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Chai C, Tang H, Yi J, Li L, Yu C, Su Y, Miao L, Ye Z, Wang Z, Luo W, Hu J, Zhang H, Miao X, Xu H, Zhou W. Establishment and characterization of DPC-X4: a novel mixed-type ampullary cancer cell line. Hum Cell 2024; 37:531-545. [PMID: 38253956 DOI: 10.1007/s13577-023-01023-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2023] [Accepted: 12/18/2023] [Indexed: 01/24/2024]
Abstract
Mixed-type ampullary cancer is a distinct subtype of ampullary cancer that manifests a merging of the biological characteristics of both intestinal and pancreaticobiliary subtypes. The absence of established cell lines specific to this subtype has resulted in a concomitant scarcity of research on its tumorigenic mechanisms and the development of novel therapeutic modalities. The present study achieved the successful establishment of a novel mixed-type ampullary cancer cell line, designated DPC-X4 through primary culture techniques. Subsequent analyses pertaining to phenotypic characteristics, molecular profiling, biomarker identification, and histological features validated the DPC-X4 cell line as a potent model for delineating the pathogenesis of mixed-type ampullary cancer and facilitating the development of new pharmacological agents. This newly established cell line was subjected to continuous cultivation for 1 year, with stable passaging for over 50 generations. Notably, the DPC-X4 cell line manifested typical morphological features associated with epithelial tumors. Furthermore, the population doubling time for the DPC-X4 cell line was determined at 70 h. Short tandem repeat (STR) analysis confirmed that the DPC-X4 cell line exhibited a high genetic concordance with the primary tumor from the patient. Karyotypic profiling indicated an abnormal sub-triploid karyotype, with representative karyotypes of 57, XXY inv (9), 14p + , 15p + , der (17), + mar. The DPC-X4 cell line demonstrated a high capacity for efficient organoid formation under suspension culture conditions. In addition, the subcutaneous inoculation of DPC-X4 cells into NXG mice led to the formation of xenografted tumors. The results of drug sensitivity testing indicated that DPC-X4 cells were sensitive to paclitaxel and resistant to oxaliplatin, 5-fluorouracil, and gemcitabine. Immunohistochemistry revealed positive expression of CK7, CK19, and CK20 in DPC-X4 cells, while CDX2 demonstrated negative expression. In addition, positive expression of E-cadherin and vimentin was identified in DPC-X4 cells, with a proliferation index indicated by Ki-67 at 70%. The findings of our study establish DPC-X4 as a novel mixed-type ampullary cancer cell line, which can serve as a potential experimental model for exploring the pathogenesis of ampullary cancer and the development of therapeutic drugs.
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Affiliation(s)
- Changpeng Chai
- The Fourth Department of General Surgery, The First Hospital of Lanzhou University, Lanzhou, 730000, China
- The Second Clinical Medical College, Lanzhou University, Lanzhou, 730000, China
| | - Huan Tang
- The Second Clinical Medical College, Lanzhou University, Lanzhou, 730000, China
| | - Jianfeng Yi
- The First Clinical Medical College, Lanzhou University, Lanzhou, 730000, China
- Department of Surgery, The First School of Clinical Medicine of Gansu University of Chinese Medicine, Lanzhou, 730000, China
| | - Lu Li
- The Fourth Department of General Surgery, The First Hospital of Lanzhou University, Lanzhou, 730000, China
| | - Cheng Yu
- The Second Clinical Medical College, Lanzhou University, Lanzhou, 730000, China
- Department of Anesthesiology, Lanzhou University Second Hospital, Lanzhou, 730000, China
| | - Yuanhui Su
- The Second Clinical Medical College, Lanzhou University, Lanzhou, 730000, China
| | - Long Miao
- The Fourth Department of General Surgery, The First Hospital of Lanzhou University, Lanzhou, 730000, China
- The Second Clinical Medical College, Lanzhou University, Lanzhou, 730000, China
| | - Zhenzhen Ye
- The Second Clinical Medical College, Lanzhou University, Lanzhou, 730000, China
- The First School of Clinical Medicine of Gansu University of Chinese Medicine, Lanzhou, 730000, China
| | - Zhengfeng Wang
- The Fourth Department of General Surgery, The First Hospital of Lanzhou University, Lanzhou, 730000, China
- The Second Clinical Medical College, Lanzhou University, Lanzhou, 730000, China
| | - Wei Luo
- The Fourth Department of General Surgery, The First Hospital of Lanzhou University, Lanzhou, 730000, China
| | - Jinjing Hu
- The Fourth Department of General Surgery, The First Hospital of Lanzhou University, Lanzhou, 730000, China
| | - Hui Zhang
- The Second Clinical Medical College, Lanzhou University, Lanzhou, 730000, China
- Department of General Surgery, Lanzhou University Second Hospital, No. 82 Cuiyingmen, Chengguan District, Lanzhou, 730000, China
| | - Xin Miao
- State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730000, China.
| | - Hao Xu
- The Fourth Department of General Surgery, The First Hospital of Lanzhou University, Lanzhou, 730000, China.
- The First Clinical Medical College, Lanzhou University, Lanzhou, 730000, China.
| | - Wence Zhou
- The Second Clinical Medical College, Lanzhou University, Lanzhou, 730000, China.
- Department of General Surgery, Lanzhou University Second Hospital, No. 82 Cuiyingmen, Chengguan District, Lanzhou, 730000, China.
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Xu H, Chai CP, Miao X, Tang H, Hu JJ, Zhang H, Zhou WC. Establishment and characterization of a new human ampullary carcinoma cell line, DPC-X1. World J Gastroenterol 2023; 29:2642-2656. [PMID: 37213400 PMCID: PMC10198051 DOI: 10.3748/wjg.v29.i17.2642] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/26/2022] [Revised: 02/17/2023] [Accepted: 04/13/2023] [Indexed: 05/23/2023] Open
Abstract
BACKGROUND An in-depth study of the pathogenesis and biological characteristics of ampullary carcinoma is necessary to identify appropriate treatment strategies. To date, only eight ampullary cancer cell lines have been reported, and a mixed-type ampullary carcinoma cell line has not yet been reported.
AIM To establish a stable mixed-type ampullary carcinoma cell line originating from Chinese.
METHODS Fresh ampullary cancer tissue samples were used for primary culture and subculture. The cell line was evaluated by cell proliferation assays, clonal formation assays, karyotype analysis, short tandem repeat (STR) analysis and transmission electron microscopy. Drug resistances against oxaliplatin, paclitaxel, gemcitabine and 5-FU were evaluated by cell counting kit-8 assay. Subcutaneous injection 1 × 106 cells to three BALB/c nude mice for xenograft studies. The hematoxylin-eosin staining was used to detect the pathological status of the cell line. The expression of biomarkers cytokeratin 7 (CK7), cytokeratin 20 (CK20), cytokeratin low molecular weight (CKL), Ki67 and carcinoembryonic antigen (CEA) were determined by immunocytochemistry assay.
RESULTS DPC-X1 was continuously cultivated for over a year and stably passaged for more than 80 generations; its population doubling time was 48 h. STR analysis demonstrated that the characteristics of DPC-X1 were highly consistent with those of the patient’s primary tumor. Furthermore, karyotype analysis revealed its abnormal sub-tetraploid karyotype. DPC-X1 could efficiently form organoids in suspension culture. Under the transmission electron microscope, microvilli and pseudopods were observed on the cell surface, and desmosomes were visible between the cells. DPC-X1 cells inoculated into BALB/C nude mice quickly formed transplanted tumors, with a tumor formation rate of 100%. Their pathological characteristics were similar to those of the primary tumor. Moreover, DPC-X1 was sensitive to oxaliplatin and paclitaxel and resistant to gemcitabine and 5-FU. Immunohistochemistry showed that the DPC-X1 cells were strongly positive for CK7, CK20, and CKL; the Ki67 was 50%, and CEA was focally expressed.
CONCLUSION Here, we have constructed a mixed-type ampullary carcinoma cell line that can be used as an effective model for studying the pathogenesis of ampullary carcinoma and drug development.
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Affiliation(s)
- Hao Xu
- The Forth Department of General Surgery, The First Hospital of Lanzhou University, Lanzhou 730000, Gansu Province, China
| | - Chang-Peng Chai
- The Forth Department of General Surgery, The First Hospital of Lanzhou University, Lanzhou 730000, Gansu Province, China
| | - Xin Miao
- State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Animal Virology of the Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730000, Gansu Province, China
| | - Huan Tang
- The Second Clinical Medical College, Lanzhou University, Lanzhou 730000, Gansu Province, China
| | - Jin-Jing Hu
- The Forth Department of General Surgery, The First Hospital of Lanzhou University, Lanzhou 730000, Gansu Province, China
| | - Hui Zhang
- Department of General Surgery, The Second Hospital of Lanzhou University, Lanzhou 730000, Gansu Province, China
| | - Wen-Ce Zhou
- The Second Clinical Medical College, Lanzhou University, Lanzhou 730000, Gansu Province, China
- Department of General Surgery, The Second Hospital of Lanzhou University, Lanzhou 730000, Gansu Province, China
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Lai ZW, Bolm L, Fuellgraf H, Biniossek ML, Makowiec F, Hopt UT, Werner M, Keck T, Bausch D, Sorio C, Scarpa A, Schilling O, Bronsert P, Wellner UF. Characterization of various cell lines from different ampullary cancer subtypes and cancer associated fibroblast-mediated responses. BMC Cancer 2016; 16:195. [PMID: 26951071 PMCID: PMC4782372 DOI: 10.1186/s12885-016-2193-5] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2015] [Accepted: 02/17/2016] [Indexed: 12/20/2022] Open
Abstract
Background Ampullary cancer is a relatively rare form of cancer and usually treated by pancreatoduodenectomy, followed by adjuvant therapy. The intestinal subtype is associated with markedly improved prognosis after resection. At present, only few cell lines are available for in vitro studies of ampullary cancer and they have not been collectively characterized. Methods We characterize five ampullary cancer cell lines by subtype maker expression, epithelial-mesenchymal transition (EMT) features, growth and invasion, drug sensitivity and response to cancer-associated fibroblast conditioned medium (CAF-CM). Results On the basis of EMT features, subtype marker expression, growth, invasion and drug sensitivity three types of cell lines could be distinguished: mesenchymal-like, pancreatobiliary-like and intestinal-like. Heterogeneous effects from the cell lines in response to CAF-CM, such as different growth rates, induction of EMT markers as well as suppression of intestinal differentiation markers were observed. In addition, proteomic analysis showed a clear difference in intestinal-like cell line from other cell lines. Conclusion Most of the available AMPAC cell lines seem to reflect a poorly differentiated pancreatobiliary or mesenchymal-like phenotype, which is consistent to their origin. We suggest that the most appropriate cell line model for intestinal-like AMPAC is the SNU869, while others seem to reflect aggressive AMPAC subtypes. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2193-5) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Zon Weng Lai
- Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany
| | - Louisa Bolm
- Clinic for Surgery, UKSH Campus Lübeck, Lübeck, Germany
| | - Hannah Fuellgraf
- Department of Pathology, University Medical Center Freiburg, Freiburg, Germany
| | - Martin L Biniossek
- Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany
| | - Frank Makowiec
- Clinic for General and Visceral Surgery, University Medical Center Freiburg, Freiburg, Germany
| | - Ulrich Theodor Hopt
- Clinic for General and Visceral Surgery, University Medical Center Freiburg, Freiburg, Germany.,Klinik für Chirurgie, Ratzeburger Allee 160, 23562, Lübeck, Germany
| | - Martin Werner
- Department of Pathology, University Medical Center Freiburg, Freiburg, Germany.,German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), D-69120, Heidelberg, Germany.,Comprehensive Cancer Center Freiburg, Freiburg, Germany
| | - Tobias Keck
- Clinic for Surgery, UKSH Campus Lübeck, Lübeck, Germany
| | - Dirk Bausch
- Clinic for Surgery, UKSH Campus Lübeck, Lübeck, Germany
| | - Claudio Sorio
- Dipartimento di Patologia, Universita di Verona, Verona, Italy
| | - Aldo Scarpa
- Dipartimento di Patologia, Universita di Verona, Verona, Italy
| | - Oliver Schilling
- Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany. .,BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany. .,German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), D-69120, Heidelberg, Germany.
| | - Peter Bronsert
- Department of Pathology, University Medical Center Freiburg, Freiburg, Germany.,German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), D-69120, Heidelberg, Germany.,Comprehensive Cancer Center Freiburg, Freiburg, Germany
| | - Ulrich Friedrich Wellner
- Clinic for Surgery, UKSH Campus Lübeck, Lübeck, Germany.,Comprehensive Cancer Center Freiburg, Freiburg, Germany.,Klinik für Chirurgie, Ratzeburger Allee 160, 23562, Lübeck, Germany
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Smith JP, Verderame MF, Ballard EN, Zagon IS. Functional significance of gastrin gene expression in human cancer cells. ACTA ACUST UNITED AC 2004; 117:167-73. [PMID: 14749036 DOI: 10.1016/j.regpep.2003.10.013] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
The gastrointestinal peptide, gastrin, stimulates the growth of human pancreatic cancer. A receptor for gastrin activity, the cholecystokinin-C (CCK-C) receptor, has been identified in binding assays, cloned and sequenced, and is a splice variant of the CCK-B receptor. The relationship of gastrin and the CCK-C receptor to the growth of cancer cells was examined in vitro and in vivo. Stable transfection of the sense cDNA of gastrin into human MDA Amp-7 ampullary cancer cells, which normally lack gastrin gene expression but possess CCK-C receptors, increased cell growth up to 10-fold over wild type (WT) and vector-transfected (VT) cells. MDA Amp-7 tumors of gastrin-transfected cells reduced latency time for a visible tumor by 35%, decreased the timetable of tumor incidence, and increased tumor size by at least 2-fold in comparison to WT and VT groups. Transfection of human BxPC-3 pancreatic cancer cells, which normally express gastrin and possess CCK-C receptors, with the antisense cDNA to human gastrin decreased cell number by 30% in culture and tumor size by 53% compared to the WT and VT groups. Transfection of sense gastrin cDNA to monkey COS-1 cells, which normally lack both the gastrin and the CCK-C receptor genes, had no effect on growth. These studies demonstrate that gastrin and the CCK-C receptor form an autocrine loop in human pancreatic cancer that plays a role in regulating growth.
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Affiliation(s)
- Jill P Smith
- Department of Medicine, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey, PA 17033, USA.
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Fernández-Salas E, Peracaula R, Frazier ML, de Llorens R. Ribonucleases expressed by human pancreatic adenocarcinoma cell lines. EUROPEAN JOURNAL OF BIOCHEMISTRY 2000; 267:1484-94. [PMID: 10691987 DOI: 10.1046/j.1432-1327.2000.01148.x] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Human ribonucleases have been considered as a possible tumor marker for pancreatic cancer, and elevated serum levels of ribonuclease activity in patients with pancreatic cancer have been reported by many authors. The reason for this elevation is unknown. In this study, we demonstrate that human pancreatic adenocarcinoma cell lines synthesize and secrete different ribonucleases. We isolated and characterized human pancreatic, or secretory, ribonuclease (RNase 1) from the conditioned media of the human pancreatic adenocarcinoma cell lines Capan-1, MDAPanc-3, IBF-CP3 and Panc-1, and the ampullary adenocarcinoma cell line MDAAmp-7, which represent a wide range of differentiation stages. Only one of these cell lines, Panc-1, produces significant amounts of nonsecretory ribonuclease. We then established a purification procedure for both secretory and nonsecretory ribonucleases, consisting of concentration of the supernatant by tangential filtration, anion-exchange and cation-exchange liquid chromatography and C4 RP-HPLC. Ribonuclease activity fractions were monitored using both the spectrophotometric and negative-staining zymogram techniques. The results of N-terminal sequence analysis, kinetic analysis and endoglycosidase digestion studies indicate that the main ribonuclease secreted by all the cell lines is the secretory-type ribonuclease and that it is composed of several differently N-glycosylated forms. Northern blot analyses confirm that some of the cell lines express secretory ribonuclease mRNA. The mRNA levels produced by Panc-1 and MDAPanc-28 are too low to be detected. Similar levels of expression of nonsecretory ribonuclease are found by Northern blot analysis in all the cell lines except Panc-1, which expresses higher levels. Here, we describe, for the first time, that several human pancreatic cancer cell lines with different degrees of differentiation express and secrete ribonucleases. This fact indicates that one origin of the elevated serum RNase levels in patients with pancreatic cancer are tumor cells. Analysis of the oligosaccharide moiety of the RNase 1 secreted by Capan-1 shows that it is highly glycosylated and its N-glycan chains are significantly different from that of the RNase 1 produced by normal pancreas. These results renew the possibility of using human serum RNase 1 determination as a tumor marker.
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Affiliation(s)
- E Fernández-Salas
- Unitat de Bioquímica, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Spain
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Smith JP, Verderame MF, Zagon IS. Antisense oligonucleotides to gastrin inhibit growth of human pancreatic cancer. Cancer Lett 1999; 135:107-12. [PMID: 10077228 DOI: 10.1016/s0304-3835(98)00279-1] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
Human pancreatic cancer is stimulated by the autocrine production of gastrin. In this study, the effects of administration of antisense oligonucleotides to gastrin on growth of pancreatic cancer were evaluated in vitro and in vivo. Log phase BxPC-3 human pancreatic cancer cells in culture were exposed to increasing concentrations (0.5-10 microM) of a synthetic 20-mer antisense phosphorothioate oligonucleotide to gastrin for 48 h and growth was assessed by the cellular proliferation assay. Growth was inhibited up to 88% by anti-gastrin oligonucleotides in a dose-related fashion compared to cells treated with diluent or a randomized sequence with the same composition as the anti-gastrin oligonucleotide. In vivo nude mice bearing BxPC-3 xenografts were treated daily for 14 days with a 0.1-ml intratumoral injection of either anti-gastrin (5 microM), the scrambled sequence control phosphorothioate oligonucleotide (5 microM), or buffer. Tumors from the anti-gastrin-treated mice were significantly smaller in volume and weight and had less gastrin detected by radioimmunoassay than either controls. These results support the role of gastrin as a stimulatory peptide for growth of human pancreatic cancer. Antisense oligonucleotide to gastrin may have a role in the future treatment of patients with pancreatic cancer.
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Affiliation(s)
- J P Smith
- Department of Medicine, The Milton S. Hershey Medical Center, Pennsylvania State University 17033, USA
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Fernández E, Fallon MJ, Frazier ML, de Llorens R, Cuchillo CM. Expression of acinar and ductal products in Capan-1 cells growing in synthetic serum and serum-free media. Cancer 1994; 73:2285-95. [PMID: 7513248 DOI: 10.1002/1097-0142(19940501)73:9<2285::aid-cncr2820730909>3.0.co;2-m] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
BACKGROUND Capan-1 is a human pancreatic adenocarcinoma cell line of presumed ductal origin. This is based on the histologic appearance of the tumor from which it arose. Yet considerable controversy exists regarding the actual cell of origin for these exocrine carcinomas. Two acinar antigens, ribonuclease and trypsin, were analyzed in cells growing in synthetic serum. METHODS Capan-1 cells were adapted to grow in basal medium supplemented with synthetic serum, because fetal bovine serum (FBS) normally used to culture cells contains bovine ribonuclease, which can interfere with measurements of the ribonuclease secretion. These cells were also adapted to grow in different serum-free media, allowing us to determine its minimal growth requirements. The presence of ribonuclease in Capan-1 and PANC-1 conditioned media was monitored by activity. Other acinar and ductal markers were monitored using Northern blot analysis. RESULTS Capan-1, PANC-1, IBF-CP3, and MDAAmp-7 cell lines were successfully adapted to grow in synthetic serum by means of the adaptation protocol reported here. The adaptation of Capan-1 to serum-free media showed that the cells are capable of growing in a medium containing insulin, transferrin, selenium, a nonprotein carrier, and lipoic and linoleic acids. Northern blot analysis showed the expression of carbonic anhydrase II, cytokeratin 18, ribonuclease, and trypsin in Capan-1 cells growing in FBS and synthetic serum. No changes in morphology, karyotype, or gene expression were observed in these cells as a result of the adaptation process. CONCLUSION The cell line Capan-1 is expressing some ductal as well as acinar products despite its supposed ductal origin. The expression of trypsin at the mRNA level and ribonuclease at mRNA and protein levels is shown in Capan-1 cells. The protein expression will be further investigated as the cell line has been adapted to grow in synthetic serum and serum-free media with no apparent changes with respect to their growth in FBS.
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Affiliation(s)
- E Fernández
- Institut de Biologia Fonamental Vicent Villar Palasi, Universitat Autònoma de Barcelona, Spain
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