Experimental Parasitology 124 (2010) 258–264

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Experimental Parasitology

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Changes in macrophage membrane properties during early Leishmania amazonensisinfection differ from those observed during established infectionand are partially explained by phagocytosis

Eduardo Quintana a,b, Yolima Torres a, Claudia Alvarez a, Angela Rojas c, María Elisa Forero a,Marcela Camacho a,d,*

a Laboratorio de Biofísica, Centro Internacional de Física, Bogotá, Colombiab Departamento de Física, Facultad de Ciencias, Universidad Nacional de Colombia, Bogotá, Colombiac Departamento de Farmacia and Doctorado en Ciencias-Química, Facultad de Ciencias, Universidad Nacional de Colombia, Colombiad Departamento de Biología, Facultad de Ciencias, Universidad Nacional de Colombia, Bogotá, Colombia

a r t i c l e i n f o

Article history:Received 23 January 2009Received in revised form 14 October 2009Accepted 19 October 2009Available online 23 October 2009

Keywords:LeishmaniaMacrophageMembrane capacitanceIon currentsDepolarizationPhagocytosis

0014-4894/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.exppara.2009.10.006

* Corresponding author. Address: Laboratorio de Bde Física, Edificio Manuel Ancizar, Ciudad UniversitariFax: +57 1 368 1517.

E-mail addresses: mmcamachon@unal.edu.co, mCamacho).

1 Abbreviations used: PG, phagolysosome; IOUT, oupotassium current; Vm, membrane potential; Cm, mem

a b s t r a c t

Understanding the impact of intracellular pathogens on the behavior of their host cells is key to designingnew interventions. We are interested in how Leishmania alters the electrical function of the plasma mem-brane of the macrophage it infects. The specific question addressed here is the impact of Leishmania infec-tion on macrophage membrane properties during the first 12 h post-infection. A decrease of 29% inmacrophage membrane capacitance at 3 h post-infection indicates that the phagolysosome membraneis donated on entry by the macrophage plasma membrane. Macrophage membrane potential depolarizedduring the first 12 h post-infection, which associated with a decreased inward potassium current density,changed in inward rectifier conductance and increased outward potassium current density. Decreasedmembrane capacitance and membrane potential, with no changes in ion current density, were foundin macrophages after phagocytosis of latex beads. Therefore we suggest that the macrophage membranechanges observed during early Leishmania infection appear to be associated with the phagocytic and acti-vation processes.

� 2009 Elsevier Inc. All rights reserved.

1. Introduction

Changes in host cell membrane permeability are known to af-fect the survival and replication of a number of intracellular patho-gens. Leishmania species are intracellular obligatory parasites ofmacrophages. After entry into its mammalian host, the parasite isphagocytosed by macrophages and confined to a lysosome-likecompartment (reviewed by Russell, 1995). Leishmania replicateswithin a phagolysosome (PG1). In other intracellular parasite sys-tems, the demands of the replicating parasite are met by incorporat-ing parasite membrane channels and transporters or by modulatingthose of the host cell (Ginsburg and Stein, 2005; Staines et al., 2003;Duranton et al., 2004). Altered calcium homeostasis in the host cellhas also been reported in Plasmodium and Trypanosoma (Tanabe,

ll rights reserved.

iofísica, Centro Internacionala, AA 4948, Bogotá, Colombia.

mcamachon@yahoo.com (M.

tward current; IKIR, inwardbrane capacitance.

1990; Olivier, 1996; Andrews, 1995) as well as in Leishmania infectedcells (Eilam et al., 1985; Olivier, 1996). However, changes in macro-phage membrane permeability may result in alterations of its abilityto activate and signal the immune system. Classical activation ofmacrophages (Stein et al., 1992; Mosser, 2003) is dependent on anincrease in outward potassium currents (IOUT) together with de-creased inward potassium currents (IKIR) (Vicente et al., 2003; Cam-acho et al., 2008) and membrane depolarization (Camacho et al.,2008). Scott et al. (2003) working with activated macrophagesshowed that Leishmania major infection or treatment with K+ chan-nel blockers suppressed nitric oxide (NO) production, consistentwith a role for potassium currents in the deactivation effect of Leish-mania. We showed that infection with Leishmania amazonensis ofnon-activated macrophage-like cells (J774A.1) is associated with in-creased IKIR activity (Forero et al., 1999), and does not alter total IOUT

density but increases the time to peak and susceptibility to TEA(Camacho et al., 2008). These changes can be interpreted as an in-crease in some potassium currents which would contribute againstactivation and would be consistent with observed suppression ofactivation by Leishmania infection (Liew et al., 1997). Moreover, inLeishmania infected macrophages, classical activation is associatedwith decreased IOUT density (Camacho et al., 2008).

E. Quintana et al. / Experimental Parasitology 124 (2010) 258–264 259

In the present study we followed the first 12 h after infection ofJ774.A1 by L. amazonensis. Our results show that early infection al-ters macrophage membrane in a manner distinct to what we haveobserved over longer periods post-infection. Most of the changesfound during early infection are similar to those observed afterthe phagocytosis of latex beads (this report) and activation (Cam-acho et al., 2008), suggesting that during early infection the mem-brane changes are related to phagocytosis and attemptedactivation by the macrophage, whereas the changes observed after24 h appear to deactivate the macrophage and to be specific toLeishmania infection.

2. Materials and methods

2.1. Cell culture

The murine macrophage-like cell line J774.A1 was obtainedfrom the European Cell line and Hybridoma Bank Collection (EE-CACC No. 91051511) and the ATTC (TIB-67) and maintained as amonolayer in 25 cm2 flasks at 34 �C, 5% CO2 for up to 4 weeks. Cellswere kept in RPMI 1640 culture medium (Invitrogen) supple-mented with 10% fetal bovine serum (FBS, Invitrogen). Suspendedcells were allowed to attach onto sterile glass cover slips kept in35 mm Petri dishes at 34 �C for 24 h prior to electrophysiologicalstudies. The medium was changed daily and again 1 h beforerecording.

2.2. Parasite culture and infection

We used a Leishmania amazonensis isolate, kindly donated byDr. Nancy Gore Saravia (FLA/BR/67/PH8, CIDEIM, Cali, Colombia).Promastigotes, at an initial concentration of 1 � 106, were cul-tured at 27 �C in 25 cm2 flasks in TC-100 or Schneider’s medium(Invitrogen) supplemented with 10% FBS (Niño and Camacho,2005). Promastigotes were allowed to reach their stationarystage (stationary growth, rosette formation and efficient infectiv-ity) and either diluted to maintain the culture or harvested forinfection.

2.3. Leishmania infection

Eighty percent confluent macrophage cultures were exposedto stationary phase promastigotes at a ratio of 1:10 and incu-bated at 34 �C, 5% CO2 for 4 h. Non-adherent promastigotes werewashed out with serum free medium and macrophages culturedin FBS supplemented media. Infected macrophages were kept forup to 12 h post-infection in the same conditions as describedabove for control cells. Cells from infection cultures were al-lowed to adhere onto sterile glass cover slips and kept in35 mm Petri dishes at 34 �C for 24 h prior to electrophysiologicalstudies.

2.4. Phagocytosis of latex beads

Eighty percent confluent macrophage cultures were exposed to3 lm latex beads (Sigma) at a ratio of 1:10 and incubated at 34 �C,5% CO2 for 4 h. Non-adherent beads were washed out with serumfree medium and macrophages cultured in FBS supplementedmedia. Post-phagocytosis macrophages were kept for 24 h in thesame conditions as described above for control cells. Cells fromphagocytosis cultures were allowed to adhere onto sterile glasscover slips and kept in 35 mm Petri dishes at 34 �C for 24 h priorto electrophysiological studies.

2.5. Electrophysiological recording and data analysis

Cover slips were placed in a recording chamber kept at roomtemperature (15–18 �C) on the stage of an inverted microscopeZeiss IM35. Cells were bathed in a solution consisting of (inmM): 145 NaCl, 5 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES-Na, 5 glucose(pH 7.34, 300 mOsm). Pipettes were pulled from non-heparin-ized hematocrit capillaries and had resistances of 2.5–3 MXwhen filled with a solution containing (in mM): 140 K gluta-mate, 2 KCl, 5 EGTA-K, 0.5 CaCl2, 4 MgCl2, 10 HEPES-K, 3ATPMg2, 0.5 GTP-Mg (pH 7.34, 300 mOsm). Voltage steps wereapplied from a holding potential of �60 mV. Data were sampledat 5 kHz, filtered at 1 kHz and digitized with a Digidata 1200interface (Molecular Devices, USA). Membrane currents were re-corded in the whole cell configuration of the patch-clamp tech-nique (Hamill et al., 1981) with an Axopatch-1C amplifier or anAxopatch 200B (Molecular Devices, USA). During whole cellrecording the series resistance was left uncompensated becauseit was never greater than 10 MX. After attaining the whole cellconfiguration, the amplifier was set to the current-clamp modeto determine the potential at which the current was zero. Thisvalue is referred to as the resting membrane potential (Vm)and was measured within the first minute. Throughout the restof the experiment, the cell was maintained under voltage-clampconditions. Data acquisition and analysis were performed withthe pCLAMP 6 software (Molecular Devices, USA) and plottedwith Origin, 7SR, (OriginLab Corporation, Northampton, MA,USA).

2.6. Membrane capacitance measurements

Membrane capacitance was measured in voltage clamp. In thewhole cell configuration 10 pulses of �10 mV and 5 ms of dura-tion, were applied, from a holding potential of �60 mV, to themacrophage plasma membrane and the capacitive transient re-corded. Membrane capacitance was calculated as Ce = s/R. Theexponential decay of the capacitive current (I) for each voltageepisode (V) was fit and the time constant (s) obtained. Resistancewas calculated from R = V/I. Mean membrane capacitance (Cm)was determined. The coefficient of variation (CV) of capacitancefor each experimental condition was calculated as CV = r(Cm/Ce) � 100.

2.7. Data analysis

Peak current densities were normalized by maximum currentdensity and fit by Eq. (1), where Imax is the maximum current,V1/2 is the voltage at which membrane achieves 50% activation,Vk is a factor associated with the slope of the curve and A0 andA1 are constants associated with the saturation of the currentdensity.

IImax¼ A0

1þ eðV�V1=2Þ=Vkþ A1 ð1Þ

2.8. Statistical analysis

The data were analyzed using Student’s t-test. Data were col-lected from three sets of independent series of experiments withan mean number of cells per group of eight for each treatmentand with a total number of cells of: untreated controls (n = 26cells), a group of macrophages after phagocytosis (n = 21) and fourinfected groups: 3 h post-infection (hpi) (n = 20), 6 hpi (n = 20), 9hpi (n = 15) and 12 hpi (n = 14).

260 E. Quintana et al. / Experimental Parasitology 124 (2010) 258–264

3. Results

3.1. Impact of Leishmania early infection on macrophage passivemembrane properties

The passive membrane properties of macrophage membrane,membrane capacitance (Cm), membrane resistance (Rm) and mem-brane potential (Vm) were determined. Membrane capacitance isan indirect measurement of membrane area. Previous reportsshowed that Cm varies as a function of time of adherence ontoglass (Gallin and Sheehy, 1985; data not shown), so time of adher-ence was kept constant at 24 h for all groups. Control macro-phages had a Cm of 15.2 ± 0.91 pF. This value is not significantlydifferent from that previously reported (Forero et al., 1999). Incontrast, a cell at 3 h post-infection (pi) had a smaller capacitativetransient compared to a control cell (Fig. 1A), and by 6 h pi, cellshad a significant Cm decrease of 26 ± 5% (p = 0.006); similar resultswere observed at up to 12 h pi (p = 0.042; Fig. 1B). Not only did Cm

decrease with infection during the first hours, but the coefficientof variation (CV) decreased as well; this variation is an indirectmeasure of the rate of vesicular fusion/fission and of macrophagevesicular traffic to the plasma membrane. Control macrophageshad a CV of 116.5 ± 129.1%; at 6 hpi this percentage decreasedto 23.1% (Fig. 1C). Rm for control macrophages was0.95 ± 0.14 GX. There were no differences between control and in-fected cells for this parameter (data not shown). The resting mem-brane potential is an indirect measure of membrane permeabilityat rest, and potentials under zero-current conditions were ex-pressed as Vm values. Control cells had a Vm of �55.7 ± 5.3 mV.

Fig. 1. Impact of Leishmania early infection on macrophage passive membrane propertievalue calculated from 10 transients. (C) CV: coefficient of variation. (D) Mean Vm. Data rep9 hpi (n = 7); 12 hpi (n = 6).

All infected groups studied were significantly depolarized com-pared to control macrophages: Vm decreased by 37 ± 8.6% (p =0.011) at 6 h pi and remained depolarized by 35 ± 7.7% (p =0.016) at 12 h pi (Fig. 1D).

3.2. Impact of Leishmania early infection on macrophage inwardcurrents

The inward rectifying potassium current (IKIR) expressed bythese cells (Fig. 2A and B), apparently the result of Kv2.1 channelactivity (Vicente et al., 2003; data not shown), has been suggestedto set the resting macrophage membrane potential (reviewed byGallin, 1991). To avoid the IKIR wash-out effect described in macro-phages (McKinney and Gallin, 1992), cells were dialyzed with a K-glutamate solution supplemented with nucleotides, HEPES bufferand EGTA. We have shown that established (more than 24 h pi)Leishmania infection increases IKIR peak current density and hyper-polarizes the macrophage (Forero et al., 1999). Macrophage IKIR

current activated at potentials below �90 mV was inactivated bya factor of 20 ± 5% (0.79 ± 0.05), consistent with Gallin and Sheehy(1985). We compared IKIR current density between control and in-fected cells (Fig. 2A and B). Mean IKIR peak current density at�130 mV was �21.3 ± 1.9 pA/pF for control macrophages. Therewas a decrease of 45.3 ± 17% in peak IKIR current density at�130 mV, during the first 6 h pi (p = 0.021; Fig. 2C and D). IKIR inac-tivation (Iss/Ip) at 3 h pi was 0.69 ± 0.06 (p = 0.037). The Boltzmannfit shows less sensitivity to voltage during the first hours post-infection (Vk in Table 1).

s. (A) Typical Cm transients of a control macrophage (left) and 3 hpi (right). (B) Cm

resent the mean value ± SE (control macrophages (n = 11); 3 hpi (n = 7); 6 hpi (n = 7);

Fig. 2. Impact of Leishmania early infection on macrophage inward currents. Typical IKIR recording of (A) a control macrophage and (B) 6 hpi. (C) Mean peak IKIR density atdifferent voltages: (h) control, (s) 3 h pi, (D) 6 h pi, (r) 9 h pi, (e) 12 h pi. (D) Mean Iss/Ip at �130 mV.

Table 1Impact of Leishmania early infection on macrophage currents.

Group IKIR IOUT

V1/2 Vk V1/2 Vk

Control �103.9 ± 0.42 10.65 ± 0.35 60.01 ± 5.40 33.09 ± 5.063 hpi �93.9 ± 1.25 19.99 ± 1.46 75.35 ± 3.54 27.71 ± 3.346 hpi �103.15 ± 1.59 15.24 ± 1.27 64.87 ± 3.15 29.27 ± 2.929 hpi �112.76 ± 7.07 19.28 ± 3.96 61.86 ± 4.37 34.46 ± 4.1512 hpi �110.1 ± 7.17 28.41 ± 5.05 75.77 ± 3.20 27.49 ± 3.02

Boltzmann activation parameters for IKIR and IOUT in control cells and after earlyhours post-infection. V1/2, Vk were calculated according to Eq. (1). Currents werenormalized (to I/Imax) and plotted against voltage.

E. Quintana et al. / Experimental Parasitology 124 (2010) 258–264 261

3.3. Impact of Leishmania early infection on macrophage outwardcurrents

These cells express partially inactivating outward currents(IOUT) (McKinney and Gallin, 1992). Minimum flux was appliedto avoid IOUT amplitude increase (Randriamampita and Traut-mann, 1987; data not shown). Because these currents are down-regulated after 24 h of adherence onto glass (data not shown),all groups tested were given the same time of adherence (20–24 h). IOUT activated at potentials higher than �10 mV and par-tially inactivated (Fig. 3A and B). Control macrophages had a meanpeak IOUT density at 90 mV of 23.6 ± 2.95 pA/pF and a Iss/Ip ratio of0.82 ± 0.04, comparable to previous reports (Camacho et al.,2008). In contrast, cells at 6 h pi had increased IOUT density to51.8 ± 16.8 pA/pF (p = 0.038), a rise of 120%. Maximum IOUT den-sity increase was detected at 9 h pi (by 187%, p = 0.030; Fig. 3C),accompanied by a 25% decrease of inactivation (p = 0.030). TheBoltzmann fit shows changes in activation during early infection(Vk in Table 1).

3.4. Impact of phagocytosis on macrophage electrical membraneproperties

To determine whether the changes detected were related to thephagocytic process, macrophage membrane properties after thephagocytosis of 3 lm latex beads were measured and comparedto those of macrophages infected with Leishmania. Membranecapacitance decreased by 17.6% (p = 0.019) following phagocytosisof latex beads and by 29% (p = 0.005) at 3 h pi (Fig. 4A). CV was sim-ilar (114.0 ± 41.4) compared to control groups. No differences weredetected in macrophage membrane resistance (data not shown),but membrane depolarization was found following phagocytosis(�40.1 ± 6.4 mV, p = 0.046) and at 3 h pi (�26 ± 3,4 mV, p = 0.008)(Fig. 4B). Significant differences (70 ± 1.7%, p = 0.01) were observedin peak IKIR current density at 3 h pi (Fig. 4C). When comparing out-ward current density we found an increase of 10% for the phago-cytic group and of around 100% (p = 0.034) at 3 h pi compared tocontrol cells (Fig. 4D).

4. Discussion

Leishmania parasites are adapted to life in the hostile endoso-ma-lysosomal environment of the PG. Though parasite entry ap-pears to be the result of a phagocytic event, the mechanismunderlying this process is not certain, as it has been postulatedto be coiling phagocytosis (Rittig et al., 1998) or a type of zippermechanism (Courret et al., 2002). There is evidence for membraneexchange between the parasite and the macrophage during inva-sion (Henriques and Souza, 2000), and for transfer of Leishmanialipophosphoglycan to the macrophage membrane (Tolson et al.,1990). Membrane capacitance changes proportional to load vol-ume have been shown in phagocytosis of latex beads (Holevinskyand Nelson, 1998; Fig 4A). To the authors’ knowledge, this is thefirst report showing a decrease of macrophage Cm after Leishmania

Fig. 3. Impact of Leishmania early infection on macrophage outward currents. (A) Typical IOUT recording of a control macrophage. (B) Mean peak IOUT density at differentvoltages: (h) control, (s) 3 h pi, (D) 6 h pi, (r) 9 h pi, (e) 12 h pi, (C) Mean Iss/Ip at �90 mV.

Fig. 4. Impact of phagocytosis on macrophage electrical membrane properties. Mean Cm, (B) mean Vm, (C) mean peak IKIR and (D) IOUT density at different voltages. Datarepresent the mean value ± SE (control macrophages (n = 15); 3 hpi (n = 13); phagocytosis of latex beads (n = 14)). (C) The control group, P: are cells after latex beadsphagocytosis, and I: represents Leishmania infected macrophages.

262 E. Quintana et al. / Experimental Parasitology 124 (2010) 258–264

E. Quintana et al. / Experimental Parasitology 124 (2010) 258–264 263

entry. This change, of around one third of the total macrophagemembrane area, suggests that to an important extent the PG mem-brane is donated by the macrophage on parasite entry. In additionto the Cm decrease upon invasion we detected decreased capaci-tance coefficient of variation during the first hours pi, which ap-pears to be specific of Leishmania infection. This result isinteresting because it suggests less endocytic/exocytic ratio thatmay compromise macrophage ability to signal. Several observa-tions may reflect altered vesicular traffic of infected macrophages:the Cm recovery after 24 h pi and the following increase (Foreroet al., 1999); evidence that lysosomal depletion is associated withL. amazonensis infection (Barbieri et al., 1985); expression in the PGof membrane markers of late endosomes and lysosomes (review byRussell, 1995); altered translocation of protein kinase C to themembrane (Olivier et al., 1992) as well as altered PG maturation(Courret et al., 2002).

We found that membrane depolarization accompanied Cm de-crease. In macrophages, depolarization has been associated withphagocytosis (Gercken et al., 1996; Fig 1C), production of oxygenradicals (Cameron et al., 1983; Holevinsky and Nelson, 1995), cellproliferation (Vicente et al., 2008), activation (Camacho et al.,2008) and Leishmania major infection of activated macrophages(Scott et al., 2003). L. amazonensis infected macrophages showedan increase of IOUT peak current density (Figs. 3C, 4D) associatedwith the depolarization. It has been suggested that appearance ofIOUT indicates readiness for antigen presentation (McKinney andGallin, 1992). Induction of IOUT has also been associated with stim-ulation by the complement factor C5a and epidermal growth factor(Ilschner et al., 1996), activation of G-proteins (McKinney and Gal-lin, 1992), oxygen radical production (Holevinsky and Nelson,1995), exposure to platelet activating factor (Ichinose et al.,1992), phagocytosis of zymosan (Berger et al., 1993) and classicalmacrophage activation by bacterial lipopolisaccharide (Nelsonet al., 1992; Vicente et al., 2003; Camacho et al., 2008) and inter-feron-c (Fisher et al., 1995; Vicente et al., 2003; Camacho et al.,2008).

However, the most important result is that early Leishmaniainfection has a distinct impact on macrophage membrane proper-ties (this report; Scott et al., 2003) compared to established infec-tion (Forero et al., 1999). Early infection results in decreasedcapacitance and membrane depolarization associated with de-creased IKIR current density and increased IOUT current density. Incontrast, established infection is accompanied by increased capac-itance, membrane hyperpolarization, increased IKIR current density(Forero et al., 1999) and increased potassium IOUT current density(Camacho et al., 2008). Membrane hyperpolarization is associatedwith less secretion (Iwanir and Reuveny, 2008), intracellular Ca2+

store refill after sustained increases of cytoplasmic Ca2+ (Mertzet al., 1992; Konig et al., 2006), oxygen radical production (Gama-ley et al., 1998), cell–cell membrane fusion (Fischer-Lougheedet al., 2001; Konig et al., 2006) and protection against apoptosis(Dallaporta et al., 1998, 1999). All of these events are relevant tothe macrophage-Leishmania relationship. Hyperpolarization isassociated with decreased release of TNF-a (Haslberger et al.,1992). Leishmania infection increases cytoplasmic Ca2+ (Eilamet al., 1985; Olivier, 1996; data not shown) and induces oxygenradical production (Sousa-Franco et al., 2006). Macrophages fuseas part of their repertoire of responses (reviewed by Vignery,2005; McNally and Anderson, 2005; Cui et al., 2006) and giant cellsare part of the granulomatous response caused by Leishmania (Par-reira de Arruda et al., 2002). Finally, Leishmania protects macro-phages against apoptosis (Aga et al., 2002; Lisi et al., 2005).

Considering these observations, we suggest that, upon invasion,macrophage vesicular traffic remains intact and the cell signalingrequired after phagocytosis is favored by membrane depolariza-tion. The electrical properties described in this study and those

of activated macrophages (Scott et al., 2003; Vicente et al., 2003;Camacho et al., 2008) suggest that the changes recorded duringthe first hours post-infection are the result of phagocytosis andearly activation. During phagocytosis, macrophages secrete TNF-a (Murray et al., 2005), which has autocrine activity on macro-phages and promotes classical activation (reviewed by Mosser,2003). Leishmania activates Toll-like receptors on entry (reviewedby Liese et al., 2008), favoring full classical activation (reviewedby Mosser, 2003). However, once Leishmania infection is estab-lished, a decrease of macrophage ability to secrete molecules forsignaling the rest of the immune system may be crucial for parasitesurvival. Therefore we suggest that the changes observed in Cm, Vm

and ion currents in macrophages during the first 12 h pi are re-sponses of a macrophage after phagocytosis and early classicalactivation. The later changes observed in macrophage membraneproperties as infection progresses (Forero et al., 1999; Camachoet al., 2008) reflect weaker activation and appear to be specific toLeishmania infection.

Acknowledgments

We acknowledge support from the Colombian agency Colcien-cias, project codes 222840820399 and 222840820384 of the Prog-rama Nacional de Ciencia y Tecnología de la Salud; from theDivisión de Investigaciones, Sede Bogotá, project codesCOL0022691 and 8003160, Universidad Nacional de Colombia;and from the Centro Internacional de Física, Bogotá, Colombia.Marcela Camacho was supported by Universidad Nacional deColombia.

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