Acknowledgements requested in publications
« This work of the Interdisciplinary Thematic Institute IMCBio, as part of the ITI 2021-2028 program of the University of Strasbourg, CNRS and Inserm, was supported by IdEx Unistra (ANR-10-IDEX-0002), and by SFRI-STRAT’US project (ANR 20-SFRI-0012) and EUR IMCBio (ANR-17-EURE-0023) under the framework of the French Investments for the Future Program. »
2023
Dufossez, Robin; Krafft, Marie-Pierre; Ursuegui, Sylvain; Mosser, Michel; Mouftakhir, Safae; Pernod, Ketty; Chaubet, Guilhem; Ryckelynck, Michael; Wagner, Alain
Microfluidic Droplet Stabilization via SPAAC Promoted Antibody Conjugation at the Water/Oil Interface Article de journal
Dans: ACS Appl Mater Interfaces, 2023, ISSN: 1944-8252.
@article{pmid37704020,
title = {Microfluidic Droplet Stabilization via SPAAC Promoted Antibody Conjugation at the Water/Oil Interface},
author = {Robin Dufossez and Marie-Pierre Krafft and Sylvain Ursuegui and Michel Mosser and Safae Mouftakhir and Ketty Pernod and Guilhem Chaubet and Michael Ryckelynck and Alain Wagner},
doi = {10.1021/acsami.3c10655},
issn = {1944-8252},
year = {2023},
date = {2023-09-01},
urldate = {2023-09-01},
journal = {ACS Appl Mater Interfaces},
abstract = {Droplet-based microfluidics is leading the development of miniaturized, rapid, and sensitive version of enzyme-linked immunosorbent assays (ELISAs), a central method for protein detection. These assays involve the use of a functionalized surface able to selectively capture the desired analyte. Using the droplet's oil water interface as a capture surface requires designing custom-perfluorinated fluorosurfactants bearing azide-containing polar groups, which spontaneously react when forming the droplet with strain-alkyne-functionalized antibodies solubilized in the aqueous phase. In this article, we present our research on the influence of the structure of surfactant's hydrophilic heads on the efficiency of SPAAC functionalization and on the effect of this antibody grafting process on droplet stability. We have shown that while short linkers lead to high grafting efficiency, long linkers lead to high stability, and that an intermediate size is required to balance both parameters. In the described family of surfactants, the optimal structure proved to be a PEG linker connecting a polar di-azide head and a per-fluoropolyether tail (Krytox). We also found that grafting an increasing amount of antibody, thus increasing interface coverage, increases droplet stability. It thus appears that such a bi-partite system with a reactive fluoro-surfactant in the oil phase and reactive antibody counterpart in the aqueous phase gives access in situ to novel surfactant construct providing unexplored interface structures and droplet functionality.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Droplet-based microfluidics is leading the development of miniaturized, rapid, and sensitive version of enzyme-linked immunosorbent assays (ELISAs), a central method for protein detection. These assays involve the use of a functionalized surface able to selectively capture the desired analyte. Using the droplet's oil water interface as a capture surface requires designing custom-perfluorinated fluorosurfactants bearing azide-containing polar groups, which spontaneously react when forming the droplet with strain-alkyne-functionalized antibodies solubilized in the aqueous phase. In this article, we present our research on the influence of the structure of surfactant's hydrophilic heads on the efficiency of SPAAC functionalization and on the effect of this antibody grafting process on droplet stability. We have shown that while short linkers lead to high grafting efficiency, long linkers lead to high stability, and that an intermediate size is required to balance both parameters. In the described family of surfactants, the optimal structure proved to be a PEG linker connecting a polar di-azide head and a per-fluoropolyether tail (Krytox). We also found that grafting an increasing amount of antibody, thus increasing interface coverage, increases droplet stability. It thus appears that such a bi-partite system with a reactive fluoro-surfactant in the oil phase and reactive antibody counterpart in the aqueous phase gives access in situ to novel surfactant construct providing unexplored interface structures and droplet functionality.
Pitolli, Martina; Cela, Marta; Paulus, Caroline; Rudinger-Thirion, Joëlle; Frugier, Magali
RNA aptamers developed against tRip: A preliminary approach targeting tRNA entry in Plasmodium Article de journal
Dans: Biochimie, 2023, ISSN: 1638-6183.
@article{pmid37414209,
title = {RNA aptamers developed against tRip: A preliminary approach targeting tRNA entry in Plasmodium},
author = {Martina Pitolli and Marta Cela and Caroline Paulus and Joëlle Rudinger-Thirion and Magali Frugier},
doi = {10.1016/j.biochi.2023.06.011},
issn = {1638-6183},
year = {2023},
date = {2023-07-01},
urldate = {2023-07-01},
journal = {Biochimie},
abstract = {Malaria is caused by Plasmodium parasites that multiply inside host cells and can be lethal when P. falciparum is involved. We identified tRip as a membrane protein that facilitates the import of exogenous transfer RNA (tRNA) into the parasite. tRip encompasses a tRNA binding domain exposed on the parasite surface. We used the SELEX approach to isolate high-affinity and specific tRip-binding RNA motifs from a library of random 25 nucleotide-long sequences. In five rounds of combined negative and positive selections, an enriched pool of aptamers was obtained; sequencing revealed that they were all different in their primary sequence; only by comparing their structure predictions did most of the selected aptamers reveal a conserved 5-nucleotide motif sequence. We showed that the integral motif is essential for tRip-binding while the rest of the molecule can be significantly reduced or mutated as long as the motif is presented in a single-stranded region. Such RNA aptamers bind in place of the original tRNA substrate and act as an efficient competitor, suggesting that they can block tRip function and slow parasite development.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Malaria is caused by Plasmodium parasites that multiply inside host cells and can be lethal when P. falciparum is involved. We identified tRip as a membrane protein that facilitates the import of exogenous transfer RNA (tRNA) into the parasite. tRip encompasses a tRNA binding domain exposed on the parasite surface. We used the SELEX approach to isolate high-affinity and specific tRip-binding RNA motifs from a library of random 25 nucleotide-long sequences. In five rounds of combined negative and positive selections, an enriched pool of aptamers was obtained; sequencing revealed that they were all different in their primary sequence; only by comparing their structure predictions did most of the selected aptamers reveal a conserved 5-nucleotide motif sequence. We showed that the integral motif is essential for tRip-binding while the rest of the molecule can be significantly reduced or mutated as long as the motif is presented in a single-stranded region. Such RNA aptamers bind in place of the original tRNA substrate and act as an efficient competitor, suggesting that they can block tRip function and slow parasite development.
Gaucherand, Lea; Iyer, Amrita; Gilabert, Isabel; Rycroft, Chris H; Gaglia, Marta M
Cut site preference allows influenza A virus PA-X to discriminate between host and viral mRNAs Article de journal
Dans: Nat Microbiol, vol. 8, no. 7, p. 1304–1317, 2023, ISSN: 2058-5276.
@article{pmid37349586,
title = {Cut site preference allows influenza A virus PA-X to discriminate between host and viral mRNAs},
author = {Lea Gaucherand and Amrita Iyer and Isabel Gilabert and Chris H Rycroft and Marta M Gaglia},
doi = {10.1038/s41564-023-01409-8},
issn = {2058-5276},
year = {2023},
date = {2023-07-01},
urldate = {2023-07-01},
journal = {Nat Microbiol},
volume = {8},
number = {7},
pages = {1304--1317},
abstract = {Many viruses block host gene expression to take over the infected cell. This process, termed host shutoff, is thought to promote viral replication by preventing antiviral responses and redirecting cellular resources to viral processes. Several viruses from divergent families accomplish host shutoff through RNA degradation by endoribonucleases. However, viruses also need to ensure expression of their own genes. The influenza A virus endoribonuclease PA-X solves this problem by sparing viral mRNAs and some host RNAs necessary for viral replication. To understand how PA-X distinguishes between RNAs, we characterized PA-X cut sites transcriptome-wide using 5' rapid amplification of complementary DNA ends coupled to high-throughput sequencing. This analysis, along with RNA structure predictions and validation experiments using reporters, shows that PA-Xs from multiple influenza strains preferentially cleave RNAs at GCUG tetramers in hairpin loops. Importantly, GCUG tetramers are enriched in the human but not the influenza transcriptome. Moreover, optimal PA-X cut sites inserted in the influenza A virus genome are quickly selected against during viral replication in cells. This finding suggests that PA-X evolved these cleavage characteristics to preferentially target host over viral mRNAs in a manner reminiscent of cellular self versus non-self discrimination.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Many viruses block host gene expression to take over the infected cell. This process, termed host shutoff, is thought to promote viral replication by preventing antiviral responses and redirecting cellular resources to viral processes. Several viruses from divergent families accomplish host shutoff through RNA degradation by endoribonucleases. However, viruses also need to ensure expression of their own genes. The influenza A virus endoribonuclease PA-X solves this problem by sparing viral mRNAs and some host RNAs necessary for viral replication. To understand how PA-X distinguishes between RNAs, we characterized PA-X cut sites transcriptome-wide using 5' rapid amplification of complementary DNA ends coupled to high-throughput sequencing. This analysis, along with RNA structure predictions and validation experiments using reporters, shows that PA-Xs from multiple influenza strains preferentially cleave RNAs at GCUG tetramers in hairpin loops. Importantly, GCUG tetramers are enriched in the human but not the influenza transcriptome. Moreover, optimal PA-X cut sites inserted in the influenza A virus genome are quickly selected against during viral replication in cells. This finding suggests that PA-X evolved these cleavage characteristics to preferentially target host over viral mRNAs in a manner reminiscent of cellular self versus non-self discrimination.
Pivard, Mariane; Caldelari, Isabelle; Brun, Virginie; Croisier, Delphine; Jaquinod, Michel; Anzala, Nelson; Gilquin, Benoît; Teixeira, Chloé; Benito, Yvonne; Couzon, Florence; Romby, Pascale; Moreau, Karen; Vandenesch, François
Complex Regulation of Gamma-Hemolysin Expression Impacts Staphylococcus aureus Virulence Article de journal
Dans: Microbiol Spectr, p. e0107323, 2023, ISSN: 2165-0497.
@article{pmid37347186,
title = {Complex Regulation of Gamma-Hemolysin Expression Impacts Staphylococcus aureus Virulence},
author = {Mariane Pivard and Isabelle Caldelari and Virginie Brun and Delphine Croisier and Michel Jaquinod and Nelson Anzala and Benoît Gilquin and Chloé Teixeira and Yvonne Benito and Florence Couzon and Pascale Romby and Karen Moreau and François Vandenesch},
doi = {10.1128/spectrum.01073-23},
issn = {2165-0497},
year = {2023},
date = {2023-06-01},
urldate = {2023-06-01},
journal = {Microbiol Spectr},
pages = {e0107323},
abstract = {Staphylococcus aureus gamma-hemolysin CB (HlgCB) is a core-genome-encoded pore-forming toxin that targets the C5a receptor, similar to the phage-encoded Panton-Valentine leucocidin (PVL). Absolute quantification by mass spectrometry of HlgCB in 39 community-acquired pneumonia (CAP) isolates showed considerable variations in the HlgC and HlgB yields between isolates. Moreover, although HlgC and HlgB are encoded on a single operon, their levels were dissociated in 10% of the clinical strains studied. To decipher the molecular basis for the variation in expression and protein production among strains, different regulation levels were analyzed in representative clinical isolates and reference strains. Both the HlgCB level and the HlgC/HlgB ratio were found to depend on promoter activity and mRNA processing and translation. Strikingly, only one single nucleotide polymorphism (SNP) in the 5' untranslated region (UTR) of mRNA strongly impaired translation in the USA300 strain, leading to a strong decrease in the level of HlgC but not in HlgB. Finally, we found that high levels of HlgCB synthesis led to mortality in a rabbit model of pneumonia, correlated with the implication of the role of HlgCB in severe S. aureus CAP. Taken together, this work illustrates the complexity of virulence factor expression in clinical strains and demonstrates a butterfly effect where subtle genomic variations have a major impact on phenotype and virulence. S. aureus virulence in pneumonia results in its ability to produce several virulence factors, including the leucocidin PVL. Here, we demonstrate that HlgCB, another leucocidin, which targets the same receptors as PVL, highly contributes to S. aureus virulence in -negative strains. In addition, considerable variations in HlgCB quantities are observed among clinical isolates from patients with CAP. Biomolecular analyses have revealed that a few SNPs in the promoter sequences and only one SNP in the 5' UTR of mRNA induce the differential expression of , drastically impacting mRNA translation. This work illustrates the subtlety of regulatory mechanisms in bacteria, especially the sometimes major effects on phenotypes of single nucleotide variation in noncoding regions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Staphylococcus aureus gamma-hemolysin CB (HlgCB) is a core-genome-encoded pore-forming toxin that targets the C5a receptor, similar to the phage-encoded Panton-Valentine leucocidin (PVL). Absolute quantification by mass spectrometry of HlgCB in 39 community-acquired pneumonia (CAP) isolates showed considerable variations in the HlgC and HlgB yields between isolates. Moreover, although HlgC and HlgB are encoded on a single operon, their levels were dissociated in 10% of the clinical strains studied. To decipher the molecular basis for the variation in expression and protein production among strains, different regulation levels were analyzed in representative clinical isolates and reference strains. Both the HlgCB level and the HlgC/HlgB ratio were found to depend on promoter activity and mRNA processing and translation. Strikingly, only one single nucleotide polymorphism (SNP) in the 5' untranslated region (UTR) of mRNA strongly impaired translation in the USA300 strain, leading to a strong decrease in the level of HlgC but not in HlgB. Finally, we found that high levels of HlgCB synthesis led to mortality in a rabbit model of pneumonia, correlated with the implication of the role of HlgCB in severe S. aureus CAP. Taken together, this work illustrates the complexity of virulence factor expression in clinical strains and demonstrates a butterfly effect where subtle genomic variations have a major impact on phenotype and virulence. S. aureus virulence in pneumonia results in its ability to produce several virulence factors, including the leucocidin PVL. Here, we demonstrate that HlgCB, another leucocidin, which targets the same receptors as PVL, highly contributes to S. aureus virulence in -negative strains. In addition, considerable variations in HlgCB quantities are observed among clinical isolates from patients with CAP. Biomolecular analyses have revealed that a few SNPs in the promoter sequences and only one SNP in the 5' UTR of mRNA induce the differential expression of , drastically impacting mRNA translation. This work illustrates the subtlety of regulatory mechanisms in bacteria, especially the sometimes major effects on phenotypes of single nucleotide variation in noncoding regions.
Husser, C.; Vuilleumier, S.; Ryckelynck, M.
FluorMango, an RNA-Based Fluorogenic Biosensor for the Direct and Specific Detection of Fluoride Article de journal
Dans: Small, vol. 19, iss. 13, p. e2205232, 2023, ISSN: 1613-6829.
@article{pmid36436882,
title = {FluorMango, an RNA-Based Fluorogenic Biosensor for the Direct and Specific Detection of Fluoride},
author = {C. Husser and S. Vuilleumier and M. Ryckelynck},
doi = {10.1002/smll.202205232},
issn = {1613-6829},
year = {2023},
date = {2023-03-29},
urldate = {2023-03-01},
journal = {Small},
volume = {19},
issue = {13},
pages = {e2205232},
abstract = {Nucleic acids are not only essential actors of cell life but also extremely appealing molecular objects in the development of synthetic molecules for biotechnological application, such as biosensors to report on the presence and concentration of a target ligand by emission of a measurable signal. In this work, FluorMango, a fluorogenic ribonucleic acid (RNA)-based biosensor specific for fluoride is introduced. The molecule consists of two RNA aptamer modules, a fluoride-specific sensor derived from the crcB riboswitch which changes its structure upon interaction with the target ion, and the light-up RNA Mango-III that emits fluorescence when complexed with a fluorogen. The two modules are connected by an optimized communication module identified by ultrahigh-throughput screening, which results in extremely high fluorescence of FluorMango in the presence of fluoride, and background fluorescence in its absence. The value and efficiency of this biosensor by direct monitoring of defluorinase activity in living bacterial cells is illustrated, and the use of this new tool in future screening campaigns aiming at discovering new defluorinase activities is discussed.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Nucleic acids are not only essential actors of cell life but also extremely appealing molecular objects in the development of synthetic molecules for biotechnological application, such as biosensors to report on the presence and concentration of a target ligand by emission of a measurable signal. In this work, FluorMango, a fluorogenic ribonucleic acid (RNA)-based biosensor specific for fluoride is introduced. The molecule consists of two RNA aptamer modules, a fluoride-specific sensor derived from the crcB riboswitch which changes its structure upon interaction with the target ion, and the light-up RNA Mango-III that emits fluorescence when complexed with a fluorogen. The two modules are connected by an optimized communication module identified by ultrahigh-throughput screening, which results in extremely high fluorescence of FluorMango in the presence of fluoride, and background fluorescence in its absence. The value and efficiency of this biosensor by direct monitoring of defluorinase activity in living bacterial cells is illustrated, and the use of this new tool in future screening campaigns aiming at discovering new defluorinase activities is discussed.
2021
Lalaouna, D; Fochesato, S; Harir, M; Ortet, P; Schmitt-Kopplin, P; Heulin, T; Achouak, W
Amplifying and Fine-Tuning Rsm sRNAs Expression and Stability to Optimize the Survival of Pseudomonas brassicacerum in Nutrient-Poor Environments Article de journal
Dans: Microorganisms, vol. 9, no. 2, 2021, ISBN: 33530561, (2076-2607 (Print) 2076-2607 (Linking) Journal Article).
@article{nokey,
title = {Amplifying and Fine-Tuning Rsm sRNAs Expression and Stability to Optimize the Survival of Pseudomonas brassicacerum in Nutrient-Poor Environments},
author = {D Lalaouna and S Fochesato and M Harir and P Ortet and P Schmitt-Kopplin and T Heulin and W Achouak},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33530561},
doi = {10.3390/microorganisms9020250},
isbn = {33530561},
year = {2021},
date = {2021-01-01},
journal = {Microorganisms},
volume = {9},
number = {2},
abstract = {In the beneficial plant root-associated Pseudomonas brassicacearum strain NFM421, the GacS/GacA two-component system positively controls biofilm formation and the production of secondary metabolites through the synthesis of rsmX, rsmY and rsmZ. Here, we evidenced the genetic amplification of Rsm sRNAs by the discovery of a novel 110-nt long sRNA encoding gene, rsmX-2, generated by the duplication of rsmX-1 (formerly rsmX). Like the others rsm genes, its overexpression overrides the gacA mutation. We explored the expression and the stability of rsmX-1, rsmX-2, rsmY and rsmZ encoding genes under rich or nutrient-poor conditions, and showed that their amount is fine-tuned at the transcriptional and more interestingly at the post-transcriptional level. Unlike rsmY and rsmZ, we noticed that the expression of rsmX-1 and rsmX-2 genes was exclusively GacA-dependent. The highest expression level and longest half-life for each sRNA were correlated with the highest ppGpp and cyclic-di-GMP levels and were recorded under nutrient-poor conditions. Together, these data support the view that the Rsm system in P. brassicacearum is likely linked to the stringent response, and seems to be required for bacterial adaptation to nutritional stress.},
note = {2076-2607 (Print)
2076-2607 (Linking)
Journal Article},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In the beneficial plant root-associated Pseudomonas brassicacearum strain NFM421, the GacS/GacA two-component system positively controls biofilm formation and the production of secondary metabolites through the synthesis of rsmX, rsmY and rsmZ. Here, we evidenced the genetic amplification of Rsm sRNAs by the discovery of a novel 110-nt long sRNA encoding gene, rsmX-2, generated by the duplication of rsmX-1 (formerly rsmX). Like the others rsm genes, its overexpression overrides the gacA mutation. We explored the expression and the stability of rsmX-1, rsmX-2, rsmY and rsmZ encoding genes under rich or nutrient-poor conditions, and showed that their amount is fine-tuned at the transcriptional and more interestingly at the post-transcriptional level. Unlike rsmY and rsmZ, we noticed that the expression of rsmX-1 and rsmX-2 genes was exclusively GacA-dependent. The highest expression level and longest half-life for each sRNA were correlated with the highest ppGpp and cyclic-di-GMP levels and were recorded under nutrient-poor conditions. Together, these data support the view that the Rsm system in P. brassicacearum is likely linked to the stringent response, and seems to be required for bacterial adaptation to nutritional stress.
2020
Riley, L G; Rudinger-Thirion, J; Frugier, M; Wilson, M; Luig, M; Alahakoon, T I; Nixon, C Y; Kirk, E P; Roscioli, T; Lunke, S; Stark, Z; Wierenga, K J; Palle, S; Walsh, M; Higgs, E; Arbuckle, S; Thirukeswaran, S; Compton, A G; Thorburn, D R; Christodoulou, J
The expanding LARS2 phenotypic spectrum: HLASA, Perrault syndrome with leukodystrophy, and mitochondrial myopathy Article de journal
Dans: Hum Mutat, vol. 41, no. 8, p. 1425-1434, 2020, ISBN: 32442335, (1098-1004 (Electronic) 1059-7794 (Linking) Journal Article Research Support, Non-U.S. Gov't).
@article{nokey,
title = {The expanding LARS2 phenotypic spectrum: HLASA, Perrault syndrome with leukodystrophy, and mitochondrial myopathy},
author = {L G Riley and J Rudinger-Thirion and M Frugier and M Wilson and M Luig and T I Alahakoon and C Y Nixon and E P Kirk and T Roscioli and S Lunke and Z Stark and K J Wierenga and S Palle and M Walsh and E Higgs and S Arbuckle and S Thirukeswaran and A G Compton and D R Thorburn and J Christodoulou},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=32442335},
doi = {10.1002/humu.24050},
isbn = {32442335},
year = {2020},
date = {2020-01-01},
journal = {Hum Mutat},
volume = {41},
number = {8},
pages = {1425-1434},
abstract = {LARS2 variants are associated with Perrault syndrome, characterized by premature ovarian failure and hearing loss, and with an infantile lethal multisystem disorder: Hydrops, lactic acidosis, sideroblastic anemia (HLASA) in one individual. Recently we reported LARS2 deafness with (ovario) leukodystrophy. Here we describe five patients with a range of phenotypes, in whom we identified biallelic LARS2 variants: three patients with a HLASA-like phenotype, an individual with Perrault syndrome whose affected siblings also had leukodystrophy, and an individual with a reversible mitochondrial myopathy, lactic acidosis, and developmental delay. Three HLASA cases from two unrelated families were identified. All were males with genital anomalies. Two survived multisystem disease in the neonatal period; both have developmental delay and hearing loss. A 55-year old male with deafness has not displayed neurological symptoms while his female siblings with Perrault syndrome developed leukodystrophy and died in their 30s. Analysis of muscle from a child with a reversible myopathy showed reduced LARS2 and mitochondrial complex I levels, and an unusual form of degeneration. Analysis of recombinant LARS2 variant proteins showed they had reduced aminoacylation efficiency, with HLASA-associated variants having the most severe effect. A broad phenotypic spectrum should be considered in association with LARS2 variants.},
note = {1098-1004 (Electronic)
1059-7794 (Linking)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
LARS2 variants are associated with Perrault syndrome, characterized by premature ovarian failure and hearing loss, and with an infantile lethal multisystem disorder: Hydrops, lactic acidosis, sideroblastic anemia (HLASA) in one individual. Recently we reported LARS2 deafness with (ovario) leukodystrophy. Here we describe five patients with a range of phenotypes, in whom we identified biallelic LARS2 variants: three patients with a HLASA-like phenotype, an individual with Perrault syndrome whose affected siblings also had leukodystrophy, and an individual with a reversible mitochondrial myopathy, lactic acidosis, and developmental delay. Three HLASA cases from two unrelated families were identified. All were males with genital anomalies. Two survived multisystem disease in the neonatal period; both have developmental delay and hearing loss. A 55-year old male with deafness has not displayed neurological symptoms while his female siblings with Perrault syndrome developed leukodystrophy and died in their 30s. Analysis of muscle from a child with a reversible myopathy showed reduced LARS2 and mitochondrial complex I levels, and an unusual form of degeneration. Analysis of recombinant LARS2 variant proteins showed they had reduced aminoacylation efficiency, with HLASA-associated variants having the most severe effect. A broad phenotypic spectrum should be considered in association with LARS2 variants.
2019
Knaap, M S; Bugiani, M; Mendes, M I; Riley, L G; Smith, D E C; Rudinger-Thirion, J; Frugier, M; Breur, M; Crawford, J; Gaalen, J; Schouten, M; Willems, M; Waisfisz, Q; Mau-Them, F T; Rodenburg, R J; Taft, R J; Keren, B; Christodoulou, J; Depienne, C; Simons, C; Salomons, G S; Mochel, F
Biallelic variants in LARS2 and KARS cause deafness and (ovario)leukodystrophy Article de journal
Dans: Neurology, vol. 92, no. 11, p. e1225-e1237, 2019, ISBN: 30737337, (1526-632X (Electronic) 0028-3878 (Linking) Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't).
@article{nokey,
title = {Biallelic variants in LARS2 and KARS cause deafness and (ovario)leukodystrophy},
author = {M S Knaap and M Bugiani and M I Mendes and L G Riley and D E C Smith and J Rudinger-Thirion and M Frugier and M Breur and J Crawford and J Gaalen and M Schouten and M Willems and Q Waisfisz and F T Mau-Them and R J Rodenburg and R J Taft and B Keren and J Christodoulou and C Depienne and C Simons and G S Salomons and F Mochel},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=30737337},
doi = {10.1212/WNL.0000000000007098},
isbn = {30737337},
year = {2019},
date = {2019-01-01},
journal = {Neurology},
volume = {92},
number = {11},
pages = {e1225-e1237},
abstract = {OBJECTIVE: To describe the leukodystrophy caused by pathogenic variants in LARS2 and KARS, encoding mitochondrial leucyl transfer RNA (tRNA) synthase and mitochondrial and cytoplasmic lysyl tRNA synthase, respectively. METHODS: We composed a group of 5 patients with leukodystrophy, in whom whole-genome or whole-exome sequencing revealed pathogenic variants in LARS2 or KARS. Clinical information, brain MRIs, and postmortem brain autopsy data were collected. We assessed aminoacylation activities of purified mutant recombinant mitochondrial leucyl tRNA synthase and performed aminoacylation assays on patients' lymphoblasts and fibroblasts. RESULTS: Patients had a combination of early-onset deafness and later-onset neurologic deterioration caused by progressive brain white matter abnormalities on MRI. Female patients with LARS2 pathogenic variants had premature ovarian failure. In 2 patients, MRI showed additional signs of early-onset vascular abnormalities. In 2 other patients with LARS2 and KARS pathogenic variants, magnetic resonance spectroscopy revealed elevated white matter lactate, suggesting mitochondrial disease. Pathology in one patient with LARS2 pathogenic variants displayed evidence of primary disease of oligodendrocytes and astrocytes with lack of myelin and deficient astrogliosis. Aminoacylation activities of purified recombinant mutant leucyl tRNA synthase showed a 3-fold loss of catalytic efficiency. Aminoacylation assays on patients' lymphoblasts and fibroblasts showed about 50% reduction of enzyme activity. CONCLUSION: This study adds LARS2 and KARS pathogenic variants as gene defects that may underlie deafness, ovarian failure, and leukodystrophy with mitochondrial signature. We discuss the specific MRI characteristics shared by leukodystrophies caused by mitochondrial tRNA synthase defects. We propose to add aminoacylation assays as biochemical diagnostic tools for leukodystrophies.},
note = {1526-632X (Electronic)
0028-3878 (Linking)
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
OBJECTIVE: To describe the leukodystrophy caused by pathogenic variants in LARS2 and KARS, encoding mitochondrial leucyl transfer RNA (tRNA) synthase and mitochondrial and cytoplasmic lysyl tRNA synthase, respectively. METHODS: We composed a group of 5 patients with leukodystrophy, in whom whole-genome or whole-exome sequencing revealed pathogenic variants in LARS2 or KARS. Clinical information, brain MRIs, and postmortem brain autopsy data were collected. We assessed aminoacylation activities of purified mutant recombinant mitochondrial leucyl tRNA synthase and performed aminoacylation assays on patients' lymphoblasts and fibroblasts. RESULTS: Patients had a combination of early-onset deafness and later-onset neurologic deterioration caused by progressive brain white matter abnormalities on MRI. Female patients with LARS2 pathogenic variants had premature ovarian failure. In 2 patients, MRI showed additional signs of early-onset vascular abnormalities. In 2 other patients with LARS2 and KARS pathogenic variants, magnetic resonance spectroscopy revealed elevated white matter lactate, suggesting mitochondrial disease. Pathology in one patient with LARS2 pathogenic variants displayed evidence of primary disease of oligodendrocytes and astrocytes with lack of myelin and deficient astrogliosis. Aminoacylation activities of purified recombinant mutant leucyl tRNA synthase showed a 3-fold loss of catalytic efficiency. Aminoacylation assays on patients' lymphoblasts and fibroblasts showed about 50% reduction of enzyme activity. CONCLUSION: This study adds LARS2 and KARS pathogenic variants as gene defects that may underlie deafness, ovarian failure, and leukodystrophy with mitochondrial signature. We discuss the specific MRI characteristics shared by leukodystrophies caused by mitochondrial tRNA synthase defects. We propose to add aminoacylation assays as biochemical diagnostic tools for leukodystrophies.
2018
Monsion, B; Incarbone, M; Hleibieh, K; Poignavent, V; Ghannam, A; Dunoyer, P; Daeffler, L; Tilsner, J; Ritzenthaler, C
Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein Article de journal
Dans: Front Plant Sci, vol. 9, p. 70, 2018, ISBN: 29449856, (1664-462X (Print) 1664-462X (Linking) Journal Article).
@article{nokey,
title = {Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein},
author = {B Monsion and M Incarbone and K Hleibieh and V Poignavent and A Ghannam and P Dunoyer and L Daeffler and J Tilsner and C Ritzenthaler},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=29449856},
doi = {10.3389/fpls.2018.00070},
isbn = {29449856},
year = {2018},
date = {2018-01-01},
journal = {Front Plant Sci},
volume = {9},
pages = {70},
abstract = {Double-stranded RNA (dsRNA) plays essential functions in many biological processes, including the activation of innate immune responses and RNA interference. dsRNA also represents the genetic entity of some viruses and is a hallmark of infections by positive-sense single-stranded RNA viruses. Methods for detecting dsRNA rely essentially on immunological approaches and their use is often limited to in vitro applications, although recent developments have allowed the visualization of dsRNA in vivo. Here, we report the sensitive and rapid detection of long dsRNA both in vitro and in vivo using the dsRNA binding domain of the B2 protein from Flock house virus. In vitro, we adapted the system for the detection of dsRNA either enzymatically by northwestern blotting or by direct fluorescence labeling on fixed samples. In vivo, we produced stable transgenic Nicotiana benthamiana lines allowing the visualization of dsRNA by fluorescence microscopy. Using these techniques, we were able to discriminate healthy and positive-sense single-stranded RNA virus-infected material in plants and insect cells. In N. benthamiana, our system proved to be very potent for the spatio-temporal visualization of replicative RNA intermediates of a broad range of positive-sense RNA viruses, including high- vs. low-copy number viruses.},
note = {1664-462X (Print)
1664-462X (Linking)
Journal Article},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Double-stranded RNA (dsRNA) plays essential functions in many biological processes, including the activation of innate immune responses and RNA interference. dsRNA also represents the genetic entity of some viruses and is a hallmark of infections by positive-sense single-stranded RNA viruses. Methods for detecting dsRNA rely essentially on immunological approaches and their use is often limited to in vitro applications, although recent developments have allowed the visualization of dsRNA in vivo. Here, we report the sensitive and rapid detection of long dsRNA both in vitro and in vivo using the dsRNA binding domain of the B2 protein from Flock house virus. In vitro, we adapted the system for the detection of dsRNA either enzymatically by northwestern blotting or by direct fluorescence labeling on fixed samples. In vivo, we produced stable transgenic Nicotiana benthamiana lines allowing the visualization of dsRNA by fluorescence microscopy. Using these techniques, we were able to discriminate healthy and positive-sense single-stranded RNA virus-infected material in plants and insect cells. In N. benthamiana, our system proved to be very potent for the spatio-temporal visualization of replicative RNA intermediates of a broad range of positive-sense RNA viruses, including high- vs. low-copy number viruses.
Bastet, A; Lederer, B; Giovinazzo, N; Arnoux, X; German-Retana, S; Reinbold, C; Brault, V; Garcia, D; Djennane, S; Gersch, S; Lemaire, O; Robaglia, C; Gallois, J L
Trans-species synthetic gene design allows resistance pyramiding and broad-spectrum engineering of virus resistance in plants Article de journal
Dans: Plant Biotechnol J, 2018, ISBN: 29504210, (1467-7652 (Electronic) 1467-7644 (Linking) Journal Article).
@article{nokey,
title = {Trans-species synthetic gene design allows resistance pyramiding and broad-spectrum engineering of virus resistance in plants},
author = {A Bastet and B Lederer and N Giovinazzo and X Arnoux and S German-Retana and C Reinbold and V Brault and D Garcia and S Djennane and S Gersch and O Lemaire and C Robaglia and J L Gallois},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=29504210},
doi = {10.1111/pbi.12896},
isbn = {29504210},
year = {2018},
date = {2018-01-01},
journal = {Plant Biotechnol J},
abstract = {To infect plants, viruses rely heavily on their host's machinery. Plant genetic resistances based on host factor modifications can be found among existing natural variability and are widely used for some but not all crops. While biotechnology can supply for the lack of natural resistance alleles, new strategies need to be developed to increase resistance spectra and durability without impairing plant development. Here, we assess how the targeted allele modification of the Arabidopsis thaliana translation initiation factor eIF4E1 can lead to broad and efficient resistance to the major group of potyviruses. A synthetic Arabidopsis thaliana eIF4E1 allele was designed by introducing multiple amino acid changes associated with resistance to potyvirus in naturally occurring Pisum sativum alleles. This new allele encodes a functional protein while maintaining plant resistance to a potyvirus isolate that usually hijacks eIF4E1. Due to its biological functionality, this synthetic allele allows, at no developmental cost, the pyramiding of resistances to potyviruses that selectively use the two major translation initiation factors, eIF4E1 or its isoform eIFiso4E. Moreover, this combination extends the resistance spectrum to potyvirus isolates for which no efficient resistance has so far been found, including resistance-breaking isolates and an unrelated virus belonging to the Luteoviridae family. This study is a proof-of-concept for the efficiency of gene engineering combined with knowledge of natural variation to generate trans-species virus resistance at no developmental cost to the plant. This has implications for breeding of crops with broad-spectrum and high durability resistance using recent genome editing techniques.},
note = {1467-7652 (Electronic)
1467-7644 (Linking)
Journal Article},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
To infect plants, viruses rely heavily on their host's machinery. Plant genetic resistances based on host factor modifications can be found among existing natural variability and are widely used for some but not all crops. While biotechnology can supply for the lack of natural resistance alleles, new strategies need to be developed to increase resistance spectra and durability without impairing plant development. Here, we assess how the targeted allele modification of the Arabidopsis thaliana translation initiation factor eIF4E1 can lead to broad and efficient resistance to the major group of potyviruses. A synthetic Arabidopsis thaliana eIF4E1 allele was designed by introducing multiple amino acid changes associated with resistance to potyvirus in naturally occurring Pisum sativum alleles. This new allele encodes a functional protein while maintaining plant resistance to a potyvirus isolate that usually hijacks eIF4E1. Due to its biological functionality, this synthetic allele allows, at no developmental cost, the pyramiding of resistances to potyviruses that selectively use the two major translation initiation factors, eIF4E1 or its isoform eIFiso4E. Moreover, this combination extends the resistance spectrum to potyvirus isolates for which no efficient resistance has so far been found, including resistance-breaking isolates and an unrelated virus belonging to the Luteoviridae family. This study is a proof-of-concept for the efficiency of gene engineering combined with knowledge of natural variation to generate trans-species virus resistance at no developmental cost to the plant. This has implications for breeding of crops with broad-spectrum and high durability resistance using recent genome editing techniques.
