Protein Profiling of Human Nonpigmented Ciliary Epithelium Cell Secretome: The Differentiation Factors Characterization for Retinal Ganglion Cell line

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Hindawi Publishing CorporationJournal of Biomedicine and BiotechnologyVolume 2011, Article ID 901329, 28 pagesdoi:10.1155/2011/901329

Research Article

Protein Profiling of Human Nonpigmented Ciliary EpitheliumCell Secretome: The Differentiation Factors Characterization forRetinal Ganglion Cell line

Ming-Hui Yang,1 Raghu R. Krishnamoorthy,2 Shiang-Bin Jong,3 Pei-Yu Chu,4

Yuan-Han Yang,5 Wen-Cheng Chen,6 Sharon Chia-Ju Chen,3 Adnan Dibas,2

Thomas Yorio,2 Tze-Wen Chung,1 and Yu-Chang Tyan3, 7, 8, 9

1 Department of Chemical and Material Engineering, National Yunlin University of Science and Technology, 123 University Road,Section 3, Douliou, Yunlin 64002, Taiwan

2 Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, USA3 Department of Medical Imaging and Radiological Sciences, Kaohsiung Medical University, 100 Shi-Chuan 1st Road,Kaohsiung 80708, Taiwan

4 Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan5 Department of Neurology, Kaohsiung Medical University Chung-Ho Memorial Hospital, Kaohsiung 80708, Taiwan6 Department of Fiber and Composite Materials, Feng Chia University, Taichung 40724, Taiwan7 National Sun Yat-Sen University and Kaohsiung Medical University Joint Research Center, Kaohsiung 80708, Taiwan8 Center for Research Resources and Development, Kaohsiung Medical University, Kaohsiung 80708, Taiwan9 Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan

Correspondence should be addressed to Tze-Wen Chung, [email protected] and Yu-Chang Tyan, [email protected]

Received 5 April 2011; Revised 10 June 2011; Accepted 13 June 2011

Academic Editor: Daniel T. Monaghan

Copyright © 2011 Ming-Hui Yang et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

The purpose of this paper was to characterize proteins secreted from the human nonpigmented ciliary epithelial (HNPE) cells,which have differentiated a rat retinal ganglion cell line, RGC-5. Undifferentiated RGC-5 cells have been shown to express severalmarker proteins characteristic of retinal ganglion cells. However, RGC-5 cells do not respond to N-methyl-D aspartate (NMDA), orglutamate. HNPE cells have been shown to secrete numbers of neuropeptides or neuroproteins also found in the aqueous humor,many of which have the ability to influence the activity of neuronal cells. This paper details the profile of HNPE cell-secretedproteins by proteomic approaches. The experimental results revealed the identification of 132 unique proteins from the HNPEcell-conditioned SF-medium. The biological functions of a portion of these identified proteins are involved in cell differentiation.We hypothesized that a differentiation system of HNPE cell-conditioned SF-medium with RGC-5 cells can induce a differentiatedphenotype in RGC-5 cells, with functional characteristics that more closely resemble primary cultures of rat retinal ganglion cells.These proteins may replace harsh chemicals, which are currently used to induce cell differentiation.

1. Introduction

Primary open angle glaucoma (POAG), a leading cause ofirreversible blindness worldwide, is an optic neuropathycharacterized by the gradual and progressive loss of retinalganglion cells (RGCs), optic nerve degeneration, and excava-tion of the optic disks [1–4]. The hypothesis has been that

larger RGCs were selectively lost in the early stage of glau-coma [5]. Although the mechanisms of optic nerve damagein glaucoma have not been completely determined, it appearsthat the optic nerve head is a major site of damage [6].

RGCs can generate action potentials that travel along theoptic fibers [7]. In general, RGCs are a mixture of morethan 20 cell subtypes. They have energy-dependent axonal

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transport functions—orthograde and retrograde transports[8]. These terminal projection areas are in the lateral genic-ulate body. RGCs can be subdivided by their morphologyand physiology, but they are usually discussed withoutclassifications.

The in vitro study of the physiology and pathophysiologyof RGCs has been limited to primary cultures. Previous stud-ies have characterized a transformed rat retinal ganglion cell-line (RGC-5), which expresses many neuronal cell markers,including Thy-1, a cell surface glycoprotein found predom-inantly in the retinal ganglion cells [6, 9, 10], and Brn-3C, a POU domain transcription factor expressed exclusivelyin the retinal ganglion cells [11]. RGC-5 cells also expressreceptors of N-methyl-D aspartate (NMDA), GABA-B, andneurotrophin [6]. However, unlike primary RGCs, thesecells were not sensitive to glutamate excitotoxicity in theirundifferentiated state. RGC-5 cells pretreated with succinylconcanavalin-A (sCon A) were sensitive to 500 μM glutamate[12]. Lacking glutamate sensitivity causes the difficulties ofusing the RGC-5 cells in experiments involving glutamate.

Ocular ciliary epithelium cells have been shown to beinvolved in the synthesis and secretion of various proteinsfound in aqueous humor [13]. Several proteins, includingneuropeptides and their processing enzymes, synthesizedand secreted by a human nonpigmented ciliary epithelial(HNPE) cell-line, have been evaluated [14], and it issuggested that these secreted proteins can act in an autocrineor paracrine manner to affect ciliary epithelial functions andother target ocular cells, such as the trabecular meshwork[13]. Because of the neuroendocrine properties of theciliary epithelium cells, the ability to confer differentiatedneuroendocrine phenotypes and the physical locations ofthese ciliary epithelium cells and RGCs [15], we hypothesizedthat factors secreted by these HNPE cells may induce theRGC-5 cells to differentiate, and possibly induce glutamateand NMDA sensitivities.

Proteomic analysis, including identification and charac-terization, is a powerful tool for determination of biologicalroles and functions of individual proteins. In the presentreport, we have utilized a system involving HNPE and RGC-5 cells, and this system may result in the morphologicaland functional differentiation of RGC-5 cells. Although theorigin of RGC-5 has been still in question, the expression ofneuronal markers was validated [16]. Proteomic approacheshave been applied to establish a map of expressed proteinsfor the characteristics of HNPE cells.

2. Materials and Methods

2.1. Cell Culture. The human non-pigmented ciliary epithe-lium cells (HNPE) were SV-40 transformed and werea gift from Dr. Miguel Coca-Prados (Yale University).HNPE were maintained at 37◦C and 5% CO2 in Dul-becco’s modified Eagle’s medium (DMEM, Gibco, GrandIsland, NY, USA) supplemented with 10% fetal bovineserum (FBS, Hyclone Laboratories, Logan, UT), 1% peni-cillin/streptomycin (Gibco, Grand Island, NY, USA) and44 mM NaHCO3. After three days, the cells were washed

with phosphate buffered saline (PBS) and the medium wasreplaced by serum-free (SF) DMEM for 12 h.

The HNPE cell conditioned SF-medium was filtered by0.22 μm filter and diluted 25 times with autoclaved Milli-Qgrade water (Millipore Co., Inc.). For each 5 kD cutoffcentrifugal tube (Amicon Ultra-15, Millipore Co., Inc.), a15 mL diluted sample was loaded. Following centrifugationat 5000×g for 20 min, the sample in the filter unit wascollected. The protein concentration of the HNPE cellconditioned SF-medium was measured by the Bio-RadBradford total protein assay kit (Bio-Rad Laboratories, Inc.).

RGC-5 cells, a secondary cell culture, were transformedrat retinal ganglion cells developed and obtained from Dr.Agarwal (University of North Texas Health Science Center).RGC-5 cells were maintained in low glucose DMEM in T-150 culture flasks supplemented with 44 mM NaHCO3, 10%FBS, and 1% penicillin/streptomycin (Gibco). DifferentiatedRGC-5 cells were obtained by using 50% HNPE cell condi-tioned SF-medium and 50% fresh DMEM (containing 10%FBS). HNPE conditioned medium, which consisted of lowglucose DMEM, was incubated with human non-pigmentedciliary epithelial cells (HNPE).

2.2. Immunocytochemistry. RGCs were grown on glass cov-erslips for 1-2 days prior to experimentation. Coverslipswere rinsed with PBS three times and then were fixed in4% paraformaldehyde for 30 min. These cells were washedwith PBS before being permeabilized in 0.1% Triton X-100for 15 min, washed with PBS, and blocked with 5% bovineserum albumin for 60 min. After rinsing with PBS, thecells were incubated with a mixture of Thy-1 (monoclonalantibodies, Chemicon, Temecula, CA, 1 : 200) and Brn-3C (polyclonal antibodies, Convance Inc, Princeton, NJ,1 : 1000) for 1.5 h at room temperature and subsequentlyincubated with a combination of secondary antibodies. AfterPBS rinses, these cells were incubated for 10 min in the darkwith 300 nM DAPI to stain nuclear regions. Cover-slideswere mounted on glass slides in antifade medium (FluorSave;Calbiochem, La Jolla, CA) and allowed to dry for 20 min inthe dark. Cells were visualized and images were taken using aZeiss LSM-410 Confocal Scanning Laser Microscope System.Controls were performed by omitting primary antibodies.

2.3. 1D SDS-PAGE. HNPE cell-secreted proteins were sep-arated under denaturing conditions in a 4–12% polyacry-lamide gel. The HNPE cell conditioned SF-medium wasresuspended in the sample buffer (Invitrogen NuPAGE SDSsample buffer), heated at 80◦C for 10 min and then storedon ice. Each well was loaded with 5 μg of sample solution.The SDS-PAGE gel was run in a Bio-Rad protean II xi cell(Richmond CA, USA) at 200 V for 1 h. After completion ofelectrophoresis, the protein bands in the gel were visualizedby silver staining and image acquired using an image scanner(Amersham Biosciences, Uppsala, Sweden), which is oper-ated by the software LabScan 5.00 (Amersham Biosciences).

2.4. Silver Staining. The gels were fixed in an aqueoussolution having 40% ethanol and 10% acetic acid overnight,

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and then incubated in a buffer solution containing 30%ethanol, 6.8% w/v sodium acetate, and 0.312% w/v sodiumthiosulfate for 30 min. After rinsing three times for 5 mineach, the gels were stained in a 0.25% w/v silver nitratesolution containing 0.02% formaldehyde for 30 min. Thedevelopment was performed for 10 min in a solution con-sisting of 2.5% sodium carbonate and 0.01% formaldehyde.An acetic acid solution (5% v/v) was used to stop thedevelopment, and the stained gels were then rinsed threetimes for 5 min each.

2.5. Protein Identification by Nano-HPLC-ESI-MS/MS. Theprotein bands were excised manually and digested usingsequence grade trypsin (V511A, Promega, USA). The proteinsamples were reduced, alkylated, and then digested withtrypsin using standard protocols [17, 18].

Reverse phase nano-high performance liquid chromatog-raphy electrospray ionization tandem mass spectrometry(RP-nano-HPLC-ESI-MS/MS) was used to identify theselected protein bands separated on the SDS-PAGE. Thepeptides obtained from the tryptic in-gel digestion were ana-lyzed using a nano-HPLC system (LC Packings, Netherlands)coupled to an ion trap mass spectrometer (LCQ Deca XPPlus, ThermoFinnigan, San Jose, CA, USA) equipped with anelectrospray ionization source. A linear acetonitrile gradientfrom 100% buffer A (5% acetonitrile/0.1% formic acid) to60% buffer B (80% acetonitrile/0.1% formic acid) was usedat a flow rate of approximately 200 nL/min for 70 min. Theseparation was performed on a C18 microcapillary column(Zorbax 300SB-C18, 3.5 μm, 75 μm I.D. ×150 mm, Agilent,Germany). Peptides eluted from the microcapillary columnwere electrosprayed into the nano-HPLC-ESI-MS/MS withthe application of a distal 1.3 kV with heated capillary atthe temperature of 200◦C. Each cycle of one full scanmass spectrum (m/z 450–2000) was followed by three data-dependent tandem mass spectra with the collision energy wasset at 35%.

2.6. Database Search. For protein identification, Mascotsoftware (Version 2.2.1, Matrix Science, London, UK) wasused to search the human protein sequence database (Swiss-Prot, Release 52.0 of 22-Feb-08). For proteolytic cleavages,only tryptic cleavage was allowed, and the number ofmaximal internal (missed) cleavage sites was set to 2. Variablemodifications of cysteine with carboxyamidomethylation,methionine with oxidation, and asparagine/glutamine withdeamidation were allowed. The mass tolerances of the pre-cursor peptide ion and fragment ion were set to 1 Da. Positiveprotein identifications were defined if the Mowse scores ofgreater than 50 were considered significant (P < 0.05).Proteins were initially annotated by similar searches usingUniProtKB/Swiss-Prot databases (Last modified September22, 2009) [19–21].

3. Results and Discussion

Cell secretome (cell-conditional medium) studies can makemajor contributions in understand biomarker discovery

and cell pathophysiological mechanisms. It is composed ofproteins that are found in the extracellular growth medium.The cell secretome consists of proteins that are secreted, shedfrom the cell surface and intracellular proteins released intothe supernatant due to cell lysis, apoptosis, and necrosis [22,23]. The secretome which consists of proteins or peptidessecreted from cells into the extracellular medium representsthe major class of molecules involved in the intercellularcommunication in multicellular organisms. It constitutesan important class of proteins that control and regulatea multitude of biological and physiological processes andindicates a clinically relevant source for biomarker andtherapeutic target discoveries [24].

Thus, secreted proteins constitute an important categoryof active molecules that play crucial roles in a number ofphysiological and pathological processes and may reflect abroad variety of pathological conditions and thus representa rich source of biomarkers. Proteomic characterization ofproteins for identification of specific biomarkers provides apowerful tool to gain deep insights into disease mechanismsin which proteins play major roles. In this study, we haveused gel electrophoresis associated with mass spectrometryfor identification of the proteome and secretome of HNPEcell conditioned SF-medium samples.

3.1. RGC-5 Cell Differentiation. The differentiation systemconsisted of RGC-5 cells on coverslips inside 6-well plates,which were exposed to the conditioned medium from HNPEcells. RGC-5 cells proliferated rapidly with a doubling timeof less than a day. Decreasing the percentage of serum in themedium may slow down proliferation. The control RGC-5cells were heterogeneous in shape. Morphological changesof RGC-5 cells were induced by HNPE cell conditionedSF-medium (Figure 1) and caused the shrinkage of thecell body with elongated neurite outgrowth (Figure 1(b)),which allows comparison with undifferentiated RGC-5 cells(Figure 1(a)). The overall morphology of RGC-5 cells afterthe treatment was similar to those seen in primary cultures ofrat retinal ganglion cells [25]. Moreover, the morphology ofRGC-5 cells differentiated by our method was similar to theones induced by a broad-spectrum protein kinase inhibitorstaurosporine [26]. Nevertheless, Frassetto and coworkersdid not conclude this to be the possible differentiationmechanism. This secretome map is a preliminary study tounveil the mechanism since the differentiation is probablythe consequence of the action of several proteins and/orenzymes. It was also noted that the differentiation treatmentled to decreased culture density compared with the controlcells. This finding is consistent with the study from Woodet al. [27]. For subsequent studies, the conditioned mediumfrom confluent flasks containing HNPE cells was used andfound to be equally effective in promoting differentiation ofRGC-5 cells.

Thy-1 expression in undifferentiated RGC-5 cells wasused as a marker to identify retinal ganglion cells [28].After treatment with HNPE cell conditioned SF-medium,RGC-5 cells have an enhanced Thy-1 expression, comparedto the undifferentiated cells (Figure 2). In the retina, the

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(a) (b)

Figure 1: Morphological changes in RGC-5 cells after treatment with HNPE conditioned SF-medium (40x) (a) before, and (b) after.The RGC-5 cells treated with HNPE conditioned SF-medium induced morphological changes, including longer axons and more neuriteoutgrowth (Figure 1(b)), compared to RGC-5 cells without treatment (Figure 1(a)).

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Figure 2: Immunocytochemical analysis of Thy-1 and Brn-3b expression in RGC-5 cells differentiated by treatment with HNPE cellconditioned SF-medium. Staining with antibodies to the cell surface glycoprotein, Thy-1, have been commonly used as a marker to identifyretinal ganglion cells. After cultivation with HNPE conditioned medium, RGC-5 cells have an enhanced Thy-1 expression, compared tothe undifferentiated cells. RGC-5 cells without cultivation with HNPE conditioned medium express Brn-3b in a different pattern comparedwith treated RGC-5 cells. Specifically, Brn-3b has a nuclear localization in RGC-5 cells without cultivation with HNPE conditioned medium;however, upon treatment, RGC-5 cells express Brn-3b in a more punctate cytosolic manner.

class IV POU domain transcription factor, Brn-3b, wasexpressed almost exclusively in subpopulations of ganglioncells and used to identify RGCs [29]. Brn-3b was regardedas a marker for differentiation of RGCs, since Brn-3 factorswere not necessary for the initial specification of sensoryneurons, but were essential for their normal differentiationand survival [30]. Specifically, Brn-3b was localized in thenuclear in RGC-5 cells; however, upon treatment with HNPE

cell conditioned SF-medium, RGC-5 cells express Brn-3b ina more punctate cytosolic manner (Figure 2).

3.2. Proteome Analysis. The SDS-PAGE followed by sil-ver staining resolved the protein bands from HNPE cellconditioned SF-medium. Figure 3 shows the silver-stained1D SDS-PAGE of secreted proteins from HNPE cells. Fivemicrograms of secreted protein was loaded on a gel for

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Figure 3: 1D SDS-PAGE image of HNPE conditioned SF-medium(5 μg/well, silver stained, left-hand side: molecular weight marker,kDa). The gel bands on the middle lane with serial numbers wereanalyzed by nano-HPLC-ESI-MS/MS. In the 30 bands, 132 proteinswere identified. The gel bands on the right-hand side were the celllysised proteins.

visualization, and more than 30 protein bands were detectedin the HNPE conditioned SF-medium using the imageanalysis software. To identify the proteins, the position ofthe 1D SDS-PAGE lane was excised from the gel, washedto remove the stain, and subjected to tryptic digestion.The resulting peptides were characterized by nano-HPLC-MS/MS for protein identification. When a protein was iden-tified by three or more unique peptides possessing MASCOTscores, no visual assessment of spectra was conducted andthe protein was considered present in the sample.

In this study, all MS/MS spectra were manually con-firmed (even if the above criteria were passed) by thevisual assessment for their overall quality. In addition, the

criteria for manual validation reported by Jaffe et al., whichrequires a readily observable series of at least four y-ions,was used [31]. Thus, the criteria should be enough for thevalidation of the identified proteins. By using this strategy,132 unique proteins with at least three unique peptidesequences matched were identified, and a summary of theprotein identifications achieved is listed in Table 1.

In this study, 47 proteins (35.6%) were known to bepresent in cytoplasm. Twenty-two proteins (16.7%) wereknown to be secreted into the extracellular space. Twenty-fiveproteins (18.9%) were known to be nuclear proteins. Elevenproteins (8.3%) were known to be membrane proteins.Ten proteins (7.6%) were known to be cytosol proteins.A few mitochondrial, endoplasmic reticulum, intracellular,cytoskeleton, and golgi apparatus proteins were also identi-fied. A considerable portion of the identified proteins (6%,8 proteins) has not been reported for their synthesizedlocations. Some proteins were described as found in differentsubcellular locations, which explains the total sum beingsubstantially larger than 100%.

Some identified proteins in the distribution of cellularlocation were not secreted proteins, but they were stillpresent in the secreted medium. To clarify the puzzle, a cellviability test was applied. The survival rate of HNPE cellswas determined by the dimethylthiazol-diphenyltetrazoliumbromide (MTT) assay, which was about 97%. Thus, thoseidentified proteins were not corresponding to releasedproteins from dead cells. Also, according the protein profilesin Figure 3, the protein patterns obtained from secretedmedium and cell lysate were very different. As a result, theseproteins identified in this study can be considered as secretedproteins, which may have been synthesized inside the cellsand transferred out.

Based on the functional categories in the Swiss-Protand TrEMBL protein database, the identified proteins wereclassified into several groups. The Swiss-Prot identifierscould be employed for linkages of proteins to defined vocab-ulary of terms describing the cellular components, biologicalprocesses, and molecular functions of known gene ontology(GO). Gene Ontology Consortium provides annotationsof each protein and its structure, which allowed us toorganize selected proteins into biologically relevant groups.These groupings can be utilized as the basis for identifyingbiological information showing correlated protein changes[20, 32]. Such protein functions were listed in Table 2.

In this study, some of the proteins secreted by HNPEcells, which were confirmed by the Western blotting method,may be candidate factors responsible for promoting differ-entiation of RGC-5 cells including thrombospondin-1, 2,3 precursor (1-2, 1-3, 1-13), galectin-3-binding protein (1-5∼1-7), neurogenic locus notch homolog protein 3 (Notch-3, 1-11), follistatin-related protein 1 precursor (1-11), sPARCprecursor (1-14), peroxiredoxin-1 (1-21, 1-22), cofilin 1 (1-24, 1-27), profilin 1 (1-27, 1-28), galectin-1 (1-28), andmyotrophin (1-30). Cell differentiation is directed by avariety of intra- and extracellular events including signalsgenerated by extracellular matrix (ECM) components, whichmediate adhesive cell-to-cell interactions and trigger a cas-cade of post-receptor intracellular signaling pathways. The

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cell

adh

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Page 7

Journal of Biomedicine and Biotechnology 7

Ta

ble

1:C

onti

nu

ed.

Seri

alN

o.Sw

issP

rot

No.

Pro

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Page 8

8 Journal of Biomedicine and Biotechnology

Ta

ble

1:C

onti

nu

ed.

Seri

alN

o.Sw

issP

rot

No.

Pro

tein

nam

eM

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Page 9

Journal of Biomedicine and Biotechnology 9T

abl

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Page 10

10 Journal of Biomedicine and Biotechnology

Ta

ble

1:C

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nu

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Seri

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o.Sw

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Pro

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Page 11

Journal of Biomedicine and Biotechnology 11

Ta

ble

1:C

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nu

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Seri

alN

o.Sw

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Page 12

12 Journal of Biomedicine and Biotechnology

Ta

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1:C

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Seri

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Pro

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Page 13

Journal of Biomedicine and Biotechnology 13

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Page 14

14 Journal of Biomedicine and Biotechnology

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Page 16

16 Journal of Biomedicine and Biotechnology

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