Accepted Manuscript

Title: Inhibition of malaria parasite Plasmodium falciparum

development by crotamine, a cell penetrating peptide from the
snake venom

Author: S. El Chamy Maluf C. Dal Mas E.B. Oliveira P.M.S.

Melo A.K. Carmona M.L. Gazarini M.A.F. Hayashi

PII: S0196-9781(16)30013-4
DOI: http://dx.doi.org/doi:10.1016/j.peptides.2016.01.013
Reference: PEP 69595

To appear in: Peptides

Received date: 4-12-2015

Revised date: 15-1-2016
Accepted date: 19-1-2016

Please cite this article as: El Chamy Maluf S, Dal Mas C, Oliveira EB, Melo PMS,
Carmona AK, Gazarini ML, Hayashi M.A.F.Inhibition of malaria parasite Plasmodium
falciparum development by crotamine, a cell penetrating peptide from the snake
venom.Peptides http://dx.doi.org/10.1016/j.peptides.2016.01.013

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Inhibition of malaria parasite Plasmodium falciparum
development by crotamine, a cell penetrating peptide from
the snake venom

El Chamy Maluf S.a, Dal Mas C.b, Oliveira E.B.c, Melo P.M.S.a, Carmona A.K.a,
Gazarini M.L.d,*, Hayashi M.A.F.b,*

a
Departamento de Biofísica, Universidade Federal de São Paulo (UNIFESP), São Paulo,
Brazil; bDepartamento de Farmacologia, Universidade Federal de São Paulo
(UNIFESP), São Paulo, Brazil; cDepartamento de Bioquímica e Imunologia,
Universidade de São Paulo (USP-RP), Ribeirão Preto, Brazil; dDepartamento de
Biociências, Universidade Federal de São Paulo (UNIFESP), Santos, SP, Brazil.

*Corresponding authors:

Mirian A. F. Hayashi, Ph.D.

Departamento de Farmacologia, Universidade Federal de São Paulo (UNIFESP), Rua 3

de maio 100, Ed. INFAR, 3rd floor, CEP 04044-020, Tel +55-11-5576 4447/FAX +55-
11-5576 4499, São Paulo, Brazil;

e-mail: mhayashi.unifesp@gmail.com or mirianhayashi@yahoo.com

and

Marcos L. Gazarini, Ph.D.

Departamento de Biociências, Universidade Federal de São Paulo (UNIFESP), Rua

Silva Jardim 136, CEP 11015-020, Santos, SP, Brazil.

e-mail: marcos.gazarini@gmail.com ou marcos.gazarini@unifesp.br

1
Graphical Abstract

Schematic figure showing the preferential affinity of crotamine for P. falciparum

infected erythrocytes, which may have increased negative charge exposure compared to
the liquid net neutral surface of uninfected erythrocytes due to the extensive host cell
remodeling mediated by parasites. The localization of crotamine in parasitophorous
vacuole (PV), as well as in acidic digestive vacuole (DV) and nucleus are also indicated.

Highlights
 Crotamine is a cationic natural peptide with anti-plasmodial activity;
 Crotamine has anti-plasmodial activity against Plasmodium falciparum;
 Crotamine inhibits the development of the P. falciparum in a dose-dependent
manner;
 Crotamine was observed in the parasite nucleus and parasitophorous vacuole;
 Crotamine may disrupt the parasite acidic compartments H+ homeostasis;

2
Abstract

We show here that crotamine, a polypeptide from the South American rattlesnake

venom with cell penetrating and selective anti-fungal and anti-tumoral properties,

presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development

of the Plasmodium falciparum parasites in a dose-dependent manner [IC50 value of 1.87

µM], and confocal microscopy analysis showed a selective internalization of

fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no

detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the

crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may

involve the disruption of parasite acidic compartments H+ homeostasis. In fact,

crotamine promoted a reduction of parasites organelle fluorescence loaded with the

lysosomotropic fluorochrome acridine orange, in the same way as previously observed

mammalian tumoral cells. Taken together, we show for the first time crotamine not only

compromised the metabolism of the P. falciparum, but this toxin also inhibited the

parasite growth. Therefore, we suggest this snake polypeptide as a promising lead

molecule for the development of potential new molecules, namely peptidomimetics,

with selectivity for infected erythrocytes and ability to inhibit the malaria infection by

its natural affinity for acid vesicles.

Abbreviations: RBC, red blood cell; iRBC, infected RBC; AO, acridine orange; Cy3,

Cyanine 3 dye;

Keywords: Plasmodium, parasites, crotamine, antimalarial, acidic compartments,

peptide trafficking.

3
1. Introduction

Malaria is a critical human infection and it is responsible for the death of nearly

a million people every year [1]. The search for new antimalarial compounds is crucial,

since drug resistance is spreading quickly in the existing parasite population [2-5]. In

contrast to the increased number of methods currently available to identify new

compounds with antimalarial activity as described in recent reports, actually very few

innovative contributions to drug discovery can be found in the field [6].

The Plasmodium life cycle involves two well-known hosts, the arthropod

mosquito vector and the vertebrate host, for the sexual and asexual stages, respectively.

During the asexual stage, which is the main target for the antimalarial studies, crucial

biochemical and physiological changes, necessary for the P. falciparum development,

are observed in erythrocytes [7-9]. The increased permeability of the erythrocyte

membrane to different metabolites during the asexual stage favors the entrance of

several inhibitors with potential as antimalarial drugs [10-15].

The acidic compartments, in addition to the endoplasmic reticulum and

mitochondria, present in Plasmodium cells, possess an important role in the intracellular

ionic homeostasis (i.e. Ca2+ and H+) [9,16-17]. They are also the key elements to

provide, during the Plasmodium development, the necessary environment for the

functions of vital enzymes [18]. For instance, the hemoglobin degradation, which is

dependent of the action of different proteases as falcipains and plasmepsinas, occurs in

acidic compartments, and therefore, the ion homeostasis is undoubtedly crucial [8, 19,

20]. The acidic compartments are also described as the local of accumulation of

antimalarials, as chloroquine and derivatives, which kill the parasite by altering the

hemoglobin metabolism and the formation of hemozoin crystals [21]. However, the

4
increasing widespread resistance to chloroquine claims the search for alternative

compounds able to inhibit the parasite development [22].

Crotamine is a polypeptide of 42 amino acid residues (sequence

YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGSG, MW = 4889.81

Da and pI of about 9.5) isolated from the venom of the South American rattlesnake

Crotalus durissus terrificus [23]. The high content of basic amino acid residues and the

presence of three disulfide bonds confer to this toxin a stable 3D structure with high

exposure of positive charges and an amphipathic characteristic [24,25]. These structural

features stimulated us to investigate the cell penetrating properties of crotamine, which

showed a unique high specificity and affinity for actively proliferating cells compared to

quiescent non-proliferating or non-tumoral mammalian cells [26- 29]. In addition,

crotamine is also capable of carrying genes and other molecules into cells [30-32].

Taking this into account, the commercial use of crotamine as a biomarker of

proliferating cells and/or its employment as a carrier of bioactive molecules into cells

was already protected by our group (PCT/BR06/000052; US-2008-0181849-A1;

1866332 EPO Bulletin No. 37/14 in Sept 10th, 2014).

Biochemical, molecular and cellular studies showed that the cell penetrating

ability of crotamine is dependent on its positive net charge and its affinity for negatively

charged surfaces [30,31]. Moreover, crotamine also shows cytotoxic effect that involves

the disruption of lysosomes and the consequent release of the vesicles contents, as the

free calcium and cathepsins, which may trigger cell apoptosis and leading us to suggest

the acid compartments of cells as the primary intracellular target of crotamine [31,33].

Crotamine also presents specificity towards the cell membranes of microorganisms,

which is also dependent on their membrane surface negative charges [27,34],

5
reinforcing again the importance of the negative net charges on surface for the

biological activities and functions of crotamine [34].

Therefore, considering that Plasmodium acidic compartments are the local of

accumulation of antimalarials and that their ionic homeostasis are crucial for the

parasite development, in the present study, the potential antimalarial effect of crotamine

was explored in a Plasmodium falciparum model. Both selective internalization into

infected red blood cells (iRBCs) and intracellular localization of crotamine were

visualized by confocal microscopy. In addition, crotamine affected parasite

development in a dose-dependent manner, more likely due to the disruption of the P.

falciparum parasites H+ homeostasis, as evaluated by flow cytometry and by the

observed changes in lysosomotropic acridine orange (AO) fluorescence in infected

erythrocytes, respectively. Therefore, we believe that the results presented here provide

interesting insight for a novel potential structural model for the development of new

antimalarial peptidomimetics based on disruption of the H+ homeostasis of P.

falciparum parasites.

2. Materials and Methods

2.1 Ethics statement

This study was approved by the Ethics Committee of the Universidade Federal

de São Paulo - UNIFESP/EPM (License number 738690/2013).

2.2. Materials

The venom of Crotalus durissus terrificus was extracted from snakes maintained

at the Faculdade de Medicina de Ribeirão Preto (FMRP) serpentarium, Universidade de

São Paulo. All chemicals and solvents were purchased from Sigma (Deisenhofen,
6
Germany or St. Louis, MO, USA). Human plasma and erythrocytes were obtained from

health volunteer donors and written informed consent was obtained from all participants

recruited. All procedures were strictly conducted according to the principles expressed

in the Helsinki Declaration.

2.3. Preparation and biochemical characterization of crotamine

Purification of native crotamine from snake venom was performed essentially as

described elsewhere [29]. Briefly, six hundred milligrams of crude dried venom were

dissolved in 5 mL of 0.25 M ammonium formate buffer pH 3.5, and the bulk of

crotoxin, the major venom component, was eliminated by slow speed centrifugation as a

heavy precipitate that formed upon slow addition of 20 mL of cold water to the solution.

Tris-base 1 M was then added dropwise to the supernatant to raise the pH to 8.8 and the

solution was applied to a CM-Sepharose FF (1.5 x 4.5 cm; GE Healthcare,

Buckinghamshire, UK) column, equilibrated with 0.04 M Tris-HC1 buffer pH 8.8,

containing 0.064 M NaCl. After washing the column with 100 mL of equilibrating

solution, crotamine was recovered as a narrow protein peak by raising the NaCl

concentration of the eluting solution to 0.64 M. The material was thoroughly dialyzed

against water (benzoylated membrane, cut-off MW = 3,000) and lyophilized. Amino

acid analysis after acid hydrolysis of a sample (4 N MeSO3H + 0.1% tryptamine; 24 h at

115oC) indicated a yield of 72 mg (14.7 mol) of crotamine and trace amounts of Thr,

Ala and Val (purity  98%). The purity of crotamine were further confirmed by

analytical C18 reversed phase HPLC, using linear gradient of 10-30% acetonitrile

containing 0.1% TFA, and the molecular masses were verified by a mass by liquid

chromatography-mass spectrometry using a LCMS-2010 EV equipped with an

electronspray ionization (ESI)-probe (Shimadzu, Tokyo, Japan), as previously described

7
[29]. Pure crotamine was then labeled with the Cy3-fluorescent dye as previously

described using the Fluorolink Cyanine 3 (Cy3) reactive dye (GE Healthcare, Little

Chalfont, UK) [32,34].

2.4. Parasites

Plasmodium falciparum chloroquine-resistant strain 3D7 was cultured in culture

bottles using RPMI 1640 medium (Atená Biotecnologia, Campinas, SP, Brazil), which

was supplemented with 10% of inactivated human plasma (human plasma and

erythrocytes were obtained from health volunteer donors) as previously described [35].

Parasitemia was verified by Giemsa-stained smears. The parasites were isolated from

the infected erythrocytes (iRBCs) by selective lysis using 10 mg/mL saponin in PBS

buffer (composed by 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM

NaH2PO4, and 1 mM CaCl2), followed by centrifugation at 2000 × g for 10 min at 4oC.

Then, the isolated parasites were washed twice in PBS buffer to remove the red cell

membranes.

2.5. Confocal microscopy

P. falciparum culture was incubated with Cy3-crotamine (10 µM) for 1 h, at

room temperature. In the last 10 min of incubation, DAPI (0.01 mg/mL) was added,

followed by washing with PBS buffer. Cells were resuspended in the same buffer and

plated on a microscopy chamber previously treated for 1 h with L-polylysine (1

mg/mL). The data acquisition was performed in a Leica TCS SP8 confocal microscope

(Leica Microsystems, Wetzlar, Germany) using an oil immersion 63  objective. The

dyes parameters used were λEX 545 nm and λEM at 590-620 nm for Cy3-crotamine, and

λEX 405 nm/λEM 420-470 nm for DAPI fluorescence.

8
 Image acquisition of H+ mobilization from acidic compartments was observed

with lysosomotropic fluorochrome acridine orange (AO) loaded iRBCs. For this, 5 µM

of AO was incubated with iRBC during 30 min. After three washes with PBS buffer,

iRBCs were resuspended in PBS buffer, and the effects on fluorescence of AO in the

presence of 20 µM of crotamine were monitored. The metachromatic dye AO

accumulates mainly in the acidic compartments, preferentially in lysosomes and

nucleus. AO shows different emission wavelengths, red or green fluorescence,

depending of the dye concentration, when excited by blue light (488 nm). Therefore, the

AO acquisition parameters were λEX 488 nm (argon laser), and λEM (green) 500-530 nm

and λEM (red)  560 nm. Lysosomal/endosomal compartments accumulate higher

concentration of AO, and consequently these compartments are labeled in red. In the

opposite side, nucleus and cytoplasm accumulates lower dye concentration emitting

green light. Analysis of merged green and red emission lights can be used to observe

simultaneously concentrated and non-concentrated compartments (uptake method). The

software-based analysis allowed measurements in the selected cells (region of interest),

as a function of time.

2.6. Flow Cytometry

Culture of iRBC containing synchronous parasites at ring stage (2.0% hematocrit

and 2.5% parasitemia) was added to a 96-wells microplate. Crotamine at five different

concentrations (from 1.25 µM to 20 µM) were added on iRBC culture. As control,

culture of red blood cells (RBC) and iRBC culture without treatment were used. The

plate was placed in an incubator set at 37oC for 48 h. Next the medium was removed

and the cells were fixed with 200 µL of 2 % formaldehyde in PBS (v/v) for 24 h at room

temperature. A solution with 0.1 % Triton X-100 in PBS (v/v), containing 1 nM

9
YOYO-1 (Invitrogen/Life Technologies, Grand Island, NY, USA) was added before the

data acquisition. Parasitemia was measured in a flow cytometer FACS Calibur (BD

Biosciences, Franklin Lakes, New Jersey, USA), with DNA stain YOYO-1 as a marker

for cell survival. The number of fluorescent events in 10,000 cells represents de

percentage of parasitemia. The IC50 values were obtained using a non-linear dose-

response curve fitting analysis via Graph Pad Prism v.5.0 software (San Diego, CA,

USA).

2.7. Spectrofluorometer measurements

Isolated parasites (107 cells mL-1) were incubated with PBS buffer in 500 µL

cuvette at 37oC to measure the intracellular proton mobilization. Cells were loaded with

5 µM of AO and the uptake of fluorescent marker by parasite was monitored. After

complete internalization of AO in the food vacuole of the parasite, crotamine was added

in steps for each concentration (1.25, 5 and 20 µM). The fluorescence was measured

continuously in a Shimadzu RF-5301 PC spectrofluorometer at λEX 495nm and λEM 530

nm. The control with chloroquine was performed in the same conditions [16].

2.8. Statistical analysis

Statistical analyses were performed using Prism5® by one-way ANOVA and

Bonferroni’s post test. The results are from three independent experiments performed on

different days .A p value < 0.05 was considered statistically significant.

10
3. Results

3.1. Localization of crotamine by confocal microscopy

After 1 h of incubation with 10 µM of Cy3-crotamine, the toxin was visualized

within the parasites inside the infected red blood cells (iRBCs) (Figure 1, supplemental

figure 1A). It is of note that crotamine was not internalized by uninfected RBC,

demonstrating the selectivity for iRBC.

Staining with DAPI also allowed demonstrating the localization of Cy3-

crotamine in the parasite nucleus, although part of the labeled crotamine remained

attached to the lipid cell membrane of the iRBCs (Figure 1A).

3.2. Toxicity of crotamine against parasites

The potential toxicity of crotamine against Plasmodium falciparum parasites was

assessed in culture, and we observed that crotamine affected the parasite development

and displayed a very potent anti-plasmodial activity, with an IC50 value of 1.87 µM

(Figure 1B). A potential anti-leishmanial activity was also described for crotamine by

others, but with no mention or discussion on the molecular mechanism of action [36].

3.2. Effects of crotamine on acidic compartments

The H+ homeostasis in isolated P. falciparum and iRBC was explored in the

presence of crotamine to verify the eventual disruption of lysosome ion maintenance, as

should be expected based on our previous studies on crotamine cytotoxic activity

[26,27]. Figure 2A shows that crotamine promotes an increase in the uptake of the

lysosomotropic fluorochrome Acridine Orange (AO) in subcellular compartments in

isolated parasites, in a dose-dependent manner. As control, the same experiment was

performed with chloroquine (a weakly basic molecule), which promotes the extrusion of
11
AO from the acidic compartments to the cytosol, presumably as a result of

alkalinization of the organelle lumen [37]. Our results suggest that crotamine acts

causing lysosomal destabilization and by interfering in the organelle and parasite cell

membrane structure (not alkalinization), as previously observed in other models [38].

Therefore, crotamine acts by a different manner of that described for chloroquine.

The H+ homeostasis destabilization by crotamine was also confirmed by

confocal microscopy (Figure 2B and 2C). We visualized an AO mobilization from acid

compartment (fluorescence channel, red) and parasite cytosol (fluorescence channel,

green). Fluorescence reduction was observed in both cases, indicating changes in

membrane structure. Thus, our data indicates that crotamine interferes with H+

homeostasis in P. falciparum parasites.

4. Discussion

Aiming to explore the mechanism of crotamine anti-plasmodial activity, we first

verified the internalization of crotamine by infected red blood cells (iRBCs). Incubation

with fluorescently labeled crotamine (Cy3-crotamine) showed the selectivity for iRBCs,

as well as no internalization was observed in uninfected RBCs (Figure 1). This result is

in good agreement with our previous findings showing no hemolytic activity for

crotamine against human erythrocytes for concentrations up to 100 µM [27], and the

importance of the negative charge on membranes for the selective affinity and activities

of native crotamine [34]. The selectivity for the iRBCs may therefore be explained by

the extensive host cell remodeling mediated by parasites, aiming to provide nutrients

from serum for their survival and export proteins and lipids to iRBC cytoplasm and

membrane [39]. As described by others, these alterations may affect the trafficking

12
routes (vesicles, channels and parasitophorous vacuole membrane extensions) and

membrane charge composition (proteins and phospholipids) [39-41].

A number of small amphipathic peptides from natural source possess both the

antimicrobial and cytotoxic effects [27], but very few of them also have cell-penetrating

properties as described for crotamine [42]. In addition, to our knowledge, among known

amphipathic peptides none of them can form complex with DNA molecules and carry

cargos into the cells as crotamine does [30,32,33]. We believe that this gives to

crotamine a unique advantage, as the anti-plasmodial activity described for crotamine

here could also be further potentiated by its combination with lethal genes specific for

malaria parasites aiming therapeutic intervention [43,44].

Crotamine was shown to completely disrupt mammalian cells lysosomes,

proving its effect on negatively charged vesicles of mammalian cells [31]. This allowed

us to suggest that crotamine effectively acts on the alteration of vesicle internal pH, as a

consequence of its abundant content of Lys and Arg residues and the resulting high net

charge (8+) [24,25]. Regulation of the internal pH is important for the survival of

parasites, as this sets the environment for the functioning of several intracellular

enzymes crucial for parasite devolpment. Confocal microscopy studies allowed

confirming that crotamine interferes with H+ homeostasis in P. falciparum parasites as

visualized by AO mobilization (fluorescence reduction) from acid compartment

(fluorescence channel, red) and parasite cytosol (fluorescence channel, green)

suggestive of changes in membrane structure, supporting its potential as a candidate for

a novel antimalarial drug or molecular template.

It is important to emphasize that the presence of crotamine in cell nucleus and its

binding to chromosomal DNA were also demonstrated by us in several cultured

13
mammalian cells [27], which is in line with the localization of Cy3-crotamine in the

parasite nucleus observed here (Figure 1, supplemental figure 1).

5. Conclusion

Taken together, we have demonstrated here that crotamine is selectively

internalized by parasite-infected erythrocytes, with co-localization with DAPI. The

dose-dependent anti-plasmodial activity of crotamine was also shown here for the first

time, and our data suggests the potential involvement of disruption of H+ homeostasis in

P. falciparum parasites in the mechanism of action for the crotamine antimalarial

activity. Our data presented here, allow us to suggest crotamine, as well as derived

peptidomimetics, as a potential molecule for the inhibition of malaria infection, and as a

powerful tool for the Plasmodium cell biology studies at the molecular and cellular

level. Aiming to innovative strategies for antimalarial therapy, crotamine activity

described here can potentially be further improved by the combination of crotamine

with therapeutic drugs and/or genes, as crotamine was also shown to be an efficient

transport vector [30,32]. Although in vivo animal model experiments with crotamine

would be now decisive for encouraging further studies for antimalarial activity, the

viability of employment of crotamine in therapy was already demonstrated by us in

vivo, at least for the antitumoral activity [29,30]. In addition, the potential of targeting

specifically infected erythrocytes versus uninfected ones and also the ability to disrupt

the acidic compartments provide a great value for crotamine as a useful new tool

compound for the Plasmodium cell biology studies.

14
6. Acknowledgments

This work was supported by the Brazilian Agencies: Fundação de Amparo

Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento

Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de

Nível Superior (CAPES). We also thank the Multiuser Multiphoton Confocal

Microscopy Laboratory (INFAR-UNIFESP) for the access to confocal microscopy

devices.

15
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Figure 1. Uptake of crotamine by parasite-infected RBCs and effect on P.
falciparum survival in vitro. (A) Plasmodium falciparum culture was incubated with
Cy3-crotamine (10 µM) for 1 h, at room temperature. In the last 10 min of incubation,
DAPI (0.01 mg/mL) was added, followed by washing with PBS buffer. Cells were
resuspended in the same buffer and plated on a microscopy chamber. The dye
parameters used were λEX 545 nm and λEm at 590-620 nm for Cy3-crotamine (red
channel), and λEX 405 nm/λEm 420-470 nm for DAPI fluorescence (blue). Scale bar = 10
µm. (B) Crotamine was added to 20, 10, 5, 2.25 and 1.25 µM in 200 µL of P.
falciparum culture (iRBC). The plate was incubated for 48 h at 37oC. YOYO-1 (1 nM)
was used as the cell nucleus dye. RBC and iRBC without treatment was used as controls
performed in the same conditions. The data were acquired via flow cytometry (FACS
Calibur, BD Biosciences). The results are from three independent experiments
performed in triplicate on different days. The combined data were compared by paired
one-way ANOVA and Boferroni’s post test * p< 0.05.

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Figure 2. Effect of chloroquine and crotamine on intracellular acridine orange
(AO) mobilization from acidic compartments of isolated P. falciparum parasites.
(A) The lysosomotropic fluorochrome AO (5 µM) were added in isolated parasites (107
cells mL-1) solution. Different concentrations of crotamine (1.25, 5, and 20 µM) were
added during AO fluorescence acquisition in spectrofluorometer cuvette. Fluorescence
intensities (arbitrary fluorescence units - AFU) represent at least three different cell
preparations. Crotamine (gray line) and chloroquine (black line). (B) The alterations of
acridine orange (AO) fluorescence was performed in confocal microscopy. iRBC was
loaded with 5 µM of AO during 30 min. After addition of 20 µM of crotamine, the
effects on fluorescence of AO were monitored. (a-f) Confocal images: (a) Brightfield
(BF); (b) Merged; (c) basal AO red channel; (d) basal AO green channel; (e-f) AO red
and green channels after crotamine addition; (C) Histogram of AO fluorescence (red
channel) with mean  SD (N = 3). The data were analyzed by paired t-test * p < 0.05.

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