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https://doi.org/10.1177/1352458519828294
https://doi.org/10.1177/1352458519828294
MULTIPLE
SCLEROSIS MSJ
JOURNAL
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Multiple Sclerosis Journal
 1 –8
DOI: 10.1177/ 
1352458519828294
© The Author(s), 2019. 
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Introduction
Progressive forms of multiple sclerosis (PMS) are 
characterized by relentless, progressive accumulation 
of disability without remission over time.1 Synaptic 
plasticity, the capacity of neuronal cells to modulate 
efficiency of synaptic transmission, is potentially able 
to limit clinical expression of brain damage.2–5 Indeed, 
efficiency of synaptic transmission can be function-
ally potentiated or depotentiated in a long-lasting 
way, a mechanism called long-term potentiation 
(LTP) or long-term depression (LTD), respectively.6
After a central nervous system (CNS) damage, LTP of 
surviving synapses may restore function of neurons 
that have lost part of their synaptic inputs and drive 
the formation of novel adaptive patterns of functional 
connectivity in the CNS.7,8
Recent studies showed that synaptic plasticity reserve 
may contrast disability progression in MS. Synaptic 
plasticity reserve can be probed in awake human sub-
jects through transcranial magnetic stimulation 
(TMS) protocols, a non-invasive and painless brain 
stimulation technique. During TMS, high power and 
short duration magnetic pulses are applied over the 
scalp to activate the underlying focal cortical region. 
Repetitive TMS pulses can induce long-lasting 
changes of cortical excitability analogous to LTP.3,4,9 
Moreover, TMS-induced synaptic plasticity appeared 
significantly compromised in patients with primary 
Oral D-Aspartate enhances synaptic plasticity 
reserve in progressive multiple sclerosis
Carolina G Nicoletti, Fabrizia Monteleone, Girolama A Marfia, Alessandro Usiello, 
Fabio Buttari, Diego Centonze and Francesco Mori
Abstract
Background: Synaptic plasticity reserve correlates with clinical recovery after a relapse in relapsing–
remitting forms of multiple sclerosis (MS) and is significantly compromised in patients with progressive 
forms of MS. These findings suggest that progression of disability in MS is linked to reduced synaptic 
plasticity reserve. D-Aspartate, an endogenous aminoacid approved for the use in humans as a dietary 
supplement, enhances synaptic plasticity in mice.
Objective: To test whether D-Aspartate oral intake increases synaptic plasticity reserve in progressive 
MS patients.
Methods: A total of 31 patients affected by a progressive form of MS received either single oral daily 
doses of D-Aspartate 2660 mg or placebo for 4 weeks. Synaptic plasticity reserve and trans-synaptic cor-
tical excitability were measured through transcranial magnetic stimulation (TMS) protocols before and 
after D-Aspartate.
Results: Both TMS-induced long-term potentiation (LTP), intracortical facilitation (ICF) and short-inter-
val ICF increased after 2 and 4 weeks of D-Aspartate but not after placebo, suggesting an enhancement of 
synaptic plasticity reserve and increased trans-synaptic glutamatergic transmission.
Conclusion: Daily oral D-Aspartate 2660 mg for 4 weeks enhances synaptic plasticity reserve in patients 
with progressive MS, opening the path to further studies assessing its clinical effects on disability pro-
gression.
Keywords: D-Aspartate, synaptic plasticity, disability, transcranial magnetic stimulation, long-term 
potentiation, theta burst stimulation
Date received: 1 October 2018; revised: 7 January 2019; accepted: 8 January 2019
Correspondence to: 
D Centonze 
Multiple Sclerosis Clinical & 
Research Center, Department 
of Systems Medicine, 
University of Rome Tor 
Vergata, Via Montpellier 1, 
00133 Rome, Italy. 
centonze@uniroma2.it
Carolina G Nicoletti 
Fabrizia Monteleone 
Girolama A Marfia 
Multiple Sclerosis Clinical & 
Research Center, Department 
of Systems Medicine, 
University of Rome Tor 
Vergata, Rome, Italy
Alessandro Usiello 
Department of 
Environmental, Biological 
and Pharmaceutical Sciences 
and Technologies, The 
Second University of Naples, 
Caserta, Italy/ Laboratory of 
Behavioral Neuroscience, 
CEINGE —Biotecnologie 
Avanzate, Naples, Italy
Fabio Buttari 
Unit of Neurology, IRCCS 
Neuromed, Pozzilli, Italy
Diego Centonze 
Francesco Mori 
Multiple Sclerosis Clinical & 
Research Center, Department 
of Systems Medicine, 
University of Rome Tor 
Vergata, Rome, Italy/ Unit 
of Neurology, IRCCS 
Neuromed, Pozzilli, Italy
828294MSJ0010.1177/1352458519828294Multiple Sclerosis JournalCG Nicoletti et al.
research-article2019
Original Research Paper
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PMS patients,3 suggesting that ineluctable progres-
sion of disability in MS is associated to reduced syn-
aptic plasticity reserve.
Based on these premises, we hypothesized that the 
enhancement of synaptic plasticity reserve may 
improve clinical progression in MS. LTP is mainly 
controlled by glutamate receptor and ion channel pro-
tein N-methyl-D-aspartate receptor (NMDAR).10 
Stimulation of NMDAR after traumatic and ischemic 
brain injury in mice limits the clinical manifestations of 
neuronal damage by inducing compensatory plasticity 
in surviving neurons.2,5 NMDA is an agonist for a sub-
set of glutamate receptors in the brain, particularly for 
the NMDAR (for a review, see Volianskis et al.10). 
NMDA can be synthesized by methylation of 
D-Aspartic acid (D-Asp) via the enzyme D-Asp 
methyl-transferase.11 D-Asp is an endogenous ami-
noacid present in the brain at high levels during embry-
onic stages and strongly decreasing after birth due to 
D-Asp oxidase (DDO) expression.12 Evidence suggests 
that D-Asp itself acts as a classical neurotransmitter as 
its biosynthesis, degradation, uptake, and release take 
place within the pre-synaptic neuron, and its release 
triggers a response in the post-synaptic neuron.13,14 
D-Asp binds the NMDAR at its L-Glu binding site,15 
hence interacting with this receptor both directly and 
indirectly. Genetic and pharmacological mouse models 
evidenced that increased D-Asp enhances hippocampal 
LTP, dendritic arborization, and spatial memory.16–18
D-Asp is approved for use in humans and commer-
cialized as a dietary supplement. In light of these 
premises, we wanted to explore in this preliminary 
study whether administration of D-Asp in patients 
affected by PMS is able to enhance LTP induction.
Subjects and methods
The study was approved by the local Ethics Committee 
of the University Hospital Tor Vergata, Rome (Italy). 
All patients signed a written informed consent before 
enrollment.
Subjects
In all, 31 consecutive patients affected by PMS, fol-
lowed at the Multiple Sclerosis Clinical Center of Tor 
Vergata University Hospital, Rome, between March 
2014 and September 2016 were enrolled in an explor-
atory, prospective, randomized, placebo-controlled, 
double-blinded, mono-center study.
Inclusion criteria were (a) diagnosis of primary or 
secondary PMS according to the 2010 revision of Mc 
Donald criteria;1 (b) Expanded Disability Status Scale 
(EDSS) score comprised between 3 and 6; and (c) age 
ranging from 18 to 65 years. Exclusion criteria were 
(a) other concomitant neurological or psychiatric dis-
orders, (b) history or the presence of any unstable 
medical condition such as malignancy or infection, 
(c) the use of medications with increased risk of sei-
zures, (d) concomitant participation to other interven-
tional pharmacological studies or within 8 weeks 
before inclusion, (e) positive pregnancy test at base-
line or active pregnancy plans during the time of the 
study, and (f) the absence of reproduciblemotor-
evoked potentials (MEP) from the first dorsal interos-
seous (FDI) muscle of the right hand.
After recruitment, patients were randomized to 
receive single daily doses containing D-Asp 2660 mg 
in oral solution or placebo (Giellepi SpA, Lissone, 
MB, Italy) in identical preparations for 4 weeks.
To randomly allocate patients to treatment group, we 
generated two separate random sequences using 
Microsoft Excel software, one for primary progres-
sive and the second for secondary progressive MS 
patients to ensure an equal distribution of the two con-
ditions in both groups. To ensure for blinding, trial 
participants and investigators involved in clinical 
assessments and data collection were unaware of the 
assigned preparation. To control the success of blind-
ing, patients and investigators were asked whether 
they were able to understand group allocation. The 
use of concomitant treatments remained unchanged 
during the entire duration of the study to avoid for 
potential confounding factors.
Neurophysiological assessment
Resting motor thresholds and active motor thresholds 
(RMT and AMT), recruitment curves, and paired-pulse 
(pp) TMS measurements of intracortical inhibition and 
facilitation were recorded to evaluate cortical functional 
integrity and trans-synaptic transmission, together with 
intermittent theta burst stimulation (iTBS), our primary 
outcome, performed to study synaptic plasticity reserve 
at each TMS experimental session.
MEPs were evoked through a figure-of-eight coil 
connected to a Magstim2002 magnetic stimulator and 
recorded from the right FDI with surface cup elec-
trodes. Coil position was adjusted to find the optimal 
scalp site to evoke motor responses in the contralat-
eral FDI.
The minimum stimulation intensity required to evoke 
an MEP from the FDI was defined as the RMT when 
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CG Nicoletti, F Monteleone et al.
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the muscle was at rest and AMT when the muscle was 
voluntarily contracted.
Average amplitudes of 10 MEPs elicited by single-
pulse TMS, with the muscle at rest, for each stimula-
tion intensity (90%, 100%, 110%, 120%, 130%, 
140%, and 150% of the RMT) were measured to cal-
culate recruitment curves of the motor cortex. Slopes 
of the entire recruitment curve were calculated in 
Microsoft Excel using linear regression.
ppTMS protocols consisted of the short-interval intra-
cortical inhibition (SICI), intracortical facilitation 
(ICF), and short-interval intracortical facilitation 
(SICF). For SICI and ICF, the average amplitude of 
10 MEPs was evoked by three different stimulation 
conditions: (a) TMS test stimulus (TS) alone, (b) TS 
preceded by a conditioning stimulus (CS) with an 
inter-stimulus interval (ISI) of 2 ms for SICI, and (c) 
10 ms for ICF.19 TMS stimulation intensity was 80% 
of AMT for the CS and 130% of RMT for the TS. 
During SICF, the TS was given alone or followed by 
a CS at two different ISIs (1.5 and 2.7 ms).20 For SICI 
and ICF, CS intensity was 80% AMT; for SICF, CS 
intensity was 90% of RMT; and TS intensity was 
130% RMT for all ppTMS experiments. The order of 
presentation of the different stimulation conditions 
during each ppTMS protocol was randomly generated 
by a computer using Signal 3 software (Cambridge 
Electronic Devices, Cambridge, UK).
iTBS protocol consisted of 200 bursts, and each burst 
composed of three TMS pulses delivered at 50 Hz. 
Trains of 10 bursts were delivered at a frequency of 
5 Hz in 2 s, for 20 times, with an inter-train time inter-
val of 8 s, over the motor “hot spot” of the right FDI 
through a Magstim Rapid2 stimulator (Magstim, 
Whitland, UK). Stimulation intensity for iTBS was 
80% of AMT. The effect of iTBS on corticospinal 
excitability was measured by calculating the ampli-
tude changes of the MEPs evoked by a constant inten-
sity (130% RMT) TMS pulse delivered over the 
motor “hot spot.” A total of 25 MEPs were collected 
before iTBS (baseline) and at two different time 
points (0 and 15 min) after the end of the stimulation 
procedure. Post iTBS, MEP amplitudes were aver-
aged and normalized to the mean baseline amplitude.
Adverse events
All adverse events (AE) were documented and 
reported in a study case report form. Monitoring of 
AE was performed for the entire duration of the study. 
Safety and tolerability were assessed by the frequency 
of reported AE.
Clinical assessment
The study was designed to test the effects of D-Asp on 
plasticity reserve; however, as exploratory endpoint, we 
also wanted to test if any difference in progression of 
clinical disability emerged over a time period of 
24 weeks after D-Asp and placebo. EDSS21 and the 
Multiple Sclerosis Functional Composite (MSFC)22 
measures were thus collected before and 12 and 
24 weeks after D-Asp or placebo. MSFC consist of 
three quantitative and objective tests which investigate 
leg function performance and deambulation (Timed 25 
Foot Walk—T25FW), upper extremity function (9-Hole 
Peg Test—9HPT), and cognitive function (Paced 
Auditory Serial Addition Test (PASAT)). Fatigue was 
assessed through Fatigue Severity Scale (FSS).23
Aim of this study was to measure the effects of D-Asp 
on synaptic plasticity. To this aim, our primary outcome 
measure was TBS-induced LTP before/after D-Asp. 
TMS recordings, TBS-induced LTP-like (primary out-
come), ppTMS, and recruitment curves (secondary out-
comes) were performed at baseline, the day before 
starting the treatment, and 2, 4, and 8 weeks after. We 
further explored possible clinical effects of D-Asp on 
disability progression by measuring EDSS and MSFC 
at baseline, 12 weeks, and 24 weeks after treatment ini-
tiation. We finally explored a possible effect on fatigue 
by measuring FSS at baseline and 4 and 8 weeks after 
treatment initiation as we hypothesized that fatigue 
would parallel cortical excitability changes.24
Statistical analysis
Sample size was calculated based on data collected in 
our laboratory during a previous study3 considering 
significant effect size of 1.25, 0.05 alpha level, and 0.9 
power. All collected variables were tested for normal-
ity and homogeneity of variance by using Kolmogorov–
Smirnov and Levene’s tests. The effect of D-Asp on 
collected variables was tested through repeated meas-
ures analysis of variance (ANOVA) using TIME as 
within-subjects and GROUP as between-subjects main 
factors, and in case of parametric variables, Friedman 
test followed by Wilcoxon post hoc pairwise compari-
sons was used alternatively in case of non-parametric 
variables. For repeated measures ANOVA, sphericity 
was tested by using Mauchly’s test and results cor-
rected by using Greenhouse–Geisser method if indi-
cated. Significance level was set at p < 0.05.
Results
In all, 16 patients, 7 females and 9 males, aged between 
33 and 61 years, disease duration = 17.5 ± 10.1 years 
(mean ± standard deviation (SD)), EDSS comprised 
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between 3.5 and 6.0, 8 diagnosed with PPMS and 8 
diagnosed with SPMS, received D-Asp at a daily dose 
of 2660 mg (D-Asp group), and 15 patients, 7 females 
and 8 males, aged between 36 and 59, disease dura-
tion = 15.6 ± 12.3, EDSS comprised between 3.0 and 
6.0, 8 diagnosed with PPMS and 7 diagnosed with 
SPMS, received an identical oral preparation contain-
ing placebo (control group; Table1).
Adverse events
One single episode of diarrhea was reported by one 
patient on the D-Asp group after D-Asp intake on day 
4. D-Asp intake was continued with no further epi-
sodes of diarrhea. Three patients complained 
increased fatigue 4 weeks after D-Asp withdrawal. 
One patient on D-Asp and two patients on placebo 
complained increased rigidity at the lower limbs 
24 weeks after treatment initiation. Four patients on 
D-Asp and three patients in the placebo arm com-
plained increased fatigue 24 weeks after treatment ini-
tiation. No other adverseevents were reported during 
treatment in the two groups.
TMS measurements
LTP reserve significantly increased after 2 and 4 weeks 
of D-Asp intake but not after placebo as shown by 
repeated measures ANOVA. Indeed, post-iTBS MEP 
mean amplitude showed a significant TIME × GROUP 
interaction (F = 3.30, p < 0.05) and a significant 
increase both after 2 (F = 4.49, p < 0.05) and 4 (F = 9.61, 
p < 0.01) weeks of D-Asp administration which 
returned to baseline values at week 8 (4 weeks after 
treatment was ended; Figure 1). ICF also increased 
during treatment in the D-Asp group but in the placebo 
group. Indeed, a significant TIME × GROUP interac-
tion emerged for both ICF (F = 5.17, p < 0.01) and 
SICF the latter measured at both 1.5 ms (F = 3.62, 
p < 0.05) and 2.7 ms (F = 3.11, p < 0.05) ISIs. Post hoc 
contrasts revealed that the increase in ICF was already 
evident after 2 weeks (F = 11.65, p < 0.01) of D-Asp 
and lasted at least until after 4 weeks (F = 7.87, p < 0.01) 
of D-Asp intake, returning to baseline values 4 weeks 
after D-Asp withdrawal. Conversely, SICF signifi-
cantly increased only after 4 weeks of D-Asp intake for 
both ISIs (F = 6.81, p < 0.05 for 1.5 ms ISI; F = 8.14, 
p < 0.01 for 2.7 ms ISI) returning to baseline 4 weeks 
after withdrawal (Figure 2(b)–(d)).
Table 1. Demographic and clinical characteristics of enrolled subjects.
Condition Sex 
(F/M)
Age (years) Disease 
duration 
(years)
EDSS SPMS PPMS DMD Concomitant 
medications
D-Aspartate 7/9 48.0 ± 8.0 17.5 ± 10.1 4.2 ± 1.5 8 8 3× 
SPMS on 
fingolimod
1× PPMS 
paroxetine 20 mg 
daily
1× PPMS 
pregabalin 75 mg 
twice daily
Placebo 7/8 45.5 ± 10.2 15.6 ± 12.3 4.6 ± 2.1 7 8 2× 
SPMS on 
fingolimod
1× PPMS 
Baclofen 
20 mg ×3 daily
1×PPMS 
amantadine 
100 mg twice 
daily
F: female; M: male; EDSS: Expanded Disability Status Scale; SPMS: secondary progressive multiple sclerosis; PPMS: primary 
progressive multiple sclerosis; DMD: disease-modifying drugs.
Figure 1. Effect of D-Aspartate on synaptic plasticity 
reserve. Magnitude of LTP induced by TMS through the 
intermittent theta burst stimulation protocol increased after 
2 and 4 weeks of D-Aspartate 2.660 mg daily doses but 
not after placebo. (*p < 0.05; pre = before treatment; 2w, 
4w, and 8w = 2, 4, and 8 weeks, respectively, after starting 
treatment.)
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No significant effect of TIME, GROUP, and 
TIME × GROUP interaction emerged for RMT, 
AMT, slopes of recruitment curves, and SICI (Figures 
2(a), 3, and 4).
Clinical assessments
EDSS and MSFC scores did not show any significant 
change in both groups (Figure 5(a)–(f)).
FSS score significantly improved after 4 weeks of 
D-Asp (F = 3.60, p < 0.05), but not after placebo, 
returning to baseline values 8 weeks after treatment 
initiation (Figure 5(e)).
Discussion
This study shows that daily D-Asp administration for 
4 weeks in people affected by PMS enhances plasticity 
reserve as measured by TMS-induced LTP. Indeed, after 
2 and 4 weeks of D-Asp, we observed an increase in 
iTBS-induced LTP but not after placebo. Furthermore, 
we observed an increase of glutamatergic synaptic trans-
mission as measured by ICF and SICF. In parallel, 
decreased fatigue, as assessed by the FSS was observed, 
while there were no changes in any other clinical score.
This negative result was expected as the study was 
specifically designed to test for an effect of D-Asp on 
TMS-induced LTP. We hypothesize that this result 
may depend on the short duration of the treatment 
Figure 2. Effect of D-Aspartate on synaptic transmission measured through paired-pulse TMS protocols. (a) Short 
intracortical inhibition did not change after D-Aspartate or Placebo. (b) Magnitude of intracortical facilitation (ICF) 
increased after 2 and 4 weeks of D-Aspartate 2.660 mg daily doses but not after placebo. Also, short-interval intracortical 
facilitation (SICF) at both (c) 1.5 and (d) 2.7 ms inter-stimulus intervals (ISI) increased after 4 weeks of D-Aspartate but 
not after placebo (*p < 0.05).
Figure 3. Effect of D-Aspartate on TMS thresholds for motor-evoked potentials. Both the (a) resting motor threshold 
(RMT) and (b) active motor threshold (AMT) remained unchanged after D-Aspartate or placebo.
Figure 4. Effect of D-Aspartate on recruitment curves 
of motor-evoked potentials. Recruitment curves were 
obtained by measuring the mean MEP amplitude evoked 
by TMS pulses of increasing intensities, 10% increments, 
ranging from 90% to 150% of resting motor thresholds 
(RMT). The graph shows the mean slopes of the entire 
recruitment curves. No significant differences emerged 
after D-Aspartate or placebo.
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with D-Asp, being 4 weeks probably insufficient to 
induce significant clinical effects that could be 
detected. The improvement of fatigue, however, may 
be interpreted both as an effect of increased corti-
cospinal excitability24 or as an effect of D-Asp on tes-
tosterone levels25,26 and muscle mass.27
D-Asp effect on LTP and cortical excitability may 
be explained by its interaction with glutamate 
receptors. D-Asp acts as neurotransmitter and as 
neuromodulator.14,17,28 It has been demonstrated that 
D-Asp activates glutamate NMDAR and modulates 
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic 
acid receptor (AMPAR). In fact, D-Asp stimulates 
NMDAR binding the same site of L-Glu and acting 
as an agonist Glu.15 Co-stimulation through L-Glu 
and D-Asp together induce a triple increase of acti-
vation time of L-Glu receptors, compared to the 
action of L-Glu alone.29
D-Asp increases LTP,16 inhibits LTD,30 and improves 
learning and memory in rats.31 Moreover, low levels 
Figure 5. Effect of D-Aspartate on neurological disability. No significant changes in the (a) Multiple Sclerosis 
Functional Composite (MSFC), nor in its single subcomponents, in the (b) Timed 25 Foot Walk (T25FW), (c) 9-Hole Peg 
Test (9HPT), (d) Paced Auditory Serial Addition Test (PASAT), and in the (e) Expanded Disability Status Scale (EDSS) 
emerged 12 and 24 weeks after starting D-Aspartate of placebo. (f) Fatigue as assessed through the Fatigue Severity Scale 
(FSS) was reduced 4 weeks after starting D-Aspartate, but not after placebo, and returned to baseline values 8 weeks after 
(*p < 0.05).
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of D-Asp were found in the brains of patients with 
Alzheimer disease, compared to healthy controls.32 
D-Asp was also reported to restore age-related loss of 
synaptic plasticity in the hippocampus of rats, sug-
gesting that it could act against signaling reduction of 
NMDAR caused by aging.17 Furthermore, D-Asp acts 
as a modulator of neurogenesis and as endogenous 
factor for growth of dendrites.18,33
MS patients show defective LTP in comparison to 
relapsing–remitting forms of multiple sclerosis 
(RRMS) and healthy controls, suggesting that the 
clinical progression is associated with reduced synap-
tic plasticity reserve.3 Our study shows that D-Asp is 
able to enhance LTP in PMS patients. Since synaptic 
plasticity is supposed to compensate clinical expres-
sion of a brain damage7,8 and indeed it has been linked 
to clinical disability in MS3,4 as well as in stroke9 and 
in Parkinson’s disease34 patients, we hypothesize that 
D-Asp may represent a therapeutic opportunity in 
order to contrast disability progression in MS, for 
which no valid therapy exists.
The small number of patients involved in this mono-
center study and the short duration of both treatment 
and follow-up as well as lack of neuroimaging data 
may limit the understanding and potential impact of 
the use of D-Asp in PMS. Further studies are thus 
warranted specifically targeted at exploring the effects 
of D-Asp on disability related to MS as well as to 
other disabling neurologicaldiseases.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of inter-
est with respect to the research, authorship, and/or 
publication of this article.
Funding
The author(s) disclosed receipt of the following finan-
cial support for the research, authorship, and/or publi-
cation of this article: This study received support from 
Fondazione Italiana Sclerosi Multipla FISM (Progetto 
Speciale 2014/PMS/2).
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