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Current Pharmaceutical Design, 2019, 25, 1-7 1
RESEARCH ARTICLE

Activation of Melanocortin-4 Receptor by a Synthetic Agonist Inhibits Ethanol-

induced Neuroinflammation in Rats

Osvaldo Flores-Bastíasa,b, Gonzalo I. Gómeza,b, Juan A. Orellanab,c and Eduardo Karahaniana,b*

a
Instituto de Ciencias Biomédicas, Facultad de Ciencias de la Salud,, Universidad Autónoma de Chile, Chile; bResearch Center for
the Study of Alcohol Drinking Behavior in Adolescents, Santiago, Chile; cDepartamento de Neurología, Escuela de Medicina and
Centro Interdisciplinario de Neurociencias, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile

ARTICLE HISTORY
Received: October 30, 2019
Accepted: December 10, 2019
DOI:
10.2174/1381612825666191216145153
Keywords: MC4R, melanocortin, α-MSH, alcohol use disorder, neuroinflammation.
Abstract: Background: High ethanol intake induces a neuroinflammatory response resulting in the subsequent
maintenance of chronic alcohol consumption. The melanocortin system plays a pivotal role in the modulation of
alcohol consumption. Interestingly, it has been shown that the activation of melanocortin-4 receptor (MC4R) in
the brain decreases the neuroinflammatory response in models of brain damage other than alcohol consumption,
such as LPS-induced neuroinflammation, cerebral ischemia, glutamate excitotoxicity, and spinal cord injury.
Objectives: In this work, we aimed to study whether MC4R activation by a synthetic MC4R-agonist peptide
prevents ethanol-induced neuroinflammation, and if alcohol consumption produces changes in MC4R expression
in the hippocampus and hypothalamus.
Methods: Ethanol-preferring Sprague Dawley rats were selected offering access to 20% ethanol on alternate days
for 4 weeks (intermittent access protocol). After this time, animals were i.p. administered an MC4R agonist pep-
tide in the last 2 days of the protocol. Then, the expression of the proinflammatory cytokines interleukin 6 (IL-6),
interleukin 1-beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) were measured in the hippocampus, hypo-
thalamus and prefrontal cortex. It was also evaluated if ethanol intake produces alterations in the expression of
MC4R in the hippocampus and the hypothalamus.
Results: Alcohol consumption increased the expression of MC4R in the hippocampus and the hypothalamus. The
administration of the MC4R agonist reduced IL-6, IL-1β and TNF-α levels in hippocampus, hypothalamus and
prefrontal cortex, to those observed in control rats that did not drink alcohol.
Conclusion: High ethanol consumption produces an increase in the expression of MC4R in the hippocampus and
hypothalamus. The administration of a synthetic MC4R-agonist peptide prevents neuroinflammation induced by
alcohol consumption in the hippocampus, hypothalamus, and prefrontal cortex. These results could explain the
effect of α-MSH and other synthetic MC4R agonists in decreasing alcohol intake through the reduction of the
ethanol-induced inflammatory response in the brain.

1. INTRODUCTION maintenance of chronic alcohol consumption and (iii) alcohol re-

According to the World Health Organization (WHO), alcohol is
the most commonly abused drug and its uncontrolled consumption,
called alcohol use disorder (AUD), is among the top five risk fac-
tors for disease and death worldwide [1, 2]. Indeed, a study in the
United Kingdom concluded that alcohol is the most harmful drug to
the society, surpassing heroin and cocaine [3]. Alcohol drinking is
associated with a risk of developing health problems such as cancer,
liver cirrhosis and various disabilities, in addition to an increased
risk of accidents [4, 5]. Data from the WHO indicate that the global
effect of noxious use of alcohol is approximately 3.3 million deaths
each year; thus, it accounts for 5.9% of all deaths worldwide [1].
Along with the various health problems mentioned above, the
excessive consumption of alcohol generates dramatical conse-
quences in the brain such as neurodegeneration [6] and deteriora-
tion of the prefrontal cortex that leads to impaired executive func-
tion (planning, thinking, and judgment) [7], depression [8], anxiety
[9] and memory impairment [10]. Although there are multiple neu-
robiological mechanisms that account for the various aspects re-
lated to AUD, i.e. (i) the acquisition of alcohol intake, (ii) the
*Address correspondence to this author at the Institute of Biomedical Sci-
ences, Faculty of Health Sciences, Universidad Autónoma de Chile, Llano
Subercaseaux 2801, San Miguel, 8910060, Santiago, Chile;
Tel: +56-2-23036664; E-mail: eduardo.karahanian@uautonoma.cl
lapse behavior, the notion that neuroinflammation and oxidative
stress would play a role in the establishment of chronic alcohol
abuse has gained importance recently [11]. An interesting hypothe-
sis is that neuroinflammation plays a critical role in the establish-
ment of addiction to several substances of abuse, including alcohol
[12, 13], particularly, through the activation of genes related to the
innate immune system. Chronic activation of the innate immune
response in the brain would produce long-lasting neurobiological
changes, e.g., increased glutamate-induced hyperexcitability and
excitotoxicity, decreased neurogenesis and behavioral changes such
as anxiety, negative affect, and depression, all of which are related
to addictive substances abuse [14].
Microglia and astrocytes are the major sources of inflammatory
mediators and ROS in the nervous system and highly sensitive to
ethanol exposure [15]. Upon ethanol stimulation, glial cells release
relevant amounts of proinflammatory cytokines, ROS and nitric
oxide (NO) - all of them crucial for the brain inflammatory re-
sponse [16]. Within the brain, the hippocampus is one of the most
pathologically affected regions during chronic ethanol intake [17,
18]. The presence of reactive astrocytes and microglia in the hippo-
campus are exacerbated in alcoholics [19]. This glial long-lasting
process promotes neuronal death in the hippocampus and other
brain regions via activation of inflammatory routes that cause exci-
totoxicity and ROS production [20]. Microglia, astrocytes and neu-
rons abundantly express NF-κB, a pivotal transcription factor in the

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2 Current Pharmaceutical Design, 2019, Vol. 25, No. 00
genesis and progression of the brain of innate immune-response
genes. NF-κB is involved in ethanol-induced neuroinflammation
[21] and can be directly activated by ROS [22]. Likewise, ethanol
increases cytochrome P4502E1 (Cyp2E1) activity [23], and in turn,
Cyp2E1 metabolism of ethanol increases ROS levels resulting in
the NF-κB-mediated production of proinflammatory mediators such
as IL-6, IL-1β and TNF-α [28]. In addition, NF-κB activation pro-
duces a greater expression of NADPH oxidase [24], an enzyme that
produces even more ROS, which creates a positive activation loop
that rapidly potentiates innate immune response [13, 14]. In accor-
dance with this, the daily administration of N-acetyl cysteine (a
precursor in the formation of the antioxidant glutathione) to rats
that have chronically consumed ethanol markedly reduces voluntary
ethanol intake [11]. In addition to the intrinsic neuronal death medi-
ated by ROS, activation of NF-κB signaling stimulates the release
of TNF-α, IL-1β, IL-6, and transforming growth factor-β (TGF-β1),
resulting in neuronal apoptosis [25]. A persistent status of neuroin-
flammation, together with the exacerbated production of ROS,
would lead to neurodegeneration of key areas involved in alcohol
consumption and addiction [13].
It has been proposed that, instead of simply being a side effect
of excessive alcohol consumption, neuronal damage associated with
drinking may actually underlie some of the mechanisms that regu-
late the development of AUD [25]. Alcohol-induced cell death in
regions such as the prefrontal cortex may lead to a lack of inhibition
in subcortical reward areas like the striatum, which in turn may
reduce behavioral inhibition and increase motivation to drink. Re-
peated stimulation of the innate immune system during chronic or
heavy alcohol consumption may facilitate this process, leading to
decreased inhibition of the mesolimbic reward system and thus,
increasing drinking [14, 29].
The hypothalamus is one of the brain regions that undergo more
physiological changes during excessive alcohol intake [26]. Within,
several orexigenic peptides (such as orexin, encephalin andgalanin)
stimulate alcohol consumption [27, 28, 29]; on the other hand,
anorexigenic peptides including corticotropin-releasing factor, the
endogenous opioid dynorphin and melanocortins, inhibit alcohol
drinking [29, 30, 31]. Melanocortins (MCs) belong to a group of
neuropeptides that are produced in the arcuate nucleus of the hypo-
thalamus (Arc) which include the α-, β-, and γ- melanocyte-
stimulating hormones (α-MSH, β-MSH and γ-MSH) [32]. α-MSH
is involved in the regulation of sexual behavior and food appetite
through its agonist activity on MC3R and MC4R melanocortin
receptors (MC3Rs and MC4Rs) expressed in the hippocampus,
paraventricular nucleus of the hypothalamus (PVN), ventral teg-
mental area (VTA) and nucleus accumbens (NAc) [33]. Of note, α-
MSH has also been implicated in the modulation of ethanol intake
[34]. The administration of a nonspecific agonist (melanotan-II) of
both MC3Rs and MC4Rs reduces voluntary ethanol consumption in
C57BL/6J mice [35] and in selectively-bred alcohol-preferring rats
[36]. However, when this agonist is administered to MC4R knock-
out mice no effect was observed, revealing that MC4Rs (and not
MC3Rs) are responsible for reducing alcohol intake [37]. Consis-
tent with this idea, the administration of a selective activator of
MC4Rs at the NAc and VTA diminished voluntary alcohol con-
sumption in rats [38]. Moreover, evidence in rats indicates that
chronic ethanol consumption reduces α-MSH levels in the hypo-
thalamus [30, 39, 40, 41], while an increase in α-MSH is observed
in acute ethanol-treated rats [42]. Nevertheless, the effect of alcohol
consumption on the expression of MC4Rs in the brain has not yet
been studied.
While chronic ethanol treatment significantly attenuates α-MSH
level in the pituitary, arcuate nucleus (ARC) and substantia nigra
(Rainero et al. 1990), the use of acute protocols elevate the hypo-
thalamic POMC mRNA expression (Rasmussen et al. 1998).
The MC system has been linked for many years with anti-
inflammatory effects in the brain [15, 43]. Interestingly, the MC4R
Flores-Bastías et al.
is highly expressed in both microglia and astrocytes [44, 45, 46],
which suggests that α-MSH may exert its anti-inflammatory effects
via MC4Rs in these key neuroinflammatory cells [47]. In fact, the
impact of MC4R activation on the reduction of neuroinflammation
has been studied in other neuropathological models of brain injury
different from ethanol intake: experimental brain inflammation
induced by LPS [48, 49, 50], cerebral ischemia damage [51, 52],
kainic acid-induced excitotoxic damage [53] and experimental spi-
nal cord injury [54, 55]. In all these studies, the neuroprotective
actions of α-MSH and other synthetic analog peptides reside in their
ability to decrease NF-κB signaling in glial cells, which is the key
transcription pathway for the induction of innate immune-response
[13]. Specifically, α-MSH and other synthetic melanocortin recep-
tor ligands prevent IκBα phosphorylation and, consequently, inhibit
NF-κB activation [56]. In agreement, α-MSH also decreases the
production of key pro-inflammatory cytokines such as TNFα [51,
52, 53, 57, 58, 59], enzymes such as iNOS and COX-2 [47] and
diverse inflammatory mediators, like NO [60, 61] in the brain.
Along with the inhibition of NF-κB activity, α-MSH also stimulates
the release of the anti-inflammatory cytokines IL-10 and TGF-β
from microglia and astrocytes, respectively, by a mechanism in-
volving the expression of the peroxisome proliferator-activated
receptor gamma (PPARγ) [62].
Given that neuroinflammation underlies the progression of chronic
alcohol intake, and because brain MC4R activation has anti-
inflammatory effects, we investigated whether heavy alcohol con-
sumption induces neuroinflammation and alteration in MC4R ex-
pression in brain areas susceptible to the greatest effects of ethanol-
induced neuroinflammation. Additionally, we evaluated if the acti-
vation of MC4R by a selective synthetic agonist-peptide prevents
the alcohol-induced neuroinflammatory response.
2. MATERIALS AND METHODS
2.1. Animals, Ethanol Consumption and Treatment with an
MC4R Agonist
The experiments were performed in male Sprague Dawley out-
bred rats purchased from the Pontificia Universidad Católica de
Chile animal facility. Rats were housed in individual cages in a
temperature-controlled room on a reversed 12-h light/12-h dark
cycle, with food and water provided ad libitum. Twenty-five days-
old rats started an intermittent-access (IA) ethanol drinking protocol
[63]: the animals were given access to 20% ethanol during three 24-
hour-sessions per week, for 4 weeks. It has been reported that 4
weeks is enough time for the animals to reach their maximum level
of consumption through the IA protocol [63]. Drinking sessions
started at the beginning of the dark cycle. Each ethanol-naive rat
was given access to one bottle of 20% v⁄v ethanol and one bottle of
water. After 24 hours, the ethanol bottle was replaced with a second
water bottle that was available for the next 24 hours. The placement
of the ethanol bottle was alternated each ethanol drinking session to
avoid for side preference. The control group drank only water ad
libitum. Ethanol and water intakes were recorded daily. As rat
strains that were not selectively bred for high preference for ethanol
are not homogeneous in the amount of ethanol ingested (such as
Long-Evans, Wistar and Sprague-Dawley) [63, 64], after the 4
weeks acquisition period we selected the animals that drank more
than 2 g ethanol/kg/day to carry out our studies (referred as ethanol-
preferring rats) [65]. Ethanol-preferring rats were separated ran-
domly in 2 groups (n = 7 per group) and in the last 2 days the IA
protocol one group received a daily i.p. dose of 180 µg/kg of a spe-
cific MC4R peptide-agonist (Cyclo(Β-Ala-His-D-Phe-Arg-Trp-
Glu)-NH2, Phoenix Pharmaceuticals, CA), while the other group
was injected with saline. The same was performed in parallel in the
respective two water-drinking control groups; (n = 7 per group).
We selected this dose based on the work of Liu et al. [66] who ad-
ministered i.p. the MC4R agonist peptide (Butir-His-D-Phe-Arg-
Trp-Sar-NH2) in a similar way to that used in this study. These
Melanocortin-4 Receptor Activation Reduces Ethanol-induced Neuroinflammation
authors reported that increased expression of IL-1β and TNF-α
evoked by a model of brain damage in rats was strongly counter-
acted with this dose of the agonist. Same doses also decreased the
expression of TNF-α when administered i.p. in a model of cerebral
ischemia in gerbils [52].
All animal procedures are in accordance with the Guide for the
Care and Use of Laboratory Animals of the National Institutes of
Health and were approved by the Bioethical and Biosafety Commit-
tee of Universidad Autónoma de Chile (Ethical Approval Record
BE003-14, 2016).
2.2. Western Blot and Enzyme-linked Immunosorbent Assay
One day after the administration of the MC4R agonist, the ani-
mals were anesthetized with ketamine/xylazine (10:1 mg/kg of
body weight, i.p.), perfused with saline, decapitated and the brains
removed. Hippocampus, hypothalamus and prefrontal cortex tissues
were extracted and homogenized with an Ultra-Turrax homogenizer
in buffer containing Tris-HCl 100 mM pH 7.4, EDTA 5 mM, SDS
1%, PMSF 1 µM and a protease inhibitor cocktail (Pierce,
Rockford, IL, USA) (1 ml lysis buffer per 0.1 g tissue). Then, the
samples were centrifuged at 14.000 x g for 10 min at 4°C to remove
tissue debris. Protein concentration was determined in the super-
natants with the Bio-Rad protein assay kit (Bio-Rad, Richmond,
CA, USA). IL-6, IL-1β and TNF-α levels were determined in the
hippocampus, hypothalamus and prefrontal cortex homogenates by
ELISA (IL-6, IL-1β and TNF-α EIA kit, Enzo Life Science, USA)
according to the manufacturer’s protocol. For the assay, 100 µL of
sample was added per ELISA plate well. The results were normal-
ized by protein amount to pg/g tissue.
Hippocampus and hypothalamus homogenates were also ana-
lyzed by Western blot with a MC4R primary antibody (diluted
1:500) (sc-55567, Santa Cruz Biotechnology, Inc.). As loading
control, β-tubulin levels were also determined in the same blots
(diluted 1:2000 primary antibody sc-9104, Santa Cruz Biotechnol-
ogy, Inc.). After the incubation with the corresponding HRP-
conjugated secondary antibodies, blotting membranes were re-
vealed for chemiluminescence with Pierce ECL Western Blotting
Substrate. The bands were quantified by densitometry with ImageJ
software.
3. RESULTS AND DISCUSSION
After 4 weeks of acquiring the preference for ethanol intake, the
rats showed high heterogeneity in their consumption levels (Fig. 1),
most likely due to idiosyncratic heterogeneity of ethanol taste per-
ception. Clearly, there were ethanol-preferring animals that initially
showed a high alcohol intake (3.66 ± 0.27 g/k/day), and this con-
sumption was increasing as the IA protocol progressed until reach-
ing an intake of 5.65 ± 0.41 g/k/day. These results are in agreement
with previous evidence reported for different strains of out-bred
rats, which also exhibit an increase in their alcohol consumption
when subjected to the IA protocol [65]. This response could repre-
sent both a mechanism of reinforcement and addiction to ethanol, as
well as a better perception of the taste of ethanol as chronic con-
sumption progresses. Tang et al. [67] demonstrated that consump-
tion of ethanol augments its oral acceptability in adolescent rats by
a mechanism mediated, at least in part, by a decrease in the respon-
siveness of the peripheral taste system to ethanol per se, rather than
to its bitter and sweet flavor components.
In contrast, in the group of rats selected as non-preferring the
initial average consumption was 2.02 ± 0.13 g/k/day and, unlike the
preferring rats, the consumption of alcohol in this group decreased
as they progressed in the IA protocol (1.2 ± 0.24 g/k/day). To our
knowledge, this effect of decreasing the voluntary consumption of
alcohol in non-preferring out-bred rats subjected to the IA protocol
has not been previously reported, and probably reflects the genera-
tion of a certain degree of aversion to ethanol intake.
Current Pharmaceutical Design, 2019, Vol. 25, No. 00 3
Fig. (1). Ethanol intake in ethanol-preferring and non-preferring rats at
the beginning and end of the 4-week IA protocol. After the first day of
access to ethanol, animals were separated into 2 groups: ethanol-non -
preferring rats if they drank a maximum of 2 g/kg/24 h (No-P first session)
and ethanol-preferring rats if they drank more than 2 g/kg/24 h (P first ses-
sion). Then, the voluntary consumption of alcohol was recorded in the last
day of the 4-week IA protocol (No-P final session and P final session).
ANOVA *** p < 0.001; * p < 0.05. (A higher resolution / colour version of
this figure is available in the electronic copy of the article).
In the last 2 days of the 4-week ethanol-acquisition stage, an
MC4R-agonist synthetic peptide was administered (180 µg/kg daily
i.p. doses) to one group of ethanol-preferring rats and to one group
of water-drinking control rats. Then, the levels of the proinflamma-
tory cytokines IL-6, IL-1β and TNF-α were determined in the hip-
pocampus, hypothalamus and prefrontal cortex homogenates by
ELISA. We chose these proinflammatory cytokines because they
are the ones that undergo major changes in the brain in response to
alcohol consumption [25]. As can be seen in Fig. 2, alcohol con-
sumption markedly increased the levels of these cytokines in the
three studied brain areas when compared to the rats that drank only
water (IL-1β: 12-fold, 22-fold and 14-fold; TNF-α: 5-fold, 4-fold
and 4-fold; IL-6: 7-fold, 6-fold and 4-fold in hippocampus, hypo-
thalamus and prefrontal cortex, respectively). Although the increase
of these pro-inflammatory cytokines may suggest their implication
in the alcohol-induced neuroinflammation, we cannot rule out other
inflammatory mediators, including inflammatory lipids such as
prostaglandins, whose synthesis is stimulated by ethanol consump-
tion [68].
Interestingly, the administration of the MC4R agonist in the last
2 days of the IA protocol decreased the levels of the three cytokines
to than water drinking animals. These results demonstrate that
MC4R activation inhibits the neuroinflammatory response induced
by heavy alcohol consumption, reported in models of neuroinflam-
mation produced by other mechanisms (e.g. LPS-induced neuroin-
flammation [48, 49, 50], cerebral ischemia [51, 52], kainic acid-
induced excitotoxic damage [53] and spinal cord injury [54, 55]).
Although not significant, the administration of the agonist also
changed cytokine levels in the non-ethanol exposed animals. How-
ever, these alterations were not consistent between the different
cytokines or the different brain areas analyzed. In fact, in some
cases the cytokine levels increase, in others, the opposite was found
or remain unchanged. Therefore, we believe that these inconsistent
alterations likely denote the inherent variability of the technique of
detection. Of note, the weight gain of the animals at the end of the
protocol showed no differences between the different experimental
groups (data not shown).
4 Current Pharmaceutical Design, 2019, Vol. 25, No. 00 Flores-Bastías et al.

Fig. (2). Levels of proinflammatory cytokines in rats that consumed alcohol, and effect of a synthetic MC4R-agonist peptide. Ethanol-preferring rats
were separated randomly in 2 groups (n = 7 per group) and in the last 2 days the IA protocol one group received a daily i.p. dose of 180 µg/kg of a specific
MC4R peptide-agonist (EtOH+Ago), while the other group was injected with saline (EtOH). The same procedure was performed in the respective two water-
drinking control groups (Ctrl+Ago and Ctrl) (n = 7 per group). One day after the administration of the MC4R agonist or saline, hippocampus, hypothalamus
and prefrontal cortex tissues were extracted, homogenized, and IL-6, IL-1β and TNF-α levels were determined by ELISA. ANOVA *** p < 0.001; ** p<0.01.
(A higher resolution / colour version of this figure is available in the electronic copy of the article).

Fig. (3). Expression of MC4R in hippocampus and hypothalamus of rats that consumed alcohol. At the end of the IA protocol, MC4R levels were deter-
mined by western blot in hippocampus (A) and hypothalamus (B) of water-drinking (Ctrl) and ethanol-drinking (EtOH) rats, that were not treated with MC4R
agonist (n = 7 per group). β-tubulin levels were determined as loading control. Three representative lanes of the western blots from each group are shown. Bars
represent MC4R/β-tubulin ratio. T-test ** p < 0.01. (A higher resolution / colour version of this figure is available in the electronic copy of the article).

Due to the marked effect of the MC4R agonist on the alcohol tions due to ethanol consumption, such as the hippocampus and the
intake-induced neuroinflammation, we examined if there were hypothalamus. As seen in Fig. 3, western blot analysis shows that
changes in the expression of MC4R in brain areas that suffer altera- alcohol consumption increases MC4R expression in both hippo-
 Melanocortin-4 Receptor Activation Reduces Ethanol-induced Neuroinflammation
campus and hypothalamus. This finding is novel since although it
was reported that α-MSH levels are decreased in the hippocampus
of rats that drink alcohol [30, 39, 40, 41], reports that account for an
alteration in the expression of MC4R are missing. It is possible that
this increase in MC4R expression reflects a brain compensatory
mechanism due to the decrease in α-MSH levels by chronic alcohol
intake or even a defense response to the inflammatory process trig-
gered by alcohol consumption. Because MC4R signaling activates
the CREB/BDNF pathway [69] which is fundamental for neuronal
plasticity and cognitive processes, we could speculate that increased
MC4R expression could have positive effects on memory and learn-
ing. In addition, considering that BDNF has been described as hav-
ing anti-inflammatory actions in the brain [70], it is likely that not
only could improve cognitive processes, but it may also decrease
the neuro-glial inflammation evoked by ethanol consumption.
CONCLUSION
In this study, we found that the i.p. administration of a synthetic
MC4R agonist prevents neuroinflammation induced by alcohol
consumption in the hippocampus, hypothalamus and prefrontal
cortex. In addition, high alcohol consumption produced an increase
in MC4R expression in the hippocampus and hypothalamus. These
results could explain the effect of α-MSH and synthetic MC4R
agonists in decreasing alcohol intake, through the reduction of the
ethanol-induced inflammatory response in the brain.
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
Not applicable.
HUMAN AND ANIMAL RIGHTS
No Animals/Humans were used for studies that are the basis of
this research.
CONSENT FOR PUBLICATION
Not applicable.
AVAILABILITY OF DATA AND MATERIALS
  ing caused by drugs of abuse: mechanisms, mediators and new
Not applicable.
FUNDING
  [13]
This work was supported by Programa de Investigación
Asociativa (PIA-CONICYT) Anillo de Ciencia y Tecnología
ACT1411 (to EK and JAO), DIUA149-2019 (to EK) and
FONDECYT 1160710 (to JAO).
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
other-wise.
ACKNOWLEDGEMENTS
Declared none.
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