediated by PI-PLC and PKC activation. Attenuation of PLD activation by platelet-derived growth factor was also observed in several oncogene-transformed cells, as well as the uncoupling of the PI-PLC pathway. Neither the co-operation with PKC activation nor the attenuation of the PLD response to growth factors in ras-transformed cells was a general consequence of cell transformation, since cells transformed by other oncogenes showed a normal response to either treatment. These results support the existence of at least two alternative signalling routes for the activation of PLD, one mediated by the PI-PLC/diacylglycerol/PKC pathway and a second one mediated by several oncogenes, independent of the PKC pathway, which synergizes with the PI-PLC/PKC-dependent pathway.

3T3 Cells↗

PubMed2001

Allograft inflammatory factor-1 augments production of interleukin-6, -10 and -12 by a mouse macrophage line.

Mouse allograft inflammatory factor-1 (AIF-1) cDNA was cloned and the AIF-1-specific monoclonal antibodies were established to examine its tissue distribution. The mouse AIF-1 was highly conserved among all reported AIF-1 from a variety of species, from invertebrates to mammals, and the cloned cDNA was in good accordance with putative expressed regions of genomic sequences in the mouse major histocompatibility complex (MHC) class III region. The messages of mouse AIF-1 were abundantly expressed in the testis, moderately in the spleen and lymph nodes and slightly in the liver and thymus of normal BALB/c mice. Immunohistological examination revealed that differentiating germ cells in the testis and presumably macrophages in the red pulp of the spleen were positive for AIF-1. To analyse the function of the AIF-1, a macrophage cell line, RAW 264.7, was transfected with mouse AIF-1 cDNA. Upon stimulation with bacterial lipopolysaccharide, the transfectants that overexpressed AIF-1 showed marked morphological changes and produced significantly large amounts of interleukin (IL)-6, IL-10 and IL-12p40 but not IL-12p70 compared with control cells. No difference was noted in production of tumour necrosis factor-alpha, transforming growth factor-beta1 and IL-1alpha. These results suggest that AIF-1 plays an important role in cells of a monocyte/macrophage lineage upon stimulation with inflammatory stimuli by augmenting particular cytokine production.

Amino Acid Sequence↗

PubMed2003

The defibrillation efficacy of high frequency alternating current sinusoidal waveforms in guinea pigs.

There have been few basic studies of alternating current (AC) defibrillation, despite growing interest in the ability of AC to terminate or alter ongoing fibrillation. Based on fibrillation threshold testing, it has been suggested that cardiac tissue is most sensitive to long duration, low strength AC stimulation at around 50 Hz. This has not been directly tested for defibrillation. Two subcutaneous electrodes were placed 40 mm apart on opposing aspects of the guinea pig thorax. Seven seconds were allowed to elapse between fibrillation initiation and defibrillation. The tested waveforms were at 50, 100, 200, 500, and 1000 Hz with 2, 4, 8, 16, and 32-cycles. The efficacy of every waveform was measured using a single stimulus in a large population of animals. Forty-one guinea pigs were used in the fixed energy group. Thirty-three guinea pigs were used in the fixed amplitude group with additional 1-cycle waveforms tested. The 200-Hz and the 2-cycle waveforms were significantly more efficacious than those at other frequencies (P < 0.02) and other durations (P < 0.001). The 50-Hz waveforms were the least successful. Amplitude, not duration or energy, was the determinate of efficacy for 2-cycle (the most efficacious) waveforms. Unlike low strength stimulation, defibrillation strength stimuli are most effective with high frequency (200 Hz) pulses (2 cycles).

Animals↗

PubMed1987

Mechanical relaxation of the hair bundle mediates adaptation in mechanoelectrical transduction by the bullfrog's saccular hair cell.

Mechanoelectrical transduction by hair cells of the frog's internal ear displays adaptation: the electrical response to a maintained deflection of the hair bundle declines over a period of tens of milliseconds. We investigated the role of mechanics in adaptation by measuring changes in hair-bundle stiffness following the application of force stimuli. Following step stimulation with a glass fiber, the hair bundle of a saccular hair cell initially had a stiffness of approximately equal to 1 mN X m-1. The stiffness then declined to a steady-state level near 0.6 mN X m-1 with a time course comparable to that of adaptation in the receptor current. The hair bundle may be modeled as the parallel combination of a spring, which represents the rotational stiffness of the stereocilia, and a series spring and dashpot, which respectively, represent the elastic element responsible for channel gating and the apparatus for adaptation.

Acclimatization↗

PubMed1997

Anti-interleukin-6 antibodies inhibit herpes simplex virus reactivation.

Herpes simplex viruses (HSVs) infect epithelial cells, become localized in neurons, and can reactivate in response to a variety of stimuli, including ultraviolet light and hyperthermia. The sequence of gene activation during viral replication is known, but the molecular linkage between exogenous stimuli and HSV reactivation has not been determined. It was hypothesized that interleukin (IL)-6 acts as a signal between exogenous stimuli and neurons, stimulating HSV reactivation from latency. Mouse corneas were infected with HSV-1, and ocular reactivation was induced 5-7 weeks later by thermal stress or corneal exposure to ultraviolet light. Anti-IL-6 monoclonal antibodies were administered to the latently infected mice 8-12 h before the reactivation stimulus. Treatment with anti-IL-6 antibodies resulted in significantly lower frequencies of ocular reactivation compared with those in mice treated with a control immunoglobulin. These results support the hypothesis that IL-6 plays a role in HSV reactivation from latency.

Animals↗

PubMed1993

Regulation of HIV production by blood mononuclear cells from HIV-infected donors: I. Lack of correlation between HIV-1 production and T cell activation.

The relationship between production of HIV-1 by peripheral blood mononuclear cells (PBMCs) from HIV-1-infected donors and the level of T cell activation by various stimuli was examined. Stimulation of PBMCs with soluble anti-CD3 antibody or staphylococcal enterotoxin/superantigen (SAg) was found to be 100-1000 times more effective at inducing production of HIV-1 than was stimulation with immobilized anti-CD3 or various other T cell activating agents. However, proliferation of CD4+ T cells and lymphokine production following stimulation with soluble anti-CD3 were less than with immobilized anti-CD3. To determine whether immobilized anti-CD3 stimulated cells may produce a factor(s) that suppresses HIV production, dual-chamber coculture experiments were performed in which soluble and immobilized anti-CD3-stimulated CD8-depleted PBMCs were separated by porous membranes. Stimulation of cells by immobilized anti-CD3 suppressed HIV-1 production by soluble anti-CD3-stimulated cells in the inner chamber, suggesting that diffusible factor(s) are involved in suppressing HIV-1 production. Experiments in which exogenous cytokines were added to cells stimulated with soluble anti-CD3 did not reveal the suppressive factor(s) produced; however, IL-7 was found to markedly increase HIV-1 production. Both T cells and monocytes were found to be required for soluble anti-CD3 to induce high levels of HIV-1 production, suggesting a role for adhesion molecules. Our results thus show that (1) soluble anti-CD3 is a powerful stimulus for HIV production, (2) there is not an absolute correlation between the level of HIV-1 production and T cell activation following stimulation of PBMCs with T cell activating agents, (3) immobilized anti-CD3 stimulation produces a factor that decreases HIV replication, and (4) T cell monocyte interactions are important for production of HIV-1 following stimulation with soluble anti-CD3.

Blood Donors↗

PubMed1991

Evidence that interleukin-1 and phorbol esters activate NF-kappa B by different pathways: role of protein kinase C.

Nuclear factor kappa B (NF-kappa B) is a ubiquitous transcription factor that affects expression of many genes, including immunoglobulin kappa (kappa), the interleukin-2 receptor alpha chain, and two genes in HIV-1. NF-kappa B can be activated by a number of stimuli, including pharmacological stimulation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) and treatment in vitro with either protein kinase C or protein kinase A. This has lead to the proposal that these kinases are key enzymes in the physiological activation of NF-kappa B as well. We have used a murine B cell line, 70Z/3, and T cell line, EL-4 6.1 C10, to study the activation of NF-kappa B by two physiological activators, interleukin-1 alpha (IL-1) and lipopolysaccharide (LPS). There are four reasons to propose that these agents activate pathways that do not include protein kinase C as a major component in these cell lines. First, the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) strongly inhibited PMA-induced activation of NF-kappa B in 70Z/3 cells but had no effect on NF-kappa B activated by IL-1 or LPS. Second, depletion of protein kinase C by prolonged growth of 70Z/3 in PMA abrogated the capacity of the cells to activate NF-kappa B in response to further PMA treatment. However, these same cells activated NF-kappa B normally after either IL-1 or LPS treatment. Third, IL-1 effectively activated NF-kappa B in EL-4 6.1 C10 cells, but PMA did not. Fourth, interferon-gamma is a potent activator of protein kinase C in 70Z/3 cells, but is completely inactive in the mobilization of NF-kappa B. These results suggest that the physiological inducers IL-1 and LPS activate NF-kappa B by pathways independent of protein kinase C in both 70Z/3 and EL-4 6.1 C10 cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1990

Induction and regulation of monocyte procoagulant activity.

Monocyte (MO) procoagulant activity (PCA) is induced by various stimuli including allogeneic stimulation, immunocomplexes, and bacterial products. Antigen-antibody complex stimulation therefore represents a pathway for MO PCA induction. Activation of MO PCA has been demonstrated in immunocomplex disease and could represent a major pathology in transplanted immunocomplex disease patients. Stimulation of monocytes via their FcRI receptor has been demonstrated to induce TNF and PGE2. This report demonstrates that stimulation of the high-density FcRI receptor-bearing (FcRI+) MO by resetting with anti-Rh coated erythrocytes also induces significant PCA levels (P less than 0.001). Muramyl dipeptide (MDP), a Gram-positive bacterial cell wall analogue, further increased PCA levels in the FcRI stimulated MO subpopulation (P less than 0.003). Although increased PCA levels were also induced in the FcRI- MO subpopulation by MDP (P less than 0.003), the FcRI+ MO responded with much greater levels of PCA and PGE2 (P less than 0.001). Greater PCA levels in the FcRI-positive MO subpopulation may indicate that stimulation of MO through their FcRI represents a different pathway from allogenic PCA activation, which can be augmented by subsequent bacterial challenge. A novel inhibitory effect of IL-4 on MO PCA induction is also demonstrated. IL-4 downregulated MO PCA levels either after isolation stimulation (55 +/- 19%), FcRI stimulation (57 +/- 12%), or FcRI plus MDP stimulation (60 +/- 13%). PCA and PGE2 levels were concomitantly downregulated by IL-4 both in the FcRI-stimulated, FcRI+ and in the MDP-stimulated FcRI- MO subpopulations. Since indomethacin blocked MDP induced MO PGE2 production without affecting MO PCA levels, PGE2 production is not required for FcRI-stimulated PCA induction.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed2006

Electroconvulsive therapy stimulus dose expressed as volume of seizure foci.

BACKGROUND: Electrical stimulus dose is typically stated as charge in millicoulombs (mC), representing the number of electrons. However, by overlooking the voltage threshold for neuronal depolarization, charge alone is inappropriate. Any charge can accumulate from subthreshold voltage over time. Stimulus energy represents heat content; without a delivery rate, it is not related to depolarization voltage. The objective was to formulate stimulus dose in accordance with seizure induction physiology. METHOD: Stimulus dose was expressed as volume of generated seizure foci, a variable dependent on current and charge. A second more detailed model considers brain voltages as related to clinical deep brain stimulation. RESULTS: For the constant current stimuli characteristic of widely available American stimulators, stimulus dose is proportional to current cubed multiplied by charge. This relationship corresponds to the 3 dimensions of a voltage field in space, where the volume above a certain voltage increases with the cube of the voltage radius. As a consequence, any particular charge at 0.9 A (ampere) current has a seizure foci dose approximately 50% higher than the same charge at 0.8 A. CONCLUSIONS: The modeling offers an explanation for published measurements comparing instruments of maximum current 0.8 and 0.9 A. The modeling also suggests compensation approaches to be applied to the age-based bilateral stimulus dosing method. An electrical dosing strategy based on "half-age" (2.5 mC/y) is reasonable for 0.9-A stimulation but a "three-quarters age" (3.7 mC/y) is more appropriate for 0.8-A stimulation. The present results can allow doses and seizure thresholds to be compared between different currents, as when substituting one electroconvulsive therapy instrument for another.

Electroconvulsive Therapy↗

PubMedDate not supplied

Massed and spaced learning in honeybees: the role of CS, US, the intertrial interval, and the test interval.

Conditioning the proboscis extension reflex of harnessed honeybees (Apis mellifera) is used to study the effect temporal spacing between successive conditioning trials has on memory. Retention is monitored at two long-term intervals corresponding to early (1 and 2 d after conditioning) and late long-term memory (3 and 4 d). The acquisition level is varied by using different conditioned stimuli (odors, mechanical stimulation, and temperature increase at the antenna), varying strengths of the unconditioned stimulus (sucrose), and various numbers of conditioning trials. How learning trials are spaced is the dominant factor both for acquisition and retention, and although longer intertrial intervals lead to better acquisition and higher retention, the level of acquisition per se does not determine the spacing effect on retention. Rather, spaced conditioning leads to higher memory consolidation both during acquisition and later, between the early and long-term memory phases. These consolidation processes can be selectively inhibited by blocking protein synthesis during acquisition.

Animals↗

PubMed1990

Neutrophil beta-2 microglobulin: an inflammatory mediator.

Beta-2 microglobulin (beta 2m) constitutes the light invariant chain of HLA class I antigen, and is a constituent of mobilizable compartments of neutrophils. Two forms of beta 2m exist: native beta 2m and proteolytically modified beta 2m (Des-Lys58-beta 2m), which shows alpha mobility in crossed radioimmuno-electrophoresis. The modification of native beta 2m can be executed by membrane-associated activity of mononuclear cells, and Des-Lys58-beta 2m augments the production of interleukin 2. In this study we present evidence that human neutrophils contain native beta 2m in specific granules, secretory vesicles, and plasma membrane. Beta 2m was released in the native form from neutrophils in response to stimulation with chemotactic stimuli and phorbol ester. The results of experiments designed to study the modification of native beta 2m by neutrophils indicated that neutrophils do not participate in the proteolysis of beta 2m. However, we demonstrated that native beta 2m following degranulation may be transformed to Des-Lys58-beta 2m by lymphocytes. We suggest that neutrophil beta 2m following exocytosis may be transformed to Des-Lys58-beta 2m, acting as an extracellular messenger between granulocytes and lymphocytes in the inflammatory focus.

Adolescent↗

PubMed1999

Responsiveness of rat substantia gelatinosa neurones to mechanical but not thermal stimuli revealed by in vivo patch-clamp recording.

1. Synaptic responses of 46 substantia gelatinosa (SG) neurones in the spinal dorsal horn to cutaneous mechanical and/or thermal stimuli were investigated in an in vivo rat preparation with whole-cell patch-clamp recordings. The clamped neurones were identified as being in the SG based on either their morphological features by intrasomatic injection of biocytin or the depth of the neurones from the surface of the spinal cord. 2. In all SG neurones examined where spontaneous EPSCs occurred, pinch (noxious) and air (innocuous) stimuli applied to the ipsilateral hindlimb elicited a barrage of EPSCs (some of which initiated an action potential under current-clamp conditions), which subsided just after cessation of the stimuli without any residual slow current (or after-discharge). The spontaneous and evoked EPSCs were reversibly abolished by a non-N-methyl-D-aspartate (non-NMDA) receptor antagonist, CNQX (20 microM). 3. Noxious (>= 45 C) or innocuous (<= 40 C) thermal stimuli did not elicit any synaptic responses in all 18 SG neurones tested which were sensitive to mechanical stimuli. Noxious cold stimulation (<= 10 C) also failed to produce any responses (n = 6). 4. It is concluded that both noxious and innocuous mechanical information to SG neurones are transmitted primarily by activation of non-NMDA receptors, probably without any involvement of slow synaptic transmission, and that thermal information is conveyed to areas of the dorsal horn other than SG.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1985

The control of atrial contraction by ventriculo-atrial pacing in the dog with AV block.

Atrial contraction in dogs with atrioventricular (AV) block was controlled by multiple atrial stimuli delivered during ventricular diastole. Acute hemodynamic changes were assessed. At a ventricular rate of 60 bpm, the spontaneous atrial rate averaged 83, and atrial cannon waves were frequent. When the atria were given two stimuli at an interval of 500 ms during ventricular diastole, the cannon waves were eliminated completely, and a fall in mean central venous pressure and a rise in systemic blood pressure were found. At a pacing rate of 90 bpm, similar changes in the cannon waves, mean central venous pressure, and systemic blood pressure were found when two atrial stimuli followed ventricular stimulation. Ventriculo-atrial multiple pacing may be useful in both clinical and experimental AV block.

Animals↗

PubMed1978

Influence of local noxious heat stimulation on sensory nerve activity in the feline dental pulp.

The present investigation was undertaken to develop an experimental model in which noxious heat stimulation was used to produce increased intradental sensory nerve activity in canine teeth of anesthetized cats. Two techniques were evaluated in which both the method of recording and the nature of the stimulus varied. Slow heating (approx 1 degree C/s) to 47 degree C of the tooth surface (combined with recording from electrodes in open dentinal cavities) did not produce any persistent nerve activity. Repeated periods of brief intense heating (approx 60 degrees C/s) (combined with recording from amalgam electrodes placed on cavity floors) resulted in an immediate response and an afterdischarge (phase 3) generally persisting for 20--60 min. Maximum phase 3 activity was characteristic for the individual cat and ranged from 0.2 to 50.2 imp/s. mean value 10.6 imp/s (S.D. +/- 9.2). A systematically higher phase 3 activity was recorded in lower compared to upper canine teeth (p less than 0.05). The maximum phase 3 response generally occurred after 3-8 stimulations; the median number of required stimuli was 3. Repeated brief heat stimulations combined with the closed cavity recording technique may be used as an experimental model by which the mechanisms behind increases in intradental sensory nerve activity associated with tissue damage can be studied.

Animals↗

PubMed1981

Parameters affecting the slow inward channel repriming process in frog atrium.

1. The time of recovery (from the inactivation) of the slow inward current was studied in the frog atrium, using the double sucrose gap voltage clamp technique. 2. The 'repriming' process was found to be distinct from the current inactivation, and to depend on experimental protocol: double pulses given at low frequencies (at 'rest') gave a faster recovery time when compared to recovery during constant stimulation, with interposed stimuli monitoring the recovery. Longer durations of the clamp pulses led to a faster recovery process. 3. Changing the holding potential of the membrane (with double pulses to the same absolute membrane potential monitoring the recovery process) greatly affect the repriming with depolarized levels slowing down the process. 4. The recovery time was fastest following clamp pulses to intermediate membrane potentials (in the plateau range). This was determined by double pulses, from a constant hold potentials, to different levels. 5. Decreasing extracellular Ca prolonged, and increasing Ca enhanced the recovery process. 6. The recovery process was markedly slowed down in Na or in K-free solutions. 7. The recovery process was enhanced in solutions with a raised concentration of Mg or H ions (lower pH). In higher Mg solutions, the inactivation of the slow inward current was slower. 8. It is proposed that the recovery process is sensitive to alterations in intracellular Ca ions and to variations in extracellular surface charges. The possible implications are discussed.

Animals↗

PubMed1988

Maintained changes in motoneuronal excitability by short-lasting synaptic inputs in the decerebrate cat.

1. During investigation of the tonic stretch reflex in the unanaesthetized decerebrate cat we observed that a short train of impulses in Ia afferents from the soleus muscle (or its synergists) may cause a prolonged activity in the soleus muscle as judged by EMG and tension recordings. This excitability increase, which outlasted the stimulus train, could stay virtually constant during long periods (even minutes), but could be terminated at any time by a train of impulses in, for example, the peroneal nerve. 2. Gradation of the strength of stimulation and the duration of the train of impulses show that the amount of maintained excitability increase depends-within some limits-on the total amount of Ia impulses. 3. In paralysed preparations a short train of impulses in Ia afferents from any part of the triceps surae, caused a maintained increase of the efferent activity in the nerves to triceps surae and a maintained increase of the triceps surae monosynaptic test reflex. These experiments demonstrate the existence of a central mechanism (in the spinal cord and/or the brain stem), which is responsible for the maintained excitability increase seen in motoneurones to the homonymous and synergic muscles. 4. In acute spinal preparations it was not possible to demonstrate any long-lasting excitability increase by a train of Ia impulses. Following intravenous administration of the serotonin precursor 5-hydroxytryptophan, mimicking the tonic activity of these pathways in the decerebrate state, it was again possible to elicit the long-lasting excitability increase by a train of impulses in Ia afferents. A subsequent I.V. injection of methysergide (a serotonin receptor blocker) abolished the long-lasting excitability increase. This set of experiments demonstrates that the basic mechanism responsible for the maintained excitability increase is located at segmental level, and involves serotonergic systems. 5. It was demonstrated that activation of several ipsilateral and crossed reflex pathways by trains of impulses in cutaneous or high-threshold muscle afferents could trigger a maintained excitability increase of those motoneurone pools which were activated by the stimulation. Trains of stimuli to facilitatory regions in the brain stem could also cause a long-lasting excitability increase of motoneurones. Furthermore, activation of all reflex pathways which mediate postsynaptic inhibition to a motor nucleus (including recurrent inhibition via Renshaw cells) could terminate the prolonged excitability increase of that particular motor nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)

5-Hydroxytryptophan↗

PubMed2001

Predicting the responses of mechanoreceptor neurons to physiological inputs by nonlinear system identification.

The nonlinear dynamic properties of action potential encoding were studied in mechanosensory neurons innervating the slits of a slit-sense organ in the tropical wandering spider, Cupiennius salei. The organ contains two types of neurons that are morphologically similar but have different dynamic properties. Type A neurons produce only one or two action potentials in response to a mechanical or electrical stimulus of any suprathreshold amplitude, while type B neurons can fire prolonged bursts of action potentials in response to similar stimuli. Neurons were stimulated with pseudorandomly modulated intracellular current while recording the resultant fluctuations in membrane potential and action potentials. A parallel cascade method was used to estimate a third-order Volterra series to describe the nonlinear dynamic relationship between membrane potential and action potentials. Kernels measured for the two types of neurons had reproducible forms that showed differences between the two neuron types. The measured kernels were able to predict the responses of the neurons to novel pseudorandomly modulated inputs with reasonable fidelity. However, the Volterra series did not adequately predict the difference in responses to step depolarizations.

Action Potentials↗

PubMed2005

Novel compartment implicated in calcium signaling--is it an "induced coupling domain"?

The endoplasmic reticulum (ER) is not simply a uniform continuous organelle, but is spatially and functionally heterogeneous with nonuniform distribution of endoplasmic Ca(2+)-handling proteins, such as Ca(2+)-binding proteins, Ca(2+) pumps, and Ca(2+)-release channels. Such nonuniform distribution of Ca(2+)-handling proteins is thought to create a spatially divided calcium store and to contribute to the generation of complex intracellular Ca(2+) dynamics. In addition to the particular distribution of these Ca(2+)-handling proteins within ER, extracellular stimuli may also stimulate the formation of dynamic new ER compartments containing Ca(2+)-handling proteins. These compartments containing Ca(2+)-handling proteins have potential roles in Ca(2+) signaling; specifically, they may function as "induced coupling domains" between the ER and plasma membrane, thereby allowing Ca(2+) entry into the ER.

Animals↗