key: cord-0712854-8jxbs7mc
authors: Shi, Chuanqin; Han, Wenwei; Zhang, Meifang; Zang, Ruochen; Du, Kaixin; Li, Li; Xu, Ximing; Li, Chunxia; Wang, Shixin; Qiu, Peiju; Guan, Huashi; Yang, Jinbo; Xiao, Shuai; Wang, Xin
title: Sulfated polymannuroguluronate TGC161 ameliorates leukopenia by inhibiting CD4(+) T cell apoptosis
date: 2020-07-06
journal: Carbohydr Polym
DOI: 10.1016/j.carbpol.2020.116728
sha: b8625f7ef9818503cf1ff8378b3b9f514c853a8b
doc_id: 712854
cord_uid: 8jxbs7mc

Polysaccharides have aroused considerable interest due to their diverse biological activities and low toxicity. In this study, we evaluated the effect of marine polysaccharide sulfated polymannuroguluronate (TGC161) on the leukopenia induced by chemotherapy. It is found that TGC161 ameliorates the leukopenia. Besides, TGC161 would promote CD4(+) T cell differentiation and maturation in the thymus, but does not have a significant effect on precursor cells in bone marrow. Furthermore, TGC161 inhibits CD4(+) T cell apoptosis in vitro. Collectively, our findings offer a natural and harmless polysaccharide to ameliorate leukopenia.

Leukopenia patients have a reduced number of white blood cells in their blood stream, which may be caused by different conditions, including viral infection, cancer, genetic and medication conditions. These patients are taking a higher risk of infections, especially for hospital infections and conditional pathogen infections (Christen, Brummendorf, & Panse, 2017) .

Although many other medical issues usually should be addressed before the white blood cell count can return to normal, medications would be used immediately to boost white blood cells when the count of white cells is very low. This requirement often happens in chemotherapies and severe viral infections. Granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF), vitamin B4 and J o u r n a l P r e -p r o o f alkylglycerols are the most often used medications to treat leukopenia, which are named as neupogen or leupogen dependent on their functional roles (Iannitti & Palmieri, 2010; Mehta, Malandra, & Corey, 2015; Tomita et al., 2016) . However, doctors might decide to stop or delay the treatment instead of administration of these drugs for leukopenia, because of the undergoing side effects of colony stimulating factors, such as fever, bone pain and nausea, or the uncertain outcomes of vitamin B4 and alkylglycerols, if the leukopenia is caused by medications. A more promised medication is required here.

Cancer is the second leading cause of deaths all over the world (Sultana, Asif, Nazar, Akhtar, & Rehman, 2014) , and chemotherapy is still the mostly often used treatment to kill fast-growing cancer cells. It believed that chemotherapy directly destroy tumor cells and has less impact on normal cells, since cancer cells grow and multiply quicker than most cells in the body, but it is undoubted that chemotherapy is also killing other normal cells, particularly for bone marrows and white blood cells, which reduces the number of white blood cells, and was named as chemotherapy induced leukopenia (Nieweg & Van, 1992) . Recently, it is found that chemotherapy triggers host immune responses to tumor cells which is essential for tumor killing (Hodge et al., 2013) . Herein, leukopenia patients not only bear an increased risk of infection, but also present an unsatisfied outcome during chemotherapies. Medications should be taken to boost white blood cells and tumor cell killing. Nevertheless, colony stimulating factors might make the condition even worse in rare cases. For example, administration of GM-CSF might promote bone metastatic in breast cancer and prostate cancer (Dai et al., 2010) . Doctors are also hesitating to use colony stimulation factors because of the risk of over-enhancement of bone marrow cell differentiation (Potosky et al., 2011) . Lentinan, an expensive polysaccharides derived from mushrooms in Lentinus family, was an alternative for some patient (Ren, J o u r n a l P r e -p r o o f lentinan in patients are also unpredictable.

Reduced number of peripheral white blood cells is also a typical symptom in viral infections, such as influenza A infection, coronavirus infection and human immunodeficiency virus (HIV) infection. Since HIV infects CD4 + T cells, it induces cellular apoptosis / pyroptosis and leads to immune exhaustion (Doitsh et al., 2014; Selliah & Finkel, 2001) . In few of HIV positive patients, viral genomic RNA is nearly undetectable in serum, but the counts of white blood cells are maintaining on a very low level (Omondi et al., 2019; Shen et al., 2015) . Herein, drugs for leukopenia, G-CSF, interleukin-12 and others, would be suggested (Maeda, Das, Kobayakawa, Tamamura, & Takeuchi, 2019) . Since the underlying mechanism of the chronic reduced CD4 + T cells is still unclear, these medications might not give expectable outcomes.

Polysaccharides have aroused considerable interest due to their immunity-enhancing activities (Li et al., 2020; Liu et al., 2016; Su et al., 2019) .

It is reported that a polysaccharide derived from Rehmannia glutimosa significantly stimulates lymphocyte proliferation (Huang et al., 2013) . EPS1-1, another polysaccharide from the liquor of Rhizopus nigricans, stimulates lymphocyte proliferation and the phagocytic function of peritoneal macrophage to enhance immunity (Yu, Kong, Zhang, Sun, & Chen, 2016) .

Sulfated polymannuroguluronate (SPMG), a heparin-like sulfated polysaccharide extracted from brown algae, could enhance T cell responses either with or without Concanavalin A stimulation (Miao, Li, Fu, Ding, & Geng, 2005) . Here, we found TGC161 which is similar to SPMG, ameliorates leukopenia in the chemotherapy model. The mechanism is that TGC161 promotes CD4 + T cell differentiation and maturation in thymus, but has no significant effect on precursor cells in bone marrow. Moreover, TGC161 also inhibits CD4 + T cell apoptosis in vitro.

J o u r n a l P r e -p r o o f 2.1. Structural analysis TGC161 was prepared following the published sulfated polymannuroguluronate preparation procedure with minor modifications (Geng et al., 2003) . Briefly, alginate powder was hydrolyzed in 0.5 mol/L HCl at 100 °C for 8 h. Subsequently, the sulfated alginate was prepared by treatment with 0.5 mol/L chlorosulfonic acid for 3 h at 70 °C. The solution was neutralized using 2 mol/L NaOH, and then precipitated with ethanol, and dried. The structure of TGC161 was identified by nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FT-IR) analysis. The 1 H-NMR (500 MHz), 13 C-NMR (125 MHz), 1 H-1 H correlated spectroscopy (COSY), heteronuclear single quantum correlation (HSQC) and total correlated spectroscopy (TOCSY) of the TGC161 were recorded at 298 K on an Agilent DD2-500 500 MHz spectrometer (Agilent, Santa Clara, US). The FT-IR spectrum was recorded on a Nicolet Nexus 470 FT-IR spectrophotometer (GMI, Ramsey, US) in KBr pellets over a wavelength range of 400-4000 cm -1 . The weight-average molecular weight (Mw) was determined by high performance gel permeation chromatography (HPGPC) using a TSK-Gel GMPW column (13 mm, 7.8 mm×300 mm) on an ultimate 3000 high performance liquid chromatography instrument.

The sulfate content analysis was performed according to the previous method (Xue et al., 2016) . In brief, TGC161 was prepared by the oxygen flask combustion (OFC) method. Then, total sulfate content and free sulfate content of TGC-161 were analyzed by ion chromatography. A series of sulfate standard solutions were measured at the same time to generate a standard calibration curve. The concentration of free sulfate and total sulfate could be calculated according to this standard calibration curve. The sulfate content % = (Ctotal sulfate − Cfree sulfate) /CTGC161×100%; Ctotal sulfate, Cfree sulfate and CTGC161

represented the concentration of total sulfate, free sulfate and TGC161, respectively.

Monosaccharide composition of TGC161 was determined using a 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization HPLC method . The PMP-labeled carbohydrates were separated by a BDS-C18 column (4.6 mm × 250 mm, 5 µm, USA) with 0.1 mol/L phosphate buffer (pH 6.0) and acetonitrile at a ratio of 83:17 (v/v, %) as a mobile phase at a flow rate of 0.8 mL/min.

Seven-week-old male C57BL/6 mice were purchased from the Southern Spleen and thymus tissues were isolated from 7-week-old C57BL/6 male mice. These tissues were ground into the cell suspension and slowly filtered through 75 μm nylon cell strainers (Corning, Corning, US). All cells were cultured in RPMI 1640 medium (Gibco, Grand Island, US) supplemented with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin at 37°C and 5% CO2 for 2 h. The culture supernatant was slowly aspirated and centrifuged to obtain cell depositions. Acquired cell depositions were re-suspended with fresh medium and planted in 24-well culture plates for 12 h. The above cell cultivation method refers to the Lefort study (Lefort & Kim, 2010) . TGC161 was distilled into cell culture medium and filtered through a 0.22 μM J o u r n a l P r e -p r o o f membrane (Merk Millipore, Darmstadt, DE) for experimental use. In some experiments, TGC161 (1, 10, 100 μg/mL) was added in thymus cell culture medium, respectively. Harvested cells were saved for following experiments.

Cytotoxicity of TGC161 was evaluated by the resazurin based assay 

The experimental mice were euthanized using CO2. Peripheral blood cell density was analyzed using the ProCyteDx Hematology Analyzer (IDEXX, Westbrook, US) following manufacture's instruction. The experiment was performed as previous described (Kourtzelis & Rafail, 2016) . Briefly, mice were injected intraperitoneally with CD4 monoclonal antibody (150 μg/per mouse, BD Bioscience, Franklin Lake, US). Fifteen days later, less than 0.5% of CD4 + T cells in peripheral blood indicated that the model was constructed successfully. CD4 + T cells in peripheral blood were detected after continuous gavage administration of TGC161 (400 mg/kg) for 15, 20, and 25 days.

Thymus cells were isolated from C57BL/6 mice and cultured according to the above method in "mice and cells". All cells were stained with carboxyfluorescein diacetate succinimidyl ester (CFDA SE, Beyotime, Shanghai, China) and seeded into 24-well plate for 12 h. Cells were collected and stained with CD4-PE (Miltenyi, Bergisch Gladbach, GER) antibody to detect proliferation by flow cytometry.

Cells were collected and stained with CD4-PE (Miltenyi, Bergisch Gladbach, GER) antibody. AnnexinⅤand PI were applied(Vazyme Biotech, Nanjing, China) to detect CD4 + T cell apoptosis by flow cytometry.

Cells were harvested and lysed in the homogenization buff er (50 mM Tris pH 7.6, 150 mM NaCl, 0.5% Triton X-100, 1 mM Na3VO4, 10mM NaF, 5 mM Na-pyrophosphate, 10 mM β-glycerophate, PMSF and protease inhibitor cocktails). Equal amounts of total protein were subjected to SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% milk for 1 h at room temperature, membranes were incubated with specific anti-Bcl2 (cat# 15071, 1:1000), anti-caspase 3 (cat# 9665, 1:1000), anti-caspase 8 (cat# 9746, 1:1000) and anti-β-Actin (cat# 3700, 1:1000) from Cell Signaling Technology (Massachusetts, China) overnight at 4°C. Then the membranes were washed by TBST and immunoblotted with HRP-conjugated secondary antibodies for 1 h at room temperature. All membranes were visualized by J o u r n a l P r e -p r o o f using the ECL western blotting reagent (Tanon, Shanghai, China).

The data were presented as mean ± standard error of the mean (mean ± SEM). One-way ANOVA was used for comparisons (SPSS 13.0 software) and the difference was considered statistically significant at P < 0.05.

The TGC161 is a sulfated polysaccharide prepared by chemical sulfation of low molecular-weight alginate. It is composed of a central backbone of sulfated poly-D-mannuronic acid (M) and sulfated poly-L-guluronic acid (G) (Fig.1A) . TGC161 had no significant inhibition on the growth of 293T cells at the concentration range of 31.3-1000 μg/mL (Fig. 1B) . The single peak in gel chromatography analysis suggested that the isolated TGC161 is homogeneous (Fig.1C ). The molecular weight (Mw) of TGC161 is 10 kDa as determined by HPGPC (as shown in Table 1 To further verify the structure information of TGC161, NMR analysis was utilized (Fig.S1-S6) . The 1 H-NMR and 13 C-NMR data assignments of TGC161 were given in Table 2 that the carbon and hydrogen signals were ascribed properly (Fig. 1E ).

Mice were injected intraperitoneally with carboplatin to induce leukopenia, and TGC161 was delivered by gavage daily since the next day of carboplatin treatment. Mostly, leukopenia was observed since the third day post carboplatin injection. Here, cell densities of leukocyte and lymphocyte were elevated on the third day after TGC161 treatment ( Fig. 2A, B and C).

Alkylglycerols which was clinically used in leukopenia patients to promote counts of white blood cell (Oh & Jadhav, 1994) , was introduced as a positive control. Noticeably, TGC161 promoted lymphocyte cell density, while alkylglycerols increased neutrophils ( Fig. 2B and D) . Compared with alkylglycerols, TGC161 increased lymphocyte numbers more efficiently which induced a considerable boost at the third day post treatment (Fig.2A) , but it only slightly increased the percentage of lymphocytes compared with carboplatin treated mice (Fig.2D ). TGC161 didn't promote neutrophils significantly in carboplatin treated mice ( Fig. 2A, E) . Both alkylglycerols and TGC161 increase lymphocytes to ameliorate leukopenia on the sixth day post administration (Fig. 2F, J) . However, TGC161 was able to increase lymphocyte numbers more efficiently because a significant boost was found at J o u r n a l P r e -p r o o f third day post treatment in TGC161 treated mice but not in alkylglycerols treated mice. This may be due to the different mechanisms of two drugs.

lymphocytes which is essential for tumor cell killing (Ostroumov, Fekete Drimusz, & Woller, 2018; Yuen, Demissie, & Pillai, 2016) . Mostly, neutrophils defend against bacterial infection in the body (Yang, Ghose, & Ismail, 2013) .

To clarify the effect of TGC161 on specific subtype of lymphocytes, cells were stained with different monoclonal fluorescent monoclonal antibodies in FACS analysis. The surface-differentiated molecule CD3 is the hallmarker of mature T lymphocytes which were divided into two populations by the cellular markers, CD4 and CD8 (Masopust & Schenkel, 2013) . CD4 T cells are helper T population expressing both CD3 and CD4 (Zhou, Chong, & Littman, 2009 ).

expressing both CD3 and CD8 (Gerritsen & Pandit, 2016) . Consistent with previous studies (Verma, Foster, Horgan, Hughes, & Carter, 2016) , T cells (CD3 + T cells) reduced significantly in cell counts and percentage after carboplatin administration (Fig. 3A) . Surprisingly, we found that total T (CD3 + )and CD4 + (CD3 + CD4 + ) T cells counts increased substantially after TGC161 administration compared with carboplatin ( Fig. 3A and B) . It indicated that TGC161 would specifically promote CD3 + CD4 + T cell populations in chemotherapy induced leukopenia, which was different from alkylglycerols. B cell, another main component in lymphocytes, also plays an important role in tumor immunity. We were wondering if TGC161 also stimulated B cells as a general immune stimulator. However, no significant difference was observed in TGC161 treated and untreated mice. The same conclusion was true when we monitor granulocytes (Fig. 3D , E, I and J). In addition, TGC161 enhanced J o u r n a l P r e -p r o o f the percentage of total T and CD3 + CD4 + T cells in leukocytes in contrast with the control group ( Fig. 3F and G) . It might attribute to the stronger killing/inhibiting effects of carboplatin on B cells and granulocytes than other leukocytes (Bisch et al., 2018; Menetrier Caux, Ray Coquard, & Caux, 2019) .

complement-mediated killing model. Viral infection usually leads to the activation of complement system (Agrawal, Nawadkar, Ojha, Kumar, & Sahu, 2017) . Complement activation might result in both acute and chronic leukopenia (Kociba, 1995) . To specify the effects of TGC161 on CD4 + T cells, CD4 antibody-dependent complementmediated killing model was utilized to kill CD4 + T cells. Apparently, no or low CD4 + T cells were found in peripheral blood when complement system was activated by CD4 + antibodies (Fig. 4A) . CD4 + T cell counts and percentage increased significantly after continuous treatment of TGC161 for 25 days (Fig.   4A , B and C). To sum up, TGC161 raises the CD4 + T cell counts in antibodydependent complement-mediated cytotoxicity killing model.

thymus, but has no significant effects on precursor cells in bone marrow.

Since all mature blood cells have a limited life span, the HSCs continuously generate new progenitor cells to maintain enough mature cells in the periphery (Bertrand et al., 2010; De Bruin, Demirel, & Nolte, 2013; De Bruin, Voermans, & Nolte, 2014) . Multipotent HSCs differentiate into common lymphoid progenitors (CLP), which then become pre-T cells and move to the thymus (Bertrand, et al., 2010; King & Goodell, 2011) . Therefore, the effect of TGC161 on T cell differentiation has important significance for further research. ST-HSC, CLP and CMP decreased in the carboplatin-treated group (Fig. 5B, C and D) . But TGC161 did not have significant effects on reversing ST-HSC, CLP and CMP cell counts (Fig. 5B, C and D) .

Positive and negative selection allow T cells to gain MHC restriction and autoimmune tolerance in the thymus (Lang et al., 2013) . Then naive T cells derived from thymus stored in the spleen and other immune organs. They play a critical role in the host defense system (Josefowicz, Lu, & Rudensky, 2012; Schwartz, 2003) . Thymic and splenic CD4 + T cell percentage increased after TGC161 administration (Fig. 5E ). TGC161 promoted the thymic and splenic CD4 + T cell numbers and percentage, and the difference was statistically significant (Fig. 5F , G, H and I). TGC161 increased CD4 + T cells in the spleen, which indirectly enhanced cellular immunity. Therefore, TGC161 facilitates the CD4 + T cell differentiation and maturation in the thymus, but has no significant effects on precursor cells in bone marrow.

To clarify the effects of TGC161 on CD4 + T cell proliferation, CFSE fluorescence intensities of CFSE in CD4 + T cells were detected by flow cytometry. TGC161 does not affect the thymus-derived CD4 + T cell proliferation (Fig. 6B) . However, TGC161 inhibited CD4 + T cell apoptosis, especially at the concentration of 10 and 100 μg/mL ( Fig. 6C and D) .

The intrinsic and extrinsic apoptosis pathways have been identified the central apoptotic pathway. Intrinsic apoptosis pathway promotes the release of cytochrome c by activating Bcl2 family proteins (Brenner & Mak, 2009; Lindsay, Esposti, & Gilmore, 2011) . Extrinsic apoptosis pathway is triggered by signals originating from death receptors on cell surface. Ligands of death receptor characteristically initiate signaling via receptor oligomerization, which results in the recruitment of specialized adaptor proteins and the activation of caspase cascades (Declercq, Vanden Berghe, & Vandenabeele, 2009 ). Then, the activated caspase 8 directly cleave and activate caspase 3 to deliver apoptosis signal (Kantari & Walczak, 2011) . In our studies, TGC161 inhibited caspase 8 and caspase 3 cleavage, but has no significant effect on Bcl2 (Fig. J o u r n a l P r e -p r o o f 7A, B). We speculated that low level of caspase 8 and caspase 3 cleavage is indicating the reduced cell apoptosis. Besides, the gray value of cleavedcaspase 8 and cleaved-caspase 3 protein bands were statistically significant ( Fig. 7C, D) . Taken together, TGC161 may inhibit CD4 + T cell apoptosis by decreasing the caspase 3 and caspase 8 cleavage (Fig. 7E) .

Nowadays, leukopenia has aroused considerable research interest.

Medications for leukopenia treatment were attracting scientists' and investors' attention. The recently developed anti-tumor medication plinabulin promotes dendritic cell maturation and ameliorates chemotherapy induced neutropenia (Bertelsen et al., 2011) , which is attracting tons of investments. In this study, we reported that a polysaccharide would benefit chemotherapy induced leukopenia patients by increasing number and percentage of CD4 + T cells in blood stream. As we know, it is the first report that polysaccharides could selectively stimulate CD4 + T cells but only have limited impact on neutrophils.

Some polysaccharides with immune activity have aroused considerable research interest. β-1,3/1,6-glucan derived from Durvillaea antarctica can increase macrophage phagocytosis and the proinflammatory cytokine secretion in vivo (Su, et al., 2019) . In addition, SPMG, which is very similar to TGC161, can enhance the T cell response without the activator stimulation (Miao, et al., 2005) . In our study, TGC161 ameliorates chemotherapy induced leukopenia. Besides, TGC161 promotes the CD4 + T cell differentiation and maturation in thymus but has less impact on precursor cells. Moreover, TGC161 may reduce caspase 8 and caspase 3 cleavage to down regulate CD4 + T cell apoptosis in vitro.

In present study, TGC161 increased CD3 + CD4 + T cells to ameliorate the leukopenia ( Fig. 3B and G) . The percentage of CD3 + CD8 + T cells elevated after TGC161 administration, but the cell numbers did not rise significantly ( Fig. 3C and H) . CD3 + CD4 + and CD3 + CD8 + T cells derived from CLP and then undergo both negative and positive selection to obtain corresponding subtypes (Zuniga Pflucker, 2004) . We speculated that the TGC161 administration may have created a special internal environment that is more conducive for CD3 + CD4 + T cell to survive. We all know that T cells' immune activity is regulated by kinds of cytokines. Interleukin-12 and interferon γ are the critical cytokines to develop Th1 CD4 + T cells (Luckheeram, Zhou, & Xia, 2012) . Interleukin-10 inhibits CD8 + T cell function by improving N-glycan branching to decrease the antigen sensitivity (Smith et al., 2018) . Therefore, we hypothesized that TGC161 may affect the secretion of cytokines to increase the number of CD3 + CD4 + T cells in peripheral blood significantly.

Exploring the effect of TGC161 on specific cytokines requires us to do further experimental verification. Taken together, our data suggested that TGC161 may be developed as a stimulator to increase CD3 + CD4 + T cell numbers and reverse leukopenia. TGC161 may be more conducive to the improvement of clinical associated diseases caused by low lymphocytes. Compared with alkylglycerols, TGC161 may affect faster in terms of elevating lymphocytes.

In vivo, TGC161 has no significant effects on precursor cells in the bone marrow (Fig. 5B, C and D) . We speculate that TGC161 may not recognize and act on these naive precursor cells, resulting in neither ST-HSC nor CMP being elevated. After positive and negative selection in pre-T cells, double positive (CD4 + CD8 + ) cells are converted to single positive (CD4 + CD8or CD4 -CD8 + ) cells. Following selection, down-regulation of co-receptor induced either naive CD4 or CD8 single positive cells that exit the thymus and circulate the periphery (Takaba & Takayanagi, 2017; Takada & Takahama, 2015) . Data showed that TGC161 promoted the differentiation and maturation of CD4 + T cells (Fig.5) . And further research is needed to determine specific isotypes of CD4 + T cell which are the TGC161 targeting.

In vitro, we found that TGC161 did not promote the proliferation of thymusderived CD4 + T cells (Fig. 6B) . However, it was inconsistent with Miao who J o u r n a l P r e -p r o o f reported that TGC161 can promote T cell proliferation (Miao, et al., 2005) .

They used MTT to measure cellular activity and made it as an indicator of cell proliferation. However, we believe that faster cell growth or fewer cell death also show a satisfactory cellular activity. To avoid interference with this factor, we labeled T cells with CFSE and detected fluorescence intensities by flow cytometry. As the cell division rising, the fluorescence intensities of CFSE will decrease. In this way, the cell proliferation ability can be properly determined.

Importantly, we were surprised to discover that TGC161 inhibited the CD4 + T cell apoptosis (Fig. 6C) . Complex apoptosis signal facilitates in the assembly of pro-caspases 8 and 3 and their autoproteolytic activation, which sends the apoptosis signal to the nucleus (Zhang, Zhang, & Xue, 2000) . Changes in cleaved caspase 3 and caspase 8 directly reflect whether apoptosis has occurred. TGC161 may inhibit caspase 3 and caspase 8 cleavage, but have no significant difference in Bcl2 ( Fig. 7A and B ). All these results showed that TGC161 may inhibit CD4 + T cell apoptosis to ameliorate leukopenia.

In summary, our data suggested that TGC161 ameliorates leukopenia caused by chemotherapy. In addition, TGC161 increases the differentiation and maturation of CD4 + T cells in the thymus, but has less impact on precursor cells in bone marrow. Moreover, TGC161 inhibits CD4 + T cell apoptosis in vitro. This research will help the development of new leukopenia treatment drugs and provide new ideas for clinical treatment. 

There are no conflict of interest exists in the present study. 

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