#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Introns mediate post-transcriptional enhancement of nuclear gene expression in the green microalga Chlamydomonas reinhardtii


Autoři: Thomas Baier aff001;  Nick Jacobebbinghaus aff001;  Alexander Einhaus aff001;  Kyle J. Lauersen aff001;  Olaf Kruse aff001
Působiště autorů: Bielefeld University, Faculty of Biology, Center for Biotechnology (CeBiTec), Universitätsstrasse, Bielefeld, Germany aff001;  Biological and Environmental Sciences and Engineering Division (BESE), King Abdullah University of Science and Technology (KAUST), Thuwal, Kingdom of Saudi Arabia aff002
Vyšlo v časopise: Introns mediate post-transcriptional enhancement of nuclear gene expression in the green microalga Chlamydomonas reinhardtii. PLoS Genet 16(7): e32767. doi:10.1371/journal.pgen.1008944
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1008944

Souhrn

Efficient nuclear transgene expression in the green microalga Chlamydomonas reinhardtii is generally hindered by low transcription rates. Introns can increase transcript abundance by a process called Intron-Mediated Enhancement (IME) in this alga and has been broadly observed in other eukaryotes. However, the mechanisms of IME in microalgae are poorly understood. Here, we identified 33 native introns from highly expressed genes in C. reinhardtii selected from transcriptome studies as well as 13 non-native introns. We investigated their IME capacities and probed the mechanism of action by modification of splice sites, internal sequence motifs, and position within transgenes. Several introns were found to elicit strong IME and found to be broadly applicable in different expression constructs. We determined that IME in C. reinhardtii exclusively occurs from introns within transcribed ORFs regardless of the promoter and is not induced by traditional enhancers of transcription. Our results elucidate some mechanistic details of IME in C. reinhardtii, which are similar to those observed in higher plants yet underly distinctly different induction processes. Our findings narrow the focus of targets responsible for algal IME and provides evidence that introns are underestimated regulators of C. reinhardtii nuclear gene expression.

Klíčová slova:

Algae – DNA transcription – Eukaryota – Gene expression – Chlamydomonas reinhardtii – Introns – Messenger RNA – Sequence motif analysis


Zdroje

1. Vannini A, Cramer P. Conservation between the RNA Polymerase I, II, and III Transcription Initiation Machineries. Mol Cell. 2012;45: 439–446. doi: 10.1016/j.molcel.2012.01.023 22365827

2. Hobert O. Gene Regulation by Transcription Factors and MicroRNAs. Science (80-). 2008;319: 1785–1786. doi: 10.1126/science.1151651 18369135

3. Chen K, Rajewsky N. The evolution of gene regulation by transcription factors and microRNAs. Nat Rev Genet. 2007;8: 93–103. doi: 10.1038/nrg1990 17230196

4. Bonasio R, Shiekhattar R. Regulation of Transcription by Long Noncoding RNAs. Annu Rev Genet. 2014;48: 433–455. doi: 10.1146/annurev-genet-120213-092323 25251851

5. Hunter T, Karin M. The regulation of transcription by phosphorylation. Cell. 1992;70: 375–387. doi: 10.1016/0092-8674(92)90162-6 1643656

6. Hernandez-Garcia CM, Finer JJ. Identification and validation of promoters and cis-acting regulatory elements. Plant Sci. 2014;217–218: 109–119. doi: 10.1016/j.plantsci.2013.12.007 24467902

7. Rose AB. Introns as Gene Regulators: A Brick on the Accelerator. Front Genet. 2019;9. doi: 10.3389/fgene.2018.00672 30792737

8. Cramer P. Organization and regulation of gene transcription. Nature. 2019;573: 45–54. doi: 10.1038/s41586-019-1517-4 31462772

9. Smale ST, Kadonaga JT. The RNA Polymerase II Core Promoter. Annu Rev Biochem. 2003;72: 449–479. doi: 10.1146/annurev.biochem.72.121801.161520 12651739

10. Kolkman JA, Stemmer WPC. Directed evolution of proteins by exon shuffling. Nat Biotechnol. 2001;19: 423–428. doi: 10.1038/88084 11329010

11. Baralle FE, Giudice J. Alternative splicing as a regulator of development and tissue identity. Nat Rev Mol Cell Biol. 2017;18: 437–451. doi: 10.1038/nrm.2017.27 28488700

12. Laxa M. Intron-Mediated Enhancement: A Tool for Heterologous Gene Expression in Plants? Front Plant Sci. 2016;7: 1977. doi: 10.3389/fpls.2016.01977 28111580

13. Shaul O. How introns enhance gene expression. Int J Biochem Cell Biol. 2017;91: 145–155. doi: 10.1016/j.biocel.2017.06.016 28673892

14. Ott CJ, Suszko M, Blackledge NP, Wright JE, Crawford GE, Harris A. A complex intronic enhancer regulates expression of the CFTR gene by direct interaction with the promoter. J Cell Mol Med. 2009;13: 680–692. doi: 10.1111/j.1582-4934.2008.00621.x 19449463

15. Wei C-L, Wu Q, Vega VB, Chiu KP, Ng P, Zhang T, et al. A Global Map of p53 Transcription-Factor Binding Sites in the Human Genome. Cell. 2006;124: 207–219. doi: 10.1016/j.cell.2005.10.043 16413492

16. Morello L, Bardini M, Sala F, Breviario D. A long leader intron of the Ostub16 rice β-tubulin gene is required for high-level gene expression and can autonomously promote transcription both in vivo and in vitro. Plant J. 2002;29: 33–44. doi: 10.1046/j.0960-7412.2001.01192.x 12060225

17. Gallegos JE, Rose AB. Intron DNA Sequences Can Be More Important Than the Proximal Promoter in Determining the Site of Transcript Initiation. Plant Cell. 2017;29: 843–853. doi: 10.1105/tpc.17.00020 28373518

18. Le Hir H. The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay. EMBO J. 2001;20: 4987–4997. doi: 10.1093/emboj/20.17.4987 11532962

19. Moabbi AM, Agarwal N, El Kaderi B, Ansari A. Role for gene looping in intron-mediated enhancement of transcription. Proc Natl Acad Sci. 2012;109: 8505–8510. doi: 10.1073/pnas.1112400109 22586116

20. Mascarenhas D, Mettler IJ, Pierce DA, Lowe HW. Intron-mediated enhancement of heterologous gene expression in maize. Plant Mol Biol. 1990;15: 913–20. doi: 10.1007/BF00039430 2103480

21. Rose AB. Requirements for intron-mediated enhancement of gene expression in Arabidopsis. RNA. 2002;8: 1444–53. doi: 10.1017/s1355838202020551 12458797

22. Sizova I, Fuhrmann M, Hegemann P. A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene. 2001;277: 221–229. doi: 10.1016/s0378-1119(01)00616-3 11602359

23. Fischer N, Rochaix JD. The flanking regions of PsaD drive efficient gene expression in the nucleus of the green alga Chlamydomonas reinhardtii. Mol Genet Genomics. 2001;265: 888–94. Available: http://www.ncbi.nlm.nih.gov/pubmed/11523806 doi: 10.1007/s004380100485 11523806

24. Neupert J, Karcher D, Bock R. Generation of Chlamydomonas strains that efficiently express nuclear transgenes. Plant J. 2009;57: 1140–1150. doi: 10.1111/j.1365-313X.2008.03746.x 19036032

25. Kurniasih SD, Yamasaki T, Kong F, Okada S, Widyaningrum D, Ohama T. UV-mediated Chlamydomonas mutants with enhanced nuclear transgene expression by disruption of DNA methylation-dependent and independent silencing systems. Plant Mol Biol. 2016;92: 629–641. doi: 10.1007/s11103-016-0529-9 27761764

26. Barahimipour R, Strenkert D, Neupert J, Schroda M, Merchant SS, Bock R. Dissecting the contributions of GC content and codon usage to gene expression in the model alga Chlamydomonas reinhardtii. Plant J. 2015;84: 704–717. doi: 10.1111/tpj.13033 26402748

27. Weiner I, Atar S, Schweitzer S, Eilenberg H, Feldman Y, Avitan M, et al. Enhancing heterologous expression in Chlamydomonas reinhardtii by transcript sequence optimization. Plant J. 2018;94: 22–31. doi: 10.1111/tpj.13836 29383789

28. Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz J, Witman GB, et al. The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions. Science (80-). 2007;318: 245–250. doi: 10.1126/science.1143609 17932292

29. Raj-Kumar P-K, Vallon O, Liang C. In silico analysis of the sequence features responsible for alternatively spliced introns in the model green alga Chlamydomonas reinhardtii. 2018;94: 253–265. doi: 10.1007/s11103-017-0605-9.In

30. Labadorf A, Link A, Rogers MF, Thomas J, Reddy AS, Ben-Hur A. Genome-wide analysis of alternative splicing in Chlamydomonas reinhardtii. BMC Genomics. 2010;11: 114. doi: 10.1186/1471-2164-11-114 20163725

31. Baier T, Wichmann J, Kruse O, Lauersen KJ. Intron-containing algal transgenes mediate efficient recombinant gene expression in the green microalga Chlamydomonas reinhardtii. Nucleic Acids Res. 2018;46: 6909–6919. doi: 10.1093/nar/gky532 30053227

32. Lumbreras V, Stevens DR, Purton S. Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. Plant J. 1998;14: 441–447. doi: 10.1046/j.1365-313X.1998.00145.x

33. Eichler-Stahlberg A, Weisheit W, Ruecker O, Heitzer M. Strategies to facilitate transgene expression in Chlamydomonas reinhardtii. Planta. 2009;229: 873–883. doi: 10.1007/s00425-008-0879-x 19127370

34. Schroda M. Good News for Nuclear Transgene Expression in Chlamydomonas. Cells. 2019;8: 1534. doi: 10.3390/cells8121534 31795196

35. Dong B, Hu HH, Li ZF, Cheng RQ, Meng DM, Wang J, et al. A novel bicistronic expression system composed of the intraflagellar transport protein gene ift25 and FMDV 2A sequence directs robust nuclear gene expression in Chlamydomonas reinhardtii. Appl Microbiol Biotechnol. 2017;101: 4227–4245. doi: 10.1007/s00253-017-8177-9 28238082

36. Purton S, Rochaix J-DD, Purton S. Characterisation of the ARG7 gene of Chlamydomonas reinhardtii and its application to nuclear transformation. Eur J Phycol. 1995;30: 141–148. doi: 10.1080/09670269500650901

37. Kindle KL. High frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA. 1990;87: 1228–1232. doi: 10.1073/pnas.87.3.1228 2105499

38. La Russa M, Bogen C, Uhmeyer A, Doebbe A, Filippone E, Kruse O, et al. Functional analysis of three type-2 DGAT homologue genes for triacylglycerol production in the green microalga Chlamydomonas reinhardtii. J Biotechnol. 2012;162: 13–20. doi: 10.1016/j.jbiotec.2012.04.006 22542934

39. Baier T, Kros D, Feiner RC, Lauersen KJ, Müller KM, Kruse O. Engineered Fusion Proteins for Efficient Protein Secretion and Purification of a Human Growth Factor from the Green Microalga Chlamydomonas reinhardtii. ACS Synth Biol. 2018;7: 2547–2557. doi: 10.1021/acssynbio.8b00226 30296377

40. Lauersen KJ, Baier T, Wichmann J, Wördenweber R, Mussgnug JH, Hübner W, et al. Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii. Metab Eng. 2016;38. doi: 10.1016/j.ymben.2016.07.013 27474353

41. Wichmann J, Baier T, Wentnagel E, Lauersen KJ, Kruse O. Tailored carbon partitioning for phototrophic production of (E)-α-bisabolene from the green microalga Chlamydomonas reinhardtii. Metab Eng. 2018;45: 211–222. doi: 10.1016/j.ymben.2017.12.010 29258965

42. Lauersen KJ, Wichmann J, Baier T, Kampranis SC, Pateraki I, Møller BL, et al. Phototrophic production of heterologous diterpenoids and a hydroxy-functionalized derivative from Chlamydomonas reinhardtii. Metab Eng. 2018;49. doi: 10.1016/j.ymben.2018.07.005 30017797

43. Perozeni F, Cazzaniga S, Baier T, Zanoni F, Zoccatelli G, Lauersen KJ, et al. Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii. Plant Biotechnol J. 2020. doi: 10.1111/pbi.13364 32096597

44. Yunus IS, Wichmann J, Wördenweber R, Lauersen KJ, Kruse O, Jones PR. Synthetic metabolic pathways for photobiological conversion of CO2 into hydrocarbon fuel. Metab Eng. 2018;49: 201–211. doi: 10.1016/j.ymben.2018.08.008 30144559

45. Crozet P, Navarro FJ, Willmund F, Mehrshahi P, Bakowski K, Lauersen KJ, et al. Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synth Biol. 2018;7. doi: 10.1021/acssynbio.8b00251 30165733

46. Lauersen KJ, Kruse O, Mussgnug JH. Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit. Appl Microbiol Biotechnol. 2015;99: 3491–3503. doi: 10.1007/s00253-014-6354-7 25586579

47. Gatignol A, Durand H, Tiraby G. Bleomycin resistance conferred by a drug-binding protein. FEBS Lett. 1988;230: 171–175. doi: 10.1016/0014-5793(88)80665-3 2450783

48. Schmollinger S, Mühlhaus T, Boyle NR, Blaby IK, Casero D, Mettler T, et al. Nitrogen-Sparing Mechanisms in Chlamydomonas Affect the Transcriptome, the Proteome, and Photosynthetic Metabolism. Plant Cell. 2014;26: 1410–1435. doi: 10.1105/tpc.113.122523 24748044

49. Parra G, Bradnam K, Rose AB, Korf I. Comparative and functional analysis of intron-mediated enhancement signals reveals conserved features among plants. Nucleic Acids Res. 2011;39: 5328–5337. doi: 10.1093/nar/gkr043 21427088

50. Rose AB, Elfersi T, Parra G, Korf I. Promoter-proximal introns in Arabidopsis thaliana are enriched in dispersed signals that elevate gene expression. Plant Cell. 2008;20: 543–51. doi: 10.1105/tpc.107.057190 18319396

51. Im CS, Grossman AR. Identification and regulation of high light-induced genes in Chlamydomonas reinhardtii. Plant J. 2002;30: 301–13. doi: 10.1046/j.1365-313x.2001.01287.x 12000678

52. Chang RL, Ghamsari L, Manichaikul A, Hom EFY, Balaji S, Fu W, et al. Metabolic network reconstruction of Chlamydomonas offers insight into light-driven algal metabolism. Mol Syst Biol. 2011;7: 518. doi: 10.1038/msb.2011.52 21811229

53. Grossman A. Acclimation of Chlamydomonas reinhardtii to its Nutrient Environment. Protist. 2000;151: 201–224. doi: 10.1078/1434-4610-00020 11079767

54. Xu D-H, Wang X, Jia Y, Wang T-Y, Tian Z, Feng X, et al. SV40 intron, a potent strong intron element that effectively increases transgene expression in transfected Chinese hamster ovary cells. J Cell Mol Med. 2018;22: 2231–2239. doi: 10.1111/jcmm.13504 29441681

55. Norris SR, Meyer SE, Callis J. The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression. Plant Mol Biol. 1993;21: 895–906. doi: 10.1007/BF00027120 8385509

56. Callis J, Fromm M, Walbot V. Introns increase gene expression in cultured maize cells. Genes Dev. 1987;1: 1183–200. doi: 10.1101/gad.1.10.1183 2828168

57. Chapman BS, Thayer RM, Vincent KA, Haigwood NL. Effect of Intron-a From Human Cytomegalovirus (Towne) Immediate-Early Gene on Heterologous Expression in Mammalian-Cells. Nucleic Acids Res. 1991;19: 3979–3986. doi: 10.1093/nar/19.14.3979 1650459

58. Tikhonov M V., Maksimenko OG, Georgiev PG, Korobko I V. Optimal artificial mini-introns for transgenic expression in the cells of mice and hamsters. Mol Biol. 2017;51: 592–595. doi: 10.1134/S0026893317040173

59. Rose AB, Last RL. Introns act post-transcriptionally to increase expression of the Arabidopsis thaliana tryptophan pathway gene PAT1. Plant J. 1997;11: 455–464. doi: 10.1046/j.1365-313x.1997.11030455.x 9107035

60. Mitsuhara I, Ugaki M, Hirochika H, Ohshima M, Murakami T, Gotoh Y, et al. Efficient Promoter Cassettes for Enhanced Expression of Foreign Genes in Dicotyledonous and Monocotyledonous Plants. Plant Cell Physiol. 1996;37: 49–59. doi: 10.1093/oxfordjournals.pcp.a028913 8720924

61. Rose AB, Carter A, Korf I, Kojima N. Intron sequences that stimulate gene expression in Arabidopsis. Plant Mol Biol. 2016;92: 337–46. doi: 10.1007/s11103-016-0516-1 27492360

62. Gallegos JE, Rose AB. An intron-derived motif strongly increases gene expression from transcribed sequences through a splicing independent mechanism in Arabidopsis thaliana. Sci Rep. 2019;9: 13777. doi: 10.1038/s41598-019-50389-5 31551463

63. Li JB, Gerdes JM, Haycraft CJ, Fan Y, Teslovich TM, May-Simera H, et al. Comparative Genomics Identifies a Flagellar and Basal Body Proteome that Includes the BBS5 Human Disease Gene. Cell. 2004;117: 541–552. doi: 10.1016/s0092-8674(04)00450-7 15137946

64. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. 2011. doi: 10.1038/msb.2011.75 21988835

65. Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, et al. MEME S UITE: tools for motif discovery and searching. 2009;37: 202–208. doi: 10.1093/nar/gkp335 19458158

66. Gallegos JE, Rose AB. The enduring mystery of intron-mediated enhancement. Plant Sci. 2015;237: 8–15. doi: 10.1016/j.plantsci.2015.04.017 26089147

67. Ferrante P, Catalanotti C, Bonente G, Giuliano G. An optimized, chemically regulated gene expression system for Chlamydomonas. PLoS One. 2008;3. doi: 10.1371/journal.pone.0003200 18787710

68. Scranton MA, Ostrand JT, Georgianna DR, Lofgren SM, Li D, Ellis RC, et al. Synthetic promoters capable of driving robust nuclear gene expression in the green alga Chlamydomonas reinhardtii. Algal Res. 2016;15: 135–142. doi: 10.1016/j.algal.2016.02.011

69. Fisher D, Lakshmanan J. Metabolism and effects of epidermal growth factor and related growth factors in mammals. Endocr Rev 1990 Aug;11(3)418–42. 1990;11: 418–442. doi: 10.1210/edrv-11-3-418 2226349

70. Alpert T, Herzel L, Neugebauer KM. Perfect timing: splicing and transcription rates in living cells. Wiley Interdiscip Rev RNA. 2017;8. doi: 10.1002/wrna.1401 27873472

71. Lin S-L, Miller JD, Ying S-Y. Intronic microRNA (miRNA). J Biomed Biotechnol. 2006;2006: 26818. doi: 10.1155/JBB/2006/26818 17057362

72. Chorev M, Carmel L. The function of introns. Front Genet. 2012;3: 55. doi: 10.3389/fgene.2012.00055 22518112

73. Fang Y, Fullwood MJ. Roles, Functions, and Mechanisms of Long Non-coding RNAs in Cancer. Genomics Proteomics Bioinformatics. 2016;14: 42–54. doi: 10.1016/j.gpb.2015.09.006 26883671

74. Ma L, Bajic VB, Zhang Z. On the classification of long non-coding RNAs. RNA Biol. 2013;10: 925–33. doi: 10.4161/rna.24604 23696037

75. Alexander RD, Innocente SA, Barrass JD, Beggs JD. Splicing-dependent RNA polymerase pausing in yeast. Mol Cell. 2010;40: 582–93. doi: 10.1016/j.molcel.2010.11.005 21095588

76. Chereji R V., Eriksson PR, Ocampo J, Prajapati HK, Clark DJ. Accessibility of promoter DNA is not the primary determinant of chromatin-mediated gene regulation. Genome Res. 2019;29: 1985–1995. doi: 10.1101/gr.249326.119 31511305

77. Fedorova E, Zink D. Nuclear architecture and gene regulation. Biochim Biophys Acta—Mol Cell Res. 2008;1783: 2174–2184. doi: 10.1016/j.bbamcr.2008.07.018 18718493

78. Jaeger D, Baier T, Lauersen KJ. Intronserter, an advanced online tool for design of intron containing transgenes. Algal Res. 2019;42: 101588. doi: 10.1016/j.algal.2019.101588

79. Lauersen KJ, Baier T, Wichmann J, Wördenweber R, Mussgnug JH, Hübner W, et al. Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii. Metab Eng. 2016;38: 331–343. doi: 10.1016/j.ymben.2016.07.013 27474353

80. López-paz C, Liu D, Geng S, Umen JG. Identification of Chlamydomonas reinhardtii endogenous genic flanking sequences for improved transgene expression. 2018;92: 1232–1244. doi: 10.1111/tpj.13731.Identification

81. Cao M., Fu Y., Guo Y et al. Chlamydomonas (Chlorophyceae) colony PCR. Protoplasma. 2009;235.

82. Higuchi R, Krummel B, Saiki R. A general method of in vitro preparation and specific mutagenesis of dna fragments: Study of protein and DNA interactions. Nucleic Acids Res. 1988;16: 7351–7367. doi: 10.1093/nar/16.15.7351 3045756

83. Gorman DS, Levine RP. Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi. Proc Natl Acad Sci. 1965;54: 1665–1669. doi: 10.1073/pnas.54.6.1665 4379719

84. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9: 671–675. doi: 10.1038/nmeth.2089 22930834

85. Cuvelier ML, Ortiz A, Kim E, Moehlig H, Richardson DE, Heidelberg JF, et al. Widespread distribution of a unique marine protistan lineage. Environ Microbiol. 2008;10: 1621–1634. doi: 10.1111/j.1462-2920.2008.01580.x 18341584

86. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. 2001;25: 402–408. doi: 10.1006/meth.2001.1262 11846609

87. Laemmli UK, Molbert E, Showe M, Kellenberger E. Form- determining function of the genes required for the assembly of the head of bacteriophage T4, Journal of molecular biology. J Mol Biol. 1970;49: 99–113. doi: 10.1016/0022-2836(70)90379-7 5450520

88. Dyballa N, Metzger S. Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels. J Vis Exp. 2009; 2–5. doi: 10.3791/1431 19684561


Článek vyšel v časopise

PLOS Genetics


2020 Číslo 7
Nejčtenější tento týden
Nejčtenější v tomto čísle
Kurzy

Zvyšte si kvalifikaci online z pohodlí domova

plice
INSIGHTS from European Respiratory Congress
nový kurz

Současné pohledy na riziko v parodontologii
Autoři: MUDr. Ladislav Korábek, CSc., MBA

Svět praktické medicíny 3/2024 (znalostní test z časopisu)

Kardiologické projevy hypereozinofilií
Autoři: prof. MUDr. Petr Němec, Ph.D.

Střevní příprava před kolonoskopií
Autoři: MUDr. Klára Kmochová, Ph.D.

Všechny kurzy
Kurzy Podcasty Doporučená témata Časopisy
Přihlášení
Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.

Přihlášení

Nemáte účet?  Registrujte se

#ADS_BOTTOM_SCRIPTS#