what type of circulatory system do birds have

1Department of Radiology, Keck School of Medicine of University of Southern California, Los Angeles, CA 90033, USA; ude.csu@drofsnal

2Department of Radiology and Developmental Neuroscience Program, Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, CA 90027, USAFind articles by

Avian embryos have been used for centuries to study development due to the ease of access. Because the embryos are sheltered inside the eggshell, a small window in the shell is ideal for visualizing the embryos and performing different interventions. The window can then be covered, and the embryo returned to the incubator for the desired amount of time, and observed during further development. Up to about 4 days of chicken development (out of 21 days of incubation), when the egg is opened the embryo is on top of the yolk, and its heart is on top of its body. This allows easy imaging of heart formation and heart development using non-invasive techniques, including regular optical microscopy. After day 4, the embryo starts sinking into the yolk, but still imaging technologies, such as ultrasound, can tomographically the embryo and its heart in vivo. Importantly, because like the human heart the avian heart develops into a four-chambered heart with valves, heart malformations and pathologies that human babies suffer can be replicated in avian embryos, allowing a unique developmental window into human congenital heart disease. Here, we review avian heart formation and provide comparisons to the mammalian heart.

Birds have been diversifying to inhabit nearly every conceivable habitat on the earth’s surface since the Cretaceous period. With this diversity, birds can flourish in numerous harsh environments, such as flying atop hypoxic Himalayan mountain ranges, swimming deeply in frigid Antarctic waters, and running across hot Mojave Desert sands [1]. To live in such extreme conditions, the bird cardiovascular system (CVS) and respiratory system evolved to systemically deliver sufficient oxygen and metabolic substrates to meet the demands of such severe niches [2,3]. The avian CVS also adapted to efficiently remove metabolic byproducts to maintain cellular function, while maintaining a bird’s body temperature [3,4].

High aerobic activities (e.g., flying) in endotherms (e.g., birds and mammals) require an efficient CVS that is afforded by four-chambered hearts, high systolic pressure and high resting metabolism [3,5]. Both the avian and human heart are located along the midline of the anterior part of the thoracic cavity. The long axis of the heart points slightly to the right of the midline in avians, and to the left of the midline in humans. A positive blood systolic pressure is required to push blood to the body tissues to meet the body’s metabolic needs. Birds have a higher metabolic rate than humans. The average body temperature of a bird is 40–41 °C, while the average body temperature of a human is 37 °C. The resting heart rate of a chicken is about 245 beats/min and can reach ~400 beats/min (the heart rate of the blue-throated hummingbird has been measured at 1260 beats/min), while the resting heart rate of a well-conditioned human is about 60–80 beats/min and may reach ~190–200 beats/min. Birds tend to have larger hearts and pump more blood per unit time than mammals (relative to body size and mass) [6,7,8]. In birds, heart mass (Mh) scales with respect to body mass (Mh) as Mh = 0.014 Mb0.91 [9], whereas in mammals, the relationship is Mh = 0.0058 Mb0.98 [10,11]. This may be due to the high aerobic power input needed to sustain flapping flight. Hummingbirds have the largest hearts relative to body mass of all birds; for 25 species of hummingbirds, Mh = 0.025 Mb0.95 [12], possibly reflecting the high aerobic requirements of hovering flight and that they are so small. Thus, cardiac output is normally greater for birds than for mammals of the same body mass. Such conditions and physiological requirements place inordinate demands on the bird heart, which has to function at a higher level than a human heart.

The avian and mammalian heart transports blood to the lungs and body in a similar manner [13]. Birds and mammals have atrial and ventricular septa, allowing separation between oxygenated and deoxygenated blood, and complete separation of the systemic and pulmonary circulations. The deoxygenated blood returns from the body to the right atrium through the large caval veins. The deoxygenated blood moves to the right ventricle, where it is pressurized for pulmonary circulation. The blood dumps its CO2 and acquires O2 via the lung capillaries. The newly oxygenated blood returns to the left atrium through four large pulmonary veins, as in mammals. The oxygenated blood moves to the left ventricle, where it is pressurized for systemic circulation. Both avian and mammalian hearts are surrounded by a thin, fibrous pericardial sac that is filled with serous fluid that lubricates the motions resulting from cardiac contractions and also confines the heart so it does not rattle around the thoracic cavity or overfill with blood.

As birds increase their activity level with flight, they must also increase oxygen delivery and thus blood supply to tissues involved with flight. The two main flight muscles, the pectoralis and supracoracoideus, originate in the sternum and insert onto the base of the humerus. Blood to the flight muscles and wings is delivered by the subclavian arteries, which branch into the pectoral (flight muscle) and brachial (wing) arteries. Flight demands lead to an increase in the number of capillary beds in the flight muscles, increasing capillary density. The greater the capillary density, the greater the surface area for gas exchange, at the expense of an increased resistance to blood flow. This requires the heart to pump harder to push the blood through all the blood vessels. Birds that migrate long distances have a greater capillary density (1935 capillaries per mm2) in their flight muscles than those of species that do not migrate or migrate only short distances (1604 capillaries per mm2) [3,14]. For an outermost example, rufous hummingbirds, which migrate from breeding grounds in Alaska and western Canada to wintering sites in Mexico, have a capillary density of 7000 per mm2 in their flight muscles [15].

The ventricles of the bird heart have more muscle mass and less chamber space than those of the human heart. The left ventricle in a bird’s heart is by far the largest heart chamber, powered by a thick cardiac muscle to pressurize the blood for transport throughout the body, and must work especially hard in birds during flapping flight. The right side of the heart only delivers blood to the lungs and the resistance of this circuit is low, thus its smaller size. Externally, the avian ventricles appear trimmer and more pointed than those of the human heart [16]. Internally, the atrial and ventricular walls are smoother than those of the human. The smoother walls and simpler valves of the bird’s heart reduce friction as the blood is pumped through; less friction means less work to pump blood.

In both birds and mammals, up to six aortic arches develop in the embryo, but only three remain in the newborn animal. In birds, the left systemic arch does not develop, and all functions are carried out by the right systemic arch. The blood vessels to the forelimb (subclavian arteries) develop from the anterior arteries supplying the head (brachiocephalic, common carotid, internal carotid arteries). In mammals, the left systemic arch develops, and the right does not. The blood vessels to the forelimbs (subclavian arteries) develop from the dorsal aorta and left systemic arch.

Studies on avian embryos date back to Aristotle, and even further back, to the Egyptians. Historically, the avian model has played an important role in establishing the foundations in circulation research. Chicken eggs were easy to obtain and could be incubated in ovens [17] to observe different periods of embryo development [18]. William Harvey, for example, used chicken eggs to watch the development of the heart and blood, and was the first to notice the directional flow of blood from the heart into the brain and body during systemic circulation [19]. The existence of capillaries that connected the veins and arteries was confirmed with the aid of a simple microscope by Marcello Malpighi, who also discovered that the heart began to beat before blood started to form [20,21].

Avian models have unique characteristics that make them invaluable for embryonic developmental studies and, in particular, heart development research. First of all, like mammals, chickens and quails are amniotes, animals whose embryos develop within an amnion and chorion, and developmental processes are highly conserved among amniotes [22]. Specifically, the development of the avian heart is similar to that of the human heart [23]. The mature avian heart consists of four chambers with valves as well as inflow and outflow connections (veins and arteries, respectively), and despite some differences, it resembles the human heart [24,25,26,27]. Importantly, cardiac defects found in humans can be recapitulated in avian embryos [26,27,28,29]. Second, like humans, avian embryos remain relatively flat from early to late gastrulation stages [30], enabling time-lapse observation of both dorsal and ventral tissues by means of whole-mount ex ovo culture techniques [31,32]. Pioneering work by María de la Cruz using iron oxide particle labelling in an ex ovo culture, for example, elucidated the details of heart tube formation and the location within the mature heart of primitive cardiac regions from the heart tube [33,34,35]. The whole-mount ex ovo culture technique for avian embryos permits studying the cell behaviors underlying heart and blood vessel morphogenesis under physiological conditions [36,37,38,39]. In addition to labeling with iron oxide particles, heart researchers have labelled cardiac progenitor cells (CPCs) in vivo with radioactive nucleotides [40,41,42,43], vital dyes [44,45,46], and fluorescent proteins [36] to understand their dynamic contributions to heart assembly. The use of transgenic quail embryos that express fluorescent proteins allows following the motion of individual CPCs, as well as extracellular matrix (ECM) components, during cardiac formation. It has been shown, for example, that the myocardium is formed by intercalated CPCs coming from the left and right heart-forming fields [23,47,48]. Third, because avian embryos develop inside the egg, they provide easy access for cell and tissue manipulation, the longitudinal follow up of manipulations, and in vivo imaging [49,50,51,52]. The topical application of pharmacological agents directly onto the heart or by injection into the circulation is common practice [26], as is hemodynamic manipulation through surgical procedures [29]. In fact, avian embryos are the best models for altering flow patterns at will (without introducing genetic modifications or drugs), while allowing follow up studies to study hemodynamic effects. Finally, avian embryos are inexpensive and the eggs are easy to store until ready for incubation. Because the embryos do not start developing until incubation, avian embryos allow easy planning and scheduling of experiments. Egg affordability and easy storage, moroever, facilitate experiments that require a large number of embryos. Avian models are therefore ideal to study cardiovascular development, from the onset of vasculogenesis and heart tube formation to the development of the four-chambered heart.

Limitations to avian model systems include the difficulties to perform high-throughput chemical mutagenesis screening, and the lack of stem cell technology that allows for efficient generation of genetically modified avian species. Nevertheless, the genomes of chickens and quails have been sequenced at deep coverage levels [53,54,55,56,57]. The genomic information obtained greatly helps with the design and execution of molecular perturbation experiments, including CRISPR [58,59,60], TALENs [61], RNAi [62,63,64,65], and transgenesis [36,66,67,68,69], to name a few.

3. Avian Development and Staging

The most popular table of normal bird development is the Hamburger and Hamilton (HH) avian staging system, which was first published in 1951 and is based on the chicken embryo [70]. It establishes 46 phases for the development of birds: HH1 is the pre-streak embryo, which is before incubation, and HH46 is the newly hatched chick. Using external characteristics that are independent of embryo size or breed, the HH staging system identifies embryos instead of depending on the imprecise and highly variable time of incubation, which varies from egg to egg. Embryos and their developmental stage can thus be consistently compared across research. Initially, prior to somites forming on day 2 of incubation (HH1 to HH6), the morphology of the streak and subsequently the formation of the head characterize the embryonic stages. Since somites are readily visible and produce a highly specific and repeatable spatiotemporal pattern [71, 72], embryos from HH7 to HH14 are staged based on the number of somite pairs present in the embryo [73]. Somotes form next to the notochord and progressively from head to tail. However, counting somites becomes challenging after HH14 (beyond 22 somite pairs), although limb development advances quickly. Afterwards, limbs (wings, legs) and visceral arches are used to stage embryos (HH15 to HHH29); later on (HH30 to HH39), feather-germs and eyelids are also helpful in this regard. HH40 to HH45 are determined by measuring the length of the third toe and beak while the embryo grows and does not undergo any other major changes. Brad Martinsen used HH stages to characterize the developmental processes of the heart in 2005 in order to draw a connection between embryonic and cardiac development [74]. Straight heart tubes are formed by CPC migration, and they initially appear in the embryo by HH8 / HH9− (6–7 somites). The tubular heart begins to beat by HH10/HH11 (10–15 somites) and loop by HH9 / HH10−. Cardiac looping continues up to HH24. Cardiac septation takes place between HH24 and HH34 (see for a comparison of mouse, human, and avian heart development timings). The heart develops further until it hatches, having four chambers and valves at HH34.

Following the establishment of the prototype avian embryo staging system by Hamburger and Hamilton [70] using chicken embryos during sequential developmental stages, Japanese quail embryos were similarly staged based on anatomical landmarks [77,78,79,80]. More recently, MRI technologies have been used to further delineate the developmental stages of the quail [81]. However, the accelerated rate of development in quail embryos makes precise registration with the chicken impossible during the mid-to-late stages of incubation [80].

The capacity to biochemically differentiate between the tissues of chickens and quails has long been used by developmental biologists to investigate developmental issues [82,83]. While chicken nucleolus-associated chromatin is not significantly stained by Schiff’s reagent, quail interphase nuclei exhibit large heterochromatic masses linked to the nucleolar RNA that stain intensely [84]. In chimeric embryos, quail cells can be separated from chicken cells thanks to differential staining. Numerous cardiac cell lineage analyses have been successfully carried out using the quail-chick chimera system [83], including the groundbreaking research on the functions of neural crest cells in cardiovascular patterning [82,83,85,86].

Can birds have heart attacks and strokes? — Mammalian platelets are small, anuclear circulating cells that form tightly adherent (i.e., sticky) thrombi (clots or plugs) to prevent blood loss after vessel injury. Platelet thrombi that form in the coronary and carotid arteries of humans can also cause common vascular diseases such as myocardial infarction (heart attacks) and stroke and are the target of drugs used to treat these diseases. Birds have high-pressure cardiovascular systems like mammals, but have nucleated thrombocytes in their blood rather than platelets. Schmaier et al. (2011) found that avian thrombocytes respond to many of the same activating stimuli as mammalian platelets (and so help stop blood loss from damaged vessels) but, unlike mammalian platelets, cannot form tighly adherent thrombi in arteries. Avian thrombocytes are larger than mammalian platelets and are less sticky (because they release different chemicals) than mammalian platelets when exposed to collagen (connective tissue to which thrombocytes and platelets are exposed when theres a break in a blood vessel). When carotid arteries of mice are damaged, platelets form thrombi that can block blood flow (check this video showing the response of human platelets when exposed to a plate covered with collagen); similar damage to the carotid arteries of Budgerigars (similar in size and speed and pressure of blood flow to the carotid arteries of mice) did not cause the formation of thrombi (check this video showing the response of chicken thrombocytes when exposed to a plate covered with collagen). These results indicate that mammalian platelets, in contrast to avian thrombocytes, will form thrombi even in arteries where blood flow is rapid and under high pressure, an essential element in human cardiovascular diseases.

Immunosenescence in certain immune components of free-living Tree Swallows – An extensive range of free-living organisms exhibit age-related increases in mortality rates and/or reductions in successful reproduction. Unfortunately, little is known about the physiological processes underlying these demographic patterns of senescence. Age-related immune function decline, or immunosenescence, has been extensively studied in both humans and lab models. It frequently results in higher disease-related morbidity and death. However, little is known about immunosenescence in free-living organisms. Palacios et al. examined immunosenescence in a population of free-living Tree Swallows (Tachycineta bicolor) by evaluating three immune system components and employing both in vitro and in vivo immunological tests. The immune system of female Tree Swallows exhibited a varied pattern as they grew older; while acquired T-cell mediated immunity decreased, neither innate nor acquired humoral immunity did. Age-related decreases in T-cell mitogen-stimulated in vitro lymphocyte proliferation raise the possibility that a mechanism underlying the immunosenescence pattern of the in vivo cell-mediated response that was recently reported for this same population may be diminished T-cell function. These findings offer the most complete explanation of immunosenescence mechanisms and patterns in a population of free-living vertebrates to date. Future studies should concentrate on the ecological effects of immunosenescence and the possible reasons why patterns vary between species.

Heart rate, diving depth and body angle of a female Common Eider (Somateria mollissima) during dives and when flying. (a, b) heart rates of 250 and 300 beats per minute, (c) heart rate ascending and descending slopes equal to or above 10 beats per minute per second (absolute values), (d) standard deviation of diving depth up to 0.1 m, and (e) change in body angle. The upward and downward pointing arrows indicate the point of take-off and landing, respectively (From: Pelletier et al. 2007).

Scanning electron microscope view of bird thrombocytes adhering to a collagen-lined plate (exposure to collagen causes bird thrombocytes, and mammalian platelets, to release chemicals that make them sticky; the chemicals released by mammalian platelets are different from those released by bird thrombocytes and make platelets stickier than thrombocytes). Avian thrombocytes are larger than mammalian platelets, have a nucleus, and, unlike mammalian platelets, do not form 3-dimensional aggregates. (Credit: Penn Medicine)

Compared to mammals, birds typically have larger hearts in terms of mass and size. It is possible that birds’ comparatively large hearts are required to meet the high metabolic demands of flight. When it comes to body mass, smaller birds tend to have larger hearts than larger ones. Hummingbirds possess the largest hearts among all birds in relation to their body mass, likely due to the high energy required for hovering.

6. Heart Pumping and Tube Looping

In developing mammals and bird species, rightward heart looping is the first evident indication of left-right asymmetry [158] (see Videos S1 and S2). The heart tube in human embryos is made up of one posterior atrium and one anterior ventricle in day 2-3 avian embryos and about four weeks in human embryos. Appropriate heart tube looping is necessary to set up the pulmonary and systemic circulatory systems as well as to arrange the heart chambers correctly. For almost a century, researchers have attempted to comprehend the mechanism of heart looping by investigating whether asymmetric changes in myocardial cells, such as those caused by cell shape, death, proliferation, or space constraints, are the source of the bending component of heart looping (reviewed by [105,159,160,161]). However, specific details are lacking in this investigation. The heart’s left-right asymmetries are caused by particular molecular signaling factors that come from Hensen’s node during gastrulation [162,163,164]. Recently, research conducted by Angela Nieto’s lab revealed that the transcription factors Pitx2 and Prrx1, which assimilate left and right information to regulate heart laterality and morphogenesis, are asymmetrically activated by the reciprocally repressed Nodal and BMP pathways converge of their respective targets [165]. In the left lateral plate mesoderm of fish and mice, the same group subsequently discovered that posterior-to-anterior Nodal signals upregulate a number of microRNAs that momentarily reduce the levels of epithelial-mesenchymal transition factors (Prrx1a and Snail1) in a Pitx2-independent manner [166]. These results provide insight into how the BMP and Nodal pathways function to appropriately balance the left-right information required for heart morphogenesis and laterality [166]. Notable additionally, Sigolđne Meilhac’s lab demonstrated via computer simulations, high-resolution episcopic microscopy, and cell labeling that looping of the growing heart tube is produced by heart tube buckling, which is caused by asymmetries at the fixed arterial and venous heart poles [153]. Substantial morphogenetic events cause the original c-shaped cardiac loop (see) to change into an s-shaped loop once the heart looping process is complete. The blueprint for the formation of the multichambered heart is established by the primitive ventricular bend, primitive conus, primitive atria, and sinus venosus found in the s-shaped heart [159]. The portion of the heart tube that will become the atria is located anterior to the portion that will become the ventricles once the looping processes are complete.

Endocardial cushions are localized thickenings of the cardiac wall that develop in the tubular heart’s atrioventricular canal (AVC) and outflow tract. The cushions come into contact with one another during myocardial contraction, completely closing the lumen and preventing reverse flow [167,168,169]. Endocardial cushions usually form on the two opposing sides of the tubular heart, giving the lumen an elliptical shape [170]. The regions surrounding the lumen perimeter, where the two cushions end, are home to tethering proteins that connect the endocardium to the myocardium [167,171]. It has been shown that the elliptical lumen shape created by the cushion is more effective for unidirectional blood pumping than a circular shape [172]. In actuality, endocardial cushions become increasingly effective at restricting reverse flow as they mature throughout the embryonic stages [173].

Septa and valves originate in the heart tube’s cardiac cushions. Initially, they are primarily made of extracellular matrix (ECM), also called cardiac jelly, which is positioned between the layers of the endocardium and myocardium. Before valve formation and septation, in the tubular stages of heart development, cardiac cushions go through an endocardial-mesenchymal transition (EndoMT). Endocardial cells delaminate from the endocardium during endomyotomy (EndoMT) and migrate into the cushion tissue, where they multiply and secrete extracellular matrix (ECM) proteins, filling and expanding the cushions. The atrioventricular valves and septum are the result of endoMT, which first develops in the AVC cushions. Later, endoMT occurs in the outflow tract cushions, where semilunar valves and a section of the interventricular septum are the result. Understanding EndoMT and cardiac valve development has been greatly aided by the use of the avian model [174,175]. Early research by Raymond Runyan and Roger Markwald removed AVC cushions from chicken embryos and cultured them in a lab setting [174,176]. They could investigate the complex signaling pathways connected to EndoMT and, consequently, valve formation, using this method. For instance, they proved that the myocardium is the source of EndoMT signals [177,178,179,180]. In both birds and mammals, endoMT cannot occur without the myocardium layer [181]. Furthermore, more recent research is demonstrating the impact of blood flow on cushion EndoMT and, consequently, valve development [182,183,184,185].

The heart gets more adept at pumping blood in tandem with the development of cushion. The maximum centerline velocity in the heart outflow tract portion increased at first and then plateaued between HH13 and HH18 (roughly two to three days of incubation) ( A) However, as developmental stages B and C progressed, volume flow rate and stroke volume increased as well, indicating the growing demands of the developing embryo. An increase in outflow tract diameter (D) was reflected in an increase in volume flow rate at constant (plateaued) centerline velocity. Interestingly, a characteristic trend appears when wall shear rate, which is proportional to wall shear stress and reflects the gradient of blood flow velocity near the wall, is approximated during the cardiac cycle’s maximum flow phase ( D). The outflow tract diameter increases in response to an increase in wall shear rate. Increasing diameter, however, decreases wall shear rate to previous values. This growth mechanism has been described for mature arteries [186, 187, 188], and it appears to be present at early stages of tubular heart development as well. In response to an increased wall shear rate, the heart wall increases its diameter, thereby decreasing wall shear rate to previous values.

Due to the ease of access for imaging, studies have used avian embryos to quantify early embryonic heart form and function under a variety of conditions. The alterations in blood flow dynamics brought about by early exposure to ethanol [189], trichloroethylene [190], and excess glucose [191,192], for instance, have been measured in certain investigations. Additionally, the effects of interventions aimed at deliberately disrupting normal blood flow conditions have been ascertained [27] and changes in flow have been measured using avian embryos [29, 193]. Overall, research indicates that during the early stages of development, the heart’s blood flow and development are highly sensitive to outside influences. Additionally, blood flow has a significant impact on how the heart develops.

Starting at HH25, the tubular heart enters the septation phase. The originally tubular heart will become a four-chamber heart with valves during septation [74]. Septation begins at the distal end of the outflow tract and splits the tube into the pulmonary trunk and aorta. Neural crest cells are derived from the early neural tube and migrate towards the heart. They are subsequently found in the aortico-pulmonary septum, which divides the outflow tract, as pioneering research by Margaret Kirby using quail-chick chimeras demonstrated [85]. Further, neural crest cell ablation abolishes outflow track septation [85]. Moreover, the semilunar valves will develop from the outflow tract endocardial cushions. The interventricular septum begins to grow in the primitive ventricle (about HH17), gradually dividing the left and right ventricles. Around HH16 (E9), an interatrial septum (the septum primum) begins to form in the primitive atrium. 5 in mice) from the atrial roof. First, this septum starts as a crescent-shape ridge. The interatrial septum and the AVC cushions have fused by HH24, dividing the AVC. The tissues from the dorsal mesenchymal protrusion, which originates from the SHF, close the atrial septum in birds and mammals in addition to the septum primum and cushions [194,195]. The division of the left and right atria, as well as the human mitral valve (left atrioventricular valve) and tricuspid valve (right atrioventricular valve), are the result of this septation [195]. On the other hand, the septal myocardium develops perforations that lead to the foramen ovale, which only fully closes to separate the two atria after hatching or birth [194]. Mammals develop an extra interatrial septum in the primitive atrium during development. The mature atrial septum is formed when the septum secumdum, also known as the secondary septum, unites with the septum primum, also known as the primary septum [195]. As a result, even though the atrial septum of birds and mammals differs slightly, both types of hearts show shunting between the left and right atria, which is required for the fetal circulation to avoid the inoperative pulmonary system [196].

The avian heart has four chambers and valves, just like the human heart, when it is fully developed. Nonetheless, compared to human anatomy, the inner walls of the atria and ventricles of birds are smoother, and their valves are simpler. The atrioventricular (AV) valves found in the hearts of birds are not the same as those found in human hearts [16]. The human fibrous tricuspid valve, which is situated between the right atrium and right ventricle, is not like the right AV valve, which is made up of a single spiral flap of myocardium attached to the inner wall of the ventricle. The left AV valve in birds, which separates the left atrium from the left ventricle, is tricuspid rather than bicuspid like the mitral valve in humans. The Purkinje system, a network of specialized conducting fibers made of electrically excitable cells, is further connected to the avian AV valves. It is through this system that the cardiac action potential, which contracts the heart muscle, is conducted. The aortic and pulmonary semilunar valves are tricuspid, just like the human heart. The larger and more muscular avian hearts compared to mammalian hearts are another distinction between the two species’ hearts. The functioning of the hearts in birds and humans is remarkably similar, despite these differences.

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