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The effects of infused materials upon the regeneration of newt limbs. III. Blastemal extracts and alkaline phosphatase in irradiated limb stumps

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The Effects of Infused Materials upon
Regeneration of Newt Limbs
Departments of Anatomy and Biology, University of Virginia,
Charlottesville, Virginia 22901
Forelimbs of adult eastern spotted newts were irradiated with
2000 or 3000 r of x-rays, and were amputated through the elbow region. Limbs
treated in this way or infused by means of a Singer microinfusion apparatus with
amphibian Ringer's solution were observed as controls. Experimental limbs were
infused at various times with (1) amphibian Ringer's solution containing material extracted from two-week-old blastemata from unirradiated limb stumps
or (2) one of four concentrations of alkaline phosphatase dissolved in Ringer's
Small polyps or blastema-like conical structures were the only regenerative
responses that followed the various infusion procedures. Blastemal extract and
Ringer's solution alone seemed to be equally effective in inducing these regenerative growths, but no such growths followed mere irradiation and amputation.
Alkaline phosphatase was more effective than either the blastemal extract or
Ringer's solution in inducing the growth of conical structures. These initial
growths in a number of cases persisted as very short cones composed mostly of
amorphous cartilage and connective tissue or as pseudoblastematous aggregations of cells. However, extensive regression of tissues and shortening of the limb
was seen rather frequently in limbs treated with alkaline phosphatase.
Ringer's solution and blastemal extracts did little to reverse the regenerationinhibiting effects of irradiation on amputated limbs. It appears that alkaline
phosphatase caused a more frequent, if still limited, reversal of these effects,
and it is suggested that a part of these inhibitory effects may result from damage
done to the production of alkaline phosphatase.
It is well known that ionizing radiation
inhibits regeneration in urodele limbs (Butler, '33; Brunst, '50). A number of investigators, however, have obtained regenerates
from irradiated limbs implanted with unirradiated tissues (Umanski, '37; Thornton,
'42; Trampusch, '51, '58; Stinson '64; et
al.). Whereas Stinson ('64) and Eggert
('66) presented evidence that such regenerates consist almost exclusively of cells
from the implant, Polezhayev ('59) and
Polezhayev and Ermakova ('60) reported
that in axolotl limbs regeneration can occur from the irradiated tissues themselves
if the animals are injected with appropriate
tissue homogenates. Polezhayev et al. ('61)
found that the ribonucleic acid and proANAT. REC.,168: 525-536.
tein fractions from such homogenates were
the most effective in restoring regenerative
ability to the irradiated tissues.
Like irradiated limbs, denervated limbs
also fail to regenerate after amputation.
Further, the histology of healing is essentially identical in both types of stump
(Schott6 and Smith, '61). Since blastemal
extracts were found to induce limited regenerative responses in denervated limbs
(Deck and Futch, '69), it seemed reasonable-especially in view of the cited works
of Polezhayev and others - to test the
capacity of blastemal extracts to restore
Received Auril 20. '70. Acceuted
6, '70.
1 Work supported-by grants from the National Science Foundation (J.D.D.) and by the Atomic Energy
Commission (J.N.D.).
growth potential in irradiated limbs. As a
companion effort, it further seemed reasonable to infuse irradiated limbs with a
specifically characterized material like
alkaline phosphatase, which has been
found in other circumstances to be involved in growth. This particular enzyme
will stimulate the development of chicken
feather germs grown in vitro (Hamilton,
'65) and is essential to a number of other
developing systems (see Hinsch, '60). It
is even more pertinent that Schmidt ('66)
has suggested that alkaline phosphatase is
a causative factor in some of the cellular
events of newt limb regeneration.
For use in this study adult newts (Notopthalamus [Triturus] v. viridescens Rafcnesque) were collected from lakes in
central Virginia. They were kept in dechlorinated water at room temperature
(23-25" C) and fed twice weekly with
pellets of lean, ground beef containing calcium phosphate and cod liver oil. Right
forelimbs were used as experimental or
control limbs.
For irradiation newts anesthetized in
0.5% MS 222 (Meta aminobenzoic acid
ethyl ester) were placed belly-down on a
turntable at a target distance of 15 cm
beneath a Picker diagnostic x-ray machine.
The body of each animal was shielded with
a sheet of lead 1.75 mm thick and the right
forelimb and adjacent shoulder region
were left exposed to irradiation. The x-ray
machine was operated at 100 Kv and 25
ma through Hvl equivalent of 1 mm Cu
to give a dose rate of 180 r/minute. Rotation of the turntable assured uniformity
of dosage. Limbs in the initial groups of
animals used received 3000 r as a regeneration-suppressing dosage. In subsequent groups, the dosage was lowered to
2000 r without causing any detectable difference in response to experimental treatment.
After recovering for at least a week,
each irradiated forelimb was amputated
through the elbow joint. The irradiated
limbs were then infused with blastemal
extract or with alkaline phosphatase by
means of a Singer microinfusion machine
(Singer et al., '55) at a rate of 0.025
ml/hour for five to six hours per day. For
the infusion, animals anesthetized with
0.5% MS 222 and held under anesthesia
with 0.05% MS 222 were placed on their
backs and connected to the infusion
machines by glass needles inserted subdennally from the shoulder to the tip of
the irradiated limb.
In a first series of experiments, irradiated limb stumps were infused with a tissue extract of macromolecules taken from
two-week regeneration blastemata. The
extract was prepared by homogenizing
blastemata in tris-buffered Ringer's solution (1:lO) at pH 7.4 and ultracentrifuging (74,000 X g) the homogenate to produce a supernatant fraction which was
then dialyzed against Ringer's solution.
Control limbs were infused with tris-buffered Ringer's solution at pH 7.4 or merely
amputated after irradiation. In the initial
experiments infusions were given on nine
occasions between days 6 and 30 postamputation. Because some of the limbs
appeared to be excessively irritated, the
frequency of infusions in later experiments
was reduced to six occasions between days
6 and 14 post-amputation. Ultimate responses of the limbs to these two frequencies of treatment were not perceptibly
In a second series, irradiated limb
stumps were infused with solutions of alkaline phosphatase made from commercial
preparations of the mammalian enzyme
(Worthington Biochemical Corp., Freehold,
N. J.). Samples of powdered enzyme were
added to tris-buffered Ringer's solution at
pH 7.4 to give concentrations of 0.01%,
O . l % , 1.0%, and 5.0% for the infusions.
Like the blastemal extracts, the enzyme
infusions were given initially on nine occasions between the sixth and thirtieth
days after amputation. To reduce irritation
and the possibility of an immune response
to the mammalian protein, additional experiments were performed using only five
periods of infusion, beginning a day or
two before amputation and concluding by
the eighth day after amputation. The 5.0%
enzyme solution was infused only by the
shorter schedule.
Several months after amputation, many
of the limbs were fixed in Bouin's fluid
for histological study. Serial sections were
cut at 10
and eosin.
and stained with hematoxylin
coverage of the amputation surface and
thus appeared to be non-regenerative (as
in fig. 3). Fifteen others developed very
small excrescences or polyps at the limb
Reactions of the irradiated limb stumps tip (as in fig. 4) and three developed
to amputation alone, or to amputation fol- conical blastema-like structures (as in fig.
lowed by infusion with saline, tissue ex- 5). Subsequently one polyp expanded and
tracts, or phosphatase were all alike dur- became a slender digitiform outgrowth
ing the first two or three weeks after am- and two others persisted as bulges at the
putation. In many cases, the soft tissues tip that consisted of a small pseudoblasteof the stump retracted and left some length matous cluster of cells and fibrous tissue
of bone exposed (fig. 1 ) . This bone was when sectioned (see fig. 14). Of the three
presently covered by epidermis and then conical structures seen in this series only
was gradually eroded away, leaving spicu- one persisted, ultimately expanding to form
lar projections that persisted for a time a complete limb nearly six months after
(fig. 2). If no significant retraction of soft amputation. Among the 22 limbs that
tissues occurred, the tip usually healed over were initially non-regenerating (table 1) ,
and presented a clubbed appearance (fig. three belatedly formed small polyps at
3). Control limbs merely amputated after their tips and two underwent internal
irradiation showed no further change.
regression and became markedly shortened.
None of the limbs which initially developed
Growth responses after infusion with
polyps or cones later underwent any sigsaline or with blastemal extract
nificant regression.
Among the 40 limbs infused with
Irradiated stumps infused with blasteRinger’s solution as a control of the ex- mal extracts did not produce results diftract infusions (table l ) , the majority ferent in quality from those infused merely
made no response beyond the epithelial with saline (table l ) , and thus the tissue
Responses o f irradiated and amputated limbs to various infusion treatments
Eventual state
of limbs
No growth
N o growth
Out- Extengrowth sive
limb tema resorbedr:fg-Hand
(small polyps)
No growth
No growth
centrations of the enzyme tended to favor
the development of early conical blastemalike structures and higher concentrations
tended to be followed by marked shortening of the limp stump. The extent of both
reactions differed significantly from the
same types of effects on irradiated limb
stumps produced by the control saline
solution (p < .0005 and p < .005, respectively).
The limited regenerative phenomena
qualitatively resembled those of the extractinfused and control limbs already described
and began to appear in the fourth week.
Conical growths developed on 14 limbs
(table 1 ) and polyps developed on another
5. These structures appeared in the greatest
proportions after application of the 0.1%
and 1.0% solutions of enzyme (table 2).
Subsequently, the short conical structures persisted for more than 14 weeks in
nine cases (table 1, fig. 6),each consisting
of a small but newly formed mass of cartilage surrounded by fibrous tissue (figs. 11,
12). Sections showed that another five of
the original cones, still evident seven weeks
or longer after amputation, were composed of fibrous tissue and scattered
blastemal cells, comprising a pseudoblastema distal to remnants of cartilage
condyles (figs. 13, 14). The five limbs
originally bearing polyps later formed two
pseudoblastemata and three much-shortened stumps.
Seven of the 12 limbs which were not
Growth responses after infusion
stimulated to grow by the enzyme infuwith alkaline phosphatase
sions gradually shortened, becoming rather
Alkaline phosphatase was infused into wrinkled externally (fig. 7), until they
31 irradiated limb stumps in four different were half-or-less their original length. This
concentrations (table 2 ) , all buffered to response of the non-growing limbs infused
pH 7.4 to parallel the pH used in infusing with enzyme differed significantly (p <
saline and blastemal extract. Lower con- .Ol) from the responses of the limbs in-
extracts were relatively ineffective in restoring growth potential. The extract-infused limbs initially had fewer polyps and
more conical blastemata, the difference
between cone development in the two cases
perhaps being significant (.1 > p > .05),
but the extract-infused limbs ultimately
had essentially the same distribution of
end-point structures as the controls (table
1). Among the 20 experimental animals,
four limbs developed polyps (fig. 4), 5 exhibited conical blastemal-like growths (fig.
5; and see fig. 14), and 11 showed no
growth of the tip (fig. 3) four weeks after
amputation. The blastemata and polyps,
however, proved to be misleading signs of
regeneration since none of these initial
structures grew normally into a regenerated
appendage. On the other hand, none of the
limbs on which polyps or cones initially
developed later underwent any regressive
shortening. In contrast, two limbs which
were initially non-regenerating were ultimately much shortened by regression. A
true blastema and hand was eventually
formed on one limb, but only after its
early conical growth and part of the stump
had been resorbed and the stump traumatized by extrusion of part of the humerus. This one case of subnormal regeneration represented the only example
of relatively complete regeneration from
irradiated limbs infused with blastemal
Responses of irradiated and amputated limbs to different concentrations
of alkaline phosphatase
,eudo- Extensive
blastema regression
fused with saline or blastemal extracts.
Sections showed that this shortening was
accompanied by extensive resorption of
the bony humerus, which was almost completely lost in some cases. Cartilaginous
condyles at the elbow, on the other hand,
seemed to be more resistant to resorption
and often remained, disconnected from a
much reduced humeral shaft. Several of
the limbs that initially developed polyps at
the tip also underwent these regressive
changes (figs. 8-10). The presence of the
polyp seemed not to prevent the subsequent shortening (compare figs. 8, lo),
and even the polyp itself was finally lost.
However, the limbs infused with enzyme
on which conical blastemata formed did
not subsequently exhibit regression (see
table 1).
Limited regenerative responses consisting of small polyps and conical blastemalike structures followed the infusion of
irradiated limb stumps with Ringer's solution, blastemal extracts or alkaline phosphatase, but did not occur after the mere
amputation of limbs irradiated with the
same dosage of x-rays. This suggests that
the limited growths were only responses of
the limbs to some irritation produced by the
injection procedure. Such a view fails to
take into account the quantitative differences in response between saline-infused
and experimental limbs, viz. mostly polypoid growths after saline but more frequently blastema-like cones after tissue extracts and enzyme solutions, particularly
after alkaline phosphatase ( p < .Ol). Histological sections showed that many of the
conical structures eventually formed cartilaginous calluses or fibrocellular pseudoblastemata, greater growth responses than
limbs usually make to 2000-3000 r of
x-rays (see Stinson, '64).
Some tissue regeneration like the polyps
or cartilage calluses not infrequently follows weaker doses such as the 1000 r dose
of x-rays used by Stinson ('63) or the low
doses of neutron irradiation used by Horn
('42). Nevertheless, the significant number of blastema-like responses after saline,
tissue extracts or enzyme infusions compared with the irradiated and amputated
control limbs (p < .025) suggests that re-
generative capacity was suppressed by the
irradiation but was somewhat restored by
the infusion procedures. An explanation
of this finding is suggested by an observation of Rose ('65), who called attention to
an excessive regression among newt limbs
irradiated with more than 2500 r. Rose
thought that such limbs as were able subsequently to regenerate did so because unirradiated cells migrated from the shielded
body into the irradiated and much shortened limb. In the present case, the pathway made by the infusion needle (repeatedly inserted from shoulder to limb tip)
may have provided a route for such cell
migration. The quantitative differences in
formation of conical blastemata between
saline controls and experimental limbs
were nevertheless significant ( p < .005)
and could have been produced by a type of
growth stimulation from the extracts and
enzyme solutions.
It seems clear, however, that material
extracted from unirradiated blastemata as
a rule did not stimulate recovery of irradiated limbs sufficiently to produce more
than a conical blastema. This fact is surprising in view of the observations of
Polezhayev and his co-workers ('59, '60)
that homogenates from various kinds of
tissues brought about limb regeneration
in irradiated axolotls. Indeed, the most active ingredients of such homogenates
(Polezhyev et al., '61) were the types of
proteins and ribonucleic acids that should
have comprised the blastemal extracts
herein used to relatively little effect. Such
extracts, that is, the supernatant portions
of homogenized blastemata, produced
blastemal cones more frequently on denervated limb stumps (Deck and Futch, '69),
but even in those limbs efforts at regrowth
were limited to early blastemata. In those
same experiments, the other portions of
the homogenized blastemata were also
tested for efficacy in growth stimulation
but were much less effective than the supernatant portion. There seemed no reason
to believe that these relatively ineffective
portions would be more effective in irradiated limbs and therefore they were not
Like the blastemal extracts, the enzyme
solutions failed to bring about complete
regeneration of irradiated limbs, but they
did provoke the collecting of cells into pseudoblastematous masses somewhat Brunst, V. V. 1950 Influence of x-rays on limb
regeneration in urodele amphibians. Quart.
more frequently than did the probably comRev. Biol., 25: 1-29.
plex mixture of materials from blastemal Butler,
E. G. 1933 The effects of x-radiation
extracts. This collecting of cells perhaps
on the regeneration of the forelimb of
came about as a consequence of the regresAmblystoma larvae. J. Exp. Zool., 65: 271-315.
sion which alkaline phosphatase seemed Deck, J. D., and C. B. Futch 1969 The effects
of infused materials upon the regeneration of
to initiate. A similar regressive effect benewt limbs. I. Blastemal extracts in denervated
gins in normal limb regeneration but dilimb stumps. Devel. Biol., 20: 332-348.
minishes as blastemal cells accumulate Eggert, R. C. 1966 The response of x-irradiated
limbs of adult urodeles to autografts of normal
and is perhaps constrained by the forming
cartilage. J. Exp. Zool., 161: 369-390.
blastema (Schottk and Butler, '44). With
Hamilton, H. L. 1965 Chemical regulation of
the 5.0% concentration of enzyme, the redevelopment in the feather. Biology of the Skin
gression in irradiated limbs went farther
and Hair Growth (Symposium in Canberra,
Australia, 1964), 313-328.
than normally, but lesser concentrations of
G. W. 1960 Alkaline phosphatase of
enzyme were apparently able not only to Hinsch,
the developing down feather: substrates, activainduce some regression in the established
tors, and inhibitors. Devel. Biol., 2: 21-41.
tissues of the limb stump but also to facili- Horn, E. C. 1942 A n analysis of neutron and
x-ray effects on regeneration of the forelimb of
tate an accumulation of a small number
larval Amblystoma. J. Morph., 71: 185-219.
of blastemal cells. Although the accumulaMoog, F. 1959 The adaptations of alkaline and
tion of cells in irradiated limbs was in genacid phosphatases in development. In: Cell, Oreral greater among animals treated with
ganism and Milieu. D. Rudnick, ed. The Ronald
Press, New York, pp. 121-155.
alkaline phosphatase than in other irradiated animals, the accumulations were still Polezhayev, L. V. 1959 Restoration of regenerative ability of the extremities of axolotlr after
inadequate to institute the explosive growth
irradiation with roentgen rays. Dokl. Akad.
pattern found in a normal blastema. On
Nauk SSSR, Biol. Sci. Sect. Transl., 127: 630634.
the other hand, it is at least interesting to
L. V., and N. I. Ermakova 1960
note that no irradiated limb on which a Polezhayev,
Restoration of regenerative capacity of the exconical blastema initially formed was later
tremities in axolotls deuressed bv roentgen
shortened by regression, in contrast to a
radiation. Dokl. Akad. N>uk SSSR; Biol. sci.
Sect. Transl., 131: 227-229.
number of the initially non-regenerating
L. V., N. A. Teplits and N. I.
limbs. It appears that limbs which were Polezhayev,
Ermakova 1961 Restoration of the regeneraable to assemble sufficient cells to form a
tive property of extremities in axolotls inhibited
kind of blastema were spared the destrucby x-ray irradiation, by means of proteins,
nucleic acids and lyophilized tissues. Dokl.
tive regression otherwise frequently inAkad. Nauk SSSR, Biol. Sci. Sect. Transl., 138:
duced by alkaline phosphatase infusions.
The roles of alkaline phosphatase in de- Rose, S. M. 1965 Regeneration versus non-revelopment (Moog, '59) and in the regengeneration. Bull. Tulane Univ. Med. Faculty,
eration of normal unirradiated limbs
24: 237-243.
(Schmidt, '66) have been established as Schmidt, A. J. 1966 The molecular basis of regeneration: enzymes. Illinois Monographs i n
highly significant, but have not been deMedical Sciences, 6: 1-78.
fined. The present findings suggest that Schott6,
0. E., and E. G. Butler 1944 Phases
the role of this particular enzyme lies within regeneration of the urodele limb and their
in the regressive phase of regeneration,
dependence upon the nervous system. J. Exp.
Zool., 97: 95121.
and that this role may be one of the factors
inhibited by the ionizing radiation which Schott6, 0. E., and C. B. Smith 1961 Effects OP
ACTH and cortisone upon amputational wound
stops growth.
healing processes in mice digits. J. Exp. Zool.,
The authors acknowledge with pleasure
the assistance of Mrs. Ruth M. Hedrick,
Mrs. Jan Redick and Miss Charlotte Holley
in the conduct and presentation of this
146: 209-230.
Singer, M., A. Weinberg and R. L. Sidman 1955
A study of limb regeneration in the adult newt,
Triturus, by infusions of dye and other substances directly into the growth. J. Exp. Zool.,
128: 185-218.
Stinson, B. D. 1963 The response of x-irradiated limbs of adult urodeles to normal tissue
grafts. I. Effects of autografts of sixty-day forearm regenerates. J. Exp. Zool., 253: 37-52.
1964 The response of x-hadiated
limbs of adult urodeles to normal tissue grafts.
Iv. Comparative
autografts and homografts of complete forearm regenerates. J. Exp.
ZOO^., 257: 159-177.
Thornton, C. S. 1942 Studies on the origin of
the regeneration blastema i n Triturus viridescens. J. Exp. Zool., 89: 375-390.
53 1
Trampusch, H. A. 1951 Regeneration inhibited
by x-rays and its recovery. Proc. Kon. Ned.
v. Wet.
c, 54: 3-15.
1958 The action of x-rays on the morphogenetic field. 11. Heterotopic skin on irradiated tails. Proc. Kon. Ned. Akad. v. Wet Ser.
Umanski, E. 1937 Untersuchungen des Regenerationsvorganges bei Amphibien mittels
Ausschaltung der einzelnen Gewebe durch
Rontgenbestrahlung. Biol. Zh., 6: 757-758.
TX, irradiated and amputated limb, infused with non-didyzable material from supernatant of blastemal homogenate.
CX, irradiated and amputated limb, infused with amphibian Ringer’s
APX, irradiated and amputated limb, infused with solution of alkaline
1 Dorsal view of limb TX 12, 29 days after amputation. Bone projects
beyond the rounded contours of the softer limb tissues. Original color
photograph from which this figure was taken showed clearly that
projecting bone was not covered with epidermis.
Dorsal view of limb TX 5,29 days after amputation. Originally wedgeshaped projection of bone, after coverage by epidermis, has been reduced to a small spicule by internal erosion of the bony tissue.
Dorsal view of limb TX 3, 21 days after amputation. Clubbed and
pigmented tip of limb presents Characteristic appearance of a limb
with limited retraction and resorption of soft, internal tissues. No
blastemal growth has occurred.
Ventral view of limb CX 25, 46 days after amputation. The small
polyp visible in the center of the rounded limb tip is characteristic of
the structures which formed on many control limbs and experimental
Dorsal view of limb TX 9, 29 days after amputation. Conical tip of
limb represents a small blastema covered by characteristically unpigmented epidermis.
Ventral view of limb APX 10, infused with a 0.1% solution of enzyme, 37 days after amputation. The transverse dark pigmentation
at the limb tip marks the approximate base of a rounded blastema
which initially appeared as shown about two weeks earlier.
7 Ventral view of limb APX 16, infused with a 1.0% solution of enzyme, 37 days after amputation. Wrinkling of the thick skin on sides
and tip mirrors a shortening of internal tissues as regressive changes
have removed bone and other tissues.
Ventral view of limb APX 5, infused with a 0.01% solution of enzyme, 23 days after amputation. Shortening of limb internally is reflected by irregular external contours of limb. Blackened mass at lower
side of limb tip in the picture was actually a bright red bulbous enlargement on the living limb.
9 Same limb APX 5, 55 days after amputation. Bulbous prominence became much larger during the month between photographs. It retained
its bright red color but evidently did not represent a common hematoma. Shortening of the limb had continued to occur slowly.
10 Same limb, APX 5, 70 days after amputation. Projection from tip became smaller and gradually lost its red coloration. Limb appears
slightly shorter in this photograph than in the preceding one (compare distance between dark pigment spots). Some four weeks after
this photograph, sections of the limb showed no trace of the distal
projection and little of the humeral shaft.
J. David Deck and James N. Dent
11 Low power view of a longitudinal section through tip of limb AF'X
54, showing a spicule of bone (center left), at the end of which lies
a fibroid mass. To the upper side of this fibroid tissue can be seen
a darkly-stained nubbin of cartilage. The cartilaginous tissue is i n
a n eccentric position with respect to the end of the bony shaft.
12 Higher power view of a n area from the preceding figure, showing
fibrous and chondroid tissue (center left) and its relation to the small
bit of cartilage a t the end of the limb stump. The chondroid tissue
with its lacunate structure and the eccentric position of the cartilage
do not resemble the usual condylar ending of the humerus, which
suggests that the cartilaginous tissue has formed after limb amputation and infusion.
13 Low power view of a longitudinal section through tip of limb APX
7, showing the darkly-stained cartilaginous tissue of the humeral
condyle, which lies in a relatively tissue-free space, and a small cellular cluster distal ( a t right) to it. The dense fibrous tissue of the limb
tip ends proximal to this cellular cluster.
Higher power view of the tip of limb shown in preceding figure. Note
the moderately large nuclei of some of the cells comprising the distal
cluster. Although the cellular accumulation is quite small, it appears
to have collected as a kind of pseudoblastema subsequent to amputation of the limb through the elbow joint. The limited-number of
these cells, and the sparsity of tissue immediately distal to the cartilage contrast with the dense cellular structure of the blastema in a
normally regenerating limb.
J. David Deck and James N. Dent
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