全部 标题 作者
关键词 摘要

OALib Journal期刊
ISSN: 2333-9721
费用：99美元
投递稿件

查看量下载量

相关文章

更多...

Transcriptome Analyses of Inhibitor-treated Schistosome Females Provide Evidence for Cooperating Src-kinase and TGFβ Receptor Pathways Controlling Mitosis and Eggshell Formation

DOI: 10.1371/journal.ppat.1003448

Full-Text   Cite this paper   Add to My Lib

Abstract:

Schistosome parasites cause schistosomiasis, one of the most prevalent parasitemias worldwide affecting humans and animals. Constant pairing of schistosomes is essential for female sexual maturation and egg production, which causes pathogenesis. Female maturation involves signaling pathways controlling mitosis and differentiation within the gonads. In vitro studies had shown before that a Src-specific inhibitor, Herbimycin A (Herb A), and a TGFβ receptor (TβR) inhibitor (TRIKI) have physiological effects such as suppressed mitoses and egg production in paired females. As one Herb A target, the gonad-specifically expressed Src kinase SmTK3 was identified. Here, we comparatively analyzed the transcriptome profiles of Herb A- and TRIKI-treated females identifying transcriptional targets of Src-kinase and TβRI pathways. After demonstrating that TRIKI inhibits the schistosome TGFβreceptor SmTβRI by kinase assays in Xenopus oocytes, couples were treated with Herb A, TRIKI, or both inhibitors simultaneously in vitro. RNA was isolated from females for microarray hybridizations and transcription analyses. The obtained data were evaluated by Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA), but also by manual classification and intersection analyses. Finally, extensive qPCR experiments were done to verify differential transcription of candidate genes under inhibitor influence but also to functionally reinforce specific physiological effects. A number of genes found to be differentially regulated are associated with mitosis and differentiation. Among these were calcium-associated genes and eggshell-forming genes. In situ hybridization confirmed transcription of genes coding for the calcium sensor hippocalcin, the calcium transporter ORAI-1, and the calcium-binding protein calmodulin-4 in the reproductive system pointing to a role of calcium in parasite reproduction. Functional qPCR results confirmed an inhibitor-influenced, varying dependence of the transcriptional activities of Smp14, Smp48, fs800, a predicted eggshell precursor protein and SmTYR1. The results show that eggshell-formation is regulated by at least two pathways cooperatively operating in a balanced manner to control egg production.

 References

[1]  Basch PF (1991) Schistosomes: development, reproduction, and host relations. New York: Oxford University Press.
[2]  Platt TR, Brooks DR (1997) Evolution of the schistosomes (Digenea: Schistosomatoidea): the origin of dioecy and colonization of the venous system. J Parasitol 83: 1035–1044. doi: 10.2307/3284358
[3]  World Health Organization: World health report 2004 - changing history. Geneva: WHO Library Cataloguing-in-Publication Data.
[4]  Steinmann P, Keiser J, Bos R, Tanner M, Utzinger J (2006) Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk. Lancet Infect Dis 6: 411–425. doi: 10.1016/s1473-3099(06)70521-7
[5]  King CH, Dickman K, Tisch DJ (2005) Reassessment of the cost of chronic helmintic infection: a meta-analysis of disability-related outcomes in endemic schistosomiasis. The Lancet 365: 1561–1569. doi: 10.1016/s0140-6736(05)66457-4
[6]  Ross AG, Bartley PB, Sleigh AC, Olds GR, Li Y, et al. (2002) Schistosomiasis. N Eng J Med 346: 1212–1220. doi: 10.1056/nejmra012396
[7]  Manson-Bahr PEC (1982) Apted FIC, Eds. Manson's tropical diseases. London: Bailliere Tindall.
[8]  Kunz W (2001) Schistosome male-female interaction: induction of germ-cell differentiation. Trends Parasitol 17: 227–231. doi: 10.1016/s1471-4922(01)01893-1
[9]  Popiel I, Basch PF (1984) Reproductive development of female Schistosoma mansoni (Digenea: Schistosomatidae) following bisexual pairing of worms and worm segements. J Exp Zool 232: 141–150. doi: 10.1002/jez.1402320117
[10]  LoVerde PT (2002) Presidential address. Sex and schistosomes: an interesting biological interplay with control implications. J Parasitol 88: 3–13. doi: 10.2307/3285383
[11]  Knobloch J, Beckmann S, Burmeister C, Quack T, Grevelding CG (2007) Tyrosine kinase and cooperative TGFbeta signaling in the reproductive organs of Schistosoma mansoni. Exp Parasitol 117: 318–336. doi: 10.1016/j.exppara.2007.04.006
[12]  LoVerde PT, Osman A, Hinck A (2007) Schistosoma mansoni: TGF-beta signaling pathways. Exp Parasitol 117: 304–317. doi: 10.1016/j.exppara.2007.06.002
[13]  Freitas TC, Jung E, Pearce EJ (2007) TGF-beta signaling controls embryo development in the parasitic flatworm Schistosoma mansoni. PLoS Pathog 3: e52. doi: 10.1371/journal.ppat.0030052
[14]  Beckmann S, Quack T, Burmeister C, Buro C, Long T, et al. (2010) Schistosoma mansoni: signal transduction processes during the development of the reproductive organs. Parasitology 137: 497–520. doi: 10.1017/s0031182010000053
[15]  Verjovski-Almeida S, Leite LC, Dias-Neto E, Menck CF, Wilson RA (2004) Schistosome transcriptome: insights and perspectives for functional genomics. Trends Parasitol 20: 304–308. doi: 10.1016/j.pt.2004.04.012
[16]  Berriman M, Haas BJ, LoVerde PT, Wilson RA, Dillon GP, et al. (2009) The genome of the blood fluke Schistosoma mansoni. Nature 460: 352–358. doi: 10.1038/nature08160
[17]  Protasio AV, Tsai IJ, Babbage A, Nichol S, Hunt M, et al. (2012) A systematically improved high quality genome and transcriptome of the human blood fluke Schistosoma mansoni. PLoS Negl Trop Dis 6: e1455. doi: 10.1371/journal.pntd.0001455
[18]  Davies SJ, Shoemaker CB, Pearce EJ (1998) A divergent member of the transforming growth factor beta receptor family from Schistosoma mansoni is expressed on the parasite surface membrane. J Biol Chem 273: 11234–11240. doi: 10.1074/jbc.273.18.11234
[19]  Forrester SG, Warfel PW, Pearce EJ (2004) Tegumental expression of a novel type II receptor serine/threonine kinase (SmRK2) in Schistosoma mansoni. Mol Biochem Parasitol 136: 149–156. doi: 10.1016/j.molbiopara.2004.03.007
[20]  Osman A, Niles EG, LoVerde PT (2004) Expression of functional Schistosoma mansoni Smad4: role in Erk-mediated transforming growth factor beta (TGF-beta) down-regulation. J Biol Chem 279: 6474–6486. doi: 10.1074/jbc.m310949200
[21]  Beall MJ, McGonigle S, Pearce EJ (2000) Functional conservation of Schistosoma mansoni Smads in TGF-beta signaling. Mol Biochem Parasitol 111: 131–142. doi: 10.1016/s0166-6851(00)00307-8
[22]  Rossi A, Pica-Mattoccia L, Cioli D, Klinkert MQ (2002) Rapamycin insensitivity in Schistosoma mansoni is not due to FKBP12 functionality. Mol Biochem Parasitol 125: 1–9. doi: 10.1016/s0166-6851(02)00207-4
[23]  Knobloch J, Rossi A, Osman A, LoVerde PT, Klinkert MQ, et al. (2004) Cytological and biochemical evidence for a gonad-preferential interplay of SmFKBP12 and SmTbetaR-I in Schistosoma mansoni. Mol Biochem Parasitol 138: 227–236. doi: 10.1016/j.molbiopara.2004.09.006
[24]  Osman A, Niles EG, LoVerde PT (2001) Identification and characterization of a Smad2 homologue from Schistosoma mansoni, a transforming growth factor-beta signal transducer. J Biol Chem 276: 100072–100082. doi: 10.1074/jbc.m005933200
[25]  Moustakas A, Heldin CH (2009) The regulation of TGFbeta signal transduction. Development 136: 3699–3714. doi: 10.1242/dev.030338
[26]  Beall MJ, Pearce EJ (2001) Human transforming growth factor-beta activates a receptor serine/threonine kinase from the intravascular parasite Schistosoma mansoni. J Biol Chem 276: 31613–31619. doi: 10.1074/jbc.m104685200
[27]  Oliveira KC, Carvalho ML, Verjovski-Almeida S, LoVerde PT (2012) Effect of human TGF-β on the gene expression profile of Schistosoma mansoni adult worms. Mol Biochem Parasitol 183: 132–139. doi: 10.1016/j.molbiopara.2012.02.008
[28]  Kapp K, Knobloch J, Schüssler P, Sroka S, Lammers R, et al. (2004) The Schistosoma mansoni Src kinase TK3 is expressed in the gonads and likely involved in cytoskeletal organization. Mol Biochem Parasitol 13: 171–182. doi: 10.1016/j.molbiopara.2004.07.010
[29]  Knobloch J, Kunz W, Grevelding CG (2006) Herbimycin A suppresses mitotic activity and egg production of female Schistosoma mansoni. International Journal for Parasitology 36: 1261–1272. doi: 10.1016/j.ijpara.2006.06.004
[30]  Sadler SE, Maller JL (1983) The development of competence for meiotic maturation during oogenesis in Xenopus laevis. Dev Biol 98: 165–172. doi: 10.1016/0012-1606(83)90345-7
[31]  Long T, Cailliau K, Beckmann S, Browaeys E, Trolet J, et al. (2010) Schistosoma mansoni Polo-like kinase 1: A mitotic kinase with key functions in parasite reproduction. Int J Parasitol 40: 1075–1086. doi: 10.1016/j.ijpara.2010.03.002
[32]  Vicogne J, Cailliau K, Tulasne D, Browaeys E, Yan YT, et al. (2004) Conservation of epidermal growth factor receptor function in the human parasitic helminth Schistosoma mansoni. J Biol Chem 279: 37407–37414. doi: 10.1074/jbc.m313738200
[33]  Beckmann S, Buro C, Dissous C, Hirzmann J, Grevelding CG (2010) The Syk kinase SmTK4 of Schistosoma mansoni is involved in the regulation of spermatogenesis and oogenesis. PLoS Pathog 6(2): e1000769. doi: 10.1371/journal.ppat.1000769
[34]  Beckmann S, Hahnel S, Cailliau K, Vanderstraete M, Browaeys E, et al. (2011) Characterization of the Src/Abl hybrid kinase SmTK6 of Schistosoma mansoni. J Biol Chem 286: 42325–42336. doi: 10.1074/jbc.m110.210336
[35]  Wieser R, Wrana JL, Massagué J (1995) GS domain mutations that constitutively activate T beta R-I, the downstream signaling component in the TGF-beta receptor complex. EMBO J 14: 2199–2208.
[36]  Verjovski-Almeida S, Venancio T M, Oliveira K C, Almeida GT, DeMarco R (2007) Use of a 44k oligoarray to explore the transcriptome of Schistosoma mansoni adult worms. Exp Parasiol 117: 236–245. doi: 10.1016/j.exppara.2007.04.005
[37]  Oliveira KC, Carvalho ML, Maracaja-Coutinho V, Kitajima JP, Verjovski-Almeida S (2011) Non-coding RNAs in schistosomes: an unexplored world. An Acad Bras Cienc 83: 673–694. doi: 10.1590/s0001-37652011000200026
[38]  Osman A, Niles E G, Verjovski-Almeida S, LoVerde PT (2006) Schistosoma mansoni TGF-beta receptor II: role in host ligand-induced regulation of a schistosome target gene. PLoS Pathog 2: e54. doi: 10.1371/journal.ppat.0020054
[39]  deWalick S, Bexkens ML, van Balkom BW, Wu YP, Smit CH, et al. (2011) The proteome of the insoluble Schistosoma mansoni eggshell skeleton. Int J Parasitol 41: 523–532. doi: 10.1016/j.ijpara.2010.12.005
[40]  Chen LL, Rekosh DM, LoVerde PT (1992) Schistosoma mansoni p48 eggshell protein gene: characterization, developmentally regulated expression and comparison to the p14 eggshell protein gene. Mol Biochem Parasitol 52: 39–52. doi: 10.1016/0166-6851(92)90034-h
[41]  Fitzpatrick JM, Hirai Y, Hirai H, Hoffman KF (2007) Schistosome egg production is dependent upon the activities of two developmentally regulated tyrosinases. FASEB J 21: 823–835. doi: 10.1096/fj.06-7314com
[42]  Dinh HK, Stavchansky S, Schuschereba ST, Stuck BE, Bowman PD (2002) Cytoprotection against thermal injury: evaluation of herbimycin A by cell viability and cDNA arrays. Pharmacogenomics J 2: 318–326. doi: 10.1038/sj.tpj.6500120
[43]  Morey JS, Ryan JC, Van Dolah FM (2006) Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR. Biol Proced Online 8: 175–193. doi: 10.1251/bpo126
[44]  K？ster B, Dargatz H, Schr？der J, Hirzmann J, Haarmann C, et al. (1988) Identification and localisation of the products of a putative eggshell precursor gene in the vitellarium of Schistosoma mansoni. Mol Biochem Parasitol 31: 183–198. doi: 10.1016/0166-6851(88)90169-7
[45]  Tzingounis AV, Kobayashi M, Takamatsu K, Nicoll RA (2007) Hippocalcin gates the calcium activation of the slow afterhyperpolarization in hippocampal pyramidal cells. Neuron 53: 487–493. doi: 10.1016/j.neuron.2007.01.011
[46]  Kim KS, Kobayashi M, Takamatsu K, Tzingounis AV (2012) Hippocalcin and KCNQ channels contribute to the kinetics of the slow afterhyperpolarization. Biophys J 103: 2446–2454. doi: 10.1016/j.bpj.2012.11.002
[47]  Rao KH (1959) Observations on the Mehlis' gland complex in the liver fluke Fasciola hepatica L. J Parasitol 45: 347–351. doi: 10.2307/3274512
[48]  Shaw JR, Erasmus DA (1981) Schistosoma mansoni: an examination of the reproductive status of females from single sex infections. Parasitology 82: 121–124. doi: 10.1017/s0031182000041913
[49]  Moczon T, Swiderski Z, Huggel H (1992) Schistosoma mansoni: the chemical nature of the secretions produced by the Mehlis' gland and ootype as revealed by cytochemical studies. Int J Parasitol 22: 65–73. doi: 10.1016/0020-7519(92)90081-u
[50]  Roberts-Thomson SJ, Peters AA, Grice DM, Monteith GR (2010) ORAI-mediated calcium entry: mechanism and roles, diseases and pharmacology. Pharmacol Ther 127: 121–130. doi: 10.1016/j.pharmthera.2010.04.016
[51]  Quack T, Knobloch J, Beckmann S, Vicogne J, Dissous C, Grevelding CG (2009) The formin-homology protein SmDia interacts with the Src kinase SmTK and the GTPase SmRho1 in the gonads of Schistosoma mansoni. PLoS One 4: e6998. doi: 10.1371/journal.pone.0006998
[52]  Downs JA, Nussenzweig MC, Nussenzweig A (2007) Chromatin dynamics and the preservation of genetic information. Nature 447: 951–958. doi: 10.1038/nature05980
[53]  Faix J, Grosse R (2006) Staying in shape with formins. Dev Cell 10: 693–706. doi: 10.1016/j.devcel.2006.05.001
[54]  Barrett LW, Fletcher S, Wilton SD (2012) Regulation of eukaryotic gene expression by the untranslated gene regions and other non-coding elements. Cell Mol Life Sci 69: 3613–3134. doi: 10.1007/s00018-012-0990-9
[55]  Karki S, Holzbaur EL (1999) Cytoplasmic dynein and dynactin in cell division and intracellular transport. Curr Opin Cell Biol 11: 45–53. doi: 10.1016/s0955-0674(99)80006-4
[56]  Nawaratna SS, McManus DP, Moertel L, Gobert GN, Jones MK (2011) Gene Atlasing of digestive and reproductive tissues in Schistosoma mansoni. PLoS Negl Trop Dis 5: e1043. doi: 10.1371/journal.pntd.0001043
[57]  Gobert GN, McManus DP, Nawaratna S, Moertel L, Mulvenna J, Jones MK (2009) Tissue specific profiling of females of Schistosoma japonicum by integrated laser microdissection microscopy and microarray analysis. PLoS Negl Trop Dis 3: e469. doi: 10.1371/journal.pntd.0000469
[58]  Wang C, Tai Y, Lisanti MP, Liao DJ (2011) c-Myc induction of programmed cell death may contribute to carcinogenesis: a perspective inspired by several concepts of chemical carcinogenesis. Canc Biol Ther 11: 615–626. doi: 10.4161/cbt.11.7.14688
[59]  Furstoss O, Dorey K, Simon V, Barilà D, Superti-Furga G, et al. (2002) c-Abl is an effector of Src for growth factor-induced c-myc expression and DNA synthesis. EMBO J 21: 514–524. doi: 10.1093/emboj/21.4.514
[60]  Aveic S, Pigazzi M, Basso G (2011) BAG1 (2011) the guardian of anti-apoptotic proteins in acute myeloid leukemia. PLoS One 6: e26097. doi: 10.1371/journal.pone.0026097
[61]  Galanti SE, Huang SC, Pearce EJ (2012) Cell death and reproductive regression in female Schistosoma mansoni. PLoS NeglTrop Dis 6: e1509. doi: 10.1371/journal.pntd.0001509
[62]  Amici M, Doherty A, Jo J, Jane D, Cho K, et al. (2009) Neuronal calcium sensors and synaptic plasticity. Biochem Soc Trans 37(PT 6): 1359–1363. doi: 10.1042/bst0371359
[63]  Yá？ez M, Gil-Longo J, Campos-Toimil M (2012) Calcium binding proteins. Advances in Exp Med Biol 740: 461–482. doi: 10.1007/978-94-007-2888-2_19
[64]  Magee RM, Fairweather I, Johnston CF, Halton DW, Shaw C (1989) Immunocytochemical demonstration of neuropeptides in the nervous system of the liver fluke, Fasciola hepatica (Trematoda, Digenea). Parasitology 98: 227–238. doi: 10.1017/s0031182000062132
[65]  Brownlee DJ, Fairweather I, Johnston CF, Thorndyke MC, Skuce PJ (1995) Immunocytochemical demonstration of a SALMFamide-like neuropeptide in the nervous system of adult and larval stages of the human blood fluke, Schistosoma mansoni. Parasitology 110: 143–153. doi: 10.1017/s0031182000063903
[66]  Venkiteswaran G, Hasan G (2009) Intracellular Ca2+ signaling and store-operated Ca2+ entry are required in Drosophila neurons for flight. Proc Natl Acad Sci USA 106: 10326–10331. doi: 10.1073/pnas.0902982106
[67]  Strange K, Yan X, Lorin-Nebel C, Xing J (2007) Physiological roles of STIM1 and Orai1 homologs and CRAC channels in the genetic model organism Caenorhabditis elegans. Cell Calcium 42: 193–203. doi: 10.1016/j.ceca.2007.02.007
[68]  Wang T, Donahoe PK (2004) The immunophilin FKBP12 a molecular guardian of the TGF-beta family type I receptors. Frontiers Biosci 9: 619–631. doi: 10.2741/1095
[69]  Bradshaw JM (2010) The Src, Syk, and Tec family kinases: distinct types of molecular switches. Cell Signal 22: 1175–1184. doi: 10.1016/j.cellsig.2010.03.001
[70]  McGinnis LK, Carroll DJ, Kinsey WH (2011) Protein tyrosine kinase signaling during oocyte maturation and fertilization. Mol Reprod Dev 78(10–11): 831–845. doi: 10.1002/mrd.21326
[71]  Freitas TC, Jung E, Pearce EJ (2008) A bone morphogenetic protein homologue in the parasitic flatworm, Schistosoma mansoni. Int J Parasitol 39: 281–287. doi: 10.1016/j.ijpara.2008.08.001
[72]  Vogt J, Traynor R, Sapkota GP (2011) The specificities of small molecule inhibitors of the TGF？ and BMP pathways. Cell Signal 23: 1831–1842. doi: 10.1016/j.cellsig.2011.06.019
[73]  Sawyer JS, Anderson BD, Beight DW, Campbell RM, Jones ML, et al. (2003) Synthesis and activity of new aryl- and heteroaryl-substituted pyrazole inhibitors of the transforming growth factor-β type receptor kinase domain. J Med Chem 46: 3953–3956. doi: 10.1021/jm0205705
[74]  Li HY, Wang Y, Heap CR, King CH, Mundla SR, et al. (2006) Dihydropyrrolopyrazole transforming growth factor-beta type I receptor kinase domain inhibitors: a novel benzimidazole series with selectivity versus transforming growth factor-beta type II receptor kinase and mixed lineage kinase-7. J. Med Chem 49: 2138–2142. doi: 10.1021/jm058209g
[75]  Zhang X, Arnott JA, Rehman S, Delong WG Jr, Sanjay A, et al. (2010) Src is a major signaling component for CTGF induction by TGF-beta1 in osteoblasts. J Cell Physiol 224: 691–701. doi: 10.1002/jcp.22173
[76]  Huang SC, Freitas TC, Amiel E, Everts B, Pearce EL, et al. (2012) Fatty acid oxidation is essential for egg production by the parasitic flatworm Schistosoma mansoni. PLoS Pathog 8: e1002996. doi: 10.1371/journal.ppat.1002996
[77]  Doenhoff MJ, Hagan P, Cioli D, Southgate V, Pica-Mattoccia L, et al. (2009) Praziquantel: its use in control of schistosomiasis in sub-Saharan Africa and current research needs. Parasitology 136: 1825–1835. doi: 10.1017/s0031182009000493
[78]  Dissous C, Grevelding CG (2011) Piggy-backing the concept of cancer drugs for schistosomiasis treatment: a tangible perspective? Trends Parasitol 27: 59–66. doi: 10.1016/j.pt.2010.09.001
[79]  G？nnert R (1955) Schistosomiasis studies I Contributions to the anatomy and histology of Schistosoma mansoni. Z Tropenmed Parasitol 6: 18–33.
[80]  Grevelding CG (1995) The female-specific W1 sequence of the Puerto Rican strain of Schistosoma mansoni occurs in both genders of a Liberian strain. Mol Biochem Parasitol 71: 269–272. doi: 10.1016/0166-6851(94)00058-u
[81]  McClelland M, Nelson M (1992) Effect of site-specific methylation on DNA modificationmethyltransferases and restriction endonucleases. Nucl Acids Res 11(20 Suppl): 2145–2157. doi: 10.1093/nar/20.suppl.2145
[82]  Quackenbush J (2002) Microarray data normalization and transformation. Nature Genet 32(Suppl): 496–501. doi: 10.1038/ng1032
[83]  Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Nat Acad Sci USA 98: 5116–5121. doi: 10.1073/pnas.091062498
[84]  Kaushal D, Naeve CW (2004) Analyzing and visualizing expression data with Spotfire. Curr Prot Bioinf Chapter 7: Unit 79. doi: 10.1002/0471250953.bi0709s7
[85]  Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, et al. (2000) Gene ontology: tool for the unification of biology; The Gene Ontology Consortium. Nature Genet 25: 25–29.
[86]  Robinson PN, Wollstein A, B？hme U, Beattie B (2004) Ontologizing gene-expression microarray data: characterizing clusters with Gene Ontology. Bioinformatics 20: 979–981. doi: 10.1093/bioinformatics/bth040
[87]  Grossmann S, Bauer S, Robinson PN, Vingron M (2007) Improved detection of overrepresentation of Gene-Ontology annotations with parent child analysis. Bioinformatics 23: 3024–3031. doi: 10.1093/bioinformatics/btm440
[88]  Benjamini Y, Drai D, Elmer G, Kafkafi N, Golani I (2001) Controlling the false discovery rate in behavior genetics research. Behav Brain Res 125: 279–284. doi: 10.1016/s0166-4328(01)00297-2
[89]  Thomas S, Bonchev D (2010) A survey of current software for network analysis in molecular biology. Human Gen 4: 353–360. doi: 10.1186/1479-7364-4-5-353
[90]  Dorak MT (2008) Real-time PCR. Oxford: Taylor & Francis, pp. 58–62.
[91]  Yun JJ, Heisler LE, Hwang II, Wilkins O, Lau SK, et al. (2006) Genomic DNA functions as a universal external standard in quantitative real-time PCR. Nucl Acids Res 34: e85. Erratum in: Nucl Acids Res 34: 6718. doi: 10.1093/nar/gkl400
[92]  Myers JL, Well AD (2003) Research Design and Statistical Analysis (2nd ed). Lawrence Erlbaum, p 508.
[93]  Wu AW, Gu J, Li ZF, Ji JF, Xu GW (2004) COX-2 expression and tumor angiogenesis in colorectal cancer. World J Gastroent 10: 2323–2326.
[94]  Neumann S, Ziv E, Lantner F, Schechter I (1992) Cloning and sequencing of an Hsp70 gene of Schistosoma mansoni. Mol Biochem Parasitol 56: 357–360. doi: 10.1016/0166-6851(92)90188-p

Full-Text

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133