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Down-regulation of HSP70 sensitizes gastric epithelial cells to apoptosis and growth retardation triggered by H. pylori
© Liu et al; licensee BioMed Central Ltd. 2011
Received: 29 August 2011
Accepted: 30 December 2011
Published: 30 December 2011
H. pylori infection significantly attenuated the expression of HSP70 in gastric mucosal cells. However, the role of HSP70 cancellation in H. pylori-associated cell damages is largely unclear.
Small interfering RNA (siRNA) was used to down-regulate HSP70 in gastric epithelial cell lines AGS. The transfected cells were then incubated with H. pylori and the functions of HSP70 suppression were observed by viability assay, cell cycle analyses and TUNEL assay. HSP70 target apoptotic proteins were further identified by Western blot.
The inhibition of HSP70 has further increased the effect of growth arrest and apoptosis activation triggered by H. pylori in gastric epithelial cells. The anti-proliferation function of HSP70 depletion was at least by up-regulating p21 and cell cycle modulation with S-phase accumulation. An increase of apoptosis-inducing factor (AIF) and cytosolic cytochrome C contributes to the activation of apoptosis following down-regulation of intracellular HSP70. Extracellular HSP70 increased cellular resistance to apoptosis by suppression the release of AIF and cytochrome c from mitochondria, as well as inhibition of p21 expression.
The inhibition of HSP70 aggravated gastric cellular damages induced by H. pylori. Induction of HSP70 could be a potential therapeutic target for protection gastric mucosa from H. pylori-associated injury.
In recent years, heat shock proteins (HSP) have been implicated to be an additional factor utilized for the gastric defence mechanisms at the intracellular level . HSP70 is generally considered to be a major molecular chaperone to accelerate the cellular recovery from different stimuli by cope with unfolded or denatured proteins , through which HSP70 might achieve efficient mucosal defence for ulcer or inflammation healing [3, 4].
Helicobacter pylori (H. pylori) infection leads to significant inflammations in the gastric mucosa, which is closely associated with development of atrophic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. Animal studies have demonstrated that H. pylori infection damages gastric mucosa by either disrupting the balance in cell apoptosis and proliferation, or decreasing migration of epithelial cells within the gastric mucosa [1, 5, 6]. Recent studies have found that H. pylori decreases the synthesis of HSP70 in gastric epithelial cells by the inactivation of heat shock factor- 1 [7–11], however, whether the inhibition of HSP70 would be the prominent event leading to the persistent damages from H. pylori in gastric epithelial cells remains unclear.
H. pylori produces ammonia in gastric mucosa with its high urease activity. Our previous animal studies have introduced ammonia solution to simulate the conditions of H. pylori infection, and succeeded in inducing atrophic gastritis in rats . Further studies demonstrated that induction of HSP70 expression is beneficial for preventing gastric atrophy and maintaining mucosal functions in gastric cells . Since the induction of HSP70 is suggested to constitute a novel therapeutic approach for the prevention or treatment of H. pylori-associated conditions, it's conceivable that deregulation of HSP70 might be a prominent cause of H. pylori-associated damages. Therefore, we investigated the correlation of HSP70 inhibition with the mucosal damages induced by H. pylori in this study.
Cell culture and transfection
Human gastric epithelial cell line AGS (CRL-1739, ATCC, USA) were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) without antibiotics at 37°C in a humidified atmosphere of 5% CO2 and 95% air.
Small interfering RNAs (siRNAs) were designed against the mRNA sequences targeting HSP70 (Genebank: NM_005345.5), siRNA1: 5'-CTTTCCAGGTGATCAACGA-3', siRNA2: 5'-AGGACGAGTTTGAGCACAA-3', siRNA3: 5'-GACTTTGCATTTCCTAGTA-3'. We used RNAi-Ready vector, which contains a neomycin resistance gene and GFP for selection of stable transfectants. In the preliminary experiments, we employed three constructs that target three distinct regions of the HSP70 gene to deplete HSP70 expression, and found siRNA2 was better than the others for the short-term inhibition of HSP70. Therefore, siRNA2 was selected for the following stable transfection. AGS cells were transfected with the HSP70 siRNA constructs by use of lipofectamine according to the manufacturer's protocol. The total amount of plasmids was adjusted by using the empty vector plasmid in each assay. Briefly, 1 ×105 cells were plated in RPMI1640 containing 10% FBS in 6-well plates 24 h before transfection. Then transfection was performed with serum-free RPMI1640 containing 2 μg plasmid constructs and 6 μl lipofectamine. After 5 h, fresh RPMI1640 containing 10% FBS was added until 2 ml of final volume. The selection with 0.4 mg/ml neomycin was started 48 h after transfection. GFP was used as a control for transfection or selection efficiency. A control sample transfected with empty vector plasmid was included. Neomycin-resistant cell pools and single cell clone were generated, in which HSP70 expression was confirmed by immunoblot analysis and real-time PCR.
Bacterial strain and coculture conditions
H. pylori expressing CagA and VacA (ATCC 700392) were grown on Columbia agar medium with 5% of fresh sheep blood under microaerobic conditions (5%O2, 10%CO2, 8%N2) at 37°C. Before the experiment, bacteria were harvested and suspended in RPMI 1640 medium (including 10% FBS but no antimicrobial agents). The bacteria were densitometrically counted according to the McFarland scale and suitable dilution was prepared for the cell culture (bacteria/cell ratio at 200:1 for most tests).
The RNA was harvested from cell culture with RNeasy columns (QIAGEN). Single stranded cDNA synthesis was made with the TaqMan RT Kit (QIAGEN) using oligo-(dT)16 primers. The cDNA originating from the transfected cells were used as template for the following PCR reaction and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was served as an internal control. Primers were designed as follows (Genebank: NM_005345.5): HSP70-for, 5'-AACACCGTGTTTGACGCGAA-3'; HSP70-rev, 5'-GGTCAGCACCATGGACGAGA-3'; HSP70-probe, 5'-FAM-CCAGGTGATCAACGACGGAGACAAGCCC-TAMRA-3'. The negative control contained the reaction mixture but no DNA. The reactions were performed with a real-time PCR machine (BIOER, Japan) with a Taq activation at 95°C for 5 min followed by 35 cycles of three segments consisting of 30 sec at 95°C, 30 sec at 55°C, and 30 sec at 72°C. The level of HSP70 mRNA was evaluated relative to that of GAPDH mRNA.
Growth curve and cell proliferation Assay
Cell growth curve or proliferation assessment was quantified using a tetrazolium salt colorimetric assay with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, final concentration of 0.5 mg/ml, Sigma-Aldrich, St. Louis, MO, USA). Briefly, the cells stably transfected with HSP70 siRNA or the empty vector were cultured in a 96-well plate for 1~6 days. In the proliferation assay, these cells were incubated with live H. pylori for 0, 24, 48 or 72 hours. The absorbance of samples was measured at 492 nm in the microplate reader.
Cell apoptosis assays
Cells suspension (2 × 104) was added to each well of 48-well plates and was incubated with H. pylori (1:200) for 24 h. We analyzed apoptosis with the use of the terminal deoyecelotibyl transferase mediated dUTP-biotin nick end labeling assay (TUNEL) kit (Cell Death Detection kit, Roche, Germany) according to the instructions provided by the manufacturer. Quantitation of apoptotic cells was accomplished by counting the number of apoptotic bodies sighted in the microscopic fields. Labeling indices were calculated as the mean number of labeled cells (from five random fields of vision) divided by total counted cells (500 cells).
Cell cycle analysis
Cells were seeded (2 × 105) on 6-well plates and were synchronized through serum starvation for 48 hours. Then the cells were incubated with H. pylori at 200:1 of bacterium to cell ratio in RPMI1640 containing 10% FBS for 0, 6, 12 or 24 hours. The treated cells were collected and fixed in 70% ethanol. Cell pellets were resuspended in 500 μl of propidium iodide buffer (10 mM Tris-Cl at PH 7.5, 50 μg/ml propidium iodide, 0.1% Triton X-100, 0.1% sodium citrate and 2 mg/ml RNase) and incubated in the dark at 4°C overnight. Stained cells were analyzed by the Beckman Coulter EPICS XL flow cytometer using the CellQuest software. At least 1 × 104 cells have been tested in each test.
Total proteins were isolated from the transfected cells and the concentration was measured by Bio-Rad Protein Assay. Mitochondrial or cytosolic protein was extracted in according to the protocol of Mitochondria/cytosol Fractionation Kit (BioVision). All procedures were performed at 4°C. The expressions of HSP70 (anti-HSP70, Sigma-Aldrich, St. Louis, MO, USA), Bax, caspase-3, caspase-6, caspase-7, cytochrome c, p21, PCNA (antibodies, Cell Signaling Technology, Danvers, MA, USA) and AIF (anti-AIF, Santa Cruz Biotechnology, Santa Cruz, CA) were assessed by immunoblotting with corresponding antibodies. The blotted membrane was visualized by chemiluminescent substrate (EZ-ECL, Kibbutz Beit Haemek Israel). The immunoblotting for β-actin (Santa Cruz Biotechnology, Santa Cruz, CA) was used as a loading control.
Any significance in differences between two data sets was determined by the Student's t-test. P values < 0.05 were considered significant in all analyses.
H. pylori infection suppressed HSP70 expression in gastric cell line AGS cells
Generation of gastric cells AGS with reduced level of HSP70
Effect of HSP70 depletion on proliferation of gastric epithelial cells with H. pylori infection
HSP70 depletion causes cell cycle arrest in S phase in gastric cells with H. pylori infection
To determine if HSP70 depletion mediated growth inhibition was the result of its cell cycle modulation, we investigated the effect of HSP70 inhibition on cell cycle distribution in H. pylori-infected AGS cells. Our results revealed a significant increase in the number of cells in the S phase in AGS/siRNAHSP70 as compared with its control at different time interval (Figure 3C).
To elucidate the molecular basis by which HSP70 modulates cell cycle in gastric epithelial cell, p21 and PCNA, the well-known genes involved in the cell cycle regulation, were analyzed. Immunoblotting analysis confirmed that p21 protein but not PCNA was expressed at higher levels in the AGS/siRNAHSP70 cells compared with control vector transfected cells (Figure 3D).
HSP70 depletion induced apoptosis and pro-apoptotic proteins in gastric cells with H. pylori infection
Identification of apoptotic genes modulated by HSP70 in AGS cells with H. pylori infection
To elucidate the molecular basis by which HSP70-suppression involved in gastric cells apoptosis, proapoptotic genes expression profile in HSP70-siRNA stably transfected AGS were analyzed by immunoblotting. The apoptosis-inducing effect by HSP70 depletion was mediated by regulating important pro-apoptotic genes including AIF and cytochrome C, as evidenced by an accumulation of AIF and the release of cytochrome c from mitochondria (Figure 4B). No considerable changes for apoptotic proteins in the down stream of the caspase-dependent apoptotic pathways, including caspase-3, caspase-6 and caspase-7 have been observed.
Extracellular HSP70 compensated for the effect of endogenous HSP70 depletion on apoptosis and proliferation
To test whether exogenously applied HSP70 might compensate for the loss of endogenous HSP70, extracellular HSP70 (50 ng/ml) were exposed to AGS/siRNAHSP70 cells with H. pylori infection for 48 hours. The exogenous HSP70 inhibited cytochrome c release from mitochondria to cytosol and AIF accumulation, as well as the expression of p21 (Figure 4C&4D).
HSP70 protects gastric mucosal cells against intrinsic and extrinsic stimuli, and maintains the proper structure and function of the gastric mucosa [14, 15], suggesting induction of HSP70 might be useful for medical treatment of diseases with mucosal damage. Tsukimi Y et al. found HSP70 may facilitate the healing of acetic acid-induced gastric ulcers in rats . Our previous study has demonstrated that up-regulation of HSP70 expression by Geranylgeranylacetone (GGA) interrupts the progression of atrophic gastritis in rats, as evidenced by the improvement of inflammation and glandular restoration in gastric mucosa . HSP70 could protect gastric mucosa from H. pylori-associated gastrointestinal diseases . H. pylori infection destroyed gastric mucosa barrier function through inducing a significant reduction of HSP70 expression in gastric epithelial cells, which was supposed to disturb gastric adaptation and facilitate H pylori to avoid host immunity . We demonstrate here that suppression of HSP70 increased the sensitivity of gastric cells to H. pylori infection with the inhibition of cellular growth and cell cycle progression, as well as induction of apoptosis.
H. pylori infection damages gastric mucosa by disturbing equilibrium between apoptosis and proliferation. HSPs could reverse these inferiorities in mucosal healing. Inhibition of endogenous HSP70 slowed cellular multiplication, and enhanced the effect of H. pylori on cell viability. The anti-proliferation role of H. pylori was more evident following the further depletion of HSP70 expression. HSP70 involving in cell growth could be a cell cycle event. Rohde M et al. reported that Hela cells transfected with siRNA against HSP70 revealed an arrest in G2/M phase of cell cycle , which resulted in growth retardation with features of cell senescence. Our previous study have found that down-regulation of HSP70 induced S-phase arrest in AGS cells. Furthermore, we investigated the effect of HSP70 on the cell cycle of AGS cells infected with H. pylori, and the results showed that depletion of HSP70 induced AGS cells accumulating in S-phase independent of H. pylori infection. Our observation that HSP70-depletion induced S-phase arrest and p21 over-expression is in agreement with the previous report that transduction of the p21 gene resulted in S-phase arrest [19, 20]. The p21 protein can inhibit DNA synthesis by interacting with PCNA , and plays a regulatory role in S phase DNA replication and DNA damage repair . Growth arrest by p21 can promote cellular differentiation, and therefore prevents cell proliferation .
HSP70 depletion could make AGS more susceptible to the cytotoxicity of H. pylori by interference with apoptotic programs. H. pylori is known to cause apoptosis of gastric epithelial cells by targeting mitochondria [24, 25]. Mitochondria respond to multiple death stimuli. It has been demonstrated that pro-apoptotic Bcl-2 family proteins such as Bax could induce mitochondrial membrane permeabilization and cause the release of mitochondria-mediated apoptosis signaling molecules including cytochrome c and AIF . Cytochrome c triggers the caspase-dependent cascade , but AIF executes cell death in the absence of caspase [28–30]. H. pylori has been reported to trigger apoptosis in AGS cells via release of cytochrome c and AIF from mitochondria . Our study demonstrated that down-regulation of HSP70 induced the further release of cytochrome c and AIF in the AGS with H. pylori infection, consistent with the hypothesis that HSP70 suppression could sensitize the gastric epithelial cells to the damage from H. pylori.
Furthermore, we evaluated the role of extracellular HSP70 in H. pylori-infected AGS cells, and demonstrated that extracellular HSP70 protein could partial compensate for the decreased intracellular HSP70 by reducing release of cytochrome c and AIF, which could block apoptosis in gastric cells with H. pylori infection. Consistently, extracellular HSP70 could also modulate the proliferation of AGS cells by inhibiting expression of p21. The exogenous HSP70 might cross the cellular plasma membrane and reduce apoptosis with the decrease of toxicity protein aggregation . Exogenous HSP70 was suggested to be a trophic factor supporting cell survival .
In conclusion, our data suggest that insufficient expression of HSP70 would render gastric epithelial cells more susceptible to H. pylori-induced damage than they would be if HSP70 were more abundant. The extracellular HSP70 may compensate for the deficit of endogenous HSP70 depletion. Designation to increase HSP70 protein may serve as a potential therapeutic strategy to improve the outcome of H. pylori-infected patients.
We thank Prof. Dai Ning who has generously provided H. pylori strain (ATCC 700392) to us. This study was supported by a grant from the National Natural Science Foundation of China (Grant No. J20111996), a joint grant from the Education Department of Zhe Jiang Province, China (Y200803495).
- Choi SR, Lee SA, Kim YJ, Ok CY, Lee HJ, Hahm KB: Role of heat shock proteins in gastric inflammation and ulcer healing. J Physiol Pharmacol. 2009, 60 (Suppl 7): 5-17.PubMedGoogle Scholar
- Basu S, Srivastava PK: Heat shock proteins: the fountainhead of innate and adaptive immune responses. Cell Stress Chaperones. 2000, 5: 443-51.View ArticlePubMedPubMed CentralGoogle Scholar
- Ishihara T, Suemasu S, Asano T, Tanaka KI, Mizushima T: Stimulation of gastric ulcer healing by heat shock protein 70. Biochem Pharmacol. 2011.Google Scholar
- Asai M, Kawashima D, Katagiri K, Takeuchi R, Tohnai G, Ohtsuka K: Protective effect of a molecular chaperone inducer, paeoniflorin, on the HCl- and ethanol-triggered gastric mucosal injury. Life Sci. 2011, 88: 350-7.View ArticlePubMedGoogle Scholar
- Nardone G, Staibano S, Rocco A, Mezza E, D'armiento FP, Insabato L, et al: Effect of helicobacter pylori infection and its eradication on cell proliferation, DNA status, and oncogene expression in patients with chronic gastritis. Gut. 1999, 44: 789-99.View ArticlePubMedPubMed CentralGoogle Scholar
- Fan XG, Kelleher D, Fan XJ, et al: Helicobacter pylori increases proliferation of gastric epithelial cells. Gut. 1996, 38: 19-22.View ArticlePubMedPubMed CentralGoogle Scholar
- Klaamas K, Kurtenkov O, von Mensdorff-Pouilly S, Shjapnikova L, Miljukhina L, Brjalin V, et al: Impact of Helicobacter pylori infection on the humoral immune response to MUC1 peptide in patients with chronic gastric diseases and gastric cancer. Immunol Invest. 2007, 36 (4): 371-86.View ArticlePubMedGoogle Scholar
- Konturek JW, Fischer H, Konturek PC, Huber V, Boknik P, Luess H, et al: Heat shock protein 70 (hsp70) in gastric adaptation to aspirin in Helicobacter pylori infection. J Physiol Pharmacol. 2001, 52: 153-64.PubMedGoogle Scholar
- Pierzchalski P, Krawiec A, Ptak-Belowska A, Baranska A, Konturek SJ, Pawlik WW: The mechanism of heat-shock protein 70 gene expression abolition in gastric epithelium caused by Helicobacter pylori infection. Helicobacter. 2006, 11 (2): 96-104.View ArticlePubMedGoogle Scholar
- Huff JL Hansen LM, Solnick JV: Gastric transcription profile of Helicobacter pylori infection in the rhesus macaque. Infect Immun. 2004, 72: 5216-26.View ArticlePubMedGoogle Scholar
- Axsen WS, Styer CM, Solnick JV: Inhibition of heat shock protein expression by helicobacter pylori. Microb Pathog. 2009, 47: 231-6.View ArticlePubMedGoogle Scholar
- Liu WL, Chen SJ, Chen Y, Sun LM, Zhang W, Zeng YM, et al: Protective effects of heat shock protein70 induced by geranyl-geranylacetone in atrophic gastritis in rats. Acta Pharmacologica Sinica. 2007, 28: 1001-6.View ArticlePubMedGoogle Scholar
- Gabai VL, Budagova KR, Sherman MY: Increased expression of the major heat shock protein Hsp72 in human prostate carcinoma cells is dispensable for their viability but confers resistance to a variety of anticancer agents. Oncogene. 2005, 24: 3328-38.View ArticlePubMedGoogle Scholar
- Suemasu S, Tanaka K, Namba T, Ishihara T, Katsu T, Fujimoto M, et al: A role for HSP70 in protecting against indomethacin-induced gastric lesions. J Biol Chem. 2009, 19705-15. 284Google Scholar
- Rokutan K: Role of heat shock proteins in gastric mucosal protection. J Gastroenterol Hepatol. 2000, 15 (Suppl): 12-9.View ArticleGoogle Scholar
- Tsukimi Y, Nakai H, Itoh S, Amagase K, Okabe S: Involvement of heat shock proteins in the healing of acetic acid-induced gastric ulcers in rats. J Physiol Pharmacol. 2001, 52: 391-406.PubMedGoogle Scholar
- Tomomitsu T, Tomoyuki S, Tomiyasu A, Masakatsu N, Daisuke Y, Masaaki O, et al: The BB genotype of heat-shock protein (HSP) 70-2 gene is associated with gastric pre-malignant condition in H. pylori-infected older patients. Anticancer Research. 2009, 29: 3453-58. 16-20Google Scholar
- Rohde M, Daugaard M, Jensen MH, et al: Members of the heat -shock protein 70 family promote cancer cell growth by distinct mechanism. Genes & Development. 2005, 19: 570-82.View ArticleGoogle Scholar
- Ogryzko VV, Wong P, Howard BH: WAF1 retards S-phase progression primarily by inhibition of cyclin-dependent kinases. Mol Cell Biol. 1997, 17: 4877-4882.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhu Hongbo, Zhang Lidong, Wu Shuhong, Teraishi Fuminori, Davis John, Jacob Dietmar, et al: Induction of S-phase arrest and p21 overexpression by a small molecule 2[[3-(2,3-dichlorophenoxy)propyl] amino] ethanol in correlation with activation of ERK. Oncogene. 2004, 23: 4984-92.View ArticlePubMedGoogle Scholar
- Chen J, Jackson PK, Kirschner MW, Dutta A: Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA. Nature. 1995, 374: 386-388.View ArticlePubMedGoogle Scholar
- Gartel AL, Radhakrishnan SK, Lost in transcription: p21 repression, mechanisms, and consequences. Cancer Res. 2005, 65 (10): 3980-5.View ArticlePubMedGoogle Scholar
- Abbas Tarek, Dutta Anindya: P21 in cancer: intricate networks and multiple activities. Nat Rev Cancer. 2009, 9 (6): 400-14.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang H, Fang DC, Lan CH, Luo YH: Helicobacter pylori infection induces apoptosis in gastric cancer cells through the mitochondrial pathway. J Gastroenterol Hepatol. 2007, 22: 1051-6.View ArticlePubMedGoogle Scholar
- Chiozzi V, Mazzini G, Oldani A, Sciullo A, Ventura U, Romano M, et al: Relationship between Vac A toxin and ammonia in Helicobacter pylori-induced apoptosis in human gastric epithelial cells. J Physiol Pharmacol. 2009, 60: 23-30.PubMedGoogle Scholar
- Yang J, Liu XS, Bhalla K, Kim CN, Ibrada AM, Cai JY, et al: Prevention of apoptosis by Bcl-2:release of cytochrome c from mitochondria blocked. Science. 1997, 275: 1129-32.View ArticlePubMedGoogle Scholar
- Garland JM, Rudin C: Cytochrome c Induces Caspase-Dependent Apoptosis in Intact Hematopoietic cells and overrides apoptosis suppression mediated by bcl-2, growth factor signaling, MAP-Kinase-Kinase, and malignant change. Blood. 1998, 92: 1235-46.PubMedGoogle Scholar
- Joza N, Susin SA, Daugas E, Stanford WL, Cho SK, Li CY, et al: Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature. 2001, 410: 549-54.View ArticlePubMedGoogle Scholar
- Susjin SA, Daugas E, Ravagnan L, Samejima K, Zamzami N, Loeffler M, et al: Two distinct pathways leading to nuclear apoptosis. J Exp Med. 2000, 192: 571-80.View ArticleGoogle Scholar
- Cregan SP, Dawson VL, Slack RS: Role of AIF in caspase-dependent and caspase-independent cell death. Oncogene. 2004, 23: 2785-96.View ArticlePubMedGoogle Scholar
- Ashkorab H, Dashwood RH, Dashwood MM, Zaidi SI, Hewitt SM, Green WR, et al: H. pylori-induced apoptosis in human gastric cancer cells mediated via the release of apoptosis-inducing factor from mitochondria. Helicobacter. 2008, 13: 506-17.View ArticleGoogle Scholar
- Novoselova TV, Margulis BA, Novoselov SS, et al: Treatment with extracellular HSP70/HSC70 protein polyglutamine toxicity and aggregation. J Neurochem. 2005, 94: 597-606.View ArticlePubMedGoogle Scholar
- Robinson MB, Tidwell JL, Gould T, et al: Extracellular heat shock protein 70: a critical component for motoneuron survival. J Neurosci. 2005, 25: 9735-45.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/11/146/prepub
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