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2021
Hees, Stijn Van; Cuypers, Bart; Bourgeois, Stefan; Groothuismink, Zwier M A; Meysman, Pieter; der Vlies, Pieter Van; de Knegt, Rob; Vonghia, Luisa; Michielsen, Peter; Francque, Sven; Laukens, Kris; Boonstra, Andre; Vanwolleghem, Thomas
Sorted B cell transcriptomes point towards actively regulated B cell responses during ongoing chronic hepatitis B infections Journal Article
In: Cellular Immunology, vol. 362, pp. 104283, 2021, ISSN: 0008-8749.
@article{VANHEES2021104283,
title = {Sorted B cell transcriptomes point towards actively regulated B cell responses during ongoing chronic hepatitis B infections},
author = {Stijn Van Hees and Bart Cuypers and Stefan Bourgeois and Zwier M A Groothuismink and Pieter Meysman and Pieter Van der Vlies and Rob de Knegt and Luisa Vonghia and Peter Michielsen and Sven Francque and Kris Laukens and Andre Boonstra and Thomas Vanwolleghem},
url = {https://www.sciencedirect.com/science/article/pii/S0008874921000022},
doi = {https://doi.org/10.1016/j.cellimm.2021.104283},
issn = {0008-8749},
year = {2021},
date = {2021-01-01},
journal = {Cellular Immunology},
volume = {362},
pages = {104283},
abstract = {The natural course of chronic hepatitis B virus (HBV) infections follows distinct clinical disease phases, characterized by fluctuating levels of serum HBV DNA and ALT. The immune cells and their features that govern these clinical disease transitions remain unknown. In the current study, we performed RNA sequencing on purified B cells from blood (n = 42) and liver (n = 10) of healthy controls and chronic HBV patients. We found distinct gene expression profiles between healthy controls and chronic HBV patients, as evidenced by 190 differentially expressed genes (DEG), but also between the clinical phenotypes of a chronic HBV infection (17\textendash110 DEG between each phase). Numerous immune pathways, including the B cell receptor pathway were upregulated in liver B cells when compared to peripheral B cells. Further investigation of the detected DEG suggested an activation of B cells during HBeAg seroconversion and an active regulation of B cell signalling in the liver.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Boullosa, Laurie Freire; Loenhout, Jinthe Van; Flieswasser, Tal; Waele, Jorrit De; Hermans, Christophe; Lambrechts, Hilde; Cuypers, Bart; Laukens, Kris; Bartholomeus, Esther; Siozopoulou, Vasiliki; Vos, Winnok H De; Peeters, Marc; Smits, Evelien L J; Deben, Christophe
In: Redox Biology, pp. 101949, 2021, ISSN: 2213-2317.
@article{BOULLOSA2021101949,
title = {Auranofin reveals therapeutic anticancer potential by triggering distinct molecular cell death mechanisms and innate immunity in mutant p53 non-small cell lung cancer},
author = {Laurie Freire Boullosa and Jinthe Van Loenhout and Tal Flieswasser and Jorrit De Waele and Christophe Hermans and Hilde Lambrechts and Bart Cuypers and Kris Laukens and Esther Bartholomeus and Vasiliki Siozopoulou and Winnok H De Vos and Marc Peeters and Evelien L J Smits and Christophe Deben},
url = {https://www.sciencedirect.com/science/article/pii/S2213231721000975},
doi = {https://doi.org/10.1016/j.redox.2021.101949},
issn = {2213-2317},
year = {2021},
date = {2021-01-01},
journal = {Redox Biology},
pages = {101949},
abstract = {Auranofin (AF) is an FDA-approved antirheumatic drug with anticancer properties that acts as a thioredoxin reductase 1 (TrxR) inhibitor. The exact mechanisms through which AF targets cancer cells remain elusive. To shed light on the mode of action, this study provides an in-depth analysis on the molecular mechanisms and immunogenicity of AF-mediated cytotoxicity in the non-small cell lung cancer (NSCLC) cell line NCI-H1299 (p53 Null) and its two isogenic derivates with mutant p53 R175H or R273H accumulation. TrxR is highly expressed in a panel of 72 NSCLC patients, making it a valid druggable target in NSCLC for AF. The presence of mutant p53 overexpression was identified as an important sensitizer for AF in (isogenic) NSCLC cells as it was correlated with reduced thioredoxin (Trx) levels in vitro. Transcriptome analysis revealed dysregulation of genes involved in oxidative stress response, DNA damage, granzyme A (GZMA) signaling and ferroptosis. Although functionally AF appeared a potent inhibitor of GPX4 in all NCI-H1299 cell lines, the induction of lipid peroxidation and consequently ferroptosis was limited to the p53 R273H expressing cells. In the p53 R175H cells, AF mainly induced large-scale DNA damage and replication stress, leading to the induction of apoptotic cell death rather than ferroptosis. Importantly, all cell death types were immunogenic since the release of danger signals (ecto-calreticulin, ATP and HMGB1) and dendritic cell maturation occurred irrespective of (mutant) p53 expression. Finally, we show that AF sensitized cancer cells to caspase-independent natural killer cell-mediated killing by downregulation of several key targets of GZMA. Our data provides novel insights on AF as a potent, clinically available, off-patent cancer drug by targeting mutant p53 cancer cells through distinct cell death mechanisms (apoptosis and ferroptosis). In addition, AF improves the innate immune response at both cytostatic (natural killer cell-mediated killing) and cytotoxic concentrations (dendritic cell maturation).},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Houtven, Joris Van; Cuypers, Bart; Meysman, Pieter; Hooyberghs, Jef; Laukens, Kris; Valkenborg, Dirk
Constrained Standardization of Count Data from Massive Parallel Sequencing Journal Article
In: Journal of Molecular Biology, pp. 166966, 2021, ISSN: 0022-2836.
@article{VANHOUTVEN2021166966,
title = {Constrained Standardization of Count Data from Massive Parallel Sequencing},
author = {Joris Van Houtven and Bart Cuypers and Pieter Meysman and Jef Hooyberghs and Kris Laukens and Dirk Valkenborg},
url = {https://www.sciencedirect.com/science/article/pii/S0022283621001674},
doi = {https://doi.org/10.1016/j.jmb.2021.166966},
issn = {0022-2836},
year = {2021},
date = {2021-01-01},
journal = {Journal of Molecular Biology},
pages = {166966},
abstract = {In high-throughput omics disciplines like transcriptomics, researchers face a need to assess the quality of an experiment prior to an in-depth statistical analysis. To efficiently analyze such voluminous collections of data, researchers need triage methods that are both quick and easy to use. Such a normalization method for relative quantitation, CONSTANd, was recently introduced for isobarically-labeled mass spectra in proteomics. It transforms the data matrix of abundances through an iterative, convergent process enforcing three constraints: (I) identical column sums; (II) each row sum is fixed (across matrices) and (III) identical to all other row sums. In this study, we investigate whether CONSTANd is suitable for count data from massively parallel sequencing, by qualitatively comparing its results to those of DESeq2. Further, we propose an adjustment of the method so that it may be applied to identically balanced but differently sized experiments for joint analysis. We find that CONSTANd can process large data sets at well over 1 million count records per second whilst mitigating unwanted systematic bias and thus quickly uncovering the underlying biological structure when combined with a PCA plot or hierarchical clustering. Moreover, it allows joint analysis of data sets obtained from different batches, with different protocols and from different labs but without exploiting information from the experimental setup other than the delineation of samples into identically processed sets (IPSs). CONSTANd’s simplicity and applicability to proteomics as well as transcriptomics data make it an interesting candidate for integration in multi-omics workflows.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Logie, Emilie; Chirumamilla, Chandra S; Perez-Novo, Claudina; Shaw, Priyanka; Declerck, Ken; Palagani, Ajay; Rangarajan, Savithri; Cuypers, Bart; Neuter, Nicolas De; Turabe, Fazil Mobashar Hussain Urf; Verma, Navin Kumar; Bogaerts, Annemie; Laukens, Kris; Offner, Fritz; Vlierberghe, Pieter Van; Ostade, Xaveer Van; Berghe, Wim Vanden
In: Cancers, vol. 13, no. 7, 2021, ISSN: 2072-6694.
@article{cancers13071618,
title = {Covalent Cysteine Targeting of Bruton’s Tyrosine Kinase (BTK) Family by Withaferin-A Reduces Survival of Glucocorticoid-Resistant Multiple Myeloma MM1 Cells},
author = {Emilie Logie and Chandra S Chirumamilla and Claudina Perez-Novo and Priyanka Shaw and Ken Declerck and Ajay Palagani and Savithri Rangarajan and Bart Cuypers and Nicolas De Neuter and Fazil Mobashar Hussain Urf Turabe and Navin Kumar Verma and Annemie Bogaerts and Kris Laukens and Fritz Offner and Pieter Van Vlierberghe and Xaveer Van Ostade and Wim Vanden Berghe},
url = {https://www.mdpi.com/2072-6694/13/7/1618},
doi = {10.3390/cancers13071618},
issn = {2072-6694},
year = {2021},
date = {2021-01-01},
journal = {Cancers},
volume = {13},
number = {7},
abstract = {Multiple myeloma (MM) is a hematological malignancy characterized by plasma cells’ uncontrolled growth. The major barrier in treating MM is the occurrence of primary and acquired therapy resistance to anticancer drugs. Often, this therapy resistance is associated with constitutive hyperactivation of tyrosine kinase signaling. Novel covalent kinase inhibitors, such as the clinically approved BTK inhibitor ibrutinib (IBR) and the preclinical phytochemical withaferin A (WA), have, therefore, gained pharmaceutical interest. Remarkably, WA is more effective than IBR in killing BTK-overexpressing glucocorticoid (GC)-resistant MM1R cells. To further characterize the kinase inhibitor profiles of WA and IBR in GC-resistant MM cells, we applied phosphopeptidome- and transcriptome-specific tyrosine kinome profiling. In contrast to IBR, WA was found to reverse BTK overexpression in GC-resistant MM1R cells. Furthermore, WA-induced cell death involves covalent cysteine targeting of Hinge-6 domain type tyrosine kinases of the kinase cysteinome classification, including inhibition of the hyperactivated BTK. Covalent interaction between WA and BTK could further be confirmed by biotin-based affinity purification and confocal microscopy. Similarly, molecular modeling suggests WA preferably targets conserved cysteines in the Hinge-6 region of the kinase cysteinome classification, favoring inhibition of multiple B-cell receptors (BCR) family kinases. Altogether, we show that WA’s promiscuous inhibition of multiple BTK family tyrosine kinases represents a highly effective strategy to overcome GC-therapy resistance in MM.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
2020
Adriaensen, Wim; Cuypers, Bart; Cordero, Carlota F; Mengasha, Bewketu; Blesson, Séverine; Cnops, Lieselotte; Kaye, Paul M; Alves, Fabiana; Diro, Ermias; van Griensven, Johan
Host transcriptomic signature as alternative test-of-cure in visceral leishmaniasis patients co-infected with HIV Journal Article
In: EBioMedicine, vol. 55, 2020, ISSN: 2352-3964.
@article{Adriaensen2020,
title = {Host transcriptomic signature as alternative test-of-cure in visceral leishmaniasis patients co-infected with HIV},
author = {Wim Adriaensen and Bart Cuypers and Carlota F Cordero and Bewketu Mengasha and S\'{e}verine Blesson and Lieselotte Cnops and Paul M Kaye and Fabiana Alves and Ermias Diro and Johan van Griensven},
url = {https://doi.org/10.1016/j.ebiom.2020.102748},
doi = {10.1016/j.ebiom.2020.102748},
issn = {2352-3964},
year = {2020},
date = {2020-05-01},
journal = {EBioMedicine},
volume = {55},
publisher = {Elsevier},
abstract = {BackgroundVisceral leishmaniasis (VL) treatment in HIV patients very often fails and is followed by high relapse and case-fatality rates. Hence, treatment efficacy assessment is imperative but based on invasive organ aspiration for parasite detection. In the search of a less-invasive alternative and because the host immune response is pivotal for treatment outcome in immunocompromised VL patients, we studied changes in the whole blood transcriptional profile of VL-HIV patients during treatment.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Deben, Christophe; Boullosa, Laurie Freire; Domen, Andreas; Wouters, An; Cuypers, Bart; Laukens, Kris; Lardon, Filip; Pauwels, Patrick
Characterization of acquired nutlin-3 resistant non-small cell lung cancer cells Journal Article
In: Cancer Drug Resistance, vol. 3, pp. [Online First], 2020, ISSN: 2578-532X.
@article{Deben2020,
title = {Characterization of acquired nutlin-3 resistant non-small cell lung cancer cells},
author = {Christophe Deben and Laurie Freire Boullosa and Andreas Domen and An Wouters and Bart Cuypers and Kris Laukens and Filip Lardon and Patrick Pauwels},
url = {https://doi.org/10.20517/cdr.2020.91},
doi = {10.20517/cdr.2020.91},
issn = {2578-532X},
year = {2020},
date = {2020-01-01},
journal = {Cancer Drug Resistance},
volume = {3},
pages = {[Online First]},
abstract = {Aim: The purpose of this manuscript is to study the potential characteristics of acquired nutlin-3 resistant non-small cell lung cancer cells (NSCLC). Nutlin-3 is an inhibitor of the murine-double minute 2 protein, the main negative regulator of wild type p53, of which several derivatives are currently in clinical development.Methods: A549 NSCLC cells were exposed to increasing concentrations of nutlin-3 for a period of 18 weeks. Monoclonal derivates were cultured, and the most resistance subclone was selected for whole transcriptome analysis. Gene set enrichment analysis was performed on differentially expressed genes between A549 nutlin-3 resistant cancer cells and the parental A549 p53 wild type cancer cells. Relevant findings were validated at the gene, protein and/or functional level.Results: All nutlin-3 resistant subclones acquired mutations in the TP53 gene, resulting in overexpression of the mutant p53 protein. The most resistant subclone was enriched for genes related to epithelial to mesenchymal transition (EMT), resulting in increased migratory and invasive potential. Furthermore, these cells were enriched in genes related to inflammation, tissue remodelling, and angiogenesis. Importantly, expression of several immune checkpoints, including PD-L1 and PD-L2, was significantly upregulated, and cisplatin-induced cell death was reduced.Conclusion: Transcriptome analysis of a highly nutlin-3 resistant A549 subclone shows the relevance of studying (1) resistance to standard of care chemotherapy; (2) secretion of immunomodulating chemo- and cytokines; (3) immune checkpoint expression; and (4) EMT and invasion in nutlin-3 resistant cancer cells in addition to acquired mutations in the TP53 gene.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
2018
Krivoshiev, Boris V; Beemster, Gerrit T S; Sprangers, Katrien; Cuypers, Bart; Laukens, Kris; Blust, Ronny; Husson, Steven J
Transcriptome profiling of HepG2 cells exposed to the flame retardant 9,10-dihydro-9-oxa-10-phosphaphenanthrene 10-oxide (DOPO) Journal Article
In: Toxicology research, vol. 7, no. 3, pp. 492-502, 2018, ISSN: 2045-452X, (c8tx00006a[PII]).
@article{Krivoshiev2018b,
title = {Transcriptome profiling of HepG2 cells exposed to the flame retardant 9,10-dihydro-9-oxa-10-phosphaphenanthrene 10-oxide (DOPO)},
author = {Boris V Krivoshiev and Gerrit T S Beemster and Katrien Sprangers and Bart Cuypers and Kris Laukens and Ronny Blust and Steven J Husson},
url = {https://doi.org/10.1039/c8tx00006a},
doi = {10.1039/c8tx00006a},
issn = {2045-452X},
year = {2018},
date = {2018-03-12},
journal = {Toxicology research},
volume = {7},
number = {3},
pages = {492-502},
publisher = {Royal Society of Chemistry},
abstract = {The flame retardant, 9,10-dihydro-9-oxa-10-phosphaphenanthrene 10-oxide (DOPO), has been receiving great interest given its superior fire protection properties, and its predicted low level of persistence, bioaccumulation, and toxicity. However, empirical toxicological data that are essential for a complete hazard assessment are severely lacking. In this study, we attempted to identify the potential toxicological modes of action by transcriptome (RNA-seq) profiling of the human liver hepatocellular carcinoma cell line, HepG2. Such insight may help in identifying compounds of concern and potential toxicological phenotypes. DOPO was found to have little cytotoxic potential, with lower effective concentrations compared to other flame retardants studied in the same cell line. Differentially expressed genes revealed a wide range of molecular effects including changes in protein, energy, DNA, and lipid metabolism, along with changes in cellular stress response pathways. In response to 250 $mu$M DOPO, the most perturbed biological processes were fatty acid metabolism, androgen metabolism, glucose transport, and renal function and development, which is in agreement with other studies that observed similar effects of other flame retardants in other species. However, treatment with 2.5 $mu$M DOPO resulted in very few differentially expressed genes and failed to indicate any potential effects on biology, despite such concentrations likely being orders of magnitude greater than would be encountered in the environment. This, together with the low levels of cytotoxicity, supports the potential replacement of the current flame retardants by DOPO, although further studies are needed to establish the nephrotoxicity and endocrine disruption of DOPO.},
note = {c8tx00006a[PII]},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Cuypers, Bart
A systems biology approach for a comprehensive understanding of molecular adaptation in** Leishmania donovani PhD Thesis
University of Antwerp, 2018.
@phdthesis{cuypers2018systems,
title = {A systems biology approach for a comprehensive understanding of molecular adaptation in** Leishmania donovani},
author = {Bart Cuypers},
year = {2018},
date = {2018-01-01},
school = {University of Antwerp},
keywords = {},
pubstate = {published},
tppubtype = {phdthesis}
}
Dumetz, F; Cuypers, B; Imamura, H; Zander, D; DtextquoterightHaenens, E; Maes, I; Domagalska, M A; Clos, J; Dujardin, J -C; Muylder, G De
Molecular Preadaptation to Antimony Resistance in Leishmania donovani on the Indian Subcontinent Journal Article
In: mSphere, vol. 3, no. 2, 2018.
@article{Dumetze00548-17,
title = {Molecular Preadaptation to Antimony Resistance in Leishmania donovani on the Indian Subcontinent},
author = {F Dumetz and B Cuypers and H Imamura and D Zander and E D{textquoteright}Haenens and I Maes and M A Domagalska and J Clos and J -C Dujardin and G De Muylder},
editor = {Margaret Phillips},
url = {https://msphere.asm.org/content/3/2/e00548-17},
doi = {10.1128/mSphere.00548-17},
year = {2018},
date = {2018-01-01},
journal = {mSphere},
volume = {3},
number = {2},
publisher = {American Society for Microbiology Journals},
abstract = {Antimonials (Sb) were used for decades for chemotherapy of visceral leishmaniasis (VL). Now abandoned in the Indian subcontinent (ISC) because of Leishmania donovani resistance, this drug offers a unique model for understanding drug resistance dynamics. In a previous phylogenomic study, we found two distinct populations of L. donovani: the core group (CG) in the Gangetic plains and ISC1 in the Nepalese highlands. Sb resistance was only encountered within the CG, and a series of potential markers were identified. Here, we analyzed the development of resistance to trivalent antimonials (SbIII) upon experimental selection in ISC1 and CG strains. We observed that (i) baseline SbIII susceptibility of parasites was higher in ISC1 than in the CG, (ii) time to SbIII resistance was higher for ISC1 parasites than for CG strains, and (iii) untargeted genomic and metabolomic analyses revealed molecular changes along the selection process: these were more numerous in ISC1 than in the CG. Altogether these observations led to the hypothesis that CG parasites are preadapted to SbIII resistance. This hypothesis was experimentally confirmed by showing that only wild-type CG strains could survive a direct exposure to the maximal concentration of SbIII. The main driver of this preadaptation was shown to be MRPA, a gene involved in SbIII sequestration and amplified in an intrachromosomal amplicon in all CG strains characterized so far. This amplicon emerged around 1850 in the CG, well before the implementation of antimonials for VL chemotherapy, and we discuss here several hypotheses of selective pressure that could have accompanied its emergence.IMPORTANCE The textquotedblleftantibiotic resistance crisistextquotedblright is a major challenge for scientists and medical professionals. This steady rise in drug-resistant pathogens also extends to parasitic diseases, with antimony being the first anti-Leishmania drug that fell in the Indian subcontinent (ISC). Leishmaniasis is a major but neglected infectious disease with limited therapeutic options. Therefore, understanding how parasites became resistant to antimonials is of commanding importance. In this study, we experimentally characterized the dynamics of this resistance acquisition and show for the first time that some Leishmania populations of the ISC were preadapted to antimony resistance, likely driven by environmental factors or by drugs used in the 19th century.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Krivoshiev, Boris V; Beemster, Gerrit T S; Sprangers, Katrien; Cuypers, Bart; Laukens, Kris; Blust, Ronny; Husson, Steven J
Toxicogenomics of the flame retardant tris (2-butoxyethyl) phosphate in HepG2 cells using RNA-seq Journal Article
In: Toxicology in Vitro, vol. 46, pp. 178 - 188, 2018, ISSN: 0887-2333.
@article{KRIVOSHIEV2018178,
title = {Toxicogenomics of the flame retardant tris (2-butoxyethyl) phosphate in HepG2 cells using RNA-seq},
author = {Boris V Krivoshiev and Gerrit T S Beemster and Katrien Sprangers and Bart Cuypers and Kris Laukens and Ronny Blust and Steven J Husson},
url = {http://www.sciencedirect.com/science/article/pii/S0887233317303089},
doi = {https://doi.org/10.1016/j.tiv.2017.10.011},
issn = {0887-2333},
year = {2018},
date = {2018-01-01},
journal = {Toxicology in Vitro},
volume = {46},
pages = {178 - 188},
abstract = {Tris (2-butoxyethyl) phosphate (TBOEP) is a compound produced at high volume that is used as both a flame retardant and a plasticizer. It is persistent and bioaccumulative, yet little is known of its toxicological modes of action. Such insight may aid risk assessment in a weight-of-evidence approach supplementing current testing strategies. We used an RNA sequencing approach as an unbiased and sensitive tool to explore potential negative health effects of sub-cytotoxic concentrations of TBOEP on the transcriptome of the human liver hepatocellular carcinoma cell line, HepG2, with the lowest concentration used potentially holding relevance to human physiological levels. Over-representation and gene set enrichment analysis corresponded well and revealed that TBOEP treatments resulted in an upregulation of genes involved in protein and energy metabolism, along with DNA replication. Such increases in cell and macromolecule metabolism could explain the increase in mitochondrial activity at lower TBOEP concentrations. In addition, TBOEP affected a wide variety of biological processes, the most notable one being the general stress response, wound healing. Finally, TBOEP showed effects on steroid hormone biosynthesis and activation, regulation, and potentiation of immune responses, in agreement with other studies. As such, this study is the first study investigating genome-wide changes in gene transcription in response to TBOEP in human cells.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
2017
Cuypers, Bart; Domagalska, Malgorzata A; Meysman, Pieter; de Muylder, Géraldine; Vanaerschot, Manu; Imamura, Hideo; Dumetz, Franck; Verdonckt, Thomas Wolf; Myler, Peter J; Ramasamy, Gowthaman; Laukens, Kris; Dujardin, Jean-Claude
Multiplexed Spliced-Leader Sequencing: A high-throughput, selective method for RNA-seq in Trypanosomatids Journal Article
In: Scientific Reports, vol. 7, no. 1, pp. 3725, 2017, ISSN: 2045-2322.
@article{Cuypers2017,
title = {Multiplexed Spliced-Leader Sequencing: A high-throughput, selective method for RNA-seq in Trypanosomatids},
author = {Bart Cuypers and Malgorzata A Domagalska and Pieter Meysman and G\'{e}raldine de Muylder and Manu Vanaerschot and Hideo Imamura and Franck Dumetz and Thomas Wolf Verdonckt and Peter J Myler and Gowthaman Ramasamy and Kris Laukens and Jean-Claude Dujardin},
url = {https://doi.org/10.1038/s41598-017-03987-0},
doi = {10.1038/s41598-017-03987-0},
issn = {2045-2322},
year = {2017},
date = {2017-06-16},
journal = {Scientific Reports},
volume = {7},
number = {1},
pages = {3725},
abstract = {High throughput sequencing techniques are poorly adapted for in vivo studies of parasites, which require prior in vitro culturing and purification. Trypanosomatids, a group of kinetoplastid protozoans, possess a distinctive feature in their transcriptional mechanism whereby a specific Spliced Leader (SL) sequence is added to the 5textasciiacutexend of each mRNA by trans-splicing. This allows to discriminate Trypansomatid RNA from mammalian RNA and forms the basis of our new multiplexed protocol for high-throughput, selective RNA-sequencing called SL-seq. We provided a proof-of-concept of SL-seq in Leishmania donovani, the main causative agent of visceral leishmaniasis in humans, and successfully applied the method to sequence Leishmania mRNA directly from infected macrophages and from highly diluted mixes with human RNA. mRNA profiles obtained with SL-seq corresponded largely to those obtained from conventional poly-A tail purification methods, indicating both enumerate the same mRNA pool. However, SL-seq offers additional advantages, including lower sequencing depth requirements, fast and simple library prep and high resolution splice site detection. SL-seq is therefore ideal for fast and massive parallel sequencing of parasite transcriptomes directly from host tissues. Since SLs are also present in Nematodes, Cnidaria and primitive chordates, this method could also have high potential for transcriptomics studies in other organisms.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Dumetz, F; Imamura, H; Sanders, M; Seblova, V; Myskova, J; Pescher, P; Vanaerschot, M; Meehan, C J; Cuypers, B; Muylder, G De; Späth, G F; Bussotti, G; Vermeesch, J R; Berriman, M; Cotton, J A; Volf, P; Dujardin, J C; Domagalska, M A
In: mBio, vol. 8, no. 3, 2017.
@article{Dumetze00599-17,
title = {Modulation of Aneuploidy in Leishmania donovani during Adaptation to Different In Vitro and In Vivo Environments and Its Impact on Gene Expression},
author = {F Dumetz and H Imamura and M Sanders and V Seblova and J Myskova and P Pescher and M Vanaerschot and C J Meehan and B Cuypers and G De Muylder and G F Sp\"{a}th and G Bussotti and J R Vermeesch and M Berriman and J A Cotton and P Volf and J C Dujardin and M A Domagalska},
editor = {Keith Gull},
url = {https://mbio.asm.org/content/8/3/e00599-17},
doi = {10.1128/mBio.00599-17},
year = {2017},
date = {2017-01-01},
journal = {mBio},
volume = {8},
number = {3},
publisher = {American Society for Microbiology},
abstract = {Aneuploidy is usually deleterious in multicellular organisms but appears to be tolerated and potentially beneficial in unicellular organisms, including pathogens. Leishmania, a major protozoan parasite, is emerging as a new model for aneuploidy, since in vitro-cultivated strains are highly aneuploid, with interstrain diversity and intrastrain mosaicism. The alternation of two life stages in different environments (extracellular promastigotes and intracellular amastigotes) offers a unique opportunity to study the impact of environment on aneuploidy and gene expression. We sequenced the whole genomes and transcriptomes of Leishmania donovani strains throughout their adaptation to in vivo conditions mimicking natural vertebrate and invertebrate host environments. The nucleotide sequences were almost unchanged within a strain, in contrast to highly variable aneuploidy. Although high in promastigotes in vitro, aneuploidy dropped significantly in hamster amastigotes, in a progressive and strain-specific manner, accompanied by the emergence of new polysomies. After a passage through a sand fly, smaller yet consistent karyotype changes were detected. Changes in chromosome copy numbers were correlated with the corresponding transcript levels, but additional aneuploidy-independent regulation of gene expression was observed. This affected stage-specific gene expression, downregulation of the entire chromosome 31, and upregulation of gene arrays on chromosomes 5 and 8. Aneuploidy changes in Leishmania are probably adaptive and exploited to modulate the dosage and expression of specific genes; they are well tolerated, but additional mechanisms may exist to regulate the transcript levels of other genes located on aneuploid chromosomes. Our model should allow studies of the impact of aneuploidy on molecular adaptations and cellular fitness.IMPORTANCE Aneuploidy is usually detrimental in multicellular organisms, but in several microorganisms, it can be tolerated and even beneficial. Leishmania\textemdasha protozoan parasite that kills more than 30,000 people each year\textemdashis emerging as a new model for aneuploidy studies, as unexpectedly high levels of aneuploidy are found in clinical isolates. Leishmania lacks classical regulation of transcription at initiation through promoters, so aneuploidy could represent a major adaptive strategy of this parasite to modulate gene dosage in response to stressful environments. For the first time, we document the dynamics of aneuploidy throughout the life cycle of the parasite, in vitro and in vivo. We show its adaptive impact on transcription and its interaction with regulation. Besides offering a new model for aneuploidy studies, we show that further genomic studies should be done directly in clinical samples without parasite isolation and that adequate methods should be developed for this.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}