Invest Clin 63(1): 7 - 18, 2022 https://doi.org/10.54817/IC.v63n1a01

Corresponding author. Yenddy Carrero. Universidad Técnica de Ambato. Facultad Ciencias de la Salud. C/ Salva-

dor entre México y Colombia, Ingahurco. Ambato 180150, Ecuador. Tel: + 59 3 33730268 ext. 5239.

E-mail: yenddycarrero@yahoo.es

Arracacia xanthorrhiza Bancr compounds

decrease β-actin, hypoxia-inducible factor-1

and nitric oxide production in HeLa cells.

Yenddy Carrero1,2, Jenny Moya2, Michael Acosta2 and Jesús Mosquera-Sulbarán3

1Universidad Técnica de Ambato. Facultad Ciencias de la Salud. Ambato, Ecuador.

2Universidad Técnica de Ambato. Facultad de Ciencias de la Salud. Laboratorio

de Biología Molecular y Celular. Ambato, Ecuador.

3Instituto de Investigaciones Clínicas “Dr. Américo Negrette”. Facultad de Medicina,

Universidad del Zulia, Maracaibo, Venezuela.

Key words: actin; nitric oxide; HIF-1; Arracacia xanthorrhiza Bancr; Hela cells.

Abstract. The treatment of cancer patients with anti-cancer drugs is often

accompanied by the presence of undesirable side effects. The use of natural

plant derivatives alone, or in conjunction with existing anti-neoplastic drugs,

has been suggested to obtain better results and decrease these side effects.

Nitric oxide (NO•), the hypoxia-inducible factor-1 (HIF-1), and decreased con-

centration of actin play important roles in cancer progression. The beneficial

effects of polyphenols in various organ disorders including cancer has been re-

ported. The aim of this study was to determine the effect of Arracacia xanthor-

rhiza Bancr extracts, white (WAXB) and red (RAXB) variants (compounds rich

in polyphenols) on the concentrations of β-actin, NO• and HIF-1 in Hela cells

cultures, to uncover possible anti-neoplastic effects. Extracts from the plant

leaves were added to Hela cell cultures at a concentration of 10-3 mg/mL, and

after 24 hours of culture, the concentrations of β-actin, NO• and HIF-1 were de-

termined by immunohistochemical, biochemical and western blot assays. Both

extracts reduced the concentrations of β-actin, NO• and HIF-1 (p<0.001), sim-

ilar to the methotrexate effect. These results suggest an antineoplastic effect

of the studied plant extracts and highlight the possibility of their use in the

treatment of neoplasms.

8 Carrero et al.

Investigación Clínica 63(1): 2022

Los componentes de la Arracacia xanthorrhiza Bancr

disminuyen las producciones de la actina β, del factor-1

inducible por la hipoxia y del óxido nítrico en células Hela.

Invest Clin 2022; 63 (1): 7 – 18

Palabras clave: actina; óxido nítrico; HIF-1; Arracacia xanthorrhiza Bancr; células Hela.

Resumen. El tratamiento de pacientes con cáncer utilizando drogas-

antineoplásicas presenta problemas relacionados con efectos colaterales inde-

seables. Se ha sugerido el uso de derivados de plantas naturales solas, o en

combinación con drogas antineoplásicas existentes para obtener mejores re-

sultados y disminuir los efectos colaterales. Así mismo, se ha reportado que

el óxido nítrico (NO•), el factor-1 inducible por hipoxia (HIF-1) y la disminu-

ción de la expresión de la actina tienen un papel en la progresión del cáncer.

También se ha reportado los efectos beneficiosos de lo polifenoles en varios

desordenes orgánicos, incluyendo el cáncer. El objetivo de este estudio fue de-

terminar los efectos de los extractos procedentes de la Arracacia xanthorrhiza

Bancr blanca (AXBB) y la variedad roja (AXBR) (compuestos ricos en polifeno-

les) en las concentraciones de la actina-beta, el NO• y el HIF-1 en cultivo de

células Hela, para destacar sus posibles efectos antineoplásicos. A los cultivos

de células Hela se les agregaron los extractos de las hojas de AXBB o AXBR

(10-3 mg/mL, concentración final) y después de 24 horas de cultivo se deter-

minaron las concentraciones de la actina-beta, el NO• y el HIF-1 por métodos

inmunohistoquímicos, bioquímicos y western blot. Ambos extractos disminuye-

ron las concentraciones de la actina-beta, el NO• y el HIF-1 (p<0,001) de una

manera similar al efecto del metotrexato. Estos resultados sugieren un efecto

antineoplásico de estos extractos y destacan la posibilidad de ser usados para el

tratamiento de las neoplasias.

Received: 13-08-2021 Accepted: 03-09-2021

INTRODUCTION

Cellular motility is the basis for can-

cer cell invasion and metastasis. Migration

involves an intricate actin branched net-

work. Regulation of cell migration is nec-

essary for development, wound healing,

and immune responses, whereas aberrant

and uncontrolled cell motility is a feature

in cancer cells 1. Therefore, compounds

capable of inducing degradation of actin,

could be important in the production of

anti-cancer drugs by decreasing the actin-

dependent cell motility. In addition to the

alterations of actin, nitric oxide (NO•), a

vasodilator, has been shown to have a role

in the regulation of cancer progression and

metastasis 2; effects that probably are medi-

ated by angiogenesis, apoptosis and cellu-

lar motility 3,4. In addition, NO• increases

the hypoxia-inducible factor-1 (HIF-1), a

transcription factor that enhances several

hypoxia-inducible genes and induces cancer

metastasis 5. Previous studies have shown

Arracacia xanthorrhiza Bancr and Hela cells 9

Vol. 63(1): 7 - 18, 2022

that methotrexate is capable of inducing

apoptosis 6, an important cytotoxic mecha-

nism of anti-cancer drugs. It is well known

that apoptosis is associated with alterations

of the reorganization and concentration of

actin 7, 8. Therefore, drugs that target ac-

tin, NO•, and HIF-1may be important in the

treatment of cancer. Thus, the aim of this

study was determining the effect of two eco-

variants of Arracacia xanthorrhiza Bancr

extracts, white (WAXB) and red (RAXB)

variants on the concentrations of β-actin,

NO• and HIF-1 in Hela cells cultures (uter-

ine cervix cancer cell line) and compared

their effects with the effect of methotrex-

ate, a well-known anti-cancer drug.

MATERIALS AND METHODS

Source of plants

Plants were obtained from the follow-

ing localities: Red Arracacia xanthorrhiza

Bancr (RAXB) from the province of Tungu-

rahua, Quinchicoto (Municipality Tisaleo),

Ecuador (Latitude: 1° 23’ 54” S; Longitude:

78° 24’ 41” W; Altitude: 3,260 meters above

sea level). White Arracacia xanthorrhiza

Bancr (WAXB) from El Triunfo (Municipal-

ity Baños), Ecuador (Latitude: 1°10’ 18” S;

Longitude: 78°32’ 33” W; Altitude: 1,683

meters above sea level). Plant samples used

for this research have been authorized by

the Ecuadorian government under the

framework of the United Nations Conven-

tion on Biodiversity.

Extract procedure

Fresh plant leaves were collected and

dried in an oven at 40°C for 24 hours until

a constant weight was obtained. The speci-

mens were stored at room temperature in

the dark prior to their extraction and subse-

quent testing. Stock solutions of RAXB and

WAXB were prepared by dissolving 60 g of

leaves in ethanol (20%) at 80°C for one hour.

Ethanol was extracted by a rotary evaporator

and active compounds remained in water.

Extracts were grinded into a powder (VirTis

Bench Top, SP Scientific, NY, USA), and then

dissolved in PBS and filtered (0.22 µm). Fil-

tered stock solutions (0.411 mg/mL) were

stored at -80°C until use. Total phenolic

content (TPC) and antioxidant activity (AA)

assays in microplates were determined. An

intra-laboratory validation was performed 9

of the Folin-Ciocalteu microplate method to

measure TPC and the 2,2-diphenyl-1-picryl-

hydrazyl (DPPH) microplate method to mea-

sure AA.

Cell culture

The human cervical cell line (HeLa) was

obtained from the American Type Culture

Collection (CCL-2). Cells were maintained

in DMEM media, supplemented with 10% fe-

tal bovine serum, 100 U/mL penicillin, and

100U/mL streptomycin. The cells were then

incubated at 37°C with 5% CO2 saturation.

Cells were used in the linear phase of growth

with a passage number of 5 to 7.

Viability assay

The cytotoxic effect of WAXB and RAXB

on HeLa cells was evaluated by MTT assay ac-

cording to the manufacturer’s instructions

(Thermo Fisher, MA, USA). The cells were

seeded at 1x104 cells per well in a 96-well

plate and incubated at 37°C for 24 h. Cells

were treated with increasing doses of WAXB

or RAXB by 2-fold dilution, starting with

10–1mg/mL until 10–11mg/mL. Cells were

treated for 24h. The MTT assay results were

obtained using a spectrophotometer plate

reader (Victor X3; Perkin Elmer, USA) at

570 nm. Half maximal inhibitory concentra-

tion (IC50: 10–3 mg/mL) was obtained by no

lineal regression analysis (Graphpad Prism

7.0 Software Inc., San Diego, CA, USA). All

experiments were performed in triplicate to

evaluate half-maximal inhibitory concentra-

tion for both plant extracts against the HeLa

cell line.

Western Blot

Untreated and WAXB or RAXB- treated

Hela cell cultures (10–3 mg/mL) and metho-

10 Carrero et al.

Investigación Clínica 63(1): 2022

trexate-treated cultures (10 µM), were lysed

with RIPA buffer for 30 min on ice. Then, cells

were centrifuged at 1200 rpm for 15 min at

4°C, and the supernatant was collected. The

protein concentration was measured by the

Bradford protein assay. Similar amount of

protein from each sample was separated by

SDS-PAGE and transferred onto PVDF mem-

branes. The membranes were blocked with

7% skimmed milk for 2 h at room tempera-

ture and incubated overnight at 4°C with a

primary anti- β actin antibody (Santa Cruz

Biotechnology Inc, Texas, USA). After wash-

ing three times with TBST(10 mM Tris-HCL,

pH 7.4, 150 mM NaCl, 0.05% Tween 20), the

membranes were incubated with the appro-

priate horseradish peroxidase-conjugated

secondary antibody (1:5000 dilution: Santa

Cruz Biotechnology Inc, Texas, USA). Ex-

periments were performed in triplicate and

at least three independent experiments were

done. Controls represent untreated HeLa

cultures.

β-actin determination

HeLa cells were seeded on coverslips at

5x105 cells per well. After 48 h of culture,

cells were treated with WAXB, RAXB (10–3

mg/mL) or methotrexate (10 µM) for addi-

tional 24 h. Cells were fixed and permeabi-

lized by a paraformaldehyde solution (4%, 15

min) and cold acetone (15 min), respectively.

Then, cells were reacted with a monoclonal

antibody against β-actin (β-Actin (C4): sc-

47778 conjugated Alexa Fluor®647; Santa

Cruz Biotechnology Inc, Texas, USA) for 30

min at 37°C. Thereafter, cells on coverslips

were analyzed through a fluorescence mi-

croscopy (Leica-DMi8, Wetzlar, Germany).

Experiments were performed in triplicate

and at least three independent experiments

were done. DAPI staining was used to iden-

tify nuclei. Results were expressed as fluo-

rescence intensity units (502/530 nm) per

x 630 field. Controls represent untreated

HeLa cultures.

Nitric oxide determination

Nitric oxide was determined by detec-

tion of nitrite formed by the spontaneous

oxidation of NO• using a Griess Reagent Kit

(G-7921: Molecular Probe Inc. Eugene, USA)

following the manufacture’s indications. Re-

sults are expressed as µM per mg of protein.

Hypoxia-inducible factor-1 determination

HIF-1 was determined in WAXB, RAXB

(10–3 mg/mL) treated Hela cell cultures us-

ing Image-iT™ Hypoxia Reagents (Invitro-

gen, Termo Fisher Scientific, Eugene, USA)

following the manufacture’s indications.

Cells on coverslips were analyzed through

a fluorescence microscopy (Leica-DMi8,

Wetzlar, Germany). Experiments were per-

formed in triplicate and at least three inde-

pendent experiments were done. DAPI stain-

ing was used to identify nuclei. Results were

expressed as fluorescence intensity units

(502/530 nm) per x 630 field. Controls rep-

resent untreated HeLa cultures.

Statistical analysis

Experiments were in triplicate and per-

formed at least three times. Results were ex-

pressed as the means ± standard deviation.

Statistical analysis was performed using one

way ANOVA and Bonferroni´s posttest us-

ing Graph Pad Prism software (version 7.0;

Graph Pad Software Inc., San Diego, CA,

USA). Statistical significance is expressed as

p<0.05.

RESULTS

The antioxidant activity of WAXB and

RAXB and their phenolic content are ob-

served in Table 1. The cytotoxicity of both

extract plants was determined by the MTT

assay. The MTT assay indicated that both

WAXB and RAXB have a half maximal inhib-

itory concentration (IC50) of 10–3 mg/mL

(Fig. 1). Western blot analysis showed de-

creased thickness and intensity of the actin

Arracacia xanthorrhiza Bancr and Hela cells 11

Vol. 63(1): 7 - 18, 2022

Table 1

Phenolic content and antioxidant activity of Arracacia xanthorrhiza Bancr

(white carrot and red carrot) extracts.

WABX RABX

Phenolic content

(mg Eq AG/g powder) 78.6 ± 0.002 59.7 ± 0.001

Antioxidant activity

(µg Eq Trolox/g powder) 0.595 ± 0.03 0.395 ± 0.02

(µmol Eq Trolox/100g powder) 237.7 ± 0.03 157.8 ± 0.02

WABX: White Arracacia xanthorrhiza Bancr; RAXB: Red Arracacia xanthorrhiza Bancr.

Fig. 1. Viability assay in Hela cell cultures. White (WABX) and red (RABX) Arracacia xanthorrhiza Bancr

leaves cytotoxic effect on HeLa cells was evaluated by MTT assay. Cells were seeded at 1x104 per well

and treated with increased doses of WAXB (A) and RAXB (B) from 10–11mg/mL to 10–1mg/mL. Expe-

riments were performed in triplicate to evaluate half-maximal inhibitory concentration (IC50: 10–3

mg/mL) for plant extracts.

12 Carrero et al.

Investigación Clínica 63(1): 2022

bands in cultures treated with WAXB, RAXB

or methotrexate compared with untreated

cultures (Fig, 2). A similar anti-actin effect

of plant extracts and methotrexate were

observed. This finding was associated to

decreased anti-β-actin antibody reactivity

on Hela cells, as shown by immunofluores-

cence analysis (Fig. 3). WAXB, RAXB and

methotrexate were capable of decreasing

the nitrite (NO•) content in Hela cell cul-

tures. Methotrexate had a higher NO• re-

ducing effect when compared to plant ex-

tracts (Fig. 4). Hela cell cultures treated

with WAXB, RAXB under culture conditions

of 5% of CO2, showed decreased expression

of HIF-1in treated cultures compared to

controls (Fig. 5).

DISCUSSION

Malignant cancer cells can invade sur-

rounding tissues, progress to intravasation

and, ultimately, to metastasize. Cellular

membrane protrusion is related to local

polymerization of actin filaments, and the

molecules that link migratory signals to the

actin cytoskeleton are upregulated in inva-

sive and metastatic cancer cells 1. Therefore,

cellular actin cytoskeleton is very important

for cancer cell migration during the forma-

tion of metastases 10. Since modifications of

the actin cytoskeleton are characteristic fea-

tures of invasive tumor cells, it is likely that

drugs inducing interactions of cytoskeletal

proteins, can be useful to induce apoptosis

Fig. 2. β-actin expression in HeLa cell cultures treated with Arracacia xanthorrhiza Bancr extracts (10–3

mg/mL). In up panel: Decreased expression of β-actin was observed in plant extract treated cultures

compared with untreated cultures. Similar effects of White Arracacia xanthorrhiza Bancr (WABX),

Red Arracacia xanthorrhiza Bancr (RAXB) and methotrexate (Mtx: 10 µM) were observed. Low panel:

Western blot analysis of β-actin in the same conditions observed in the upper panel. One way ANOVA

with Bonferroni´s post-test.

Arracacia xanthorrhiza Bancr and Hela cells 13

Vol. 63(1): 7 - 18, 2022

Fig. 3. β-actin expression in HeLa cell cultures treated with Arracacia xanthorrhiza Bancr extract. A) Decrea-

sed fluorescence intensity units were observed in White Arracacia xanthorrhiza Bancr (WABX), Red

Arracacia xanthorrhiza Bancr (RAXB) (10–3 mg/mL) and methotrexate (Mtx: 10 µM) treated cultures

compared with control without treatment. Low panel: β-actin positive cells in HeLa cell cultures. B)

Control (untreated). C) WABX. D) RAXB. E) Methotrexate (10 µM). Monoclonal antibody against

β-actin (β-Actin (C4): sc-47778 conjugated Alexa Fluor®647. One way ANOVA with Bonferroni´s post-

test. Magnification X1000.

14 Carrero et al.

Investigación Clínica 63(1): 2022

or inhibit cancer cell metastasis. In this re-

gard, increased attention has been observed

for drugs capable of modifying the state of

actin (polymerization, microfilament organi-

zation) 11.

In this study, extracts from WAXB and

RAXB could induce decreased expression of

B-actin on Hela cells. The effects of WAXB

and RAXB were like that observed using

methotrexate. Methotrexate is an anti-folate

used in the treatment of cancers and auto-

immune diseases 12. In previous a communi-

cation it has been reported that methotrex-

ate is an actin affecting drug, acting mainly

on its polymerized form 13. In this study, all

studied extracts and drug decreased the re-

activity to an anti- β-actin antibody in Hela

cells, suggesting an actin molecular altera-

tion. We could not observe alterations in the

actin cytoskeleton organization; however,

some similar effects related to actin reorga-

nization between methotrexate and extracts

were observed. Methotrexate can induce al-

terations of the actin cytoskeleton in uterine

cervix cancer cell line (CaSki) 6 as observed

in Hela cells (uterine cervix cancer cell line)

using extracts and methotrexate. Apoptosis

is the major cytotoxic mechanism of anti-

cancer therapies 8, and it is known that mor-

phology alterations during apoptosis 7 cor-

relate with the reorganization of the actin

microfilaments and concentration of actin;

in this study both, AXB extracts and metho-

trexate are capable of decreasing the actin

concentration. These data suggest that the

anticancer effects of WAXB and RAXB are

like those observed in methotrexate. WAXB

and RAXB actin degradation mechanisms re-

main unknown. Previous reports have shown

that actin initiates apoptosis and that the fi-

nal degradation of actin filaments enhances

the apoptosis signaling 14. A decreased actin

band was observed in western blot analysis,

suggesting degradation of actin molecule.

Since plant extracts and methotrexate are

capable of inducing apoptosis, activation of

proteases during the apoptotic process may

act on actin leading to its degradation. In

Fig. 4. Nitrite (NO•) production by HeLa cell cultures treated with Arracacia xanthorrhiza Bancr extracts. De-

creased nitrite production was observed in White Arracacia xanthorrhiza Bancr (WABX), Red Arracacia

xanthorrhiza Bancr (RAXB) (10-3 mg/mL) and methotrexate (Mtx: 10 µM) treated cultures compared with

control (untreated). Methotrexate had higher reducer NO• effect compared to plant extracts. Griess Rea-

gent Kit (G-7921: Molecular Probe Inc. Eugene, USA). One way ANOVA with Bonferroni´s post-test.

Arracacia xanthorrhiza Bancr and Hela cells 15

Vol. 63(1): 7 - 18, 2022

Fig. 5. Hypoxia-inducible factor-1expression (HIF-1)in HeLa cell cultures treated with Arracacia xanthorr-

hiza Bancr extracts. A) Decreased fluorescence intensity units were observed in White Arracacia

xanthorrhiza Bancr (WABX) and Red Arracacia xanthorrhiza Bancr (RAXB) (10–3 mg/mL) treated

cultures compared with control (untreated). Low panel: HIF-1positive cells in HeLa cell cultures.

B) Control. C) WABX. D) RAXB. Image-iT™ Hypoxia Reagents (Invitrogen, Termo Fisher Scientific,

Eugene, USA ). One way ANOVA with Bonferroni´s post-test. Magnification X1000.

16 Carrero et al.

Investigación Clínica 63(1): 2022

this regard, interleukin-1b-converting en-

zyme (ICE)-like family of proteases such as

Nedd2 / Ich-1, CPP32 / Apopain / Yama, TX

/ ICErelII, TY / ICErelIII, Mch 2, Mch 3 / ICE-

LAP3, Mch 4, and FLICE are implicated in

actin degradation during apoptosis 15.

Nitric oxide has been involved in the

cancer biology probably by induction of

angiogenesis, apoptosis, increased HIF-

1 and alterations of cellular motility 3-5.

Some of these effects can induces cancer

metastasis, spread and growth of tumor

cells through angiogenesis and high pro-

duction of HIF-1; however, other effects

such as apoptosis and alterations of actin

may impede or eliminate metastatic pro-

gression 2. Angiogenesis is the growth of

new blood vessels and is essential for tu-

mor progression and metastasis 16. It has

been reported that increased activity of

isoform enzyme iNOS (inducible nitric ox-

ide synthase) facilitates tumor cell angio-

genesis and metastasis 3, probably medi-

ated by interleukin-33 17.

HIF-1 is a transcription factor that reg-

ulates hundreds of genes, and it is activated

by hypoxia (reduced oxygen availability) 18.

HIF-1 is stabilized by hypoxia-dependent or

independent pathways and associated with

pro-carcinogenic effects 19. Stabilization of

HIF-1 leads to changes in glycolysis, nutri-

ent uptake, angiogenesis, cell migration and

apoptosis promoting survival of tumor and

metastasis 20, 21. However, HIF can be acti-

vated by non-hypoxic pathways. In this re-

gard, NO• can induce this transcription fac-

tor that enhances many hypoxia-inducible

genes, via the PI3k/Akt pathway 5.

Previous studies have shown that plant

extracts and polyphenolic compounds may

have a beneficial role in several diseases

including cancer 22-26. Mechanisms related

to the regulation of immune system, inac-

tivation of arachidonic acids pathways, in-

activation of NF-κB (nuclear factor kappa-

light-chain-enhancer of activated B cells),

suppression of toll-like receptor and antioxi-

dant activity 27 may be involved. Biochemical

analysis of WAXB and RAXB shows that both

are phenolic compounds and the effects pre-

viously reported for phenolic compounds

probably are implicated in ours results. In

this regard, WAXB and RAXB have antioxi-

dant activity as shown by the Trolox assay.

In addition, these plant extracts induced a

decreased content of NO•, suggesting that

these extract plants can act on nitrogen re-

active species and may represent one of their

anti-cancer mechanisms.

In conclusion, in this study Hela cells

were capable of producing NO• and HIF-1,

compounds that are related to cancer pro-

gression. WABX and RABX extracts induced

decreased expressions of NO• and HIF-1 and

actin-β in treated cultures. These effects as-

sociated with anti-cancer progression were

similar to the effect of methotrexate, a

well-known anti-cancer drug. As an impor-

tant point, not included in this study, it is

necessary to determine the effect of WAXB

and RWABX on other types of cancer to de-

termine if these plant extracts have a gen-

eral effect on cancer. Further investigation

should explore the clinical benefits of WAXB

and RWABX (dietary or alcoholic extract) in

human cancer.

ACKNOWLEDGMENTS AND FUNDING

The authors express their gratitude to

the Dirección de Investigación de la Univer-

sidad Técnica de Ambato (DIDE) for fund-

ing the Project ESTUDIO ETNOBOTÁNICO

Y BIOENSAYOS DE PLANTAS MEDICINALES

RELACIONADAS CON EL TRATAMIENTO

DE CÁNCER DE CÉRVIX EN LA CIUDAD DE

AMBATO, PROVINCIA DE TUNGURAHUA,

ECUADOR, with resolution 0904-CU-P-2018.

Declaration of conflict

of interest

The manuscript has no conflict of in-

terest.

Arracacia xanthorrhiza Bancr and Hela cells 17

Vol. 63(1): 7 - 18, 2022

Authors’ ORCID numbers

• Yenddy Carrero (YC)

0000-0003-4050-4468

• Jenny Moya (JM)

0000-0002-9846-0122

• Michael Acosta (MA)

0000-0001-9437-828X

• Jesús Mosquera-Sulbaran (JM-S)

0000-0002-1496-5511

Authors’ Contribution

• Conception and design of the work

YC/JM/JM-S

• Experimental work YC/JM/MA

• Data Collection/obtaining results

YC/JM/MA

• Data analysis and interpretation YC/

JM/MA/JM-S

• Drafting the article. YC/JM-S

• Critical revision of the article YC/

JM-S

• Final approval of the version to be

published. YC/JM/MA/JM-S.

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