Abstract
Echinococcus multilocularis is the etiological agent of alveolar echinococcosis (AE), a serious parasitic disease in the Northern Hemisphere. The E. multilocularis primary cell cultivation system, together with E. multilocularis genome data and a range of pioneering molecular-based tools have advanced the research on this and other cestodes. RNA interference (RNAi) and microRNA knock-down have recently contributed to the study of the cellular and molecular basis of tapeworm development and host-parasite interaction. These, as well as other techniques, normally involve an electroporation step for the delivery of RNA, DNA, peptides, and small molecules into cells. Using transcriptome data and bioinformatic analyses, we herein report a genome-wide comparison between primary cells of E. multilocularis and primary cells under electroporated conditions after 48 h of culture. We observed that ~ 15% of genes showed a significant variation in expression level, including highly upregulated genes in electroporated cells, putatively involved in detoxification and membrane remodeling. Furthermore, we found genes related to carbohydrate metabolism, proteolysis, calcium ion binding and microtubule processing significantly altered, which could explain the cellular dispersion and the reduced formation of cellular aggregates observed during the first 48 h after electroporation.
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Data availability
The RNA-seq data are available in the European Nucleotide Archive (ENA) accession number ERP106379.
Code availability
All third party bioinformatics tools are available for academic use and can be downloaded in their respective repositories. In-house R code, as well as raw gene expression data and annotations are available at GitHub (https://github.com/natinreg/PR_Emultilocularis_PC).
References
Akbarzadeh A, Rezaei-Sadabady R, Davaran S, Joo SW, Zarghami N, Hanifehpour Y, Samiei M, Kouhi M, Nejati-Koshki K (2013) Liposome: classification, preparation, and applications. Nanoscale Res Lett 8(1):102. https://doi.org/10.1186/1556-276X-8-102
Allen MA, Hillier LW, Waterston RH, Blumenthal T (2011) A global analysis of C. elegans trans-splicing. Genome Res 21(2):255–64. https://doi.org/10.1101/gr.113811.110
Alvite G, Esteves A (2012) Lipid binding proteins from parasitic platyhelminthes. Front Physiol 3:363. https://doi.org/10.3389/fphys.2012.00363
Bélgamo JA, Alberca LN, Pórfido JL, Romero FNC, Rodriguez S, Talevi A, Córsico B, Franchini GR (2020) Application of target repositioning and in silico screening to exploit fatty acid binding proteins (FABPs) from Echinococcus multilocularis as possible drug targets. J Comput Aided Mol Des. 34(12):1275–1288. https://doi.org/10.1007/s10822-020-00352-8
Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics (Oxford, England) 30(15):2114–2120. https://doi.org/10.1093/bioinformatics/btu170
Boyle EI, Weng S, Gollub J, Jin H, Botstein D, Cherry M, Sherlock G (2004) GO:TermFinder—open source software for accessing Gene Ontology information and finding significantly enriched Gene Ontology terms associated with a list of genes. Bioinformatics 20(18):3710–3715. https://doi.org/10.1093/bioinformatics/bth456
Brehm K, Koziol U (2014) On the importance of targeting parasite stem cells in anti-echinococcosis drug development. Parasite 21:72. https://doi.org/10.1051/parasite/2014070
Britton C, Winter AD, Marks ND, Gu H, McNeilly TN, Gillan V, Devaney E (2015) Application of small RNA technology for improved control of parasitic helminths. Vet Parasitol 212(1–2):47–53. https://doi.org/10.1016/j.vetpar.2015.06.003
Brunetti E, Kern P, Vuitton DA (2010) Writing Panel for the WHO-IWGE Expert consensus for the diagnosis and treatment of cystic and alveolar echinococcosis in humans. Acta Trop 114(1):1–16. https://doi.org/10.1016/j.actatropica.2009.11.001
Canoy RJ, André F, Shmakova A, Wiels J, Lipinski M, Vassetzky Y, Germini D (2020) Easy and robust electrotransfection protocol for efficient ectopic gene expression and genome editing in human B cells. Gene Ther. https://doi.org/10.1038/s41434-020-00194-x
Chopra S, Ruzgys P, Maciulevičius M, Jakutavičiūtė M, Šatkauskas S (2020) Investigation of plasmid DNA delivery and cell viability dynamics for optimal cell electrotransfection in vitro. Appl Sci 10(17):6070. https://doi.org/10.3390/app10176070
Craig P (2003) Echinococcus multilocularis. Curr Opin Infect Dis 16(5):437–44. https://doi.org/10.1097/00001432-200310000-00010
Da’dara AA, Skelly PJ (2015) Gene suppression in schistosomes using RNAi. Methods Mol Biol 1201:143–64. https://doi.org/10.1007/978-1-4939-1438-8_8
Dalzell JJ, McVeigh P, Warnock ND, Mitreva M, Bird DM, Abad P, Fleming CC, Day TA, Mousley A, Marks NJ, Maule AG (2011) RNAi effector diversity in nematodes. PLoS Negl. Trop. Dis 5. https://doi.org/10.1371/journal.pntd.0001176
Dang Z, Yagi K, Oku Y et al (2009) Evaluation of Echinococcus multilocularis tetraspanins as vaccine candidates against primary alveolar echinococcosis. Vaccine 27:7339–7345. https://doi.org/10.1016/j.vaccine.2009.09.045
Durinck S, Spellman PT, Birney E, Huber W (2009) Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt. Nat Protoc 4(8):1184–91. https://doi.org/10.1038/nprot.2009.97
Eckert J, Deplazes P (2004) Biological, epidemiological, and clinical aspects of echinococcosis, a zoonosis of increasing concern. Clin Microbiol Rev 17(1):107–135. https://doi.org/10.1128/CMR.17.1.107-135.2004
Eckert J, Thompson RCA, Mehlhorn H (1983) Proliferation and metastases formation of larval Echinococcus multilocularis. Z Parasitenkd 69:737–748. https://doi.org/10.1007/BF00927423
Ewels P, Magnusson M, Lundin S, Käller M (2016) MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics 32(19):3047–8. https://doi.org/10.1093/bioinformatics/btw354
Farley EK, Gale E, Chambers D. et al (2011) Effects of in ovo electroporation on endogenous gene expression: genome-wide analysis. Neural Dev 6:17. 0-doi-org.brum.beds.ac.uk/https://doi.org/10.1186/1749-8104-6-17
Felleisen R, Gottstein B (1993) Echinococcus multilocularis: molecular and immunochemical characterization of diagnostic antigen II/3–10. Parasitology 107(Pt 3):335–42. https://doi.org/10.1017/s0031182000079300
Förster S, Koziol U, Schäfer T, Duvoisin R, Cailliau K, Vanderstraete M, Dissous C, Brehm K (2019) The role of fibroblast growth factor signalling in Echinococcus multilocularis development and host-parasite interaction. PLoS Negl Trop Dis 13(3):e0006959. https://doi.org/10.1371/journal.pntd.0006959
Gehl J (2003) Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research. Acta Physiol Scand 177(4):437–447. https://doi.org/10.1046/j.1365-201X.2003.01093
Hancock K, Pattabhi S, Whitfield FW et al (2006) Characterization and cloning of T24, a Taenia solium antigen diagnostic for cysticercosis. Mol Biochem Parasitol 147:109–117. https://doi.org/10.1016/j.molbiopara.2006.02.004
Hemer S, Konrad C, Spiliotis M, Koziol U, Schaack D, Forster S et al (2014) Host insulin stimulates Echinococcus multilocularis insulin signalling pathways and larval development. BMC Biol 12. https://doi.org/10.1186/1741-7007-12-5
Huang F, Dang Z, Suzuki Y, Horiuchi T, Yagi K, Kouguchi H, Irie T, Kim K, Oku Y (2016) Analysis on gene expression profile in oncospheres and early stage metacestodes from Echinococcus multilocularis. PLoS Negl Trop Dis 10(4):e0004634. https://doi.org/10.1371/journal.pntd.0004634
Jiang B, Liang P, Deng G, Tu Z, Liu M, Xiao X (2011) Increased stability of Bcl-2 in HSP70-mediated protection against apoptosis induced by oxidative stress. Cell Stress & Chaperones 16:143–152. https://doi.org/10.1007/s12192-010-0226-6
Jordan ET, Collins M, Terefe J, Ugozzoli L, Rubio T (2008) Optimizing electroporation conditions in primary and other difficult-to-transfect cells. J Biomol Tech 19(5):328–334
Kalinna BH, Brindley PJ (2007) Manipulating the manipulators: advances in parasitic helminth transgenesis and RNAi. Trends Parasitol 23(5):197–204. https://doi.org/10.1016/j.pt.2007.03.007
Kassambara A, Mundt F (2020) Factoextra: extract and visualize the results of multivariate data analyses. https://CRAN.R-project.org/package=factoextra
Kim TK, Eberwine JH (2010) Mammalian cell transfection: the present and the future. Anal Bioanal Chem 397(8):3173–3178. https://doi.org/10.1007/s00216-010-3821-6
Kolde R (2019) pheatmap: Pretty Heatmaps. R package version 1.0.12. 2019. https://CRAN.R-project.org/package=pheatmap.
Kotnik T, Rems L, Tarek M, Miklavčič D (2019) Membrane electroporation and electropermeabilization: mechanisms and models. Annu Rev Biophys 48:63–91. https://doi.org/10.1146/annurev-biophys-052118-115451
Koziol U (2017) Evolutionary developmental biology (evo-devo) of cestodes. Exp Parasitol 180:84–100. https://doi.org/10.1016/j.exppara.2016.12.004
Koziol U, Rauschendorfer T, Zanon Rodriguez L, Krohne G, Brehm K (2014) The unique stem cell system of the immortal larva of the human parasite Echinococcus multilocularis. EvoDevo 5:10. https://doi.org/10.1186/2041-9139-5-10
Lazarev VF, Nikotina AD, Mikhaylova ER, Nudler E, Polonik SG, Guzhova IV, Margulis BA (2016) Hsp70 chaperone rescues C6 rat glioblastoma cells from oxidative stress by sequestration of aggregating GAPDH, Biochemical and Biophysical Research Communications, Volume 470, Issue 3, 2016, Pages 766–771, ISSN0006–291X, https://doi.org/10.1016/j.bbrc.2015.12.076.
Luan X, Sansanaphongpricha K, Myers I, Chen H, Yuan H, Sun D (2017) Engineering exosomes as refined biological nanoplatforms for drug delivery. Acta Pharmacol Sin 38(6):754–763. https://doi.org/10.1038/aps.2017.12
Lê S, Josse J, Husson F (2008) FactoMineR: an R package for multivariate analysis. J Stat Softw 25(1):1–18
Leducq R, Gabrion C (1992) Developmental changes of Echinococcus multilocularis metacestodes revealed by tegumental ultrastructure and lectin-binding sites. Parasitology 104(Pt 1):129–41. https://doi.org/10.1017/s003118200006087x
Liebau E, Müller V, Lucius R, Walter RD, Henkle-Dührsen K (1996) Molecular cloning, expression and characterization of a recombinant glutathione S-transferase from Echinococcus multilocularis. Mol Biochem Parasitol 77(1):49–56. https://doi.org/10.1016/0166-6851(96)02578-9
Liew A, André FM, Lesueur LL, De Ménorval MA, O’Brien T, Mir LM (2013) Robust, efficient, and practical electrogene transfer method for human mesenchymal stem cells using square electric pulses. Human Gene Therapy Methods 24(5):289–297. https://doi.org/10.1089/hgtb.2012.159
Louvet-Vallée S (2000) ERM proteins: from cellular architecture to cell signaling. Biol Cell 92:305–316. https://doi.org/10.1016/S0248-4900(00)01078-9
Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome BioL 15(12):550. https://doi.org/10.1186/s13059-014-0550-8
Luft C, Ketteler R (2015) Electroporation knows no boundaries: the use of electrostimulation for siRNA delivery in cells and tissues. J Biomol Screen 20(8):932–42. https://doi.org/10.1177/1087057115579638
Mali S (2013) Delivery systems for gene therapy. Ind J Hum Genet 19(1):3–8. https://doi.org/10.4103/0971-6866.112870
Maule AG, Marks NJ (2006) Parasitic flatworms: molecular biology, biochemistry, immunology and physiology. CABI, Wallingford
McCoy CJ, Warnock ND, Atkinson LE, Atcheson E, Martin RJ, Robertson AP, Maule AG, Marks NJ, Mousley A (2015) RNA interference in adult Ascaris suum–an opportunity for the development of a functional genomics platform that supports organism-, tissue- and cell-based biology in a nematode parasite. Int J Parasitol 45(11):673–8. https://doi.org/10.1016/j.ijpara.2015.05.003
Molnar MJ, Gilbert R, Lu Y, Liu AB, Guo A, Larochelle N, Orlopp K, Lochmuller H, Petrof BJ, Nalbantoglu J, Karpati G (2004) Factors influencing the efficacy, longevity, and safety of electroporation-assisted plasmid-based gene transfer into mouse muscles. Mol Ther 10(3):447–455. https://doi.org/10.1016/j.ymthe.2004.06.642
Mousavi SM, Afgar A, Mohammadi MA, Mortezaei S, Faridi A, Sadeghi B, Fasihi Harandi M (2020) Biological and morphological consequences of dsRNA-induced suppression of tetraspanin mRNA in developmental stages of Echinococcus granulosus. Parasit Vectors. 13(1):190. https://doi.org/10.1186/s13071-020-04052-y
Mehlhorn H, Eckert J, Thompson RCA (1983) Proliferation and metastases formation of larval Echinococcus multilocularis. Z Parasitenkd 69:749–763. https://doi.org/10.1007/BF00927423
Misra S, Gupta J, Misra-Bhattacharya S (2017) RNA interference mediated knockdown of Brugia malayi UDP-galactopyranose mutase severely affects parasite viability, embryogenesis and in vivo development of infective larvae. Parasit Vectors 10(1):34. https://doi.org/10.1186/s13071-017-1967-1
Mizukami C, Spiliotis M, Gottstein B, Yagi K, Katakura K, Oku Y (2010) Gene silencing in Echinococcus multilocularis protoscoleces using RNA interference. Parasitol Int 59(4):647–52. https://doi.org/10.1016/j.parint.2010.08.010
Mlakar V, Todorovic V, Cemazar M, Glavac D, Sersa G (2009) Electric pulses used in electrochemotherapy and electrogene therapy do not significantly change the expression profile of genes involved in the development of cancer in malignant melanoma cells. BMC Cancer 9:299. https://doi.org/10.1186/1471-2407-9-299
Mousavi SM, Afgar A, Mohammadi MA, Mortezaei S, Sadeghi B, Harandi MF (2019) Calmodulin-specific small interfering RNA induces consistent expression suppression and morphological changes in Echinococcus granulosus. Sci Rep 9(1):3894. https://doi.org/10.1038/s41598-019-40656-w
Mühlschlegel F, Frosch P, Castro A et al (1995) Molecular cloning and characterization of an Echinococcus multilocularis and Echinococcus granulosus stress protein homologous to the mammalian 78 kDa glucose regulated protein. Mol Biochem Parasitol 74:245–250. https://doi.org/10.1016/0166-6851(95)02501-4
Nono JK, Lutz MB, Brehm K (2014) EmTIP, a T-Cell immunomodulatory protein secreted by the tapeworm Echinococcus multilocularis is important for early metacestode development. Dalton JP, editor. PLoS Negl Trop Dis 8(1):e2632. https://doi.org/10.1371/journal.pntd.0002632
Nono JK, Lutz MB, Brehm K (2020) Expansion of host regulatory T cells by secreted products of the tapeworm Echinococcus multilocularis. Front Immunol. 2020;11:798. Published 2020 May 8. https://doi.org/10.3389/fimmu.2020.00798
Parkinson J, Wasmuth JD, Salinas G, Bizarro CV, Sanford C, Berriman M, Ferreira HB, Zaha A, Blaxter ML, Maizels RM, Fernández C (2012) A transcriptomic analysis of Echinococcus granulosus larval stages: implications for parasite biology and host adaptation. PLoS Negl Trop Dis 6(11):e1897. https://doi.org/10.1371/journal.pntd.0001897
Pérez MG, Spiliotis M, Rego N, Macchiaroli N, Kamenetzky L, Holroyd N, Cucher MA, Brehm K, Rosenzvit MC (2019) Deciphering the role of miR-71 in Echinococcus multilocularis early development in vitro. PLoS Negl Trop Dis. 13(12):e0007932. https://doi.org/10.1371/journal.pntd.0007932
Pertea M, Kim D, Pertea GM, Leek JT, Salzberg SL (2016) Transcript-level expression analysis of RNA-seq experiments with HISAT. StringTie and Ballgown. Nat Protoc 11(9):1650–67. https://doi.org/10.1038/nprot.2016.095
Phung LT, Chaiyadet S, Hongsrichan N, Sotillo J, Dieu HDT, Tran CQ, Brindley PJ, Loukas A, Laha T (2019) Recombinant Opisthorchis viverrini tetraspanin expressed in Pichia pastoris as a potential vaccine candidate for opisthorchiasis. Parasitol Res. 118(12):3419–3427. https://doi.org/10.1007/s00436-019-06488-3
Pierson L, Mousley A, Devine L, Marks NJ, Day TA, Maule AG (2010) RNA interference in a cestode reveals specific silencing of selected highly expressed gene transcripts. Int J Parasitol 40(5):605–15. https://doi.org/10.1016/j.ijpara.2009.10.012
Pinton P, Giorgi C, Siviero R, Zecchini E, Rizzuto R (2008) Calcium and apoptosis: ER-mitochondria Ca2+ transfer in the control of apoptosis. Oncogene 27(50):6407–18. https://doi.org/10.1038/onc.2008.308
Piñero J, López-Baena M, Ortiz T, Cortés F (1997) Apoptotic and necrotic cell death are both induced by electroporation in HL60 human promyeloid leukaemia cells. Apoptosis: an international journal on programmed cell death 2(3):330–336. https://doi.org/10.1023/a:1026497306006.
Plessis L, Škunca N, Dessimoz C (2011) The what, where, how and why of gene ontology—a primer for bioinformaticians. Briefings in Bioinformatics 12(6):723–735. https://doi.org/10.1093/bib/bbr002
Potter H. (2003). Transfection by electroporation. Current protocols in molecular biology, Chapter 9, Unit–9.3. https://doi.org/10.1002/0471142727.mb0903s62.
Pórfido JL, Alvite G, Silva V, Kennedy MW, Esteves A, Corsico B (2012) Direct interaction between EgFABP1, a fatty acid binding protein from Echinococcus granulosus, and phospholipid membranes. PLoS Negl Trop Dis 6(11):e1893. https://doi.org/10.1371/journal.pntd.0001893
Pórfido JL, Herz M, Kiss F et al (2020) Fatty acid-binding proteins in Echinococcus spp.: the family has grown. Parasitol Res 119:1401–1408. https://doi.org/10.1007/s00436-020-06631-5
Pouchkina-Stantcheva NN, Cunningham LJ, Hrčkova G, Olson PD (2013) RNA-mediated gene suppression and in vitro culture in Hymenolepis microstoma. Int J Parasitol 43(8):641–6. https://doi.org/10.1016/j.ijpara.2013.03.004
Qian W, Zhang J (2008) Evolutionary dynamics of nematode operons: easy come, slow go. Genome Res 18(3):412–421. https://doi.org/10.1101/gr.7112608
Rausch, (1954) Studies on the helminth fauna of Alaska. XX. The histogenesis of the alveolar larva of Echinococcus species. J Int Dis 94:178–186
Rols MP, Teissié J (1990) Electropermeabilization of mammalian cells. Quantitative analysis of the phenomenon. Biophysical Journal 58:1089–98. https://doi.org/10.1093/infdis/94.2.178
Seigneuret M, et al (2013) Structural bases for tetraspanin functions. Tetraspanins: Springer; 2013; p1–29. https://doi.org/10.1007/978-94-007-6070-7_1
Silva-Álvarez V, Franchini GR, Pórfido JL, Kennedy MW, Ferreira AM, Córsico B (2015) Lipid-free antigen B subunits from Echinococcus granulosus: oligomerization, ligand binding, and membrane interaction properties. PLoS Negl Trop Dis 9(3):e0003552. https://doi.org/10.1371/journal.pntd.0003552.
Smyth JD, McManus DP (2007) The physiology and biochemistry of cestodes. Cambridge University Press
Solana J, Kao D, Mihaylova Y, Jaber-Hijazi F, Malla S, Wilson R, Aboobaker A (2012) Defining the molecular profile of planarian pluripotent stem cells using a combinatorial RNAseq. RNA interference and irradiation approach. Genome Biol 13:R19. https://doi.org/10.1186/gb-2012-13-3-r19
Spiliotis M, Brehm K (2009) Axenic in vitro cultivation of Echinococcus multilocularis metacestode vesicles and the generation of primary cell cultures. Methods Mol Biol 470:245–62. https://doi.org/10.1007/978-1-59745-204-5_17
Spiliotis M, Lechner S, Tappe D, Scheller C, Krohne G, Brehm K (2008) Transient transfection of Echinococcus multilocularis primary cells and complete in vitro regeneration of metacestode vesicles. Int J Parasitol 38(8–9):1025–1039. https://doi.org/10.1016/j.ijpara.2007.11.002
Spiliotis M, Mizukami C, Oku Y, Kiss F, Brehm K, Gottstein B (2010) Echinococcus multilocularis primary cells: improved isolation, small-scale cultivation and RNA interference. Mol Biochem Parasitol 174(1):83–87. https://doi.org/10.1016/j.molbiopara.2010.07.001
Stroh T, Erben U, Kühl AA, Zeitz M, Siegmund B (2010) Combined pulse electroporation–a novel strategy for highly efficient transfection of human and mouse cells. PLoS ONE 5(3):e9488. https://doi.org/10.1371/journal.pone.0009488
Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 102(43):15545–50. https://doi.org/10.1073/pnas.0506580102
Teissié J, Rols MP (1993) An experimental evaluation of the critical potential difference inducing cell membrane electropermeabilization. Biophys J 65:409–413
Tsai IJ, Zarowiecki M, Holroyd N, Garciarrubio A, Sanchez-Flores A, Brooks KL et al (2013) The genomes of four tapeworm species reveal adaptations to parasitism. Nature 496(7443):57–63. https://doi.org/10.1038/nature12031
Wagner DE, Wang IE, Reddien PW (2011) Clonogenic neoblasts are pluripotent adult stem cells that underlie planarian regeneration. Science (New York, N.Y.), 332(6031), 811–816. https://doi.org/10.1126/science.1203983
Wagner GP, Kin K, Lynch VJ (2012) Measurement of mRNA abundance using RNA-seq data: RPKM measure is inconsistent among samples. Theory Biosci 131(4):281–5. https://doi.org/10.1007/s12064-012-0162-3
Wang B, Lee J, Li P, Saberi A, Yang H, Liu C, Zhao M, Newmark PA (2018) Stem cell heterogeneity drives the parasitic life cycle of Schistosoma mansoni. eLife 7:e35449. https://doi.org/10.7554/eLife.35449
Wang W, Wan P, Lai F, Zhu T, Fu Q (2018b) Double-stranded RNA targeting calmodulin reveals a potential target for pest management of Nilaparvata lugens. Pest Manag Sci 74:1711–1719. https://doi.org/10.1002/ps.4865
Xu M, Joo H-J, Paik Y-K (2011) Novel functions of lipid-binding protein 5 in Caenorhabditis elegans fat metabolism. J Biol Chem 286:28111–28118. https://doi.org/10.1074/jbc.M111.227165
You H, Jones MK, Whitworth DJ, McManus DP (2021) Innovations and advances in schistosome stem cell research. Front Immunol 12:599014. https://doi.org/10.3389/fimmu.2021.599014
Yu G (2020). enrichplot: Visualization of functional enrichment result. R package version 1.8.1, https://github.com/GuangchuangYu/enrichplot.
Yu G, Wang LG, Han Y, He QY (2012) clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16(5):284–7. https://doi.org/10.1089/omi.2011.0118
Zhu A, Ibrahim JG, Love MI (2019) Heavy-tailed prior distributions for sequence count data: removing the noise and preserving large differences. Bioinformatics 35(12):2084–2092. https://doi.org/10.1093/bioinformatics/bty895
Zhu Y, Ren J, Da’dara A, et al (2004) The protective effect of a Schistosoma japonicum Chinese strain 23kDa plasmid DNA vaccine in pigs is enhanced with IL-12. Vaccine 23:78–83. https://doi.org/10.1016/j.vaccine.2004.04.031
Zügeli U, Kaufmann SHE (1999) Immune response against heat shock proteins in infectious diseases. Immunobiology 201:22–35. https://doi.org/10.1016/S0171-2985(99)80044-8
Acknowledgements
We thank Matt Berriman, Nancy Holroyd, and the Parasite Genomics group for providing the sequencing data for this study.
Funding
This work was supported by ERANET LAC Project ELAC2015/T080544 (to MCR and KB); the Wellcome Trust (https://wellcome.ac.uk/), grant 107475/Z/15/Z (to KB; FUGI), and grant WT 098051; Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), Argentina, Projects PICT 2017–2966 and the Agencia Nacional de Promoción Científica y Técnológica, Argentina (grant numbers PICT 2013 N°2121 and PICT 2019 N°3367) (to MCR); Consultant Laboratory for Echinococcosis of the Robert Koch Institute; Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Project: PIP 2015 (to MCR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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All experiments in animals were carried out in accordance with European and German regulations on the protection of animals (Tierschutzgesetz, Sect. 6). Ethical approval of the study was obtained by the local ethics committee of the government of Lower Franconia (permit no. 55.2–2531.01–61/13).
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Supplementary Information
Below is the link to the electronic supplementary material.
436_2022_7427_MOESM1_ESM.xlsx
Supplementary file1 TPM values for 7,448 genes where average TPM is above 1 in at least one condition. Columns A-G show TPM estimations per gene and sample. TPM.mean.EPC shows the average TPM for electroporated samples (EPC). TPM.mean.PC shows the average TPM value for non-electroporated primary cell samples (PC). Columns K-P show gene annotation obtained with biomaRt from WormBase Parasite. Those 577 genes with TPM>=50 in the non-electroporated primary cell samples are shown with a blue background. (XLSX 1138 KB)
436_2022_7427_MOESM2_ESM.xlsx
Supplementary file2 Functional analysis of genes expressed in the primary cell samples. Gene Set Enrichment Analysis for 7,324 genes with average TPM above 1 in the primary cell samples without any treatment. Over Representation Analysis of 577 top expressed genes (TPM>=50) in the primary cell samples without any treatment. Enrichment analysis was performed for the Gene Ontology terms, including the three domains: Biological Process, Molecular Function, and Cellular Component. (XLSX 21 KB)
436_2022_7427_MOESM3_ESM.xlsx
Supplementary file3 DESeq2 output for 9,197 genes are kept after a minimum pre-filtering of at least ten reads per gene. Columns A-G show the standard DESeq2 analysis (results function). Columns G-H show the output of the log2FC shrinkage by apeglm and the s-value when assessed at a threshold of 1 for the abs(log2FC). Column J (LFC1) shows the p-value associated with the specific test abs(log2FC) >=1 (during the initial Wald tests). Columns K-Q show gene information obtained with biomaRt from WormBase Parasite. (XLSX 1527 KB)
436_2022_7427_MOESM4_ESM.xlsx
Supplementary file4 Functional analysis of expression changes in the primary cell culture after electroporation treatment. Over Representation Analysis was performed on the 1,645 differentially expressed genes (adjusted p-value <= 0.05 without log2 Fold Change thresholds). ORA was also realized on the list of genes showing at least double or half gene expression under electroporation. Gene Set Enrichment Analysis was performed on the whole gene set, using the shrunken log2 fold change estimations in decreasing order as input (for 8,992 genes where DESeq2 was able to estimate p-values). Enrichment analysis was performed for the Gene Ontology terms, including the three domains: Biological Process, Molecular Function, and Cellular Component.(XLSX 24 KB)
Supplementary Fig. 1
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High Resolution Image Expression heatmaps for different genes of interest. Expression values correspond to TPMs. Expression change after electroporation is shown to the left as raw log2 fold changes and as shrunken values by apeglm. a. Expression heatmap for seven Antigen B genes. The “genes'' legend shows gene annotation as provided by WormBase Parasite. b. Expression heatmap for 37 heat shock proteins (HSPs). To the right, HSP category is indicated based on gene description provided by WormBase Parasite. PC with light blue label: biological replicates of primary cell culture without any treatment. EPC with coral label: biological replicates of primary cell culture with electroporation treatment. c. Expression heatmap for 39 tetraspanins (TSPs). The “genes'' legend shows gene annotation as provided by WormBase Parasite. (EPS 150 KB)
Supplementary Fig. 2
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High Resolution Image Histogram of p-values from the Wald tests in the differential expression analysis. The p-values follow a uniform distribution with an overabundance of low p-values, indicating a sufficiently powered experiment. To reduce the noise, only results for genes with a minimum expression level (baseMean>1) were taken into account. (EPS 15 KB)
Supplementary Fig. 3
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High Resolution Image Gene Ontology terms of the Biological Process domain describing the functional features of E. multilocularis primary cells affected by electroporation treatment. Enriched GO:BP terms were obtained by ORA of the 1,645 genes showing differential expression (adjusted p-value <=0.05). In the heatplot, for each GO:BP enriched term in the x-axis, the gene color represents its log2 fold change after apeglm shrinkage. (EPS 31 KB)
Supplementary Fig. 4
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High Resolution Image a. Ridgeplot of the GO:MF terms defined by the GSEA of 8,992 genes in decreasing order of log2 fold change after apeglm shrinkage. It shows the density distributions of log2 fold changes within each enriched GO:MF term, which helps to interpret the up- or down-regulation of the term. X-axis represents log2 fold change in expression for genes present in each GO:MF term, with positive values indicating up-regulated expression after electroporation and negative values down-regulation in electroporated samples. Peaks are colored by adjusted p-value per GO:MF term. b. Same as a., but for GO:CC terms. In both cases (GO:MF and GO:CC), after electroporation of primary cell culture, a trend in down-regulation is observed for the shown molecular function terms. (EPS 258 KB)
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Pérez, M.G., Rego, N., Spiliotis, M. et al. Transcriptional effects of electroporation on Echinococcus multilocularis primary cell culture. Parasitol Res 121, 1155–1168 (2022). https://doi.org/10.1007/s00436-022-07427-5
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DOI: https://doi.org/10.1007/s00436-022-07427-5