112 results on '"Rouet R"'
Search Results
2. Epitope-based selection of SARS-CoV-2 neutralizing antibodies from convalescent patients
- Author
-
Langley, D.B., primary, Christ, D., additional, and Rouet, R., additional
- Published
- 2022
- Full Text
- View/download PDF
3. Decreased susceptibility to arrhythmias in hypertrophied hearts of physically trained rats
- Author
-
Bélichard, P., Pruneau, D., Salzmann, J. L., and Rouet, R.
- Published
- 1992
- Full Text
- View/download PDF
4. Electrophysiological effects of morphine in an in vitro model of the ‘border zone’ between normal and ischaemic–reperfused guinea-pig myocardium
- Author
-
Yvon, A., Hanouz, J.-L., Terrien, X., Ducouret, P., Rouet, R., Bricard, H., and Gérard, J.-L.
- Published
- 2002
5. Anti recombinant prolactin receptor scFv
- Author
-
Langley, D.B., primary, Rouet, R., additional, and Christ, D., additional
- Published
- 2019
- Full Text
- View/download PDF
6. Retrofitting antibodies with stabilizing mutations. Herceptin VL mutant F53D.
- Author
-
Langley, D.B., primary, Rouet, R., additional, and Christ, D., additional
- Published
- 2015
- Full Text
- View/download PDF
7. Crystal Structure of anti-MSP2 Fv fragment (mAb6D8)in complex with MSP2 11-23
- Author
-
Morales, R.A.V., primary, MacRaild, C.A., additional, Seow, J., additional, Bankala, K., additional, Drinkwater, N., additional, McGowan, S., additional, Rouet, R., additional, Christ, D., additional, Anders, R.F., additional, and Norton, R.S., additional
- Published
- 2015
- Full Text
- View/download PDF
8. Crystal Structure of anti-MSP2 Fv fragment (mAb6D8) in complex with 3D7-MSP2 14-30
- Author
-
Morales, R.A.V., primary, MacRaild, C.A., additional, Seow, J., additional, Bankala, K., additional, Drinkwater, N., additional, McGowan, S., additional, Rouet, R., additional, Christ, D., additional, Anders, R.F., additional, and Norton, R.S., additional
- Published
- 2015
- Full Text
- View/download PDF
9. Crystal Structure of anti-MSP2 Fv fragment (mAb6D8)in complex with FC27-MSP2 14-30
- Author
-
Morales, R.A.V., primary, MacRaild, C.A., additional, Seow, J., additional, Bankala, K., additional, Drinkwater, N., additional, McGowan, S., additional, Rouet, R., additional, Christ, D., additional, Anders, R.F., additional, and Norton, R.S., additional
- Published
- 2015
- Full Text
- View/download PDF
10. Human heavy-chain domain antibody in complex with hen egg-white lysozyme
- Author
-
Christ, D., primary, Langley, D.B., additional, and Rouet, R., additional
- Published
- 2015
- Full Text
- View/download PDF
11. Structure of a human VH antibody domain binding to the cleft of hen egg lysozyme
- Author
-
Rouet, R., primary, Langley, D.B., additional, and Christ, D., additional
- Published
- 2015
- Full Text
- View/download PDF
12. Structural evidence for antigen receptor evolution
- Author
-
Langley, D.B., primary, Rouet, R., additional, Roome, B., additional, Stock, D., additional, and Christ, D., additional
- Published
- 2014
- Full Text
- View/download PDF
13. A general strategy for the generation of human antibody variable domains with increased aggregation resistance
- Author
-
Dudgeon, K., primary, Rouet, R., additional, Kokmeijer, I., additional, Langley, D.B., additional, and Christ, D., additional
- Published
- 2012
- Full Text
- View/download PDF
14. KATP channels and 'border zone' arrhythmias: role of the repolarization dispersion between normal and ischaemic ventricular regions.
- Author
-
Picard, Sandra, Rouet, René, Ducouret, Pierre, Puddu, Paolo Emilio, Flais, Frédéric, Criniti, Anna, Monti, Francesco, Gérard, Jean-Louis, Picard, S, Rouet, R, Ducouret, P, Puddu, P E, Flais, F, Criniti, A, Monti, F, and Gérard, J L
- Published
- 1999
- Full Text
- View/download PDF
15. In Vitro and In Vivo Electrophysiologic Effects of Indecainide Hydrochloride: Role of the Use-Dependent Depression of the Sodium Channel in the Alterations of Electrophysiologic Characteristics After Intravenous Administration in Humans.
- Author
-
Libersa, C. C., Rouet, R., Caron, J. F., Werquin, S., Kacet, S., Dupuis, B. A., and Lekieffre, J. P.
- Abstract
Examines the intracellular electrophysiologic effects of a novel antiarrhythmic agent, indecainide hydrochloride, in guinea pig papillary muscle preparations using standard microelectrode techniques. Electrophysiologic effects observed after intravenous infusion in patients undergoing electrophysiologic study; Classification of indecainide as a slow class IC antiarrhythmic agent with promising electrophysiologic properties.
- Published
- 1988
16. Interest and Limitations of Phenotype Determination of Hydroxylation Ability in Patients Treated with Propafenone.
- Author
-
Libersa, C., Caron, J., Bechtel, P., Kacet, S., Dagano, J., Rouet, R., Marchand, X., Dupuis, B., and Lekieffre, J.
- Abstract
In order to confirm that variations in the plasma levels of propafenone in patients receiving the same daily dose are polymorphically distributed in relation to oxidative metabolism, the relationship between propafenone administration and debrisoquine metabolic phenotype was studied in two series of Caucasian patients with chronic supraventricular or ventricular arrhythmias. In a first series of 16 Caucasian patients treated with propafenone, the poor metabolizer phenotype appeared to he more than 90%. In a second series of 13 Caucasian patients, two debrisoquine tests were performed: the first phenotype determination of debrisoquine hydroxylation ability was done without propafenone administration and the second determination when a steady state concentration of propafenone was reached. Of the 10 extensive metabolizers (determined without propafenone treatment), 7 patients became apparent poor metabolizers while considering the debrisoquine test during propafenone treatment with a general shift of the 13 studied patients to the upper values of log of the metabolic ratio debrisoquine/4-OH debrisoquine suggesting deficiency in metabolizing debrisoquine. This study seems to indicate the presence of a common hydroxylation for propafenone and debrisoquine, which probably compete in the pathway. This metabolic competition should therefore be taken into consideration during phenotypic determination of hydroxylation ability. [ABSTRACT FROM AUTHOR]
- Published
- 1987
- Full Text
- View/download PDF
17. Comparative Electrophysiological Effects of Propafenone, 5-Hydroxy-Propafenone, and N-Depropylpropafenone on Guinea Pig Ventricular Muscle Fibers.
- Author
-
Rouet, R., Libersa, C. C., Broly, F., Caron, J. F., Adamantidis, M. M., Honore, E., Wajman, A., and Dupuis, B. A.
- Published
- 1989
- Full Text
- View/download PDF
18. 071 - PAR1 contribution in acute electrophysiological properties of oral anticoagulants in rabbit pulmonary vein sleeve preparations.
- Author
-
Font, J., Simeon, M., Simard, C., Allouche, S., Plane, A., Brionne-François, M., Rouet, R., Manrique, A., Puddu, P., Milliez, P., and Alexandre, J.
- Published
- 2017
- Full Text
- View/download PDF
19. Bradykinin and adenosine receptors mediate desflurane induced postconditioning in human myocardium: role of reactive oxygen species
- Author
-
Gérard Jean-Louis, Massetti Massimo, Babatasi Gérard, Ivascau Calin, Rouet René, Buléon Clément, Lemoine Sandrine, and Hanouz Jean-Luc
- Subjects
Anesthesiology ,RD78.3-87.3 - Abstract
Abstract Background Desflurane during early reperfusion has been shown to postcondition human myocardium, in vitro. We investigated the role of adenosine and bradykinin receptors, and generation of radical oxygen species in desflurane-induced postconditioning in human myocardium. Methods We recorded isometric contraction of human right atrial trabeculae hanged in an oxygenated Tyrode's solution (34 degrees Celsius, stimulation frequency 1 Hz). After a 30-min hypoxic period, desflurane 6% was administered during the first 5 min of reoxygenation. Desflurane was administered alone or with pretreatment of N-mercaptopropionylglycine, a reactive oxygen species scavenger, 8-(p-Sulfophenyl)theophylline, an adenosine receptor antagonist, HOE140, a selective B2 bradykinin receptor antagonist. In separate groups, adenosine and bradykinin were administered during the first minutes of reoxygenation alone or in presence of N-mercaptopropionylglycine. The force of contraction of trabeculae was recorded continuously. Developed force at the end of a 60-min reoxygenation period was compared (mean ± standard deviation) between the groups by a variance analysis and post hoc test. Results Desflurane 6% (84 ± 6% of baseline) enhanced the recovery of force after 60-min of reoxygenation as compared to control group (51 ± 8% of baseline, P < 0.0001). N-mercaptopropionylglycine (54 ± 3% of baseline), 8-(p-Sulfophenyl)theophylline (62 ± 9% of baseline), HOE140 (58 ± 6% of baseline) abolished desflurane-induced postconditioning. Adenosine (80 ± 9% of baseline) and bradykinin (83 ± 4% of baseline) induced postconditioning (P < 0.0001 vs control), N-mercaptopropionylglycine abolished the beneficial effects of adenosine and bradykinin (54 ± 8 and 58 ± 5% of baseline, respectively). Conclusions In vitro, desflurane-induced postconditioning depends on reactive oxygen species production, activation of adenosine and bradykinin B2 receptors. And, the cardioprotective effect of adenosine and bradykinin administered at the beginning of reoxygenation, was mediated, at least in part, through ROS production.
- Published
- 2010
- Full Text
- View/download PDF
20. R003 Effets du sevoflurane et du desflurane sur une preparation d'oreillette humaine in vitro
- Author
-
Bessodes, A, Hanouz, J.L., Babatasi, G, M.A.ssetti, M, Khayat, A, Bricard, H, Rouet, R, Ducouret, P, and Gérard, J.L.
- Published
- 1998
- Full Text
- View/download PDF
21. A.137 Elelctrophysiological effect bupivacaine studied an in vitro model of ischaemia
- Author
-
Gérard, J.L., Picard, S., Rouet, R., Fiais, F., and Ducouret, P.
- Published
- 1996
- Full Text
- View/download PDF
22. Human genomic DNA is widely interspersed with i-motif structures.
- Author
-
Peña Martinez CD, Zeraati M, Rouet R, Mazigi O, Henry JY, Gloss B, Kretzmann JA, Evans CW, Ruggiero E, Zanin I, Marušič M, Plavec J, Richter SN, Bryan TM, Smith NM, Dinger ME, Kummerfeld S, and Christ D
- Abstract
DNA i-motif structures are formed in the nuclei of human cells and are believed to provide critical genomic regulation. While the existence, abundance, and distribution of i-motif structures in human cells has been demonstrated and studied by immunofluorescent staining, and more recently NMR and CUT&Tag, the abundance and distribution of such structures in human genomic DNA have remained unclear. Here we utilise high-affinity i-motif immunoprecipitation followed by sequencing to map i-motifs in the purified genomic DNA of human MCF7, U2OS and HEK293T cells. Validated by biolayer interferometry and circular dichroism spectroscopy, our approach aimed to identify DNA sequences capable of i-motif formation on a genome-wide scale, revealing that such sequences are widely distributed throughout the human genome and are common in genes upregulated in G0/G1 cell cycle phases. Our findings provide experimental evidence for the widespread formation of i-motif structures in human genomic DNA and a foundational resource for future studies of their genomic, structural, and molecular roles., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
23. Temporally resolved proteomics identifies nidogen-2 as a cotarget in pancreatic cancer that modulates fibrosis and therapy response.
- Author
-
Pereira BA, Ritchie S, Chambers CR, Gordon KA, Magenau A, Murphy KJ, Nobis M, Tyma VM, Liew YF, Lucas MC, Naeini MM, Barkauskas DS, Chacon-Fajardo D, Howell AE, Parker AL, Warren SC, Reed DA, Lee V, Metcalf XL, Lee YK, O'Regan LP, Zhu J, Trpceski M, Fontaine ARM, Stoehr J, Rouet R, Lin X, Chitty JL, Porazinski S, Wu SZ, Filipe EC, Cadell AL, Holliday H, Yang J, Papanicolaou M, Lyons RJ, Zaratzian A, Tayao M, Da Silva A, Vennin C, Yin J, Dew AB, McMillan PJ, Goldstein LD, Deveson IW, Croucher DR, Samuel MS, Sim HW, Batten M, Chantrill L, Grimmond SM, Gill AJ, Samra J, Jeffry Evans TR, Sasaki T, Phan TG, Swarbrick A, Sansom OJ, Morton JP, Pajic M, Parker BL, Herrmann D, Cox TR, and Timpson P
- Subjects
- Animals, Humans, Mice, Calcium-Binding Proteins metabolism, Calcium-Binding Proteins genetics, Cancer-Associated Fibroblasts metabolism, Cancer-Associated Fibroblasts pathology, Cell Adhesion Molecules, Cell Line, Tumor, Deoxycytidine analogs & derivatives, Deoxycytidine pharmacology, Disease Models, Animal, Fibrosis, Gemcitabine, Carcinoma, Pancreatic Ductal metabolism, Carcinoma, Pancreatic Ductal pathology, Carcinoma, Pancreatic Ductal genetics, Pancreatic Neoplasms metabolism, Pancreatic Neoplasms pathology, Pancreatic Neoplasms genetics, Proteomics methods
- Abstract
Pancreatic ductal adenocarcinoma (PDAC) is characterized by increasing fibrosis, which can enhance tumor progression and spread. Here, we undertook an unbiased temporal assessment of the matrisome of the highly metastatic KPC ( Pdx1-Cre , LSL-Kras
G12D/+ , LSL-Trp53R172H/+ ) and poorly metastatic KPfl C ( Pdx1-Cre , LSL-KrasG12D/+ , Trp53fl/+ ) genetically engineered mouse models of pancreatic cancer using mass spectrometry proteomics. Our assessment at early-, mid-, and late-stage disease reveals an increased abundance of nidogen-2 (NID2) in the KPC model compared to KPfl C, with further validation showing that NID2 is primarily expressed by cancer-associated fibroblasts (CAFs). Using biomechanical assessments, second harmonic generation imaging, and birefringence analysis, we show that NID2 reduction by CRISPR interference (CRISPRi) in CAFs reduces stiffness and matrix remodeling in three-dimensional models, leading to impaired cancer cell invasion. Intravital imaging revealed improved vascular patency in live NID2-depleted tumors, with enhanced response to gemcitabine/Abraxane. In orthotopic models, NID2 CRISPRi tumors had less liver metastasis and increased survival, highlighting NID2 as a potential PDAC cotarget.- Published
- 2024
- Full Text
- View/download PDF
24. Broadly neutralizing SARS-CoV-2 antibodies through epitope-based selection from convalescent patients.
- Author
-
Rouet R, Henry JY, Johansen MD, Sobti M, Balachandran H, Langley DB, Walker GJ, Lenthall H, Jackson J, Ubiparipovic S, Mazigi O, Schofield P, Burnett DL, Brown SHJ, Martinello M, Hudson B, Gilroy N, Post JJ, Kelleher A, Jäck HM, Goodnow CC, Turville SG, Rawlinson WD, Bull RA, Stewart AG, Hansbro PM, and Christ D
- Subjects
- Humans, Female, Animals, Mice, Broadly Neutralizing Antibodies, Leukocytes, Mononuclear, Antibodies, Viral, Antibodies, Monoclonal, Antibodies, Neutralizing, Epitopes, Spike Glycoprotein, Coronavirus genetics, Neutralization Tests, SARS-CoV-2, COVID-19
- Abstract
Emerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice; broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells of convalescent patients using SARS-CoV-2 receptor binding domains carrying epitope-specific mutations. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and 5). Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (S309) by orders of magnitude. They also provide prophylactic and therapeutic in vivo protection of female hACE2 mice against viral challenge. Our results indicate that exposure to SARS-CoV-2 induces antibodies that maintain broad neutralization against emerging VOCs using two unique strategies: either by targeting the divergent class 1 epitope in a manner resistant to VOCs (ACE2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14); or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). Our results provide guidance for next generation monoclonal antibody development and vaccine design., (© 2023. The Author(s).)
- Published
- 2023
- Full Text
- View/download PDF
25. Tracking the clonal dynamics of SARS-CoV-2-specific T cells in children and adults with mild/asymptomatic COVID-19.
- Author
-
Khoo WH, Jackson K, Phetsouphanh C, Zaunders JJ, Alquicira-Hernandez J, Yazar S, Ruiz-Diaz S, Singh M, Dhenni R, Kyaw W, Tea F, Merheb V, Lee FXZ, Burrell R, Howard-Jones A, Koirala A, Zhou L, Yuksel A, Catchpoole DR, Lai CL, Vitagliano TL, Rouet R, Christ D, Tang B, West NP, George S, Gerrard J, Croucher PI, Kelleher AD, Goodnow CG, Sprent JD, Powell JE, Brilot F, Nanan R, Hsu PS, Deenick EK, Britton PN, and Phan TG
- Subjects
- Humans, Adult, SARS-CoV-2, CD4-Positive T-Lymphocytes, Immunity, Cellular, Lymphocyte Activation, Antibodies, Viral, COVID-19
- Abstract
Children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop less severe coronavirus disease 2019 (COVID-19) than adults. The mechanisms for the age-specific differences and the implications for infection-induced immunity are beginning to be uncovered. We show by longitudinal multimodal analysis that SARS-CoV-2 leaves a small footprint in the circulating T cell compartment in children with mild/asymptomatic COVID-19 compared to adult household contacts with the same disease severity who had more evidence of systemic T cell interferon activation, cytotoxicity and exhaustion. Children harbored diverse polyclonal SARS-CoV-2-specific naïve T cells whereas adults harbored clonally expanded SARS-CoV-2-specific memory T cells. A novel population of naïve interferon-activated T cells is expanded in acute COVID-19 and is recruited into the memory compartment during convalescence in adults but not children. This was associated with the development of robust CD4
+ memory T cell responses in adults but not children. These data suggest that rapid clearance of SARS-CoV-2 in children may compromise their cellular immunity and ability to resist reinfection., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2023
- Full Text
- View/download PDF
26. High titre neutralizing antibodies in response to SARS-CoV-2 infection require RBD-specific CD4 T cells that include proliferative memory cells.
- Author
-
Phetsouphanh C, Khoo WH, Jackson K, Klemm V, Howe A, Aggarwal A, Akerman A, Milogiannakis V, Stella AO, Rouet R, Schofield P, Faulks ML, Law H, Danwilai T, Starr M, Munier CML, Christ D, Singh M, Croucher PI, Brilot-Turville F, Turville S, Phan TG, Dore GJ, Darley D, Cunningham P, Matthews GV, Kelleher AD, and Zaunders JJ
- Subjects
- Humans, SARS-CoV-2, Antibodies, Neutralizing, Epitopes, T-Lymphocyte, CD4-Positive T-Lymphocytes, COVID-19
- Abstract
Background: Long-term immunity to SARS-CoV-2 infection, including neutralizing antibodies and T cell-mediated immunity, is required in a very large majority of the population in order to reduce ongoing disease burden., Methods: We have investigated the association between memory CD4 and CD8 T cells and levels of neutralizing antibodies in convalescent COVID-19 subjects., Findings: Higher titres of convalescent neutralizing antibodies were associated with significantly higher levels of RBD-specific CD4 T cells, including specific memory cells that proliferated vigorously in vitro . Conversely, up to half of convalescent individuals had low neutralizing antibody titres together with a lack of receptor binding domain (RBD)-specific memory CD4 T cells. These low antibody subjects had other, non-RBD, spike-specific CD4 T cells, but with more of an inhibitory Foxp3+ and CTLA-4+ cell phenotype, in contrast to the effector T-bet+, cytotoxic granzymes+ and perforin+ cells seen in RBD-specific memory CD4 T cells from high antibody subjects. Single cell transcriptomics of antigen-specific CD4+ T cells from high antibody subjects similarly revealed heterogenous RBD-specific CD4+ T cells that comprised central memory, transitional memory and Tregs, as well as cytotoxic clusters containing diverse TCR repertoires, in individuals with high antibody levels. However, vaccination of low antibody convalescent individuals led to a slight but significant improvement in RBD-specific memory CD4 T cells and increased neutralizing antibody titres., Interpretation: Our results suggest that targeting CD4 T cell epitopes proximal to and within the RBD-region should be prioritized in booster vaccines., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Phetsouphanh, Khoo, Jackson, Klemm, Howe, Aggarwal, Akerman, Milogiannakis, Stella, Rouet, Schofield, Faulks, Law, Danwilai, Starr, Munier, Christ, Singh, Croucher, Brilot-Turville, Turville, Phan, Dore, Darley, Cunningham, Matthews, Kelleher and Zaunders.)
- Published
- 2022
- Full Text
- View/download PDF
27. The retroelement Lx9 puts a brake on the immune response to virus infection.
- Author
-
Bartonicek N, Rouet R, Warren J, Loetsch C, Rodriguez GS, Walters S, Lin F, Zahra D, Blackburn J, Hammond JM, Reis ALM, Deveson IW, Zammit N, Zeraati M, Grey S, Christ D, Mattick JS, Chtanova T, Brink R, Dinger ME, Weatheritt RJ, Sprent J, and King C
- Subjects
- Animals, CRISPR-Cas Systems genetics, Evolution, Molecular, Mice, RNA, Untranslated genetics, Regulatory Sequences, Nucleic Acid genetics, DNA Transposable Elements genetics, DNA Transposable Elements immunology, Host Microbial Interactions genetics, Host Microbial Interactions immunology, Immunity genetics, Retroelements genetics, Retroelements immunology, Virus Diseases genetics, Virus Diseases immunology
- Abstract
The notion that mobile units of nucleic acid known as transposable elements can operate as genomic controlling elements was put forward over six decades ago
1,2 . However, it was not until the advancement of genomic sequencing technologies that the abundance and repertoire of transposable elements were revealed, and they are now known to constitute up to two-thirds of mammalian genomes3,4 . The presence of DNA regulatory regions including promoters, enhancers and transcription-factor-binding sites within transposable elements5-8 has led to the hypothesis that transposable elements have been co-opted to regulate mammalian gene expression and cell phenotype8-14 . Mammalian transposable elements include recent acquisitions and ancient transposable elements that have been maintained in the genome over evolutionary time. The presence of ancient conserved transposable elements correlates positively with the likelihood of a regulatory function, but functional validation remains an essential step to identify transposable element insertions that have a positive effect on fitness. Here we show that CRISPR-Cas9-mediated deletion of a transposable element-namely the LINE-1 retrotransposon Lx9c11-in mice results in an exaggerated and lethal immune response to virus infection. Lx9c11 is critical for the neogenesis of a non-coding RNA (Lx9c11-RegoS) that regulates genes of the Schlafen family, reduces the hyperinflammatory phenotype and rescues lethality in virus-infected Lx9c11-/- mice. These findings provide evidence that a transposable element can control the immune system to favour host survival during virus infection., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2022
- Full Text
- View/download PDF
28. Antibody-mediated delivery of CRISPR-Cas9 ribonucleoproteins in human cells.
- Author
-
Ubiparipovic S, Christ D, and Rouet R
- Subjects
- Humans, Ribonucleoproteins genetics, Ribonucleoproteins metabolism, Gene Editing, Antibodies, Monoclonal genetics, Antibodies, Monoclonal metabolism, CRISPR-Cas Systems, RNA, Guide, CRISPR-Cas Systems genetics, RNA, Guide, CRISPR-Cas Systems metabolism
- Abstract
The CRISPR genome editing technology holds great clinical potential for the treatment of monogenetic disorders such as sickle cell disease. The therapeutic in vivo application of the technology relies on targeted delivery methods of the Cas9 and gRNA complex to specific cells or tissues. However, such methods are currently limited to direct organ delivery, preventing clinical application. Here, we show that monoclonal antibodies can be employed to deliver the Cas9/gRNA complex directly into human cells via cell-surface receptors. Using the SpyCatcher/SpyTag system, we conjugated the Fab fragment of the therapeutic antibodies Trastuzumab and Pertuzumab directly to the Cas9 enzyme and observed HER2-specific uptake of the ribonucleoprotein in a human HER2 expressing cell line. Following cellular uptake in the presence of an endosomolytic peptide, modest gene editing was also observed. This finding provides a blueprint for the targeted delivery of the CRISPR technology into specific cells using monoclonal antibodies., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2022
- Full Text
- View/download PDF
29. Immunizations with diverse sarbecovirus receptor-binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability.
- Author
-
Burnett DL, Jackson KJL, Langley DB, Aggrawal A, Stella AO, Johansen MD, Balachandran H, Lenthall H, Rouet R, Walker G, Saunders BM, Singh M, Li H, Henry JY, Jackson J, Stewart AG, Witthauer F, Spence MA, Hansbro NG, Jackson C, Schofield P, Milthorpe C, Martinello M, Schulz SR, Roth E, Kelleher A, Emery S, Britton WJ, Rawlinson WD, Karl R, Schäfer S, Winkler TH, Brink R, Bull RA, Hansbro PM, Jäck HM, Turville S, Christ D, and Goodnow CC
- Subjects
- Animals, Antibodies, Neutralizing metabolism, Antibodies, Viral metabolism, Conserved Sequence genetics, Evolution, Molecular, Humans, Immunization, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Protein Binding, Protein Domains genetics, Spike Glycoprotein, Coronavirus genetics, Spike Glycoprotein, Coronavirus immunology, Vaccine Development, Betacoronavirus physiology, COVID-19 Vaccines immunology, Coronavirus Infections immunology, Severe acute respiratory syndrome-related coronavirus physiology, Spike Glycoprotein, Coronavirus metabolism
- Abstract
Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies., Competing Interests: Declaration of interests All affiliations are listed on the title page of the manuscript. The authors and their immediate family members have no financial interests to declare. The authors have lodged a patent (Australian provisional patent application no. 2021901716) based on this work., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
30. NSG-Pro mouse model for uncovering resistance mechanisms and unique vulnerabilities in human luminal breast cancers.
- Author
-
Sun Y, Yang N, Utama FE, Udhane SS, Zhang J, Peck AR, Yanac A, Duffey K, Langenheim JF, Udhane V, Xia G, Peterson JF, Jorns JM, Nevalainen MT, Rouet R, Schofield P, Christ D, Ormandy CJ, Rosenberg AL, Chervoneva I, Tsaih SW, Flister MJ, Fuchs SY, Wagner KU, and Rui H
- Abstract
Most breast cancer deaths are caused by estrogen receptor-α–positive (ER
+ ) disease. Preclinical progress is hampered by a shortage of therapy-naïve ER+ tumor models that recapitulate metastatic progression and clinically relevant therapy resistance. Human prolactin (hPRL) is a risk factor for primary and metastatic ER+ breast cancer. Because mouse prolactin fails to activate hPRL receptors, we developed a prolactin-humanized Nod-SCID-IL2Rγ (NSG) mouse (NSG-Pro) with physiological hPRL levels. Here, we show that NSG-Pro mice facilitate establishment of therapy-naïve, estrogen-dependent PDX tumors that progress to lethal metastatic disease. Preclinical trials provide first-in-mouse efficacy of pharmacological hPRL suppression on residual ER+ human breast cancer metastases and document divergent biology and drug responsiveness of tumors grown in NSG-Pro versus NSG mice. Oncogenomic analyses of PDX lines in NSG-Pro mice revealed clinically relevant therapy-resistance mechanisms and unexpected, potently actionable vulnerabilities such as DNA-repair aberrations. The NSG-Pro mouse unlocks previously inaccessible precision medicine approaches for ER+ breast cancers.- Published
- 2021
- Full Text
- View/download PDF
31. Long-term persistence of RBD + memory B cells encoding neutralizing antibodies in SARS-CoV-2 infection.
- Author
-
Abayasingam A, Balachandran H, Agapiou D, Hammoud M, Rodrigo C, Keoshkerian E, Li H, Brasher NA, Christ D, Rouet R, Burnet D, Grubor-Bauk B, Rawlinson W, Turville S, Aggarwal A, Stella AO, Fichter C, Brilot F, Mina M, Post JJ, Hudson B, Gilroy N, Dwyer D, Sasson SC, Tea F, Pilli D, Kelleher A, Tedla N, Lloyd AR, Martinello M, and Bull RA
- Subjects
- Adult, Aged, Aged, 80 and over, Antibodies, Neutralizing immunology, Asymptomatic Diseases, COVID-19 immunology, COVID-19 virology, Female, Humans, Limit of Detection, Male, Middle Aged, Neutralization Tests, Protein Domains immunology, SARS-CoV-2 isolation & purification, Severity of Illness Index, Spike Glycoprotein, Coronavirus immunology, Spike Glycoprotein, Coronavirus metabolism, Time Factors, Young Adult, Antibodies, Neutralizing blood, COVID-19 pathology, Memory B Cells metabolism, SARS-CoV-2 metabolism, Spike Glycoprotein, Coronavirus chemistry
- Abstract
Considerable concerns relating to the duration of protective immunity against severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) exist, with evidence of antibody titers declining rapidly after infection and reports of reinfection. Here, we monitor the antibody responses against SARS-CoV-2 receptor-binding domain (RBD) for up to 6 months after infection. While antibody titers are maintained, ∼13% of the cohort's neutralizing responses return to background. However, encouragingly, in a selected subset of 13 participants, 12 have detectable RBD-specific memory B cells and these generally are increasing out to 6 months. Furthermore, we are able to generate monoclonal antibodies with SARS-CoV-2 neutralizing capacity from these memory B cells. Overall, our study suggests that the loss of neutralizing antibodies in plasma may be countered by the maintenance of neutralizing capacity in the memory B cell repertoire., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)
- Published
- 2021
- Full Text
- View/download PDF
32. Potent SARS-CoV-2 binding and neutralization through maturation of iconic SARS-CoV-1 antibodies.
- Author
-
Rouet R, Mazigi O, Walker GJ, Langley DB, Sobti M, Schofield P, Lenthall H, Jackson J, Ubiparipovic S, Henry JY, Abayasingam A, Burnett D, Kelleher A, Brink R, Bull RA, Turville S, Stewart AG, Goodnow CC, Rawlinson WD, and Christ D
- Subjects
- Antibody Specificity, Cross Reactions, Humans, Mutagenesis, Site-Directed, Antibodies, Neutralizing immunology, Antibodies, Viral immunology, COVID-19 immunology, Severe acute respiratory syndrome-related coronavirus immunology, SARS-CoV-2 immunology
- Abstract
Antibodies against coronavirus spike protein potently protect against infection and disease, but whether such protection can be extended to variant coronaviruses is unclear. This is exemplified by a set of iconic and well-characterized monoclonal antibodies developed after the 2003 SARS outbreak, including mAbs m396, CR3022, CR3014 and 80R, which potently neutralize SARS-CoV-1, but not SARS-CoV-2. Here, we explore antibody engineering strategies to change and broaden their specificity, enabling nanomolar binding and potent neutralization of SARS-CoV-2. Intriguingly, while many of the matured clones maintained specificity of the parental antibody, new specificities were also observed, which was further confirmed by X-ray crystallography and cryo-electron microscopy, indicating that a limited set of VH antibody domains can give rise to variants targeting diverse epitopes, when paired with a diverse VL repertoire. Our findings open up over 15 years of antibody development efforts against SARS-CoV-1 to the SARS-CoV-2 field and outline general principles for the maturation of antibody specificity against emerging viruses.
- Published
- 2021
- Full Text
- View/download PDF
33. CAF hierarchy driven by pancreatic cancer cell p53-status creates a pro-metastatic and chemoresistant environment via perlecan.
- Author
-
Vennin C, Mélénec P, Rouet R, Nobis M, Cazet AS, Murphy KJ, Herrmann D, Reed DA, Lucas MC, Warren SC, Elgundi Z, Pinese M, Kalna G, Roden D, Samuel M, Zaratzian A, Grey ST, Da Silva A, Leung W, Mathivanan S, Wang Y, Braithwaite AW, Christ D, Benda A, Parkin A, Phillips PA, Whitelock JM, Gill AJ, Sansom OJ, Croucher DR, Parker BL, Pajic M, Morton JP, Cox TR, and Timpson P
- Subjects
- Animals, Cell Line, Tumor, Cell Movement, Cell Proliferation, Gene Expression Regulation, Neoplastic genetics, Mice, Mice, Inbred BALB C, Neoplasm Invasiveness pathology, Pancreas pathology, Pancreatic Neoplasms genetics, Signal Transduction physiology, Tumor Suppressor Protein p53 genetics, Cancer-Associated Fibroblasts pathology, Drug Resistance, Neoplasm genetics, Heparan Sulfate Proteoglycans metabolism, Pancreatic Neoplasms pathology, Tumor Suppressor Protein p53 metabolism
- Abstract
Heterogeneous subtypes of cancer-associated fibroblasts (CAFs) coexist within pancreatic cancer tissues and can both promote and restrain disease progression. Here, we interrogate how cancer cells harboring distinct alterations in p53 manipulate CAFs. We reveal the existence of a p53-driven hierarchy, where cancer cells with a gain-of-function (GOF) mutant p53 educate a dominant population of CAFs that establish a pro-metastatic environment for GOF and null p53 cancer cells alike. We also demonstrate that CAFs educated by null p53 cancer cells may be reprogrammed by either GOF mutant p53 cells or their CAFs. We identify perlecan as a key component of this pro-metastatic environment. Using intravital imaging, we observe that these dominant CAFs delay cancer cell response to chemotherapy. Lastly, we reveal that depleting perlecan in the stroma combined with chemotherapy prolongs mouse survival, supporting it as a potential target for anti-stromal therapies in pancreatic cancer.
- Published
- 2019
- Full Text
- View/download PDF
34. Human Antibody Bispecifics through Phage Display Selection.
- Author
-
Luthra A, Langley DB, Schofield P, Jackson J, Abdelatti M, Rouet R, Nevoltris D, Mazigi O, Crossett B, Christie M, and Christ D
- Subjects
- Amino Acid Sequence, Antibodies, Bispecific chemistry, Antibody Affinity, Humans, Immunoglobulin Heavy Chains chemistry, Immunoglobulin Heavy Chains immunology, Immunoglobulin Light Chains chemistry, Immunoglobulin Light Chains immunology, Models, Molecular, Antibodies, Bispecific immunology, Peptide Library
- Abstract
We developed a repertoire approach to generate human antibody bispecifics. Using phage display selection of antibody heavy chains in the presence of a competitor light chain and providing a cognate light chain with an affinity handle, we identified mutations that prevent heavy/light chain mispairing. The strategy allows for the selection of human antibody chains that autonomously assemble into bispecifics.
- Published
- 2019
- Full Text
- View/download PDF
35. Efficient Intracellular Delivery of CRISPR-Cas Ribonucleoproteins through Receptor Mediated Endocytosis.
- Author
-
Rouet R and Christ D
- Subjects
- Asialoglycoprotein Receptor chemistry, Asialoglycoprotein Receptor metabolism, Biological Transport, CRISPR-Associated Proteins metabolism, Cell Line, Gene Editing methods, Humans, Signal Transduction, CRISPR-Associated Proteins genetics, CRISPR-Cas Systems physiology, Endocytosis physiology, Oligopeptides metabolism, Ribonucleoproteins metabolism
- Abstract
We recently reported a new delivery system harnessing surface receptors for targeted uptake of CRISPR-Cas9 ribonucleoprotein into mammalian cells (Rouet et al., JACS 2018). For this purpose, Cas9 protein was labeled with the small molecule ligand ASGRL, specific for the asialoglycoprotein receptor, enabling endosomal uptake of the ribonucleoprotein into human cells expressing the receptor. However, detailed mechanistic insights had remained unknown and editing efficiency low. Here we investigate the mechanism of endosomal escape as mediated by the ppTG21 endosomolytic peptide and outline the development of novel Cas9 or Cas12a ribonucleoprotein complexes with increased editing efficiency.
- Published
- 2019
- Full Text
- View/download PDF
36. Engineering CRISPR-Cas9 RNA-Protein Complexes for Improved Function and Delivery.
- Author
-
Rouet R, de Oñate L, Li J, Murthy N, and Wilson RC
- Abstract
The use of CRISPR-derived, RNA-guided nucleases for genome editing has shown great promise for addressing genetic disease. Encouraging work in cell culture and animal models has demonstrated the capability of genome-editing enzymes to correct disease-causing loci, but clinical translation can only proceed once the corrective enzymes have been rendered safe, effective, and capable of being delivered to the appropriate cells. To address these needs, there has been rapid and extensive progress in the engineering of the RNA and protein components of Cas9, the most widely-used genome editor. Here we review advances in engineering of the enzyme Cas9 by altering its chemical or molecular composition. Such efforts have enhanced enzyme stability, improved capacity for delivery, augmented specificity, and broadened the horizons of genome manipulation.
- Published
- 2018
- Full Text
- View/download PDF
37. Optimized CRISPR-Cpf1 system for genome editing in zebrafish.
- Author
-
Fernandez JP, Vejnar CE, Giraldez AJ, Rouet R, and Moreno-Mateos MA
- Subjects
- Animals, Clostridiales genetics, DNA End-Joining Repair genetics, Genome genetics, RNA, Guide, CRISPR-Cas Systems genetics, Recombinational DNA Repair genetics, Bacterial Proteins genetics, Clustered Regularly Interspaced Short Palindromic Repeats genetics, Endonucleases genetics, Gene Editing methods, Zebrafish genetics
- Abstract
The impact of the CRISPR-Cas biotechnological systems has recently broadened the genome editing toolbox available to different model organisms further with the addition of new efficient RNA-guided endonucleases. We have recently optimized CRISPR-Cpf1 (renamed Cas12a) system in zebrafish. We showed that (i) in the absence of Cpf1 protein, crRNAs are unstable and degraded in vivo, and CRISPR-Cpf1 RNP complexes efficiently mutagenize the zebrafish genome; and (ii) temperature modulates Cpf1 activity especially affecting AsCpf1, which experiences a reduced performance below 37 °C. Here, we describe a step-by-step protocol on how to easily design and generate crRNAs in vitro, purify recombinant Cpf1 proteins, and assemble ribonucleoprotein complexes to carry out efficient mutagenesis in zebrafish in a constitutive and temperature-controlled manner. Finally, we explain how to induce Cpf1-mediated homology-directed repair using single-stranded DNA oligonucleotides. In summary, this protocol includes the steps to efficiently modify the zebrafish genome and other ectothermic organisms using the CRISPR-Cpf1 system., (Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
38. PAR1 contribution in acute electrophysiological properties of oral anticoagulants in rabbit pulmonary vein sleeve preparations.
- Author
-
Font J, Simeon M, Simard C, Allouche S, Plane AF, Ferchaud V, Brionne M, Rouet R, Nowoczyn M, Manrique A, Puddu PE, Milliez P, and Alexandre J
- Subjects
- Administration, Oral, Animals, Anti-Arrhythmia Agents pharmacology, Atrial Fibrillation drug therapy, Atrial Fibrillation metabolism, Female, Pulmonary Veins metabolism, Pyrazoles pharmacology, Pyridones pharmacology, Rabbits, Anticoagulants pharmacology, Electrophysiological Phenomena drug effects, Pulmonary Veins drug effects, Receptor, PAR-1 metabolism
- Abstract
Whether oral anticoagulants, vitamin K antagonists (VKAs), and nonvitamin K oral anticoagulant (NOACs) frequently prescribed to atrial fibrillation (AF) patients, do themselves have a pro- or anti-arrhythmic effect have never been addressed. Transmembrane action potentials were recorded in an acute rabbit model of superfused pulmonary veins (PVs) sleeves preparations using standard microelectrode technique. Fluindione 10 μm (n = 6) increased the AP (action potential) duration (APD), induced a significantly V
max depression (from 95 ± 14 to 53 ± 5 V/s, P < 0.05), and 2 : 1 blocks during rapid atrial pacing thus evoking class I anti-arrhythmic properties, and prevented spontaneous trigger APs. Apixaban 10 μm (n = 6) increased the APD, significantly prolonged the effective refractory period (from 56.3 ± 4.2 to 72.0 ± 8.6 ms, P < 0.05), and prevented triggered APs occurrence. Fluindione and apixaban effects were suppressed with the addition of the protease-activated receptors 1 (PAR 1) agonist SFLLR-NH2 . Warfarin 10 μm (n = 6) significantly abbreviated the early refractory period (from 56.3 ± 4.2 to 45.0 ± 2.2 ms, P < 0.05) and increased triggered APs occurrence that were successfully prevented by nifedipine but not by the addition of the protease-activated receptors 1 agonist SFLLR-NH2 . In this acute rabbit PVs model, VKAs and NOACs, at physiological concentrations, exhibited very different pharmacological properties that influence PVs electrophysiology, implying PAR1, with fluindione and apixaban which exhibited more anti-arrhythmic properties, whereas warfarin exhibited more pro-arrhythmic properties., (© 2018 Société Française de Pharmacologie et de Thérapeutique.)- Published
- 2018
- Full Text
- View/download PDF
39. I-motif DNA structures are formed in the nuclei of human cells.
- Author
-
Zeraati M, Langley DB, Schofield P, Moye AL, Rouet R, Hughes WE, Bryan TM, Dinger ME, and Christ D
- Subjects
- Cell Cycle, DNA immunology, Genome, Human, HeLa Cells, Humans, Hydrogen-Ion Concentration, Immunoglobulin Fragments immunology, MCF-7 Cells, Promoter Regions, Genetic, Telomere, Cell Nucleus chemistry, DNA chemistry, Nucleic Acid Conformation
- Abstract
Human genome function is underpinned by the primary storage of genetic information in canonical B-form DNA, with a second layer of DNA structure providing regulatory control. I-motif structures are thought to form in cytosine-rich regions of the genome and to have regulatory functions; however, in vivo evidence for the existence of such structures has so far remained elusive. Here we report the generation and characterization of an antibody fragment (iMab) that recognizes i-motif structures with high selectivity and affinity, enabling the detection of i-motifs in the nuclei of human cells. We demonstrate that the in vivo formation of such structures is cell-cycle and pH dependent. Furthermore, we provide evidence that i-motif structures are formed in regulatory regions of the human genome, including promoters and telomeric regions. Our results support the notion that i-motif structures provide key regulatory roles in the genome.
- Published
- 2018
- Full Text
- View/download PDF
40. Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing.
- Author
-
Rouet R, Thuma BA, Roy MD, Lintner NG, Rubitski DM, Finley JE, Wisniewska HM, Mendonsa R, Hirsh A, de Oñate L, Compte Barrón J, McLellan TJ, Bellenger J, Feng X, Varghese A, Chrunyk BA, Borzilleri K, Hesp KD, Zhou K, Ma N, Tu M, Dullea R, McClure KF, Wilson RC, Liras S, Mascitti V, and Doudna JA
- Subjects
- Cell Line, Tumor, Endonucleases genetics, Hep G2 Cells, Humans, Molecular Structure, Protein Engineering, CRISPR-Cas Systems, Endonucleases metabolism, Gene Editing
- Abstract
CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.
- Published
- 2018
- Full Text
- View/download PDF
41. Next-Generation Sequencing of Antibody Display Repertoires.
- Author
-
Rouet R, Jackson KJL, Langley DB, and Christ D
- Subjects
- Antibody Affinity, Bacteriophages, Epitope Mapping, Humans, Antibodies, High-Throughput Nucleotide Sequencing
- Abstract
In vitro selection technology has transformed the development of therapeutic monoclonal antibodies. Using methods such as phage, ribosome, and yeast display, high affinity binders can be selected from diverse repertoires. Here, we review strategies for the next-generation sequencing (NGS) of phage- and other antibody-display libraries, as well as NGS platforms and analysis tools. Moreover, we discuss recent examples relating to the use of NGS to assess library diversity, clonal enrichment, and affinity maturation.
- Published
- 2018
- Full Text
- View/download PDF
42. Expression of IgG Monoclonals with Engineered Immune Effector Functions.
- Author
-
Vazquez-Lombardi R, Nevoltris D, Rouet R, and Christ D
- Subjects
- Cell Line, Chromatography, Affinity, DNA metabolism, Genetic Vectors metabolism, Humans, Immunoglobulin G chemistry, Immunoglobulin Variable Region chemistry, Plasmids metabolism, Protein Domains, Recombinant Proteins metabolism, Transfection, Antibodies, Monoclonal metabolism, Immunoglobulin G metabolism, Protein Engineering methods
- Abstract
The therapeutic development of monoclonal antibodies requires robust and reliable methods for their recombinant expression and characterization. In this context, an increasingly important aspect in the antibody development process is to determine the contribution of Fc-mediated immune effector functions to therapeutic activity. Here we describe steps for the cloning and mammalian expression of mouse and human IgG monoclonals with reduced immune effector functions, based on mutation of Fc-gamma receptor and complement-binding sites. The resulting antibody preparations contain low levels of endotoxin and are suitable for testing in animal models of disease.
- Published
- 2018
- Full Text
- View/download PDF
43. CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing.
- Author
-
Moreno-Mateos MA, Fernandez JP, Rouet R, Vejnar CE, Lane MA, Mis E, Khokha MK, Doudna JA, and Giraldez AJ
- Subjects
- Animals, Bacterial Proteins genetics, Endonucleases genetics, Humans, Models, Genetic, Mutagenesis, Temperature, Xenopus genetics, Zebrafish genetics, Bacterial Proteins metabolism, CRISPR-Cas Systems, Endonucleases metabolism, Gene Editing methods, Recombinational DNA Repair
- Abstract
Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus. We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms. We capitalize on this property to show that temporal control of the temperature allows post-translational modulation of Cpf1-mediated genome editing. Finally, we determine that LbCpf1 significantly increases homology-directed repair in zebrafish, improving current approaches for targeted DNA integration in the genome. Together, we provide a molecular understanding of Cpf1 activity in vivo and establish Cpf1 as an efficient and inducible genome engineering tool across ectothermic species.
- Published
- 2017
- Full Text
- View/download PDF
44. Argon Exposure Induces Postconditioning in Myocardial Ischemia-Reperfusion.
- Author
-
Lemoine S, Blanchart K, Souplis M, Lemaitre A, Legallois D, Coulbault L, Simard C, Allouche S, Abraini JH, Hanouz JL, Rouet R, Sallé L, Guinamard R, and Manrique A
- Subjects
- Animals, Atrial Appendage drug effects, Atrial Appendage physiopathology, Guinea Pigs, Humans, Male, Myocardial Reperfusion Injury physiopathology, Organ Culture Techniques, Rats, Rats, Wistar, Argon administration & dosage, Ischemic Postconditioning methods, Myocardial Reperfusion methods, Myocardial Reperfusion Injury prevention & control
- Abstract
Background and Purpose: Cardioprotection against ischemia-reperfusion (I/R) damages remains a major concern during prehospital management of acute myocardial infarction. Noble gases have shown beneficial effects in preconditioning studies. Because emergency proceedings in the context of myocardial infarction require postconditioning strategies, we evaluated the effects of argon in such protocols on mammalian cardiac tissue., Experimental Approaches: In rat, cardiac I/R was induced in vivo by transient coronary artery ligature and cardiac functions were evaluated by magnetic resonance imaging. Hypoxia-reoxygenation (H/R)-induced arrhythmias were evaluated in vitro using intracellular microelectrodes on both rat-isolated ventricle and a model of border zone in guinea pig ventricle. Hypoxia-reoxygenation loss of contractile force was assessed in human atrial appendages. In those models, postconditioning was induced by 5 minutes application of argon at the time of reperfusion., Key Results: In the in vivo model, I/R produced left ventricular ejection fraction decrease (24%) and wall motion score increase (36%) which was prevented when argon was applied in postconditioning. In vitro, argon postconditioning abolished H/R-induced arrhythmias such as early after depolarizations, conduction blocks, and reentries. Recovery of contractile force in human atrial appendages after H/R was enhanced in the argon group, increasing from 51% ± 2% in the nonconditioned group to 83% ± 7% in the argon-treated group ( P < .001). This effect of argon was abolished in the presence of wortmannin and PD98059 which inhibit prosurvival phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) and MEK/extracellular receptor kinase 1/2 (ERK 1/2), respectively, or in the presence of the mitochondrial permeability transition pore opener atractyloside, suggesting the involvement of the reperfusion injury salvage kinase pathway., Conclusion and Implications: Argon has strong cardioprotective properties when applied in conditions of postconditioning and thus appears as a potential therapeutic tool in I/R situations.
- Published
- 2017
- Full Text
- View/download PDF
45. Structural reconstruction of protein ancestry.
- Author
-
Rouet R, Langley DB, Schofield P, Christie M, Roome B, Porebski BT, Buckle AM, Clifton BE, Jackson CJ, Stock D, and Christ D
- Subjects
- Animals, Crystallography, X-Ray, Immunoglobulin Heavy Chains chemistry, Immunoglobulin Heavy Chains genetics, Immunoglobulin kappa-Chains chemistry, Immunoglobulin kappa-Chains genetics, Muramidase chemistry, Receptors, Polymeric Immunoglobulin genetics, Vertebrates genetics, Vertebrates immunology, Evolution, Molecular, Phylogeny, Receptors, Polymeric Immunoglobulin chemistry
- Abstract
Ancestral protein reconstruction allows the resurrection and characterization of ancient proteins based on computational analyses of sequences of modern-day proteins. Unfortunately, many protein families are highly divergent and not suitable for sequence-based reconstruction approaches. This limitation is exemplified by the antigen receptors of jawed vertebrates (B- and T-cell receptors), heterodimers formed by pairs of Ig domains. These receptors are believed to have evolved from an extinct homodimeric ancestor through a process of gene duplication and diversification; however molecular evidence has so far remained elusive. Here, we use a structural approach and laboratory evolution to reconstruct such molecules and characterize their interaction with antigen. High-resolution crystal structures of reconstructed homodimeric receptors in complex with hen-egg white lysozyme demonstrate how nanomolar affinity binding of asymmetrical antigen is enabled through selective recruitment and structural plasticity within the receptor-binding site. Our results provide structural evidence in support of long-held theories concerning the evolution of antigen receptors, and provide a blueprint for the experimental reconstruction of protein ancestry in the absence of phylogenetic evidence.
- Published
- 2017
- Full Text
- View/download PDF
46. Structure and Characterisation of a Key Epitope in the Conserved C-Terminal Domain of the Malaria Vaccine Candidate MSP2.
- Author
-
Seow J, Morales RA, MacRaild CA, Krishnarjuna B, McGowan S, Dingjan T, Jaipuria G, Rouet R, Wilde KL, Atreya HS, Richards JS, Anders RF, Christ D, Drinkwater N, and Norton RS
- Subjects
- Animals, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal immunology, Antibodies, Protozoan chemistry, Antibodies, Protozoan immunology, Antigens, Protozoan chemistry, Calorimetry, Crystallography, X-Ray, Epitopes chemistry, Malaria Vaccines chemistry, Malaria Vaccines genetics, Malaria Vaccines immunology, Mice, Models, Molecular, Protein Binding, Protein Conformation, Protozoan Proteins chemistry, Surface Plasmon Resonance, Antigens, Protozoan genetics, Antigens, Protozoan immunology, Epitopes genetics, Epitopes immunology, Plasmodium falciparum genetics, Plasmodium falciparum immunology, Protozoan Proteins genetics, Protozoan Proteins immunology
- Abstract
Merozoite surface protein 2 (MSP2) is an intrinsically disordered antigen that is abundant on the surface of the malaria parasite Plasmodium falciparum. The two allelic families of MSP2, 3D7 and FC27, differ in their central variable regions, which are flanked by highly conserved C-terminal and N-terminal regions. In a vaccine trial, full-length 3D7 MSP2 induced a strain-specific protective immune response despite the detectable presence of conserved region antibodies. This work focuses on the conserved C-terminal region of MSP2, which includes the only disulphide bond in the protein and encompasses key epitopes recognised by the mouse monoclonal antibodies 4D11 and 9H4. Although the 4D11 and 9H4 epitopes are overlapping, immunofluorescence assays have shown that the mouse monoclonal antibody 4D11 binds to MSP2 on the merozoite surface with a much stronger signal than 9H4. Understanding the structural basis for this antigenic difference between these antibodies will help direct the design of a broad-spectrum and MSP2-based malaria vaccine. 4D11 and 9H4 were reengineered into antibody fragments [variable region fragment (Fv) and single-chain Fv (scFv)] and were validated as suitable models for their full-sized IgG counterparts by surface plasmon resonance and isothermal titration calorimetry. An alanine scan of the 13-residue epitope 3D7-MSP2
207-222 identified the minimal binding epitope of 4D11 and the key residues involved in binding. A 2.2-Å crystal structure of 4D11 Fv bound to the eight-residue epitope NKENCGAA provided valuable insight into the possible conformation of the C-terminal region of MSP2 on the parasite. This work underpins continued efforts to optimise recombinant MSP2 constructs for evaluation as potential vaccine candidates., (Copyright © 2017 Elsevier Ltd. All rights reserved.)- Published
- 2017
- Full Text
- View/download PDF
47. TRPM4 non-selective cation channels influence action potentials in rabbit Purkinje fibres.
- Author
-
Hof T, Sallé L, Coulbault L, Richer R, Alexandre J, Rouet R, Manrique A, and Guinamard R
- Subjects
- Animals, Calcium metabolism, Cells, Cultured, Female, Myocytes, Cardiac physiology, Potassium metabolism, Purkinje Fibers cytology, Purkinje Fibers physiology, Rabbits, Sodium metabolism, Action Potentials, Myocytes, Cardiac metabolism, Purkinje Fibers metabolism, TRPM Cation Channels metabolism
- Abstract
Key Points: The transient receptor potential melastatin 4 (TRPM4) inhibitor 9-phenanthrol reduces action potential duration in rabbit Purkinje fibres but not in ventricle. TRPM4-like single channel activity is observed in isolated rabbit Purkinje cells but not in ventricular cells. The TRPM4-like current develops during the notch and early repolarization phases of the action potential in Purkinje cells., Abstract: Transient receptor potential melastatin 4 (TRPM4) Ca(2+)-activated non-selective cation channel activity has been recorded in cardiomyocytes and sinus node cells from mammals. In addition, TRPM4 gene mutations are associated with human diseases of cardiac conduction, suggesting that TRPM4 plays a role in this aspect of cardiac function. Here we evaluate the TRPM4 contribution to cardiac electrophysiology of Purkinje fibres. Ventricular strips with Purkinje fibres were isolated from rabbit hearts. Intracellular microelectrodes recorded Purkinje fibre activity and the TRPM4 inhibitor 9-phenanthrol was applied to unmask potential TRPM4 contributions to the action potential. 9-Phenanthrol reduced action potential duration measured at the point of 50 and 90% repolarization with an EC50 of 32.8 and 36.1×10(-6) mol l(-1), respectively, but did not modulate ventricular action potentials. Inside-out patch-clamp recordings were used to monitor TRPM4 activity in isolated Purkinje cells. TRPM4-like single channel activity (conductance = 23.8 pS; equal permeability for Na(+) and K(+); sensitivity to voltage, Ca(2+) and 9-phenanthrol) was observed in 43% of patches from Purkinje cells but not from ventricular cells (0/16). Action potential clamp experiments performed in the whole-cell configuration revealed a transient inward 9-phenanthrol-sensitive current (peak density = -0.65 ± 0.15 pA pF(-1); n = 5) during the plateau phases of the Purkinje fibre action potential. These results show that TRPM4 influences action potential characteristics in rabbit Purkinje fibres and thus could modulate cardiac conduction and be involved in triggering arrhythmias., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)
- Published
- 2016
- Full Text
- View/download PDF
48. Cardiotoxic Electrophysiological Effects of the Herbicide Roundup(®) in Rat and Rabbit Ventricular Myocardium In Vitro.
- Author
-
Gress S, Lemoine S, Puddu PE, Séralini GE, and Rouet R
- Subjects
- Action Potentials, Animals, Arrhythmias, Cardiac metabolism, Arrhythmias, Cardiac physiopathology, Calcium metabolism, Calcium Channel Agonists pharmacology, Calcium Channels, L-Type drug effects, Calcium Channels, L-Type metabolism, Dose-Response Relationship, Drug, Enzyme Inhibitors pharmacology, Female, Glycine toxicity, Heart Conduction System metabolism, Heart Conduction System physiopathology, Heart Ventricles metabolism, Heart Ventricles physiopathology, In Vitro Techniques, Male, Myocytes, Cardiac metabolism, Rabbits, Rats, Sprague-Dawley, Risk Assessment, Sodium-Potassium-Exchanging ATPase antagonists & inhibitors, Sodium-Potassium-Exchanging ATPase metabolism, Time Factors, Glyphosate, Arrhythmias, Cardiac chemically induced, Glycine analogs & derivatives, Heart Conduction System drug effects, Heart Ventricles drug effects, Herbicides toxicity, Myocytes, Cardiac drug effects
- Abstract
Roundup (R), a glyphosate (G)-based herbicide (GBH), containing unknown adjuvants is widely dispersed around the world. Used principally by farmers, intoxications have increasingly been reported. We have studied R effects (containing 36 % of G) on right ventricular tissues (male Sprague-Dawley rats, up to 20,000 ppm and female New Zealand rabbits, at 25 and 50 ppm), to investigate R cardiac electrophysiological actions in vitro. We tested the reduced Ca(++) intracellular uptake mechanism as one potential cause of the electrical abnormalities after GBH superfusion, using the Na(+)/K(+)-ATPase inhibitor ouabain or the 1,4-dihydropyridine L-type calcium channel agonist BAY K 8644 which increases I Ca. R concentrations were selected based on human blood ranges found after acute intoxication. The study showed dose-dependent V max, APD50 and APD90 variations during 45 min of R superfusion. At the highest concentrations tested, there was a high incidence of conduction blocks, and 30-min washout with normal Tyrode solution did not restore excitability. We also observed an increased incidence of arrhythmias at different doses of R. Ouabain and BAY K 8644 prevented V max decrease, APD90 increase and the cardiac inexcitability induced by R 50 ppm. Glyphosate alone (18 and 180 ppm) had no significant electrophysiological effects. Thus, the action potential prolonging effect of R pointing to I Ca interference might explain both conduction blocks and proarrhythmia in vitro. These mechanisms may well be causative of QT prolongation, atrioventricular conduction blocks and arrhythmias in man after GBH acute intoxications as reported in retrospective hospital records.
- Published
- 2015
- Full Text
- View/download PDF
49. Aldosterone and testosterone: two steroid hormones structurally related but with opposite electrophysiological properties during myocardial ischemia-reperfusion.
- Author
-
Alexandre J, Milliez P, Rouet R, Manrique A, Allouche S, Piccirillo G, Schiariti M, and Puddu PE
- Subjects
- Action Potentials drug effects, Animals, Atrioventricular Block drug therapy, Atrioventricular Block physiopathology, Dose-Response Relationship, Drug, Electrophysiological Phenomena drug effects, Female, Heart Conduction System drug effects, Heart Ventricles drug effects, Myocardial Contraction drug effects, Myocardial Reperfusion Injury physiopathology, Rabbits, Ventricular Premature Complexes drug therapy, Aldosterone pharmacology, Myocardial Reperfusion Injury drug therapy, Testosterone pharmacology
- Abstract
Steroid hormones appear to be a key factor in the gender differences in the rates and severity of cardiovascular diseases. Aldosterone and testosterone have typical steroid ring structure, but despite this, they demonstrate very different properties. During acute myocardial ischemia-reperfusion, the deleterious impact of aldosterone is now well established. Conversely, the electrophysiological impact of testosterone in this context remained unknown. We used female rabbit in vitro models and standard microelectrode technique including right ventricle mimicking the 'border zone' existing between normal and ischemic/reperfused areas and isolated right ventricle experiments to assess the acute electrophysiological impact of both aldosterone and testosterone. During ischemia-reperfusion, acute superfusion of 10 and 100 nmol/L testosterone decreased normoxic and reperfused action potential duration at 90% (APD90 ), systematically induced conduction blocks, and decreased APD90 dispersion between ischemic and nonischemic areas (from 98 ± 4 to 57 ± 7 ms and 66 ± 3 ms, for, respectively, testosterone 10 and 100 nmol/L, P < 0.05). Testosterone 10 and 100 nmol/L concomitantly decreased sustained premature ventricular contraction (PVC) occurrence (from 55 to 0%, P < 0.05). Conversely, aldosterone 10 and 100 nmol/L increased normoxic and reperfused APD90 , APD90 dispersion, and reperfusion-induced PVCs. Furthermore, testosterone demonstrated cycle length-dependent effects on APD90 for high heart rate, whereas aldosterone did not exhibit any significant effect compared with controls. During acute myocardial ischemia-reperfusion, acute superfusion of physiological concentrations of testosterone seemed to be anti-arrhythmic by removing a pro-arrhythmic substrate (APD90 dispersion), inducing conduction blocks and decreasing triggered activities (PVC occurrence). Further experiments are warranted to confirm our results., (© 2015 Société Française de Pharmacologie et de Thérapeutique.)
- Published
- 2015
- Full Text
- View/download PDF
50. Rapid and MR-Independent IK1 Activation by Aldosterone during Ischemia-Reperfusion.
- Author
-
Alexandre J, Hof T, Puddu PE, Rouet R, Guinamard R, Manrique A, Beygui F, Sallé L, and Milliez P
- Subjects
- Animals, Female, Heart Ventricles pathology, Male, Myocardial Infarction metabolism, Myocardial Infarction pathology, Myocardial Reperfusion Injury pathology, Myocytes, Cardiac pathology, Rabbits, Aldosterone pharmacology, Heart Ventricles metabolism, Membrane Potentials drug effects, Myocardial Contraction drug effects, Myocardial Reperfusion Injury metabolism, Myocytes, Cardiac metabolism
- Abstract
In ST elevation myocardial infarction (STEMI) context, clinical studies have shown the deleterious effect of high aldosterone levels on ventricular arrhythmia occurrence and cardiac mortality. Previous in vitro reports showed that during ischemia-reperfusion, aldosterone modulates K+ currents involved in the holding of the resting membrane potential (RMP). The aim of this study was to assess the electrophysiological impact of aldosterone on IK1 current during myocardial ischemia-reperfusion. We used an in vitro model of "border zone" using right rabbit ventricle and standard microelectrode technique followed by cell-attached recordings from freshly isolated rabbit ventricular cardiomyocytes. In microelectrode experiments, aldosterone (10 and 100 nmol/L, n=7 respectively) increased the action potential duration (APD) dispersion at 90% between ischemic and normoxic zones (from 95±4 ms to 116±6 ms and 127±5 ms respectively, P<0.05) and reperfusion-induced sustained premature ventricular contractions occurrence (from 2/12 to 5/7 preparations, P<0.05). Conversely, potassium canrenoate 100 nmol/L and RU 28318 1 μmol/l alone did not affect AP parameters and premature ventricular contractions occurrence (except Vmax which was decreased by potassium canrenoate during simulated-ischemia). Furthermore, aldosterone induced a RMP hyperpolarization, evoking an implication of a K+ current involved in the holding of the RMP. Cell-attached recordings showed that aldosterone 10 nmol/L quickly activated (within 6.2±0.4 min) a 30 pS K+-selective current, inward rectifier, with pharmacological and biophysical properties consistent with the IK1 current (NPo =1.9±0.4 in control vs NPo=3.0±0.4, n=10, P<0.05). These deleterious effects persisted in presence of RU 28318, a specific MR antagonist, and were successfully prevented by potassium canrenoate, a non specific MR antagonist, in both microelectrode and patch-clamp recordings, thus indicating a MR-independent IK1 activation. In this ischemia-reperfusion context, aldosterone induced rapid and MR-independent deleterious effects including an arrhythmia substrate (increased APD90 dispersion) and triggered activities (increased premature ventricular contractions occurrence on reperfusion) possibly related to direct IK1 activation.
- Published
- 2015
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.