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567 results on '"CIPF"'

1. Guía de vigilancia

Author: Secretaría de la CIPF and Secretaría de la CIPF
Abstract: La vigilancia es una obligación de las organizaciones nacionales de protección fitosanitaria (ONPF) y proporciona una base técnica para múltiples medidas fitosanitarias, como los requisitos fitosanitarios de importación, las áreas libres de plagas, y la notificación y erradicación de plagas. Esta guía presenta los enfoques y las aplicaciones de la vigilancia.
Published: 2022

2. Protein misfolding and clearance in the pathogenesis of a new infantile onset ataxia caused by mutations in PRDX3

Author: Dolores Martínez-Rubio, Ángela Rodríguez-Prieto, Paula Sancho, Carmen Navarro-González, Nerea Gorría-Redondo, Javier Miquel-Leal, Clara Marco-Marín, Alison Jenkins, Mario Soriano-Navarro, Alberto Hernández, Belén Pérez-Dueñas, Pietro Fazzari, Sergio Aguilera-Albesa, Carmen Espinós, Instituto de Salud Carlos III, European Commission, Ministerio de Economía y Competitividad (España), Fundació La Marató de TV3, Generalitat Valenciana, Ministerio de Ciencia e Innovación (España), Marco-Marín, Clara, Institut Català de la Salut, [Martínez-Rubio D] Rare Neurodegenerative Diseases Laboratory, Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain. Joint Unit CIPF-IIS La Fe Rare Diseases, Valencia, Spain. [Rodríguez-Prieto Á, Navarro-González C, Miquel-Leal J] Cortical Circuits in Health and Disease Laboratory, Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain. [Sancho P] Rare Neurodegenerative Diseases Laboratory, Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain. [Gorría-Redondo N] Pediatric Neurology Unit, Department of Pediatrics, Complejo Hospitalario de Navarra, Navarrabiomed, Pamplona, Spain. [Pérez-Dueñas B] Servei de Neurologia Pediàtrica, Vall d’Hebron Hospital Universitari, Barcelona, Spain. Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain, and Vall d'Hebron Barcelona Hospital Campus
Subjects: Pediatria, Peroxiredoxin III, Cerebel - Degeneració, Cerebellar Ataxia, Otros calificadores::Otros calificadores::/genética [Otros calificadores], aminoácidos, péptidos y proteínas::proteínas::proteínas mitocondriales [COMPUESTOS QUÍMICOS Y DROGAS], General Medicine, Atàxia - Aspectes genètics, Mitochondrial Proteins, Mice, enfermedades del sistema nervioso::enfermedades del sistema nervioso central::enfermedades cerebrales::enfermedades cerebelosas::degeneraciones espinocerebelosas [ENFERMEDADES], enfermedades del sistema nervioso::manifestaciones neurológicas::discinesias::ataxia [ENFERMEDADES], Amino Acids, Peptides, and Proteins::Proteins::Mitochondrial Proteins [CHEMICALS AND DRUGS], Mutation, Other subheadings::Other subheadings::/genetics [Other subheadings], Nervous System Diseases::Central Nervous System Diseases::Brain Diseases::Cerebellar Diseases::Spinocerebellar Degenerations [DISEASES], Genetics, Humans, Animals, Ataxia, Molecular Biology, Genetics (clinical), Nervous System Diseases::Neurologic Manifestations::Dyskinesias::Ataxia [DISEASES], HeLa Cells, Spinocerebellar Degenerations
Abstract: 17 páginas, 8 figuras, Peroxiredoxin 3 (PRDX3) encodes a mitochondrial antioxidant protein, which is essential for the control of reactive oxygen species homeostasis. So far, PRDX3 mutations are involved in mild-to-moderate progressive juvenile onset cerebellar ataxia. We aimed to unravel the molecular bases underlying the disease in an infant suffering from cerebellar ataxia that started at 19 months old and presented severe cerebellar atrophy and peripheral neuropathy early in the course of disease. By whole exome sequencing, we identified a novel homozygous mutation, PRDX3 p.D163E, which impaired the mitochondrial ROS defense system. In mouse primary cortical neurons, the exogenous expression of PRDX3 p.D163E was reduced and triggered alterations in neurite morphology and in mitochondria. Mitochondrial computational parameters showed that p.D163E led to serious mitochondrial alterations. In transfected HeLa cells expressing the mutation, mitochondria accumulation was detected by correlative light electron microscopy. Mitochondrial morphology showed severe changes, including extremely damaged outer and inner membranes with a notable cristae disorganization. Moreover, spherical structures compatible with lipid droplets were identified, which can be associated with a generalized response to stress and can be involved in the removal of unfolded proteins. In the patient's fibroblasts, PRDX3 expression was nearly absent. The biochemical analysis suggested that the mutation p.D163E would result in an unstable structure tending to form aggregates that trigger unfolded protein responses via mitochondria and endoplasmic reticulum. Altogether, our findings broaden the clinical spectrum of the recently described PRDX3-associated neurodegeneration and provide new insight into the pathological mechanisms underlying this new form of cerebellar ataxia., The Instituto de Salud Carlos III (ISCIII)—Subdirección General de Evaluación y Fomento de la Investigación within the framework of the National R + D + I Plan cofunded with European Regional Development Funds (ERDF) (grants PI18/00147 and PI21/00103 to C.E.); the Spanish Ministry of Economy and Competitiveness (grant SAF2017-89020-R to P.F.); the Fundació La Marató TV3 (grants 20143130 and 20143131 to B.P.-D. and C.E.) and the Generalitat Valenciana (grant PROMETEO/2018/135 to C.E.). Part of the equipment employed in this work was funded by Generalitat Valenciana and co-financed with ERDF (OP ERDF of Comunitat Valenciana 2014-2020). P.F. and A.R.-P. are supported by the Spanish Ministry of Science and Innovation (grants RyC-2014-16410 to P.F. and PRE2018-083562 to A.R.-P.).
Published: 2022

3. Mutations, Genes, and Phenotypes Related to Movement Disorders and Ataxias

Author: Dolores Martínez-Rubio, Isabel Hinarejos, Paula Sancho, Nerea Gorría-Redondo, Raquel Bernadó-Fonz, Cristina Tello, Clara Marco-Marín, Itxaso Martí-Carrera, María Jesús Martínez-González, Ainhoa García-Ribes, Raquel Baviera-Muñoz, Isabel Sastre-Bataller, Irene Martínez-Torres, Anna Duat-Rodríguez, Patrícia Janeiro, Esther Moreno, Leticia Pías-Peleteiro, Mar O’Callaghan Gordo, Ángeles Ruiz-Gómez, Esteban Muñoz, Maria Josep Martí, Ana Sánchez-Monteagudo, Candela Fuster, Amparo Andrés-Bordería, Roser Maria Pons, Silvia Jesús-Maestre, Pablo Mir, Vincenzo Lupo, Belén Pérez-Dueñas, Alejandra Darling, Sergio Aguilera-Albesa, Carmen Espinós, Institut Català de la Salut, [Martínez-Rubio D, Hinarejos I] Rare Neurodegenerative Diseases Laboratory, Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain. Joint Unit CIPF-IIS La Fe Rare Diseases, Valencia, Spain. [Sancho P, Tello C] Rare Neurodegenerative Diseases Laboratory, Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain. [Gorría-Redondo N, Bernadó-Fonz R] Paediatric Neurology Unit, Department of Paediatrics, Hospital Universitario de Navarra, Navarrabiomed, Pamplona, Spain. [Pérez-Dueñas B] Servei de Neurologia Pediàtrica, Vall d’Hebron Hospital Universitari, Barcelona, Spain. Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain, Vall d'Hebron Barcelona Hospital Campus, Instituto de Salud Carlos III, European Commission, Fundació La Marató de TV3, Generalitat Valenciana, Ministerio de Educación, Cultura y Deporte (España), Fundació per Amor a L'Art, Marco-Marín, Clara [0000-0002-8813-3515], and Marco-Marín, Clara
Subjects: Iron, Genetic Phenomena::Genetic Variation::Mutation [PHENOMENA AND PROCESSES], Kinesins, Catalysis, Atàxia - Aspectes genètics, Inorganic Chemistry, cerebellar atrophy, gene panel, Other subheadings::Other subheadings::/genetics [Other subheadings], Humans, movement disorders, ataxia, neurodegeneration with brain iron accumulation (NBIA), exome sequencing, Physical and Theoretical Chemistry, Molecular Biology, fenómenos genéticos::variación genética::mutación [FENÓMENOS Y PROCESOS], Spectroscopy, Movement Disorders, Otros calificadores::Otros calificadores::/genética [Otros calificadores], Organic Chemistry, Brain, Neurodegenerative Diseases, General Medicine, Computer Science Applications, Fenotip, Phosphotransferases (Alcohol Group Acceptor), Anomalies cromosòmiques, Phenotype, enfermedades del sistema nervioso::manifestaciones neurológicas::discinesias::ataxia [ENFERMEDADES], Mutation, Genetic Phenomena::Phenotype [PHENOMENA AND PROCESSES], Ataxia, fenómenos genéticos::fenotipo [FENÓMENOS Y PROCESOS], Nervous System Diseases::Neurologic Manifestations::Dyskinesias::Ataxia [DISEASES]
Abstract: 26 páginas, 4 figuras, 3 tablas, Our clinical series comprises 124 patients with movement disorders (MDs) and/or ataxia with cerebellar atrophy (CA), many of them showing signs of neurodegeneration with brain iron accumulation (NBIA). Ten NBIA genes are accepted, although isolated cases compatible with abnormal brain iron deposits are known. The patients were evaluated using standardised clinical assessments of ataxia and MDs. First, NBIA genes were analysed by Sanger sequencing and 59 patients achieved a diagnosis, including the detection of the founder mutation PANK2 p.T528M in Romani people. Then, we used a custom panel MovDisord and/or exome sequencing; 29 cases were solved with a great genetic heterogeneity (34 different mutations in 23 genes). Three patients presented brain iron deposits with Fe-sensitive MRI sequences and mutations in FBXO7, GLB1, and KIF1A, suggesting an NBIA-like phenotype. Eleven patients showed very early-onset ataxia and CA with cortical hyperintensities caused by mutations in ITPR1, KIF1A, SPTBN2, PLA2G6, PMPCA, and PRDX3. The novel variants were investigated by structural modelling, luciferase analysis, transcript/minigenes studies, or immunofluorescence assays. Our findings expand the phenotypes and the genetics of MDs and ataxias with early-onset CA and cortical hyperintensities and highlight that the abnormal brain iron accumulation or early cerebellar gliosis may resembling an NBIA phenotype., This work was supported by the Instituto de Salud Carlos III (ISCIII)—Subdirección General de Evaluación y Fomento de la Investigación within the framework of the National R + D+I Plan co-funded with European Regional Development Funds (ERDF) [Grants PI18/00147 and PI21/00103 to CE]; the Fundació La Marató TV3 [Grants 20143130 and 20143131 to BPD and CE]; and by the Generalitat Valenciana [Grant PROMETEO/2018/135 to CE]. Part of the equipment employed in this work was funded by Generalitat Valenciana and co-financed with ERDF (OP ERDF of Comunitat Valenciana 2014–2020). PS had an FPU-PhD fellowship funded by the Spanish Ministry of Education, Culture and Sport [FPU15/00964]. IH has a PFIS-PhD fellowship [FI19/00072]. ASM has a contract funded by the Spanish Foundation Per Amor a l’Art (FPAA)
Published: 2022
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4. Generation of three human iPSC lines from PLAN (PLA2G6-associated neurodegeneration) patients

Author: José Antonio Sánchez-Alcázar, Carmen Espinós, Alejandra Darling, Irene Villalón-García, Slaven Erceg, Belén Pérez-Dueñas, Candela Machuca, Deyanira García-Navas, Marta Correa-Vela, Institut Català de la Salut, [Machuca C] Unit of Rare Neurodegenerative Diseases, Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain. Rare Diseases Joint Units, CIPF-IIS La Fe & INCLIVA, Valencia, Spain. Stem Cells Therapies in Neurodegenerative Diseases Lab, Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain. [Correa-Vela M] Servei de Neurologia Pediàtrica, Vall d’Hebron Hospital Universitari, Barcelona, Spain. Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. [García-Navas D] Department of Pediatric Neurology. Hospital Universitario San Pedro de Alcántara, Cáceres, Spain. [Darling A] Unit of Pediatric Movement Disorders, Hospital Sant Joan de Déu, Barcelona, Spain. [Villalón-García I, Sánchez-Alcázar JA] Centro Andaluz de Biología del Desarrollo (CABD-CSIC), Universidad Pablo de Olavide, Seville, Spain. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain. [Pérez-Dueñas B] Servei de Neurologia Pediàtrica, Vall d’Hebron Hospital Universitari, Barcelona, Spain. Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. Universitat Autònoma de Barcelona, Bellaterra, Spain, Vall d'Hebron Barcelona Hospital Campus, Instituto de Salud Carlos III, and Generalitat Valenciana
Subjects: 0301 basic medicine, QH301-705.5, Cellular differentiation, Induced Pluripotent Stem Cells, Neuroaxonal Dystrophies, Cells::Stem Cells::Adult Stem Cells::Induced Pluripotent Stem Cells [ANATOMY], Biology, medicine.disease_cause, células::células madre::células madre adultas::células madre pluripotentes inducidas [ANATOMÍA], Sistema nerviós - Degeneració, Cell Line, Dermal fibroblast, Group VI Phospholipases A2, 03 medical and health sciences, Kruppel-Like Factor 4, 0302 clinical medicine, SOX2, medicine, Humans, enfermedades del sistema nervioso::enfermedades neurodegenerativas [ENFERMEDADES], Biology (General), Induced pluripotent stem cell, Mutation, Neurodegeneration, Cell Differentiation, Cell Biology, General Medicine, medicine.disease, Cellular Reprogramming, 030104 developmental biology, KLF4, Nervous System Diseases::Neurodegenerative Diseases [DISEASES], Cancer research, Malalties rares, Reprogramming, 030217 neurology & neurosurgery, Genètica, Developmental Biology
Abstract: © 2021 The Authors., The human iPSC cell lines, PLANFiPS1-Sv4F-1 (RCPFi004-A), PLANFiPS2-Sv4F-1 (RCPFi005-A), PLANFiPS3-Sv4F-1 RCPFi006-A), derived from dermal fibroblast from three patients suffering PLAN (PLA2G6-associated neurodegeneration; MIM 256600) caused by mutations in the PLA2G6 gene, was generated by non-integrative reprogramming technology using OCT3/4, SOX2, CMYC and KLF4 reprogramming factors. The pluripotency was assessed by immunocytochemistry and RT-PCR. Differentiation capacity was verified in vitro. This iPSC line can be further differentiated toward affected cells to better understand molecular mechanisms of disease and pathophysiology., This work was supported by the Instituto de Salud Carlos III (ISCIII) - Subdireccion ´ General de Evaluacion ´ y Fomento de la Investigacion ´ [PI18/00147to CE and PI18/01319 to BPD], and by the Generalitat Valenciana [PROMETEO/2018/135], within the framework of the National R + D + I Plan co-funded with ERDF funds. CM has a CIPF-PhD fellowship [P.I.06/2017]. Part of the equipment employed in this work has been funded by Generalitat Valenciana and co-financed with ERDF funds (OP ERDF of Comunitat Valenciana 2014–2020).
Published: 2021

5. Expanding the β-III Spectrin-Associated Phenotypes toward Non-Progressive Congenital Ataxias with Neurodegeneration

Author: Sancho, Paula, Andrés-Bordería, Amparo, Gorría-Redondo, Nerea, Llano, Katia, Martínez-Rubio, Dolores, Yoldi-Petri, María Eugenia, Blumkin, Luba, Rodríguez de la Fuente, Pablo, Gil-Ortiz, Fernando, Fernández-Murga, Leonor, Sánchez-Monteagudo, Ana, Lupo, Vincenzo, Pérez-Dueñas, Belén, Espinós, Carmen, Aguilera-Albesa, Sergio, Universitat Autònoma de Barcelona, Institut Català de la Salut, [Sancho P, Martínez-Rubio D] Unit of Rare Neurodegenerative Diseases, Centro de Investigación Príncipe Felipe (CIPF), 46012 Valencia, Spain. [Andrés-Bordería A] Unit of Rare Neurodegenerative Diseases, Centro de Investigación Príncipe Felipe (CIPF), 46012 Valencia, Spain. Department of Physiology, Faculty of Medicine and Dentistry, University of Valencia, 46010 Valencia, Spain. [Gorría-Redondo N, Yoldi-Petri ME] Pediatric Neurology Unit, Department of Pediatrics, Complejo Hospitalario de Navarra, 31008 Pamplona, Spain. [Pérez-Dueñas B] Grup de Recerca en Neurologia Pediàtrica, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain. Universitat Autònoma de Barcelona, Bellaterra, Spain, and Vall d'Hebron Barcelona Hospital Campus
Subjects: Male, 0301 basic medicine, Proband, Pathology, Protein Conformation, Sequence Homology, SPTBN2 gene, b-III spectrin, 030105 genetics & heredity, Fluid-attenuated inversion recovery, Cohort Studies, lcsh:Chemistry, Non-progressive congenital ataxia, 0302 clinical medicine, β-III spectrin, Spectrin, enfermedades del sistema nervioso::enfermedades neurodegenerativas [ENFERMEDADES], Age of Onset, Child, lcsh:QH301-705.5, Spectroscopy, Otros calificadores::Otros calificadores::/genética [Otros calificadores], Neurodegeneration, neurodegeneration, Neurodegenerative Diseases, non-progressive congenital ataxia, Syndrome, General Medicine, Phenotype, Hypotonia, Computer Science Applications, Nervous System Diseases::Neurodegenerative Diseases [DISEASES], Spinocerebellar ataxia, medicine.symptom, medicine.medical_specialty, congenital, hereditary, and neonatal diseases and abnormalities, Cerebellar Ataxia, Neuroimaging, Biology, Catalysis, Article, Inorganic Chemistry, 03 medical and health sciences, Nervous System Diseases::Central Nervous System Diseases::Brain Diseases::Cerebellar Diseases::Cerebellar Ataxia [DISEASES], Other subheadings::Other subheadings::/genetics [Other subheadings], medicine, Humans, Amino Acid Sequence, Physical and Theoretical Chemistry, Molecular Biology, Genetic Association Studies, Organic Chemistry, medicine.disease, Hyperintensity, Sistema nerviós - Degeneració - Aspectes genètics, lcsh:Biology (General), lcsh:QD1-999, enfermedades del sistema nervioso::enfermedades del sistema nervioso central::enfermedades cerebrales::enfermedades cerebelosas::ataxia cerebelosa [ENFERMEDADES], Mutation, 030217 neurology & neurosurgery
Abstract: (1) Background: A non-progressive congenital ataxia (NPCA) phenotype caused by b-III spectrin (SPTBN2) mutations has emerged, mimicking spinocerebellar ataxia, autosomal recessive type 14 (SCAR14). The pattern of inheritance, however, resembles that of autosomal dominant classical spinocerebellar ataxia type 5 (SCA5). (2) Methods: In-depth phenotyping of two boys studied by a customized gene panel. Candidate variants were sought by structural modeling and protein expression. An extensive review of the literature was conducted in order to better characterize the SPTBN2-associated NPCA. (3) Results: Patients exhibited an NPCA with hypotonia, developmental delay, cerebellar syndrome, and cognitive deficits. Both probands presented with progressive global cerebellar volume loss in consecutive cerebral magnetic resonance imaging studies, characterized by decreasing midsagittal vermis relative diameter measurements. Cortical hyperintensities were observed on fluid-attenuated inversion recovery (FLAIR) images, suggesting a neurodegenerative process. Each patient carried a novel de novo SPTBN2 substitution: c.193A &gt, G (p.K65E) or c.764A &gt, G (p.D255G). Modeling and protein expression revealed that both mutations might be deleterious. (4) Conclusions: The reported findings contribute to a better understanding of the SPTBN2-associated phenotype. The mutations may preclude proper structural organization of the actin spectrin-based membrane skeleton, which, in turn, is responsible for the underlying disease mechanism.
Published: 2021

6. NR4A2 Mutations Can Cause Intellectual Disability and Language Impairment With Persistent Dystonia-Parkinsonism

Author: Silvia Jesús, Astrid D Adarmes, Isabel Hinarejos, Pablo Mir, Fátima Carrillo, Carmen Espinós, Daniel Macías-García, Dolores Martínez-Rubio, Belén Pérez-Dueñas, Vincenzo Lupo, Ana Sánchez-Monteagudo, Institut Català de la Salut, [Jesús S, Carrillo F, Macías-García D] Unidad de Trastornos del Movimiento, Servicio de Neurología y Neurofisiología Clínica, Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Spain. Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas (CIBERNED), Spain. [Hinarejos I, Martínez-Rubio D, Sánchez-Monteagudo A] Unit of Genetics and Genomics of Neuromuscular and Neurodegenerative Disorders, Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain. Joint Units INCLIVA and IIS La Fe Rare Diseases, Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain. [Pérez-Dueñas B] Servei de Neurologia Pediàtrica, Vall d'Hebron Hospital Universitari, Barcelona, Spain. Universitat Autònoma de Barcelona, Bellaterra, Spain, and Vall d'Hebron Barcelona Hospital Campus
Subjects: 0301 basic medicine, enfermedades del sistema nervioso::manifestaciones neurológicas::manifestaciones neuroconductuales::trastornos de la comunicación::trastornos del lenguaje [ENFERMEDADES], Genetic Phenomena::Genetic Variation::Mutation [PHENOMENA AND PROCESSES], Substantia nigra, Nervous System Diseases::Neurologic Manifestations::Neurobehavioral Manifestations::Intellectual Disability [DISEASES], Nervous System Diseases::Neurologic Manifestations::Neurobehavioral Manifestations::Communication Disorders::Language Disorders [DISEASES], 03 medical and health sciences, Motor tics, 0302 clinical medicine, Intellectual disability, Discapacitats - Comunicació, medicine, Trastorns del llenguatge - Aspectes genètics, Clinical phenotype, Clinical/Scientific Notes, fenómenos genéticos::variación genética::mutación [FENÓMENOS Y PROCESOS], Genetics (clinical), business.industry, Mutació (Biologia), Language impairment, medicine.disease, Phenotype, nervous system diseases, Ventral tegmental area, 030104 developmental biology, medicine.anatomical_structure, Rapid onset, enfermedades del sistema nervioso::manifestaciones neurológicas::manifestaciones neuroconductuales::discapacidad intelectual [ENFERMEDADES], Neurology (clinical), business, Neuroscience, 030217 neurology & neurosurgery
Abstract: Dystonia; Parkinson's disease/Parkinsonism; Genetic linkage Malaltia de Parkinson/Parkinsonisme; Vinculació genètica; Distonia Enfermedad de Parkinson/Parkinsonismo; Enlace genético; Distonía This work was supported by the Health Institute Carlos III—General Subdirectorate for Research Evaluation and Promotion (PI16/01575, PI18/01898, PI18/00147, PI19/01576), the Spanish Ministry of Economy and Competitiveness (SAF2007-60700), the Ministry of Economy, Innovation, Science and Business of the Government of Andalucía (CVI-02526, CTS-7685), the Ministry of Health and Social Welfare of the Government of Andalucía (PI-0459-2018, PE-0210-2018, PE-0186-2019) and by the Valencian Government (PROMETEO/2018/135), within the framework of the National Research and Development Plan co-funded with European Regional Development Funds. Part of the equipment employed in this study was funded by the Valencian Government and co-financed with European Regional Development Funds (OP ERDF of Valencian Community 2014-2020). I. Hinarejos has a PFIS-PhD fellowship (FI19/00072), S. Jesús has a contract “Acción B Clínicos-Investigadores” (Action B Clinicians-Researchers) contract (B-0007-2019) funded by the Ministry of Health and Family of the Government of Andalucía, and D. Macías-García has a Río Hortega contract (CM18/00142) funded by the Health Institute Carlos III.
Published: 2021

7. Phase 2 Trial (POLA Study) of Lurbinectedin plus Olaparib in Patients with Advanced Solid Tumors: Results of Efficacy, Tolerability, and the Translational Study

Author: Andres Poveda, Raquel Lopez-Reig, Ana Oaknin, Andres Redondo, Maria Jesus Rubio, Eva Guerra, Lorena Fariñas-Madrid, Alejandro Gallego, Victor Rodriguez-Freixinos, Antonio Fernandez-Serra, Oscar Juan, Ignacio Romero, Jose A. Lopez-Guerrero, Institut Català de la Salut, [Poveda A] Oncogynecologic Department, Initia Oncology, Hospital Quironsalud, Valencia, Spain. [Lopez-Reig R] Laboratory of Molecular Biology, Fundación Instituto Valenciano de Oncología, Valencia, Spain. IVO-CIPF Joint Research Unit of Cancer, Príncipe Felipe Research Center (CIPF), Valencia, Spain. [Oaknin A, Fariñas-Madrid L] Servei d’Oncologia Mèdica, Vall d’Hebron Hospital Universitari, Barcelona, Spain. Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain. [Redondo A] Medical Oncology Department, Hospital Universitario La Paz-IdiPAZ, Universidad Autónoma de Madrid (UAM), Madrid, Spain. [Rubio MJ] Medical Oncology Department, Universitary Hospital Reina Sofia, Cordoba, Spain. [Guerra E] Medical Oncology, Hospital Universitario Ramón y Cajal, Madrid, Spain. [Rodriguez-Freixinos V] Servei d’Oncologia Mèdica, Vall d’Hebron Hospital Universitari, Barcelona, Spain. Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain. Department of Medical Oncology and Hematology, Odette Cancer Centre, Sunnybrook Health Sciences Centre, Toronto, Canada, Vall d'Hebron Barcelona Hospital Campus, and UAM. Departamento de Medicina
Subjects: Genomic instability, Lurbinectedin, Cancer Research, Medicina, Càncer - Tractament, Otros calificadores::Otros calificadores::/farmacoterapia [Otros calificadores], Other subheadings::Other subheadings::/drug therapy [Other subheadings], Neoplasms [DISEASES], neoplasias [ENFERMEDADES], Olaparib, Endometrial cancer, Oncology, Ovarian cancer, ovarian cancer, endometrial cancer, lurbinectedin, olaparib, genomic instability, Avaluació de resultats (Assistència sanitària)
Abstract: Endometrial cancer; Genomic instability; Olaparib Cáncer endometrial; Inestabilidad genómica; Olaparib Càncer d'endometri; Inestabilitat genòmica; Olaparib We hypothesized that the combination of olaparib and lurbinectedin maximizes DNA damage, thus increasing its efficacy. The POLA phase 1 trial established the recommended phase 2 dose of lurbinectedin as being 1.5 mg (day 1) and that of olaparib as being 250 mg/12 h (days 1–5) for a 21-day cycle. In phase 2, we explore the efficacy of the combination in terms of clinical response and its correlation with mutations in the HRR genes and the genomic instability (GI) parameters. Results: A total of 73 patients with high-grade ovarian (n = 46), endometrial (n = 26), and triple-negative breast cancer (n = 1) were treated with lurbinectedin and olaparib. Most patients (62%) received ≥3 lines of prior therapy. The overall response rate (ORR) and disease control rate (DCR) were 9.6% and 72.6%, respectively. The median progression-free survival (PFS) was 4.54 months (95% CI 3.0–5.2). Twelve (16.4%) patients were considered long-term responders (LTR), with a median PFS of 13.3 months. No clinical benefit was observed for cases with HRR gene mutation. In ovarian LTRs, although a direct association with GI and a total loss of heterozygosity (LOH) events was observed, the association did not reach statistical significance (p = 0.055). Globally, the total number of LOHs might be associated with the ORR (p =0.074). The most common grade 3–4 toxicities were anemia and thrombocytopenia, in 6 (8.2%) and 3 (4.1%) patients, respectively. Conclusion: The POLA study provides evidence that the administration of lurbinectedin and olaparib is feasible and tolerable, with a DCR of 72.6%. Different GI parameters showed associations with better responses. This trial was sponsored by AstraZeneca and PharmaMar, including supply of the drugs used in this study.
Published: 2022
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8. Cutting-edge multi-level analytical and structural characterization of antibody-drug conjugates: present and future

Author: Oscar Hernandez-Alba, Emmanuel Leize-Wagner, Alain Beck, Yannis François, Davy Guillarme, Szabolcs Fekete, Valentina D'Atri, Sarah Cianférani, Anthony Ehkirch, Rabah Gahoual, Centre d'Immunologie Pierre Fabre, CIPF, School of Pharmaceutical Science, Université de Lausanne (UNIL)-University of Geneva [Switzerland], Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Tectonique Moléculaire du Solide (TMS), Chimie de la matière complexe (CMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Chimie de Coordination Organique, Laboratoire d'Electrochimie et de Chimie Analytique (LECA), Université Pierre et Marie Curie - Paris 6 (UPMC)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS), University of Geneva [Switzerland], Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg (UNISTRA), Centre d'Immunologie Pierre Fabre (CIPF), PIERRE FABRE, University of Geneva [Switzerland]-Université de Lausanne (UNIL), Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Unité de Technologies Chimiques et Biologiques pour la Santé (UTCBS - UM 4 (UMR 8258 / U1022)), Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Center of Immunology Pierre Fabre, Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5)-Ecole Nationale Supérieure de Chimie de Paris- Chimie ParisTech-PSL (ENSCP), and Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Drug, Electrophoresis, Bioanalysis, Immunoconjugates, Computer science, media_common.quotation_subject, Nanotechnology, inotuzumab ozogamicin, Biochemistry, Inotuzumab ozogamicin, Sacituzumab govitecan, Mass Spectrometry, 03 medical and health sciences, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, gemtuzumab ozogamicin, [CHIM]Chemical Sciences, Humans, Gemtuzumab ozogamicin, Amino Acid Sequence, Molecular Biology, Trastuzumab emtansine, ComputingMilieux_MISCELLANEOUS, media_common, trastuzumab emtansine, ddc:615, Chromatography, 030102 biochemistry & molecular biology, small-protein fragment-DCs, 3. Good health, Characterization (materials science), body regions, 030104 developmental biology, 3G-ADCs, Brentuximab vedotin, Fragment-DCs, Small-protein, Hydrophobic and Hydrophilic Interactions, Conjugate
Abstract: International audience; Introduction : The development and optimization of antibody drug conjugates (ADCs) rely on improving their analytical and bioanalytical characterization, by assessing critical quality attributes (CQAs). Among the CQAs, the glycoprofile, drug load distribution (DLD), the amountof unconjugated antibody (D0), the average drug-to-antibody ratio (DAR), the drug conjugation sites and the residual drug-linker and related product proportions (SMDs) in addition to high and low molecular weight species (H/LMWS) are the most important ones.Areas covered : The analytical and structural toolbox for the characterization of 1st, 2d and 3d generation ADCs was significantly extended in the last 3 years. Here, we reviewed state-ofthe-art techniques, such as liquid chromatography, high resolution native and ion mobility mass spectrometry, multidimensional LC and capillary electrophoresis hyphenated to mass spectrometry, reported mainly since 2016.Expert commentary : These emerging techniques allow a deep insight into important CQAs that are related to ADC Chemistry Manufacturing and Control (CMC) as well as an improved understanding of in vitro and in vivo ADC biotransformations. This knowledge and the development of quantitative bioanalytical assays will continue to contribute to earlydevelopability assessment for the optimization of all the ADC components (i.e., antibody, drug, and linker) and help to bring next-generation candidate ADC into the clinic and hopefully to the marke
Published: 2019
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9. Monoclonal antibody N-glycosylation profiling using capillary electrophoresis – Mass spectrometry: Assessment and method validation

Author: Elsa Wagner-Rousset, Emmanuelle Leize-Wagner, Alain Beck, Jérémie Giorgetti, Olivier Colas, Davy Guillarme, Antony Lechner, Julie Canonge, Yannis-Nicolas François, Valentina D'Atri, Tectonique Moléculaire du Solide (TMS), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), School of Pharmaceutical Science, University of Geneva [Switzerland]-Université de Lausanne (UNIL), University of Geneva [Switzerland], Center of Immunology Pierre Fabre, Centre d'Immunologie Pierre Fabre, CIPF, Laboratoire de synthèses métallo-induites, Institut de Chimie de Strasbourg-Dynamique et structure moléculaire par spectrométrie de masse (LDSM2), Chimie de la matière complexe (CMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de spectrométrie de masse des interactions et des systèmes, Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie Pierre Fabre (CIPF), and PIERRE FABRE
Subjects: 0301 basic medicine, Monoclonal antibody, Glycan, Spectrometry, Mass, Electrospray Ionization, Glycosylation, medicine.drug_class, IgG glycosylation, Computational biology, Glycopeptide, 01 natural sciences, Capillary electrophoresis–mass spectrometry, Analytical Chemistry, HILIC-FD, 03 medical and health sciences, chemistry.chemical_compound, Protein structure, N-linked glycosylation, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, medicine, Effector functions, ComputingMilieux_MISCELLANEOUS, Chromatography, ddc:615, biology, Chemistry, Hydrophilic interaction chromatography, 010401 analytical chemistry, Glycopeptides, Antibodies, Monoclonal, Electrophoresis, Capillary, Reproducibility of Results, 0104 chemical sciences, carbohydrates (lipids), 030104 developmental biology, biology.protein, lipids (amino acids, peptides, and proteins), Capillary electrophoresis-mass spectrometry
Abstract: Characterization of therapeutic proteins represents a major challenge for analytical sciences due to their heterogeneity caused by post-translational modifications (PTM). Among these PTM, glycosylation which is possibly the most prominent, require comprehensive identification because of their major influence on protein structure and effector functions of monoclonal antibodies (mAbs). As a consequence, glycosylation profiling must be deeply characterized. For this application, several analytical methods such as separation-based or MS-based methods, were evaluated. However, no CE-ESI-MS approach has been assessed and validated. Here, we illustrate how the use of CE-ESI-MS method permits the comprehensive characterization of mAbs N-glycosylation at the glycopeptide level to perform relative quantitation of N-glycan species. Validation of the CE-ESI-MS method in terms of robustness and reproducibility was demonstrated through the relative quantitation of glycosylation profiles for ten different mAbs produced in different cell lines. Glycosylation patterns obtained for each mAbs were compared to Hydrophilic Interaction Chromatography of 2-aminobenzamide labelled glycans with fluorescence detector (HILIC-FD) analysis considered as a reference method. Very similar glycoprofiling were obtained with the CE-ESI-MS and HILIC-FD demonstrating the attractiveness of CE-ESI-MS method to characterize and quantify the glycosylation heterogeneity of a wide range of therapeutic mAbs with high accuracy and precision.
Published: 2018
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10. The Scarface Score: Deciphering Response to DNA Damage Agents in High-Grade Serous Ovarian Cancer—A GEICO Study

Author: Fernandez-Serra, Antonio, López-Reig, Raquel, Márquez, Raúl, Gallego, Alejandro, De Sande-González, Luis Miguel, Yubero Esteban, Alfonso, OAKNIN, ANA, Institut Català de la Salut, [Fernández-Serra A, López-Reig R] Molecular Biology Lab, Molecular Biology Department, Instituto Valenciano de Oncologia, Valencia, Spain. Joint IVO-CIPF Cancer Research Unit, Valencia, Spain. [Márquez R] Medical Oncology Department, MD Anderson Cancer Center, Madrid, Spain. [Gallego A] Medical Oncology Department, Hospital Universitario La Paz, Madrid, Spain. [de Sande LM] Medical Oncology Department, Hospital Universitario de León, León, Spain. [Yubero A] Medical Oncology Department, Hospital Clínico Universitario Lozano Blesa, Zaragoza, Spain. [Oaknin A] Servei d’Oncologia Mèdica, Vall d’Hebron Hospital Universitari, Barcelona, Spain, and Vall d'Hebron Barcelona Hospital Campus
Subjects: Genòmica, Anomalies cromosòmiques, Neoplasms::Neoplasms by Site::Endocrine Gland Neoplasms::Ovarian Neoplasms [DISEASES], Genetic Phenomena::Genetic Variation::Mutation::Genetic Phenomena::Genomic Instability [PHENOMENA AND PROCESSES], Ovaris - Càncer - Aspectes genètics, neoplasias::neoplasias por localización::neoplasias de las glándulas endocrinas::neoplasias ováricas [ENFERMEDADES], fenómenos genéticos::variación genética::mutación::fenómenos genéticos::inestabilidad genómica [FENÓMENOS Y PROCESOS]
Abstract: Genomic instability; Machine learning Inestabilidad genómica; Aprendizaje automático Inestabilitat genòmica; Aprenentatge automàtic Genomic Instability (GI) is a transversal phenomenon shared by several tumor types that provide both prognostic and predictive information. In the context of high-grade serous ovarian cancer (HGSOC), response to DNA-damaging agents such as platinum-based and poly(ADP-ribose) polymerase inhibitors (PARPi) has been closely linked to deficiencies in the DNA repair machinery by homologous recombination repair (HRR) and GI. In this study, we have developed the Scarface score, an integrative algorithm based on genomic and transcriptomic data obtained from the NGS analysis of a prospective GEICO cohort of 190 formalin-fixed paraffin-embedded (FFPE) tumor samples from patients diagnosed with HGSOC with a median follow up of 31.03 months (5.87–159.27 months). In the first step, three single-source models, including the SNP-based model (accuracy = 0.8077), analyzing 8 SNPs distributed along the genome; the GI-based model (accuracy = 0.9038) interrogating 28 parameters of GI; and the HTG-based model (accuracy = 0.8077), evaluating the expression of 7 genes related with tumor biology; were proved to predict response. Then, an ensemble model called the Scarface score was found to predict response to DNA-damaging agents with an accuracy of 0.9615 and a kappa index of 0.9128 (p < 0.0001). The Scarface Score approaches the routine establishment of GI in the clinical setting, enabling its incorporation as a predictive and prognostic tool in the management of HGSOC. This research was partially funded by GVA Grants “Subvencions per a la realització de projectes d’i+d+i desenvolupats per grups d’investigació emergents (GV/2020/158)” and “Ayudas para la contratación de personal investigador en formación de carácter predoctoral” (ACIF/2016/008) and “Beca de investigación traslacional Andrés Poveda 2020” from GEICO group. This study was awarded the Prize “Antonio Llombart Rodriguez-FINCIVO 2020” from the Royal Academy of Medicine of the Valencian Community.
Published: 2023

11. Improving the management of Inherited Retinal Dystrophies by targeted sequencing of a population-specific gene panel

Author: Guillermo Antiñolo, Nereida Bravo-Gil, Salud Borrego, Cristina Méndez-Vidal, Enrique Rodríguez de la Rúa, Joaquín Dopazo, Laura Romero-Pérez, María González del Pozo, [Bravo-Gil,N, Méndez-Vidal,C, Romero-Pérez,L, González-Del Pozo,M, Borrego,S, Antiñolo,G] Department of Genetics, Reproduction and Fetal Medicine, Institute of Biomedicine of Seville, University Hospital Virgen del Rocío/CSIC/University of Seville, Seville, Spain. [Bravo-Gil,N, Dopazo,J, Antiñolo,G] Centre for Biomedical Network Research on Rare Diseases (CIBERER), Spain. [Rodríguez-de la Rúa,E] Department of Ophthalmology, University Hospital Virgen Macarena, Seville, Spain. [Dopazo,J] Computational Genomics Department, Centro de Investigación Príncipe Felipe (CIPF).Functional Genomics Node, (INB) at CIPF, Valencia, Spain., This work was supported by the Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness, Spain (PI11-02923), CIBERER ACCI, CDTI FEDER-Innterconecta (EXP00052887/ITC-20111037), Regional Ministry of Economy, Innovation, Science and Employment of the Autonomous Government of Andalusia (CTS-1664) and the Foundation Ramon Areces (CIVP16A1856). The CIBERER is an initiative of the ISCIII, Spanish Ministry of Economy and Competitiveness. NB-G is supported by fellowship FI12/00545 from ISCIII., Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (España), European Commission, Centro de Investigación Biomédica en Red Enfermedades Raras (España), Centro para el Desarrollo Tecnológico Industrial (España), Junta de Andalucía, and Fundación Ramón Areces
Subjects: 0301 basic medicine, Proband, Phenomena and Processes::Genetic Phenomena::Phenotype [Medical Subject Headings], Heterogeneidad genética, DNA Mutational Analysis, Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Sequence Analysis::High-Throughput Nucleotide Sequencing [Medical Subject Headings], medicine.disease_cause, Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Genetic Association Studies [Medical Subject Headings], Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Primates::Haplorhini::Catarrhini::Hominidae::Humans [Medical Subject Headings], Exoma, Exome sequencing, Genetics, education.field_of_study, Mutation, Multidisciplinary, High-Throughput Nucleotide Sequencing, Disciplines and Occupations::Natural Science Disciplines::Biological Science Disciplines::Biology::Genetics::Genetics, Medical::Genetic Counseling [Medical Subject Headings], Phenotype, Distrofias retinianas, Phenomena and Processes::Genetic Phenomena::Genotype [Medical Subject Headings], Fenotipo, Retinal Dystrophies, Diseases::Eye Diseases::Retinal Diseases::Retinal Degeneration::Retinal Dystrophies [Medical Subject Headings], Phenomena and Processes::Genetic Phenomena::Genetic Variation::Mutation [Medical Subject Headings], DNA Copy Number Variations, Genetic counseling, Population, Biology, Phenomena and Processes::Genetic Phenomena::Genetic Structures::Genome::Exome [Medical Subject Headings], Article, 03 medical and health sciences, Genetic Heterogeneity, medicine, Humans, Computer Simulation, Allele, education, Eye Proteins, Alleles, Genetic Association Studies, Gene Library, Genetic heterogeneity, Estudios de asociación genética, Asesoramiento genético, Genetic Therapy, 030104 developmental biology, Phenomena and Processes::Genetic Phenomena::Genetic Variation::Genetic Heterogeneity [Medical Subject Headings], Genotipo
Abstract: Next-generation sequencing (NGS) has overcome important limitations to the molecular diagnosis of Inherited Retinal Dystrophies (IRD) such as the high clinical and genetic heterogeneity and the overlapping phenotypes. The purpose of this study was the identification of the genetic defect in 32 Spanish families with different forms of IRD. With that aim, we implemented a custom NGS panel comprising 64 IRD-associated genes in our population, and three disease-associated intronic regions. A total of 37 pathogenic mutations (14 novels) were found in 73% of IRD patients ranging from 50% for autosomal dominant cases, 75% for syndromic cases, 83% for autosomal recessive cases, and 100% for X-linked cases. Additionally, unexpected phenotype-genotype correlations were found in 6 probands, which led to the refinement of their clinical diagnoses. Furthermore, intra- and interfamilial phenotypic variability was observed in two cases. Moreover, two cases unsuccessfully analysed by exome sequencing were resolved by applying this panel. Our results demonstrate that this hypothesis-free approach based on frequently mutated, population-specific loci is highly cost-efficient for the routine diagnosis of this heterogeneous condition and allows the unbiased analysis of a miscellaneous cohort. The molecular information found here has aid clinical diagnosis and has improved genetic counselling and patient management., This work was supported by the Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness, Spain (PI11-02923), CIBERER ACCI, CDTI FEDER-Innterconecta (EXP00052887/ITC-20111037), Regional Ministry of Economy, Innovation, Science and Employment of the Autonomous Government of Andalusia (CTS-1664) and the Foundation Ramon Areces (CIVP16A1856). The CIBERER is an initiative of the ISCIII, Spanish Ministry of Economy and Competitiveness. NB-G is supported by fellowship FI12/00545 from ISCIII.
Published: 2016

12. Prediction of human population responses to toxic compounds by a collaborative competition

Author: Eduati, F, Mangravite, L, Wang, T, Tang, Hongxiang, Bare, J, Huang, R, Norman, T, Kellen, M, Menden, M, Yang, J, Zhan, X, Zhong, R, Xiao, G, Xia, M, Abdo, N, Kosyk, O, Friend, S, Stolovitzky, G, Dearry, A, Tice, R, Simeonov, S, Rusyn, I, Wright, F, Xie, Y, Alaimo, S, Amadoz, A, Ammad-ud-din, M, Chloé-Azencott, A, Bacardit, J, Barron, P, Bernard, E, Beyer, A, Bin, S, van Bömmel, A, Borgwardt, K, Brys, A, Caffrey, B, Chang, J, Christodoulou, E, Clément-Ziza, M, Cohen, T, Cowherd, M, Demeyer, S, Dopazo, J, Elhard, J, Falcao, A, Ferro, A, Friedenberg, D, Giugno, R, Gong, Y, Gorospe, J, Granville, C, Grimm, D, Heinig, M, Hernansaiz, R, Hochreiter, S, Liang-Huang, C, Huska, M, Jiao, Y, Klambauer, G, Kuhn, M, Kursa, M, Kutum, R, Lazzarini, N, Lee, I, Leung, M, K W, Lim, Liu, C, F L, López, Mammana, A, Mayr, A, Michoel, T, Mongiovì, M, Moore, J, Narasimhan, R, Opiyo, S, Pandey, G, Peabody, A, Perner, J, Pulvirenti, A, Rawlik, K, Reinhardt, S, Riffle, C, Ruderfer, D, Sander, A, Savage, R, Scornet, E, Sebastian-Leon, P, Sharan, R, Simon-Gabriel, C, Stoven, V, Sun, J, Tang, H, Teixeira, A, Tenesa, A, Jean-Vert, P, Vingron, M, Walter, T, Whalen, S, Wiśniewska, Z, Wu, Y, Xu, H, Zhang, S, Zhao, J, W Zheng J, Ziwei, D, Simeonov, A, Raymond, R Tice, Saez-Rodriguez, J., NIEHS-NCATS-UNC DREAM Toxicogenetics Collaboration, Institut de Recherche pour le Développement (IRD), National Center for Advancing Translational Sciences (NCATS), National Institutes of Health [Bethesda] (NIH), Data Storage Institute - A*STAR, Capital Normal University [Beijing], Southwest University of China, sans affiliation, Shanghai Ocean University, Centre de Bioinformatique (CBIO), MINES ParisTech - École nationale supérieure des mines de Paris-PSL Research University (PSL), Cancer et génôme: Bioinformatique, biostatistiques et épidémiologie d'un système complexe, MINES ParisTech - École nationale supérieure des mines de Paris-Institut Curie-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de l'Information Géographique et Forestière [IGN] (IGN), Systèmes Productifs, Logistique, Organisation des Transports et Travail (IFSTTAR/AME/SPLOTT), Communauté Université Paris-Est-Institut Français des Sciences et Technologies des Transports, de l'Aménagement et des Réseaux (IFSTTAR), Biotechnology Center [Dresden] (BIOTEC), Technische Universität Dresden (TUD), Laboratoire National de Métrologie et d'Essais [Trappes] (LNE ), Centre for Biomedical Network Research on Rare Diseases (CIBERER), Department of Computational Genomics, Centro de Investigación Príncipe Felipe (CIPF), Functional Genomics Node (INB), CIPF, Computational Intelligence Research Group (CA3), Centre of Technology and Systems (CTS), Faculty of Sciences and Technology (FCT NOVA), Universidade Nova de Lisboa (NOVA)-Universidade Nova de Lisboa (NOVA)-Faculty of Sciences and Technology (FCT NOVA), Universidade Nova de Lisboa (NOVA)-Universidade Nova de Lisboa (NOVA)-Faculdade de Ciências e Tecnologia (FCT NOVA), Universidade Nova de Lisboa (NOVA), ACEEE, The Institute of Doctors Engineers and Scientists - IDES, Laboratoire Traitement et Communication de l'Information (LTCI), Télécom ParisTech-Institut Mines-Télécom [Paris] (IMT)-Centre National de la Recherche Scientifique (CNRS), Laboratory of Information, Network and Communication Sciences (LINCS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de Recherche en Informatique et en Automatique (Inria)-Institut Mines-Télécom [Paris] (IMT), Institute of Computational Biology, Helmholtz-Zentrum München (HZM), Stony Brook University [SUNY] (SBU), State University of New York (SUNY), University of Pensylvania, Institute for Infocomm Research - I²R [Singapore], Institut de recherche en informatique de Toulouse (IRIT), Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées, Department of Mechanical and Aerospace Engineering [Davis], University of California [Davis] (UC Davis), University of California-University of California, Commonwealth Scientific and Industrial Research Organisation [Canberra] (CSIRO), Ludwig-Maximilians-Universität München (LMU), Laboratoire Architecture, Ville, Urbanisme, Environnement (LAVUE), École nationale supérieure d'architecture de Paris-La Villette (ENSAPLV)-École nationale supérieure d'architecture de Paris Val-de-Seine (ENSA PVDS)-Université Paris 8 Vincennes-Saint-Denis (UP8)-Ministère de la Culture (MC)-Université Paris Nanterre (UPN)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Statistique Théorique et Appliquée (LSTA), Université Pierre et Marie Curie - Paris 6 (UPMC), Agricultural Technological Extensive Station of Luntai County in Xinjiang, Medstar Research Institute, Huawei Technologies [Shanghaï], School of Economics and Business Administration (School of Economics and Business Administration), Beijing Normal University, National Center for Mathematics and Interdisciplinary Sciences [Beijing] (NCMIS), Academy of Mathematics and Systems Science (AMSS), Chinese Academy of Sciences [Beijing] (CAS)-Chinese Academy of Sciences [Beijing] (CAS), Division of Biostatistics, Helmholtz-Institute for Biomedical Engineering [RWTH Aachen University], European Bioinformatics Institute [Hinxton] (EMBL-EBI), EMBL Heidelberg, Agricultural Information Institute (AII), Chinese Academy of Agricultural Sciences (CAAS), Department of Computer Science - Lafayette College, Lafayette College [Easton], MINES ParisTech - École nationale supérieure des mines de Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Cancer et génome: Bioinformatique, biostatistiques et épidémiologie d'un système complexe, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Français des Sciences et Technologies des Transports, de l'Aménagement et des Réseaux (IFSTTAR)-Communauté Université Paris-Est, Technische Universität Dresden = Dresden University of Technology (TU Dresden), Faculdade de Ciências e Tecnologia = School of Science & Technology (FCT NOVA), Universidade Nova de Lisboa = NOVA University Lisbon (NOVA)-Universidade Nova de Lisboa = NOVA University Lisbon (NOVA)-Faculdade de Ciências e Tecnologia = School of Science & Technology (FCT NOVA), Universidade Nova de Lisboa = NOVA University Lisbon (NOVA)-Universidade Nova de Lisboa = NOVA University Lisbon (NOVA), Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Paris 8 Vincennes-Saint-Denis (UP8)-École nationale supérieure d'architecture de Paris-La Villette (ENSAPLV)-Université Paris Nanterre (UPN)-École nationale supérieure d'architecture de Paris Val-de-Seine (ENSA PVDS)-Centre National de la Recherche Scientifique (CNRS)-Ministère de la Culture (MC), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Beijing Normal University (BNU), Sans affiliation, Mines Paris - PSL (École nationale supérieure des mines de Paris), Helmholtz Zentrum München = German Research Center for Environmental Health, Université Fédérale Toulouse Midi-Pyrénées-Toulouse Mind & Brain Institut (TMBI), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse III - Paul Sabatier (UT3), Department of Mechanical and Aerospace Engineering [Univ California Davis] (MAE - UC Davis), University of California (UC)-University of California (UC), Université Paris 8 Vincennes-Saint-Denis (UP8)-École nationale supérieure d'architecture de Paris-La Villette (ENSAPLV), HESAM Université - Communauté d'universités et d'établissements Hautes écoles Sorbonne Arts et métiers université (HESAM)-HESAM Université - Communauté d'universités et d'établissements Hautes écoles Sorbonne Arts et métiers université (HESAM)-Université Paris Nanterre (UPN)-École nationale supérieure d'architecture de Paris Val-de-Seine (ENSA PVDS)-Centre National de la Recherche Scientifique (CNRS)-Ministère de la Culture (MC), Centre National de la Recherche Scientifique (CNRS)-Université Paris Nanterre (UPN)-Ministère de la Culture (MC)-Université Paris 8 Vincennes-Saint-Denis (UP8)-École nationale supérieure d'architecture de Paris Val-de-Seine (ENSA PVDS)-École nationale supérieure d'architecture de Paris-La Villette (ENSAPLV), Institut Mines-Télécom [Paris] (IMT)-Institut National de Recherche en Informatique et en Automatique (Inria)-Université Pierre et Marie Curie - Paris 6 (UPMC), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Toulouse Mind & Brain Institut (TMBI), Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT), and MINES ParisTech - École nationale supérieure des mines de Paris-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Bioinformatics, MESH: Risk Assessment, Applied Microbiology and Biotechnology, MESH: Dose-Response Relationship, Drug, Genotype, MESH: Incidence, MESH: Models, Genetic, Cytotoxicity, ComputingMilieux_MISCELLANEOUS, media_common, education.field_of_study, High-throughput screening, MESH: Genetic Predisposition to Disease, drug, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], [INFO.INFO-TI]Computer Science [cs]/Image Processing [eess.IV], Trait, Molecular Medicine, Health occupations, Predictive medicine, Risk factors, Risk assessment, Biotechnology, data mining, expression, MESH: High-Throughput Screening Assays, media_common.quotation_subject, Population, Biomedical Engineering, MESH: Genetics, Population, Bioengineering, Computational biology, Biology, Competition (biology), Article, MESH: Computer Simulation, ddc:570, 1000 Genomes Project, education, MESH: Toxicity Tests, ta113, MESH: Humans, [INFO.INFO-CV]Computer Science [cs]/Computer Vision and Pattern Recognition [cs.CV], MESH: Hazardous Substances, MESH: Lymphocytes, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], RA
Abstract: The ability to computationally predict the effects of toxic compounds on humans could help address the deficiencies of current chemical safety testing. Here, we report the results from a community-based DREAM challenge to predict toxicities of environmental compounds with potential adverse health effects for human populations. We measured the cytotoxicity of 156 compounds in 884 lymphoblastoid cell lines for which genotype and transcriptional data are available as part of the Tox21 1000 Genomes Project. The challenge participants developed algorithms to predict interindividual variability of toxic response from genomic profiles and population-level cytotoxicity data from structural attributes of the compounds. 179 submitted predictions were evaluated against an experimental data set to which participants were blinded. Individual cytotoxicity predictions were better than random, with modest correlations (Pearson's r < 0.28), consistent with complex trait genomic prediction. In contrast, predictions of population-level response to different compounds were higher (r < 0.66). The results highlight the possibility of predicting health risks associated with unknown compounds, although risk estimation accuracy remains suboptimal., Nature Biotechnology, 33 (9), ISSN:1546-1696, ISSN:1087-0156
Published: 2015
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13. Benefits and Limitations of High-Resolution Cyclic IM-MS for Conformational Characterization of Native Therapeutic Monoclonal Antibodies

Author: Evolène Deslignière, Simon Ollivier, Alain Beck, David Ropartz, Hélène Rogniaux, Sarah Cianférani, Laboratoire de Spectrométrie de Masse BioOrganique [Strasbourg] (LSMBO), Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Infrastructure Nationale de Protéomique, FR2048 ProFI, Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Centre Régional de Mesures Physiques de l'Ouest (CRMPO), Université de Rennes (UR), Institut des Sciences Chimiques de Rennes (ISCR), Université de Rennes (UR)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Ecole Nationale Supérieure de Chimie de Rennes (ENSCR)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Synthèse Caractérisation Analyse de la Matière (ScanMAT), Université de Rennes (UR)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie Pierre Fabre (CIPF), PIERRE FABRE, Unité de recherche sur les Biopolymères, Interactions Assemblages (BIA), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), University of Strasbourg., and ANR-18-CE29-0006,ALGAIMS,Analyses de polysaccharides comportant des unités Galf par IRMPD et MS-mobilité ionique(2018)
Subjects: DYNAMICS, HUMAN IGG, INSIGHTS, GLYCOSYLATION, IMMUNOGLOBULIN, ION MOBILITY, [CHIM]Chemical Sciences, MOBILITY MASS-SPECTROMETRY, [SDV.IMM.IMM]Life Sciences [q-bio]/Immunology/Immunotherapy, Analytical Chemistry
Abstract: International audience; Monoclonal antibodies (mAbs) currently represent the main class of therapeutic proteins. mAbs approved by regulatory agencies are selected from IgG1, IgG2, and IgG4 subclasses, which possess different interchain disulfide connectiv-ities. Ion mobility coupled to native mass spectrometry (IM-MS) has emerged as a valuable approach to tackle the challenging characterization of mAbs' higher order structures. However, due to the limited resolution of first-generation IM-MS instruments, subtle conformational differences on large proteins have long been hard to capture. Recent technological developments have aimed at increasing available IM resolving powers and acquisition mode capabilities, namely, through the release of high-resolution IM-MS (HR-IM-MS) instruments, like cyclic IM-MS (cIM-MS). Here, we outline the advantages and drawbacks of cIM-MS for better conformational characterization of intact mAbs (similar to 150 kDa) in native conditions compared to first-generation instruments. We first assessed the extent to which multipass cIM-MS experiments could improve the separation of mAbs' conformers. These initial results evidenced some limitations of HR-IM-MS for large native biomolecules which possess rich conformational landscapes that remain challenging to decipher even with higher IM resolving powers. Conversely, for collision-induced unfolding (CIU) approaches, higher resolution proved to be particularly useful (i) to reveal new unfolding states and (ii) to enhance the separation of coexisting activated states, thus allowing one to apprehend gas-phase CIU behaviors of mAbs directly at the intact level. Altogether, this study offers a first panoramic overview of the capabilities of cIM-MS for therapeutic mAbs, paving the way for more widespread HR-IM-MS/CIU characterization of mAb-derived formats.
Published: 2023
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14. Laboratory Cross-Comparison and Ring Test Trial for Tumor BRCA Testing in a Multicenter Epithelial Ovarian Cancer Series: The BORNEO GEICO 60-0 Study

Author: Zaida Garcia-Casado, Ana Oaknin, Marta Mendiola, Gorka Alkorta-Aranburu, Jose Ramon Antunez-Lopez, Gema Moreno-Bueno, Jose Palacios, Alfonso Yubero, Raul Marquez, Alejandro Gallego, Ana Beatriz Sanchez-Heras, Jose Antonio Lopez-Guerrero, Cristina Perez-Segura, Pilar Barretina-Ginesta, Jesus Alarcon, Lydia Gaba, Antonia Marquez, Judit Matito, Juan Cueva, Isabel Palacio, Maria Iglesias, Angels Arcusa, Luisa Sanchez-Lorenzo, Eva Guerra-Alia, Ignacio Romero, Ana Vivancos, Institut Català de la Salut, [Garcia-Casado Z] Molecular Biology Department, Fundacion Instituto Valenciano de Oncologia, Valencia, Spain. [Oaknin A] Medical Oncology Department, Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain. [Mendiola M] Instituto de Investigacion Biomedica del Hospital La Paz (IdiPAZ), Madrid, Spain. Centro de Investigacion Biomedica en Red de Cáncer (CIBERONC) Instituto de Salud Carlos III, Madrid, Spain. [Alkorta-Aranburu G] CIMA LAB Diagnostics/Universidad de Navarra, Pamplona, Spain. [Antunez-Lopez JR] Molecular Biology Department, Hospital Clinico Universitario Santiago, Santiago, Spain. [Moreno-Bueno G] Centro de Investigacion Biomedica en Red de Cáncer (CIBERONC) Instituto de Salud Carlos III, Madrid, Spain. Fundacion MD Anderson, Madrid, Spain. Departamento de Bioquímica, Instituto de Investigaciones Biomedicas ‘Alberto Sols. Conexion Cancer (UAM-CSIC), Universidad Autonoma de Madrid (UAM), IdiPAZ, Madrid, Spain, Vall d'Hebron Barcelona Hospital Campus, AstraZeneca, [Garcia-Casado, Zaida] Fdn Inst Valenciano Oncol, Mol Biol Dept, Valencia 46009, Spain, [Antonio Lopez-Guerrero, Jose] Fdn Inst Valenciano Oncol, Mol Biol Dept, Valencia 46009, Spain, [Oaknin, Ana] Vall dHebron Inst Oncol, Med Oncol Dept, Barcelona 08035, Spain, [Mendiola, Marta] Inst Invest Biomed Hosp La Paz IdiPAZ, Madrid 28029, Spain, [Mendiola, Marta] Inst Salud Carlos III, Ctr Invest Biomed Red Canc CIBERONC, Madrid 28029, Spain, [Moreno-Bueno, Gema] Inst Salud Carlos III, Ctr Invest Biomed Red Canc CIBERONC, Madrid 28029, Spain, [Palacios, Jose] Inst Salud Carlos III, Ctr Invest Biomed Red Canc CIBERONC, Madrid 28029, Spain, [Alkorta-Aranburu, Gorka] Univ Navarra, CIMA LAB Diagnost, Pamplona 31008, Spain, [Ramon Antunez-Lopez, Jose] Hosp Clin Univ Santiago, Mol Biol Dept, Santiago 15706, Spain, [Moreno-Bueno, Gema] Fdn MD Anderson, Madrid 28033, Spain, [Marquez, Raul] Fdn MD Anderson, Madrid 28033, Spain, [Moreno-Bueno, Gema] Univ Autonoma Madrid UAM, Inst Invest Biomed Alberto Sols Conex Canc UAM CS, Dept Bioquim, IdiPAZ, Madrid 28029, Spain, [Palacios, Jose] Hosp Univ Ramon Y Cajal, Pathol Dept, Madrid 28034, Spain, [Palacios, Jose] Alcala Univ, Fac Med, Madrid 28801, Spain, [Palacios, Jose] Inst Ramon Y Cajal Hlth Res IRYCIS, Madrid 28034, Spain, [Yubero, Alfonso] Hosp Clin Univ Lozano Blesa, Med Oncol Dept, Zaragoza 50009, Spain, [Gallego, Alejandro] Hosp Univ La Paz, Med Oncol Dept, Madrid 28029, Spain, [Beatriz Sanchez-Heras, Ana] Hosp Gen Univ Elche, Med Oncol Dept, Elche 03203, Spain, [Antonio Lopez-Guerrero, Jose] Univ Catolica Valencia, Valencia 46001, Spain, [Antonio Lopez-Guerrero, Jose] Unidad Mixta Invest Canc IVO CIPF, Valencia 46009, Spain, [Perez-Segura, Cristina] Hosp St Pau & Santa Tecla, Med Oncol Dept, Tarragona 43003, Spain, [Barretina-Ginesta, Pilar] Inst Catala dOncol Girona, Med Oncol Dept, Girona 17007, Spain, [Alarcon, Jesus] Hosp Univ Son Espases, Med Oncol Dept, Palma De Mallorca 07120, Spain, [Gaba, Lydia] Hosp Clin Barcelona, Med Oncol Dept, Barcelona 08036, Spain, [Marquez, Antonia] Reg & Virgen Victoria Univ Hosp, Med Oncol Interctr Unit, IBIMA, Malaga 29010, Spain, [Matito, Judit] Vall dHebron Inst Oncol VHIO, Canc Genom Lab, Barcelona 08035, Spain, [Vivancos, Ana] Vall dHebron Inst Oncol VHIO, Canc Genom Lab, Barcelona 08035, Spain, [Cueva, Juan] Hosp Clin Univ Santiago, Med Oncol Dept, Santiago 15706, Spain, [Palacio, Isabel] Hosp Univ Cent Asturias, Med Oncol Dept, Oviedo 33011, Spain, [Iglesias, Maria] Hosp Univ Son LLatzer, Med Oncol Dept, Palma De Mallorca 07198, Spain, [Arcusa, Angels] Hosp Terrassa, Med Oncol Dept, Terrassa 08227, Spain, [Sanchez-Lorenzo, Luisa] Clin Univ Navarra, Med Oncol Dept, Pamplona 31008, Spain, [Guerra-Alia, Eva] Hosp Univ Ramon Y Cajal, Med Oncol Dept, Madrid 28034, Spain, [Romero, Ignacio] Inst Valenciano Oncol, Med Oncol Dept, Valencia 46009, Spain, and Astra Zeneca Farmaceutica Spain SA
Subjects: Standards, Germline, Survival, Medicine (miscellaneous), Ovaris - Càncer - Aspectes genètics, Guidelines, neoplasias::neoplasias por localización::neoplasias de las glándulas endocrinas::neoplasias ováricas [ENFERMEDADES], Sequence variants, Association, Diagnosis::Diagnostic Techniques and Procedures::Clinical Laboratory Techniques::Genetic Testing [ANALYTICAL, DIAGNOSTIC AND THERAPEUTIC TECHNIQUES, AND EQUIPMENT], Somatic mutations, Ovarian cancer, Other subheadings::Other subheadings::/genetics [Other subheadings], Chemotherapy, Joint-consensus-recommendation, Ring Test Trial, Otros calificadores::Otros calificadores::/genética [Otros calificadores], Neoplasms::Neoplasms by Site::Endocrine Gland Neoplasms::Ovarian Neoplasms [DISEASES], Cromosomes humans - Anomalies - Diagnòstic, diagnóstico::técnicas y procedimientos diagnósticos::técnicas de laboratorio clínico::pruebas genéticas [TÉCNICAS Y EQUIPOS ANALÍTICOS, DIAGNÓSTICOS Y TERAPÉUTICOS], College, BRCA mutations, BRCA testing, ovarian cancer, NGS
Abstract: Germline and tumor BRCA testing constitutes a valuable tool for clinical decision-making in the management of epithelial ovarian cancer (EOC) patients. Tissue testing is able to identify both germline (g) and somatic (s) BRCA variants, but tissue preservation methods and the widespread implementation of NGS represent pre-analytical and analytical challenges that need to be managed. This study was carried out on a multicenter prospective GEICO cohort of EOC patients with known gBRCA status in order to determine the inter-laboratory reproducibility of tissue sBRCA testing. The study consisted of two independent experimental approaches, a bilateral comparison between two reference laboratories (RLs) testing 82 formalin-paraffin-embedded (FFPE) EOC samples each, and a Ring Test Trial (RTT) with five participating clinical laboratories (CLs) evaluating the performance of tissue BRCA testing in a total of nine samples. Importantly, labs employed their own locally adopted next-generation sequencing (NGS) analytical approach. BRCA mutation frequency in the RL sub-study cohort was 23.17%: 12 (63.1%) germline and 6 (31.6%) somatic. Concordance between the two RLs with respect to BRCA status was 84.2% (gBRCA 100%). The RTT study distributed a total of nine samples (three commercial synthetic human FFPE references, three FFPE, and three OC DNA) among five CLs. The median concordance detection rate among them was 64.7% (range: 35.3–70.6%). Analytical discrepancies were mainly due to the minimum variant allele frequency thresholds, bioinformatic pipeline filters, and downstream variant interpretation, some of them with consequences of clinical relevance. Our study demonstrates a wide range of concordance in the identification and interpretation of BRCA sequencing data, highlighting the relevance of establishing standard criteria for detecting, interpreting, and reporting BRCA variants., This research was funded by Astra Zéneca Farmacéutica Spain SA (Grant Number GEICO60-0).
Published: 2022

15. High-Resolution IMS–MS to Assign Additional Disulfide Bridge Pairing in Complementarity-Determining Regions of an IgG4 Monoclonal Antibody

Author: Elsa Wagner-Rousset, Olivier Colas, Kevin Giles, Sarah Cianférani, Thomas Botzanowski, Evolène Deslignière, Oscar Hernandez-Alba, Alain Beck, Dale Cooper-Shepherd, Hélène Diemer, Guillaume Béchade, Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Infrastructure Nationale de Protéomique, FR2048 ProFI, Waters Corporation, Centre d'Immunologie Pierre Fabre (CIPF), and PIERRE FABRE
Subjects: medicine.drug_class, Complementarity determining region, Monoclonal antibody, Immunoglobulin light chain, 01 natural sciences, Mass Spectrometry, 03 medical and health sciences, chemistry.chemical_compound, Structural Biology, Ion Mobility Spectrometry, medicine, Humans, [CHIM]Chemical Sciences, Disulfides, Spectroscopy, 030304 developmental biology, 0303 health sciences, Dipeptide, biology, Chemistry, Immunogenicity, 010401 analytical chemistry, Antibodies, Monoclonal, Complementarity Determining Regions, 0104 chemical sciences, Biochemistry, Immunoglobulin G, biology.protein, Antibody, Critical quality attributes, Cysteine
Abstract: International audience; Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases, including cancers and immunological disorders. Disulfide bonds play a pivotal role in therapeutic antibody structure and activity relationships. Disulfide connectivity and cysteine-related variants are considered as critical quality attributes (CQAs) that must be monitored during mAb manufacturing and storage, as non-native disulfide bridges and aggregates might be responsible for loss of biological function and immunogenicity. The presence of cysteine residues in the Complementarity-Determining Regions (CDRs) is rare in human antibodies but may be critical for the antigen-binding or deleterious for therapeutic antibody development. Consequently, in-depth characterization of their disulfide network is a prerequisite for mAb developability assessment. Mass spectrometry (MS) techniques represent powerful tools for accurate identification of disulfide connectivity. We report here on the MS-based characterization of an IgG4 comprising two additional cysteine residues in the CDR of its light chain. Classical bottom-up approaches after trypsin digestion first allowed identification of a dipeptide containing two disulfide bridges. To further investigate the conformational heterogeneity of the disulfide-bridged dipeptide, we performed ion mobility spectrometry-mass spectrometry (IMS-MS) experiments. Our results highlight benefits of high resolution IMS-MS to tackle the conformational landscape of disulfide peptides generated after trypsin digestion of a humanized IgG4 mAb under development. By comparing arrival time distributions of the mAb collected peptide and synthetic peptides, cyclic IMS afforded unambiguous assessment of disulfide bonds. In addition to classical peptide mapping, qualitative high-resolution IMS-MS can be of great interest to identify disulfide bonds within therapeutic mAbs.
Published: 2021
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16. Genome-scale mechanistic modeling of signaling pathways made easy: A bioconductor/cytoscape/web server framework for the analysis of omic data

Author: Kinza Rian, Marta R. Hidalgo, Cankut Çubuk, Matias M. Falco, Joaquín Dopazo, Carlos Loucera, Maria Peña-Chilet, Inmaculada Alamo-Alvarez, Marina Esteban-Medina, Ministerio de Economía y Competitividad (España), European Commission, [Rian,K, Çubuk,C, Falco,MM, Loucera,C, Esteban-Medina,M, Alamo-Alvarez,I, Peña-Chilet,M, Dopazo,J] Clinical Bioinformatics Area, Fundación Progreso y Salud (FPS), Hospital Virgen del Rocío, Sevilla, Spain. [Rian,K] Laboratory of Innovative Technologies (LTI), National School of Applied Sciences in Tangier, UAE, Morocco. [Hidalgo,MR] Bioinformatics and Biostatistics Unit, Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain. [Falco,MM, Dopazo,J] Bioinformatics in RareDiseases (BiER), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Sevilla, Spain. [Loucera,C, Dopazo,J] Computational Systems Medicine. Institute of Biomedicine of Seville (IBiS), Sevilla, Spain. [Dopazo,J] Functional Genomics Node (INB-ELIXIR-es), Sevilla, Spain., and This work is supported by grants SAF2017-88908-R from the Spanish Ministry of Economy and Competitiveness and PT17/0009/0006 and PI20/01305 from the ISCIII, both co-funded with European Regional Development Funds (ERDF) as well as H2020 Programme of the European Union grants Marie Curie Inno vative Training Network ‘‘Machine Learning Frontiers in Precision Medicine' (MLFPM) (GA 813533) and ‘‘ELIXIR-EXCELERATE fast track ELIXIR implementation and drive early user exploitation across the life sciences' (GA 676559) to JD. Funding for open access charge: from the Spanish Ministry of Economy and Competitive ness / SAF2017-88908-R
Subjects: Web server, Phenomena and Processes::Genetic Phenomena::Phenotype [Medical Subject Headings], Phenomena and Processes::Genetic Phenomena::Genetic Variation::Mutation [Medical Subject Headings], Computer science, Genomic data, Phenomena and Processes::Genetic Phenomena::Genetic Processes::Gene Expression::Transcription, Genetic::Transcriptome [Medical Subject Headings], Biophysics, Genome scale, Genomics, Computational biology, computer.software_genre, Biochemistry, Web tool, Bioconductor, 03 medical and health sciences, 0302 clinical medicine, Structural Biology, Genetics, Plug-in, 030304 developmental biology, Sistema de señalización, 0303 health sciences, Mutación, Mathematical modelling, Signaling pathway, Potential effect, Phenomena and Processes::Chemical Phenomena::Biochemical Phenomena::Biochemical Processes::Signal Transduction [Medical Subject Headings], Method Article, Computer Science Applications, Causalidad, Causality, Transcriptomic, 030220 oncology & carcinogenesis, Disciplines and Occupations::Natural Science Disciplines::Biological Science Disciplines::Biology::Genetics::Genomics [Medical Subject Headings], Phenomena and Processes::Genetic Phenomena::Genotype [Medical Subject Headings], computer, Mutations, TP248.13-248.65, Biotechnology
Abstract: Genome-scale mechanistic models of pathways are gaining importance for genomic data interpretation because they provide a natural link between genotype measurements (transcriptomics or genomics data) and the phenotype of the cell (its functional behavior). Moreover, mechanistic models can be used to predict the potential effect of interventions, including drug inhibitions. Here, we present the implementation of a mechanistic model of cell signaling for the interpretation of transcriptomic data as an R/Bioconductor package, a Cytoscape plugin and a web tool with enhanced functionality which includes building interpretable predictors, estimation of the effect of perturbations and assessment of the effect of mutations in complex scenarios., This work is supported by grants SAF2017-88908-R from the Spanish Ministry of Economy and Competitiveness and PT17/0009/0006 and PI20/01305 from the ISCIII, both co-funded with European Regional Development Funds (ERDF) as well as H2020 Programme of the European Union grants Marie Curie Innovative Training Network “Machine Learning Frontiers in Precision Medicine” (MLFPM) (GA 813533) and “ELIXIR-EXCELERATE fast-track ELIXIR implementation and drive early user exploitation across the life sciences” (GA 676559) to JD. Funding for open access charge: from the Spanish Ministry of Economy and Competitiveness / SAF2017-88908-R.
Published: 2021

17. Developmental Disruption of Erbb4 in Pet1+ Neurons Impairs Serotonergic Sub-System Connectivity and Memory Formation

Author: Candela Barettino, Álvaro Ballesteros-Gonzalez, Andrés Aylón, Xavier Soler-Sanchis, Leticia Ortí, Selene Díaz, Isabel Reillo, Francisco García-García, Francisco José Iborra, Cary Lai, Nathalie Dehorter, Xavier Leinekugel, Nuria Flames, Isabel Del Pino, Centro de Investigación Principe Felipe, Institut de Biologie Valrose (IBV), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Centro de Investigación Príncipe Felipe (CIPF), Indiana University [Bloomington], Indiana University System, Institut de Neurobiologie de la Méditerranée [Aix-Marseille Université] (INMED - INSERM U1249), Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Generalitat Valenciana, Ministerio de Ciencia, Innovación y Universidades (España), European Research Council, Flames, Nuria, pellegrino, Christophe, and Flames, Nuria [0000-0003-0961-0609]
Subjects: Serotonin, 0303 health sciences, Neuromodulation, QH301-705.5, neurodevelopmental disorders, Neurodevelopmental disorders, Cell Biology, serotonin, memory, Cell and Developmental Biology, 03 medical and health sciences, ErbB4, 0302 clinical medicine, Memory, neuromodulation, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Biology (General), 030217 neurology & neurosurgery, NRG, Original Research, 030304 developmental biology, Developmental Biology
Abstract: 17 páginas, 6 figuras, 1 tabla. Material suplementario de este artículo online en: https://www.frontiersin.org/articles/10.3389/fcell.2021.770458/full#supplementary-material, The serotonergic system of mammals innervates virtually all the central nervous system and regulates a broad spectrum of behavioral and physiological functions. In mammals, serotonergic neurons located in the rostral raphe nuclei encompass diverse sub-systems characterized by specific circuitry and functional features. Substantial evidence suggest that functional diversity of serotonergic circuits has a molecular and connectivity basis. However, the landscape of intrinsic developmental mechanisms guiding the formation of serotonergic sub-systems is unclear. Here, we employed developmental disruption of gene expression specific to serotonergic subsets to probe the contribution of the tyrosine kinase receptor ErbB4 to serotonergic circuit formation and function. Through an in vivo loss-of-function approach, we found that ErbB4 expression occurring in a subset of serotonergic neurons, is necessary for axonal arborization of defined long-range projections to the forebrain but is dispensable for the innervation of other targets of the serotonergic system. We also found that Erbb4-deletion does not change the global excitability or the number of neurons with serotonin content in the dorsal raphe nuclei. In addition, ErbB4-deficiency in serotonergic neurons leads to specific behavioral deficits in memory processing that involve aversive or social components. Altogether, our work unveils a developmental mechanism intrinsically acting through ErbB4 in subsets of serotonergic neurons to orchestrate a precise long-range circuit and ultimately involved in the formation of emotional and social memories., This work was supported by the CIDEGenT excellence research program of the Valencian government (CIDEGENT/2019/044) and by the Spanish Ministry of Science, Innovation and Universities (RTI 2018-100872-J-I00) as well as by the Young IBRO Regions Connecting Award to IDP and the ERC Consolidator Grant 101002203 to NF. Part of the equipment employed in this work has been funded by Generalitat Valenciana and co-financed with ERDF funds (OP ERDF of Comunitat Valenciana 2014-2020).
Published: 2021
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18. The compass to follow: Focal adhesion turnover

Author: Manos Mavrakis, M. Angeles Juanes, MOSAIC (MOSAIC), Institut FRESNEL (FRESNEL), Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Centre National de la Recherche Scientifique (CNRS), Centro de Investigación Príncipe Felipe (CIPF), Teesside University, Middlesbrough TS1 3BX, United Kingdom, CIDEGENT Excellent Research Program of the Valencian regional government CIDEGENT/2021/026, and Academy of Medical Science/the Wellcome Trust/ the Government Department of Business, Energy and Industrial Strategy/the British Heart Foundation/Diabetes UK Springboard Award [SBF006\1070]
Subjects: [SDV]Life Sciences [q-bio], Cell Biology
Abstract: International audience; How cells move is a fundamental biological question. The directionality of adherent migrating cells depends on the assembly and disassembly (turnover) of focal adhesions (FAs). FAs are micron-sized actin-based structures that link cells to the extracellular matrix. Traditionally, microtubules have been considered key to triggering FA turnover. Through the years, advancements in biochemistry, biophysics, and bioimaging tools have been invaluable for many research groups to unravel a variety of mechanisms and molecular players that contribute to FA turnover, beyond microtubules. Here we discuss recent discoveries of key molecular players that affect the dynamics and organization of the actin cytoskeleton to enable timely FA turnover and consequently proper directed cell migration.
Published: 2023
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19. Transplantation of Human-Fetal-Spinal-Cord-Derived NPCs Primed with a Polyglutamate-Conjugated Rho/Rock Inhibitor in Acute Spinal Cord Injury

Author: Esther Giraldo, Pablo Bonilla, Mara Mellado, Pablo Garcia-Manau, Carlota Rodo, Ana Alastrue, Eric Lopez, Elena Carreras Moratonas, Ferran Pellise, Snežana Đorđević, María J. Vicent, Victoria Moreno Manzano, Institut Català de la Salut, [Giraldo E] Neuronal and Tissue Regeneration Laboratory, Centro de Investigación Príncipe Felipe, Valencia, Spain. Department of Biotechnology. Universitat Politècnica de València, Valencia, Spain. UPV-CIPF Joint Research Unit Disease Mechanisms and Nanomedicine, Centro de Investigación Príncipe Felipe, Valencia, Spain. [Bonilla P, Mellado M, Alastrue A] Neuronal and Tissue Regeneration Laboratory, Centro de Investigación Príncipe Felipe, Valencia, Spain. [Garcia-Manau P, Rodo C, Carreras Moratonas E] Unitat de Medicina i Cirurgia Fetal, Vall d’Hebron Hospital Universitari, Barcelona, Spain. [Pellise F] Unitat de Lesionats Medul·lars, Vall d’Hebron Hospital Universitari, Barcelona, Spain, and Vall d'Hebron Barcelona Hospital Campus
Subjects: Neurons, Prostaglandins A, rho-Associated Kinases, Cell Transplantation, Teràpia cel·lular, Therapeutics::Biological Therapy::Cell- and Tissue-Based Therapy::Cell Transplantation [ANALYTICAL, DIAGNOSTIC AND THERAPEUTIC TECHNIQUES, AND EQUIPMENT], Other subheadings::/therapy [Other subheadings], General Medicine, Medul·la espinal - Ferides i lesions - Tractament, Rats, Nervous System Diseases::Central Nervous System Diseases::Spinal Cord Diseases::Spinal Cord Injuries [DISEASES], Mice, terapéutica::terapia biológica::tratamientos basados en células y tejidos::trasplante de células [TÉCNICAS Y EQUIPOS ANALÍTICOS, DIAGNÓSTICOS Y TERAPÉUTICOS], human fetal neural precursor, NPC transplantation, Rho/ROCK kinase inhibition, cell priming, spinal cord injury, Polyglutamic Acid, Animals, Humans, enfermedades del sistema nervioso::enfermedades del sistema nervioso central::enfermedades de la médula espinal::traumatismos de la médula espinal [ENFERMEDADES], Spinal Cord Injuries, Otros calificadores::/terapia [Otros calificadores]
Abstract: NPC transplantation; Cell priming; Human fetal neural precursor Trasplante de NPC; Cebado celular; Precursor neuronal fetal humano Trasplantament de NPC; Cebament cel·lular; Precursor neuronal fetal humà Neural precursor cell (NPC) transplantation represents a promising therapy for treating spinal cord injuries (SCIs); however, despite successful results obtained in preclinical models, the clinical translation of this approach remains challenging due, in part, to the lack of consensus on an optimal cell source for human neuronal cells. Depending on the cell source, additional limitations to NPC-based therapies include high tumorigenic potential, alongside poor graft survival and engraftment into host spinal tissue. We previously demonstrated that NPCs derived from rat fetal spinal cords primed with a polyglutamate (PGA)-conjugated form of the Rho/Rock inhibitor fasudil (PGA-SS-FAS) displayed enhanced neuronal differentiation and graft survival when compared to non-primed NPCs. We now conducted a similar study of human-fetal-spinal-cord-derived NPCs (hfNPCs) from legal gestational interruptions at the late gestational stage, at 19–21.6 weeks. In vitro, expanded hfNPCs retained neural features, multipotency, and self-renewal, which supported the development of a cell banking strategy. Before transplantation, we established a simple procedure to prime hfNPCs by overnight incubation with PGA-SS-FAS (at 50 μM FAS equiv.), which improved neuronal differentiation and overcame neurite-like retraction after lysophosphatidic-acid-induced Rho/Rock activation. The transplantation of primed hfNPCs into immune-deficient mice (NU(NCr)-Foxn1nu) immediately after the eighth thoracic segment compression prompted enhanced migration of grafted cells from the dorsal to the ventral spinal cord, increased preservation of GABAergic inhibitory Lbx1-expressing and glutamatergic excitatory Tlx3-expressing somatosensory interneurons, and elevated the numbers of preserved, c-Fos-expressing, activated neurons surrounding the injury epicenter, all in a low percentage. Overall, the priming procedure using PGA-SS-FAS could represent an alternative methodology to improve the capabilities of the hfNPC lines for a translational approach for acute SCI treatment. This research was funded by Fundació Marató TV3 2017/refs.20172230, 20172231, Agencia Valenciana de Innovación (AVI) (INNVAL10/19/047 and Grants RTI2018-095872-B-C21 and PDI2021-1243590B-I00/ERDF funded by MCIN/AEI//10.13039/501100011033 and by ERDF A way of making Europe). This project was also funded by Project 964562 (RISEUP), H2020 FetOpen program.
Published: 2022
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20. State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

Author: Jonathan Sjögren, Alain Beck, Hanna Toftevall, Oscar Hernandez-Alba, Sarah Cianférani, Bastiaan L. Duivelshof, Evolène Deslignière, Valentina D'Atri, Anthony Ehkirch, Davy Guillarme, Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), University of Geneva [Switzerland], Genovis AB, Centre d'Immunologie Pierre Fabre (CIPF), and PIERRE FABRE
Subjects: 0301 basic medicine, Glycan, medicine.drug_class, native mass spectrometry, Size-exclusion chromatography, Pharmaceutical Science, Monoclonal antibody, 01 natural sciences, Article, 03 medical and health sciences, sitespecific conjugation, Pharmacy and materia medica, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Drug Discovery, medicine, ddc:615, Bioconjugation, biology, Chemistry, 010401 analytical chemistry, site-specific conjugation, [SDV.SP]Life Sciences [q-bio]/Pharmaceutical sciences, Combinatorial chemistry, 0104 chemical sciences, body regions, RS1-441, 030104 developmental biology, Forced degradation, antibody-drug conjugate (ADC), Click chemistry, biology.protein, Molecular Medicine, collision-induced unfolding (CIU), Medicine, size-exclusion chromatography (SEC), ion mobility-mass spectrometry (IM-MS), Linker, Conjugate
Abstract: International audience; Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratio (DAR). Sitespecific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs. We report here the characterization of a sitespecific DAR2 ADC generated with the GlyCLICK three-step process, which involves glycan-based enzymatic remodeling and click chemistry, using state-of-the-art native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS), which offered a straightforward identification and quantification of all reaction products, providing a direct snapshot of the ADC homogeneity. Benefits of SEC-nMS were further demonstrated for forced degradation studies, for which fragments generated upon thermal stress were clearly identified, with no deconjugation of the drug-linker observed for the T-GlyGLICK-DM1 ADC. Lastly, innovative ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior of compounds along the conjugation process, highlighting an increased resistance of the mAb against gas-phase unfolding upon drug conjugation. Altogether, these state-of-the-art nMS methods represent innovative approaches to investigate drug loading and distribution of last generation ADCs, their evolution during the bioconjugation process and their impact on solution and gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation empowered mAbderived products such as engineered nanobodies, bispecific ADCs or immunocytokines.
Published: 2021
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21. CSVS, a crowdsourcing database of the Spanish population genetic variability

Author: Peña-Chilet, María, Roldán Gema, Perez-Florido, Javier, Ortuño, Francisco M., Carmona, Rosario, Aquino, Virginia, Lopez-Lopez, Daniel, Loucera, Carlos, Fernandez-Rueda, Jose L., Gallego, Asunción, García-García, Francisco, González-Neira, Anna, Pita, Guillermo, Núñez-Torres, Rocío, Santoyo-López, Javier, Ayuso, Carmen, Minguez, Pablo, Avila-Fernandez, Almudena, Corton, Marta, Moreno-Pelayo, Miguel Ángel, Morin, Matías, Gallego-Martinez, Alvaro, Lopez-Escamez, Jose A., Borrego, Salud, Antiñolo, Guillermo, Amigo, Jorge, Salgado-Garrido, Josefa, Pasalodos-Sanchez, Sara, Morte, Beatriz, The Spanish Exome Crowdsourcing Consortium, Carracedo Álvarez, Ángel, Alonso, Ángel, Dopazo, Joaquín, Grinberg Vaisman, Daniel Raúl, [Peña-Chilet,M, Roldán,G, Perez-Florido,J, Ortuño,FM, Carmona,R, Aquino,V, Lopez-Lopez,D, Loucera,C, Fernandez-Rueda,JL, Dopazo,J] Clinical Bioinformatics Area, Fundacion Progreso y Salud (FPS), Hospital Virgen del Rocío, Sevilla, Spain. [Peña-Chilet,M, Dopazo,J] Bioinformatics in Rare Diseases (BiER), Center for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Sevilla, Spain. [Peña-Chilet,M, Dopazo,J] Computational Systems Medicine group, Institute of Biomedicine of Seville (IBIS) Hospital Virgen del Rocío, Sevilla, Spain. [Perez-Florido,J, Dopazo,J] Functional Genomics Node, FPS/ELIXIR-ES, Hospital Virgen del Rocío, Sevilla, Spain. [Gallego,A] Sistemas Genomicos, Paterna, Valencia, Spain. [García-Garcia,F] Unidad de Bioinformatica y Bioestadística, Centro de Investigacion Príncipe Felipe (CIPF), Valencia, Spain. [González-Neira,A, Pita,G, Núñez-Torres,R] Human Genotyping Unit–Centro Nacional de Genotipado (CEGEN), Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain. [Santoyo-López,J] Edinburgh Genomics, The University of Edinburgh, Edinburgh, UK. [Ayuso,C, Minguez,P, Avila-Fernandez,A, Corton,M] Department of Genetics, Instituto de Investigacion Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autonoma de Madrid (IIS-FJD, UAM), Madrid, Spain. [Minguez,P] Center for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Madrid, Spain. [Moreno-Pelayo,MÁ, Morin,M] Servicio de Genetica, Ramón y Cajal Institute of Health Research (IRYCIS) and Biomedical Network Research Centre on Rare Diseases (CIBERER), Madrid, Spain, [Gallego-Martinez,A, Lopez-Escamez,JA] Otology & Neurotology Group CTS 495, Department of Genomic Medicine, Centre for Genomics and Oncological Research (GENYO), Pfizer University of Granada, Granada, Spain. [Gallego-Martinez,A, Lopez-Escamez,JA] Department of Otolaryngology, Instituto de Investigacion Biosanitaria, IBS. GRANADA, Hospital Universitario Virgen de las Nieves, Universidad de Granada, Granada, Spain. [Borrego,S, Antiñolo,G] Department of Maternofetal Medicine, Genetics and Reproduction, Institute of Biomedicine of Seville (IBIS), University Hospital Virgen del Rocío /CSIC/University of Seville, Seville, Spain. [Borrego,S, Antiñolo,G] Centre for Biomedical Network Research on Rare Diseases (CIBERER), Seville, Spain. [Amigo,J, Carracedo,Á] Fundacion Pública Galega de Medicina Xenómica, SERGAS, IDIS, Santiago de Compostela, Spain. [Salgado-Garrido,J, Pasalodos-Sanchez,S, Alonso,Á] Navarrabiomed-IdiSNA, Complejo Hospitalario de Navarra, Universidad Publica de Navarra (UPNA), IdiSNA (Navarra Institute for Health Research), Pamplona, Navarra, Spain. [Morte,B] Undiagnosed Rare Diseases Programme (ENoD). Center for Biomedical Research on Rare Diseases (CIBERER), ISCIII, Madrid, Spain. [Carracedo,Á] Grupo de Medicina Xenomica, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), CIMUS, Universidade de Santiago de Compostela, Santiago de Compostela, España., Spanish Ministry of Economy and Competitiveness [SAF2017-88908-R, PT17/0009/0006 to J.D., PI19/00321 and CIBERER ACCI-06/07/0036 to C.A., PI14-948, PI17-1659 and CIBERER ACCI-06/07/0036 to M.A.M.P.], Regional Government of Madrid, RAREGenomics CM [B2017/BMD-3721 to C.A. and B2017/BMD3721 to M.A.M.P.], all co-funded with European Regional Development Funds (ERDF) as well as EU H2020-INFRADEV-1-2015-1 ELIXIR-EXCELERATE [676559], University Chair UAM-IIS-FJD of Genomic Medicine and the Ramon Areces Foundation also supported this work. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [SAF2017-88908-R]., Ministerio de Economía, Industria y Competitividad (España), Comunidad de Madrid, European Commission, Fundación Ramón Areces, Ministerio de Economía y Competitividad (España), Centro de Investigación Biomédica en Red Enfermedades Raras (España), and Universidad Autónoma de Madrid
Subjects: Genetics, population, Població, AcademicSubjects/SCI00010, computer.software_genre, Bases de dades, Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Primates::Haplorhini::Catarrhini::Hominidae::Humans [Medical Subject Headings], 0302 clinical medicine, Gene Frequency, Genética de poblaciones, Databases, Genetic, Database Issue, Exome, Precision Medicine, 0303 health sciences, education.field_of_study, Database, Disciplines and Occupations::Natural Science Disciplines::Biological Science Disciplines::Biology::Genetics::Genetics, Population [Medical Subject Headings], Chromosome Mapping, Genomics, Bases de datos genéticas, Phenomena and Processes::Genetic Phenomena::Genetic Structures::Genome::Genome, Human [Medical Subject Headings], 3. Good health, Databases, genetic, Information Science::Information Science::Data Collection::Crowdsourcing [Medical Subject Headings], Crowdsourcing, Disciplines and Occupations::Natural Science Disciplines::Biological Science Disciplines::Biology::Genetics::Genomics [Medical Subject Headings], Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Chromosome Mapping::Physical Chromosome Mapping [Medical Subject Headings], Phenomena and Processes::Genetic Phenomena::Genetic Variation [Medical Subject Headings], Participación colectiva, Population, Proveïment participatiu, Biology, Variación genética, Phenomena and Processes::Genetic Phenomena::Genetic Structures::Genome::Exome [Medical Subject Headings], Genètica de poblacions humanes, 03 medical and health sciences, Databases, Information Science::Information Science::Information Storage and Retrieval::Databases as Topic::Databases, Factual::Databases, Genetic [Medical Subject Headings], Genetic variation, Genetics, Humans, Genetic variability, Espanya, education, Allele frequency, Alleles, Phenomena and Processes::Genetic Phenomena::Genetic Structures::Genome::Genome Components::Genes::Alleles [Medical Subject Headings], 030304 developmental biology, Geographical Locations::Geographic Locations::Europe::Spain [Medical Subject Headings], Internet, Genoma humà -- Espanya, business.industry, Genome, Human, Genetic Variation, Human population genetics, Gene frequency, Frecuencia génica, Genetics, Population, Spain, Personalized medicine, business, Phenomena and Processes::Genetic Phenomena::Gene Frequency [Medical Subject Headings], computer, 030217 neurology & neurosurgery, Imputation (genetics), Software
Abstract: The knowledge of the genetic variability of the local population is of utmost importance in personalized medicine and has been revealed as a critical factor for the discovery of new disease variants. Here, we present the Collaborative Spanish Variability Server (CSVS), which currently contains more than 2000 genomes and exomes of unrelated Spanish individuals. This database has been generated in a collaborative crowdsourcing effort collecting sequencing data produced by local genomic projects and for other purposes. Sequences have been grouped by ICD10 upper categories. A web interface allows querying the database removing one or more ICD10 categories. In this way, aggregated counts of allele frequencies of the pseudo-control Spanish population can be obtained for diseases belonging to the category removed. Interestingly, in addition to pseudo-control studies, some population studies can be made, as, for example, prevalence of pharmacogenomic variants, etc. In addition, this genomic data has been used to define the first Spanish Genome Reference Panel (SGRP1.0) for imputation. This is the first local repository of variability entirely produced by a crowdsourcing effort and constitutes an example for future initiatives to characterize local variabilityworldwide. CSVS is also part of the GA4GH Beacon network., Spanish Ministry of Economy and Competitiveness SAF2017-88908-R PT17/0009/0006 PI19/00321 CIBERER ACCI-06/07/0036 PI14-948 PI171659, Regional Government of Madrid, RAREGenomicsCM B2017/BMD3721 B2017/BMD-3721, European Union (EU), European Union (EU) 676559, University Chair UAM-IIS-FJD of Genomic Medicine, Ramon Areces Foundation
Published: 2020
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22. Hyphenation of size exclusion chromatography to native ion mobility mass spectrometry for the analytical characterization of therapeutic antibodies and related products

Author: Sarah Cianférani, Oscar Hernandez-Alba, Anthony Ehkirch, Alain Beck, Olivier Colas, Davy Guillarme, Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie Pierre Fabre (CIPF), PIERRE FABRE, School of Pharmaceutical Science, and Université de Lausanne (UNIL)-University of Geneva [Switzerland]
Subjects: Monoclonal antibody, 0301 basic medicine, Aggregates, native mass spectrometry, medicine.drug_class, Ion-mobility spectrometry, Ion mobility, Clinical Biochemistry, Size-exclusion chromatography, High resolution, Mass spectrometry, 01 natural sciences, Biochemistry, Mass Spectrometry, antibody-drug conjugate, Analytical Chemistry, Protein Aggregates, 03 medical and health sciences, ion mobility, Native mass spectrometry, Size exclusion chromatography, medicine, [CHIM]Chemical Sciences, Sample preparation, Antibody-drug conjugate, ddc:615, Chromatography, Chemistry, 010401 analytical chemistry, Antibodies, Monoclonal, Cell Biology, General Medicine, 0104 chemical sciences, Characterization (materials science), 030104 developmental biology, monoclonal antibody, aggregates, Chromatography, Gel, Mass spectrum
Abstract: International audience; Mass spectrometry performed in non-denaturing conditions (native MS), and its hyphenation to ion mobility spectrometry (IM-MS), have gained interest for the qualitative and quantitative characterization of intact monoclonal antibody-related (mAb) products. However, one main drawback is the manual sample preparation, which hampers its routine use in high throughput automated environments. Size exclusion chromatography (SEC) appears as an interesting technique to perform online buffer exchange in an automated way. We present here an exhaustive and systematic evaluation of the possibilities and versatility of SEC direct hyphenation to native MS or IM-MS (SEC-nativeMS/IM-MS) for the characterization of a variety of mAb-formats (IgGs, ADCs, bispecific mAbs and Fc-fusion proteins). First, online SEC-native MS allows automated sample preparation, resulting in high resolution mass spectra and improved mass accuracies (
Published: 2018
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23. Glycan-Mediated Technology for Obtaining Homogeneous Site-Specific Conjugated Antibody–Drug Conjugates: Synthesis and Analytical Characterization by Using Complementary Middle-up LC/HRMS Analysis

Author: Davy Guillarme, Valentina D'Atri, Sarah Cianférani, Evolène Deslignière, Alain Beck, Jonathan Sjögren, Bastiaan L. Duivelshof, Oscar Hernandez-Alba, Anthony Ehkirch, Hanna Toftevall, School of Pharmaceutical Science, Université de Lausanne (UNIL)-University of Geneva [Switzerland], Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Genovis AB, Centre d'Immunologie Pierre Fabre (CIPF), and PIERRE FABRE
Subjects: ddc:615, Glycan, Bioconjugation, Chromatography, Immunoconjugates, biology, Chemistry, Hydrophilic interaction chromatography, 010401 analytical chemistry, Molecular Conformation, Antibodies, Monoclonal, Reversed-phase chromatography, Conjugated system, 010402 general chemistry, Mass spectrometry, 01 natural sciences, Mass Spectrometry, 0104 chemical sciences, Analytical Chemistry, Polysaccharides, biology.protein, [CHIM]Chemical Sciences, Critical quality attributes, Conjugate, Chromatography, Liquid
Abstract: International audience; Conventional antibody–drug conjugate (ADC) manufacturing methods are based on the nonselective bioconjugation of cytotoxic drugs to lysine and cysteine residues. This results in highly heterogeneous mixtures of different drug–antibody ratios (DAR) that can significantly affect the safety and efficacy of the ADC product. Recently, an innovative procedure named GlyCLICK was suggested, consisting of a two-step enzymatic procedure to transform Fc-glycans present on IgG mAbs into two site-specific anchor points for the conjugation of any alkyne-containing payload of choice. Here, we evaluated the conjugation process by comparing trastuzumab and trastuzumab conjugated with DM1, following the GlyCLICK procedure. Complementary reversed phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) coupled to high-resolution mass spectrometry (HRMS) were used to analyze the protein subunits (ca. 25–100 kDa) obtained after different levels of enzymatic digestion and chemical reduction. Our results demonstrated that the hydrophobic character of the drug molecule allows to rapidly confirm the Fc-drug conjugation at the chromatographic level. Furthermore, the hyphenation to MS detection provided accurate mass information on the ADC subunits and facilitated the DAR determination of 2.0. Therefore, this work illustrates how middle-up analysis using LC/HRMS can provide accurate and complementary information on the critical quality attributes of these novel site-specific ADC products.
Published: 2020
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24. Drug Loading and Distribution of ADCs After Reduction or IdeS Digestion and Reduction

Author: Alain Beck, Elsa Wagner-Rousset, Olivier Colas, Yannis-Nicolas François, Sarah Cianférani, Sabine Heinisch, Davy Guillarme, Centre d'Immunologie Pierre Fabre (CIPF), PIERRE FABRE, Chimie de la matière complexe (CMC), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), School of Pharmaceutical Science, University of Geneva [Switzerland]-Université de Lausanne (UNIL), Institut Pluridisciplinaire Hubert Curien (IPHC), Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), and Centre d'Immunologie Pierre Fabre
Subjects: Mass spectrometry, 01 natural sciences, Reduction (complexity), 03 medical and health sciences, chemistry.chemical_compound, [CHIM]Chemical Sciences, Distribution (pharmacology), HIC, 030304 developmental biology, 0303 health sciences, ddc:615, Fabricator, Chromatography, Hydrophilic interaction chromatography, 010401 analytical chemistry, IdeS, DAR, 0104 chemical sciences, Monomer, chemistry, Covalent bond, Yield (chemistry), DLD, Brentuximab vedotin, Cysteine
Abstract: International audience; High-resolution native mass spectrometry (MS) provides accurate mass measurements (within 30 ppm) of intact ADCs and can also yield drug load distribution (DLD) and average drug to antibody ratio (DAR) in parallel with hydrophobic interaction chromatography (HIC). Native MS is furthermore unique in its ability to simultaneously detect covalent and noncovalent species in a mixture and for HIC peak identity assessment offline or online.As an orthogonal method described in this chapter, LC-MS following ADC reduction or IdeS (Fabricator) digestion and reduction can also be used to measure the DLD of light chain and Fd fragments for hinge native cysteine residues such as brentuximab vedotin. Both methods allow also the measurement of average DAR for both monomeric and multimeric species. In addition, the Fc fragments can be analyzed in the same run, providing a complete glycoprofile and the demonstration or absence of additional conjugation of this subdomain involved in FcRn and Fc-gammaR binding.
Published: 2020
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25. Middle level IM-MS and CIU experiments for improved therapeutic immunoglobulin subclass fingerprinting ACS Paragon Plus Environment Analytical Chemistry

Author: Botzanowski, T., Hernandez-Alba, O., Malissard, M., Wagner-Rousset, E., Desligniere, E., Colas, O., Haeuw J., F., Beck, A., Cianferani, S., Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie Pierre Fabre (CIPF), and PIERRE FABRE
Subjects: [CHIM]Chemical Sciences, ComputingMilieux_MISCELLANEOUS
Abstract: International audience
Published: 2020
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26. Differential modulation of Quorum-Sensing signaling through QslA in Pseudomonas

Author: Sana, Thibault Gery, Lomas, Rodrigo, Gimenez, Maxime Rémi, Laubier, Aurélie, Soscia, Chantal, Chauvet, Claire, Conesa, Ana, Voulhoux, Romé, Ize, Bérengère, BLEVES, Sophie, Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Centro de Investigación Príncipe Felipe (CIPF), University of Florida [Gainesville] (UF), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and BLEVES, Sophie
Subjects: [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, [SDV.BC.IC] Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], biochemical phenomena, metabolism, and nutrition, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, bacterial infections and mycoses, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology
Abstract: International audience; Two clinical isolates of the opportunist pathogen Pseudomonas aeruginosa named PAO1 and PA14 are commonly studied in research laboratories. Despite being closely related, PA14 exhibits increased virulence compared to PAO1. To determine which players are responsible for the hypervirulence phenotype of the PA14 strain, we elected for a transcriptomic approach through RNA sequencing. We found 2029 genes that are differentially expressed between the two strains, including several genes that are involved with or regulated by Quorum-Sensing (QS), known to control most of the virulence factors in P. aeruginosa. Among them, we chose to focus our study on QslA, an anti-activator of QS whose expression was barely detectable in the PA14 strain according our data. We hypothesized that lack of expression of qslA in PA14 could be responsible for higher QS expression in the PA14 strain, possibly explaining its hyper-virulence phenotype. After confirming Qs lA protein was highly produced in PAO1 but not in the PA14 strain, we provided evidence showing that a PAO1 deletion strain of qslA has faster QS gene expression kinetics compared to PA14. Moreover, known virulence factors activated by QS such as (i) pyocyanin production, (ii) H2-T6SS(Type VI Secretion System) gene expression, and (iii) Xcp-T2SS (Type II Secretion System) machinery production and secretion were all lower in PAO1 compared to PA14 strain, due to higher qslAexpression. However, biofilm formation and cytotoxicity towards macrophages, although increased in PA14 compared to PAO1, were independent of QslA control. Altogether, our findings implicated differential qslA expression as a major determinant of virulence factor expression in P. aeruginos astrains PAO1 and PA14.
Published: 2019

27. Analysis of ADCs by Native Mass Spectrometry

Author: Oscar Hernandez-Alba, Anthony Ehkirch, Alain Beck, Sarah Cianférani, Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie Pierre Fabre (CIPF), PIERRE FABRE, and L. Nathan Tumey
Subjects: 0303 health sciences, Antibody-drug conjugate, Chromatography, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Ion-mobility spectrometry, Chemistry, 010401 analytical chemistry, Size-exclusion chromatography, Ms analysis, Load distribution, Mass spectrometry, 01 natural sciences, 0104 chemical sciences, body regions, 03 medical and health sciences, Qualitative analysis, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], 030304 developmental biology
Abstract: International audience; Mass spectrometry performed in nondenaturing conditions (native MS) has proven its utility for the quantitative and qualitative analysis of antibody-drug conjugates (ADCs), especially when ADCs’ subunits involve noncovalent interactions (i.e., cysteine-conjugated ADCs). Its hyphenation to ion mobility spectrometry (IM-MS) allows differentiation of gas-phase ions based on their rotationally averaged collision cross section providing an additional dimension of conformational characterization of ADCs. More recently, size exclusion chromatography (SEC) appeared as an interesting technique to perform online buffer exchange in an automated way prior to native MS/IM-MS analysis. Online SEC-native MS/IM-MS allows the global structural characterization of ADCs and the assessment of some critical quality attributes (CQAs) required for ADC release on the market, such as drug load distribution (DLD), drug-to-antibody ratio (DAR), the average DAR (DARav), and the relative amount of unconjugated mAb.
Published: 2019
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28. Intact monoclonal antibodies separation and analysis by sheathless capillary electrophoresis-mass spectrometry

Author: Yannis-Nicolas François, Jérémie Giorgetti, Emmanuelle Leize-Wagner, Elise Del Nero, Alain Beck, Antony Lechner, Laboratoire de spectrométrie de masse des interactions et des systèmes, Chimie de la matière complexe (CMC), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie Pierre Fabre, Tectonique Moléculaire du Solide (TMS), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg (UNISTRA), CIPF, Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de synthèses métallo-induites, and Institut de Chimie de Strasbourg-Dynamique et structure moléculaire par spectrométrie de masse (LDSM2)
Subjects: Glycosylation, medicine.drug_class, 010402 general chemistry, Monoclonal antibody, Mass spectrometry, 01 natural sciences, Capillary electrophoresis–mass spectrometry, Mass Spectrometry, Protein structure, Isomerism, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Aspartic acid, medicine, Asparagine, Deamidation, ComputingMilieux_MISCELLANEOUS, Spectroscopy, Chromatography, Chemistry, Biopharmaceutics, 010401 analytical chemistry, Electrophoresis, Capillary, General Medicine, Trastuzumab, Atomic and Molecular Physics, and Optics, 0104 chemical sciences, Protein Processing, Post-Translational
Abstract: Capillary electrophoresis mass spectrometry coupling (CE-MS) is a growing technique in biopharmaceutics characterization. Assessment of monoclonal antibodies (mAbs) is well known at middle-up and bottom-up levels to obtain information about the sequence, post-translational modifications (PTMs) and degradation products. Intact protein analysis is an actual challenge to be closer to the real protein structure. At this level, actual techniques are time consuming or cumbersome processes. In this work, a 20 minutes separation method has been developed to optimize characterization of intact mAbs. Thus, separation have been done on a positively-charged coated capillary (PEI) with optimized volatile background electrolyte (BGE) and sample buffer (SB). A sheathless interface allowed to hyphenate CE to a quadrupole-time-of-flight mass spectrometer (Q-TOF) which parameters has been tuned to improve the high masses detection and identification of intact mAbs. Three world-wide health authorities approved mAbs have been used to set up a rapid and ease of use method. Intact trastuzumab, rituximab and palivizumab isoforms have been partially separated with this method in less than 20 minutes under denaturing conditions. For each mAb, 2X-glycosylated and 1X-glycosylated structures have been identified and separated. Concerning basic and acidic variants potential Iso-Asp modification and Asn deamidation have been observed. Accurate mass determination for high-mass molecular species remains a challenge, but the progress in intact mAbs separation appears very promising for biopharmaceutics characterization.
Published: 2019
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29. A Novel Online Four-Dimensional SEC×SEC-IM×MS Methodology for Characterization of Monoclonal Antibody Size Variants

Author: Alexandre Goyon, Florent Rouvière, Oscar Hernandez-Alba, Cyrille Dreyfus, Anthony Ehkirch, Alain Beck, Valentina D'Atri, Hélène Diemer, Jean-François Haeuw, Sarah Cianférani, Sabine Heinisch, Davy Guillarme, Institut Pluridisciplinaire Hubert Curien (IPHC), Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), School of Pharmaceutical Science, University of Geneva [Switzerland]-Université de Lausanne (UNIL), Chromatography & Hyphenated Techniques - Chromatographie et techniques couplées, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie Pierre Fabre (CIPF), PIERRE FABRE, We thank GIS IBiSA and Région Alsace for financial support in purchasing a Synapt G2 HDMS instrument. Agilent Technologies is acknowledged for the loan of AdvanceBio SEC columns. A.E. acknowledges the 'Association Nationale de la Recherche et de la Technologie' (ANRT) and Syndivia for funding his Ph.D. fellowship. This work was supported by the CNRS, the Université de Strasbourg, the Université de Lyon, the Agence Nationale de la Recherche (ANR) and the French Proteomic Infrastructure (ProFI, ANR-10-INBS-08-03), and the Swiss National Science Foundation (fellowship 31003A_159494)., and ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010)
Subjects: 0301 basic medicine, Chromatography, medicine.drug_class, Phosphate salts, Chemistry, 010401 analytical chemistry, Antibodies, Monoclonal, Context (language use), Mass spectrometry, Monoclonal antibody, Antibodies, Monoclonal, Humanized, 01 natural sciences, Mass Spectrometry, 0104 chemical sciences, Analytical Chemistry, Characterization (materials science), Bevacizumab, 03 medical and health sciences, 030104 developmental biology, Antineoplastic Agents, Immunological, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, medicine, Chromatography, Gel, Drug product, ComputingMilieux_MISCELLANEOUS
Abstract: The determination of size variants is a major critical quality attribute of a therapeutic monoclonal antibody (mAb that may affect the drug product safety, potency, and efficacy. Size variant characterization often relies on size-exclusion chromatography (SEC), which could be hampered by difficult identification of peaks. On the other hand, mass spectrometry (MS)-based techniques performed in nondenaturing conditions have proven to be valuable for mAb-related compound characterization. On the basis of the observation that limited SEC performance was observed in nondenaturing MS compatible ammonium acetate buffer compared with classical phosphate salts, a multidimensional analytical approach was proposed. It combines comprehensive online two-dimensional chromatography (SEC×SEC), with ion mobility and mass spectrometry (IM-MS) in nondenaturing conditions for the characterization of a variety of mAbs. We first exemplify the versatility of our approach for simultaneous detection, identification, and quantitation of adalimumab size variants. Benefits of the SEC×SEC-native IM×MS were further highlighted on forced degraded pembrolizumab and bevacizumab samples, for which the 4D setup was mandatory to obtain an extensive and unambiguous identification, and accurate quantitation of unexpected high/low molecular weight species (HMWS and LMWS). In this specific context, monomeric conformers were detected by IM-MS as HMWS or LMWS. Altogether, our results emphasize the power of comprehensive 2D LC×LC setups hyphenated to IM×MS in nondenaturing conditions with unprecedented performance including: (i) maintaining optimal SEC performance (under classical nonvolatile salt conditions), (ii) performing online native MS identification, and (iii) providing IM-MS conformational characterization of all separated size variants.
Published: 2018
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30. Native Mass Spectrometry, Ion Mobility, and Collision-Induced Unfolding for Conformational Characterization of IgG4 Monoclonal Antibodies

Author: Alain Beck, Oscar Hernandez-Alba, Sarah Cianférani, Elsa Wagner-Rousset, Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie Pierre Fabre (CIPF), and PIERRE FABRE
Subjects: Models, Molecular, 0301 basic medicine, Subfamily, Protein Conformation, medicine.drug_class, Computational biology, Antibodies, Monoclonal, Humanized, Mass spectrometry, Monoclonal antibody, medicine.disease_cause, 01 natural sciences, Mass Spectrometry, Analytical Chemistry, 03 medical and health sciences, Protein structure, Ion Mobility Spectrometry, parasitic diseases, medicine, Humans, Point Mutation, [CHIM]Chemical Sciences, Protein Unfolding, Mutation, Protein Stability, Chemistry, Natalizumab, Point mutation, 010401 analytical chemistry, 0104 chemical sciences, Nivolumab, 030104 developmental biology, Immunoglobulin G, Monoclonal, Function (biology)
Abstract: International audience; Although the majority of FDA and EMA approved therapeutic monoclonal antibodies (mAbs) are IgG1, the number of IgG4-based formats reaching the market is increasing. IgG4 differs from other mAb isotypes by its specificity to form half mAbs that recombine into bispecific (bsAbs) molecules, through a process termed fab-arm exchange (FAE). We report here the complementarity of native mass spectrometry (MS), ion mobility (IM), and collision-induced unfolding (CIU) experiments for the structural characterization of members of the IgG4 subfamily (wild-type (wt), hinge-stabilized (hs, S228P mutation), and the resulting bsAb IgG4s). Native MS allows confirming/invalidating the occurrence of FAE as a function of these different types of IgG4. While IM-MS was unable to distinguish iso-cross-section IgG4 species, CIU experiments provide unique specific structural signatures of each individual IgG4 based on their specific unfolding pathways. Common CIU features of IgG4 formats include the observation of three conformational states and two transitions. In addition, CIU experiments demonstrated that S228P mutation stabilizes gas phase conformations of hsIgG4, in agreement with increased stability related to more rigid hinge regions. CIU patterns also appear to be more informative than IM-MS for bsAb structural characterization, unfolding signature of the bsAb being intermediate to the ones of the former parent wt-IgG4s, highlighting that bsAb CIU profiles keep the memory of their origins. Altogether, our results demonstrate that CIU patterns can serve as mAb specific structural signatures and are mature to be included in MS-based analytical workflows for conformational/structural characterization of mAb formats in early development phases and for multiple attribute monitoring.
Published: 2018
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31. Genomics of the origin and evolution of Citrus

Author: Manuel Talon, Joaquín Dopazo, Francisco R. Tadeo, Concha Domingo, Dongliang Du, Estela Pérez-Román, Victoria Ibanez, Guohong Albert Wu, José Carbonell-Caballero, Carles Borredá, Patrick Ollitrault, Daniel S. Rokhsar, Javier Terol, Antonio López-García, Roberto Alonso, Mikeal L. Roose, Frederick G. Gmitter, Franck Curk, Wu, Guohong Albert, Rokhsar, Daniel S., Talon, Manuel, United States Department of Energy, Centro de Genómica - Centre de Genòmica [IVIA], Instituto Valenciano de Investigaciones Agrarias - Institut Valencià d'Investigacions Agraries - Valencian Institute for agricultural Research (IVIA), Centro de Investigación Príncipe Felipe (CIPF), Amélioration Génétique et Adaptation des Plantes méditerranéennes et Tropicales Corse - Antenne Corse (AGAP-Corse), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA), University of South Florida [Tampa] (USF), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad), Department of Botany and Plant Sciences, University of California, Spanish National Bioinformatics Institute (INB), University of Florida [Gainesville] (UF), IFAS, Unite de recherche migrations et sociétés (URMIS), Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), University of California [Berkeley], Technology Graduate University, and Okinawa Institute of Science and Technology Graduate University (OIST)
Subjects: 0301 basic medicine, Citrus, Phylogénie, [SDV]Life Sciences [q-bio], Plant genetics, Biodiversity, Évolution, F30 - Génétique et amélioration des plantes, Génétique des populations, Asia, Southeastern, History, Ancient, Phylogeny, Multidisciplinary, Domestication des plantes, food and beverages, Genomics, F70 - Taxonomie végétale et phytogéographie, Crop Production, Provenance, Genome, Plant, Citrus hybrids, Heterozygote, Genetic Speciation, Human Migration, Introgression, Biology, Southeast asian, génomique, Evolution, Molecular, 03 medical and health sciences, Variation génétique, Phylogenetics, Botany, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, Domestication, Hybrid, Génome, 15. Life on land, biology.organism_classification, 030104 developmental biology, Haplotypes, Hybridization, Genetic
Abstract: The genus Citrus, comprising some of the most widely cultivated fruit crops worldwide, includes an uncertain number of species. Here we describe ten natural citrus species, using genomic, phylogenetic and biogeographic analyses of 60 accessions representing diverse citrus germ plasms, and propose that citrus diversified during the late Miocene epoch through a rapid southeast Asian radiation that correlates with a marked weakening of the monsoons. A second radiation enabled by migration across the Wallace line gave rise to the Australian limes in the early Pliocene epoch. Further identification and analyses of hybrids and admixed genomes provides insights into the genealogy of major commercial cultivars of citrus. Among mandarins and sweet orange, we find an extensive network of relatedness that illuminates the domestication of these groups. Widespread pummelo admixture among these mandarins and its correlation with fruit size and acidity suggests a plausible role of pummelo introgression in the selection of palatable mandarins. This work provides a new evolutionary framework for the genus Citrus. The origin, evolution and domestication of Citrus and the genealogy of the most important wild and cultivated citrus varieties. Citrus fruits are one of the most cultivated crops worldwide, yet the evolutionary relationships among citrus species remain uncertain. Daniel Rokhsar, Manuel Talon and colleagues analyse the genomes of 60 accessions that represent a diverse range of citrus species, including 30 newly sequenced citrus genomes. They characterize the diversity and evolution of citrus at the species level and identify interspecific citrus hybrids and admixtures—genetic mixing between previously isolated populations—that could be the result of human activities such as migration and agriculture. The authors identify 10 progenitor species and suggest that citrus originated in southeast Asia, diversifying during the late Miocene epoch through a rapid southeast Asian radiation that correlated with a changing climate, including the weakening of the monsoons. They also find extensive relatedness among mandarins and sweet oranges, showing a complex history of admixture during the domestication of these groups.
Published: 2018
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32. An Online Four-Dimensional HIC×SEC-IM×MS Methodology for Proof-of-Concept Characterization of Antibody Drug Conjugates

Author: Oscar Hernandez-Alba, Florent Rouvière, Sabine Heinisch, Davy Guillarme, Olivier Colas, Sarah Cianférani, Valentina D'Atri, Alexandre Goyon, Anthony Ehkirch, Alain Beck, Morgan Sarrut, Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), School of Pharmaceutical Science, University of Geneva [Switzerland]-Université de Lausanne (UNIL), Chromatography & Hyphenated Techniques - Chromatographie et techniques couplées, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie Pierre Fabre (CIPF), PIERRE FABRE, This work was also supported by the 'Agence Nationale de la Recherche' (ANR) and the French Proteomic Infrastructure (ProFI, ANR-10-INBS-08-03). The authors thank GIS IBiSA and Region Alsace for financial support in purchasing a Synapt G2 HDMS instrument. A.E. acknowledges the 'Association Nationale de la Recherche et de la Technologie' (ANRT) and Syndivia for funding his Ph.D. fellowship. The authors would like to thank David Lascoux (Waters) for his kind support and valuable assistance. The authors acknowledge Liz Bevan and Tony Edge from Agilent Technologies, for providing the AdvanceBioSEC column. Finally, the authors wish to thank Waters for the loan of 2D-LC instrumentation used in this work., and ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010)
Subjects: 0301 basic medicine, ddc:615, Chromatography, Immunoconjugates, Chemistry, Hydrophilic interaction chromatography, 010401 analytical chemistry, Mass spectrometry, 01 natural sciences, Mass measurement, Mass Spectrometry, 0104 chemical sciences, Analytical Chemistry, Characterization (materials science), 03 medical and health sciences, 030104 developmental biology, Proof of concept, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Chromatography, Gel, [CHIM]Chemical Sciences, Critical quality attributes, Hydrophobic and Hydrophilic Interactions, Conjugate
Abstract: International audience; There are currently two main techniques allowing the analytical characterization of interchain cysteine-linked antibody drug conjugates (ADCs) under native conditions, namely, hydrophobic interaction chromatography (HIC) and native mass spectrometry (MS). HIC is a chromatographic technique allowing the evaluation of drug load profile and calculation of average drug-to-antibody ratio (DAR) in quality control laboratories. Native MS offers structural insights into multiple ADC critical quality attributes, thanks to accurate mass measurement. However, both techniques can lead to misinterpretations or incomplete characterization when used as standalone methods. Online coupling of both techniques can thus potentially be of great interest, but the presence of large amounts of nonvolatile salts in HIC mobile phases makes it not easily directly compatible with native MS. Here, we present an innovative multidimensional analytical approach combining comprehensive online two-dimensional (2D)-chromatography that consists of HIC and size-exclusion chromatography (SEC), to ion mobility and mass spectrometry (IM-MS) for performing analytical characterization of ADCs under nondenaturing conditions. This setup enabled comprehensive and streamlined characterization of both native and forced degraded ADC samples. The proposed 4D methodology might be more generally adapted for online all-in-one HIC×SEC-IM×MS analysis of single proteins or analysis of protein complexes in nondenaturing conditions.
Published: 2017
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33. Viva Europa, a Land of Excellence in Research and Innovation for Health and Wellbeing

Author: Sonia Abdelhak, Mehmet Ozturk, Bento Soares, Bernd Schmeck, Samir K. Brahmachari, Andrey Lisistsa, Leroy Hood, Philippe Sabatier, Carole Goble, Pierre Hainaut, Marion Koopmans, Alain Cozzone, Christian Bréchot, Nathan Price, Vitaly Volpert, David Supple, Elissa Epel, Peter Hunter, Bartha Maria Knoppers, Ursula Klingmüller, Timothy Radstake, Zhu Chen, Mathias Uhlén, Johann Pellet, Steve Webb, Anna Norrby-Teglund, Vincent Lotteau, Dominique Charron, Josep Roca, Jean-Marie Lehn, Maria Manuela Nogueira, François Gros, Françoise Argoul, Ferran Sanz, Martine Raes, Antoine Magnan, Peter J. M. Openshaw, Alain Fischer, Shlomo Sasson, Peter J. Sterk, Francis Lévi, Hiroaki Kitano, Susanna Palkonen, Ross Arena, Albert-László Barabási, Michel Goldman, Ian M. Adcock, Andrea Gelemanovic, Sai-Juan Chen, Christian Pristipino, Jacques Demotes, Jesper Tegnér, Anders Bjartell, Laurent P. Nicod, Yves Moreau, Walter Kolch, Laurent Nottale, Emiel F.M. Wouters, Ismail Serageldin, Ratko Djukanovic, Jean-François Deleuze, Anita Simonds, Alfredo Cesario, Ana Conesa, Stylianos E. Antonarakis, David Harrison, Hans Hoffmann, Sylvie van der Werf, Yves Jacob, Marta Cascante, Andres Metspalu, James N’Dow, Alvar Agusti, Herman Goossens, Bertrand Boutron, Rudi Balling, Francisco Nobrega, Jacques S. Beckmann, Menno de Jong, Margarida Amaral, Alberto Di Meglio, Niklas Blomberg, Fan Chung, Denis Noble, Doron Lancet, Charles Auffray, Giulio Superti-Furga, Takashi Gojobori, Christopher E. Brightling, Thomas Bourgeron, Ugur Dogrusoz, Damjana Rozman, Karine Clément, Yi-Ke Guo, Bongani M. Mayosi, Ozren Polasek, Martine Laville, Michael Sagner, Manlio Vinciguerra, Pablo Villoslada, Silvio Parodi, European Institute for Systems Biology and Medicine (EISBM), University of Illinois [Chicago] (UIC), University of Illinois System, Institut Pasteur de Tunis, Réseau International des Instituts Pasteur (RIIP), National Heart and Lung Institute [London] (NHLI), Imperial College London-Royal Brompton and Harefield NHS Foundation Trust, University of Barcelona, Universidade de Lisboa, Université de Genève = University of Geneva (UNIGE), Université de Bordeaux (UB), Center for Systems Biomedicine [Falkensee], Central European University [Budapest, Hongrie] (CEU), Université de Lausanne = University of Lausanne (UNIL), University of Lund, Lund University [Lund], ELIXIR Hub [Cambridge], Génétique humaine et fonctions cognitives - Human Genetics and Cognitive Functions (GHFC (UMR_3571 / U-Pasteur_1)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), CSIR Institute of Genomics and Integrative Biology [New Delhi] (IGIB), Institut Pasteur [Paris] (IP), University of Leicester, Institute of Biomedicine of University of Barcelona, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona (UB), Hopital Saint-Louis [AP-HP] (AP-HP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Shangaï Jiao Tong University [Shangaï], Imperial College London, CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Centro de Investigación Príncipe Felipe (CIPF), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, Fondation Jean Dausset CEPH, European Clinical Research Infrastructures Network [Paris] (ECRIN), Ecrin, CHU Necker - Enfants Malades [AP-HP], Collège de France (CdF (institution)), Académie des Sciences [Paris], Institut de France, Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Hospices Civils de Lyon (HCL), Université de Strasbourg (UNISTRA), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre hospitalier universitaire de Nantes (CHU Nantes), Observatoire de Paris - Site de Meudon (OBSPM), Observatoire de Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS), Génétique Moléculaire des Virus à ARN - Molecular Genetics of RNA Viruses (GMV-ARN (UMR_3569 / U-Pasteur_2)), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut Camille Jordan (ICJ), École Centrale de Lyon (ECL), Université de Lyon-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Jean Monnet - Saint-Étienne (UJM)-Centre National de la Recherche Scientifique (CNRS), Modélisation mathématique, calcul scientifique (MMCS), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Jean Monnet - Saint-Étienne (UJM)-Centre National de la Recherche Scientifique (CNRS)-École Centrale de Lyon (ECL)
Subjects: 0301 basic medicine, 03 medical and health sciences, 030104 developmental biology, 0302 clinical medicine, SISTA, Excellence, 030220 oncology & carcinogenesis, media_common.quotation_subject, Political science, [SDV]Life Sciences [q-bio], General Medicine, Public administration, media_common
Abstract: ispartof: Progress in preventive medicine pages:1-2 status: published
Published: 2017
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34. Advanced Antibody-Drug Conjugate Structural Characterization by Sheathless Capillary Electrophoresis-Tandem Mass Spectrometry Using Complementary Approaches

Author: Said, Nassur, Giorgetti, Jérémie, Kuhn, Lauriane, Beck, Alain, Leize-Wagner, Emmanuelle, Gahoual, Rabah, Francois, Yannis-Nicolas, Chimie de la matière complexe (CMC), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Plateforme Protéomique Strasbourg - Esplanade (IBMC / CNRS FRC1589 / UNIV Strasbourg), Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Moléculaire et Cellulaire [Strasbourg] (IBMC), Centre d'Immunologie Pierre Fabre (CIPF), PIERRE FABRE, and dhotel, helene
Subjects: [SDV] Life Sciences [q-bio], [SDV.SP] Life Sciences [q-bio]/Pharmaceutical sciences, [SDV.GEN]Life Sciences [q-bio]/Genetics, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, [SDV]Life Sciences [q-bio], [SDV.GEN] Life Sciences [q-bio]/Genetics, [SDV.SP]Life Sciences [q-bio]/Pharmaceutical sciences, ComputingMilieux_MISCELLANEOUS, [SDV.BIO] Life Sciences [q-bio]/Biotechnology
Abstract: International audience
Published: 2017

35. An online four-dimensional HICxSEC-IMxMS methodology for in-depth characterization of antibody drug conjugates

Author: Anthony Ehkirch, Atri, Valentina D., Florent Rouvière, Oscar Hernandez-Alba, Alexandre Goyon, Olivier Colas, Morgan Sarrut, Alain Beck, Davy Guillarme, Sabine Heinisch, Sarah Cianferani, Bussy, Agnès, Institut Pluridisciplinaire Hubert Curien (IPHC), Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), School of Pharmaceutical Sciences, Chromatography & Hyphenated Techniques - Chromatographie et techniques couplées, Institut des Sciences Analytiques (ISA), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), School of Pharmaceutical Science, Université de Lausanne (UNIL)-University of Geneva [Switzerland], Centre d'Immunologie Pierre Fabre (CIPF), and PIERRE FABRE
Subjects: [CHIM.ANAL] Chemical Sciences/Analytical chemistry, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, [CHIM] Chemical Sciences, [CHIM]Chemical Sciences
Abstract: https://smmap2017.sciencesconf.org/; International audience; Antibody Drug Conjugates (ADCs) are tripartite molecules consisting of a monoclonal antibody (mAb) onto which highly cytotoxic small molecules are conjugated by cleavable or non-cleavable link- ers. They show better efficiency than canonical unconjugated mAbs, due to the synergic effect of mAb specificity for its target and the efficacy of the highly cytotoxic drug .There are currently two main techniques allowing the analytical characterization of cysteine linked anti- body drug conjugates under non denaturing conditions, namely hydrophobic interaction chromatography (HIC) and native high resolution mass spectrometry.HIC is a chromatographic technique allowing the evaluation of drug load profile and calculation of av- erage drug to antibody ratio (DAR). High resolution mass spectrometry (MS) offers a wealth of information on the biochemical and biophysical properties of ADCs, thanks to accurate mass measurement. On-line coupling of both techniques can potentially be of great interest, but the presence of large amounts of non-volatile salts in HIC mobile phases make them non compatible with MS.Here, we present an innovative multidimensional analytical approach combining comprehensive on-line two dimensional chromatography (HICxSEC) to ion mobility and mass spectrometry (IM-MS) for per- forming analytical characterization of ADCs under non-denaturing conditions. Online hyphenation of non-denaturing 2D chromatography to 2D IM-MS enabled comprehensive and streamlined characteriza- tion of both native and stressed ADC samples.
Published: 2017

36. Analysis of antibody-drug conjugates by comprehensive on-line two-dimensional hydrophobic interaction chromatography x reversed phase liquid chromatography hyphenated to high resolution mass spectrometry. II- Identification of sub-units for the characterization of even and odd load drug species

Author: Szabolcs Fekete, Morgan Sarrut, Alain Beck, Marie-Claire Janin-Bussat, Olivier Colas, Davy Guillarme, Sabine Heinisch, Chromatography & Hyphenated Techniques - Chromatographie et techniques couplées, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), School of Pharmaceutical Science, Université de Lausanne (UNIL)-University of Geneva [Switzerland], Centre d'Immunologie Pierre Fabre (CIPF), PIERRE FABRE, and Davy Guillarme wishes to acknowl- edge the Swiss National Science Foundation for support through a fellowship to Szabolcs Fekete (31003A 159494).
Subjects: 0301 basic medicine, Antibody-drug conjugate, Immunoconjugates, Clinical Biochemistry, Analytical chemistry, Mass spectrometry, 01 natural sciences, Biochemistry, Mass Spectrometry, Analytical Chemistry, 03 medical and health sciences, Isomerism, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Structural isomer, Cysteine, Brentuximab Vedotin, Chromatography, Reverse-Phase, Chromatography, Chemistry, Hydrophilic interaction chromatography, 010401 analytical chemistry, Cell Biology, General Medicine, Reversed-phase chromatography, Single injection, 0104 chemical sciences, Characterization (materials science), 030104 developmental biology, Hydrophobic and Hydrophilic Interactions, Conjugate
Abstract: This paper is the second part of a two-part series dedicated to the development of an on-line comprehensive HICxRPLC-UV/MS method for the characterization of a commercial inter-chain cysteine-linked ADC (brentuximab vedotin, Adcetris(®)). The first part focused on the optimization of the chromatographic conditions. In the second part of this series of papers, the structural characterization of the Brentuximab Vedotin was extensively discussed. With the combination of HIC and RPLC-MS data, the average DAR was easily measured in HIC and, at the same time, the predominant positional isomers were identified in RPLC-MS in one single injection. It was also demonstrated that the retention data obtained in the first and second dimensions was particularly useful to assist ADC characterization through the identification of sub-units. Using this methodology, the presence of odd DARs (1, 3 and 5) and their relative abundance was assessed by a systematic evaluation of HIC x RPLC-UV/MS data for both commercial and stressed ADC samples. Finally, once the exhaustive characterization of ADC was completed, MS could be conveniently replaced by UV detection to quickly assess the conformity of different ADCs batches.
Published: 2016
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37. Cutting-edge mass spectrometry methods for the multi-level structural characterization of antibody-drug conjugates

Author: Olivier Colas, Sarah Cianférani, Alain Beck, Marie-Claire Janin-Bussat, Julien Marcoux, Elsa Wagner-Rousset, Guillaume Terral, François Debaene, Alain Van Dorsselaer, Centre d'Immunologie Pierre Fabre, Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie Pierre Fabre (CIPF), PIERRE FABRE, Laboratoire de Spectrométrie de Masse BioOrganique [Strasbourg] (LSMBO), and Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC)
Subjects: 0301 basic medicine, critical quality attribute, Immunoconjugates, T-DM1, capillary electrophoresis-sodium dodecyl sulphate, 01 natural sciences, Biochemistry, CQA, Mass Spectrometry, trastuzumab emtansine Abbreviation List MS, chemistry.chemical_compound, UHPLC, Brentuximab vedotin, media_common, higher order structure, Chemistry, IEF, Expert Review of Proteomics Antibody drug conjugate, HOS, Antibodies, Monoclonal, size exclusion chromatography, SEC, hydrophobic interaction chromatography, rpHPLC, CE, 3. Good health, ion mobility MS, ultra-high performance liquid chromatography, SMCC, Critical quality attributes, native MS, medicine.drug, Drug, Bioanalysis, Antibody-drug conjugate, medicine.drug_class, hydrogen/deuterium exchange, IgG, media_common.quotation_subject, bioanalysis, electrospray ionization, capillary electrophoresis, Monoclonal antibody, CE-SDS, 03 medical and health sciences, brentuximab vedotin, human immunoglobilin G, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, HER2, ESI, tandem mass spectrometry, medicine, HDX, MS/MS, BV, Molecular Biology, HIC, trastuzumab emtansine, mAb, drug to antibody ratio, 010401 analytical chemistry, IdeS, human epidermal growth factor receptor 2, DAR, Combinatorial chemistry, 0104 chemical sciences, 030104 developmental biology, ADC, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, Trastuzumab emtansine, monoclonal antibody, drug-to-antibody ratio, reversed-phase high performance liquid chromatography, antibody-drug-conjugate, Conjugate
Abstract: International audience; Antibody drug conjugates (ADCs) are highly cytotoxic drugs covalently attached via conditionally stable linkers to monoclonal antibodies (mAbs) and are among the most promising next-generation empowered biologics for cancer treatment. ADCs are more complex than naked mAbs, as the heterogeneity of the conjugates adds to the inherent microvariability of the biomolecules. The development and optimization of ADCs rely on improving their analytical and bioanalytical characterization by assessing several critical quality attributes, namely the distribution and position of the drug, the amount of naked antibody, the average drug to antibody ratio, and the residual druglinker and related product proportions. Here brentuximab vedotin (Adcetris®) and trastuzumab emtansine (Kadcyla®), the first and gold-standard hinge-cysteine and lysine drug conjugates, respectively, were chosen to develop new mass spectrometry (MS) methods and to improve multiplelevel structural assessment protocols.
Published: 2015
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38. Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine-linked antibody drug conjugate

Author: Marcoux, Julien, Champion, Thierry, Colas, Olivier, Wagner-Rousset, Elsa, Corvaïa, Nathalie, Van Dorsselaer, Alain, Beck, Alain, Cianférani, Sarah, Laboratoire de Spectrométrie de Masse BioOrganique [Strasbourg] (LSMBO), Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC), Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Institut de Mathématiques de Toulon - EA 2134 (IMATH), Université de Toulon (UTLN), Centre d'Immunologie Pierre Fabre (CIPF), PIERRE FABRE, Centre de Recherche Pierre Fabre (Centre de R&D Pierre Fabre), and Centre d'Immunologie Pierre Fabre
Subjects: [CHIM.ANAL]Chemical Sciences/Analytical chemistry, ComputingMilieux_MISCELLANEOUS
Abstract: International audience
Published: 2015
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39. Nanodevice-Mediated Immune Cell Recruitment: Targeting Senescent Cells via MMP-3-Responsive CXCL12-Coated Nanoparticles.

Author: Escriche-Navarro B, Garrido E, Clara-Trujillo S, Labernadie A, Sancenon F, García-Fernández A, and Martínez-Máñez R
Subjects: Humans, Killer Cells, Natural drug effects, Killer Cells, Natural immunology, Jurkat Cells, Chemokine CXCL12 metabolism, Chemokine CXCL12 pharmacology, Chemokine CXCL12 chemistry, Nanoparticles chemistry, Cellular Senescence drug effects, Matrix Metalloproteinase 3 metabolism, Silicon Dioxide chemistry
Abstract: Senescent cells are involved in age-related disorders in different organs and are therapeutic targets for fibrotic and chronic pathologies. Immune-modulating agents, able to enhance senescent cell detection and elimination by endogenous immune cells, have emerged as pharmacological strategies. We report herein a nanoparticle for immune cell-mediated senolytic therapy designed to recruit immune cells in response to specific enzymatic matrix metalloproteinase-3 (MMP-3) activity in the senescence-associated secretory phenotype. For this, mesoporous silica nanoparticles (MSNs) are coated with a peptide substrate of the metalloproteinase MMP-3, and the peptide is decorated with chemokine CXCL12 that enhances immune cell recruitment (NPs@CXCL12). Controlled release studies confirmed the progressive and specific release of CXCL12 in the presence of MMP-3. The ability of immune cell recruitment in response to a senescent microenvironment (senescent WI-38 fibroblasts) is confirmed by Transwell migration assays with green fluorescent Jurkat T-cells, showing NPs@CXCL12 has an enhanced chemotaxis effect toward senescent cells compared to free CXCL12 (2-fold). Moreover, the cytotoxic capacity of human primary natural killer (NK) cells over senescent WI-38 is also confirmed, and their migration trajectories in response to NPs@CXCL12 or free CXCL12 are monitored by using a microfluidic device. Results confirm the ability of NPs@CXCL12 to generate a chemotactic gradient able to attract NK cells. When compared with free CXCL12, the NPs@CXCL12 system showed a reduction of up to 15.56% in the population of NK cells migrating toward free CXCL12 under competitive conditions. This study demonstrates the potential of designing nanoparticles to recruit immune cells under specific responses to eliminate senescent cells. Results confirm that NPs@CXCL12 can effectively establish a chemotactic gradient to attract NK cells.
Published: 2025
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40. Modulation of Mitochondria-Endoplasmic Reticulum Contacts (MERCs) by Small Molecules as a New Strategy for Restoring Lipid Metabolism in an Amyotrophic Lateral Sclerosis Model.

Author: Etxebeste-Mitxeltorena M, Flores-Romero H, Ramos-Inza S, Masiá E, Nenchova M, Montesinos J, Martinez-Gonzalez L, Porras G, Orzáez M, Vicent MJ, Gil C, Area-Gomez E, Garcia-Saez AJ, and Martinez A
Subjects: Humans, Cholesterol metabolism, HCT116 Cells, Mitochondria Associated Membranes, Amyotrophic Lateral Sclerosis metabolism, Amyotrophic Lateral Sclerosis drug therapy, Endoplasmic Reticulum metabolism, Endoplasmic Reticulum drug effects, Mitochondria metabolism, Mitochondria drug effects, Lipid Metabolism drug effects, Small Molecule Libraries pharmacology, Small Molecule Libraries chemistry
Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease without effective treatment. The progressive motoneuron death in ALS is associated with alterations in lipid metabolism. As its regulation occurs in mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), modulation of mitochondria-ER contacts (MERCs) is emerging as a crucial factor in MAM formation and lipid metabolism control. Using the MERLIN biosensor in a high-throughput screening within the EU-OPENSCREEN ERIC, we discovered small molecules that increase MERCs in HCT116 cells, enhancing their ability to uptake cholesterol. We demonstrated that cholesterol trafficking is decreased in an ALS patient-derived cell model, and this trafficking is restored after treatment with the discovered MERC modulator 24 . Electron microscopy revealed that treatment with compound 24 increases MERCs, promotes lipid droplet formation, and restores mitochondrial cristae. Overall, the brain-permeable MERC modulator, compound 24 , may serve as a valuable pharmacological tool for studying MAM function and holds potential for in vivo studies in ALS and other MAM dysfunction diseases.
Published: 2025
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41. Outcomes of a Pilot Newborn Screening Program for Spinal Muscular Atrophy in the Valencian Community.

Author: Berzal-Serrano A, García-Bohórquez B, Aller E, Jaijo T, Pitarch-Castellano I, Rausell D, García-García G, and Millán JM
Abstract: Spinal muscular atrophy (SMA) is a degenerative neuromuscular condition resulting from a homozygous deletion of the survival motor neuron 1 ( SMN1 ) gene in 95% of patients. A timely diagnosis via newborn screening (NBS) and initiating treatment before the onset of symptoms are critical for improving health outcomes in affected individuals. We carried out a screening test by quantitative PCR (qPCR) to amplify the exon seven of SMN1 using dried blood spot (DBS) samples. From October 2021 to August 2024, a total of 31,560 samples were tested in the Valencian Community (Spain) and 4 of them were positive for SMA, indicating an incidence of 1/7890. Genetic confirmation was performed using multiplex ligation-dependent probe amplification (MLPA) and AmplideX PCR/CE SMN1/2 Plus kit, in parallel obtaining concordant results in survival motor neuron 2 ( SMN2 ) gene copy number. Within the first few weeks of their lives, two of the four patients detected by NBS showed signs of severe hypotonia, becoming ineligible for treatment. The other two patients were the first presymptomatic patients with two copies of SMN2 to receive treatment with Risdiplam in Spain. In order to treat positive cases in their early stages, we conclude that the official deployment of SMA newborn screening is necessary.
Published: 2025
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42. Lipid Oxidation at the Crossroads: Oxidative Stress and Neurodegeneration Explored in Caenorhabditis elegans .

Author: Tortajada-Pérez J, Carranza ADV, Trujillo-Del Río C, Collado-Pérez M, Millán JM, García-García G, and Vázquez-Manrique RP
Abstract: Lipid metabolism plays a critical role in maintaining cellular integrity, especially within the nervous system, where lipids support neuronal structure, function, and synaptic plasticity. However, this essential metabolic pathway is highly susceptible to oxidative stress, which can lead to lipid peroxidation, a damaging process induced by reactive oxygen species. Lipid peroxidation generates by-products that disrupt many cellular functions, with a strong impact on proteostasis. In this review, we explore the role of lipid oxidation in protein folding and its associated pathological implications, with a particular focus on findings in neurodegeneration from Caenorhabditis elegans studies, an animal model that remains underutilized. Additionally, we highlight the effectiveness of different methodologies applied in this nematode to deepen our understanding of this intricate process. In the nervous system of any animal, including mammals and invertebrates, lipid oxidation can disturb the delicate balance of cellular homeostasis, leading to oxidative stress, the build-up of toxic by-products, and protein misfolding, key factors in neurodegenerative diseases. This disruption contributes to the pathogenesis of neurodegenerative disorders such as Alzheimer's, Parkinson's, or Huntington's disease. The findings from Caenorhabditis elegans studies offer valuable insights into these complex processes and highlight potential avenues for developing targeted therapies to mitigate neurodegenerative disease progression.
Published: 2025
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43. Renal-clearable probes for disease detection and monitoring.

Author: Domínguez M, García-Fernández A, Martí-Centelles V, Sancenón F, Blandez JF, and Martínez-Máñez R
Abstract: The demand for novel, minimally invasive, cost-effective, and easily readable diagnostic tools, primarily designed for the longitudinal monitoring of diseases and their treatments, has promoted the development of diagnostic systems that selectively target cells, tissues, or organs, at the same time minimizing their nonspecific accumulation, thus reducing the risk of toxicity and side effects. In this review, we explore the development of renal-clearable systems in non-invasive or minimally invasive detection protocols, all with the objective of minimizing nonspecific accumulation and its associated toxicity effects through quick renal excretion. These probes can identify molecules of interest or different healthy states of the patients through the direct analysis of urine (urinalysis). As we discuss, these diagnostics systems hold significant treatment monitoring potential., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Ltd.)
Published: 2024
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44. Peroxynitrite is involved in the mitochondrial dysfunction induced by Sorafenib in liver cancer cells.

Author: Cruz-Ojeda P, Navarro-Villarán E, Fuertes-Agudo M, Mata A, López-Lluch G, Navas P, Cadenas S, Casado M, and Muntané J
Abstract: Background: Sorafenib is a tyrosine kinase inhibitor (TKI) that belongs to the landscape of treatments for advanced stages of hepatocellular carcinoma (HCC). The induction of cell death and cell cycle arrest by Sorafenib has been associated with mitochondrial dysfunction in liver cancer cells. Our research aim was to decipher underlying oxidative and nitrosative stress induced by Sorafenib leading to mitochondrial dysfunction in liver cancer cells., Methods: MnTBAP, catalase and the scavenger of peroxynitrite FeTPPs were administered to Sorafenib (0-10 μM)-treated HepG2 cells. Oxygen consumption and glycolytic flux were determined in cultured cells. Mitochondrial complex activities were measured in mitochondrial fraction and cell lysates. The protein and mRNA expression of subunits of electron transport chain (ETC) were assessed by immunoblot and RNA-seq., Results: Sorafenib (10 μM) increased nitric oxide (NO) and superoxide anion (O 2 .- ) leading to peroxynitrite generation, and drastically reduced oxygen consumption. Moreover, Sorafenib led to mitochondrial network disorganization and loss of membrane potential. The administration of FeTPPs influenced the recovery of mitochondrial network and oxygen consumption, as well as associated ATP production. Sorafenib downregulated the mRNA expression of all mitochondrial-encoded subunits of ETC and, at to a lesser extent, nuclear-encoded mitochondrial genes. The protein expression of complex I, complex III and complex IV was greatly affected by Sorafenib. Furthermore, Sorafenib diminished the activity of complex I in in-gel assays, whose expression and activity were restored by FeTPPs. However, Sorafenib did not affect the assembly of mitochondrial supercomplexes. Sorafenib altered glycolysis and reduced Krebs cycle intermediates and increased NAD/NADH ratio., Conclusions: The induction of cell death by Sorafenib was associated with peroxynitrite generation, which impacted the expression of ETC subunits and mitochondrial functionality in liver cancer cells., Competing Interests: Declarations of interest None., (Copyright © 2025 Elsevier Inc. All rights reserved.)
Published: 2024
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45. Characterization of Mesenchymal and Neural Stem Cells Response to Bipolar Microsecond Electric Pulses Stimulation.

Author: Innamorati G, Sanchez-Petidier M, Bergafora G, Codazzi C, Palma V, Camera F, Merla C, André FM, Pedraza M, Moreno Manzano V, Caramazza L, Colella M, Marracino P, Balucani M, Apollonio F, Liberti M, and Consales C
Subjects: Cell Differentiation, Animals, Humans, Cells, Cultured, Neural Stem Cells cytology, Neural Stem Cells metabolism, Mesenchymal Stem Cells cytology, Mesenchymal Stem Cells metabolism, Electric Stimulation methods, Cell Proliferation, Apoptosis, Cell Cycle
Abstract: In the tissue regeneration field, stem cell transplantation represents a promising therapeutic strategy. To favor their implantation, proliferation and differentiation need to be controlled. Several studies have demonstrated that stem cell fate can be controlled by applying continuous electric field stimulation. This study aims to characterize the effect of a specific microsecond electric pulse stimulation (bipolar pulses of 100 µs + 100 µs, delivered for 30 min at an intensity of 250 V/cm) to induce an increase in cell proliferation on mesenchymal stem cells (MSCs) and induced neural stem cells (iNSCs). The effect was evaluated in terms of (i) cell counting, (ii) cell cycle, (iii) gene expression, and (iv) apoptosis. The results show that 24 h after the stimulation, cell proliferation, cell cycle, and apoptosis are not affected, but variation in the expression of specific genes involved in these processes is observed. These results led us to investigate cell proliferation until 72 h from the stimulation, observing an increase in the iNSCs number at this time point. The main outcome of this study is that the microsecond electric pulses can modulate stem cell proliferation.
Published: 2024
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46. Copper-cobalt peroxide nanoparticles: a biomimetic cascade reaction for enhanced Fenton-like therapy at physiologically relevant pH.

Author: Farrokhnia M, Manoochehri H, Shirkani M, Martínez-Máñez R, and Karimi S
Subjects: Hydrogen-Ion Concentration, Cell Line, Tumor, Humans, Iron chemistry, Peroxides chemistry, Nanoparticles chemistry, Nanoparticles therapeutic use, Mice, Cell Proliferation drug effects, Animals, Metal Nanoparticles chemistry, Metal Nanoparticles therapeutic use, Biomimetic Materials chemistry, Biomimetic Materials pharmacology, Tumor Microenvironment drug effects, Catalysis, Hydroxyl Radical chemistry, Hydroxyl Radical metabolism, Copper chemistry, Copper pharmacology, Hydrogen Peroxide chemistry, Cobalt chemistry
Abstract: Traditional Fenton-like reactions, commonly employed in chemodynamic therapy (CDT) for cancer treatment, face limitations due to the mildly acidic tumor microenvironment (TME) and scarce H 2 O 2 availability. Aiming to overcome these hurdles, we report herein the preparation of copper-cobalt peroxide (CCp) nanoparticles, a novel catalyst that enables a pH-activated, self-supplying H 2 O 2 -mediated cascade reaction. In the slightly acidic TME (pH 6.5-7.0), CCp nanoparticles degrade, generating H 2 O 2 in situ . This intrinsic H 2 O 2 production eliminates the need for external H 2 O 2 sources and enables activation in a significantly higher pH range. Simultaneously, released Cu and Co ions, primarily in lower oxidation states, synergistically drive a catalytic loop for sustained hydroxyl radical (˙OH) production. The non-ferrous bimetallic approach exhibits exquisite pH sensitivity and self-sufficiency, surpassing traditional Fenton reactions. Comparative studies highlight CCp's superior performance against copper-based bimetallic peroxides containing Fe and Ce, confirming the synergistic power of Cu-Co pairing. In vitro experiments demonstrate that the synthesized CCp-NPs exhibit greater toxicity toward breast cancer cells (4T1) than towards non-cancerous cells, showcasing their therapeutic potential. Furthermore, CCp-NPs outperform other nanoparticles in inhibiting cancer cell proliferation, colony formation, and migration. Density Functional Theory (DFT) calculations suggest that Co doping enhances CCp's ability to participate in Fenton reactions. Overall, this work is pioneering in relation to the design of a new class of smart nanoparticles for CDT. The combination of self-generated H 2 O 2 , high pH activation, and synergistic metal effects in CCp opens the door for next-generation cancer theranostic nanoparticles with unprecedented efficiency and precision, minimizing side effects.
Published: 2024
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47. Variants in the AGBL5 gene are responsible for autosomal recessive Retinitis pigmentosa with hearing loss.

Author: Karali M, García-García G, Kaminska K, AlTalbishi A, Cancellieri F, Testa F, Barillari MR, Panagiotou ES, Psillas G, Vaclavik V, Tran VH, Janeschitz-Kriegl L, Scholl HP, Salameh M, Barberán-Martínez P, Rodríguez-Muñoz A, Armengot M, Scarpato M, Zeuli R, Quinodoz M, Simonelli F, Rivolta C, Banfi S, and Millán JM
Abstract: The AGBL5 gene encodes for the Cytoplasmic Carboxypeptidase 5 (CCP5), an α-tubulin deglutamylase that cleaves the γ-carboxyl-linked branching point of glutamylated tubulin. To date, pathogenic variants in AGBL5 have been associated only with isolated retinitis pigmentosa (RP). Hearing loss has not been reported in AGBL5-caused retinal disease. In this study, we performed exome sequencing in probands of eight unrelated families from Italy, Spain, Palestine, Switzerland, and Greece. All subjects had a clinical diagnosis of (suspected) Usher syndrome type II for the concurrent presence of RP and post-verbal sensorineural hearing loss (SNHL) that ranged from mild to moderate.We identified biallelic sequence variants in AGBL5 in all analysed subjects. Four of the identified variants were novel. The variants co-segregated with the retinal and auditory phenotypes in additional affected family members. We did not detect any causative variants in known deafness or Usher syndrome genes that could explain the patients' hearing loss. We therefore conclude that SNHL is a feature of a syndromic presentation of AGBL5 retinopathy. This study provides the first evidence that mutations in AGBL5 can cause syndromic RP forms associated with hearing loss, probably due to dysfunction of sensory cilia in the retina and the inner ear., Competing Interests: Competing interests: The authors declare no competing interests. Ethical approval: All procedures adhered to the tenets of the Declaration of Helsinki and were approved by the Ethics Committees of the participating institutes. An informed consent was obtained by all patients., (© 2024. The Author(s).)
Published: 2024
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48. Orbitofrontal cortex hypergyrification in hallucinating schizophrenia patients: Surface ratio as a promising brain biomarker.

Author: Núñez C, Stephan-Otto C, Roldán A, Grasa EM, Escartí MJ, Aguilar García-Iturrospe EJ, García-Martí G, de la Iglesia-Vaya M, Nacher J, Portella MJ, and Corripio I
Subjects: Humans, Male, Female, Adult, Biomarkers, Middle Aged, Young Adult, Schizophrenia diagnostic imaging, Schizophrenia pathology, Schizophrenia metabolism, Hallucinations diagnostic imaging, Hallucinations pathology, Magnetic Resonance Imaging methods, Prefrontal Cortex diagnostic imaging, Prefrontal Cortex pathology
Abstract: The study of brain gyrification may provide useful information on the cytoarchitecture and connectivity of the brain. One of the methods that have been developed to estimate brain gyrification, known as surface ratio (SR), has not yet been studied in schizophrenia. Here we aimed to assess whether SR could provide new insights on the brain structure of schizophrenia patients and the severity of symptoms. We also computed a more established brain gyrification measure, namely absolute mean curvature (AMC). We analyzed 63 magnetic resonance images, 25 from schizophrenia patients with treatment-resistant auditory verbal hallucinations (SCH-H), 18 from schizophrenia patients without hallucinations (SCH-NH), and 20 from healthy controls (HC). The SR measure revealed that SCH-H patients had a more folded orbitofrontal cortex than SCH-NH patients and HC. Gyrification in this region was also negatively associated with positive symptoms, specifically with the delusions and conceptual disorganization items, only in the SCH-H group. Regarding the AMC measure, we identified two areas where HC showed more gyrification than SCH-H patients, but no relationships arose with symptoms. The hypergyrification of the orbitofrontal cortex displayed by SCH-H patients, as captured by the SR measure, suggests aberrant and/or excessive wiring in these patients, which in turn could give rise to auditory verbal hallucinations. Alternatively, we comment on potential compensatory mechanisms that may better explain the negative association between orbitofrontal gyrification and positive symptomatology. The SR measure captured the most relevant differences and associations, making it a promising biomarker in schizophrenia., Competing Interests: Conflicts of interest Dr. Roldán has served as advisor or speaker for the companies Otsuka, Rovi and Angelini (unrelated to the present work). The rest of the authors have nothing to disclose., (Copyright © 2024. Published by Elsevier B.V.)
Published: 2024
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49. Disruption of CAD Oligomerization by Pathogenic Variants.

Author: Del Caño-Ochoa F, Ramadane-Morchadi L, Eixerés L, Moreno-Morcillo M, Fernández-Leiro R, and Ramón-Maiques S
Subjects: Humans, Pentosyltransferases genetics, Pentosyltransferases metabolism, Pentosyltransferases chemistry, Crystallography, X-Ray, Protein Conformation, Protein Domains, Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing), Protein Multimerization, Aspartate Carbamoyltransferase metabolism, Aspartate Carbamoyltransferase genetics, Aspartate Carbamoyltransferase chemistry, Mutation, Missense, Dihydroorotase metabolism, Dihydroorotase genetics, Dihydroorotase chemistry, Models, Molecular
Abstract: CAD, the multi-enzymatic protein essential for initiating the de novo biosynthesis of pyrimidine nucleotides, forms large hexamers whose structure and function are not fully understood. Defects in CAD cause a severe neurometabolic disorder that is challenging to diagnose. We developed a cellular functional assay to identify defective CAD variants, and in this study, we characterized five pathogenic missense mutations in CAD's dihydroorotase (DHO) and aspartate transcarbamoylase (ATC) domains. All mutations impaired enzymatic activities, with two notably disrupting the formation of DHO dimers and ATC trimers. Combining crystal structures and AlphaFold predictions, we modeled the hexameric CAD complex, highlighting the central role of the DHO and ATC domains in its assembly. Our findings provide insight into CAD's stability, function, and organization, revealing that correct oligomerization of CAD into a supramolecular complex is required for its function in nucleotide synthesis and that mutations affecting this assembly are potentially pathogenic., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
Published: 2024
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50. Predictive and Dynamic Signature for Antiangiogenics in Combination with a PD1 Inhibitor in Soft-Tissue Sarcoma: Correlative Studies Linked to the IMMUNOSARC Trial.

Author: Moura DS, Lopez-Marti JM, Benesova I, de Andrea C, di Lernia D, Lacerenza S, Mondaza-Hernandez JL, Martin-Ruiz M, Ramirez-Calvo M, Grignani G, Martinez-Trufero J, Redondo A, Valverde C, Stacchiotti S, Lopez-Pousa A, Lopez-Guerrero JA, Gutierrez A, Encinas-Tobajas V, Hindi N, Sangiolo D, Lopez-Martin JA, Strizova ZO, and Martin-Broto J
Subjects: Humans, Female, Male, Angiogenesis Inhibitors therapeutic use, Angiogenesis Inhibitors administration & dosage, Middle Aged, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Immune Checkpoint Inhibitors therapeutic use, Immune Checkpoint Inhibitors pharmacology, Programmed Cell Death 1 Receptor antagonists & inhibitors, Nivolumab therapeutic use, Nivolumab administration & dosage, CD8-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes drug effects, CD8-Positive T-Lymphocytes metabolism, Adult, Aged, Lymphocytes, Tumor-Infiltrating immunology, Lymphocytes, Tumor-Infiltrating metabolism, Lymphocytes, Tumor-Infiltrating drug effects, Biomarkers, Tumor, Tumor Microenvironment drug effects, Tumor Microenvironment immunology, Prognosis, Sarcoma drug therapy, Sarcoma pathology, Sarcoma genetics
Abstract: Purpose: The IMMUNOSARC trial combined an antiangiogenic agent (sunitinib) with a PD1 inhibitor (nivolumab) in advanced sarcomas. Here, we present the first correlative studies of the soft-tissue sarcoma cohort enrolled in this trial., Experimental Design: Formalin-fixed paraffin-embedded and peripheral blood samples were collected at baseline and week 13. Formalin-fixed paraffin-embedded samples were used for transcriptomics and multiplex immunofluorescence, whereas peripheral blood samples were used for multiplexed immunoassays. Flow cytometry and Luminex assays were performed to validate translational findings in tumor-isolated cells and peripheral blood mononuclear cells derived from patients., Results: The density of intratumoral CD8+ T cells, measured by multiplexed immunophenotyping, was significantly increased after treatment. This augment was accompanied by the dynamic significant increase in the gene expressions of CD86, CHI3L1, CXCL10, CXCL9, LAG3, and VCAM1 and the decrease in the expression levels of NR4A1. In peripheral blood, 12 proteins were significantly modulated by treatment at week 13. A score integrating the dynamic expression of the 7 genes and the 12 soluble factors separated 2 groups with distinct progression-free survival (PFS): 4.1 months [95% confidence interval, 3.5-not reached (NR)] versus 17 months (95% confidence interval, 12.0-NR), P = 0.014. This molecular score was predictive of PFS when applied to the normalized data determined in the baseline samples., Conclusions: Treatment with sunitinib and nivolumab inflamed the sarcoma microenvironment, increasing CD8+ T-cell density and the expression of several genes/proteins with relevance in the response to PD1 inhibitors. A molecular signature identified two groups of patients with distinct PFS for the combination of antiangiogenics plus PD1 inhibitor therapy., (©2024 American Association for Cancer Research.)
Published: 2024
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