extfieldsofstudy: Biology Medicine

text:
Activation of the PI3K/mTOR Pathway Is Involved in Cystic Proliferation of Cholangiocytes of the PCK Rat

The polycystic kidney (PCK) rat is an animal model of Caroli’s disease as well as autosomal recessive polycystic kidney disease (ARPKD). The signaling pathways involving the mammalian target of rapamycin (mTOR) are aberrantly activated in ARPKD. This study investigated the effects of inhibitors for the cell signaling pathways including mTOR on cholangiocyte proliferation of the PCK rat. Cultured PCK cholangiocytes were treated with rapamycin and everolimus [inhibitors of mTOR complex 1 (mTOC1)], LY294002 [an inhibitor of phosphatidylinositol 3-kinase (PI3K)] and NVP-BEZ235 (an inhibitor of PI3K and mTORC1/2), and the cell proliferative activity was determined in relation to autophagy and apoptosis. The expression of phosphorylated (p)-mTOR, p-Akt, and PI3K was increased in PCK cholangiocytes compared to normal cholangiocytes. All inhibitors significantly inhibited the cell proliferative activity of PCK cholangiocytes, where NVP-BEZ235 had the most prominent effect. NVP-BEZ235, but not rapamycin and everolimus, further inhibited biliary cyst formation in the three-dimensional cell culture system. Rapamycin and everolimus induced apoptosis in PCK cholangiocytes, whereas NVP-BEZ235 inhibited cholangiocyte apoptosis. Notably, the autophagic response was significantly induced following the treatment with NVP-BEZ235, but not rapamycin and everolimus. Inhibition of autophagy using siRNA against protein-light chain3 and 3-methyladenine significantly increased the cell proliferative activity of PCK cholangiocytes treated with NVP-BEZ235. In vivo, treatment of the PCK rat with NVP-BEZ235 attenuated cystic dilatation of the intrahepatic bile ducts, whereas renal cyst development was unaffected. These results suggest that the aberrant activation of the PI3K/mTOR pathway is involved in cystic proliferation of cholangiocytes of the PCK rat, and inhibition of the pathway can reduce cholangiocyte proliferation via the mechanism involving apoptosis and/or autophagy.

Introduction
Caroli's disease is characterized by the progressive, multiple cystic dilatation of intrahepatic bile ducts, and is frequently associated with portal fibrosis corresponding to congenital hepatic fibrosis [1]. It belongs to a group of congenital hepatorenal fibrocystic syndrome, and is a hepatic manifestation of autosomal recessive polycystic kidney disease (ARPKD) [2]. The polycystic kidney (PCK) rat is an established animal model of Caroli's disease with congenital hepatic fibrosis as well as ARPKD [3]. Using the PCK rat, potential therapeutic strategies for Caroli's disease and ARPKD have been analyzed. However, no effective therapy applicable to human disease has been established [4].
The mammalian target of rapamycin (mTOR) has attracted attention because of its involvement in a variety of diseases including cancer [5]. The mTOR functions as two distinct multiprotein complexes, mTOR complex 1 (mTORC1) and complex 2 (mTORC1). The mTORC1 phosphorylates S6 kinase (S6) and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), and regulates cell growth, proliferation, and survival. The mTORC2 has been suggested that it lies downstream of phosphatidylinositol 3-kinase (PI3K) signaling and phosphorylates Akt on Ser473, although it is less well studied in contrast to mTORC1.
Rapamycin and its analog, everolimus, are inhibitors of mTORC1, and have antitumor effects in various cancers by inhibiting cell proliferation, and by affecting apoptosis and autophagy. The rapamycin-sensitive mTORC1 contains phosphorylated (p)-Ser2448, which is consistent with Ser2448 phosphorylation being sensitive to acute rapamycin treatment. The rapamycin-insensitive mTORC2 complex contains p-Ser2481, which is consistent with Ser2481 being a rapamycin-insensitive autophosphorylation site [6].
Rapamycin have been shown to be effective in rodent models of autosomal dominant PKD (ADPKD), which is consistent with the findings showing that the PI3K/Akt/mTOR pathway is aberrantly activated in ADPKD [7][8][9][10][11]. However, in clinical trials, rapamycin and everolimus have not shown benefit in patients with ADPKD [12,13]. Similarly, rapamycin failed to attenuate progression of kidney and liver lesions in the PCK rat in vivo, although the activation of Akt/mTOR pathway has been observed in patients with ARPKD [14,15]. Thus, the clinical application of mTORC1 inhibitors to PKD patients seems to be limited of use.
Recently, multi-targeting approach against the PI3K/mTOR pathway has provided novel tools for elucidation of the roles of mTOR and mTOR-based therapeutic strategy. NVP-BEZ235 belongs to the class of imidazoquinolines, and is a novel inhibitor of PI3K and mTORC1/2 [5]. In a certain type of human neoplastic cells, NVP-BEZ235 suppresses cell proliferation by inducing apoptosis or autophagy [16,17], but its therapeutic benefits for the pathogenesis of PKD have not been tested. Overexpression of p-mTOR, p-Akt, p-S6, and PI3K in PCK cholangiocytes. The immunohistochemical expression of p-mTOR (Ser2448) and p-Akt (Ser473) was examined using liver sections of 10-month-old rats. Increased expression of p-mTOR (Ser2448) and p-Akt (Ser473) was observed in bile duct epithelium of the PCK liver compared to that of the normal liver (A). Western blot analysis using protein extracts from cultured cholangiocytes showed that the expression p-mTOR (Ser2448), p-mTOR (Ser2481), p-Akt (Ser473), p-S6, PI3K p110a, and PI3K p85 was increased in PCK cholangiocytes compared to normal cholangiocytes (B Disturbance in the balance between apoptosis and cell proliferation is a pathologic feature of PKD. Increased apoptosis has been observed in human ADPKD as well as the rodent models of PKD [3,[18][19][20][21]. Activation of caspase signaling and dysregulation of antiapoptotic bcl-2 protein have been described in PKD. Recently, the role of autophagy has been investigated in mouse models of PKD [22]. However, the pathologic significance of autophagy in the PCK rat is largely unknown.
In this study, the effects of the PI3K and/or mTORC1/2 inhibitors were investigated using the PCK rat to clarify the mechanism of cystic dilatation of the intrahepatic bile ducts and their therapeutic usefulness for the inhibition of bile duct dilatation, particularly focusing on the cell proliferative activity in relation to apoptotic and autophagic responses.

Animals
The PCK rats were maintained at the laboratory animal institute of Kanazawa University Graduate School of Medicine. Normal (Crj:CD) rats were purchased from Charles River Japan (Sagamihara, Japan). The protocol was approved by the committee of Institute for Experimental Animals, Kanazawa University Advanced Science Research Center (Permit Number: AP-122551). Animal studies were performed in accordance with the guidelines for the Care and Use of laboratory Animals at Takara-machi Campus of Kanazawa University.

Liver Specimens
For immunohistochemical analysis, livers were removed from normal and PCK rats. The liver tissues were immersed in 10% formalin neutral buffer solution (pH 7.4), and embedded in paraffin. More than 10 serial sections, 4 mm thick, were cut from each paraffin block, and were subjected to the analysis.

Cell Culture of Cholangiocytes
Cholangiocytes were isolated, purified, and cultured from the intrahepatic large bile ducts of normal and PCK and rats as described previously [23,24]. The cells were set on cell culture dishes covered with a standard medium composed of Dulbecco's modified Eagle's medium/F-12 (Gibco, Grand Island, NY) containing 10% bovine growth serum (HyClone, Logan, UT), 5 mmol/L forskolin (Wako Pure Chemical Industries, Osaka, Japan), 20 ng/ml of epidermal growth factor (Upstate Biotechnology, Lake Placid, NY) and 1% antibiotics-antimycotic (Life Technologies) at 37 o C in an atmosphere of 5% CO 2 .

Cell Proliferation Assay
Proliferative activity of cholangiocytes was determined using the WST-1 assay according to the manufacturer's instructions (Roche, Mannheim, Germany). The cells were seeded in 96-well tissue culture plates and maintained for 24 hours with the standard medium. Then the cells were treated with rapamycin, everolimus, NVP-BEZ235 and LY294002 at the concentrations and at the intervals indicated. The WST-1 reagent was added and incubated for 1 hour before reading the plate. Each assay was conducted in six sets.

Three-dimensional Cell Culture
Biliary cyst formation were examined using the three-dimensional cell culture system with the use of the growth factor reduced Matrigel (BD Biosciences, San Jose, CA). On day 6 after the beginning of the cell culture, the culture medium was changed to that containing rapamycin (100 ng/ml), everolimus (100 nM) and NVP-BEZ235 (100 nM). Incubation was continued for further 72 hours. The morphological changes were recorded using a digital camera under phase-contrast microscopy. More than 6 fields at x40 magnification were recorded for each experimental group, and the size of well-developed cysts was analyzed within the field. The size of the same cyst was determined at day 0 (before the treatment) and day 3 (72 hours after the treatment) on the digital images, and the growth rate was calculated.

Quantitative Real-time PCR
Reverse transcriptase-PCR was performed using total RNA (1 mg) extracted from the cholangiocytes. Total RNA was extracted using an RNA extraction kit (RNeasy mini; Qiagen, Tokyo, Japan) and was used to synthesize cDNA with reverse transcriptase (ReverTra Ace; Toyobo, Osaka, Japan). Quantitative real-time PCR was performed according to a standard protocol using the SYBR Green PCR Master Mix (Toyobo Co.) and Mx3000P&Mx3005P Real-Time QPCR System (Applied Biosystems, CA). The sequences of the primers (59-39) used were: bcl-2; forward, CTGGCATCTTCTCCTTCCAG; reverse, CGGTAGCGACGAGAGAAGTC: glyceraldehyde-3-phosphate dehydrogenase (GAPDH); forward, GAGTCAACG-GATTTGGTCGT; reverse, TTGATTTTGGAGGGATCTC. Cycling conditions were incubated at 62uC for 2 minutes, 95uC for 10 minutes, 40 cycles of 95uC for 15 seconds and 60uC for 1 minute. Fold difference compared with GAPDH was calculated. Each assay was conducted in three sets. In vitro effects of PI3K and mTOR inhibitors on cholangiocytes. Normal and PCK cholangiocytes were treated with the PI3K and/or mTOR inhibitors, and the cell proliferative activity was determined using the WST-1 assay as described in the Materials and Methods. Rapamycin, everolimus, NVP-BEZ235 and LY294002 significantly inhibited the cell proliferative activity of normal (A) and PCK (B) cholangiocytes in a dosedependent fashion, where the most prominent inhibitory effect was observed in both cholangiocytes treated with NVP-BEZ235 for 120 hours. Rictor siRNA significantly reduced the cell proliferative activity of the PCK cholangiocytes when it was used in combination with rapamycin and LY 294002 (C). Western blot analysis showed that the expression of p-Akt (Se473) and p-S6 was reduced following 24 hour-treatment with rapamycin, everolimus and NVP-BEZ235 in a dose-dependent fashion in PCK cholangiocytes (D). The results of the semiquantitative analysis of Western blotting are shown in E. Note that the expression of p-Akt (Ser473) and p-4E-BP1 was markedly decreased in PCK cholangiocytes following the treatment with 500 nM NVP-BEZ235 (D and E). *, p,0.01; **, p,0.05 (vs. untreated control). doi:10.1371/journal.pone.0087660.g002 PI3K/mTOR Pathway in PCK Cholangiocyte PLOS ONE | www.plosone.org

Apoptosis Assay
Flow cytometric analysis of apoptosis was performed by the quantitative detection of phosphatidylserine of the cell surface using Annexin V/FITC and PI apoptosis detection kit (BD Biosciences, San Jose, CA). Cultured cholangiocytes were treated with rapamycin (100 ng/ml), everolimus (100 nM), and NVP-BEZ235 (500 nM) for 24 hours. The cells were washed twice with cold phosphate-buffered saline, and suspended in binding buffer. The cell suspension (100 ml) was incubated with Annexin V-FITC (5 ml) and propidium iodide (PI) (2 ml) for 15 minutes at room temperature. Then binding buffer (400 ml) was added in the cell suspension, and the samples were analyzed within 1 hour using flow cytometer (JASAN, Bay bioscience, Kobe, Japan). Apoptotic cells were expressed by the percentage of Annexin V-FITC positive-, PI negative-cells of total gated cells. The experiments were performed in three sets for each condition. Apoptosis positive control cells were treated with 200 mM H 2 O 2 for 24 hours.
For the detection of apoptotic cells in paraffin-embedded tissue sections, a terminal dUTP nick-end labeling (TUNEL) method was used. After proteinase K digestion and endogenous peroxidase blocking, the sections were stained by using a commercial kit (TdT-FragEL DNA fragmentation detection kit, Calbiochem). After color development with DAB, sections were counterstained with methyl green.

Transfection of siRNA
Synthetic siRNA for LC3, caspase 3 and Rictor, and nonsilencing siRNA (negative control) were purchased from Qiagen (Tokyo, Japan). Transfections of siRNA were performed using HiPerFect Transfection Reagent (Qiagen) according to the manufacturer's instructions. Cholangiocytes were incubated for 24 hour with the standard medium. After removal of the medium, the cells were incubated with Dulbecco's modified Eagle's medium/F-12 (Gibco) containing premixed siRNA (10 nM) and 3 ml of HiPerFect Transfection Reagent, and were further incubated for 72 hours.

Immunofluorescence Confocal Microscopy
Cholangiocytes were seeded in slide chamber, and were treated with rapamycin (100 ng/ml), everolimus (100 nM), and NVP-BEZ235 (500 nM) for 24 hours. They were fixed with 4% paraformaldehyde for 15 minutes, and were permeabilized for 10 minutes with 0.1% Triton X-100. After blocking, the cells were incubated with a primary antibody against LC3 (1:200; Cell Signaling Technology) for overnight at 4uC. Alexa-488 (10 mg/ml; Molecular Probes, Eugene, OR) was used as a secondary antibody, and nuclei were stained with 496-diamidino-2-phenylindole. Positive control of autophagy was the cells incubated with serum-free medium for 24 hours.

In vivo Administration of NVP-BEZ235
A total of 22 male rats were used. At 4 weeks of age, 11 normal and 11 PCK rats were divided into control and experimental groups. The experimental groups were intraperitoneally administrated 20 mg/kg NVP-BEZ235 daily between 4 and 8 weeks of age. The dosage was determined based on the previous report [25]. The control group received vehicle (2% Tween 80 and 1% methylcellulose in water) alone. At 8 weeks of age, rats were weighed and anesthetized with diethyl ether. Blood was obtained by puncture of inferior vena cava for hematological analysis. The liver and kidney were weighed and immersed in 10% formalin neutral buffer solution (pH 7.4), and the tissues were embedded in paraffin for histological analysis.

Histological Assessment
Paraffin-embedded sections were prepared for the liver and kidney, and whole tissue sections were used to measure cyst volumes and liver fibrosis. Cyst volumes of the liver and kidney were assessed using hematoxylin-eosin stained sections. Liver Figure 4. Effects of mTOR inhibitors on cholangiocyte autophagy in vitro. PCK cholangiocytes were treated with rapamycin, everolimus and NVP-BEZ235 for 24 hours, and autophagy of the cells was detected by the conversion of LC3 I to LC3 II using Western blot analysis. NVP-BEZ235 induced a dose-dependent increase in the autophagy-specific LC3 II form, while rapamycin and everolimus did not significantly induce autophagy in PCK cholangiocytes (A). The results of the semiquantitative analysis of Western blotting are shown in B. Immunocytochemistry of LC3 showed the induction of autophagy by NVP-BEZ235 in PCK cholangiocytes (C). Silencing of LC3 was performed using LC3 siRNA in PCK cholangiocytes, and immunocytochemistry (D) and Western blot analysis (E) showed that LC3 siRNA reduced LC3 II formation in the cholangiocytes. When PCK cholangiocytes were treated with NVP-BEZ235 (100 nM) for 48 hours, the cell proliferative activity of the LC3 siRNA-treated cells was significantly high than those without LC3 siRNA treatment that was determined using the WST-1 assay (F). Following 48-hour treatment of PCK cholangiocytes with 3methyladenine (500 nM) together with NVP-BEZ235 (100 nM), the cell proliferative activity was significantly increased compared to those with NVP-BEZ235 alone (G). Original magnifications, x1000 (C and D). *, p,0.01; **, p,0.05 (vs. untreated control). doi:10.1371/journal.pone.0087660.g004 PI3K/mTOR Pathway in PCK Cholangiocyte PLOS ONE | www.plosone.org fibrosis was assessed using picrosirius red staining. Stained sections were visualized under a light microscope, and the digital images were acquired and reproduced on a computer. Image analysis was performed in software using Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA). A color threshold was applied at a level that separated cysts from non-cystic tissue or the picrosirius red stained material from the background to calculate the volume of the cysts or fibrosis. The areas of interest were expressed as a percentage of the total tissue. Evaluation of apoptosis was performed using liver sections stained using the TUNEL method. More than 500 biliary epithelial cells were surveyed in the sections, and the percentage of biliary epithelial cells positive for TUNEL was expressed as the TUNEL-labeling index.
To evaluate the cell proliferative activity, Ki-67 protein-positive signals were similarly counted for biliary epithelial cells of the Ki-67-immunostained liver sections, and the percentage of biliary epithelial cells positive for Ki-67 protein were expressed as the Ki-67-labeling index.

Statistics
The mean 6 SD was calculated for all parameters. Statistical differences were determined using the T-test and analysis of variance. A p value ,0.05 was accepted as the level of statistical significance.

Overexpression of p-mTOR, p-Akt, p-S6 and PI3K in PCK Cholangiocytes
The immunohistochemical expression of p-mTOR (Ser2448) and p-Akt (Ser473) was examined using liver sections of 10-monthold rats. In the normal rat, the expression of p-mTOR (Ser2448) in bile duct epithelium was faint or negligible, and that of p-Akt (Ser473) was weak ( Figure 1A, arrows). In the PCK liver, the expression of both p-mTOR (Ser2448) and p-Akt (Ser473) in bile duct epithelium was markedly increased ( Figure 1A).

In vitro Effects of PI3K and mTOR Inhibitors on Cholangiocytes
Normal and PCK cholangiocytes were treated with rapamycin (mTORC1 inhibitor), everolimus (mTORC1 inhibitor), NVP-BEZ235 (PI3K and mTORC1/2 inhibitor) and LY294002 (PI3K inhibitor), and the cell proliferative activity was determined using the WST-1 assay. Each inhibitor significantly inhibited the cell proliferative activity of normal ( Figure 2A) and PCK ( Figure 2B) cholangiocytes in a dose-dependent fashion. Above all, NVP-BEZ235 had most prominent inhibitory effects on the cell proliferative activity of both cholangiocytes, and at the concentration of 500 nM, it reduced the cell proliferative activity of both cholangiocytes to less than 30% compared to that of untreated groups at 120 hours (Figure 2A and 2B). Higher concentrations of rapamycin (10 mg/ml) and everolimus (10 mM) showed almost same inhibitory effects on the cell proliferative activity as those seen in 100 ng/ml rapamycin and 100 nM everolimus, respectively (data not shown).
To address the role of mTORC2 in cholangiocyte proliferation, the experiment of depletion of Rictor using siRNA was performed. Western blot analysis confirmed the gene silencing for Rictor was efficiently conducted ( Figure 2C, upper panel). Although the Rictor siRNA alone did not affect the cell proliferative activity of PCK cholangiocytes, the cell proliferative activity was further reduced when the siRNA was used in combination with rapamycin and LY294002 ( Figure 2C). Thus, mTORC2 was partially involved in the increased cell proliferation of PCK cholangiocytes.
Western blot analysis showed that the expression of p-Akt (Se473) and p-S6 was reduced following the treatment with rapamycin, everolimus and NVP-BEZ235 in a dose-dependent fashion in PCK cholangiocytes, and NVP-BEZ235 further reduced the expression of p-4E-BP1 ( Figure 2C and 2D). Higher concentration (500 nM) of NVP-BEZ235 almost completely diminished the expression of p-Akt (Ser473).

Effects of mTOR Inhibitors on Cholangiocyte Apoptosis in vitro
Apoptosis was determined following the treatment of PCK cholangiocytes with rapamycin, everolimus and NVP-BEZ235. Flow cytometric analysis showed that rapamycin and everolimus significantly induced apoptosis in PCK cholangiocytes ( Figure 3A). By contrast, apoptosis was significantly inhibited by the treatment with NVP-BEZ235 ( Figure 3A).
In relation to the inhibition of apoptosis, NVP-BEZ235 significantly reduced the expression of cleaved caspase 3 in PCK cholangiocytes, while it tended to be unchanged or rather increased following the treatment with everolimus (Figure 3B  Figure 3D). In consistent with the inhibition of apoptosis by NVP-BEZ235, the analysis with quantitative real-time PCR showed that treatment with NVP-BEZ235 significantly increased the expression of bcl-2 mRNA in PCK cholangiocytes ( Figure 3E).

Effects of mTOR Inhibitors on Cholangiocyte Autophagy in vitro
The occurrence of autophagy in cholangiocytes was detected by the conversion of LC3 I to LC3 II. Western blot analysis showed that NVP-BEZ235 induced a dose-dependent increase in the autophagy-specific LC3 II form, while rapamycin and everolimus did not significantly induce autophagy in PCK cholangiocytes ( Figure 4A and 4B). Immunocytochemistry of LC3 confirmed the induction of autophagy by NVP-BEZ235 ( Figure 4C).
To further determine the involvement of autophagy in the inhibition of the cell proliferative activity by NVP-BEZ235, silencing of LC3 was performed using LC3 siRNA in PCK cholangiocytes, and the cell proliferative activity was compared for the cells with and without NVP-BEZ235 treatment. The gene silencing was monitored using immunocytochemistry and Western blotting for PCK cholangiocytes maintained with serum-free medium, and the analysis showed that LC3 siRNA reduced LC3 II formation in the cholangiocytes, although their expression was not completely diminished ( Figure 4D and 4E). As expected, when PCK cholangiocytes were treated with NVP-BEZ235, the cell proliferative activity of the LC3 siRNA-treated cells was significantly high than those without LC3 siRNA treatment ( Figure 4F). When PCK cholangiocytes were treated with 3MA, a specific inhibitor of autophagy, together with NVP-BEZ235, the cell proliferative activity was significantly increased compared to those with NVP-BEZ235 alone ( Figure 4G), confirming that autophagy was involved in the growth inhibition by NVP-BEZ235 in PCK cholangiocytes.

Inhibition of Biliary Cyst Formation by mTOR Inhibitors in vitro
The effects of mTOR inhibitors on biliary cyst formation were examined using three-dimensional cell culture system. PCK cholangiocytes grew more rapidly in a spheroidal form in the Matrigel compared to normal cholangiocytes under normal condition ( Figure 5A). NVP-BEZ235 significantly inhibited cystic growth of both normal and PCK cholangiocytes ( Figure 5A and 5B). Knockdown of LC3 with siRNA of PCK cholangiocytes that were treated with NVP-BEZ235 significantly increased the biliary cyst formation (Figure 5C), showing further evidence of the involvement of autophagy in these processes. By contrast, rapamycin and everolimus did not have significant inhibitory effects on biliary cyst formation of both normal and PCK cholangiocytes ( Figure 5A and 5B).

In vivo Administration of NVP-BEZ235
Finally, the effects of in vivo administration of NVP-BEZ235 on the progression of liver and renal diseases in the PCK rat were examined. Hematological analysis showed that liver and renal functions were preserved following the treatment, but retardation in body weight increase was noted in both normal and PCK rats by the treatment ( Table 1). As expected, the treatment reduced the extent of dilatation of intrahepatic bile ducts of the PCK rat, whereas no apparent histological changes were observed in the liver of normal rats ( Figure 6A). On the contrary, no beneficial effects were observed on renal cyst development because of the treatment as evaluated using routine histological sections ( Figure 6A). Histomorphometric analysis using whole liver and kidney tissues confirmed that the treatment significantly reduced liver cyst index, but not kidney cyst index ( Figure 6B).
In the liver of the PCK rat, apoptosis of biliary epithelial cells was significantly inhibited by the treatment (Figure 6C), while the cell proliferative activity of the cells was significantly reduced ( Figure 6D), which were in consistent with the results of in vitro experiments. In addition, the treatment significantly improved the extent of liver fibrosis of the PCK rat ( Figure 6E).
Immunohistochemical analysis confirmed that the expression of p-Akt (Ser473), p-mTOR (Ser2448) and p-S6 was reduced in bile duct epithelium of the PCK rat treated with NVP-BEZ235 ( Figure 7A). The treatment increased immunohistochemical expression of LC3 in bile duct epithelium of the PCK rat ( Figure 7B), suggesting the occurrence of autophagy.
In the kidney, the expression of p-Akt (Ser473), p-mTOR (Ser2448) and p-S6 was increased in collecting tubule-derived cyst epithelium of the PCK rat without NVP-BEZ235 treatment compared to those in the renal tubules of normal rat ( Figure 7C). Although the treatment had no significant effect on kidney cyst index, the immunohistochemical expression of these molecules appeared to be reduced in the kidney of the PCK rat following the treatment ( Figure 7C).

Discussion
This study demonstrated that the PI3K/Akt/mTOR pathway was activated in PCK cholangiocytes. The treatment with NVP-BEZ235 effectively inhibited cholangiocyte cystic proliferation by enhancing autophagy and inhibiting apoptosis in vitro, and the inhibitory effects were also observed in the PCK rat treated with NVP-BEZ235 in vivo. Rapamycin and everolimus induced apoptosis of the cells, but they could not inhibit cystic growth of the cells in the three-dimensional cell culture system.
A recent study showed that in vivo administration of rapamycin failed to inhibit cystic dilatation of the bile ducts of the PCK rat, and the authors considered that this might be due to intrinsic or acquitted rapamycin resistance [14]. In clinical trials where rapamycin and everolimus did not show beneficial effects in patients with ADPKD, low tissue concentrations of the drugs at clinically tolerable doses have been considered as one of the causes [12,13]. Our data suggest that the inhibition of mTORC1 alone may be insufficient for the inhibition of cell proliferation of PCK cholangiocytes, because other complementary signaling pathways Figure 6. Effects of in vivo administration of NVP-BEZ235 on liver and renal diseases of the PCK rat. Normal and PCK rats were treated with NVP-BEZ235 or vehicle alone daily between 4 and 8 weeks of age. Liver and kidney sections stained with hematoxylin-eosin showed that NVP-BEZ235 improved dilatation of intrahepatic bile ducts of the PCK rat, but no beneficial effects were observed on kidney lesions (A). For the PCK rat, liver and kidney cyst index (B), TUNEL-labeling index of the biliary epithelial cells (C), Ki-67-labeling index of the biliary epithelial cells (D), and liver fibrosis score (E) were determined as described in the Materials and Methods. Treatment with NVP-BEZ235 significantly reduced liver cyst index, TUNEL-labeling index, Ki-67-labeling index and liver fibrosis score of the PCK rat, whereas kidney cyst index was unaffected. Original magnifications: x200 (A, upper panel; E); x400 (D). Bars, 2 mm (A, lower panel). *, p,0.01. doi:10.1371/journal.pone.0087660.g006 PI3K/mTOR Pathway in PCK Cholangiocyte Figure 7. Immunohistochemical analysis of the the liver and kidney of the PCK rat treated with NVP-BEZ235. Treatment with NVP-BEZ235 reduced the expression of p-Akt (Ser473), p-mTOR (Ser2448) and p-S6 in bile duct epithelium of the PCK rat (A). The treatment increased immunohistochemical expression of LC3 in bile duct epithelium of the PCK rat (B). In the kidney, the expression of p-Akt (Ser473), p-mTOR (Ser2448) and p-S6 was increased in collecting tubule-derived cyst epithelium of the PCK rat without NVP-BEZ235 treatment compared to those in the renal tubules of normal rat, and their expression appeared to be reduced in the kidney of the PCK rat following the treatment (C). Arrows indicate interlobular bile ducts of the normal liver. Original magnifications; x400 (A-C). doi:10.1371/journal.pone.0087660.g007 were activated in PCK cholangiocytes. More prominent inhibitory effects of NVP-BEZ235 rather than rapamycin and everolimus on the cell proliferation of PCK cholangiocytes support that the inhibition of other signaling pathways such as PI3K may be important for inhibiting the cell proliferative activity.
Drug-induced growth inhibition is often associated with increased apoptosis in PKD cells, and apoptosis has been implicated in strategies for therapeutic intervention [21]. However, the studies of the effects of rapamycin on apoptosis in PKD are conflicting [26], and the present study showed that rapamycin and everolimus induced apoptosis in PCK cholangiocytes. By contrast, NVP-BEZ235 inhibited apoptosis by reducing the expression of cleaved caspase 3 and by inducing bcl-2 expression. In several cancer cell lines, it has been shown that NVP-BEZ235 induced apoptosis and cell cycle arrest accompanied by increased caspase 3 activity, and bcl-2 abrogated the effects of NVP-BEZ235 [27][28][29]. Regarding the effects of NVP-BEZ235 on apoptosis in PKD cells, limited data are available, and further detailed studies are required to clarify the mechanism of the inhibition of apoptosis in PCK cholangiocytes by NVP-BEZ235.
In the kidney, a recent study showed an increase in LC3 II and beclin 1 expression in the late stages of PKD in rodent models of ADPKD [22]. In consistent with the facts that autophagy has been described as hypoxia-inducible factor-1a-dependent adaptive response, HIF-1a was also highly expressed in the late stages of PKD in relation to the occurrence of autophagy [22]. In their studies, however, the hypoxia-inducible factor-1a inhibitor had no significant effects on kidney lesions in vivo. To date, published data on the involvement of autophagy is unavailable for the disease pathogenesis of kidney and liver lesions of ARPKD as well as Caroli's disease and their rodent models.
The mTOC1 is a negative regulator of autophagy, directly by phosphorylating ULK1 and preventing ULK1-Atg13-FIP200 complex formation, and indirectly by phosphorylating S6 and 4E-BP1 [30]. Recently, reports on the involvement of mTORC2 in autophagy have started to emerge, and mTORC2 regulates autophagy by affecting forkhead box O and regulating some key autophagy genes such as LC3 and beclin 1 [31]. In these conditions, rapamycin is unable to induce autophagy. The regulation of autophagy process by mTORC1/2 suggests that the inhibition of mTOR kinase activity, by having an impact on both mTOR complexes, may have a greater effect on autophagy, compared to rapamycin alone. Indeed, NVP-BEZ235, not rapamycin and everolimus, significantly induced autophagy in PCK cholangiocytes.
In this study, rapamycin, everolimus and NVP-BEZ235 blocked the phosphorylation of Akt (Ser473) and the downstream effectors of mTORC1/2, S6 and 4E-BP1, in PCK cholangiocytes. The inhibition of the phosphorylation of Akt (Ser473) by the mTORC1 inhibitors was consistent with the reported findings showing that higher concentration of rapamycin might target mTORC2 [32]. It was of note that higher concentration (500 nM) of NVP-BEZ235 markedly reduced the expression of p-Akt (Ser473) and p-4E-BP1 in PCK cholangiocytes. Since recent studies have shown that 4E-BP1 is a potential new target molecule for anti-cancer therapy [33], the marked inhibitory effects of NVP-BEZ235 on the phosphorylation of 4E-BP1 as well as Akt (Ser473) further support its applicability as a therapeutic agent.
As expected, the treatment of the PCK rats with NVP-BEZ235 significantly inhibited cystic dilatation of the intrahepatic bile ducts in vivo, whereas renal cyst development was not affected by the treatment. Similarly, an epidermal growth factor tyrosine kinase inhibitor (gefitinib) significantly has been shown to reduce cystic bile duct dilatation of the PCK rat in vivo, but kidney lesions were unaffected or rather worsened according to our previous study [34]. These results suggest that the mechanism of cyst progression of the PCK rat may be different between the liver and kidney. In addition, in vivo administration of NVP-BEZ235 caused retardation in body weight increase in the rats, indicating the limitation of clinical application of this agent for the patients with ARPKD, at least at the dosage and the treatment period used in this study.
In conclusion, this study showed the involvement of aberrant activation of the PI3K/mTOR pathway in the dilatation of intrahepatic bile ducts of the PCK rat. Although the PI3K/Akt/ mTOR signaling pathway represents a complex network, inhibition of the pathway using specific inhibitors attenuates cystic proliferation of PCK cholangiocytes via the mechanism involving apoptosis and/or autophagy of the cells, which is preferably further addressed for therapeutic purpose.


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Development of Enzyme-Linked Immunosorbent Assays Using 2 Truncated ORF2 Proteins for Detection of IgG Antibodies Against Hepatitis E Virus

Background Without appropriate culture systems for hepatitis E virus (HEV), sufficient natural viral proteins are difficult to generate for use in serological tests. Therefore, it is important to produce large amounts of HEV recombinant proteins in an economical way. The present study developed ELISAs using 2 truncated forms of the HEV open reading frame (ORF) 2 protein in order to detect anti-HEV IgG in serum samples. Methods Two truncated forms of the ORF2 protein were expressed in Escherichia coli and were purified by Ni2+-chelate-affinity chromatography (Qiagen, Germany). Two ELISAs were developed using these proteins and were compared with DIA.PRO HEV IgG ELISA kit (DIA.PRO. Italy) in 220 serum samples. Results High yields of the target proteins were obtained through codon optimization. The concentration and purity of the proteins were improved with Amicon filters (EMD Millipore, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis of the resultant proteins showed a protein band of approximately 60 kDa corresponding to ORF2.1 (amino acids 112-660) and a protein band of approximately 55 kDa corresponding to ORF2.2 (amino acids 112-607). Positive agreement, negative agreement, and concordance of the 2 in-house ELISAs compared with DIA.PRO HEV IgG ELISA kit were 87%, 99.5%, and 98.1%, respectively (kappa=0.899, P=0.625). Conclusions The newly developed ELISAs are useful for detecting anti-HEV IgG in serum samples and are highly concordant with DIA.PRO HEV IgG ELISA kit.

INTRODUCTION
Hepatitis E virus (HEV) causes acute, self-limited hepatitis [1,2] and is endemic in many developing countries, especially in South and Southwest Asia, North Africa, and the Middle East [3]. HEV belongs to the Hepeviridae family [4]. Its genome consists of a positive single-stranded RNA containing 3 overlapping open reading frames (ORFs), ORF1, ORF2, and ORF3 [5,6]. According to the phylogenetic analysis, HEV has 5 genotypes with different geographical distributions and hosts [7]. Humans may cause serious consequences in pregnant women and patients with chronic liver disease [2,7]. The infection is lifethreatening during pregnancy, causing fatal and fulminant hepatitis with a high rate of miscarriage and premature birth [7]. Epidemiological studies have reported a mortality rate of up to 30% in pregnant women [7].
Consequently, HEV is a serious public-health threat, and rapid diagnosis is essential. HEV viremia is limited to the acute phase of the infection; therefore, diagnosis depends on serological assays [10]. Unfortunately, sufficient amounts of natural viral proteins are not available for serological diagnosis, because there are no appropriate culture systems for HEV. Therefore, production of recombinant proteins is required to overcome this problem. Such proteins can then be used for specific antibody detection [10,11].
Among the HEV genes, ORF1 encodes non-structural proteins, which are not targeted by antibodies [12]. Antibodies formed against the protein encoded by ORF3 are transient, making ORF3 an unsuitable antigen for serological diagnosis of HEV [13]. ORF2 encodes a capsid protein of 72kDa (660 amino acids). It is suitable for serological diagnosis of HEV and is a candidate for a vaccine against HEV infection, because it is immune-dominant and highly conserved among HEV species, and induces long-lived immunity [9,13,14]. When expressed, the full-length capsid protein (72 kDa) is not a suitable diagnostic target, because the important epitopes are relatively hydrophobic, insoluble, and therefore masked. However, truncated forms of the capsid protein are considered diagnostic antigens. Among several truncated forms of the full-length ORF2 protein, the 56-kDa form is more stable and is highly active in the detection of HEV antibodies [15,16].
Since HEV grows poorly in cell culture, the ORF2 gene or fragments thereof have been cloned and expressed in different expression systems, such as prokaryotes, insect cells, animal cells, and transgenic plants [6]. Recently, different recombinant antigens have been used in the design of assays for diagnosing HEV; these assays have optimized the sensitivity and specificity of the antigens in order to provide the best diagnostic test. In addition to antigen sensitivity and specificity, economical largescale production of the target proteins is an important goal [11]. Satisfactory expression of ORF2 proteins can be achieved in bacterial and animal cells, but proteins produced in animal cells are not cost effective. Therefore, an economical method of highyield production of ORF2 proteins is expression in a prokaryotic expression vector system in E. coli cells [6].
In the present study, we described the simple and low-cost development of 2 ELISAs using 2 truncated forms of the HEV ORF2 proteins. We then evaluated the ability of these ELISAs to detect anti-HEV IgG in serum samples and compared our results to those obtained with DIA.PRO HEV IgG ELISA kit (DIA. PRO, Milan, Italy).

Gene optimization and synthesis
The nucleotide sequence of the truncated ORF2 gene (ORF2.1, encoding amino acids 112-660) of HEV genotype 1, isolate sar-55 (Gen-Bank accession number AF444002.1) was analyzed using GenScript Rare Codon Analysis Tool (GenScript USA Inc., Piscataway, NJ, USA). The sequence data was submitted to GenScript, and the optimal gene was designed using GenScript's OptimumGene Gene Design tool (GenScript USA Inc.) for expression in E. coli. To subclone it into the pET30a (+) vector, 2 restriction-enzyme digestion sites, NdeI and XhoI, were placed in the codon-optimized gene. The pET system is one of the best tools for the cloning and expression of recombinant proteins in E. coli BL21 cells, and was, therefore, used for subcloning [17]. Another truncated form of the ORF2 gene was constructed (ORF2.2, encoding amino acids 112-608) from the previously truncated form by using 2 digestion sites for NheI; the first was at amino acid 608, and the second was added after the stop codon. After the second NheI digestion site, an 8-His tag and 2 stop codons were added. To confirm our design, in silico digestion was performed using Clone Manager Basic software version 9 (Sci-Ed Software, Cary, NC, USA), and the translated protein sequences were aligned using sar55 strain by MEGA software version 4.0 (Biodesign Institute, Tempe, AZ, USA) [18]. The optimized coding sequence was then synthesized and cloned into the commercial cloning vector, pBluescript II SK (+) by Biomatik Company (Biomatik Corporation, Cambridge, Canada). (New England BioLab). After ligation, the first recombinant plasmid pET30a-ORF2.1 was generated and transformed into E. coli DH5α competent cells by electroporation as described previously [19]. Transformed E. coli cells were selected on Luria-Bertani (LB) medium (HiMedia, Mumbai, India) agar plates containing kanamycin (50 mg/L). Several colonies were assayed by colony PCR using universal primers for the T7 promoter and T7 terminator. After selecting recombinant clones, the plasmid DNA was extracted from cells cultured overnight by using the High Pure Plasmid Isolation Kit (Roche). The second recombinant plasmid, pET30a-ORF2.2, was obtained by deleting a 193-nucleotide fragment from the first recombinant plasmid by digestion with NheI. The fragment was then ligated using T4 DNA ligase. Finally, the plasmid DNA was extracted and confirmed by PCR, restriction-enzyme digestion, and DNA sequencing. In order to enhance protein expression, the recombinant plasmids were each transformed into competent E. coli BL21 (DE3) cells separately.

Expression and purification of recombinant proteins
E. coli BL21 (DE3) cells containing the first recombinant plasmid pET30a-truncated ORF2.1 were cultured overnight, inoculated in Terrific Broth (HiMedia) supplemented with kanamycin (50 mg/L) in a 1:100 volumetric ratio, and grown with shaking at 250 rpm at 37°C until the optical density at 600 nm (OD600) reached 0.5. Protein expression was induced by adding various concentrations (0.1-1 mM) of isopropyl-β-D-thiogalactopyranoside (IPTG) to the bacterial culture, and the cells were incubated with shaking for different induction times (2, 4, 6, 8, and 16 hr) at different induction temperatures (37°C, 30°C, and 25°C) to optimize the protein expression. The optimal conditions were determined and large-scale protein expression studies were carried out. Purification by Ni 2+chelate-affinity chromatography (Qiagen, Hilden, Germany) under denaturing conditions was carried out according to the manufacturer's protocol. The induced cells from a 200 mL culture were harvested by centrifugation at 4,000 rpm at 4°C for 20 min. The cell pellet was suspended in 10 mL of phosphatebuffered saline (PBS) and then lysed by 3 cycles of freezethawing in liquid nitrogen and cold water (4°C) and sonication 3 times in 10 sec burst. The lysate was centrifuged at 15,000 rpm at 4°C for 30 min. To solubilize the inclusion body proteins, the pellet was resuspended in lysis buffer (6 M guanidine HCl, 20 mM NaH2PO4, 500 mM NaCl, pH 8.0) and stirred for 30 min at room temperature. The suspension was cleared by centrifugation at 15,000 rpm for 30 min at 4°C. One milliliter of Ni-NTA agarose (Qiagen) was equilibrated with the lysis buffer and added to the clear supernatant. The agarose-sample suspension was gently shaken at room temperature for 30 min to allow the protein to bind to the agarose, and then centrifuged at 1,000 rpm for 2 min. The supernatant was removed, and the agarose was sequentially washed 3 times with 10 volumes of binding buffer (8 M urea, 20 mM NaH2PO4, 500 mM NaCl, pH 8.0). The agarose was then transferred to a column and sequentially washed twice with 3 volumes of wash buffer (binding buffer with a linear pH gradient of 8.0, 6.5, and 6.0). The protein bound to Ni-NTA agarose was eluted with 4 volumes of elution buffer (binding buffer with a linear pH gradient of 5.0, 4.5, and 4.0). The eluate was refolded by dialysis in 500 mL of PBS containing 10% glycerol at 4°C for 4 hr and concentrated using Amicon Ultra-4 Centrifugal Filter Unit with a molecular weight of 30 kDa (EMD Millipore, Billerica, MA, USA).
The presence of the target protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. SDS-PAGE was performed according to the procedure described previously [20]. For western blotting, protein extracts separated by SDS-PAGE were transferred to a 0.45-µm pore (polyvinylidene difluoride (PVDF) membrane (Roche) by using a Semi-Dry Transfer Cell (Bio-Rad, Hercules, CA, USA). The expression of the truncated ORF2 protein was detected by immunodetection with anti-His6-peroxidase (Roche) using the manufacturer's protocol. Finally, the target protein was visualized with 3,3'-diaminobenzidine (DAB) as a substrate [21]. Concentration of the target protein was determined by the Bradford method [22]. The second truncated form of the ORF2 protein (ORF2.2) was expressed and purified using the same procedure.

ELISA
The diagnostic utility of the 2 purified truncated ORF2 proteins was evaluated by developing 2 ELISAs for the detection of HEV IgG antibody. Checker Board Titration and calculation of P/N value (absorbance value of the test sample/absorbance value of the negative control) were used to determine the optimal dilution of the antigen, serum, and conjugate. Briefly, ELISA plates (MAXISOR; Nunc, Roskilde, Denmark) were coated with 50 µL/ well of serial dilutions of the 2 purified proteins separately (0.3-10 µg) in 50 mM carbonate/bicarbonate buffer (pH 9.6), incubated overnight at 4°C, and incubated at 37°C for 1 hr. Two uncoated wells were used as blanks. The plates were washed 3 times with PBS containing 0.05% Tween-20 (PBST, pH 7.4) and tapped dry; then, the plates were blocked with 200 µL/well of 2% casein hydrolysate and 1% bovine serum albumin (BSA) in PBS at 37°C for 1 hr. The well contents were emptied completely and the wash step was repeated as described above, and the wells were tapped dry. At a concentration of 100 µL/well, each serum sample was diluted in sample diluent (1% BSA in 10 mM sodium citrate buffer, pH 6.0, 0.5% Tween 20, 0.09% sodium azide), added to the wells in duplicate, and incubated at 37°C for 1 hr. The plate was washed as described above, and 100 µL of an horseradish peroxidase (HRP)-conjugated goat anti-human IgG secondary antibody (Abcam company, Cambridge, MA, USA) diluted 1:15,000 in diluent buffer (1% BSA in 150 mM NaCl and 10 mM Tris buffer, pH 8.0, 0.05% Tween 20) was added to each well and incubated at 37°C for 1 hr. After incubation, the plates were washed as described above. The substrate solution (100 µL/well, H2O2 and 10 mg of tetramethylbenzidine [TMB] in 1 mL of dimethylsulfoxide [DMSO] and 100 mL of citrate buffer, pH 6.0) was added, and the plates were incubated in a dark place at room temperature for 20 min. Finally, 100 µL/well of 1 M H2SO4 was added to stop the reaction.
The absorbance of the wells was read at 450 nm by an ELISA reader (Tecan Sunrise ELISA Reader; Tecan Trading AG, Mannedorf, Switzerland). The results were analyzed, and the cut-off value was determined as the mean absorbance value of 42 negative serum samples plus 2 standard deviations. All 42 negative serum samples were tested by a commercially available kit (Genlabs Diagnostics Inc., Irvine, CA, USA) for HEV IgG antibodies All 42 negative serum samples were negative for HEV IgG antibody by DIA.PRO HEV IgG ELISA kit. The 42 verified-negative serum samples were tested in duplicate in 3 separate coated plates for each in-house ELISA. On the basis of the obtained results, a cut-off value was determined for each in-house ELISA. The difference in cut-off values between the 3 plates was less than 0.02; therefore, the mean cut-off value of the 3 plates was used. After calculating the cut-off value, a pooled negative serum was prepared from the 42 negative serum samples, which was used as a negative control in the evaluation of 220 serum samples by the 2 in-house ELISA using 4 replicates each. These 220 serum samples were randomly selected from samples collected in the laboratory of Imam Khomeini Hospital in Ahvaz city. Ethical consent as written form was obtained from all participants.
The 42 negative serum samples as well as 26 weakly positive and 32 highly positive serum samples were tested to determine reproducibility (intra-assay and inter-assay precisions), sensitivity, and specificity. To determine within-run or intra-assay precision, each sample was tested in 24 replicates in a single day, and the coefficient of variation for each sample was determined.
To determine between-run or inter-assay precision, each sample was tested in 8 replicates on 3 sequential days, and the coefficient of variation for each sample was determined. All serum samples were obtained from the virology reference lab of Jundishapour University. Regarding inter-assay precision, the mean coefficients of variation for the highly positive serum samples, weakly positive serum samples, and negative serum samples were 5.13%, 6.77%, and 6.18%, respectively. Regarding intra-assay precision, the mean coefficients of variation for the pooled positive serum samples and the pooled negative serum samples were 8.05% and 11.5%, respectively. Thus, there was acceptable reproducibility.
The 220 serum samples were tested for anti-HEV IgG antibodies by using the 2 in-house ELISAs and DIA.PRO HEV IgG ELISA kit. Briefly, the following steps were carried out for the 2 in-house ELISAs. All 220 serum samples and the negative and positive controls were diluted 1:21 in sample diluents, and 100 µL of each diluted sample was added in duplicate to coated wells of the ELISA plates. The ELISA plates were incubated at 37°C for 60 min, washed 5 times with PBST, pH 7.4. One hundred microliters of diluted HRP-conjugated goat anti-human IgG secondary antibody was added to each well, and the plates were incubated at 37°C for 60 min. The wash step was carried out as described above. One hundred microliters of chromogensubstrate mixture was added to each well, and the ELISA plates were incubated at room temperature in a dark place for 20 min. Finally, 100 µL of 1 M sulfuric acid was added to each well to stop the enzymatic reaction. The absorbance of the wells was read at 450 nm by the ELISA reader. The results of the tests were analyzed by comparison with the cut-off value. DIA.PRO HEV IgG ELISA kit for the detection of anti-HEV IgG antibodies was used according to the manufacturer's instructions.

Optimization of expression of the truncated ORF2 gene
The truncated ORF2 gene was optimized for expression in E. coli by using GenScript software (GenScript USA Inc.) without changing the amino acid sequence by analyzing the codon adaptation index (CAI) before and after optimization. The level of protein expression is correlated with the CAI value, where a CAI > 0.8 is ideal for expression in the target host. In our study, a CAI value of 0.85 was determined for the optimized gene. The sequences of the native ORF2 gene and the optimized gene are shown in Fig. 1

Construction of the recombinant plasmid
After subcloning, the recombinant plasmids were confirmed by PCR, restriction enzyme digestion, and DNA sequencing. PCR amplification using universal plasmid primers followed by electrophoresis on 1.2% agarose gel revealed a PCR product of 1,943 bp for ORF2.1 and 1,750 bp for ORF2.2 (Fig. 2).

Protein expression and purification
The optimal conditions for protein expression were induction with 1 mM IPTG with shaking at 37°C for 4 hr. The highly expressed target proteins were purified by Ni 2+ -chelate-affinity chromatography (Qiagen). The concentration and purity of the proteins were maximized by Amicon Ultra-4 Centrifugal Filter Unit (EMD Millipore) with a molecular weight of 30 kDa. The expression and purification of the truncated ORF2 protein was confirmed by SDS-PAGE and western blotting, which revealed a protein band of approximately 60 kDa for ORF2.1 and approximately 55 kDa for ORF2.2 (Fig. 3). The abundant target proteins were eluted with the elution buffer at pH 4.5. The yield of the purified protein was approximately 1 mg/L of the culture medium. Western blotting was carried out to confirm the presence of the ORF2.1 and ORF2.2 proteins in E. coli BL21 (DE3) (Fig. 4).

ELISA based on the 2 truncated ORF2 proteins
The ELISA was optimized by examining different antigen concentrations and serum dilutions. The optimal concentration of the coating ORF2.1 protein for detecting HEV IgG antibody was 2 µg/mL. The optimal blocking buffer was 2% casein and 1% BSA in PBS. The optimal serum dilution was 1:21, and the optimal conjugate dilution was 1:15,000. The optimal conditions for the ORF2.2 protein were identical. The cut-off values for ORF2.1 and ORF2.2 were 0.35 and 0.4, respectively. Samples were considered positive, if the OD was above the cut-off value.
The 220 serum samples were tested anti-HEV IgG antibodies by using the 2 in-house ELISAs and the results were compared with DIA.PRO HEV IgG ELISA kit (Fig. 5). Among the 220 serum samples analyzed by the 2 in-house ELISAs, 21 were positive for anti-HEV IgG antibodies; 23 of the samples tested positive by using DIA.PRO HEV IgG ELISA kit (

DISCUSSION
HEV antibodies are indicative of past infection. For seroepidemiological studies, recombinant ORF2 protein is needed in order to detect HEV antibodies. Many studies have investigated lowcost methods for over-expressing recombinant proteins as diagnostic tools [10,11,23]. In our study, 2 truncated forms of HEV ORF2 protein were produced on a large scale for the development of 2 ELISAs that could detect anti-HEV IgG. The truncated forms (especially the 56-kDa form) of ORF2 protein are more stable and useful for the detection of antibodies against HEV [11,15,16]. Therefore, we expressed and purified 2 truncated forms of the ORF2 protein in E. coli. The advantages of expressing proteins in E. coli are cost effectiveness, ease of handling, short reproductive cycle, and low pollution [24]. However, expressing proteins in E. coli can result in low-level or failed expression because of some problems including high GC content and rare codon in the gene sequence [24]. To overcome poor expression, we optimized our gene sequence for expression in E. coli; this provided several advantages, including elimination of polyadenylation sites, splicing sites, killer motifs, repeated sequences, RNA secondary structure, and high GC content [25,26]. The expression level can be improved through using host-favorite codons and lowering the GC content, since rare codons and high GC content can decrease or even prevent expression [27]. There are several approaches for expressing the HEV structural gene [21,28]. However, no previous study has utilized codon-optimized expression of the ORF2 protein. Our results indicate that codon optimization resulted in ef-fective expression in E. coli with high protein yield.
For the positive serum samples, the mean OD values were higher when tested by the in-house ELISA coated with ORF2.2 as compared to the in-house ELISA coated with ORF2.1 and DIA.PRO HEV IgG ELISA kit. When tested by the in-house ELISA coated with ORF2.1 protein, the mean OD values of the negative serum samples were close to the cut-off value for this ELISA. This is a limitation of this in-house ELISA as compared with the in-house ELISA coated with ORF2.2 protein and DIA. PRO HEV IgG ELISA kit.
Since E. coli was used to express these 2 proteins, the proteins could have been contaminated with bacterial proteins during purification. Subsequently, this contamination could have led to false-positive results for serum samples containing antibodies against E. coli. To eliminate this possibility, Toxin Eraser Endotoxin Removal kit (Genscript Inc.) was used to remove bacterial proteins from the expressed proteins.
An approximately 4 g of bacterial pellet was obtained from each liter of culture media, which yielded approximately 1 mg of purified protein. One hundred nanograms of the purified protein per well was used in each in-house ELISA to detect anti-HEV IgG. It is estimated that approximately 10,000 serum samples could be screened using the protein obtained from 1 L of bacterial culture. In conclusion, high yields of the highly purified proteins were obtained and could be used as a diagnostic antigen in ELISA-based detection of anti-HEV IgG in serum. Moreover, both in-house ELISAs are highly concordant with DIA.PRO HEV IgG ELISA kit.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

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Preparation, Characterization, In Vitro Release and Degradation of Cathelicidin-BF-30-PLGA Microspheres

Cathelicidin-BF-30 (BF-30), a water-soluble peptide isolated from the snake venom of Bungarus fasciatus containing 30 amino acid residues, was incorporated in poly(D,L-lactide-co-glycolide) (PLGA) 75∶25 microspheres (MS) prepared by a water in oil in water W/O/W emulsification solvent extraction method. The aim of this work was to investigate the stability of BF-30 after encapsulation. D-trehalose was used as an excipient to stabilize the peptide. The MS obtained were mostly under 2 µm in size and the encapsulation efficiency was 88.50±1.29%. The secondary structure of the peptide released in vitro was determined to be nearly the same as the native peptide using Circular Dichroism (CD). The ability of BF-30 to inhibit the growth of Escherichia coli was also maintained. The cellular relative growth and hemolysis rates were 92.16±3.55% and 3.52±0.45% respectively.

Introduction
BF-30 is a 30-residue peptide isolated from the venom of the snake Bungarus fasciatus, which exhibits broad antimicrobial activity against bacteria and fungi. The peptide has strong antibacterial activity that has been identified in vitro against Pseudomonas aeruginosain in infected burns [1,2]. BF-30 has a short onset of action when acting against P. aeruginosa and the drug-resistant bacteria, S. aureus. However, the peptide has a short half-life in serum [3], which may be caused by proteases present in serum [4]. Attempts have been made to increase the stability of BF-30 by chemical modification of the peptide through pegylation to extend the half-life [5,6]. However, pegylation is a chemical modification method for increasing the stability of peptide by conjugating PEG to peptide or protein, the process may have an effect on the therapeutic effect of the peptide [3]. An alternative route to improve stability is to formulate the chemically intact peptide in a suitable carrier vehicle [3].
Microsphere (MS)-based peptide polymer conjugation has proved to be a good delivery system for the encapsulation of other similar animal-based peptides [7,8]. Hence this system was investigated as a potential method to encapsulate BF-30 for use in inhibiting the growth of bacteria. Figure 1 shows the preparation procedure of BF-30 loaded microspheres by W/O/W solvent extraction method, which containing twice emulsification and then solvent extraction by isopropanol solution.
Biomacromolecular therapeutics are a type of improved drugs that have been developed in recent years and biodegradable polymers such as polylactide have been used widely in protein and peptide delivery systems [7,8,9,10]. As most biomolecular materials are hydrophilic, the MS are made by the water/oil/water emulsification solvent extraction method. In recent years, several kinds of novel polymers have been made to be used in drug delivery systems [11]. Of these polymers, poly(D,L-lactide-coglycolide) (PLGA) has been in use the longest and has been approved by the US FDA for medical use [12]. In addition, MS containing luteinizing hormone-releasing hormone (LH-RH) formed using PLGA have been commercialized [9].
The aim of this research is to encapsulate BF-30 in PLGA MS, allowing a steady release of the peptide, while maintaining its activity. Preparation of BF-30 loaded PLGA MS BF-30 MS were prepared by dispersion followed by solvent extraction and evaporation according to the methods described in early articles by Ogawa and He [9,13]. Briefly, 0.50 mL BF-30 solution in water (20 mg/mL) with excipient trehalose was slowly added to a solution of 5 mL of 16% (w/v) PLGA in dichloromethane (DCM) in an ice-bath under high-speed homogenization with Tween80 as surfactant. Then, the obtained emulsion was injected into a 1% PVA solution (the continuous phase) under the conditions described above. After the second emulsion process, the double emulsion was added to a 5% isopropanol solution to solidify the MS, then the MS were obtained by centrifugation for 10 min at 5000 rpm and washed with 0.30% mannitol/water solution three times.

Surface morphology and size analysis
The surface morphology of the MS was analyzed using a JEM-100CX (HATACHI S-3000N, Japan) Scanning Electronic Microscope (SEM) and E-1010 Ion sputtering apparatus (HATA-CHI, Japan). Freeze-dried MS were placed on a metal stub, which was coated with conducting resin. The particle size and mean diameter distribution (polydispersity index) of the MS were measured by laser diffraction analysis using a Mastersizer 2000 (Malvern, UK). The preparation of the sample was as follows: the microparticles were dispersed in distilled water, then a dilute solution of uniformly dispersed microspheres were obtained by ultrasonic vibration. The samples were measured three times to calculate the mean diameter and polydispersity index (PDI).

Encapsulation efficiency (EE) and microspheres yield rate
High encapsulation efficiency of MS was a key index of an excellent MS delivery system, and was the ratio between the amount of peptide entrapment and peptide added in the process of preparing MS. The amount of peptide entrapment was measured by dissolving 50 mg MS in 2.00 mL DCM (n = 3), then extracting the peptide twice with 2.0 mL Phosphate Buffer Solution containing Tween (PBST) (pH 7.40, 10 mM, containing 0.02% NaN 3 , 0.02% Tween80, and 150 mM NaCl) according to the method of OGAWA [9]. The concentration of peptide in the water phase was measured by HPLC, compared with a standard curve data of known concentrations of BF-30 solutions. In order to determine the amount of peptide loss during the inefficient extraction process, a certain amount (m 1 ) of peptide solution was added into PLGA/DCM solution with the same concentration of MS preparing process. The BF-30 concentration in extraction liquid was measured in triplicate for three different batches, and different calibration values for inefficient extraction were detected for each sample as the method of Li [14]. The yield rate is another index of a delivery system which relates to the amount of microspheres obtained. The peptide entrapment efficiency was calculated according to the equation below.
Encapsulation Efficiency (%) Extraction Efficiency (%) Peptide in extraction solution(mg) m 1 (mg) |100% ð2Þ In vitro release study The MS loaded with BF-30 were incubated in a capped centrifugal tube containing 10 mL PBST (n = 3), kept at 37 uC and shaken at 120 rpm. At predetermined intervals, the tubes were removed and the samples were centrifuged at 3000 rpm for 10 min. Supernatant (1 mL) was extracted and an equal amount of fresh PBST was added. The peptide concentration in the supernatant was determined by the method described above.
Q: cumulative release (mg) C n : concentration of the release medium (mg/mL) at time t V t :volume of the release medium, V t = 10 mL V s : volume of solution obtained from the release medium for testing, V s = 1 mL The release profile was obtained by plotting the cumulative release rate (Q/m 2 6100%) versus the release time.
Each experiment was repeated three times and the result is presented as the mean value of the three samples. The error bars in the plot show the standard deviation of the data.

In vitro degradation study
The in vitro degradation study was similar to the release study, except that at the predetermined intervals, the degradation medium in the tubes was removed and the degraded MS were washed with distilled water to rinse off the buffer salt.

HPLC and CD analysis
The concentration of the peptide extracted in the water phase was measured using reverse-phase HPLC (Agilent Technologies 1200 Series, Waters, xTerra, RP18, 5 mm, 4.66250 mm column). The elution phase was A: water with 0.10% (v/v) trifluoroacetic acid and B: acetonitrile with 0.10% (v/v) trifluoroacetic acid; UV detection was at 220 nm [15]. The concentration of the acetonitrile phase was raised from 21.00% to 46.00% over 25 min using a linear gradient, and the sample size was 25 mL.
The peptide conformational change during the release period was measured on a far-UV (190-280 nm) Circular Dichroism Spectrometer Chirascan (Applied Photophysics Limited) at ambi-ent temperature in a quartz cell (path length 0.05 cm) [16,17] and compared with the native peptide.

In vitro cytotoxicity analysis
The in vitro cytotoxicity of the peptide-loaded PLGA MS was characterized by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method, inendothelial cells at a concentration of 120 mg/mL (n = 5) [18,19], under the conditions described by Shen [19]. The MTT method is based on the theory that succinate dehydrogenase in the mitochondria reduces dissolved MTT to a purple, water-insoluble formazan product in living cells, but not in dead cells. In detail, endothelial cells were grown at 37 uC under a 5% CO 2 atmosphere in RPMI-1640 medium in a 96-well plate, supplemented with 10% calf serum in a fully humidified incubator. After 24 h, the culture medium was removed, MTT was added, and the samples were incubated for another 4 h. DMSO was added to dissolve the formazan crystals, and the optical density at 570 nm was measured on Multiskan Go spectrophotometer (Thermo, USA) with PBST as negative control. Each sample was prepared in triplicate. The cellular relative growth rate (RGR) indicating the ratio of living cells during the test was calculated according to the following equation:   In vitro hemolysis test A hemolysis test was carried out to measure the ability of the biomaterial to destroy red blood cells when contacted with blood. The rate was quantified by measuring the suspension absorbance at 541 nm, which was consistent with the ferrohemoglobin released by destroyed red cells [20]. The hemolysis activity of BF-30-loaded PLGA MS was tested according to the method of Shen [19]. The hemolysis was quantified spectrophotometrically according to the method of Shen [19]and Fischer [16]. Blood obtained from rabbit pericardium was collected in heparinized tubes (n = 3), then centrifuged at 1500 rpm for 10 min on Eppendorf centrifuges (Germany, 5417R) to acquire rabbit red blood cells (RRBCs). Discarding the serum above by suction, the RRBCs were found deep in the centrifuge tube. Suspensions of the red blood cells were obtained by washing the RRBCs with a sterile physiological saline solution until the absorbance value of the positive control supernatant was located in the range 0.5-0.6 at 541 nm.
The hemolysis rate was calculated according to the following equation: Hemolysis rate(%)~A bs material {Abs negative control Abs positive control {Abs negative control |100% ð6Þ The sterile physiological saline solution and distilled water were the negative and positive controls respectively, and the corresponding hemolysis rates were 0% and 100% respectively.

Biological activity
The ability of the released peptide to inhibit the growth of Escherichia coli was investigated according to the method of Kim [21]. The freeze-dried Escherichia coli, purchased from Guangdong Huankai Microbial SCI & Tech. Co. Ltd. (Cat. No. FSCC 149005, Lot. No. A0144B), were reconstituted in a brain-heart infusion broth. The bacterial solution was diluted to between 0.10 and 0.20 at OD625, an absorbance of half the McFarland standard. The absorbance value at 625 nm of the bacteria solution is proportional to the concentration of the bacteria in the solution [21]. Diluted bacterial solution (0.10 mL) was added to 96-well plates with 0.10 mL of the released peptide solution (n = 3), then incubated on a shaker at a constant temperature of 37uC and a rotation speed of 80 rpm. The absorbances at 625 nm were measured after 5 and 24 h. The percentage of bacterial inhibition was calculated according to the absorbance difference between the bacteria solution with and without the peptide after 5 and 24 h incubation using the equation:  A bsc and A bss stand for the absorbance at 625 nm of the control group solution and the release medium with peptide, respectively.

Morphology characterization
Microspheres prepared by the W/O/W emulsion/solvent evaporation/extraction method with a high yield of 76.6468.07% according to the equation (3). SEM was used to investigate the morphology of the microspheres. Figure 2 shows the SEM images of lyophilized PLGA MS; most of the MS had spherical and smooth surfaces, but a few MS appeared pitted. As seen in Figure 2 (b), PLGA (75:25) had little nonmicrosphereforming ratio.
The MS size and size distribution were studied in a microsphere-water solution using Mastersizer 2000. As shown in Figure 3, the size of all microspheres obtained ranged from 500 to 3000 nm, with a mean diameter of 1460614.02 nm. The majority of MS had a mean diameter between 800 and 1500 nm, in the submicro range [22], and a PDI of 0.24, which was,0.30. This means a narrow size distribution.MS with a diameter under 50 mm contribute to a steady release rate and decrease the burst release, thus the release kinetics would be close to a zero-order release mode [23,24].

Encapsulation and in vitro release of BF-30 loaded PLGA MS
In our study, the concentration of the peptide had a liner range of 55.50 to 333 mg/mL, as measured by HPLC according to the standard curve equation: y = 4.88x-74.80, R 2 = 0.9991; where y is the peak area and x is the concentration of the peptide (mg/mL). The encapsulation efficiency indicates the ratio of the actual amount of peptide encapsulated in the MS. In this study, the encapsulation efficiency was 88.5061.29%, which was the calibration value according to inefficient extraction ratio 62.6660.86%. Figure 4 shows the release profile of the BF-30 loaded PLGA MS. The release profile indicated the burst release was nearly 40% over the first release day, which may relate to peptide adsorbed on the surface of the MS [25,26]. The peptide entrapped in the MS was released gradually with the degradation of polymer. The release process of the peptide containing three phases: the first burst release, lag and the second burst release. The second burst release often due to the erosion of polymer matrix [27]. At the end, 76.6061.70% of the peptide encapsulated in the MS had been released.
In vitro degradation and erosion of PLGA MS Figure 5 shows a continued degradation of PLGA MS over 60 days with a significant drop in the pH value of the release medium and the dry mass (%). MS dry mass and Mw decreased during the first few degradation days were apparent, dry mass loss from 100% to 91% in the first day, the Mw of the BF-30 loaded microspheres dropped visibly from 18.50 to 17.60 KDa, which indicated that MS had undergone apparent degradation. As a result of acidic degradation products of PLGA-(lactic acid and glycolic acid) monomers and oligomers [24], pH value of the degradation medium dropped from 7.40 to 7.26, followed by a slower degradation process over the remaining 60 days.

HPLC and CD analysis
As shown in Figure 6, the HPLC elution curve of the peptide released on the 11 th day(b) is similar to the native peptide(a), with the same retention time of 12.30 min. Curve (c) represents the peptide released in the 10 th day, which containing two peaks, peptide and acylated peptide. The HPLC results indicated that the peptide released on the 11 th day has the same polarity as the native peptide, but produced some acylated peptide on the 10 th day [28].
PLGA degraded to acidic compounds, including lactic and glycolic acids over time, which resulted in the observed decrease in the pH value of the degradation medium. Peptide would precipitate and assemble when the pH value of the medium is close to the isoelectric point. Fortunately, the isoelectric point of 11.79 for BF-30 is far above the observed pH value of the degradation medium. Thus the peptide in the MS would not precipitate and assemble and so most peptide encapsulated would be released [29]. Furthermore, Figure 7 shows that the far-UV CD spectrum of the peptide in the release medium of the 1 st day and of the later period of the release process, the 12 th and 13 th days, matching that of the native peptide. This demonstrates that the released BF-30 retains its a-helical secondary structure [30], indicating that the structure of BF-30 has not changed during the release process.  Antimicrobial activity of the released peptide To test the biological effect of the released peptide, we investigated the activity of release medium on the 1 st , 10 th , 11 th ,and 12 th days against Escherichia coli, which is sensitive to BF-30 [1,2]. As shown in Figure 8, the released medium of the 1 st , 10 th , 11 th and 12 th days were efficient in inhibiting the growth of Escherichia coli.  10,19.4760.10 mg/mL on days 1, 10, 11, and 12 respectively, while the concentration of native peptide for antimicrobial test was 35 mg/mL. The inhibition ratio of the native peptide was higher than the 1 st , 10 th , 11 th , 12 th days in all, and the 10 th day was lower than the 11 th and the 12 th day; this may be caused by a lower peptide concentration in the 10 th day, the acylated peptide, and the degradation of the MS, which makes the system unstable. We can see from Figure 6 that the 10 th day containing acylated peptide with lower concentration, and the 11 th day containing unbroken peptide compared with native peptide HPLC curves, but a little change in the 12 th day according to Figure 7. We can see from Figure 4 that the 11 th and the 12 th day were during the second burst release process. The peptide concentration in the release medium and the inhibition ratio on the 11 th day were higher than on the 10 th and 12 th days. Also, peptide in release medium of the 1 st day maintained its bioactivity according to Figure 8. The ability of the release medium to inhibit the growth of Escherichia was apparent after 24 h incubation with dilute bacteria solution. This means that the peptide has retained its bioactivity during the release period and the PLGA MS delivery system has the potential to protect the fragile peptide from adverse conditions.

In vitro cytotoxicity analysis
A concentration of 120 mg/mL was used for cytotoxicity testing based on the amount of peptide encapsulated in the microspheres and the minimal inhibitory concentration (MIC) of 0.15 mg/mL for Escherichia coli [1].
After 48 h, the water-insoluble formazan formed by MTT in living cells was dissolved in dimethyl sulfoxide and the absorbance value of the samples at 570 nm was measured by absorbance spectroscopy on a Multiskan Go spectrophotometer (Thermo, USA). Figure 9 shows the results of the MTT assay, using PBST as the negative control. The BF-30-loaded microspheres showed 92.1663.55% RGR, which indicated that no toxicity occurred at the concentration tested (120 mg/mL).
In vitro hemolysis Figure 10 shows the hemolysis level of microspheres at different concentrations. The OD value of the negative control, corresponding to 0% hemolysis, was 0.0460.0003 and the value for the positive control, corresponding to 100% hemolysis, was 0.3960.005. The hemolysis rate was 3.5260.45%, which was lower than the ISO criterion of 5% [13], when the concentration of the microspheres was 125 mg/mL. These results indicate that  the BF-30-loaded PLGA microspheres are safe when in contact with blood at the efficient concentration of 125 mg/mL.

Conclusions
The antimicrobial peptide BF-30 of animal origin, which has antimicrobial activity, has been encapsulated into PLGA microspheres by the W/O/W double emulsion method with high encapsulation efficiency. The surface morphology of the MS was found to be spherical with a narrow size distribution. The delivery system released peptide over more than 15 days. The peptide retained its a-helix secondary structure during the release period. The BF-30 peptide released from the microspheres was found to be effective in inhibiting the growth of Escherichia coli, which means that the PLGA microsphere delivery system protected the peptide in terms of both structural stability and antimicrobial activity. In addition, the cytotoxicity and biocompatibility of the MS system was found to be within acceptable limits. In summary, the BF-30-PLGA microspheres hold great potential for use as an antimicrobial agent and the system may be useful for encapsulating other sensitive peptide-based drugs.