79 results on '"Gilboa T"'
Search Results
2. EPILEPSY IN PRADER-WILLI SYNDROME; THE ISRAELI EXPERIENCE: p519
- Author
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Gilboa, T. and Gross-Tsur, V.
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- 2012
3. Epilepsy in Prader-Willi syndrome; the Israeli experience: A4–P2
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GROSS-TSUR, V and GILBOA, T
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- 2012
4. Lymphoid follicle formation and human vaccination responses recapitulated in an organ-on-a-chip
- Author
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Goyal, G., primary, Prabhala, P., additional, Mahajan, G., additional, Bausk, B., additional, Gilboa, T., additional, Xie, L., additional, Zhai, Y., additional, Lazarovits, R., additional, Mansour, A., additional, Kim, Min Sun, additional, Curran, D., additional, Long, J. M., additional, Sharma, S., additional, Cohen, L., additional, Levy, O., additional, Prantil-Baun, R., additional, Walt, D.R., additional, and Ingber, D.E., additional
- Published
- 2019
- Full Text
- View/download PDF
5. Newly diagnosed and growing subependymal giant cell astrocytoma in adults with tuberous sclerosis complex: Results from the International TOSCA Study
- Author
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Jansen, A.C. Belousova, E. Benedik, M.P. Carter, T. Cottin, V. Curatolo, P. D'Amato, L. D'Augères, G.B. De Vries, P.J. Ferreira, J.C. Feucht, M. Fladrowski, C. Hertzberg, C. Jozwiak, S. Lawson, J.A. MacAya, A. Marques, R. Nabbout, R. O'Callaghan, F. Qin, J. Sander, V. Sauter, M. Shah, S. Takahashi, Y. Touraine, R. Youroukos, S. Zonnenberg, B. Kingswood, J.C. Fladrowsk, C. Shinohara, N. Horie, S. Kubota, M. Tohyama, J. Imai, K. Kaneda, M. Kaneko, H. Uchida, Y. Kirino, T. Endo, S. Inoue, Y. Uruno, K. Serdaroglu, A. Yapici, Z. Anlar, B. Altunbasak, S. Lvova, O. Belyaev, O.V. Agranovich, O. Levitina, E.V. Maksimova, Y.V. Karas, A. Jiang, Y. Zou, L. Xu, K. Zhang, Y. Luan, G. Zhang, Y. Wang, Y. Jin, M. Ye, D. Liao, W. Zhou, L. Liu, J. Liao, J. Yan, B. Deng, Y. Jiang, L. Liu, Z. Huang, S. Li, H. Kim, K. Chen, P.-L. Lee, H.-F. Tsai, J.-D. Chi, C.-S. Huang, C.-C. Riney, K. Yates, D. Kwan, P. Likasitwattanakul, S. Nabangchang, C. Krisnachai Chomtho, L.T. Katanyuwong, K. Sriudomkajorn, S. Wilmshurst, J. Segel, R. Gilboa, T. Tzadok, M. Fattal-Valevski, A. Papathanasopoulos, P. Papavasiliou, A.S. Giannakodimos, S. Gatzonis, S. Pavlou, E. Tzoufi, M. Vergeer, A.M.H. Dhooghe, M. Verhelst, H. Roelens, F. Nassogne, M.C. Defresne, P. De Waele, L. Leroy, P. Demonceau, N. Legros, B. Van Bogaert, P. Ceulemans, B. Dom, L. Castelnau, P. De Saint Martin, A. Riquet, A. Milh, M. Cances, C. Pedespan, J.-M. Ville, D. Roubertie, A. Auvin, S. Berquin, P. Richelme, C. Allaire, C. Gueden, S. The Tich, S.N. Godet, B. Ruiz Falco Rojas, M.L. Planas, J.C. Bermejo, A.M. Dura, P.S. Aparicio, S.R. Martinez Gonzalez, M.J. Pison, J.L. Blanco Barca, M.O. Laso, E.L. Luengo, O.A. Aguirre Rodriguez, F.J. Dieguez, I.M. Salas, A.C. Carrera, I.M. Salcedo, E.M. Yoldi Petri, M.E. Candela, R.C. Da Conceicao Carrilho, I. Vieira, J.P. Da Silva Oliveira Monteiro, J.P. Santos De Oliveira Ferreira Leao, M.J. Marceano Ribeiro Luis, C.S. Mendonca, C.P. Endziniene, M. Strautmanis, J. Talvik, I. Canevini, M.P. Gambardella, A. Pruna, D. Buono, S. Fontana, E. Dalla Bernardina, B. Burloiu, C. Bacos Cosma, I.S. Vintan, M.A. Popescu, L. Zitterbart, K. Payerova, J. Bratsky, L. Zilinska, Z. Gruber-Sedlmayr, U. Baumann, M. Haberlandt, E. Rostasy, K. Pataraia, E. Elmslie, F. Johnston, C.A. Crawford, P. Uldall, P. Dahlin, M. Uvebrant, P. Rask, O. Bjoernvold, M. Brodtkorb, E. Sloerdahl, A. Solhoff, R. Gilje Jaatun, M.S. Mandera, M. Radzikowska, E.J. Wysocki, M. Fischereder, M. Kurlemann, G. Wilken, B. Wiemer-Kruel, A. Budde, K. Marquard, K. Knuf, M. Hahn, A. Hartmann, H. Merkenschlager, A. Trollmann, R. on behalf of TOSCA Consortium TOSCA Investigators
- Abstract
The onset and growth of subependymal giant cell astrocytoma (SEGA) in tuberous sclerosis complex (TSC) typically occurs in childhood. There is minimal information on SEGA evolution in adults with TSC. Of 2,211 patients enrolled in TOSCA, 220 of the 803 adults (27.4%) ever had a SEGA. Of 186 patients with SEGA still ongoing in adulthood, 153 (82.3%) remained asymptomatic, and 33 (17.7%) were reported to ever have developed symptoms related to SEGA growth. SEGA growth since the previous scan was reported in 39 of the 186 adults (21%) with ongoing SEGA. All but one patient with growing SEGA had mutations in TSC2. Fourteen adults (2.4%) were newly diagnosed with SEGA during follow-up, and majority had mutations in TSC2. Our findings suggest that surveillance for new or growing SEGA is warranted also in adulthood, particularly in patients with mutations in TSC2. © 2019 Jansen, Belousova, Benedik, Carter, Cottin, Curatolo, D'Amato, Beaure d'Augères, de Vries, Ferreira, Feucht, Fladrowski, Hertzberg, Jozwiak, Lawson, Macaya, Marques, Nabbout, O'Callaghan, Qin, Sander, Sauter, Shah, Takahashi, Touraine, Youroukos, Zonnenberg and Kingswood.
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- 2019
6. Epilepsy in tuberous sclerosis complex: Findings from the TOSCA Study
- Author
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Nabbout, R, Belousova, E, Benedik, Mp, Carter, T, Cottin, V, Curatolo, P, Dahlin, M, Damato, L, D'Augeres, Gb, de Vries, Pj, Ferreira, Jc, Feucht, M, Fladrowski, C, Hertzberg, C, Jozwiak, S, Lawson, Ja, Macaya, A, Marques, R, O'Callaghan, F, Qin, J, Sander, V, Sauter, M, Shah, S, Takahashi, Y, Touraine, R, Youroukos, S, Zonnenberg, B, Jansen, A, Kingswood, Jc, Shinohara, N, Horie, S, Kubota, M, Tohyama, J, Imai, K, Kaneda, M, Kaneko, H, Uchida, Y, Endo, S, Inoue, Y, Uruno, K, Serdaroglu, A, Yapici, Z, Anlar, B, Altunbasak, S, Lvova, O, Valeryevich Belyaev, O, Agranovich, O, Vladislavovna Levitina, E, Vladimirovna Maksimova, Y, Karas, A, Jiang, Y, Zou, L, Xu, K, Zhang, Y, Luan, G, Wang, Y, Jin, M, Ye, D, Liao, W, Zhou, L, Liu, J, Liao, J, Yan, B, Deng, Y, Jiang, L, Liu, Z, Huang, S, Li, H, Kim, K, Chen, P, Lee, H, Tsai, J, Chi, C, Huang, C, Riney, K, Yates, D, Kwan, P, Likasitwattanakul, S, Nabangchang, C, Thampratankul Krisnachai Chomtho, L, Katanyuwong, K, Sriudomkajorn, S, Wilmshurst, J, Segel, R, Gilboa, T, Tzadok, M, Fattal-Valevski, A, Papathanasopoulos, P, Syrigou Papavasiliou, A, Giannakodimos, S, Gatzonis, S, Pavlou, E, Tzoufi, M, Dhooghe, M, Verhelst, H, Roelens, F, Cecile Nassogne, M, Defresne, P, De Waele, L, Leroy, P, Demonceau, N, Van Bogaert, P, Ceulemans, B, Dom, L, Castelnau, P, De Saint Martin, A, Riquet, A, Milh, M, Cances, C, Pedespan, J, Ville, D, Roubertie, A, Auvin, S, Berquin, P, Richelme, C, Allaire, C, Gueden, S, Nguyen The Tich, S, Godet, B, Rojas, Mlrf, Planas, Jc, Bermejo, Am, Dura, Ps, Aparicio, Sr, Gonzalez, Mjm, Pison, Jl, Blanco Barca, Mo, Laso, El, Luengo, Oa, Rodriguez, Fja, Dieguez, Im, Salas, Ac, Carrera, Im, Salcedo, Em, Petri, Mey, Candela, Rc, Carrilho, Idc, Vieira, Jp, Monteiro, Jpdso, Leao, Mjsdof, Luis, Csmr, Pires Mendonca, C, Endziniene, M, Strautmanis, J, Talvik, I, Canevini, Mp, Gambardella, A, Pruna, D, Buono, S, Fontana, E, Bernardina, Bd, Burloiu, C, Cosma, Isb, Vintan, Ma, Popescu, L, Zitterbart, K, Payerova, J, Bratsky, L, Zilinska, Z, Gruber-Sedlmayr, U, Haberlandt, E, Rostasy, K, Pataraia, E, Elmslie, F, Ann Johnston, C, Crawford, P, Uldall, P, Uvebrant, P, Rask, O, Bjoernvold, M, Sloerdahl, A, Solhoff, R, Jaatun, Msg, Mandera, M, Radzikowska, Ej, Wysocki, M, Fischereder, M, Kurlemann, G, Wilken, B, Wiemer-Kruel, A, Budde, K, Marquard, K, Knuf, M, Hahn, A, Hartmann, H, Merkenschlager, A, and Trollmann, R
- Subjects
0301 basic medicine ,medicine.medical_specialty ,Pediatrics ,Neurology ,Disease ,tuberous sclerosis complex ,030105 genetics & heredity ,registry ,03 medical and health sciences ,Tuberous sclerosis ,Epilepsy ,0302 clinical medicine ,Intellectual disability ,medicine ,Seizure control ,TOSCA ,business.industry ,epilepsy ,medicine.disease ,Settore MED/39 - Neuropsichiatria Infantile ,3. Good health ,medicine.anatomical_structure ,Cohort ,Full‐length Original Research ,Neurology (clinical) ,TSC1 ,business ,030217 neurology & neurosurgery - Abstract
Summary Objective To present the baseline data of the international TuberOus SClerosis registry to increase disease Awareness (TOSCA) with emphasis on the characteristics of epilepsies associated with tuberous sclerosis complex (TSC). Methods Retrospective and prospective patients’ data on all aspects of TSC were collected from multiple countries worldwide. Epilepsy variables included seizure type, age at onset, type of treatment, and treatment outcomes and association with genotype, seizures control, and intellectual disability. As for noninterventional registries, the study protocol did not specify any particular clinical instruments, laboratory investigations, or intervention. Evaluations included those required for diagnosis and management following local best practice. Results Epilepsy was reported in 83.6% of patients (1852/2216) at baseline; 38.9% presented with infantile spasms and 67.5% with focal seizures. The mean age at diagnosis of infantile spasms was 0.4 year (median
- Published
- 2019
7. Identification of a novel alternatively spliced variant endothelin converting enzyme-1 lacking a transmembrane domain
- Author
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Klipper, E., Levy, N., Gilboa, T., laurent Muller, Meidan, R., Centre interdisciplinaire de recherche en biologie (CIRB), Labex MemoLife, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), and Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)
- Subjects
Umbilical Veins ,[SDV]Life Sciences [q-bio] ,MESH: Metalloendopeptidases ,MESH: Umbilical Veins ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Endothelin-Converting Enzymes ,Cell Line ,MESH: Protein Structure, Tertiary ,Corpus Luteum ,Animals ,Aspartic Acid Endopeptidases ,Humans ,MESH: Animals ,MESH: Endothelin-Converting Enzymes ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,RNA, Messenger ,MESH: Corpus Luteum ,Cells, Cultured ,MESH: RNA, Messenger ,MESH: Humans ,MESH: Alternative Splicing ,MESH: Molecular Weight ,Metalloendopeptidases ,MESH: Cell Line ,Protein Structure, Tertiary ,Isoenzymes ,Molecular Weight ,MESH: Cattle ,Alternative Splicing ,MESH: Isoenzymes ,Cattle ,Female ,MESH: Endothelium, Vascular ,MESH: Aspartic Acid Endopeptidases ,Endothelium, Vascular ,MESH: Female ,MESH: Cells, Cultured - Abstract
International audience; Endothelin-converting enzyme (ECE)-1 cleaves big endothelins, as well as bradykinin and beta-amyloid peptide. Several isoforms of ECE-1 (ECE-1a, 1b, 1c, and 1d) have been identified to date, they differ only in their amino terminus and share the catalytic domain located in the C-terminal end. In addition to full-length ECE-1 forms, we identified novel, alternatively spliced messenger RNAs (mRNAs) of ECE-1b, 1c, and 1d. These splice variants (SVs) lack exon 3', which codes for the transmembrane (TM) region and is present in full-length forms. SV mRNAs were highly expressed in endothelial cells (EC) derived from macrovascular and microvascular beds. Analyses of ECE-1d and its SV forms in stably transfected human embryonic kidney (HEK)-293 cells revealed that both proteins were recognized by antibodies to C-terminal ECE-1, but an antibody to the N-terminal only bound ECE-1d. The novel protein, designated ECE-1sv, has an apparent molecular weight of 75 kDa. ECE-1sv lacks the TM sequence (or signal peptide) and, therefore, is expected to remain cytosolic. Presence of ECE-1sv in different cellular compartments than the full-length forms of the ECE-1 may suggest a distinct physiologic role for these proteins.
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- 2006
8. 654 Prolonged Video-Eeg Necessary to Identify Non-Convulsive Status Epilepticus in Infants Admitted to Icus
- Author
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Gilboa, T, primary
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- 2010
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9. Invasive mechanical ventilation: risk factor for death in patients with cystic fibrosis admitted to intensive care unit – Outcome and prolonged follow up
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Efrati, O., primary, Bylin, I., additional, Segal, E., additional, Vilozni, D., additional, Gilboa, T., additional, Shalev, K., additional, Barak, A., additional, Sofer, Y., additional, Szeinberg, A., additional, Yahav, Y., additional, and Paret, G., additional
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- 2008
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10. PROLONGED VIDEOEEG NECESSARY TO IDENTIFY NONCONVULSIVE STATUS EPILEPTICUS IN INFANTS ADMITTED TO ICUS
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Gilboa, T.
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- 2010
11. Loss-of-function mutations in UDP-Glucose 6-Dehydrogenase cause recessive developmental epileptic encephalopathy
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Ali Al Asmari, Emmanuelle Szenker-Ravi, Carine Bonnard, Bruno Reversade, Laura Schultz-Rogers, I. Kraegeloh-Mann, Maha Abdulrahim, Hesham Aldhalaan, Byrappa Venkatesh, Célia Bosso-Lefèvre, Aida Telegrafi, Hiyam M. Marzouqa, Gunaseelan Narayanan, Sha Tang, Sonal Mahida, Melanie A. Simpson, Fowzan S. Alkuraya, Michelle Eio, Eissa Faqeih, Renske Oegema, Sarah Weckhuysen, George Grady, Joseph J. Barycki, Mohammed Al-Owain, Lamyaa A. Jad, David A. Koolen, Marjon van Slegtenhorst, Tyler Mark Pierson, Marisa V. Andrews, Rebecca Schüle, Reinhard Keimer, Amber Begtrup, Sateesh Maddirevula, Michael Muriello, Sakkubai Naidu, Damien Haye, Adel A H Mahmoud, Brian Ciruna, Abdullah Tamim, Thong Teck Tan, Rolph Pfundt, Peter Bauer, Jiin Ying Lim, Ali Awaji, Marco Tartaglia, Meral Gunay-Aygun, Eric W. Klee, Marcia C. Willing, Monica Yau, Angelika Riess, Diego Martinelli, Sabina Barresi, Sumanty Tohari, Werner Deigendesch, Dirk Lefeber, Saumya Shekhar Jamuar, Ludger Schöls, Ralitza H. Gavrilova, Alvin Yu Jin Ng, Hannah Stamberger, Suleyman Gulsuner, Adam Claridge-Chang, Élise Lebigot, Moeenaldeen Al-Sayed, Ee Shien Tan, Kagistia Hana Utami, Sarah B. Pierce, Helene Verhelst, Hankun Li, James C. Stewart, Ingo Helbig, Tal Gilboa, Mahmoud A. Pouladi, Hagar Mor-Shaked, Boris Keren, Ajay S. Mathuru, Holger Hengel, Michèl A.A.P. Willemsen, Nader Handal, Tahsin Stefan Barakat, Sulwan M. Algain, Terrence Thomas, Lance H. Rodan, Mais Hashem, Wendy G. Mitchell, Center for Reproductive Medicine, ARD - Amsterdam Reproduction and Development, ACS - Diabetes & metabolism, Clinical Genetics, Reversade, Bruno, Hengel, H., Bosso-Lefèvre, C., Grady, G., Szenker-Ravi, E., Li, H., Pierce, S., Lebigot, É., Tan, T.-T., Eio, M.Y., Narayanan, G., Utami, K.H., Yau, M., Handal, N., Deigendesch, W., Keimer, R., Marzouqa, H.M., Gunay-Aygun, M., Muriello, M.J., Verhelst, H., Weckhuysen, S., Mahida, S., Naidu, S., Thomas, T.G., Lim, J.Y., Tan, E.S., Haye, D., Willemsen, M.A.A.P., Oegema, R., Mitchell, W.G., Pierson, T.M., Andrews, M.V., Willing, M.C., Rodan, L.H., Barakat, T.S., van Slegtenhorst, M., Gavrilova, R.H., Martinelli, D., Gilboa, T., Tamim, A.M., Hashem, M.O., AlSayed, M.D., Abdulrahim, M.M., Al-Owain, M., Awaji, A., Mahmoud, A.A.H., Faqeih, E.A., Asmari, A.A., Algain, S.M., Jad, L.A., Aldhalaan, H.M., Helbig, I., Koolen, D.A., Riess, A., Kraegeloh-Mann, I., Bauer, P., Gulsuner, S., Stamberger, H., Ng, A.Y.J., Tang, S., Tohari, S., Keren, B., Schultz-Rogers, L.E., Klee, E.W., Barresi, S., Tartaglia, M., Mor-Shaked, H., Maddirevula, S., Begtrup, A., Telegrafi, A., Pfundt, R., Schüle, R., Ciruna, B., Bonnard, C., Pouladi, M.A., Stewart, J.C., Claridge-Chang, A., Lefeber, D.J., Alkuraya, F.S., Mathuru, A.S., Venkatesh, B., Barycki, J.J., Simpson, M.A., Jamuar, S.S., Schöls, L, and School of Medicine
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0301 basic medicine ,Male ,Glycobiology ,General Physics and Astronomy ,VARIANTS ,Encephalopathy ,Neurodegenerative ,Germline ,0302 clinical medicine ,UDP-GLUCOSE DEHYDROGENASE ,Loss of Function Mutation ,Medicine and Health Sciences ,EMBRYOGENESIS ,2.1 Biological and endogenous factors ,UGDH protein, human ,Aetiology ,Child ,lcsh:Science ,Zebrafish ,UTILITY ,Genetics ,pathology [Organoids] ,Multidisciplinary ,Uridine diphosphate glucose dehydrogenase ,Uridine diphosphate ,DP-glucuronic acid ,Syndrome ,Disorders of movement Donders Center for Medical Neuroscience [Radboudumc 3] ,Hypotonia ,3. Good health ,Pedigree ,DEFICIENCY ,genetics [Loss of Function Mutation] ,Organoids ,genetics [Uridine Diphosphate Glucose Dehydrogenase] ,Child, Preschool ,Neurological ,Medicine ,Female ,ddc:500 ,medicine.symptom ,Oxidoreductases ,Engineering sciences. Technology ,Rare cancers Radboud Institute for Health Sciences [Radboudumc 9] ,ENZYME ,Adolescent ,CONGENITAL DISORDER ,Science ,Intellectual and Developmental Disabilities (IDD) ,genetics [Epilepsy] ,chemistry [Oxidoreductases] ,Genetics and Molecular Biology ,Genes, Recessive ,Biology ,Uridine Diphosphate Glucose Dehydrogenase ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Protein Domains ,medicine ,Animals ,Humans ,Recessive ,Clinical genetics ,Allele ,Preschool ,Gene ,Loss function ,Alleles ,HEPARAN-SULFATE ,Phenocopy ,genetics [Oxidoreductases] ,Neurodevelopmental disorders Donders Center for Medical Neuroscience [Radboudumc 7] ,Epilepsy ,GLYCOSYLATION ,Neurosciences ,Infant ,General Chemistry ,biology.organism_classification ,medicine.disease ,Brain Disorders ,carbohydrates (lipids) ,Kinetics ,030104 developmental biology ,Genes ,General Biochemistry ,Neuronal development ,lcsh:Q ,Human medicine ,030217 neurology & neurosurgery ,Congenital disorder - Abstract
Developmental epileptic encephalopathies are devastating disorders characterized by intractable epileptic seizures and developmental delay. Here, we report an allelic series of germline recessive mutations in UGDH in 36 cases from 25 families presenting with epileptic encephalopathy with developmental delay and hypotonia. UGDH encodes an oxidoreductase that converts UDP-glucose to UDP-glucuronic acid, a key component of specific proteoglycans and glycolipids. Consistent with being loss-of-function alleles, we show using patients’ primary fibroblasts and biochemical assays, that these mutations either impair UGDH stability, oligomerization, or enzymatic activity. In vitro, patient-derived cerebral organoids are smaller with a reduced number of proliferating neuronal progenitors while mutant ugdh zebrafish do not phenocopy the human disease. Our study defines UGDH as a key player for the production of extracellular matrix components that are essential for human brain development. Based on the incidence of variants observed, UGDH mutations are likely to be a frequent cause of recessive epileptic encephalopathy., German Research Foundation (DFG); European Union (European Union); NEUROMICS Network; International Coordination Action (ICA); Fund for Scientific Research Flanders (FWO); Netherlands Organization for Scientific Research (ZONMW VIDI); National Medical Research Council, Singapore; A Strategic Positioning Fund on Genetic Orphan Diseases (GODAFIT); Industry Alignment Fund on Singapore Childhood Undiagnosed Diseases Program (SUREKids); Biomedical Research Council, A*STAR; Diana and Steve Marienhoff Fashion Industries Guild Endowed Fellowship in Pediatric Neuromuscular Diseases; Fondazione Bambino Gesù (Vite Coraggiose); Canadian Institutes of Health Research; Natural Sciences and Engineering Research Council of Canada; Eurocores Program EuroEPINOMICS; University of Antwerp Research Fund; FRAXA Foundation; Brain & Behavior Research Foundation, NARSAD Young Investigator Grant
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- 2020
12. Economic aspects of prolonged home video-EEG monitoring: a simulation study.
- Author
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Vander T, Bikmullina R, Froimovich N, Stroganova T, Nissenkorn A, Gilboa T, Eliashiv D, Ekstein D, and Medvedovsky M
- Abstract
Introduction: Video EEG monitoring (VEM) is an important tool for characterizing clinical events suspected as seizures. It is also used for pre-surgical workups in patients with drug-resistant epilepsy (DRE). In-hospital VEM high cost, long admission waiting periods and some other inconveniences led to an interest in home VEM (HVEM). However, because antiseizure medications cannot be reduced at home, HVEM may require longer monitoring. While the economic aspect is one of the main motivations for HVEM, the cost of HVEM lasting several weeks has not been assessed., Methods: We modeled the cost of HVEM for 8 weeks and compared it to the cost of 1-week in-hospital VEM. Additionally, we modeled the per-patient cost for a combination of HVEM and in-hospital VEM, considering that if in a proportion of patients HVEM fails to achieve its goal, they should undergo in-hospital VEM with drug reduction., Results: The average cost of HVEM up to 4-6 weeks of monitoring was lower than that for the 1-week in-hospital VEM. Combining the 3-week HVEM with 1-week in-hospital VEM (if needed) reduced the per-patient cost by 6.6-28.6% as compared to the situation when all the patients with DRE were referred to the in-hospital VEM., Conclusions: A prolonged intermittent HVEM can be cost-effective, especially if the minimal seizure frequency is about one seizure per week. The study findings support directing efforts into clinical trials and technology development., (© 2024. The Author(s).)
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- 2024
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13. Autoimmune encephalitis in Israeli children - A retrospective nationwide study.
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Sachs N, Zohar-Dayan E, Ben Zeev B, Gilboa T, Kurd M, Latzer IT, Meirson H, Krause I, Dizitzer Y, and Cohen EG
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- Humans, Female, Male, Child, Israel epidemiology, Retrospective Studies, Child, Preschool, Adolescent, Infant, Magnetic Resonance Imaging, Electroencephalography, Hashimoto Disease, Immunoglobulins, Intravenous therapeutic use, Infant, Newborn, Encephalitis immunology, Encephalitis diagnosis
- Abstract
Immune-mediated or autoimmune encephalitis (AE) is a relatively new, rare and elusive form of encephalitis in children. We retrospectively collected seropositive children (0-18 years old) with well characterized antibodies through 3 reference laboratories in Israel. Clinical symptoms, MRI and EEG findings and treatment courses were described. A total of 16 patients were included in the study, with 10 females. Anti NMDA encephalitis was most common followed by anti HU and anti mGLuR1. Psychiatric symptoms, abnormal movements, seizures and behavioral changes were the most common presentation. Pathological MRI and EEG findings were described in 37% and 56% of children, respectively. Treatment with corticosteroids, Intravenous immunoglobulins (IVIG) was first line in most children. Following inadequate response children were treated with plasmapheresis and/or rituximab. Two patients relapsed following both first and second line protocols. In terms of long term prognosis, 9 children (56%) had one or more residual behavioral, psychiatric or neurologic findings. Three children required hospitalization for rehabilitation. AE remains a rare diagnosis with variable presenting symptoms, requiring a high index of suspicion. Consensus recommended treatment is generally effective in the pediatric population. Female gender was associated with a higher chance of severe disease. Larger cohorts would be needed to identify prognostic factors in the pediatric population., (© 2024 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.)
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- 2024
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14. Validation of the 2023 international diagnostic criteria for MOGAD in a pediatric cohort.
- Author
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Kurd M, Pratt LT, Gilboa T, Fattal-Valevski A, Vaknin-Dembinsky A, Gadoth A, Hacohen Y, and Meirson H
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- Humans, Child, Female, Male, Child, Preschool, Adolescent, Infant, Autoantibodies blood, Cohort Studies, Sensitivity and Specificity, Myelin-Oligodendrocyte Glycoprotein immunology
- Abstract
Objective: To validate the recently published diagnostic criteria for Myelin Oligodendrocyte Glycoprotein-antibody associated disease (MOGAD) in real-world cohort of children with acquired demyelinating syndromes., Methods: Patients <18yrs presenting with demyelinating disease to Pediatric neuroimmunology clinics at two Israeli tertiary centers who had MOG antibodies (MOG-Abs) tested between 01/07/2017 and 15/08/2023 were included. Diagnostic criteria for MOGAD were applied and sensitivity and specificities were calculated., Results: MOG-Abs were detected in 28/63 (44 %). Median age at onset for all patients was 11.4 yrs (range 1.1-17.6 yrs) and 41 (65 %) were female. Of the patients testing negative, ADEM was the most common diagnosis (n = 11) followed by MS (n = 8). No patients without MOG-Abs were diagnosed with MOGAD. All patients with a clinical diagnosis of MOGAD had positive MOG-Abs and fulfilled the 2023 international diagnostic criteria for MOGAD. Sensitivity, specificity, positive predictive value, and negative predictive value were 100 %. We found no difference between younger (<10yrs old) and older (>10 yrs old) children in the number of supportive criteria fulfilled at onset (median 2 vs. 2.5, p = 0.4) The number of supporting features was higher in patients with relapsing (n = 5) vs. monophasic (n = 23) disease course at onset (median 3 vs. 2, p = 0.03) and at final follow-up (median 5 vs. 2, p = 0.004)., Conclusion: Recent MOGAD diagnostic criteria had excellent performance in this pediatric cohort but did not add to the diagnostic accuracy of the antibody test alone., Competing Interests: Declaration of competing interest Mohammad Kurd received funding from the European Paediatric Neurology Society., (© 2024 Published by Elsevier Ltd on behalf of European Paediatric Neurology Society.)
- Published
- 2024
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15. Toward the quantification of α-synuclein aggregates with digital seed amplification assays.
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Gilboa T, Swank Z, Thakur R, Gould RA, Ooi KH, Norman M, Flynn EA, Deveney BT, Chen A, Borberg E, Kuzkina A, Ndayisaba A, Khurana V, Weitz DA, and Walt DR
- Subjects
- Humans, alpha-Synuclein, Antibodies, Multiple System Atrophy, Parkinson Disease, Synucleinopathies
- Abstract
The quantification and characterization of aggregated α-synuclein in clinical samples offer immense potential toward diagnosing, treating, and better understanding neurodegenerative synucleinopathies. Here, we developed digital seed amplification assays to detect single α-synuclein aggregates by partitioning the reaction into microcompartments. Using pre-formed α-synuclein fibrils as reaction seeds, we measured aggregate concentrations as low as 4 pg/mL. To improve our sensitivity, we captured aggregates on antibody-coated magnetic beads before running the amplification reaction. By first characterizing the pre-formed fibrils with transmission electron microscopy and size exclusion chromatography, we determined the specific aggregates targeted by each assay platform. Using brain tissue and cerebrospinal fluid samples collected from patients with Parkinson's Disease and multiple system atrophy, we demonstrated that the assay can detect endogenous pathological α-synuclein aggregates. Furthermore, as another application for these assays, we studied the inhibition of α-synuclein aggregation in the presence of small-molecule inhibitors and used a custom image analysis pipeline to quantify changes in aggregate growth and filament morphology., Competing Interests: Competing interests statement:D.R.W. is a founder and equity holder in Quanterix. His interests were reviewed and are managed by BWH and Partners HealthCare in accordance with their conflict of interest policies.
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- 2024
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16. The manifestations of metabolic acidosis during acetazolamide treatment in a cohort of pediatric idiopathic intracranial hypertension.
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Bulkowstein Y, Nitzan-Luques A, Schnapp A, Barnoy N, Reif S, Gilboa T, and Volovesky O
- Subjects
- Humans, Child, Acetazolamide adverse effects, Carbonic Anhydrase Inhibitors adverse effects, Retrospective Studies, Pseudotumor Cerebri drug therapy, Pseudotumor Cerebri diagnosis, Acidosis chemically induced, Acidosis drug therapy
- Abstract
Background: Idiopathic intracranial hypertension is characterized by increased intracranial pressure with unidentified pathology. Despite its use as the first-line treatment, data on acetazolamide's effectiveness and safety in pediatric idiopathic intracranial hypertension is sparse. This study's objective was to assess those issues and the need for routine blood gas monitoring during treatment., Methods: Retrospective observational cohort study, based on multicenter computerized medical charts of pediatric patients with idiopathic intracranial hypertension diagnosed between 2007-2018 in three medical centers serving one metropolitan area (an estimated population of 400,000 children). Clinical and laboratory data of children up to 18 years old, fulfilling the Friedman criteria and taking acetazolamide, were collected and analyzed., Results: Sixty-eight patients were included with a mean acetazolamide treatment duration of 8.5 months and a median maximal dose 18 mg/kg/d. Sixty-two children had mild (76%), moderate (13%), or severe (1.5%) metabolic acidosis. At least one adverse effect (neurologic, gastrointestinal, renal) was recorded among 27% of patients. No significant difference was found between the mean pH of children with or without clinical adverse effects (p = 0.35). No correlation was found between laboratory acidosis and adverse effect severity (p = 0.3), or between median acetazolamide dose and acidosis level (p = 0.57)., Conclusions: Although laboratory finding of metabolic acidosis is common among patients with idiopathic intracranial hypertension treated with acetazolamide, it is not correlated with clinics. Therefore, we recommend sending blood tests during acetazolamide treatment based on clinical judgment. A higher resolution version of the Graphical abstract is available as Supplementary information., (© 2023. The Author(s), under exclusive licence to International Pediatric Nephrology Association.)
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- 2024
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17. Real-Life Experience With Purified Cannabidiol Treatment for Refractory Epilepsy: A Multicenter Retrospective Study.
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Tzadok M, Gur-Pollack R, Florh H, Michaeli Y, Gilboa T, Lezinger M, Heyman E, Chernuha V, Gudis I, Nissenkorn A, Lerman-Sagie T, Ben Zeev B, and Uliel-Sibony S
- Subjects
- Child, Young Adult, Humans, Female, Male, Anticonvulsants therapeutic use, Retrospective Studies, Quality of Life, Seizures drug therapy, Cannabidiol adverse effects, Drug Resistant Epilepsy drug therapy, Drug Resistant Epilepsy diagnosis, Lennox Gastaut Syndrome drug therapy
- Abstract
Background: Drug-resistant epilepsy (DRE) affects the development and quality of life of children and young adults. We analyzed the effectiveness and safety of purified CBD in this population., Methods: A retrospective analysis of medical records of 139 children and young adults (54.7% female, median age 12.0 years) with DRE treated with purified CBD from 2018 to 2022 at five medical centers in Israel., Results: The most common diagnosis was Lennox-Gastaut syndrome (37.4%) followed by Dravet syndrome (16.5%) and tuberous sclerosis complex (16.5%). Median purified CBD dose was 12.5 mg/kg (range 2.5 to 20.0), and median treatment duration was 9.0 months (range 0.5 to 48.0). Most patients (92.2%) had a reduced seizure frequency following treatment initiation; 41.1% had >50% reduction. Fifty-three patients (38.1%) had positive effects: improved alertness (31.7%), improved speech (10.1%), and achievement of new developmental milestones (2.2%). A multivariate linear model assessing predictive factors for seizure reduction demonstrated that patients previously treated with CBD oils, especially those with >50% seizure reduction on prior treatment, were also more likely to have a reduced seizure frequency while they were treated with purified CBD (P = 0.01, P < 0.0001). Development, diagnosis, age, purified CBD dose (0 to 10 mg/kg/day vs 10 to 20 mg/kg/day), and concomitant treatment with clobazam, valproic acid, or everolimus did not affect seizure reduction by purified CBD. The most common adverse events were irritability (20.9%) and drowsiness (12.9%)., Conclusion: Purified CBD is well-tolerated and effective in reducing seizure frequency in children and young adults with DRE., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Michal Tzadok reports a relationship with LivaNova Plc that includes consulting or advisory. Rotem Pollack reports a relationship with Neopharm Israel that includes employment. Statistical analysis and medical writing support were provided by Medistat Ltd and MIMS Pte. Ltd and were funded by Jazz Pharmaceuticals Inc., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2024
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18. Novel homozygous variants in PRORP expand the genotypic spectrum of combined oxidative phosphorylation deficiency 54.
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Smith TB, Rea A, Thomas HB, Thompson K, Oláhová M, Maroofian R, Zamani M, He L, Sadeghian S, Galehdari H, Lotan NS, Gilboa T, Herman KC, McCorvie TJ, Yue WW, Houlden H, Taylor RW, Newman WG, and O'Keefe RT
- Subjects
- Female, Humans, Genotype, Homozygote, RNA, Transfer, Hearing Loss, Sensorineural genetics, Mitochondrial Diseases genetics, Ribonuclease P genetics
- Abstract
Biallelic hypomorphic variants in PRORP have been recently described as causing the autosomal recessive disorder combined oxidative phosphorylation deficiency type 54 (COXPD54). COXPD54 encompasses a phenotypic spectrum of sensorineural hearing loss and ovarian insufficiency (Perrault syndrome) to leukodystrophy. Here, we report three additional families with homozygous missense PRORP variants with pleiotropic phenotypes. Each missense variant altered a highly conserved residue within the metallonuclease domain. In vitro mitochondrial tRNA processing assays with recombinant TRMT10C, SDR5C1 and PRORP indicated two COXPD54-associated PRORP variants, c.1159A>G (p.Thr387Ala) and c.1241C>T (p.Ala414Val), decreased pre-tRNA
Ile cleavage, consistent with both variants impacting tRNA processing. No significant decrease in tRNA processing was observed with PRORP c.1093T>C (p.Tyr365His), which was identified in an individual with leukodystrophy. These data provide independent evidence that PRORP variants are associated with COXPD54 and that the assessment of 5' leader mitochondrial tRNA processing is a valuable assay for the functional analysis and clinical interpretation of novel PRORP variants., (© 2023. The Author(s).)- Published
- 2023
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19. A recurrent de novo variant in NUSAP1 escapes nonsense-mediated decay and leads to microcephaly, epilepsy, and developmental delay.
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Mo A, Paz-Ebstein E, Yanovsky-Dagan S, Lai A, Mor-Shaked H, Gilboa T, Yang E, Shao DD, Walsh CA, and Harel T
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- Humans, Mutation genetics, Epilepsy, Microcephaly genetics, Microcephaly pathology, Nervous System Malformations, Neurodevelopmental Disorders genetics
- Abstract
NUSAP1 encodes a cell cycle-dependent protein with key roles in mitotic progression, spindle formation, and microtubule stability. Both over- and under-expression of NUSAP1 lead to dysregulation of mitosis and impaired cell proliferation. Through exome sequencing and Matchmaker Exchange, we identified two unrelated individuals with the same recurrent, de novo heterozygous variant (NM_016359.5 c.1209C > A; p.(Tyr403Ter)) in NUSAP1. Both individuals had microcephaly, severe developmental delay, brain abnormalities, and seizures. The gene is predicted to be tolerant of heterozygous loss-of-function mutations, and we show that the mutant transcript escapes nonsense mediated decay, suggesting that the mechanism is likely dominant-negative or toxic gain of function. Single-cell RNA-sequencing of an affected individual's post-mortem brain tissue indicated that the NUSAP1 mutant brain contains all main cell lineages, and that the microcephaly could not be attributed to loss of a specific cell type. We hypothesize that pathogenic variants in NUSAP1 lead to microcephaly possibly by an underlying defect in neural progenitor cells., (© 2023 The Authors. Clinical Genetics published by John Wiley & Sons Ltd.)
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- 2023
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20. Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins.
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Ter-Ovanesyan D, Gilboa T, Budnik B, Nikitina A, Whiteman S, Lazarovits R, Trieu W, Kalish D, Church GM, and Walt DR
- Subjects
- Humans, Apolipoprotein B-100 analysis, Apolipoprotein B-100 metabolism, Chromatography, Gel, Enzyme-Linked Immunosorbent Assay, Lipoproteins metabolism, Extracellular Vesicles metabolism
- Abstract
Extracellular vesicles (EVs) are released by all cells into biofluids such as plasma. The separation of EVs from highly abundant free proteins and similarly sized lipoproteins remains technically challenging. We developed a digital ELISA assay based on Single Molecule Array (Simoa) technology for ApoB-100, the protein component of several lipoproteins. Combining this ApoB-100 assay with previously developed Simoa assays for albumin and three tetraspanin proteins found on EVs (Ter-Ovanesyan, Norman et al., 2021), we were able to measure the separation of EVs from both lipoproteins and free proteins. We used these five assays to compare EV separation from lipoproteins using size exclusion chromatography with resins containing different pore sizes. We also developed improved methods for EV isolation based on combining several types of chromatography resins in the same column. We present a simple approach to quantitatively measure the main impurities of EV isolation in plasma and apply this approach to develop novel methods for enriching EVs from human plasma. These methods will enable applications where high-purity EVs are required to both understand EV biology and profile EVs for biomarker discovery., Competing Interests: DT DT has filed IP on methods for EV isolation and analysis, TG TG has filed IP on methods for EV isolation and analysis, BB BB has filed IP on methods for EV isolation and analysis, AN, SW, RL, WT No competing interests declared, DK DK has filed IP on methods for EV isolation and analysis, GC GMC has filed IP on methods for EV isolation and analysis, DW DRW has filed IP on methods for EV isolation and analysis, (© 2023, Ter-Ovanesyan, Gilboa et al.)
- Published
- 2023
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21. Disulfide Bonds Are Not Necessary for Intrinsic TNSALP Activity.
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Melkonian AV, Gilboa T, and Walt DR
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- Animals, Chlorocebus aethiops, Humans, COS Cells, Mutation, Disulfides chemistry, Alkaline Phosphatase chemistry, Alkaline Phosphatase genetics, Alkaline Phosphatase metabolism, Hypophosphatasia genetics, Hypophosphatasia metabolism
- Abstract
Recent developments in single-molecule enzymology (SME) have allowed for the observation of subpopulations present in enzyme ensembles. Tissue-nonspecific alkaline phosphatase (TNSALP), a homodimeric monophosphate esterase central to bone metabolism, has become a model enzyme for SME studies. TNSALP contains two internal disulfide bonds that are critical for its effective dimerization; mutations in its disulfide bonding framework have been reported in patients with hypophosphatasia, a rare disease characterized by impaired bone and tooth mineralization. In this paper, we present the kinetics of these mutants and show that these disulfide bonds are not crucial for TNSALP enzymatic function. This surprising result reveals that the enzyme's active conformation does not rely on its disulfide bonds. We posit that the signs and symptoms seen in hypophosphatasia are likely not primarily due to impaired enzyme function, but rather decreased enzyme expression and trafficking.
- Published
- 2023
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22. The yield of ophthalmoscopy as a screening tool for intracranial pathology in pediatric headache.
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Kawar RA, Gross I, Biro Y, Guzner N, Peyser-Rosenberg M, Azulai S, Mechulam H, Gilboa T, Cohen H, and Hashavya S
- Subjects
- Humans, Child, Retrospective Studies, Ophthalmoscopy, Headache diagnosis, Headache etiology, Papilledema diagnosis, Papilledema pathology, Pseudotumor Cerebri, Brain Neoplasms complications, Brain Neoplasms diagnosis
- Abstract
Headache is a common complaint in children who present at the pediatric emergency department (PED). Serious conditions such as intracranial tumors and idiopathic intracranial hypertension (IIH) should be rapidly ruled out. Ophthalmoscopy for the presence of papilledema has long been considered critical to the assessment of headaches in children; however, the yield of this procedure is poorly validated. This retrospective study implemented a computerized search of the medical records of a single tertiary center to identify all children aged 2-18 years who presented at the PED complaining of headache between 2007 and 2017. The clinical, demographic, radiographic, and laboratory data were analyzed. Of the 948 children aged 2-18 years who presented at the PED complaining of headache, 536 had an ophthalmoscopy examination carried out by an ophthalmologist. Forty-one had papilledema, of whom 7 had an intracranial tumor, 15 had IIH, and 9 had optic nerve head drusen. Of the 495 children without papilledema, 3 had intracranial tumor, and 11 had IIH. The sensitivity and specificity of papilledema for the diagnosis of intracranial tumor were 70% and 93.5%, respectively, with an NPV and PPV of 99.4% and 17.1%, respectively. The sensitivity and specificity of papilledema for the diagnosis of intracranial pathology in general were 61.1% and 96.2%, respectively, with an NPV and PPV of 97.2% and 53.7%, respectively. Conclusion: Assessment by ophthalmoscopy for papilledema in children presenting to the PED with headache had high sensitivity and high specificity, thus reinforcing the importance of ophthalmoscopy as a screening tool in these children. What is Known: • Headache is a common complaint in children. Serious intracranial pathologies need to be rapidly excluded. • Ophthalmoscopy for the presence of papilledema is commonly used as a screening tool for intracranial pathology, but this procedure is poorly validated. What is New: • Ophthalmoscopy for the assessment of papilledema in children who present with headache to the pediatric emergency department is shown to exhibit sensitivity and specificity for the diagnosis of intracranial pathology., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)
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- 2023
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23. Delineating the phenotype and genetic basis of AMPD2-related pontocerebellar hypoplasia.
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Gilboa T, Elefant N, Meiner V, and Hacohen N
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- Humans, Cerebellum abnormalities, Phenotype, Mutation, Cerebellar Diseases genetics, Microcephaly genetics, AMP Deaminase genetics
- Abstract
Pontocerebellar hypoplasia is a group of disorders with a wide range of presentations. We describe here the genetic and phenotypic features of PCH type 9 due to mutations in AMPD2. All patients have severe intellectual disability, and the vast majority manifest abnormal tone, cortical blindness, and microcephaly. Almost all have agenesis of the corpus callosum and severe cerebellar hypoplasia. The course is not progressive, however, few die in the first decade of life. Mutations are spread throughout the gene, and no hot spot can be identified. One of the mutations we report here is the most distal truncating variant known in this gene and is predicted to result in a truncated protein. The phenotype is severe in all cases; thus, no clear genotype-phenotype correlation can be established., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)
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- 2023
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24. Improving pediatric idiopathic intracranial hypertension care: a retrospective cohort study.
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Nitzan-Luques A, Bulkowstein Y, Barnoy N, Aran A, Reif S, and Gilboa T
- Subjects
- Humans, Child, Retrospective Studies, Quality of Life, Headache complications, Pseudotumor Cerebri diagnosis, Pseudotumor Cerebri therapy, Intracranial Hypertension complications
- Abstract
To describe the clinical course and prognosis of pediatric idiopathic intracranial hypertension (IIH) and examine the preferred management setting. IIH is characterized by increased intracranial pressure and is often associated with headaches and visual complaints. IIH is a preventable cause of vision loss in children. Hence, a rapid diagnosis followed by prompt treatment and follow-up is essential. However, standardization of the management of IIH in the pediatric population is not well established. Computerized medical charts of all 82 pediatric (< 18 years) patients diagnosed with IIH between 2007 and 2018 in the metropolitan area of Jerusalem were reviewed. Comparison was made between children followed in a multidisciplinary clinic in tertiary centers and those followed elsewhere. Detailed demographic and clinical data, as well as data regarding the follow-up setting and clinical course of the disease, were collected and analyzed. Recurrent IIH-related hospital returns were selected as a measurable marker for the uncontrolled disease. Recurrent IIH-related hospital return rate was significantly lower and occurred later among children followed by multidisciplinary teams compared to individual experts. Follow-up in multidisciplinary clinics improve the quality of life, and financial burden and may prevent permanent visual impairment in children with IIH., (© 2022. The Author(s).)
- Published
- 2022
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25. What is the optimal duration of home-video-EEG monitoring for patients with <1 seizure per day? A simulation study.
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Vander T, Stroganova T, Doufish D, Eliashiv D, Gilboa T, Medvedovsky M, and Ekstein D
- Abstract
Ambulatory "at home" video-EEG monitoring (HVEM) may offer a more cost-effective and accessible option as compared to traditional inpatient admissions to epilepsy monitoring units. However, home monitoring may not allow for safe tapering of anti-seizure medications (ASM). As a result, longer periods of monitoring may be necessary to capture a sufficient number of the patients' stereotypic seizures. We aimed to quantitatively estimate the necessary length of HVEM corresponding to various diagnostic scenarios in clinical practice. Using available seizure frequency statistics, we estimated the HVEM duration required to capture one, three, or five seizures on different days, by simulating 100,000 annual time-courses of seizure occurrence in adults and children with more than one and <30 seizures per month (89% of adults and 85% of children). We found that the durations of HVEM needed to record 1, 3, or 5 seizures in 80% of children were 2, 5, and 8 weeks (median 2, 12, and 21 days), respectively, and significantly longer in adults -2, 6, and 10 weeks (median 3, 14, and 26 days; p < 10
-10 for all comparisons). Thus, longer HVEM than currently used is needed for expanding its clinical value from diagnosis of nonepileptic or very frequent epileptic events to a presurgical tool for patients with drug-resistant epilepsy. Technical developments and further studies are warranted., Competing Interests: Authors MM and DEk are the inventors of patents related to HVEM development. Authors DEl, MM, and DEk are involved with VIRDA startup company, which develops HVEM systems. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vander, Stroganova, Doufish, Eliashiv, Gilboa, Medvedovsky and Ekstein.)- Published
- 2022
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26. Single-molecule studies reveal method for tuning the heterogeneous activity of alkaline phosphatase.
- Author
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Gilboa T, Ogata AF, Reilly CB, and Walt DR
- Subjects
- Alkaline Phosphatase chemistry, Alkaline Phosphatase metabolism, Animals, COS Cells, Chlorocebus aethiops, Hypophosphatasia
- Abstract
Single-molecule-enzymology (SME) methods have enabled the observation of heterogeneous catalytic activities within a single enzyme population. Heterogeneous activity is hypothesized to originate from conformational changes in the enzyme that result from changes in the local environment leading to catalytically active substates. Here, we use SME to investigate the mechanisms of heterogeneous activity exhibited by tissue nonspecific alkaline phosphatase (TNSALP), which reveals two subpopulations with different catalytic activities. We show the effect of pH and temperature on the distribution of TNSALP activity and confirm the presence of two subpopulations attributed to half- and fully active TNSALP substates. We provide mechanistic insight about protein structure using molecular dynamic simulations and show pH- and temperature-dependent conformational transitions that corroborate experimentally observed changes in TNSALP activity. These results show the utility of SME to understand heterogeneous enzyme activity and demonstrate a simple approach using pH and temperature to tune catalytic activity within an enzyme population., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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27. Ectopic Lymphoid Follicle Formation and Human Seasonal Influenza Vaccination Responses Recapitulated in an Organ-on-a-Chip.
- Author
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Goyal G, Prabhala P, Mahajan G, Bausk B, Gilboa T, Xie L, Zhai Y, Lazarovits R, Mansour A, Kim MS, Patil A, Curran D, Long JM, Sharma S, Junaid A, Cohen L, Ferrante TC, Levy O, Prantil-Baun R, Walt DR, and Ingber DE
- Subjects
- Animals, Antibodies, Viral, Humans, Lab-On-A-Chip Devices, Seasons, Vaccination, Influenza Vaccines, Influenza, Human prevention & control, Tertiary Lymphoid Structures
- Abstract
Lymphoid follicles (LFs) are responsible for generation of adaptive immune responses in secondary lymphoid organs and form ectopically during chronic inflammation. A human model of ectopic LF formation will provide a tool to understand LF development and an alternative to non-human primates for preclinical evaluation of vaccines. Here, it is shown that primary human blood B- and T-lymphocytes autonomously assemble into ectopic LFs when cultured in a 3D extracellular matrix gel within one channel of a two-channel organ-on-a-chip microfluidic device. Superfusion via a parallel channel separated by a microporous membrane is required for LF formation and prevents lymphocyte autoactivation. These germinal center-like LFs contain B cells expressing Activation-Induced Cytidine Deaminase and exhibit plasma cell differentiation upon activation. To explore their utility for seasonal vaccine testing, autologous monocyte-derived dendritic cells are integrated into LF Chips. The human LF chips demonstrate improved antibody responses to split virion influenza vaccination compared to 2D cultures, which are enhanced by a squalene-in-water emulsion adjuvant, and this is accompanied by increases in LF size and number. When inoculated with commercial influenza vaccine, plasma cell formation and production of anti-hemagglutinin IgG are observed, as well as secretion of cytokines similar to vaccinated humans over clinically relevant timescales., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)
- Published
- 2022
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28. High-Sensitivity Single Molecule Array Assays for Pathological Isoforms in Parkinson's Disease.
- Author
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Norman M, Gilboa T, and Walt DR
- Subjects
- Biomarkers cerebrospinal fluid, Cross-Sectional Studies, Humans, Protein Isoforms, Parkinson Disease diagnosis, alpha-Synuclein cerebrospinal fluid
- Abstract
Background: Clinical trials for neurodegenerative diseases are increasingly utilizing measurements of post-translational modifications (PTMs) and pathological isoforms as surrogate markers of target engagement and therapeutic efficacy. These isoforms, however, tend to exist at femtomolar concentrations, well below the detection limit of conventional immunoassays. Therefore, highly sensitive and well-validated assays for these isoforms are needed., Methods: We developed a novel panel of single molecule array assays for pathological isoforms and PTMs implicated in the development and pathophysiology of Parkinson's disease. We validated this panel by measuring these analytes in the cerebrospinal fluid of a cross-sectional cohort of 100 patients with Parkinson's disease and 100 healthy controls., Results: When comparing patients with Parkinson's disease to healthy controls, alpha synuclein, pSer129 alpha synuclein, DJ-1, and C-reactive protein were shown to be reduced in patients with Parkinson's disease while p396 tau and neurofilament light chain were shown to be increased. A random forest analysis produced an area under the curve of 0.70 for the panel., Conclusions: Measurement of post-translational modifications and pathological isoforms in patients with Parkinson's disease improved diagnostic accuracy above that of total protein measurements, demonstrating the potential utility of these assays for monitoring patients in clinical trials., (© American Association for Clinical Chemistry 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)
- Published
- 2022
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29. Severe Acute Respiratory Syndrome Coronavirus 2 Antigens as Targets of Antibody Responses.
- Author
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Ogata AF, Lazarovits R, Uwamanzu-Nna A, Gilboa T, Cheng CA, and Walt DR
- Subjects
- Antibodies, Viral, Antibody Formation, Child, Humans, Immunity, Humoral, COVID-19, SARS-CoV-2
- Abstract
Humoral immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during acute infection and convalescence has been widely studied since March 2020. In this review, the authors summarize literature on humoral responses to SARS-CoV-2 antigens with a focus on spike, nucleocapsid, and the receptor-binding domain as targets of antibody responses. They highlight serologic studies during acute SARS-CoV-2 infection and discuss the clinical relevance of antibody levels in COVID-19 progression. Antibody responses in pediatric COVID-19 patients are also reviewed. Finally, the authors discuss antibody responses during convalescence and their role in protection from SARS-CoV-2 reinfection., Competing Interests: Disclosure D.R. Walt has a financial interest in Quanterix Corporation, a company that develops an ultrasensitive digital immunoassay platform. He is an inventor of the Simoa technology, founder of the company, and serves on its Board of Directors. Dr D.R. Walt’s interests were reviewed and are managed by Brigham and Women’s Hospital and Partners HealthCare in accordance with their conflict-of-interest policies., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2022
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30. Zonulin Antagonist, Larazotide (AT1001), As an Adjuvant Treatment for Multisystem Inflammatory Syndrome in Children: A Case Series.
- Author
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Yonker LM, Swank Z, Gilboa T, Senussi Y, Kenyon V, Papadakis L, Boribong BP, Carroll RW, Walt DR, and Fasano A
- Abstract
Objectives: A recent study suggests that Multisystem Inflammatory Syndrome in Children (MIS-C) is triggered by gastrointestinal breach of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles from the gut lumen into systemic circulation. The virus remains in the gut weeks to months after respiratory infection, causing zonulin release from the intestinal epithelial cells. Zonulin loosens tight junctions, permitting trafficking of highly inflammatory viral particles into circulation. Current MIS-C treatments target the subsequent immune hyperactivation, not the causative loss of mucosal barrier integrity. Larazotide, a zonulin inhibitor, prevents breakdown of tight junctions, limiting antigen trafficking., Design: Children with MIS-C were treated with larazotide as an adjuvant to steroid/intravenous immunoglobulin therapy. Clinical outcomes, SARS-CoV-2 antigenemia, and cytokine profiles are reported. Outcomes were compared with children with MIS-C receiving steroids and/or IVIG therapy alone., Patients: Four children with MIS-C, ages 3-17 years, were enrolled., Interventions: Patients were treated with open label larazotide 10 mcg/kg (maximum 500 mcg/dose) orally four times daily for 21 days., Measurements and Main Results: All four patients tolerated larazotide without adverse effects and displayed reduction in Spike antigenemia to undetectable levels. When compared with 22 children with MIS-C receiving steroids and/or intravenous immunoglobulin therapy alone, larazotide-treated patients reported significantly improved time to resolution of gastrointestinal symptoms ( p = 0.03), and time to clearance of Spike antigenemia ( p = 0.04), plus a trend towards shorter length of stay., Conclusions: Larazotide appears safe and well-tolerated and may offer potential benefit as an adjuvant to immune-targeted therapies. Expansion of clinical trials is urgently needed to ascertain the clinical impact of larazotide on MIS-C., Competing Interests: Dr. Fasano is co-founder and stockholder of Alba Therapeutics. Dr. Walt has a financial interest in Quanterix Corporation, a company that develops an ultrasensitive digital immunoassay platform. He is an inventor of the Simoa technology, a founder of the company and also serves on its Board of Directors. Drs. Walt’s and Fasano’s interests were reviewed and are managed by Brigham and Women's Hospital (to Dr. Walt), Massachusetts General Hospital (to Dr. Fasano), and Partners HealthCare (both) in accordance with their conflict of interest policies. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2022 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine.)
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- 2022
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31. Single-Molecule Enzymology for Diagnostics: Profiling Alkaline Phosphatase Activity in Clinical Samples.
- Author
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Gilboa T, Ogata AF, and Walt DR
- Subjects
- Alkaline Phosphatase metabolism, Biomarkers analysis, Biomarkers metabolism, Cardiovascular Diseases metabolism, Humans, Neoplasms metabolism, Neurodegenerative Diseases metabolism, Optical Imaging, Alkaline Phosphatase analysis, Cardiovascular Diseases diagnostic imaging, Neoplasms diagnostic imaging, Neurodegenerative Diseases diagnostic imaging, Single Molecule Imaging
- Abstract
Enzymes can be used as biomarkers for a variety of diseases. However, profiling enzyme activity in clinical samples is challenging due to the heterogeneity in enzyme activity, and the low abundance of the target enzyme in biofluids. Single-molecule methods can overcome these challenges by providing information on the distribution of enzyme activities in a sample. Here, we describe the concept of using the single-molecule enzymology (SME) method to analyze enzymatic activity in clinical samples. We present recent work focused on measuring alkaline phosphatase isotypes in serum samples using SME. Future work will involve improving and simplifying this technology, and applying it to other enzymes for diagnostics., (© 2021 Wiley-VCH GmbH.)
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- 2022
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32. A SARS-CoV-2 Neutralization Assay Using Single Molecule Arrays.
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Gilboa T, Cohen L, Cheng CA, Lazarovits R, Uwamanzu-Nna A, Han I, Griswold K Jr, Barry N, Thompson DB, Kohman RE, Woolley AE, Karlson EW, and Walt DR
- Subjects
- Angiotensin-Converting Enzyme 2 immunology, Angiotensin-Converting Enzyme 2 metabolism, Antibodies, Viral immunology, Antigen-Antibody Reactions, COVID-19 pathology, COVID-19 virology, COVID-19 Vaccines administration & dosage, COVID-19 Vaccines immunology, Case-Control Studies, Enzyme-Linked Immunosorbent Assay, Humans, SARS-CoV-2 isolation & purification, SARS-CoV-2 metabolism, Spike Glycoprotein, Coronavirus immunology, Spike Glycoprotein, Coronavirus metabolism, Antibodies, Neutralizing immunology, COVID-19 immunology, Neutralization Tests methods, SARS-CoV-2 immunology
- Abstract
Coronavirus disease 2019 (COVID-19) manifests with high clinical variability and warrants sensitive and specific assays to analyze immune responses in infected and vaccinated individuals. Using Single Molecule Arrays (Simoa), we developed an assay to assess antibody neutralization with high sensitivity and multiplexing capabilities based on antibody-mediated blockage of the ACE2-spike interaction. The assay does not require live viruses or cells and can be performed in a biosafety level 2 laboratory within two hours. We used this assay to assess neutralization and antibody levels in patients who died of COVID-19 and patients hospitalized for a short period of time and show that neutralization and antibody levels increase over time. We also adapted the assay for SARS-CoV-2 variants and measured neutralization capacity in pre-pandemic healthy, COVID-19 infected, and vaccinated individuals. This assay is highly adaptable for clinical applications, such as vaccine development and epidemiological studies., (© 2021 Wiley-VCH GmbH.)
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- 2021
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33. Seizures as the main presenting manifestation of acute SARS-CoV-2 infection in children.
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Kurd M, Hashavya S, Benenson S, and Gilboa T
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- Adult, Child, Female, Humans, Infant, Retrospective Studies, SARS-CoV-2, Seizures diagnosis, Seizures drug therapy, Seizures epidemiology, COVID-19, Status Epilepticus
- Abstract
Objectives: To explore the rate, characteristics, risk factors, and prognosis of children presenting with seizures as the main symptom of acute COVID-19 (coronavirus disease 2019)., Methods: We conducted a systematic retrospective study to identify all children who presented to the emergency departments of a tertiary academic medical center between March 1st and December 31st 2020 and had a SARS-CoV-2 infection based on RT-PCR (reverse transcription-polymerase chain reaction) from nasopharyngeal swab. Clinical and demographic data were extracted from the electronic medical records and reviewed., Results: Total of 175 children were diagnosed with acute SARS-CoV-2 infection in the emergency departments during the study period. Of those, 11 presented with seizures. Age ranged from six months to 17 years and 4 were girls. Five presented with status epilepticus and responded to loading doses of anti-seizure medications. Six had fever. Seven had prior history of neurological disorder. Full recovery was the rule., Significance: Unlike in adults, seizures occur early and may be the main manifestation of acute COVID-19 in children. Seizures, including status epilepticus, may occur without fever even in children with no history of epilepsy and are not associated with severe disease. A high index of suspicion is required for early diagnosis thus infection control measures can be taken., (Copyright © 2021. Published by Elsevier Ltd.)
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- 2021
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34. Evaluation of Three Commercial and Two Non-Commercial Immunoassays for the Detection of Prior Infection to SARS-CoV-2.
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Nilles EJ, Karlson EW, Norman M, Gilboa T, Fischinger S, Atyeo C, Zhou G, Bennett CL, Tolan NV, Oganezova K, Walt DR, Alter G, Simmons DP, Schur P, Jarolim P, Woolley AE, and Baden LR
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- Antibodies, Viral, COVID-19 Serological Testing, Humans, Immunoassay, Immunoglobulin G, Immunoglobulin M, Sensitivity and Specificity, COVID-19, SARS-CoV-2
- Abstract
Background: Serological testing provides a record of prior infection with SARS-CoV-2, but assay performance requires independent assessment., Methods: We evaluated 3 commercial (Roche Diagnostics pan-IG, and Epitope Diagnostics IgM and IgG) and 2 non-commercial (Simoa and Ragon/MGH IgG) immunoassays against 1083 unique samples that included 251 PCR-positive and 832 prepandemic samples., Results: The Roche assay registered the highest specificity 99.6% (3/832 false positives), the Ragon/MGH assay 99.5% (4/832), the primary Simoa assay model 99.0% (8/832), and the Epitope IgG and IgM 99.0% (8/830) and 99.5% (4/830), respectively. Overall sensitivities for the Simoa, Roche pan-IG, Epitope IgG, Ragon/MGH IgG, and Epitope IgM were 92.0%, 82.9%, 82.5%, 64.5% and 47.0%, respectively. The Simoa immunoassay demonstrated the highest sensitivity among samples stratified by days postsymptom onset (PSO), <8 days PSO (57.69%) 8-14 days PSO (93.51%), 15-21 days PSO (100%), and > 21 days PSO (95.18%)., Conclusions: All assays demonstrated high to very high specificities while sensitivities were variable across assays., (© American Association for Clinical Chemistry 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)
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- 2021
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35. A Modular Biomaterial Scaffold-Based Vaccine Elicits Durable Adaptive Immunity to Subunit SARS-CoV-2 Antigens.
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Langellotto F, Dellacherie MO, Yeager C, Ijaz H, Yu J, Cheng CA, Dimitrakakis N, Seiler BT, Gebre MS, Gilboa T, Johnson R, Storm N, Bardales S, Graveline A, White D, Tringides CM, Cartwright MJ, Doherty EJ, Honko A, Griffiths A, Barouch DH, Walt DR, and Mooney DJ
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- Adaptive Immunity, Antibodies, Viral, Biocompatible Materials, COVID-19 Vaccines, Humans, COVID-19, SARS-CoV-2
- Abstract
The coronavirus disease 2019 (COVID-19) pandemic demonstrates the importance of generating safe and efficacious vaccines that can be rapidly deployed against emerging pathogens. Subunit vaccines are considered among the safest, but proteins used in these typically lack strong immunogenicity, leading to poor immune responses. Here, a biomaterial COVID-19 vaccine based on a mesoporous silica rods (MSRs) platform is described. MSRs loaded with granulocyte-macrophage colony-stimulating factor (GM-CSF), the toll-like receptor 4 (TLR-4) agonist monophosphoryl lipid A (MPLA), and SARS-CoV-2 viral protein antigens slowly release their cargo and form subcutaneous scaffolds that locally recruit and activate antigen-presenting cells (APCs) for the generation of adaptive immunity. MSR-based vaccines generate robust and durable cellular and humoral responses against SARS-CoV-2 antigens, including the poorly immunogenic receptor binding domain (RBD) of the spike (S) protein. Persistent antibodies over the course of 8 months are found in all vaccine configurations tested and robust in vitro viral neutralization is observed both in a prime-boost and a single-dose regimen. These vaccines can be fully formulated ahead of time or stored lyophilized and reconstituted with an antigen mixture moments before injection, which can facilitate its rapid deployment against emerging SARS-CoV-2 variants or new pathogens. Together, the data show a promising COVID-19 vaccine candidate and a generally adaptable vaccine platform against infectious pathogens., (© 2021 Wiley-VCH GmbH.)
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- 2021
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36. Digital Semiology: A Prototype for Standardized, Computer-Based Semiologic Encoding of Seizures.
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Benoliel T, Gilboa T, Har-Shai Yahav P, Zelker R, Kreigsberg B, Tsizin E, Arviv O, Ekstein D, and Medvedovsky M
- Abstract
Video-EEG monitoring (VEM) is imperative in seizure classification and presurgical assessment of epilepsy patients. Analysis of VEM is currently performed in most institutions using a freeform report, a time-consuming process resulting in a non-standardized report, limiting the use of this essential diagnostic tool. Herein we present a pilot feasibility study of our experience with "Digital Semiology" (DS), a novel seizure encoding software. It allows semiautomated annotation of the videos of suspected events from a predetermined, hierarchal set of options, with highly detailed semiologic descriptions, somatic localization, and timing. In addition, the software's semiologic extrapolation functions identify characteristics of focal seizures and PNES, sequences compatible with a Jacksonian march, and risk factors for SUDEP. Sixty episodes from a mixed adult and pediatric cohort from one level 4 epilepsy center VEM archives were analyzed using DS and the reports were compared with the standard freeform ones, written by the same epileptologists. The behavioral characteristics appearing in the DS and freeform reports overlapped by 78-80%. Encoding of one episode using DS required an average of 18 min 13 s (standard deviation: 14 min and 16 s). The focality function identified 19 out of 43 focal episodes, with a sensitivity of 45.45% (CI 30.39-61.15%) and specificity of 87.50% (CI 61.65-98.45%). The PNES function identified 6 of 12 PNES episodes, with a sensitivity of 50% (95% CI 21.09-78.91%) and specificity of 97.2 (95% CI 88.93-99.95%). Eleven events of GTCS triggered the SUDEP risk alert. Overall, these results show that video recordings of suspected seizures can be encoded using the DS software in a precise manner, offering the added benefit of semiologic alerts. The present study represents an important step toward the formation of an annotated video archive, to be used for machine learning purposes. This will further the goal of automated VEM analysis, ultimately contributing to wider utilization of VEM and therefore to the reduction of the treatment gap in epilepsy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Benoliel, Gilboa, Har-Shai Yahav, Zelker, Kreigsberg, Tsizin, Arviv, Ekstein and Medvedovsky.)
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- 2021
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37. Protective heterologous T cell immunity in COVID-19 induced by the trivalent MMR and Tdap vaccine antigens.
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Mysore V, Cullere X, Settles ML, Ji X, Kattan MW, Desjardins M, Durbin-Johnson B, Gilboa T, Baden LR, Walt DR, Lichtman AH, Jehi L, and Mayadas TN
- Subjects
- Humans, Mumps Vaccine, Receptors, Antigen, T-Cell, Rubella Vaccine, SARS-CoV-2, Spike Glycoprotein, Coronavirus, T-Lymphocytes, COVID-19 prevention & control, Measles, Whooping Cough
- Abstract
Background: T cells control viral infection, promote vaccine durability, and in coronavirus disease 2019 (COVID-19) associate with mild disease. We investigated whether prior measles-mumps-rubella (MMR) or tetanus-diphtheria-pertussis (Tdap) vaccination elicits cross-reactive T cells that mitigate COVID-19., Methods: Antigen-presenting cells (APC) loaded ex vivo with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), MMR, or Tdap antigens and autologous T cells from COVID-19-convalescent participants, uninfected individuals, and COVID-19 mRNA-vaccinated donors were co-cultured. T cell activation and phenotype were detected by interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assays and flow cytometry. ELISAs (enzyme-linked immunosorbant assays) and validation studies identified the APC-derived cytokine(s) driving T cell activation. TCR clonotyping and single-cell RNA sequencing (scRNA-seq) identified cross-reactive T cells and their transcriptional profile. A propensity-weighted analysis of COVID-19 patients estimated the effects of MMR and Tdap vaccination on COVID-19 outcomes., Findings: High correlation was observed between T cell responses to SARS-CoV-2 (spike-S1 and nucleocapsid) and MMR and Tdap proteins in COVID-19-convalescent and -vaccinated individuals. The overlapping T cell population contained an effector memory T cell subset (effector memory re-expressing CD45RA on T cells [T
EMRA ]) implicated in protective, anti-viral immunity, and their detection required APC-derived IL-15, known to sensitize T cells to activation. Cross-reactive TCR repertoires detected in antigen-experienced T cells recognizing SARS-CoV-2, MMR, and Tdap epitopes had TEMRA features. Indices of disease severity were reduced in MMR- or Tdap-vaccinated individuals by 32%-38% and 20%-23%, respectively, among COVID-19 patients., Conclusions: Tdap and MMR memory T cells reactivated by SARS-CoV-2 may provide protection against severe COVID-19., Funding: This study was supported by a National Institutes of Health (R01HL065095, R01AI152522, R01NS097719) donation from Barbara and Amos Hostetter and the Chleck Foundation., Competing Interests: M.L.S., B.D.-J., M.D., L.R.B., A.H.L., X.J., M.W.K., and L.J. declare that they have no competing interests to disclose. D.R.W. has a financial interest in Quanterix Corporation, a company that develops an ultra-sensitive digital immunoassay platform. He is an inventor of the Simoa technology, founder of the company, and serves on its Board of Directors. The anti-SARS-CoV-2 Simoa assays in this publication have been licensed by Brigham and Women’s Hospital to Quanterix Corporation. T.G. receives royalty payments from Brigham and Women’s Hospital for the antibody assay technology. T.N.M. has a financial interest in neuAPC Therapeutics, a company that will develop AAC for the generation of nAPCs and serves as one of its scientific advisors. V.M., X.C., and T.N.M. have a provisional patent on the generation of immunogenic nAPCs and methods of use thereof., (© 2021 The Authors.)- Published
- 2021
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38. Activity of mRNA COVID-19 vaccines in patients with lymphoid malignancies.
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Crombie JL, Sherman AC, Cheng CA, Ryan CE, Zon R, Desjardins M, Baker P, McDonough M, Izaguirre N, Bausk B, Krauss J, Gilboa T, Senussi Y, Walt DR, Davids MS, Brown JR, Armand P, Baden LR, and Issa N
- Subjects
- COVID-19 Vaccines, Humans, RNA, Messenger, SARS-CoV-2, COVID-19, Neoplasms
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- 2021
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39. Fast and Deterministic Fabrication of Sub-5 Nanometer Solid-State Pores by Feedback-Controlled Laser Processing.
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Zvuloni E, Zrehen A, Gilboa T, and Meller A
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- Feedback, Silicon Compounds, Lasers, Nanopores
- Abstract
Nanopores are single-molecule sensors capable of detecting and quantifying a broad range of unlabeled biomolecules including DNA and proteins. Nanopores' generic sensing principle has permitted the development of a vast range of biomolecular applications in genomics and proteomics, including single-molecule DNA sequencing and protein fingerprinting. Owing to their superior mechanical and electrical stability, many of the recent studies involved synthetic nanopores fabricated in thin solid-state membranes such as freestanding silicon nitride. However, to date, one of the bottlenecks in this field is the availability of a fast, reliable, and deterministic fabrication method capable of repeatedly forming small nanopores (i.e., sub 5 nm) in situ . Recently, it was demonstrated that a tightly focused laser beam can induce controlled etching of silicon nitride membranes suspended in buffered aqueous solutions. Herein, we demonstrate that nanopore laser drilling (LD) can produce nanopores deterministically to a prespecified size without user intervention. By optimizing the optical apparatus, and by designing a multistep control algorithm for the LD process, we demonstrate a fully automatic fabrication method for any user-defined nanopore size within minutes. The LD process results in a double bowl-shaped structure having a typical size of the laser point-spread function (PSF) at its openings. Numerical simulations of the characteristic LD nanopore shape provide conductance curves that fit the experimental result and support the idea that the pore is produced at the thinnest area formed by the back-to-back facings bowls. The presented LD fabrication method significantly enhances nanopore fabrication throughput and accuracy and hence can be adopted for a large range of biomolecular sensing applications.
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- 2021
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40. Multisystem inflammatory syndrome in children is driven by zonulin-dependent loss of gut mucosal barrier.
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Yonker LM, Gilboa T, Ogata AF, Senussi Y, Lazarovits R, Boribong BP, Bartsch YC, Loiselle M, Rivas MN, Porritt RA, Lima R, Davis JP, Farkas EJ, Burns MD, Young N, Mahajan VS, Hajizadeh S, Lopez XIH, Kreuzer J, Morris R, Martinez EE, Han I, Griswold K Jr, Barry NC, Thompson DB, Church G, Edlow AG, Haas W, Pillai S, Arditi M, Alter G, Walt DR, and Fasano A
- Subjects
- Adolescent, Antigens, Viral blood, Biomarkers blood, COVID-19 virology, Case-Control Studies, Child, Child, Preschool, Female, Haptoglobins antagonists & inhibitors, Humans, Infant, Infant, Newborn, Intestinal Mucosa drug effects, Intestinal Mucosa virology, Male, Oligopeptides pharmacology, Permeability drug effects, Proof of Concept Study, Protein Precursors antagonists & inhibitors, Protein Precursors blood, Spike Glycoprotein, Coronavirus blood, Spike Glycoprotein, Coronavirus immunology, Systemic Inflammatory Response Syndrome virology, Young Adult, COVID-19 etiology, COVID-19 physiopathology, Haptoglobins physiology, Intestinal Mucosa physiopathology, Protein Precursors physiology, SARS-CoV-2 immunology, SARS-CoV-2 pathogenicity, Systemic Inflammatory Response Syndrome etiology, Systemic Inflammatory Response Syndrome physiopathology
- Abstract
BACKGROUNDWeeks after SARS-CoV-2 infection or exposure, some children develop a severe, life-threatening illness called multisystem inflammatory syndrome in children (MIS-C). Gastrointestinal (GI) symptoms are common in patients with MIS-C, and a severe hyperinflammatory response ensues with potential for cardiac complications. The cause of MIS-C has not been identified to date.METHODSHere, we analyzed biospecimens from 100 children: 19 with MIS-C, 26 with acute COVID-19, and 55 controls. Stools were assessed for SARS-CoV-2 by reverse transcription PCR (RT-PCR), and plasma was examined for markers of breakdown of mucosal barrier integrity, including zonulin. Ultrasensitive antigen detection was used to probe for SARS-CoV-2 antigenemia in plasma, and immune responses were characterized. As a proof of concept, we treated a patient with MIS-C with larazotide, a zonulin antagonist, and monitored the effect on antigenemia and the patient's clinical response.RESULTSWe showed that in children with MIS-C, a prolonged presence of SARS-CoV-2 in the GI tract led to the release of zonulin, a biomarker of intestinal permeability, with subsequent trafficking of SARS-CoV-2 antigens into the bloodstream, leading to hyperinflammation. The patient with MIS-C treated with larazotide had a coinciding decrease in plasma SARS-CoV-2 spike antigen levels and inflammatory markers and a resultant clinical improvement above that achieved with currently available treatments.CONCLUSIONThese mechanistic data on MIS-C pathogenesis provide insight into targets for diagnosing, treating, and preventing MIS-C, which are urgently needed for this increasingly common severe COVID-19-related disease in children.
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- 2021
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41. Correction to: Evaluation of serological lateral flow assays for severe acute respiratory syndrome coronavirus-2.
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Trombetta BA, Kandigian SE, Kitchen RR, Grauwet K, Webb PK, Miller GA, Jennings CG, Jain S, Miller S, Kuo Y, Sweeney T, Gilboa T, Norman M, Simmons DP, Ramirez CE, Bedard M, Fink C, Ko J, De León Peralta EJ, Watts G, Gomez-Rivas E, Davis V, Barilla RM, Wang J, Cunin P, Bates S, Morrison-Smith C, Nicholson B, Wong E, El-Mufti L, Kann M, Bolling A, Fortin B, Ventresca H, Zhou W, Pardo S, Kwock M, Hazra A, Cheng L, Ahmad QR, Toombs JA, Larson R, Pleskow H, Luo NM, Samaha C, Pandya UM, De Silva P, Zhou S, Ganhadeiro Z, Yohannes S, Gay R, Slavik J, Mukerji SS, Jarolim P, Walt DR, Carlyle BC, Ritterhouse LL, and Suliman S
- Published
- 2021
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42. Evaluation of serological lateral flow assays for severe acute respiratory syndrome coronavirus-2.
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Trombetta BA, Kandigian SE, Kitchen RR, Grauwet K, Webb PK, Miller GA, Jennings CG, Jain S, Miller S, Kuo Y, Sweeney T, Gilboa T, Norman M, Simmons DP, Ramirez CE, Bedard M, Fink C, Ko J, De León Peralta EJ, Watts G, Gomez-Rivas E, Davis V, Barilla RM, Wang J, Cunin P, Bates S, Morrison-Smith C, Nicholson B, Wong E, El-Mufti L, Kann M, Bolling A, Fortin B, Ventresca H, Zhou W, Pardo S, Kwock M, Hazra A, Cheng L, Ahmad QR, Toombs JA, Larson R, Pleskow H, Luo NM, Samaha C, Pandya UM, De Silva P, Zhou S, Ganhadeiro Z, Yohannes S, Gay R, Slavik J, Mukerji SS, Jarolim P, Walt DR, Carlyle BC, Ritterhouse LL, and Suliman S
- Subjects
- Adult, Aged, COVID-19 blood, Female, Humans, Limit of Detection, Male, Middle Aged, Predictive Value of Tests, Prevalence, Sensitivity and Specificity, User-Centered Design, User-Computer Interface, Antibodies, Viral blood, COVID-19 diagnosis, COVID-19 Serological Testing methods, Immunoglobulin G blood, Immunoglobulin M blood, SARS-CoV-2 immunology
- Abstract
Background: COVID-19 has resulted in significant morbidity and mortality worldwide. Lateral flow assays can detect anti-Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) antibodies to monitor transmission. However, standardized evaluation of their accuracy and tools to aid in interpreting results are needed., Methods: We evaluated 20 IgG and IgM assays selected from available tests in April 2020. We evaluated the assays' performance using 56 pre-pandemic negative and 56 SARS-CoV-2-positive plasma samples, collected 10-40 days after symptom onset, confirmed by a molecular test and analyzed by an ultra-sensitive immunoassay. Finally, we developed a user-friendly web app to extrapolate the positive predictive values based on their accuracy and local prevalence., Results: Combined IgG + IgM sensitivities ranged from 33.9 to 94.6%, while combined specificities ranged from 92.6 to 100%. The highest sensitivities were detected in Lumiquick for IgG (98.2%), BioHit for both IgM (96.4%), and combined IgG + IgM sensitivity (94.6%). Furthermore, 11 LFAs and 8 LFAs showed perfect specificity for IgG and IgM, respectively, with 15 LFAs showing perfect combined IgG + IgM specificity. Lumiquick had the lowest estimated limit-of-detection (LOD) (0.1 μg/mL), followed by a similar LOD of 1.5 μg/mL for CareHealth, Cellex, KHB, and Vivachek., Conclusion: We provide a public resource of the accuracy of select lateral flow assays with potential for home testing. The cost-effectiveness, scalable manufacturing process, and suitability for self-testing makes LFAs an attractive option for monitoring disease prevalence and assessing vaccine responsiveness. Our web tool provides an easy-to-use interface to demonstrate the impact of prevalence and test accuracy on the positive predictive values.
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- 2021
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43. Abundance of P -glycoprotein and Breast Cancer Resistance Protein Measured by Targeted Proteomics in Human Epileptogenic Brain Tissue.
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Brukner AM, Billington S, Benifla M, Nguyen TB, Han H, Bennett O, Gilboa T, Blatch D, Fellig Y, Volkov O, Unadkat JD, Ekstein D, and Eyal S
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- ATP Binding Cassette Transporter, Subfamily B analysis, ATP Binding Cassette Transporter, Subfamily B metabolism, ATP Binding Cassette Transporter, Subfamily G, Member 2 analysis, Adolescent, Adult, Anticonvulsants therapeutic use, Drug Resistant Epilepsy pathology, Drug Resistant Epilepsy surgery, Female, Hippocampus metabolism, Hippocampus surgery, Humans, Male, Neoplasm Proteins analysis, Young Adult, ATP Binding Cassette Transporter, Subfamily G, Member 2 metabolism, Anticonvulsants pharmacokinetics, Drug Resistant Epilepsy drug therapy, Hippocampus pathology, Neoplasm Proteins metabolism
- Abstract
Our goal was to measure the absolute differential abundance of key drug transporters in human epileptogenic brain tissue and to compare them between patients and at various distances from the epileptogenic zone within the same patient. Transporter protein abundance was quantified in brain tissue homogenates from patients who underwent epilepsy surgery, using targeted proteomics, and correlations with clinical and tissue characteristics were assessed. Fourteen brain samples (including four epileptogenic hippocampal samples) were collected from nine patients. Among the quantifiable drug transporters, the abundance (median, range) ranked: breast cancer resistance protein (ABCG2/BCRP; 0.55, 0.01-3.26 pmol/g tissue) > P -glycoprotein (ABCB1/MDR1; 0.30, 0.02-1.15 pmol/g tissue) > equilibrative nucleoside transporter 1 (SLC29A1/ENT1; 0.06, 0.001-0.35 pmol/g tissue). The ABCB1/ABCG2 ratio (mean 0.27, range 0.08-0.47) was comparable with literature values from nonepileptogenic brain tissue (mean 0.5-0.8). Transporter abundance was lower in the hippocampi than in the less epileptogenic neocortex of the same patients. ABCG2/BCRP and ABCB1/MDR1 expression strongly correlated with that of glucose transporter 1 (SLC2A1/GLUT1) ( r = 0.97, p < 0.001; r = 0.90, p < 0.01, respectively). Low transporter abundance was found in patients with overt vascular pathology, whereas the highest abundance was seen in a sample with normally appearing blood vessels. In conclusion, drug transporter abundance highly varies across patients and between epileptogenic and less epileptogenic brain tissue of the same patient. The strong correlation in abundance of ABCB1/MDR1, ABCG2/BCRP, and SLC2A1/GLUT1 suggests variation in the content of the functional vasculature within the tissue samples. The epileptogenic tissue can be depleted of key drug transport mechanisms, warranting consideration when selecting treatments for patients with drug-resistant epilepsy.
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- 2021
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44. Protective heterologous T cell immunity in COVID-19 induced by MMR and Tdap vaccine antigens.
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Mysore V, Cullere X, Settles ML, Ji X, Kattan MW, Desjardins M, Durbin-Johnson B, Gilboa T, Baden LR, Walt DR, Lichtman A, Jehi L, and Mayadas TN
- Abstract
T cells are critical for control of viral infection and effective vaccination. We investigated whether prior Measles-Mumps-Rubella (MMR) or Tetanus-Diphtheria-pertussis (Tdap) vaccination elicit cross-reactive T cells that mitigate COVID-19. Using co-cultures of antigen presenting cells (APC) loaded with antigens and autologous T cells, we found a high correlation between responses to SARS-CoV-2 (Spike-S1 and Nucleocapsid) and MMR and Tdap vaccine proteins in both SARS-CoV-2 infected individuals and individuals immunized with mRNA-based SARS-CoV-2 vaccines. The overlapping T cell population contained effector memory T cells (TEMRA) previously implicated in anti-viral immunity and their activation required APC-derived IL-15. TCR- and scRNA-sequencing detected cross-reactive clones with TEMRA features among the cells recognizing SARS-CoV-2, MMR and Tdap epitopes. A propensity-weighted analysis of 73,582 COVID-19 patients revealed that severe disease outcomes (hospitalization and transfer to intensive care unit or death) were reduced in MMR or Tdap vaccinated individuals by 38-32% and 23-20% respectively. In summary, SARS-CoV-2 re-activates memory T cells generated by Tdap and MMR vaccines, which may reduce disease severity.
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- 2021
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45. Nanopore Identification of Single Nucleotide Mutations in Circulating Tumor DNA by Multiplexed Ligation.
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Burck N, Gilboa T, Gadi A, Patkin Nehrer M, Schneider RJ, and Meller A
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- Animals, Biomarkers, Tumor genetics, DNA, Neoplasm genetics, Female, Humans, Mice, Mutation, Nucleotides, Breast Neoplasms diagnosis, Breast Neoplasms genetics, Circulating Tumor DNA genetics, Nanopores
- Abstract
Background: Circulating tumor DNAs (ctDNAs) are highly promising cancer biomarkers, potentially applicable for noninvasive liquid biopsy and disease monitoring. However, to date, sequencing of ctDNAs has proven to be challenging primarily due to small sample size and high background of fragmented cell-free DNAs (cfDNAs) derived from normal cells in the circulation, specifically in early stage cancer., Methods: Solid-state nanopores (ssNPs) have recently emerged as a highly efficient tool for single-DNA sensing and analysis. Herein, we present a rapid nanopore genotyping strategy to enable an amplification-free identification and classification of ctDNA mutations. A biochemical ligation detection assay was used for the creation of specific fluorescently-labelled short DNA reporter molecules. Color conjugation with multiple fluorophores enabled a unique multi-color signature for different mutations, offering multiplexing potency. Single-molecule readout of the fluorescent labels was carried out by electro-optical sensing via solid-state nanopores drilled in titanium oxide membranes., Results: As proof of concept, we utilized our method to detect the presence of low-quantity ERBB2 F310S and PIK3Ca H1047R breast cancer mutations from both plasmids and xenograft mice blood samples. We demonstrated an ability to distinguish between a wild type and a mutated sample, and between the different mutations in the same sample., Conclusions: Our method can potentially enable rapid and low cost ctDNA analysis that completely circumvents PCR amplification and library preparation. This approach will thus meet a currently unmet demand in terms of sensitivity, multiplexing and cost, opening new avenues for early diagnosis of cancer., (© American Association for Clinical Chemistry 2021.)
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- 2021
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46. Ultrasensitive Measurement of Both SARS-CoV-2 RNA and Antibodies from Saliva.
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Ter-Ovanesyan D, Gilboa T, Lazarovits R, Rosenthal A, Yu X, Li JZ, Church GM, and Walt DR
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- COVID-19 virology, Female, Humans, Limit of Detection, Male, Real-Time Polymerase Chain Reaction, Reproducibility of Results, SARS-CoV-2 immunology, Antibodies, Viral analysis, COVID-19 diagnosis, COVID-19 Testing methods, RNA, Viral analysis, SARS-CoV-2 genetics, Saliva immunology
- Abstract
Tests for COVID-19 generally measure SARS-CoV-2 viral RNA from nasal swabs or antibodies against the virus from blood. It has been shown, however, that both viral particles and antibodies against those particles are present in saliva, which is more accessible than both swabs and blood. We present methods for highly sensitive measurements of both viral RNA and antibodies from the same saliva sample. We developed an efficient saliva RNA extraction method and combined it with an ultrasensitive antibody test based on single molecule array (Simoa) technology. We apply our test to the saliva of patients who presented to the hospital with COVID-19 symptoms, some of whom tested positive with a conventional RT-qPCR nasopharyngeal swab test. We demonstrate that combining viral RNA detection by RT-qPCR with antibody detection by Simoa identifies more patients as infected than either method alone. Our results demonstrate the utility of combining viral RNA and antibody testing from saliva, a single easily accessible biofluid.
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- 2021
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47. Sequential Protein Capture in Multiplex Single Molecule Arrays: A Strategy for Eliminating Assay Cross-Reactivity.
- Author
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Gilboa T, Maley AM, Ogata AF, Wu C, and Walt DR
- Subjects
- COVID-19 blood, COVID-19 virology, Calibration, Cytokines blood, Humans, Reproducibility of Results, SARS-CoV-2 physiology, Biological Assay, Cross Reactions immunology, Proteins analysis
- Abstract
Measurements of multiple biomolecules within the same biological sample are important for many clinical applications to enable accurate disease diagnosis or classification. These disease-related biomarkers often exist at very low levels in biological fluids, necessitating ultrasensitive measurement methods. Single-molecule arrays (Simoa), a bead-based digital enzyme-linked immunosorbent assay, is the current state of the art for ultrasensitive protein detection and can detect sub-femtomolar protein concentrations, but its ability to achieve high-order multiplexing without cross-reactivity remains a challenge. Here, a sequential protein capture approach for multiplex Simoa assays is implemented to eliminate cross-reactivity between binding reagents by sequentially capturing each protein analyte and then incubating each capture bead with only its corresponding detection antibody. This strategy not only reduces cross-reactivity to background levels and significantly improves measurement accuracies, but also enables higher-order multiplexing. As a proof of concept, the sequential multiplex Simoa assay is used to measure five different cytokines in plasma samples from Coronavirus Disease 2019 (COVID-19) patients. The ultrasensitive sequential multiplex Simoa assays will enable the simultaneous measurements of multiple low-abundance analytes in a time- and cost-effective manner and will prove especially critical in many cases where sample volumes are limited., (© 2020 Wiley-VCH GmbH.)
- Published
- 2021
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48. Ultra-Sensitive Serial Profiling of SARS-CoV-2 Antigens and Antibodies in Plasma to Understand Disease Progression in COVID-19 Patients with Severe Disease.
- Author
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Ogata AF, Maley AM, Wu C, Gilboa T, Norman M, Lazarovits R, Mao CP, Newton G, Chang M, Nguyen K, Kamkaew M, Zhu Q, Gibson TE, Ryan ET, Charles RC, Marasco WA, and Walt DR
- Subjects
- Adult, Aged, Aged, 80 and over, COVID-19 blood, COVID-19 Serological Testing, Coronavirus Nucleocapsid Proteins blood, Female, Hospitalization, Humans, Intensive Care Units, Intubation, Limit of Detection, Male, Middle Aged, Phosphoproteins blood, Prognosis, Protein Subunits blood, Spike Glycoprotein, Coronavirus blood, Antibodies, Viral blood, Antigens, Viral blood, COVID-19 diagnosis, Disease Progression, SARS-CoV-2 chemistry, SARS-CoV-2 immunology
- Abstract
Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 21 million people worldwide since August 16, 2020. Compared to PCR and serology tests, SARS-CoV-2 antigen assays are underdeveloped, despite their potential to identify active infection and monitor disease progression., Methods: We used Single Molecule Array (Simoa) assays to quantitatively detect SARS-CoV-2 spike, S1 subunit, and nucleocapsid antigens in the plasma of patients with coronavirus disease (COVID-19). We studied plasma from 64 patients who were COVID-19 positive, 17 who were COVID-19 negative, and 34 prepandemic patients. Combined with Simoa anti-SARS-CoV-2 serological assays, we quantified changes in 31 SARS-CoV-2 biomarkers in 272 longitudinal plasma samples obtained for 39 patients with COVID-19. Data were analyzed by hierarchical clustering and were compared to longitudinal RT-PCR test results and clinical outcomes., Results: SARS-CoV-2 S1 and N antigens were detectable in 41 out of 64 COVID-19 positive patients. In these patients, full antigen clearance in plasma was observed a mean ± 95% CI of 5 ± 1 days after seroconversion and nasopharyngeal RT-PCR tests reported positive results for 15 ± 5 days after viral-antigen clearance. Correlation between patients with high concentrations of S1 antigen and ICU admission (77%) and time to intubation (within 1 day) was statistically significant., Conclusions: The reported SARS-CoV-2 Simoa antigen assay is the first to detect viral antigens in the plasma of patients who were COVID-19 positive to date. These data show that SARS-CoV-2 viral antigens in the blood are associated with disease progression, such as respiratory failure, in COVID-19 cases with severe disease., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)
- Published
- 2020
- Full Text
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49. Ultrasensitive high-resolution profiling of early seroconversion in patients with COVID-19.
- Author
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Norman M, Gilboa T, Ogata AF, Maley AM, Cohen L, Busch EL, Lazarovits R, Mao CP, Cai Y, Zhang J, Feldman JE, Hauser BM, Caradonna TM, Chen B, Schmidt AG, Alter G, Charles RC, Ryan ET, and Walt DR
- Subjects
- Aged, Aged, 80 and over, Antibodies immunology, Humans, Immunoglobulin G immunology, Male, Middle Aged, Pandemics prevention & control, SARS-CoV-2 immunology, Sensitivity and Specificity, COVID-19 immunology, Immunoassay methods, Seroconversion physiology
- Abstract
Sensitive assays are essential for the accurate identification of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we report a multiplexed assay for the fluorescence-based detection of seroconversion in infected individuals from less than 1 µl of blood, and as early as the day of the first positive nucleic acid test after symptom onset. The assay uses dye-encoded antigen-coated beads to quantify the levels of immunoglobulin G (IgG), IgM and IgA antibodies against four SARS-CoV-2 antigens. A logistic regression model trained using samples collected during the pandemic and samples collected from healthy individuals and patients with respiratory infections before the first outbreak of coronavirus disease 2019 (COVID-19) was 99% accurate in the detection of seroconversion in a blinded validation cohort of samples collected before the pandemic and from patients with COVID-19 five or more days after a positive nasopharyngeal test by PCR with reverse transcription. The high-throughput serological profiling of patients with COVID-19 allows for the interrogation of interactions between antibody isotypes and viral proteins, and should help us to understand the heterogeneity of clinical presentations.
- Published
- 2020
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50. Quantification of mRNA Expression Using Single-Molecule Nanopore Sensing.
- Author
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Rozevsky Y, Gilboa T, van Kooten XF, Kobelt D, Huttner D, Stein U, and Meller A
- Subjects
- Betacoronavirus genetics, Biosensing Techniques standards, HCT116 Cells, Humans, RNA, Messenger genetics, RNA, Messenger metabolism, RNA, Viral genetics, RNA, Viral metabolism, S100 Calcium-Binding Protein A4 genetics, S100 Calcium-Binding Protein A4 metabolism, SARS-CoV-2, Single Molecule Imaging standards, Trans-Activators genetics, Trans-Activators metabolism, Biosensing Techniques methods, Nanopores, RNA, Messenger analysis, Single Molecule Imaging methods
- Abstract
RNA quantification methods are broadly used in life science research and in clinical diagnostics. Currently, real-time reverse transcription polymerase chain reaction (RT-qPCR) is the most common analytical tool for RNA quantification. However, in cases of rare transcripts or inhibiting contaminants in the sample, an extensive amplification could bias the copy number estimation, leading to quantification errors and false diagnosis. Single-molecule techniques may bypass amplification but commonly rely on fluorescence detection and probe hybridization, which introduces noise and limits multiplexing. Here, we introduce reverse transcription quantitative nanopore sensing (RT-qNP), an RNA quantification method that involves synthesis and single-molecule detection of gene-specific cDNAs without the need for purification or amplification. RT-qNP allows us to accurately quantify the relative expression of metastasis-associated genes MACC1 and S100A4 in nonmetastasizing and metastasizing human cell lines, even at levels for which RT-qPCR quantification produces uncertain results. We further demonstrate the versatility of the method by adapting it to quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA against a human reference gene. This internal reference circumvents the need for producing a calibration curve for each measurement, an imminent requirement in RT-qPCR experiments. In summary, we describe a general method to process complicated biological samples with minimal losses, adequate for direct nanopore sensing. Thus, harnessing the sensitivity of label-free single-molecule counting, RT-qNP can potentially detect minute expression levels of RNA biomarkers or viral infection in the early stages of disease and provide accurate amplification-free quantification.
- Published
- 2020
- Full Text
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