Conformationally constrained human calcitonin (hCt) analogues
reveal a critical role of sequence 17–21 for the oligomerization
state and bioactivity of hCt
Athanasios Kazantzis
1
, Michaela Waldner
1
, John W. Taylor
2
and Aphrodite Kapurniotu
1
1
Physiological-chemical Institute, Department of Physical Biochemistry, University of Tu
¨
bingen, Germany;
2
Rutgers University,
Department of Chemistry and Chemical Biology, Piscataway, NJ, USA
Calcitonin (Ct) is a 32-residue peptide hormone that is
mainly known for its hypocalcemic effect and the inhibition
of bone resorption. Our p revious studies have led to poten t,
side-chain lactam-bridged human Ct (hCt) analogues
[Kapurniotu, A. Kayed, R. , Taylor, J.W. & Voelter W.
(1999) Eur. J. Biochem. 265, 606–618; Kapurniotu, A. &
Taylor, J .W. (1995) J. Med. Chem. 38, 836–847]. We have
hypothesized that a possibly type I b turn/b sheet confor-
mation in the region 1 7–21 may play an important role in
hCt bioactivity. To investigate this hypothesis, analogues of
the potent hCt agonist cyclo17,21-[Asp17,Lys21]hCt (1)
bearing type I (and II¢)orIIb turn-promoting substituents at
positions 18 and 19 were designed, synthesized and their

relationships of hCt and to design novel hCt agonists for the
treatment of osteoporosis and other bone-disorder-related
diseases.
Keywords: Human calcitonin; b turn/b sheet conformation;
dimerization; receptor binding; hypocalcemic activity.
Calcitonins (Ct) are peptide hormones of 32 amino-acid
residues that have been mainly known for their hypocalce-
mic effect and the inhibition of bone-resorption [1,2].
Calcitonins are u sed therapeutically for the treatment of
osteoporosis and other with bone disorder-related diseases
[1,2]. A marked species-specific differenc e in hypocalcemic
potencies is observed for the Cts. C ts of ultimobranchial
origin, i.e. salmon Ct ( sCt), are the most potent ones,
whereas the human hormone (hCt) h as a strongly reduced
potency [1,2]. Therefore, sCt is the main Ct to be applied
therapeutically to date. However, there is only a 50%
sequence homology between sCt and hCt, which is the cause
for immunogenic reactions in humans w hen t reated with
sCt [3]. Therefore, the development of hCt analogues
bearing high bioactivity and a close structural similarity to
the hCt sequence still remains an important task.
The b iologically active conformation of the Cts yet
remain to be identified. It has been long proposed that the
propensity of the Cts to f orm an amphiphilic a helix in the
region 8–22 might strongly correlate with their bioactivit ies
[4–10]. H owever, while several reports have suggested t hat
this might b e the case for sCt, no evidence h as been
presented for a direct l ink between helicity and bioactivity
for t he human sequence. In contrast, there is increasing
evidence, suggesting that other factors, including a b turn/

mational flexibility, their b ioactive conformation may b e
completely different from t hat observed in the media used i n
the N MR studies [22]. Introduction of conformational
constraints has been often proven to be a necessary strategy
towards ÔlockingÕ a p eptide into a bioactive conform ation
[22,23]. The (i,i + 4) side chain-to-side ch ain c yclization
approach has been successfully used for the stabilization o f
bioactive a helical conformations of several medium-size
peptide hormones [ 24,25]. We have previously applied this
approach to constrain t he potentially bioactive, a helical
conformation of hCt [14]. These studies unexpectedly led to
the discovery of the potent but nonhelical hCt agonist,
cyclo17,21-[Asp17,Lys21]hCt (1). Base d on our structure-
activity results and previously published NMR data [19], we
have suggested that a type I bturn/ b sheet conformation
between residues 17 and 21, t hat might have been stabilize d
by the introduced lactam bridge, may play an essential role
in hCt bioactivity [14]. T his b turn could be centered at
amino acids Lys18 and Phe19 a ccording t o the NMR data
[14,19].
To investigate t he importance o f the conformational and
topological features of the region between amino-acid
residues 17 and 21 for hCt bioactivity and the b turn
hypothesis, we have followed two strategies: in the first one,
we prepared a s eries of ring-size analogues of 1 to study the
effect of ring-size o n b turn/ b sheet stabilization a nd hCt
bioactivity [ 15]. T hese studies led v ery r ecently to the
discovery of the superpotent b turn/b sheet-stabilized, hCt-
agonist cyclo17,21-[Asp17,Orn21]hCt [15]. In the second
strategy, w hich we present in this r eport, we designed and

a
-Boc-protected amino
acids [14,15,25]. F ollowing deprotection and cleavage from
the resin using HF and scavengers a ccording to our recently
published procedure disulfide bridge formation was
achieved by air oxidation of the crude peptides at 10
)4
M
in 0.1
M
NH
4
CO
3
[15] in the presence of 0 .5–1
M
GdnHCl to
improve solubilities and oxidation yields and its c ompletion
was followed b y HPLC. Cru de, oxidized peptides were
purified by reverse phase HPLC on a C
18
Nucleosil 250/8
column (Grom) with a length of 25 cm, an internal diameter
of 8 mm and a 7-lm particle size. The flow rate was
2.0 mLÆmin
)1
and e luting buffers were: A, 0 .058% (v/v)
trifluoroacetic acid in water and B , 0.05% (v/v) trifluoro-
acetic acid in 90% (v/v) C H
3

N,N,N¢,N¢-tetramethyluronium tetrafluoroborate (TBTU;
fourfold excess) and DIEA (sixfold excess). The final
cleavage of the peptide and t he side chain p rotecting groups
from the resin was performed with trifluoroacetic a cid/H
2
O/
thioanisol/EDT/phenol (10/0.5/0.5/0.25/0.5) (v/v w ith the
exception o f p henol) [ 27]. F ormation of the d isulfide bridges
of the crude peptides and HPLC purification were
performed as described above.
Identity of the HPLC purified synthetic peptides 1–6 was
verified by matrix a ssisted laser desorption ionization mass
spectrometry (MALDI-MS) with a Kratos Compact
MALDI I (Shimadzu Europe, D uisburg, Germany) and
a-cyano-4-hydroxycinnamic acid as matrix. Purity of the
HPLC purified peptides were also confirmed by analytical
HPLC analyses. The fo llowing results of MALDI-MS
were obtained for the synthesized peptides by the Boc-
and the Fmoc- protection strategy, respectively:
Cyclo17,21-[Asp17,Lys21]hCt (1): MH+ of 3427.5 (calcu-
lated 3428.9); cyclo17, 21-[Asp17,
D
-Phe19,Lys21]hCt (2):
MH+ o f 3427.1 (3428.1, respectively) (calculated 3428.9);
cyclo17, 21-[ Asp17,Aib18,Lys21]hCt (3): MH+ of 3387.3
(3386.5, respectively) (calculated 3386.9); cyclo17,
21-[Asp17,
D
-Lys18,Lys21]hCt (4): MH+ of 3427.0 (3449.3
(Na+ adduct), respectively) (calculated MH+ 3428.9 and

(3a): MH+ of
906.4 (calculated 906.5).
Far-UV CD spectropolarimetry
CD spectra were obtained with a J-720 spectropolarimeter
(JASCO) at room temperature. Spectra were measured at
0.2 intervals (0.5 n m for the p artial sequence peptides), with
a spectral ban d width of 1 nm, a scan speed of 20 nmÆmin
)1
(50 nmÆmin
)1
for the partial sequence peptides), a response
time of 4 s (8 s for the partial peptides), and represent the
average of three s cans in the range of 195–250 nm (185–
250 nm for the p artial sequence p eptides). Spectra were
measured in 10 m
M
aqueous sodium phosphate buffer
(pH 7.4) and in 10 m
M
sodium phosphate buffer (pH 7.4)
diluted 1/1 (v/v) with TFE and peptides were diluted directly
from their stock solutions into the buffer at the indicated
concentrations. UV a bsorbance a t 274.5 nm was used to
exactly determine the concentrations of the stock solutions
of analogues 1–6 ( 500 l
M
)in1m
M
HCl, using
e

. For SDS/PAGE,
peptide s tock solutions (500 l
M
in 1 m
M
HCl ( see under
CD part) were diluted i nto 1 0 m
M
sodium phosphate
buffer, pH 7.4, at a concentration of 100 l
M
,aswasalso
done for the CD experiments. The peptide solutions were
then diluted with sample buffer [ 30] to a final concentration
of 50 l
M
, boiled for 5 min, and electrophoresed.
Cell culture
T47D cells were obtained f rom the American Tissue Culture
Collection and were cultured in RPMI 1640 containing
10% heat inactivated fetal bovine serum, 1% streptomycin/
penicillin, 0.1 l
M
insulin, and 0.1 l
M
hydrocortisone in 5%
CO
2
and 37 °C. The latter hormones were omitted from the
medium when subculturing cells that were to be used for the

(50 lL) of different concentrations of the peptides in assay
buffer were added to t he cells, and following gentle mixing
cells were incubated for 1 h at room temperature. Peptide
solutions were freshly m ade p rior to each experiment by
782 A. Kazantzis et al. (Eur. J. Biochem. 269) Ó FEBS 2002
diluting peptide s tocks ( 500 l
M
in 1 m
M
HCl [14]) in
assay buffer. Binding was terminated by aspiration of the
medium and w ashing of the cells with NaCl/P
i
three times.
Cells were then removed from the wells by s hort treatment
(1 min) with 0.5
M
NaOH (2 · 0.5 mL) and bound radio-
activity was assessed by c-counting (counter efficiency
 70%). Nonspecific binding was determined a s t he binding
of 100 n
M
sCt. This was assessed from 13 independent
experiments to b e 12.94% ( ± 3.59). Specific binding was
the difference between total binding (tracer alone) and
nonspecific binding.
In vivo
hypocalcemic assay
The in vivo hypocalcemic assay in mice w as performed as
described previously [14,15]. Hypocalcemic activities are

effects of 1, 3, and 4 at doses of 10–0.1 ng that corresponded
to the linear parts of the curves as compared to t he respective
hCt effects, whereas the effects o f 2, 5, and 6 were very
similar t o h Ct. O f note, the low maximum hypocalcemic
effects of 2 and 5 also differed significantly from the
maximum effect of hCt (P < 0.05 and < 0.01, respectively).
In addition, the effects of the 100 ng doses of 2 and 5 also
differed significantly from the effect of hCt (P <0.05).
Effective concentrations at 50% o f the maximal e ffect
(EC
50
) were estimated by nonlinear regression analyses of
the data using the software
PRISM
(GraphPad Software, Inc.)
RESULTS AND DISCUSSION
Design of the analogues
A b turn conformation strongly depends on the n ature a nd
chirality of the amino-acid residues a t its corner positions
and even small changes of these residues may dramatically
affect the type a nd stability o f the turn [34,35].
To investigate the importance of t he type of the
postulated turn for hCt b ioactivity, the i +1turn-residue
L
-Phe19 of 1 was r eplaced by
D
-Phe19 t o giv e cy clo17,21-
[Asp17,
D
-Phe19,Lys21]hCt (2) ( Scheme 1). This substitu-

D
-Phe19]hCt (5)and[
D
-Lys18]hCt (6)were
also synthesized and s tudied (Scheme 1).
CD spectroscopy describes the average conformation
of polypeptides and the contribution o f a local confor-
mational feature such as a f our-residue b turn to the CD
spectrum of a polypeptide of 32 amino acids will usually
remain unrecognised due to o ther secondary s tructure
elements [41]. Therefore, t o be able to obtain more detailed
information about a potential bturn stabilization we also
synthesized a nd studied the conformation of cyclo17,21-
[Asp17,Lys21]hCt(16–22)-NH
2
(1a), cyclo17,21-[Asp17,
D
-Phe19,Lys21]hCt(16–22)-NH
2
(2a), cyclo17,21-[Asp17,
Aib19, Lys21]hCt(16–22)-NH
2
(3a), and [Asp17,Lys21]
hCt(16–22)-NH
2
(1b) that comprise mainly the lactam
bridge-containing region 16–2 2 of analogues 1, 2 and 3,
respectively, and als o a linear control peptide for 1a ,
analogue 1b.
Conformational analyses by CD: studies of hCt

reference s pectra: one class C CD spectrum [42] (a negative
band between 200 and 210 nm, a weak negative b and at
about 220 nm, and a possitive band between 180 and
195 nm), one spectrum correponding to an op en or ÔZÕ
conformation (one minimum at 195–200 nm) [43], and the
third component could be the type I and II bturn spe ctrum
according t o Brahms a nd Brahms [28] that exhibits a
characteristic minimum at a bout 225 nm and a maximum
at 210–220 nm.
The spectra of 1a and 1b were very similar to each other.
The spectra of both peptides showed positive bands at
about 220 nm (Fig. 1B) th at most likely arise from coupling
between the phenylalanyl (there are three P he residues in
sequence 1 6–22) and the amide chromophores [44,45]. This
suggestion w as further s upported by t he observation t hat
the intensity of the 220 band, that most likely corresponds to
the phenylalanyl La band [42,45], was significantly le ss in 1b
and its maximum was blue shifted compared to 1a [44,45].
The similarity between the CD curves of 1a and 1b
suggested that the lactam bridge of 1a did not significantly
constrain t he aqueous conformation of 1b . However, the
intense positive band of 1a at 220 nm suggest ed that
cyclization may have resulted in topological changes of the
side chain of one or more Phe r esidue(s). Interestingly, these
positive bands were not present in 2a and 3a suggesting a
significant effect of residues
D
-Phe19 and Aib18 on back-
bone conformation and t opography of the Phe side chain(s)
in sequence 16–22. The s pectrum of 3a had, except for t he

M
(for hCt and 1–6)and1m
M
(for 1a, 1b, 2a ,and3a) and at room temperature.
784 A. Kazantzis et al. (Eur. J. Biochem. 269) Ó FEBS 2002
As shown in F ig. 1C, TFE had a strong structuring and
a helix inducing effect on hCt and the nonbridged peptides 5
and 6 that conta ined 40–50% a helical components accord-
ing to s econdary structure a nalysis by t he reference s pectra
of Brahms and Brahms [28]. This was consistent with the
long described a helix-forming propensity of the middle
region of the calcitonin sequence [4,16,52,53]. In contrast,
nearly no a helical contents were found for all bridged
analogues w hich contained i nstead 40–50% b sheet struc-
ture, t he rest being mainly r andom coil. Thu s, it appears
that the a helix-inducing e ffect of TFE on 1–4 was not as
strong as on hCt, 5, and 6, most likely b ecause 1–4 were
already significantly constrained by t he lactam bridges. Of
note, the CD spectra of 1–4 were very similar to each other.
Taken together, the results in 50% TFE were consistent
to the ones under pure aqueous conditions and suggested
that the introduction of the substituents at positions 18 and
19 of analogue 1 did not affect its o verall con formation. In
addition, the studies in 50% TFE confirmed our earlier
observations [14] that the 20- membered Asp17 t o Lys21
lactam bridge had an a helix-destabilizing a nd a bsheet-
stabilizing effect on hCt. It has been reported that (i,i +4)
Asp/Lys bridges may result in both stabilization [ 54] and
destabilization [55] of a helices. Together with t hese reports,
our results suggest that the effect of such bridges on a helix

as low a s 5 l
M
were applied which are clo se to physio-
logically relevant concentrations. Confirming previous
findings [14,15], no con centration dependence of t he CD
spectra or aggregation was found between 5 and 100 l
M
for
hCt and also for 1 in aq ueous buffer, pH 7.4. This suggested
that the conformations observed by CD were adopted by
monomeric peptides. However, there was a striking con-
centration dependence of the CD spectra o f 2–4 between
5 and 100 l
M
(Fig. 2 A), that was indicative of peptide self
association [5]. The mean residue ellipticities at 202 nm
([h]
202
), that corresponded t o the minima of the C D spectra,
Fig. 2. Studies on t he oligomerization propensity o f h Ct and 1–6.
(A) CD concentration dependence studies: the concent ration depen-
dence ( 5–100 l
M
) of t he mean residue ellipticity at 202 nm ([ h]
202
)
for analogues 2, 3,and4 in aqueous buffer is shown. In the inset
the line ar regression analysis of the data points of 2 th at were intro-
duced to t he eq uation [([ h]
202(observed)

phosphate
buffer, pH 7.4 w ere diluted 1 : 1 with sample buffer containing 2 %
SDS, boiled and electrophoresed as described under Materials and
methods.
Ó FEBS 2002 Conformationally constrained hCt analogues (Eur. J. Biochem. 269) 785
decreased with increasing concentrations, s uggesting that
the peptides b ecame more ordered during s elf association
[42]. Plateau values were reached at 50 l
M
(Fig. 2 A). The
change of [h]
202
was b est fitted to an equation describing
peptide cooperative dimerization [5].
Dimerization was also confirmed by SDS/PAGE
(see below). [h]
202
for the monome rs ([h]
202(mono)
)were
obtained at 5 or 1 l
M
, where all analogues were essentially
monome ric, and were )6059 (2), ) 6802 (3)and) 7680
deg.cm
2
/dmol (4). Plots of the observed [ h]
202(obs)
vs.
{([h]

As observ ed for hCt a nd 1, CD concentration depen-
dence studies of 5 and 6 showed that these analogues also
did not aggregate. This suggested that self-assembly of 2, 3,
and 4 was related to both the confor mational r estriction, i.e.
the b turn/b sheet stabilization that had been achieved by
the lactam bridge, and the topological features of the side
chains of residues 18 a nd 19. It has b een previously
suggested that several hydrophobic residues, that may
occupy the one face of the putative a helical region 8–21 of
hCt, participate in the initial helix–helix association step
[61]. This s tep i s then followed by f ormation of b sheet
aggregates [61]. Thus, a reason for the increased b sheet
formation and oligomerization propensity of 2–4 could be
the changed topogr aphy of the side chains of r esidues 18
and 19 in 2–4. This may have led to formation of an
hydrophobic face in the lac tam bridge-stabilized b sheet a n d
an increase d dimerization and oligomerization propensity
[55,62]. Association of b sheets i nto multimers and fibrils
would be consistent with models of hCt fibrils [56,57].
To further study the self-assembly states of 2–4,50l
M
solutions of hCt and the analogues were next subjected to
SDS/PAGE analysis (Fig. 2B,C). B ased on the results of the
CD studies, 2, 3, and 4 were expected to predominantly
consist o f (noncovalent) dimers. Stability of the dimers
towards S DS treatment conditions (2% S DS, 100 m
M
2-mercaptoethanol, 100 °C f or 5 min [30]) was not known.
In fact, mixtures of peptide monomers and dimers at a ratio
of about 40/60 were observed in 2, 3, and 4 (Fig. 2B),

I-labelled
sCt as described under Materials and meth-
ods. Specific r adioligand binding is plotted vs.
the concentration of c ompeting sCt, hCt, and
1–6 as indicated. Data for 1–6 represent the
mean ± SD for three to five independen t
experiments and data for h Ct and sCt are t he
mean of 13 and 1 4, respectively, assays.
786 A. Kazantzis et al. (Eur. J. Biochem. 269) Ó FEBS 2002
specific binding of the r adioligand
125
I-labelled sCt that
binds with high affinity and selectivity to the Ct receptors of
this cell line [33]. As shown in Fig. 3, 1 sh owed increased
binding affinity compared to hCt. Receptor binding affinity
of 1 (IC
50
¼ 2n
M
) was threefold lower than the a ffinity of
sCt (IC
50
¼ 630 p
M
), that was about 6 times more potent
than hCt ( IC
50
¼ 4n
M
) [33]. The higher binding affinity of

Asp17,Lys21-lactam br idge leads to a partial inversement of
the latter effect.
In vivo
hypocalcemic activities
To directly assess the biological relevance of the introduced
substitutions, we n ext studied in vivo hyp ocalcemic potencies
in mice [1–3,14,15]. Analogues 3 and 4 exhib ited identical
bioactivity to 1 [14], which was 5 times more potent than
hCt (Fig. 4). However, 2 was co mpletely devoid of the
increased bioactivity of 1.Analogue2 hadanEC
50
of 25 ng
that corresponded to a hypocalcemic potency that was even
lower than t he potency of hCt (EC
50
, 20 ng). Of note, 2 was
unable to reach the maximum hypocalcemic effect o f hCt
(20%) ( caused by 2 lg hCt), even when 10-fold higher doses
were applied. The maximum effect of 2 was caused by the
20-lg dose a nd was at 16.1%. A nalogue 5 had t he same
dose–response c urve as 2. This i ndicated that t he confor-
mational restriction was not capable of r eversing the
negative e ffect of the inversement of ch irality of Phe19 on
in vivo bioactivity of 1. In contrast, 6 had the same potency
and maximum effe ct as hCt which suggested that inverse-
ment of chirality of L ys18 was w ell tolerated.
Correlation of solution conformations with receptor
binding affinities and
in vivo
bioactivities of hCt

measured in gro ups of 3–10 mice pe r dose and
3–8 control mice 1 h a fter subcutane ous
injection of the peptide s olution or vehicle
alone. Hypocalce mic activities of ea ch dose
are expressed as percent reduction of calcium
(mean ± S EM) caused by the p eptide relative
to control.
Ó FEBS 2002 Conformationally constrained hCt analogues (Eur. J. Biochem. 269) 787
Furthermore, 2 was t he only analogue with reduced in vivo
activity as compared to hCt, suggesting a crucial role of the
topological features of the side chain of Phe19 i n confor-
mation and in vivo bioactivity o f hCt.
In the T47D receptor bindin g studies, only 1 showed a
higher binding affinity than hCt, whereas 2–4 were equally
potent t o hCt. Analogues 5 and 6 showed decreased binding
affinities as compared to hCt. Thus, t hese studies demon-
strated the cr ucial role of residues 18 a nd 19 and their
chirality for human receptor binding. M oreover, these
findings suggested that the side chains of residues 18 and 19
and/or of other residues i n r egion 1 7–21 may be d irectly
involved in receptor binding. T hese results we re consistent
with a recent model of ligand–Ct-receptor interaction and
activation. According to this model, all three regions of the
Ct sequence, including the N-terminal l oop 1–7, the
potential ahelica l region 8 –22, and t he C-terminal region
22–32 interact with distinct domains of the Ct receptor
[10,67]. Accordingly, even small or local changes in confor-
mational and topographical features in Ct, may r esult i n
dramatic changes in binding affinities an d efficacies [10].
Taken together, our CD and bioactivity studies suggested

bioactivity of the Cts is d etermined by many different
factors including receptor binding, signal transduction,
receptor regulation, as also bioavailability and biodegrada-
bility of the ligand [9,10,68]. The analogues 2–6 presented
here differ from 1 and hCt only in the chirality o f residues 19
and/or 18 and/or the p resence of Aib instead of Lys18.
Thus, these analogues are expected to have in vivo ahigher
proteolytic stability t han 1 and hCt [69]. Therefore, the
Table 1. S ummary of the results of the CD s tudies, the receptor binding affinities, and the hypocalcemic potencies in vivo of hCt, sCt, analogues 1–6
and/or the partial sequence analogues 1a, 1b, 2a, and 3a. The CD data of the partial sequence analogues 1a, 1b, 2a,and3a are presented, because
there w ere no differences between the spectra of the respective complete s equence peptides. CD spectra were measured in 1 0 m
M
phosphate buffer,
pH 7.4 and in 50% TFE in 10 m
M
phosphate buffer, pH 7.4, at room temperature. Peptide concentrations were 1 m
M
(for 1a, 1b, 2a,and3a)and
5 l
M
(for hCt, sCt, and 1–6). Exp. turn, expected stabilized turn based o n the analogue design strategy. Min., minimum of CD spectrum; max.,
maximumofCDspectrum.
Analogue
(exp. turn)
Conformational analysis of partial sequence peptides by CD
Receptor binding
affinities (in vivo)
Hypocalcemic potencies
In aqueous solution In 50% aqueous TFE
1 Min., 190 nm; max., 220 nm:

less maximum at 220 than in 1a
Spectrum of 1b: very
weak bands
Sixfold lower than sCt the strongest potency;
sCt sCt: mainly random coil
a
sCt: a helix
(more than hCt)
a
The strongest binding
(IC
50
¼ 630 p
M
)
95-fold lower than sCt;
95-fold higher than hCt [15]
a
The sCt data were not shown in this work (see also references [4,12]).
788 A. Kazantzis et al. (Eur. J. Biochem. 269) Ó FEBS 2002
results of our studies support the notion that the in vivo
hypocalcemic potency of the C ts is directly associated to a
distinct bioactive con formeric population r ather than t o
differences in proteolytic degradation rates [1,7,14,15,18,
67,70–72].
In conclusion, our structure activity studies supported the
suggestion that a type I bturn/ b sheet conformation in the
region 17–21 may play a n important role in hCt b ioactivity
and showed that the conformation and the topological
features of the side chains o f amino acid residues 18 a nd 19

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