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  1. Article ; Online: Evaluation of a viral transcriptome Next Generation Sequencing assay as an alternative to animal assays for viral safety testing of cell substrates

    Beurdeley-Fehlbaum, Pascale / Pennington, Matthew / Hégerlé, Nicolas / Albert, Mélanie / Bennett, Amy / Cheval, Justine / Joe, Allison Clark / Cruveiller, Stéphane / Desbrousses, Céline / Frederick, Janalyn / Gros, Edwige / Hunter, Kathryn / Jaber, Tareq / Gaiser, Madison / Jouffroy, Ophélie / Lamamy, Arnaud / Melkowski, Mickael / Moro, Jennifer / Niksa, Paula /
    Pillai, Shenba / Eloit, Marc / Ruppach, Horst

    Vaccine. 2023 Aug., v. 41, no. 37 p.5383-5391

    2023  

    Abstract: The viral safety of biological products is ensured by tests throughout the production chain, and, for certain products, by steps in the manufacturing process enabling the elimination or inactivation of viruses. Current testing programs include sample ... ...

    Abstract The viral safety of biological products is ensured by tests throughout the production chain, and, for certain products, by steps in the manufacturing process enabling the elimination or inactivation of viruses. Current testing programs include sample inoculation in animals and embryonic eggs. Following the 3Rs principles of replacement, reduction, and refinement of animal-use methods, such techniques are intended to be replaced not only for ethical reasons but also because of their inherent technical limitations, their long turnaround times, and their limits in virus detection. Therefore, we have compared the limit and range of sensitivity of in vivo tests used for viral testing of cells with a transcriptomic assay based on Next Generation Sequencing (NGS). Cell cultures were infected with a panel of nine (9) viruses, among them only five (5) were detected, with variable sensitivity, by in vivo tests. The transcriptomic assay was able to detect one (1) infected cell among 10³ to 10⁷ non-infected cells for all viruses assessed, including those not detected by the conventional in vivo tests. Here we show that NGS extends the breath of detection of viral contaminants compared to traditional testing. Collectively, these results support the replacement of the conventional in vivo tests by an NGS-based transcriptomic assay for virus safety testing of cell substrates.
    Keywords animal use alternatives ; animals ; ethics ; transcriptome ; transcriptomics ; vaccines ; viral contamination ; viruses ; Adventitious virus ; Next Generation Sequencing ; In vivo assay ; Cell substrates
    Language English
    Dates of publication 2023-08
    Size p. 5383-5391.
    Publishing place Elsevier Ltd
    Document type Article ; Online
    Note Pre-press version
    ZDB-ID 605674-x
    ISSN 1873-2518 ; 0264-410X
    ISSN (online) 1873-2518
    ISSN 0264-410X
    DOI 10.1016/j.vaccine.2023.07.019
    Database NAL-Catalogue (AGRICOLA)

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  2. Article ; Online: Evaluation of a viral transcriptome Next Generation Sequencing assay as an alternative to animal assays for viral safety testing of cell substrates.

    Beurdeley-Fehlbaum, Pascale / Pennington, Matthew / Hégerlé, Nicolas / Albert, Mélanie / Bennett, Amy / Cheval, Justine / Clark, Allison / Cruveiller, Stéphane / Desbrousses, Céline / Frederick, Janalyn / Gros, Edwige / Hunter, Kathryn / Jaber, Tareq / Gaiser, Madison / Jouffroy, Ophélie / Lamamy, Arnaud / Melkowski, Mickael / Moro, Jennifer / Niksa, Paula /
    Pillai, Shenba / Eloit, Marc / Ruppach, Horst

    Vaccine

    2023  Volume 41, Issue 37, Page(s) 5383–5391

    Abstract: The viral safety of biological products is ensured by tests throughout the production chain, and, for certain products, by steps in the manufacturing process enabling the elimination or inactivation of viruses. Current testing programs include sample ... ...

    Abstract The viral safety of biological products is ensured by tests throughout the production chain, and, for certain products, by steps in the manufacturing process enabling the elimination or inactivation of viruses. Current testing programs include sample inoculation in animals and embryonic eggs. Following the 3Rs principles of replacement, reduction, and refinement of animal-use methods, such techniques are intended to be replaced not only for ethical reasons but also because of their inherent technical limitations, their long turnaround times, and their limits in virus detection. Therefore, we have compared the limit and range of sensitivity of in vivo tests used for viral testing of cells with a transcriptomic assay based on Next Generation Sequencing (NGS). Cell cultures were infected with a panel of nine (9) viruses, among them only five (5) were detected, with variable sensitivity, by in vivo tests. The transcriptomic assay was able to detect one (1) infected cell among 10
    MeSH term(s) Animals ; Transcriptome ; High-Throughput Nucleotide Sequencing ; Viruses/genetics ; Cell Culture Techniques ; Biological Products
    Chemical Substances Biological Products
    Language English
    Publishing date 2023-07-18
    Publishing country Netherlands
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 605674-x
    ISSN 1873-2518 ; 0264-410X
    ISSN (online) 1873-2518
    ISSN 0264-410X
    DOI 10.1016/j.vaccine.2023.07.019
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: A microarray configuration to quantify expression levels and relative abundance of splice variants.

    Fehlbaum, Pascale / Guihal, Caroline / Bracco, Laurent / Cochet, Olivier

    Nucleic acids research

    2005  Volume 33, Issue 5, Page(s) e47

    Abstract: Over the past decade, alternative RNA splicing has raised a great interest appearing to be of high importance in the generation of expression diversity. This regulatory process plays a critical role in the normal development and its impact on the ... ...

    Abstract Over the past decade, alternative RNA splicing has raised a great interest appearing to be of high importance in the generation of expression diversity. This regulatory process plays a critical role in the normal development and its impact on the initiation and development of human disorders as well as on the pharmacological properties of drugs is increasingly being recognized. Only few studies describe specific alternative splicing expression profiling. Microarray strategies have been conceived to address alternative splicing events but with very few experimental data related to their abilities to provide true quantification values. We have developed a specific microarray configuration relying on a few, well optimized probes per splice event. Basically, five probes of 24mer are used to fully characterize a splice event. These probes are of two types, exon probes and junction probes, and are either specific to a splice event or not. The performances of such a 'splice array' were validated on synthetic model systems and on complex biological materials. The results indicate that DNA chips based on this design combining exon and junction derived probes enable the detection and, absolute and relative quantification of splice variants. In addition, this strategy is compatible with all the microarrays that use oligonucleotide probes.
    MeSH term(s) Alternative Splicing ; Cell Line ; Exons ; Female ; Gene Expression Profiling/methods ; Humans ; Introns ; Oligonucleotide Array Sequence Analysis/methods ; Oligonucleotide Probes/chemistry ; Protein Isoforms/analysis ; Protein Isoforms/genetics
    Chemical Substances Oligonucleotide Probes ; Protein Isoforms
    Language English
    Publishing date 2005-03-10
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Validation Study
    ZDB-ID 186809-3
    ISSN 1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
    ISSN (online) 1362-4962 ; 1362-4954
    ISSN 0301-5610 ; 0305-1048
    DOI 10.1093/nar/gni047
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article: Protein phosphatase 1 binds to the RNA recognition motif of several splicing factors and regulates alternative pre-mRNA processing.

    Novoyatleva, Tatyana / Heinrich, Bettina / Tang, Yesheng / Benderska, Natalya / Butchbach, Matthew E R / Lorson, Christian L / Lorson, Monique A / Ben-Dov, Claudia / Fehlbaum, Pascale / Bracco, Laurent / Burghes, Arthur H M / Bollen, Mathieu / Stamm, Stefan

    Human molecular genetics

    2007  Volume 17, Issue 1, Page(s) 52–70

    Abstract: Alternative splicing emerges as one of the most important mechanisms to generate transcript diversity. It is regulated by the formation of protein complexes on pre-mRNA. We demonstrate that protein phosphatase 1 (PP1) binds to the splicing factor ... ...

    Abstract Alternative splicing emerges as one of the most important mechanisms to generate transcript diversity. It is regulated by the formation of protein complexes on pre-mRNA. We demonstrate that protein phosphatase 1 (PP1) binds to the splicing factor transformer2-beta1 (tra2-beta1) via a phylogenetically conserved RVDF sequence located on the RNA recognition motif (RRM) of tra2-beta1. PP1 binds directly to tra2-beta1 and dephosphorylates it, which regulates the interaction between tra2-beta1 and other proteins. Eight other proteins, including SF2/ASF and SRp30c, contain an evolutionary conserved PP1 docking motif in the beta-4 strand of their RRMs indicating that binding to PP1 is a new function of some RRMs. Reducing PP1 activity promotes usage of numerous alternative exons, demonstrating a role of PP1 activity in splice site selection. PP1 inhibition promotes inclusion of the survival of motoneuron 2 exon 7 in a mouse model expressing the human gene. This suggests that reducing PP1 activity could be a new therapeutic principle to treat spinal muscular atrophy and other diseases caused by missplicing events. Our data indicate that the binding of PP1 to evolutionary conserved motifs in several RRMs is the link between known signal transduction pathways regulating PP1 activity and pre-mRNA processing.
    MeSH term(s) Alternative Splicing ; Amino Acid Motifs ; Amino Acid Sequence ; Animals ; Base Sequence ; Binding Sites ; Cell Line ; Conserved Sequence ; Cyclic AMP Response Element-Binding Protein/chemistry ; Cyclic AMP Response Element-Binding Protein/genetics ; Cyclic AMP Response Element-Binding Protein/metabolism ; DNA Primers/genetics ; Evolution, Molecular ; Exons ; Humans ; Molecular Sequence Data ; Nerve Tissue Proteins/chemistry ; Nerve Tissue Proteins/genetics ; Nerve Tissue Proteins/metabolism ; Phylogeny ; Protein Phosphatase 1/metabolism ; RNA Precursors/metabolism ; RNA-Binding Proteins/chemistry ; RNA-Binding Proteins/genetics ; RNA-Binding Proteins/metabolism ; Recombinant Proteins/chemistry ; Recombinant Proteins/genetics ; Recombinant Proteins/metabolism ; SMN Complex Proteins ; Sequence Homology, Amino Acid ; Serine-Arginine Splicing Factors
    Chemical Substances Cyclic AMP Response Element-Binding Protein ; DNA Primers ; Nerve Tissue Proteins ; RNA Precursors ; RNA-Binding Proteins ; Recombinant Proteins ; SMN Complex Proteins ; TRA2B protein, human ; Serine-Arginine Splicing Factors (170974-22-8) ; Protein Phosphatase 1 (EC 3.1.3.16)
    Language English
    Publishing date 2007-10-02
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 1108742-0
    ISSN 1460-2083 ; 0964-6906
    ISSN (online) 1460-2083
    ISSN 0964-6906
    DOI 10.1093/hmg/ddm284
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article: Role of chemotherapy resistance genes in outcome of neuroblastoma.

    de Cremoux, Patricia / Jourdan-Da-Silva, Nathalie / Couturier, Jérôme / Tran-Perennou, Carine / Schleiermacher, Gudrun / Fehlbaum, Pascale / Doz, François / Mosseri, Véronique / Delattre, Olivier / Klijanienko, Jerzy / Vielh, Philippe / Michon, Jean

    Pediatric blood & cancer

    2007  Volume 48, Issue 3, Page(s) 311–317

    Abstract: Background: Neuroblastoma is a heterogeneous pediatric disease. Most patients with localized disease usually have a favorable prognosis, but patients with advanced disease have a poor prognosis despite combination chemotherapy. Treatment failure may be ... ...

    Abstract Background: Neuroblastoma is a heterogeneous pediatric disease. Most patients with localized disease usually have a favorable prognosis, but patients with advanced disease have a poor prognosis despite combination chemotherapy. Treatment failure may be attributable to resistance to cytotoxic drugs.
    Procedure: Using quantitative RT-PCR, we investigated the clinical significance of the level of mRNA expression of multidrug resistance genes (MDR1, MRP1, MRP5, LRP) in a series of 29 advanced neuroblastoma samples.
    Results: At the end of induction chemotherapy, 48% of patients achieved a clinical complete response, 28% achieved a partial response or stable disease, and 24% presented progressive disease. MDR1 mRNA overexpression (i.e., mRNA level >2 copies of MDR1 gene) was observed in 74% of samples, and MRP1, MRP5, LRP overexpression was observed less frequently (30, 33, and 33% of samples, respectively). None of these parameters were predictive of response, relapse, or survival. However, clinical response to treatment was highly predictive of relapse-free survival and overall survival.
    Conclusions: High expression of these multidrug resistance genes in advanced neuroblastoma is not the main parameter of response to cytotoxic drugs; clinical response to treatment remains the most important parameter in predicting the prognosis of patients with advanced neuroblastoma, until other relevant laboratory parameters have been identified.
    MeSH term(s) ATP-Binding Cassette, Sub-Family B, Member 1/biosynthesis ; ATP-Binding Cassette, Sub-Family B, Member 1/genetics ; ATP-Binding Cassette, Sub-Family B, Member 1/physiology ; Adolescent ; Antineoplastic Combined Chemotherapy Protocols/pharmacology ; Antineoplastic Combined Chemotherapy Protocols/therapeutic use ; Carboplatin/administration & dosage ; Carboplatin/pharmacology ; Cell Line, Tumor/drug effects ; Cell Line, Tumor/metabolism ; Child ; Child, Preschool ; Chromosome Deletion ; Chromosomes, Human, Pair 1/ultrastructure ; Cisplatin/administration & dosage ; Cisplatin/pharmacology ; Computer Systems ; Cyclophosphamide/administration & dosage ; Cyclophosphamide/pharmacology ; Disease-Free Survival ; Doxorubicin/administration & dosage ; Doxorubicin/pharmacology ; Drug Resistance, Multiple/genetics ; Drug Resistance, Neoplasm/genetics ; Etoposide/administration & dosage ; Etoposide/pharmacology ; Female ; Gene Expression Profiling ; Genes, MDR ; Genes, myc ; Humans ; Infant ; Kaplan-Meier Estimate ; Male ; Multidrug Resistance-Associated Proteins/biosynthesis ; Multidrug Resistance-Associated Proteins/genetics ; Multidrug Resistance-Associated Proteins/physiology ; Neoplasm Proteins/biosynthesis ; Neoplasm Proteins/genetics ; Neoplasm Proteins/physiology ; Neuroblastoma/drug therapy ; Neuroblastoma/genetics ; Neuroblastoma/metabolism ; Neuroblastoma/mortality ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology ; RNA, Messenger/biosynthesis ; RNA, Neoplasm/biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; S Phase ; Treatment Outcome ; Vault Ribonucleoprotein Particles/biosynthesis ; Vault Ribonucleoprotein Particles/genetics ; Vault Ribonucleoprotein Particles/physiology ; Vincristine/administration & dosage ; Vincristine/pharmacology
    Chemical Substances ABCC5 protein, human ; ATP-Binding Cassette, Sub-Family B, Member 1 ; Multidrug Resistance-Associated Proteins ; Neoplasm Proteins ; RNA, Messenger ; RNA, Neoplasm ; Vault Ribonucleoprotein Particles ; major vault protein ; Vincristine (5J49Q6B70F) ; Etoposide (6PLQ3CP4P3) ; Doxorubicin (80168379AG) ; Cyclophosphamide (8N3DW7272P) ; Carboplatin (BG3F62OND5) ; Cisplatin (Q20Q21Q62J) ; multidrug resistance-associated protein 1 (Y49M64GZ4Q)
    Language English
    Publishing date 2007-03
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2131448-2
    ISSN 1545-5017 ; 1545-5009
    ISSN (online) 1545-5017
    ISSN 1545-5009
    DOI 10.1002/pbc.20853
    Database MEDical Literature Analysis and Retrieval System OnLINE

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