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Article: Methods behind neoantigen prediction for personalized anticancer vaccines.

Godazandeh, Kiyana / Van Olmen, Lies / Van Oudenhove, Lore / Lefever, Steve / Bogaert, Cedric / Fant, Bruno

2023 Volume 183, Page(s) 161–186

Abstract: Next to conventional cancer therapies, immunotherapies such as immune checkpoint inhibitors have broadened the cancer treatment landscape over the past decades. Recent advances in next generation sequencing and bioinformatics technologies have made it ... ...

Abstract	Next to conventional cancer therapies, immunotherapies such as immune checkpoint inhibitors have broadened the cancer treatment landscape over the past decades. Recent advances in next generation sequencing and bioinformatics technologies have made it possible to identify a patient's own immunogenic neoantigens. These cancer neoantigens serve as important targets for personalized immunotherapy which has the benefit of being more active and effective in targeting cancer cells. This paper is a step-by-step guide discussing the different analyses and challenges encountered during in-silico neoantigen prediction. The protocol describes all the tools and steps required for the identification of immunogenic neoantigens.
MeSH term(s)	Humans ; Antigens, Neoplasm/genetics ; Cancer Vaccines/genetics ; Cancer Vaccines/therapeutic use ; Neoplasms/genetics ; Neoplasms/therapy ; Computational Biology ; Immunotherapy/methods
Chemical Substances	Antigens, Neoplasm ; Cancer Vaccines
Language	English
Publishing date	2023-09-11
Publishing country	United States
Document type	Journal Article
ISSN	0091-679X
ISSN	0091-679X
DOI	10.1016/bs.mcb.2023.05.002
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Article ; Online: Validation of Circular RNAs Using RT-qPCR After Effective Removal of Linear RNAs by Ribonuclease R.

Vromman, Marieke / Yigit, Nurten / Verniers, Kimberly / Lefever, Steve / Vandesompele, Jo / Volders, Pieter-Jan

Current protocols

2021 Volume 1, Issue 7, Page(s) e181

Abstract: Circular RNAs (circRNAs) are a class of endogenous noncoding RNAs that have been shown to play a role in normal development, homeostasis, and disease, including cancer. CircRNAs are formed through a process called back-splicing, which results in a ... ...

Abstract	Circular RNAs (circRNAs) are a class of endogenous noncoding RNAs that have been shown to play a role in normal development, homeostasis, and disease, including cancer. CircRNAs are formed through a process called back-splicing, which results in a covalently closed loop with a nonlinear back-spliced junction (BSJ). In general, circRNA BSJs are predicted in RNA sequencing data using one of numerous circRNA detection algorithms. Selected circRNAs are then typically validated using an orthogonal method such as reverse transcription quantitative PCR (RT-qPCR) with circRNA-specific primers. However, linear transcripts originating from endogenous trans-splicing can lead to false-positive signals both in RNA sequencing and in RT-qPCR experiments. Therefore, it is essential to perform the RT-qPCR validation step only after linear RNAs have been degraded using an exonuclease such as ribonuclease R (RNase R). Several RNase R protocols are available for circRNA detection using RNA sequencing or RT-qPCR. These protocols-which vary in enzyme concentration, RNA input amount, incubation times, and cleanup steps-typically lack a detailed validated standard protocol and fail to provide a range of conditions that deliver accurate results. As such, some protocols use RNase R concentrations that are too high, resulting in partial degradation of the target circRNAs. Here, we describe an optimized workflow for circRNA validation, combining RNase R treatment and RT-qPCR. First, we outline the steps for circRNA primer design and qPCR assay validation. Then, we describe RNase R treatment of total RNA and, importantly, a subsequent essential buffer cleanup step. Lastly, we outline the steps to perform the RT-qPCR and discuss the downstream data analyses. © 2021 Wiley Periodicals LLC. Basic Protocol 1: CircRNA primer design and qPCR assay validation Basic Protocol 2: RNase R treatment, cleanup, and RT-qPCR.
Language	English
Publishing date	2021-04-19
Document type	Journal Article
ISSN	2691-1299
ISSN (online)	2691-1299
DOI	10.1002/cpz1.181
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Article ; Online: Performance Evaluation of Three DNA Sample Tracking Tools in a Whole Exome Sequencing Workflow.

Wils, Gertjan / Helsmoortel, Céline / Volders, Pieter-Jan / Vereecke, Inge / Milazzo, Mauro / Vandesompele, Jo / Coppieters, Frauke / De Leeneer, Kim / Lefever, Steve

Molecular diagnosis & therapy

2022 Volume 26, Issue 4, Page(s) 411–419

Abstract: Introduction: Next-generation sequencing applications are becoming indispensable for clinical diagnostics. These experiments require numerous wet- and dry-laboratory steps, each one increasing the probability of a sample swap or contamination. Therefore, ...

Abstract	Introduction: Next-generation sequencing applications are becoming indispensable for clinical diagnostics. These experiments require numerous wet- and dry-laboratory steps, each one increasing the probability of a sample swap or contamination. Therefore, identity confirmation at the end of the process is recommended to ensure the right data are used for each patient. Methods: We tested three commercially available, single nucleotide polymorphism (SNP)-based sample tracking kits in a diagnostic workflow to evaluate their ease of use and performance. The coverage uniformity, on-target specificity, sample identification, and genotyping performance were determined to assess the reliability and cost effectiveness of each kit. Results and discussion: Hands-on time and manual steps are almost identical for the kits from pxlence and Nimagen. The Swift kit has an extra purification step, making it the longest and most demanding protocol. Furthermore, the Swift kit failed to correctly genotype 26 of the 46 samples. The Nimagen kit identified all but one sample and the pxlence kit unambiguously identified all samples, making it the most reliable and robust kit of this evaluation. The Nimagen kit showed poor on-target mapping rates, resulting in deeper sequencing needs and higher sequencing costs compared with the other two kits. Conclusion: Our conclusion is that the Human Sample ID kit from pxlence is the most cost effective of the three tested tools for DNA sample tracking and identification.
MeSH term(s)	DNA ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Reproducibility of Results ; Whole Exome Sequencing ; Workflow
Chemical Substances	DNA (9007-49-2)
Language	English
Publishing date	2022-05-28
Publishing country	New Zealand
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2232796-4
ISSN	1179-2000 ; 1177-1062
ISSN (online)	1179-2000
ISSN	1177-1062
DOI	10.1007/s40291-022-00585-3
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Article ; Online: How long noncoding RNAs enforce their will on mitochondrial activity: regulation of mitochondrial respiration, reactive oxygen species production, apoptosis, and metabolic reprogramming in cancer.

De Paepe, Boel / Lefever, Steve / Mestdagh, Pieter

Current genetics

2018 Volume 64, Issue 1, Page(s) 163–172

Abstract: The cellular transcriptome contains a wide diversity of untranslated RNAs, of which the class of regulatory long noncoding RNAs (lncRNAs) has only recently been recognized. Evidence swiftly accumulates of lncRNAs influencing mitochondrial activities of ... ...

Abstract	The cellular transcriptome contains a wide diversity of untranslated RNAs, of which the class of regulatory long noncoding RNAs (lncRNAs) has only recently been recognized. Evidence swiftly accumulates of lncRNAs influencing mitochondrial activities of eukaryotic cells, and perturbed expression is conspicuously associated with human diseases. In this review, we describe the multifaceted effects of lncRNAs on mitochondrial function, more particularly on the balance between oxidative phosphorylation and glycolysis, on the production of reactive oxygen species, and on apoptosis in human cells. Emphasis is placed on the involvement of lncRNAs in cancer metabolism, as tumor cells rely heavily on modifications of mitochondrial functioning as an essential component for sustained tumorigenesis and cancer progression. From the nonexhaustive list of lncRNAS described in this review, ANRIL, AScmtRNA, H19, HOTAIR, LincRNA-p21, MALAT1, RMRP, SAMMSON, and VL30 have emerged as potent regulators of mitochondrial metabolism. Due to their key role in cancer progression, they represent potential targets of innovative lncRNA-based treatment strategies.
MeSH term(s)	Animals ; Apoptosis/genetics ; Cell Respiration ; Energy Metabolism ; Gene Expression Regulation ; Humans ; Inactivation, Metabolic/genetics ; Mitochondria/genetics ; Mitochondria/metabolism ; Neoplasms/genetics ; Neoplasms/metabolism ; Oxidative Phosphorylation ; RNA/genetics ; RNA, Long Noncoding/genetics ; Reactive Oxygen Species/metabolism ; Signal Transduction
Chemical Substances	RNA, Long Noncoding ; RNA, mitochondrial ; Reactive Oxygen Species ; RNA (63231-63-0)
Language	English
Publishing date	2018-02
Publishing country	United States
Document type	Journal Article ; Review
ZDB-ID	282876-5
ISSN	1432-0983 ; 0172-8083
ISSN (online)	1432-0983
ISSN	0172-8083
DOI	10.1007/s00294-017-0744-1
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Article ; Online: Challenges in neoantigen-directed therapeutics.

Lybaert, Lien / Lefever, Steve / Fant, Bruno / Smits, Evelien / De Geest, Bruno / Breckpot, Karine / Dirix, Luc / Feldman, Steven A / van Criekinge, Wim / Thielemans, Kris / van der Burg, Sjoerd H / Ott, Patrick A / Bogaert, Cedric

Cancer cell

2022 Volume 41, Issue 1, Page(s) 15–40

Abstract: A fundamental prerequisite for the efficacy of cancer immunotherapy is the presence of functional, antigen-specific T cells within the tumor. Neoantigen-directed therapy is a promising strategy that aims at targeting the host's immune response against ... ...

Abstract	A fundamental prerequisite for the efficacy of cancer immunotherapy is the presence of functional, antigen-specific T cells within the tumor. Neoantigen-directed therapy is a promising strategy that aims at targeting the host's immune response against tumor-specific antigens, thereby eradicating cancer cells. Initial forays have been made in clinical environments utilizing vaccines and adoptive cell therapy; however, many challenges lie ahead. We provide an in-depth overview of the current state of the field with an emphasis on in silico neoantigen discovery and the clinical aspects that need to be addressed to unlock the full potential of this therapy.
MeSH term(s)	Humans ; Cancer Vaccines/therapeutic use ; Neoplasms/drug therapy ; Antigens, Neoplasm ; Immunotherapy ; T-Lymphocytes
Chemical Substances	Cancer Vaccines ; Antigens, Neoplasm
Language	English
Publishing date	2022-11-10
Publishing country	United States
Document type	Journal Article ; Review ; Comment
ZDB-ID	2078448-X
ISSN	1878-3686 ; 1535-6108
ISSN (online)	1878-3686
ISSN	1535-6108
DOI	10.1016/j.ccell.2022.10.013
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Article: Antisense Oligonucleotide-Based Downregulation of the G56R Pathogenic Variant Causing

Naessens, Sarah / Ruysschaert, Laurien / Lefever, Steve / Coppieters, Frauke / De Baere, Elfride

Genes

2019 Volume 10, Issue 5

Abstract: The recurrent missense variant in Nuclear Receptor Subfamily 2 Group E Member 3 (NR2E3), c.166G>A, p.(Gly56Arg) or G56R, underlies 1%-2% of cases with autosomal dominant retinitis pigmentosa (adRP), a frequent, genetically heterogeneous inherited retinal ...

Abstract	The recurrent missense variant in Nuclear Receptor Subfamily 2 Group E Member 3 (NR2E3), c.166G>A, p.(Gly56Arg) or G56R, underlies 1%-2% of cases with autosomal dominant retinitis pigmentosa (adRP), a frequent, genetically heterogeneous inherited retinal disease (IRD). The mutant NR2E3 protein has a presumed dominant negative effect (DNE) by competition for dimer formation with Cone-Rod Homeobox (CRX) but with abolishment of DNA binding, acting as a repressor in trans. Both the frequency and DNE of G56R make it an interesting target for allele-specific knock-down of the mutant allele using antisense oligonucleotides (AONs), an emerging therapeutic strategy for IRD. Here, we designed gapmer AONs with or without a locked nucleic acid modification at the site of the mutation, which were analyzed for potential off-target effects. Next, we overexpressed wild type (WT) or mutant NR2E3 in RPE-1 cells, followed by AON treatment. Transcript and protein levels of WT and mutant NR2E3 were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot respectively. All AONs showed a general knock-down of mutant and WT NR2E3 on RNA and protein level, showing the accessibility of the region for AON-induced knockdown. Further modifications are needed however to increase allele-specificity. In conclusion, we propose the first proof-of-concept for AON-mediated silencing of a single nucleotide variation with a dominant negative effect as a therapeutic approach for NR2E3-associated adRP.
MeSH term(s)	Cell Line ; Down-Regulation ; Genes, Dominant ; Humans ; Microtubule-Associated Proteins/genetics ; Microtubule-Associated Proteins/metabolism ; Mutation ; Mutation, Missense ; Oligonucleotides/pharmacology ; Oligonucleotides, Antisense/genetics ; Orphan Nuclear Receptors/genetics ; Orphan Nuclear Receptors/metabolism ; Polymorphism, Single Nucleotide/genetics ; Retina/pathology ; Retinitis Pigmentosa/genetics ; Retinitis Pigmentosa/metabolism
Chemical Substances	Microtubule-Associated Proteins ; NR2E3 protein, human ; Oligonucleotides ; Oligonucleotides, Antisense ; Orphan Nuclear Receptors ; RP1 protein, human ; locked nucleic acid
Language	English
Publishing date	2019-05-10
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2527218-4
ISSN	2073-4425
ISSN	2073-4425
DOI	10.3390/genes10050363
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Article ; Online: Comprehensive RNA dataset of tissue and plasma from patients with esophageal cancer or precursor lesions.

Schoofs, Kathleen / Philippron, Annouck / Avila Cobos, Francisco / Koster, Jan / Lefever, Steve / Anckaert, Jasper / De Looze, Danny / Vandesompele, Jo / Pattyn, Piet / De Preter, Katleen

Scientific data

2022 Volume 9, Issue 1, Page(s) 86

Abstract: In the past decades, the incidence of esophageal adenocarcinoma has increased dramatically in Western populations. Better understanding of disease etiology along with the identification of novel prognostic and predictive biomarkers are urgently needed to ...

Abstract	In the past decades, the incidence of esophageal adenocarcinoma has increased dramatically in Western populations. Better understanding of disease etiology along with the identification of novel prognostic and predictive biomarkers are urgently needed to improve the dismal survival probabilities. Here, we performed comprehensive RNA (coding and non-coding) profiling in various samples from 17 patients diagnosed with esophageal adenocarcinoma, high-grade dysplastic or non-dysplastic Barrett's esophagus. Per patient, a blood plasma sample, and a healthy and disease esophageal tissue sample were included. In total, this comprehensive dataset consists of 102 sequenced libraries from 51 samples. Based on this data, 119 expression profiles are available for three biotypes, including miRNA (51), mRNA (51) and circRNA (17). This unique resource allows for discovery of novel biomarkers and disease mechanisms, comparison of tissue and liquid biopsy profiles, integration of coding and non-coding RNA patterns, and can serve as a validation dataset in other RNA landscaping studies. Moreover, structural RNA differences can be identified in this dataset, including protein coding mutations, fusion genes, and circular RNAs.
MeSH term(s)	Adenocarcinoma/blood ; Adenocarcinoma/genetics ; Barrett Esophagus/blood ; Barrett Esophagus/genetics ; Biomarkers ; Disease Progression ; Esophageal Neoplasms/blood ; Esophageal Neoplasms/genetics ; Humans ; MicroRNAs/genetics ; Plasma/metabolism
Chemical Substances	Biomarkers ; MicroRNAs
Language	English
Publishing date	2022-03-14
Publishing country	England
Document type	Dataset ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2775191-0
ISSN	2052-4463 ; 2052-4463
ISSN (online)	2052-4463
ISSN	2052-4463
DOI	10.1038/s41597-022-01176-x
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Article: How long noncoding RNAs enforce their will on mitochondrial activity: regulation of mitochondrial respiration, reactive oxygen species production, apoptosis, and metabolic reprogramming in cancer

De Paepe, Boel / Steve Lefever / Pieter Mestdagh

Current genetics. 2018 Feb., v. 64, no. 1

2018

Abstract	The cellular transcriptome contains a wide diversity of untranslated RNAs, of which the class of regulatory long noncoding RNAs (lncRNAs) has only recently been recognized. Evidence swiftly accumulates of lncRNAs influencing mitochondrial activities of eukaryotic cells, and perturbed expression is conspicuously associated with human diseases. In this review, we describe the multifaceted effects of lncRNAs on mitochondrial function, more particularly on the balance between oxidative phosphorylation and glycolysis, on the production of reactive oxygen species, and on apoptosis in human cells. Emphasis is placed on the involvement of lncRNAs in cancer metabolism, as tumor cells rely heavily on modifications of mitochondrial functioning as an essential component for sustained tumorigenesis and cancer progression. From the nonexhaustive list of lncRNAS described in this review, ANRIL, AScmtRNA, H19, HOTAIR, LincRNA-p21, MALAT1, RMRP, SAMMSON, and VL30 have emerged as potent regulators of mitochondrial metabolism. Due to their key role in cancer progression, they represent potential targets of innovative lncRNA-based treatment strategies.
Keywords	apoptosis ; carcinogenesis ; disease course ; eukaryotic cells ; glycolysis ; human diseases ; humans ; mitochondria ; neoplasm cells ; neoplasms ; non-coding RNA ; oxidative phosphorylation ; reactive oxygen species ; transcriptome
Language	English
Dates of publication	2018-02
Size	p. 163-172.
Publishing place	Springer Berlin Heidelberg
Document type	Article
Note	Review
ZDB-ID	282876-5
ISSN	1432-0983 ; 0172-8083
ISSN (online)	1432-0983
ISSN	0172-8083
DOI	10.1007/s00294-017-0744-1
Database	NAL-Catalogue (AGRICOLA)

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Article: A Combined RNA Preservation and Extraction Protocol for Gene Expression Studies in Cacao Beans.

De Wever, Jocelyn / Tulkens, Dieter / Verwaeren, Jan / Everaert, Helena / Rottiers, Hayley / Dewettinck, Koen / Lefever, Steve / Messens, Kathy

Frontiers in plant science

2020 Volume 11, Page(s) 992

Abstract: Despite the high economic importance of cacao beans, few RNA-based studies have been conducted on this plant material and hence no optimal RNA-extraction has been reported. Moreover, extraction of high-quality RNA from recalcitrant cacao bean tissue has ... ...

Abstract	Despite the high economic importance of cacao beans, few RNA-based studies have been conducted on this plant material and hence no optimal RNA-extraction has been reported. Moreover, extraction of high-quality RNA from recalcitrant cacao bean tissue has shown many difficulties and requires optimization. Furthermore, cacao beans are mostly found at remote and under-resourced locations, which pressures the outsourcing of such analysis and thereby demands RNA-stable preservation and transportation of cacao beans. This study aims to select an appropriate RNA extraction and preservation/transportation method for cacao beans. For this purpose, three sample homogenization and five extraction protocols on cacao beans were compared. In addition, 13 preservation conditions-differing in tissue crushing degree, preservation method, duration, and temperature-were compared and evaluated. A comparative analysis revealed that CTAB-based homogenization and extraction outcompeted all tested commercial protocols in RNA yield and integrity, respectively. Preservation at -80°C affected RNA quality the least, whereas freeze-drying was most suitable for transportation at room temperature for maximum 1 week. The cacao bean RNA obtained from the selected methods were compatible for downstream applications. The results of this study will facilitate on-field sampling and transportation of genetically sensitive cacao material prior to cacao bean transcriptomic studies. In addition, valuable insights on sample homogenization, extraction, preservation, and transportation have been provided, which is of interest to every plant geneticist.
Language	English
Publishing date	2020-06-30
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2711035-7
ISSN	1664-462X
ISSN	1664-462X
DOI	10.3389/fpls.2020.00992
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Article ; Online: Comprehensive RNA dataset of tissue and plasma from patients with esophageal cancer or precursor lesions

Kathleen Schoofs / Annouck Philippron / Francisco Avila Cobos / Jan Koster / Steve Lefever / Jasper Anckaert / Danny De Looze / Jo Vandesompele / Piet Pattyn / Katleen De Preter

Scientific Data, Vol 9, Iss 1, Pp 1-

2022 Volume 12

Abstract: Measurement(s) mRNA Sequencing • MicroRNA Sequencing Technology Type(s) sequencer Sample Characteristic - Organism Homo sapiens Sample Characteristic - Location ... ...

Abstract	Measurement(s) mRNA Sequencing • MicroRNA Sequencing Technology Type(s) sequencer Sample Characteristic - Organism Homo sapiens Sample Characteristic - Location Belgium
Keywords	Science ; Q
Language	English
Publishing date	2022-03-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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