32 results on '"Neal JT"'
Search Results
2. Test Characteristics of Cardiac Point-of-Care Ultrasound in Children With Preexisting Cardiac Conditions.
- Author
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Hoffmann RM, Neal JT, Arichai P, Gravel CA, Neuman MI, Monuteaux MC, Levy JA, and Miller AF
- Subjects
- Humans, Child, Point-of-Care Systems, Ultrasonography, Heart, Emergency Service, Hospital, Pericardial Effusion diagnostic imaging, Ventricular Dysfunction, Left diagnostic imaging
- Abstract
Objective: The aim of the study is to assess diagnostic performance of cardiac point-of-care ultrasound (POCUS) performed by pediatric emergency medicine (PEM) physicians in children with preexisting cardiac disease., Methods: We evaluated the use of cardiac POCUS performed by PEM physicians among a convenience sample of children with preexisting cardiac disease presenting to a tertiary care pediatric ED. We assessed patient characteristics and the indication for POCUS. The test characteristics of the sonologist interpretation for the assessment of both pericardial effusion as well as left ventricular systolic dysfunction were compared with expert POCUS review by PEM physicians with POCUS fellowship training., Results: A total of 104 children with preexisting cardiac disease underwent cardiac POCUS examinations between July 2015 and December 2017. Among children with preexisting cardiac disease, structural defects were present in 72%, acquired conditions in 22%, and arrhythmias in 13% of patients. Cardiac POCUS was most frequently obtained because of chest pain (55%), dyspnea (18%), tachycardia (17%), and syncope (10%). Cardiac POCUS interpretation compared with expert review had a sensitivity of 100% (95% confidence interval [CI], 85.7-100) for pericardial effusion and 100% (95% CI, 71.5-100) for left ventricular systolic dysfunction; specificity was 97.5% (95% CI, 91.3.1-99.7) for pericardial effusion and 98.9% (95% CI, 93.8-99.8) for left ventricular systolic dysfunction., Conclusions: Cardiac POCUS demonstrates good sensitivity and specificity in diagnosing pericardial effusion and left ventricular systolic dysfunction in children with preexisting cardiac conditions when technically adequate studies are obtained. These findings support future studies of cardiac POCUS in children with preexisting cardiac conditions presenting to the ED., Competing Interests: Disclosure: The authors declare no conflict of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)
- Published
- 2024
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3. A genome-wide atlas of human cell morphology.
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Ramezani M, Bauman J, Singh A, Weisbart E, Yong J, Lozada M, Way GP, Kavari SL, Diaz C, Haghighi M, Batista TM, Pérez-Schindler J, Claussnitzer M, Singh S, Cimini BA, Blainey PC, Carpenter AE, Jan CH, and Neal JT
- Abstract
A key challenge of the modern genomics era is developing data-driven representations of gene function. Here, we present the first unbiased morphology-based genome-wide perturbation atlas in human cells, containing three genome-scale genotype-phenotype maps comprising >20,000 single-gene CRISPR-Cas9-based knockout experiments in >30 million cells. Our optical pooled cell profiling approach (PERISCOPE) combines a de-stainable high-dimensional phenotyping panel (based on Cell Painting
1,2 ) with optical sequencing of molecular barcodes and a scalable open-source analysis pipeline to facilitate massively parallel screening of pooled perturbation libraries. This approach provides high-dimensional phenotypic profiles of individual cells, while simultaneously enabling interrogation of subcellular processes. Our atlas reconstructs known pathways and protein-protein interaction networks, identifies culture media-specific responses to gene knockout, and clusters thousands of human genes by phenotypic similarity. Using this atlas, we identify the poorly-characterized disease-associated transmembrane protein TMEM251/LYSET as a Golgi-resident protein essential for mannose-6-phosphate-dependent trafficking of lysosomal enzymes, showing the power of these representations. In sum, our atlas and screening technology represent a rich and accessible resource for connecting genes to cellular functions at scale., Competing Interests: Conflicts of interest C.H.J. and J.Y. are employees of Calico Life Sciences LLC. S.S. and A.E.C. serve as scientific advisors for companies that use image-based profiling and Cell Painting (A.E.C: Recursion, SyzOnc, S.S.: Waypoint Bio, Dewpoint Therapeutics) and receive honoraria for occasional talks at pharmaceutical and biotechnology companies. P.C.B. is a consultant to or holds equity in 10X Genomics, General Automation Lab Technologies/Isolation Bio, Celsius Therapeutics, Next Gen Diagnostics, Cache DNA, Concerto Biosciences, Stately, Ramona Optics, Bifrost Biosystems, and Amber Bio. P.C.B.’s laboratory receives research funding from Merck and Genentech for work related to genetic screening. The Broad Institute and MIT may seek to commercialize aspects of this work, and related applications for intellectual property have been filed including WO2019222284A1 In situ cell screening methods and systems. All other authors declare no competing interests.- Published
- 2023
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4. An Atlas of Variant Effects to understand the genome at nucleotide resolution.
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Fowler DM, Adams DJ, Gloyn AL, Hahn WC, Marks DS, Muffley LA, Neal JT, Roth FP, Rubin AF, Starita LM, and Hurles ME
- Subjects
- Humans, Genome, Human, High-Throughput Nucleotide Sequencing, Precision Medicine, Genetic Variation, Genomics
- Abstract
Sequencing has revealed hundreds of millions of human genetic variants, and continued efforts will only add to this variant avalanche. Insufficient information exists to interpret the effects of most variants, limiting opportunities for precision medicine and comprehension of genome function. A solution lies in experimental assessment of the functional effect of variants, which can reveal their biological and clinical impact. However, variant effect assays have generally been undertaken reactively for individual variants only after and, in most cases long after, their first observation. Now, multiplexed assays of variant effect can characterise massive numbers of variants simultaneously, yielding variant effect maps that reveal the function of every possible single nucleotide change in a gene or regulatory element. Generating maps for every protein encoding gene and regulatory element in the human genome would create an 'Atlas' of variant effect maps and transform our understanding of genetics and usher in a new era of nucleotide-resolution functional knowledge of the genome. An Atlas would reveal the fundamental biology of the human genome, inform human evolution, empower the development and use of therapeutics and maximize the utility of genomics for diagnosing and treating disease. The Atlas of Variant Effects Alliance is an international collaborative group comprising hundreds of researchers, technologists and clinicians dedicated to realising an Atlas of Variant Effects to help deliver on the promise of genomics., (© 2023. The Author(s).)
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- 2023
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5. Refining sonographic criteria for paediatric appendicitis: combined effects of age-based appendiceal size and secondary findings.
- Author
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Neal JT, Monuteaux MC, Rangel SJ, Barnewolt CE, and Bachur RG
- Subjects
- Child, Humans, Retrospective Studies, Sensitivity and Specificity, Ultrasonography, Appendicitis diagnostic imaging, Appendix diagnostic imaging
- Abstract
Objective: Appendiceal diameter is a primary sonographic determinant of paediatric appendicitis. We sought to determine if the diagnostic performance of outer appendiceal diameter differs based on age or with the addition of secondary sonographic findings., Methods: We retrospectively reviewed patients aged less than 19 years who presented to the Boston Children's Hospital ED and had an ultrasound (US) for the evaluation of appendicitis between November 2015 and October 2018. Our primary outcome was the presence of appendicitis. We analysed the cases to evaluate the optimal outer appendiceal diameter as a predictor for appendicitis stratified by age (<6, 6 to <11, 11 to <19 years), and with the addition of one or more secondary sonographic findings., Results: Overall, 945 patients met criteria for inclusion, of which 43.9% had appendicitis. Overall, appendiceal diameter as a continuous measure demonstrated excellent test performance across all age groups (area under the curve (AUC) >0.95) but was most predictive of appendicitis in the youngest age group (AUC=0.99 (0.98-1.00)). Although there was no significant difference in optimal diameter threshold between age groups, both 7- and 8-mm thresholds were more predictive than 6 mm across all groups (p<0.001). The addition of individual (particularly appendicolith or echogenic fat) or combinations of secondary sonographic findings increased the diagnostic value for appendicitis above diameter alone., Conclusions: Appendiceal diameter as a continuous measure was more predictive of appendicitis in the youngest group. Across all age groups, the optimal diameter threshold was 7 mm for the diagnosis of paediatric appendicitis. The addition of individual or combination secondary sonographic findings increases diagnostic performance., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)
- Published
- 2022
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6. The Effect of COVID-19 Stay-At-Home Orders on the Rate of Pediatric Foreign Body Ingestions.
- Author
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Neal JT, Monuteaux MC, Porter JJ, and Hudgins JD
- Subjects
- Adolescent, Child, Humans, Child, Preschool, Pandemics, Communicable Disease Control, Retrospective Studies, Emergency Service, Hospital, Eating, COVID-19 epidemiology, Foreign Bodies epidemiology, Foreign Bodies therapy
- Abstract
Background: Foreign body ingestions are a common presentation in the emergency department (ED), particularly in young children., Objective: We sought to determine whether the COVID-19 pandemic lockdowns had an effect on the proportion of foreign body ingestions., Methods: We performed a retrospective review of the Pediatric Health Information System for patients younger than 19 years who were identified by International Classification of Diseases, Tenth Revision codes for foreign body ingestion. We analyzed patients in the following three groups: young children (younger than 5 years), school-aged children (5-12 years), and adolescents (13 years and older), using an interrupted time series analysis. Our primary outcome was the difference in proportion of foreign body ingestions. We compared 1 year after the declaration of the COVID-19 pandemic (March 13, 2020 to March 31, 2021) with the previous 3 years (March 1, 2017 to March 12, 2020)., Results: Total pediatric ED encounters decreased in the post period (p < 0.01); 4902 patients per year presented for foreign body ingestion pre-COVID-19 shutdown vs. 5235 patients per year post-COVID-19 shutdown. In all three age groups (young children, school-age children, and adolescents), there was a higher proportion of foreign body ingestions post-COVID-19 shutdown (p < 0.01, p < 0.01, and p = 0.028, respectively), driven primarily by the decrease in total ED encounters. In the youngest age group (younger than 5 years), there was also a significant increase in slope for foreign body ingestions post-COVID-19 (p = 0.010)., Conclusions: The proportion of foreign body ingestions increased after the declaration of the COVID-19 pandemic, primarily driven by an overall decrease in total ED volume., (Copyright © 2022 Elsevier Inc. All rights reserved.)
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- 2022
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7. Author Correction: Massively parallel phenotyping of coding variants in cancer with Perturb-seq.
- Author
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Ursu O, Neal JT, Shea E, Thakore PI, Jerby-Arnon L, Nguyen L, Dionne D, Diaz C, Bauman J, Mosaad MM, Fagre C, Lo A, McSharry M, Giacomelli AO, Ly SH, Rozenblatt-Rosen O, Hahn WC, Aguirre AJ, Berger AH, Regev A, and Boehm JS
- Published
- 2022
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8. Reducing Pediatric Emergency Department Prescription Errors.
- Author
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Devarajan V, Nadeau NL, Creedon JK, Dribin TE, Lin M, Hirsch AW, Neal JT, Stewart A, Popovsky E, Levitt D, Hoffmann JA, Lee M, Perron C, Shah D, Eisenberg MA, and Hudgins JD
- Subjects
- Anti-Bacterial Agents therapeutic use, Child, Drug Prescriptions, Emergency Service, Hospital, Humans, Medical Order Entry Systems, Medication Errors prevention & control
- Abstract
Background: Prescription errors are a significant cause of iatrogenic harm in the health care system. Pediatric emergency department (ED) patients are particularly vulnerable to error. We sought to decrease prescription errors in an academic pediatric ED by 20% over a 24-month period by implementing identified national best practice guidelines., Methods: From 2017 to 2019, a multidisciplinary, fellow-driven quality improvement (QI) project was conducted using the Model for Improvement. Four key drivers were identified including simplifying the electronic order entry into prescription folders, improving knowledge of dosing by indication, increasing error feedback to prescribers, and creating awareness of common prescription pitfalls. Four interventions were subsequently implemented. Outcome measures included prescription errors per 1000 prescriptions written for all medications and top 10 error-prone antibiotics. Process measures included provider awareness and use of prescription folders; the balancing measure was provider satisfaction. Differences in outcome measures were assessed by statistical process control methodology. Process and balancing measures were analyzed using 1-way analysis of variance and χ2 testing., Results: Before our interventions, 8.6 errors per 1000 prescriptions written were identified, with 62% of errors from the top 10 most error-prone antibiotics. After interventions, error rate per 1000 prescriptions decreased from 8.6 to 4.5 overall and from 20.1 to 8.8 for top 10 error-prone antibiotics. Provider awareness of prescription folders was significantly increased., Conclusion: QI efforts to implement previously defined best practices, including simplifying and standardizing computerized provider order entry (CPOE), significantly reduced prescription errors. Synergistic effect of educational and technological efforts likely contributed to the measured improvement., (Copyright © 2022 by the American Academy of Pediatrics.)
- Published
- 2022
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9. Massively parallel phenotyping of coding variants in cancer with Perturb-seq.
- Author
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Ursu O, Neal JT, Shea E, Thakore PI, Jerby-Arnon L, Nguyen L, Dionne D, Diaz C, Bauman J, Mosaad MM, Fagre C, Lo A, McSharry M, Giacomelli AO, Ly SH, Rozenblatt-Rosen O, Hahn WC, Aguirre AJ, Berger AH, Regev A, and Boehm JS
- Subjects
- Chromosome Mapping, Humans, Phenotype, Lung Neoplasms genetics, Proto-Oncogene Proteins p21(ras) genetics
- Abstract
Genome sequencing studies have identified millions of somatic variants in cancer, but it remains challenging to predict the phenotypic impact of most. Experimental approaches to distinguish impactful variants often use phenotypic assays that report on predefined gene-specific functional effects in bulk cell populations. Here, we develop an approach to functionally assess variant impact in single cells by pooled Perturb-seq. We measured the impact of 200 TP53 and KRAS variants on RNA profiles in over 300,000 single lung cancer cells, and used the profiles to categorize variants into phenotypic subsets to distinguish gain-of-function, loss-of-function and dominant negative variants, which we validated by comparison with orthogonal assays. We discovered that KRAS variants did not merely fit into discrete functional categories, but spanned a continuum of gain-of-function phenotypes, and that their functional impact could not have been predicted solely by their frequency in patient cohorts. Our work provides a scalable, gene-agnostic method for coding variant impact phenotyping, with potential applications in multiple disease settings., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2022
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10. Genome-wide analysis of somatic noncoding mutation patterns in cancer.
- Author
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Dietlein F, Wang AB, Fagre C, Tang A, Besselink NJM, Cuppen E, Li C, Sunyaev SR, Neal JT, and Van Allen EM
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- DNA Mutational Analysis, Gene Expression Regulation, Neoplastic, Humans, Male, Mutation, Oncogenes, Regulatory Sequences, Nucleic Acid, X-Box Binding Protein 1, Neoplasms genetics, Neoplasms pathology, Promoter Regions, Genetic
- Abstract
We established a genome-wide compendium of somatic mutation events in 3949 whole cancer genomes representing 19 tumor types. Protein-coding events captured well-established drivers. Noncoding events near tissue-specific genes, such as ALB in the liver or KLK3 in the prostate, characterized localized passenger mutation patterns and may reflect tumor-cell-of-origin imprinting. Noncoding events in regulatory promoter and enhancer regions frequently involved cancer-relevant genes such as BCL6 , FGFR2 , RAD51B , SMC6 , TERT , and XBP1 and represent possible drivers. Unlike most noncoding regulatory events, XBP1 mutations primarily accumulated outside the gene's promoter, and we validated their effect on gene expression using CRISPR-interference screening and luciferase reporter assays. Broadly, our study provides a blueprint for capturing mutation events across the entire genome to guide advances in biological discovery, therapies, and diagnostics.
- Published
- 2022
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11. Significance of Sonographic Subcentimeter, Subpleural Consolidations in Pediatric Patients Evaluated for Pneumonia.
- Author
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Gravel CA, Neuman MI, Monuteaux MC, Neal JT, Miller AF, and Bachur RG
- Subjects
- Child, Child, Preschool, Humans, Lung diagnostic imaging, Prospective Studies, Ultrasonography, Emergency Medicine, Pneumonia diagnostic imaging, Pneumonia epidemiology
- Abstract
Objectives: To investigate the rates of radiographic pneumonia and clinical outcomes of children with suspected pneumonia and subcentimeter, subpleural consolidations on point-of-care lung ultrasound., Study Design: We enrolled a prospective convenience sample of children aged 6 months to 18 years undergoing chest radiography (CXR) for pneumonia evaluation in a single tertiary-care pediatric emergency department. Point-of-care lung ultrasound was performed by an emergency medicine physician with subsequent expert review. We determined rates of radiographic pneumonia and clinical outcomes in the children with subcentimeter, subpleural consolidations, stratified by the presence of larger (>1 cm) sonographic consolidations. The children were followed prospectively for 2 weeks to identify a delayed diagnosis of pneumonia., Results: A total of 188 patients, with a median age of 5.8 years (IQR, 3.5-11.0 years), were evaluated. Of these patients, 62 (33%) had subcentimeter, subpleural consolidations on lung ultrasound, and 23 (37%) also had larger (>1 cm) consolidations. Patients with subcentimeter, subpleural consolidations and larger consolidations had the highest rates of definite radiographic pneumonia (61%), compared with 21% among children with isolated subcentimeter, subpleural consolidations. Overall, 23 children with isolated subcentimeter, subpleural consolidations (59%) had no evidence of pneumonia on CXR. Among 16 children with isolated subcentimeter, subpleural consolidations and not treated with antibiotics, none had a subsequent pneumonia diagnosis within the 2-week follow-up period., Conclusions: Children with subcentimeter, subpleural consolidations often had radiographic pneumonia; however, this occurred most frequently when subcentimeter, subpleural consolidations were identified in combination with larger consolidations. Isolated subcentimeter, subpleural consolidations in the absence of larger consolidations should not be viewed as synonymous with pneumonia; CXR may provide adjunctive information in these cases., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2022
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12. Development of a Consensus-Based Definition of Focused Assessment With Sonography for Trauma in Children.
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Kornblith AE, Addo N, Plasencia M, Shaahinfar A, Lin-Martore M, Sabbineni N, Gold D, Bellman L, Berant R, Bergmann KR, Brenkert TE, Chen A, Constantine E, Deanehan JK, Dessie A, Elkhunovich M, Fischer J, Gravel CA, Kharasch S, Kwan CW, Lam SHF, Neal JT, Pade KH, Rempell R, Shefrin AE, Sivitz A, Snelling PJ, Tessaro MO, and White W
- Subjects
- Child, Consensus, Delphi Technique, Humans, Reproducibility of Results, Ultrasonography, Focused Assessment with Sonography for Trauma
- Abstract
Importance: The wide variation in the accuracy and reliability of the Focused Assessment With Sonography for Trauma (FAST) and the extended FAST (E-FAST) for children after blunt abdominal trauma reflects user expertise. FAST and E-FAST that are performed by experts tend to be more complete, better quality, and more often clinically valuable., Objective: To develop definitions of a complete, high-quality, and accurate interpretation for the FAST and E-FAST in children with injury using an expert, consensus-based modified Delphi technique., Design, Setting, and Participants: This consensus-based qualitative study was conducted between May 1 to June 30, 2021. It used a scoping review and iterative Delphi technique and involved 2 rounds of online surveys and a live webinar to achieve consensus among a 26-member panel. This panel consisted of international experts in pediatric emergency point-of-care ultrasonography., Main Outcomes and Measures: Definitions of complete, high-quality, and accurate FAST and E-FAST studies for children after injury., Results: Of the 29 invited pediatric FAST experts, 26 (15 men [58%]) agreed to participate in the panel. All 26 panelists completed the 2 rounds of surveys, and 24 (92%) participated in the live and asynchronous online discussions. Consensus was reached on FAST and E-FAST study definitions, and the panelists rated these 5 anatomic views as important and appropriate for a complete FAST: right upper-quadrant abdominal view, left upper-quadrant abdominal view, suprapubic views (transverse and sagittal), and subxiphoid cardiac view. For E-FAST, the same FAST anatomic views with the addition of the lung or pneumothorax view were deemed appropriate and important. In addition, the panelists rated a total of 32 landmarks as important for assessing completeness. Similarly, the panelists rated 14 statements on quality and 20 statements on accurate interpretation as appropriate., Conclusions and Relevance: This qualitative study generated definitions for complete FAST and E-FAST studies with high image quality and accurate interpretation in children with injury. These definitions are similar to those in adults with injury and may be used for future education, quality assurance, and research. Future research may focus on interpretation of trace volumes of abdominal free fluid and the use of serial FAST.
- Published
- 2022
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13. Use of Cardiac Point-of-Care Ultrasound in the Pediatric Emergency Department.
- Author
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Miller AF, Arichai P, Gravel CA, Vieira RL, Neal JT, Neuman MI, Monuteaux MC, and Levy JA
- Subjects
- Child, Emergency Service, Hospital, Humans, Point-of-Care Systems, Ultrasonography, Emergency Medicine, Pediatric Emergency Medicine
- Abstract
Objectives: We sought to describe the test characteristics of cardiac point-of-care ultrasound (POCUS) performed by pediatric emergency medicine (PEM) physicians after structured cardiac POCUS training., Methods: We evaluated the use of clinically indicated cardiac POCUS by PEM physicians in a single tertiary care pediatric emergency department after implementation of a focused cardiac POCUS training curriculum. The test characteristics of the sonologist interpretation were compared with expert POCUS review, by PEM physicians who have completed PEM POCUS fellowship training, for the assessment of both pericardial effusion and left ventricular systolic dysfunction., Results: A total of 1241 cardiac POCUS examinations were performed between July 2015 and December 2017, of which 456 were clinically indicated and underwent expert POCUS review and comprised the study sample. These examinations were performed by 33 different PEM attending sonologists. Chest pain (52%), dyspnea (20%), and tachycardia (18%) were the most common indications for cardiac POCUS. Prevalence of pericardial effusion and global systolic dysfunction based on expert POCUS review were 11% (48/443) and 4% (16/435), respectively. Real-time cardiac POCUS interpretation had a sensitivity and specificity of 100% and 99.5%, respectively, for both pericardial effusion and left ventricular systolic dysfunction when compared with expert POCUS review., Conclusions: Cardiac POCUS is both sensitive and specific for identifying pericardial effusion and left ventricular systolic dysfunction when performed by PEM attendings with focused training., Competing Interests: Disclosure: The authors declare no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)
- Published
- 2022
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14. CHK1 protects oncogenic KRAS-expressing cells from DNA damage and is a target for pancreatic cancer treatment.
- Author
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Klomp JE, Lee YS, Goodwin CM, Papke B, Klomp JA, Waters AM, Stalnecker CA, DeLiberty JM, Drizyte-Miller K, Yang R, Diehl JN, Yin HH, Pierobon M, Baldelli E, Ryan MB, Li S, Peterson J, Smith AR, Neal JT, McCormick AK, Kuo CJ, Counter CM, Petricoin EF 3rd, Cox AD, Bryant KL, and Der CJ
- Subjects
- Animals, Apoptosis, Carcinoma, Pancreatic Ductal genetics, Carcinoma, Pancreatic Ductal metabolism, Carcinoma, Pancreatic Ductal pathology, Cell Proliferation, Checkpoint Kinase 1 genetics, Checkpoint Kinase 1 metabolism, Humans, Mice, Pancreatic Neoplasms genetics, Pancreatic Neoplasms metabolism, Pancreatic Neoplasms pathology, Proto-Oncogene Proteins p21(ras) metabolism, Xenograft Model Antitumor Assays, Carcinoma, Pancreatic Ductal drug therapy, Checkpoint Kinase 1 antagonists & inhibitors, DNA Damage, Mutation, Pancreatic Neoplasms drug therapy, Protein Kinase Inhibitors pharmacology, Proto-Oncogene Proteins p21(ras) genetics
- Abstract
We apply genetic screens to delineate modulators of KRAS mutant pancreatic ductal adenocarcinoma (PDAC) sensitivity to ERK inhibitor treatment, and we identify components of the ATR-CHK1 DNA damage repair (DDR) pathway. Pharmacologic inhibition of CHK1 alone causes apoptotic growth suppression of both PDAC cell lines and organoids, which correlates with loss of MYC expression. CHK1 inhibition also activates ERK and AMPK and increases autophagy, providing a mechanistic basis for increased efficacy of concurrent CHK1 and ERK inhibition and/or autophagy inhibition with chloroquine. To assess how CHK1 inhibition-induced ERK activation promotes PDAC survival, we perform a CRISPR-Cas9 loss-of-function screen targeting direct/indirect ERK substrates and identify RIF1. A key component of non-homologous end joining repair, RIF1 suppression sensitizes PDAC cells to CHK1 inhibition-mediated apoptotic growth suppression. Furthermore, ERK inhibition alone decreases RIF1 expression and phenocopies RIF1 depletion. We conclude that concurrent DDR suppression enhances the efficacy of ERK and/or autophagy inhibitors in KRAS mutant PDAC., Competing Interests: Declaration of interests C.J.D. is a consultant/advisory board member for Anchiano Therapeutics, Deciphera Pharmaceuticals, Mirati Therapeutics, and Revolution Medicines. C.J.D. has received research funding support from SpringWorks Therapeutics, Mirati Therapeutics, and Deciphera Pharmaceuticals and has consulted for Ribometrix, Sanofi, Jazz Therapeutics, Turning Point Therapeutics, and Eli Lilly. A.D.C. has consulted for Eli Lilly and Mirati Therapeutics. E.F.P. and M.P. receive royalties from Avant Diagnostics. E.F.P. is a consultant to and shareholder in Avant Diagnostics, Inc., and Perthera, Inc., and received funding support from Mirati Therapeutics, Genentech, Inc., and Abbvie, Inc., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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15. Does age affect the test performance of secondary sonographic findings for pediatric appendicitis?
- Author
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Neal JT, Monuteaux MC, Rangel SJ, Bachur RG, and Barnewolt CE
- Subjects
- Child, Humans, Retrospective Studies, Sensitivity and Specificity, Ultrasonography, Appendicitis diagnostic imaging, Appendix diagnostic imaging
- Abstract
Background: Secondary sonographic findings of appendicitis can aid image analysis and support diagnosis with and without visualization of an appendix., Objective: We sought to determine if age affected the test performance of secondary findings for pediatric appendicitis., Materials and Methods: We performed a medical record review of emergency department patients younger than 19 years of age who had a sonogram for suspected appendicitis. Our primary patient outcome was appendicitis, as determined by pathology or by image-confirmed perforation/abscess. Our primary analysis was test performance of secondary sonographic findings as recorded by sonographers on the final diagnosis of appendicitis stratified by age (<6 years, 6 to <11 years, 11 to <19 years)., Results: A total of 1,219 patients with suspected appendicitis were evaluated by ultrasound, and 1,147 patients met the criteria for analysis. Of the 1,147 patients, 431 (37.6%) had a final diagnosis of appendicitis. Across all age groups, echogenic fat was the most accurate secondary finding (92.5% [95% confidence interval (CI): 91.0, 94.0]) and free fluid was the least accurate secondary finding (54.7% [95% CI: 51.8, 57.5]). There was no significant difference in the age-stratified test performance of secondary sonographic findings except that (1) appendicolith was a more accurate predictor in patients <6 years old than in the middle group (P<0.001) or the oldest group (P<0.001), and (2) free fluid was a more accurate predictor in the middle group than in the oldest group (P=0.02)., Conclusion: There are no significant differences in the age-stratified test performance of secondary sonographic findings in the prediction of pediatric appendicitis except that appendicolith is more predictive in younger patients., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)
- Published
- 2021
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16. Yield of Plain Radiography in Addition to Ultrasound Among Children with Hip Pain.
- Author
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Gravel CA, Lynn AQ, Hannon M, Miller AF, Neal JT, Neuman MI, and Vieira RL
- Subjects
- Child, Emergency Service, Hospital, Humans, Radiography, Retrospective Studies, Ultrasonography, Pain, Point-of-Care Systems
- Abstract
Background: Children with limp or hip pain often undergo radiographs and ultrasound as part of their initial evaluation. Previous research suggests that hip radiography may have limited utility, and early use of ultrasound may safely reduce the use of radiographs., Objectives: We sought to assess the utility of radiography in addition to ultrasound by evaluating the rate of bony abnormalities present on hip radiographs among children with and without effusion on ultrasound. We also assessed the agreement of point-of-care and Radiology-performed ultrasounds for the detection of effusion., Methods: This is a retrospective cohort study of children presenting to a pediatric emergency department with acute atraumatic limp or hip pain. Data from patients who received both hip ultrasound and hip radiography as part of their evaluation were analyzed. We included both point-of-care and Radiology-performed hip ultrasounds., Results: We identified 134 patients who received both hip ultrasound and hip radiographs. Sixty-eight patients (51%) had a hip effusion present on ultrasound and none of these had bony abnormalities on radiography (0%, 95% confidence interval 0-5.3%). Of the 66 patients (49%) who had no effusion on hip ultrasound, 2 patients were found to have a bony abnormality (3%, 95% confidence interval 0.4-10.5%). For patients who received both point-of-care and Radiology-performed ultrasound, the overall agreement for diagnosis of effusion was 92.6% (kappa = 0.82)., Conclusions: We observed that no children with an effusion on ultrasound had bony pathology on plain radiography, suggesting that the routine performance of hip radiography may not be indicated in all children. Future studies are needed to evaluate the negative predictive value of effusion in larger numbers of patients with known bony abnormalities., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
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17. Massively parallel assessment of human variants with base editor screens.
- Author
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Hanna RE, Hegde M, Fagre CR, DeWeirdt PC, Sangree AK, Szegletes Z, Griffith A, Feeley MN, Sanson KR, Baidi Y, Koblan LW, Liu DR, Neal JT, and Doench JG
- Subjects
- Alleles, BRCA1 Protein genetics, BRCA2 Protein genetics, Base Sequence, Catalytic Domain, Cell Line, Tumor, Humans, Loss of Function Mutation, Mutagenesis genetics, Myeloid Cell Leukemia Sequence 1 Protein genetics, Point Mutation genetics, Poly(ADP-ribose) Polymerases chemistry, Poly(ADP-ribose) Polymerases genetics, Reproducibility of Results, Selection, Genetic, bcl-X Protein genetics, Gene Editing, Genetic Variation, High-Throughput Nucleotide Sequencing
- Abstract
Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here, we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens, identifying known loss-of-function mutations in BRCA1 and BRCA2 with high precision. To demonstrate the utility of base editor screens to probe small molecule-protein interactions, we screen against BH3 mimetics and PARP inhibitors, identifying point mutations that confer drug sensitivity or resistance. We also create a library of single guide RNAs (sgRNAs) predicted to generate 52,034 ClinVar variants in 3,584 genes and conduct screens in the presence of cellular stressors, identifying loss-of-function variants in numerous DNA damage repair genes. We anticipate that this screening approach will be broadly useful to readily and scalably functionalize genetic variants., Competing Interests: Declaration of interests J.G.D. consults for Agios, Maze Therapeutics, Microsoft Research, and Pfizer and consults for, and has equity in, Tango Therapeutics. D.R.L. is a consultant and co-founder of Prime Medicine, Beam Therapeutics, Pairwise Plants, and Editas Medicine, companies that use genome editing, and has previously filed patents on base editing technology. The interests of J.G.D. and D.R.L. were reviewed and are managed by the Broad Institute in accordance with its conflict of interest policies., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
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- View/download PDF
18. A Young Boy with Fever and Grunting.
- Author
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Shapiro DJ and Neal JT
- Abstract
Case Presentation: A 16-month-old boy presented with a temperature of 99°Fahrenheit (F) (down from 102°F at home after antipyretics), grunting, and tachypnea. On examination, he was tachycardic, tachypneic, and ill-appearing with abdominal distention and diffuse tenderness. A plain film abdominal radiograph showed moderate free air, and emergent laparoscopy revealed perforated Meckel's diverticulitis with peritonitis., Discussion: Although tachypnea and grunting in preverbal or nonverbal patients are often considered to be signs of respiratory illness, these findings may reflect intra-abdominal emergencies. Perforated Meckel's diverticulitis is an important differential consideration in patients with pneumoperitoneum.
- Published
- 2021
- Full Text
- View/download PDF
19. Young Girl With Headache.
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Marshall GD and Neal JT
- Subjects
- Astrocytoma complications, Brain Neoplasms complications, Child, Female, Headache diagnostic imaging, Humans, Hydrocephalus diagnostic imaging, Hydrocephalus etiology, Magnetic Resonance Imaging, Tomography, X-Ray Computed, Astrocytoma diagnostic imaging, Brain diagnostic imaging, Brain Neoplasms diagnostic imaging, Headache etiology
- Published
- 2020
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- View/download PDF
20. The Variability of Preferred Infant Lumbar Puncture Insertion Site Between Novice and Experienced Physicians.
- Author
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Neal JT, Levy JA, Rempell RG, and Vieira RL
- Abstract
Background: We sought to determine if vertebral interspace selection for performance of infant lumbar puncture (LP) varies between less experienced trainees and more experienced pediatric emergency medicine (PEM) attending physicians., Methods: We performed an observational prospective study using a convenience sample of infants aged 0 to 12 months presenting to a single emergency department. Trainees with limited LP experience (defined as less than 10 infant LPs performed) marked their preferred LP insertion site with an invisible ultraviolet pen. PEM attending physicians subsequently marked their preferred LP insertion site with a visible pen. A trained sonographer then performed a bedside ultrasound to confirm interspace concordance or discordance. Our primary outcome was the proportion of concordant marked insertion sites., Results: Of the 110 patients enrolled, 102 (92.8%) completed study procedures. Trainee and PEM attending LP interspace markings were concordant in 27% of cases. Trainees marked a preferred interspace below the level of the attending in 55% of patients: 29 (28.4%) marked one spot inferior, 20 (19.6%) marked two spots inferior, and seven (6.9%) marked three spots inferior in relation to the attending., Conclusions: There is variability of preferred LP insertion site based on provider experience. Trainees with limited LP experience tended to mark insertion spaces more caudal than those marked by the attending physicians in an area where the subarachnoid space is slightly smaller., (© 2019 by the Society for Academic Emergency Medicine.)
- Published
- 2019
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- View/download PDF
21. Neonate With Abdominal Distention.
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Lynn AQ, Toce MS, and Neal JT
- Subjects
- Ablation Techniques methods, Endoscopy methods, Humans, Hydronephrosis diagnostic imaging, Infant, Newborn, Infant, Premature, Male, Point-of-Care Testing, Rupture, Spontaneous diagnostic imaging, Treatment Outcome, Urethra diagnostic imaging, Urethra surgery, Urethral Obstruction diagnostic imaging, Urethral Obstruction surgery, Urethra abnormalities, Urethral Obstruction etiology
- Published
- 2019
- Full Text
- View/download PDF
22. Organoid Modeling of the Tumor Immune Microenvironment.
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Neal JT, Li X, Zhu J, Giangarra V, Grzeskowiak CL, Ju J, Liu IH, Chiou SH, Salahudeen AA, Smith AR, Deutsch BC, Liao L, Zemek AJ, Zhao F, Karlsson K, Schultz LM, Metzner TJ, Nadauld LD, Tseng YY, Alkhairy S, Oh C, Keskula P, Mendoza-Villanueva D, De La Vega FM, Kunz PL, Liao JC, Leppert JT, Sunwoo JB, Sabatti C, Boehm JS, Hahn WC, Zheng GXY, Davis MM, and Kuo CJ
- Subjects
- Animals, B7-H1 Antigen immunology, Coculture Techniques, Female, Humans, Immunotherapy, Male, Mice, Mice, Inbred BALB C, Neoplasm Proteins immunology, Neoplasms, Experimental pathology, Neoplasms, Experimental therapy, Organoids pathology, Models, Immunological, Neoplasms, Experimental immunology, Organoids immunology, Receptors, Antigen, T-Cell immunology, Tumor Microenvironment immunology
- Abstract
In vitro cancer cultures, including three-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. Here, an air-liquid interface (ALI) method propagated patient-derived organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages). Robust droplet-based, single-cell simultaneous determination of gene expression and immune repertoire indicated that PDO TILs accurately preserved the original tumor T cell receptor (TCR) spectrum. Crucially, human and murine PDOs successfully modeled immune checkpoint blockade (ICB) with anti-PD-1- and/or anti-PD-L1 expanding and activating tumor antigen-specific TILs and eliciting tumor cytotoxicity. Organoid-based propagation of primary tumor epithelium en bloc with endogenous immune stroma should enable immuno-oncology investigations within the TME and facilitate personalized immunotherapy testing., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
23. Adolescent With Chest Pain.
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Neal JT and Rempell RG
- Subjects
- Adolescent, Angina Pectoris diagnostic imaging, Cardiac Tamponade diagnostic imaging, Humans, Male, Pericardial Effusion diagnostic imaging, Pericarditis diagnostic imaging, Point-of-Care Systems, Ultrasonography, Angina Pectoris etiology, Cardiac Tamponade complications, Pericardial Effusion complications, Pericarditis complications
- Published
- 2017
- Full Text
- View/download PDF
24. The Effect of Bedside Ultrasonographic Skin Marking on Infant Lumbar Puncture Success: A Randomized Controlled Trial.
- Author
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Neal JT, Kaplan SL, Woodford AL, Desai K, Zorc JJ, and Chen AE
- Subjects
- Anatomic Landmarks diagnostic imaging, Emergency Service, Hospital, Female, Humans, Infant, Male, Point-of-Care Systems, Spinal Puncture methods, Ultrasonography, Interventional methods
- Abstract
Study Objective: Lumbar puncture is a commonly performed procedure, although previous studies have documented low rates of successful completion in infants. Ultrasonography can visualize the anatomic landmarks for lumbar puncture and has been shown in some studies to reduce the failure rate of lumbar puncture in adults. We seek to determine whether ultrasonography-assisted site marking increases success for infant lumbar punctures., Methods: This was a prospective, randomized, controlled trial in an academic pediatric emergency department (ED). We enrolled a convenience sample of infants younger than 6 months between June 2014 and February 2016 and randomized them to either a traditional lumbar puncture arm or an ultrasonography-assisted lumbar puncture arm. Infants in the ultrasonography arm received bedside ultrasonography of the spine by one of 3 study sonographers before lumbar puncture, during which the conus medullaris and most appropriate intervertebral space were identified and marked. The lumbar puncture was then performed by the predetermined ED provider. Our primary outcome was successful first-attempt lumbar puncture. Subjects were considered to have a successful lumbar puncture if cerebrospinal fluid was obtained and RBC counts were less than 1,000/mm
3 . All outcomes were assessed by intention-to-treat analysis., Results: One hundred twenty-eight patients were enrolled, with 64 in each arm. No differences between the 2 arms were found in the baseline characteristics of the study subjects and providers, except for sex and first-attempt position. The first-attempt success rate was higher for the ultrasonography arm (58%) versus the traditional arm (31%) (absolute risk difference 27% [95% CI 10% to 43%]). Success within 3 attempts was also higher for the ultrasonography arm (75%) versus the traditional arm (44%) (absolute risk difference 31% [95% CI 15% to 47%]). On average, performing bedside ultrasonography on 4 patients (95% CI 2.1 to 6.6) resulted in 1 additional successful lumbar puncture., Conclusion: Ultrasonography-assisted site marking improved infant lumbar puncture success in a tertiary care pediatric teaching hospital. This method has the potential to reduce unnecessary hospitalizations and exposures to antibiotics in this vulnerable population., (Copyright © 2016 American College of Emergency Physicians. Published by Elsevier Inc. All rights reserved.)- Published
- 2017
- Full Text
- View/download PDF
25. Organoids as Models for Neoplastic Transformation.
- Author
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Neal JT and Kuo CJ
- Subjects
- Animals, Humans, Cell Transformation, Neoplastic pathology, Models, Biological, Neoplasms pathology, Organoids pathology
- Abstract
Cancer models strive to recapitulate the incredible diversity inherent in human tumors. A key challenge in accurate tumor modeling lies in capturing the panoply of homo- and heterotypic cellular interactions within the context of a three-dimensional tissue microenvironment. To address this challenge, researchers have developed organotypic cancer models (organoids) that combine the 3D architecture of in vivo tissues with the experimental facility of 2D cell lines. Here we address the benefits and drawbacks of these systems, as well as their most recent advances. In particular, we focus on the application of such models to the discovery of novel cancer drivers, the study of tumor biology, and the development of novel therapeutic approaches for the treatment of cancer.
- Published
- 2016
- Full Text
- View/download PDF
26. Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture.
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Li X, Nadauld L, Ootani A, Corney DC, Pai RK, Gevaert O, Cantrell MA, Rack PG, Neal JT, Chan CW, Yeung T, Gong X, Yuan J, Wilhelmy J, Robine S, Attardi LD, Plevritis SK, Hung KE, Chen CZ, Ji HP, and Kuo CJ
- Subjects
- Animals, Gastrointestinal Neoplasms pathology, Mice, Mice, Inbred C57BL, Organ Culture Techniques, Cell Transformation, Neoplastic genetics, Gastrointestinal Tract pathology, Oncogenes
- Abstract
The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here we used a single air-liquid interface culture method without modification to engineer oncogenic mutations into primary epithelial and mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia as a result of expression of Kras carrying the G12D mutation (Kras(G12D)), p53 loss or both and readily generated adenocarcinoma after in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, Kras(G12D) and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), as compared to the more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the IGF2 (insulin-like growth factor-2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues.
- Published
- 2014
- Full Text
- View/download PDF
27. H. pylori virulence factor CagA increases intestinal cell proliferation by Wnt pathway activation in a transgenic zebrafish model.
- Author
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Neal JT, Peterson TS, Kent ML, and Guillemin K
- Subjects
- Adenocarcinoma pathology, Aging pathology, Alleles, Animals, Animals, Genetically Modified, Cell Proliferation, Disease Models, Animal, Epithelium metabolism, Epithelium microbiology, Epithelium pathology, Helicobacter Infections metabolism, Helicobacter Infections microbiology, Helicobacter Infections pathology, Hyperplasia, Intestinal Mucosa metabolism, Intestinal Neoplasms metabolism, Intestinal Neoplasms pathology, Phosphorylation, Transcription Factor 4, Transcription Factors metabolism, Transgenes, Tumor Suppressor Protein p53 metabolism, Zebrafish Proteins metabolism, beta Catenin metabolism, Antigens, Bacterial metabolism, Bacterial Proteins metabolism, Helicobacter pylori pathogenicity, Intestines microbiology, Intestines pathology, Virulence Factors metabolism, Wnt Signaling Pathway, Zebrafish microbiology
- Abstract
Infection with Helicobacter pylori is a major risk factor for the development of gastric cancer, and infection with strains carrying the virulence factor CagA significantly increases this risk. To investigate the mechanisms by which CagA promotes carcinogenesis, we generated transgenic zebrafish expressing CagA ubiquitously or in the anterior intestine. Transgenic zebrafish expressing either the wild-type or a phosphorylation-resistant form of CagA exhibited significantly increased rates of intestinal epithelial cell proliferation and showed significant upregulation of the Wnt target genes cyclinD1, axin2 and the zebrafish c-myc ortholog myca. Coexpression of CagA with a loss-of-function allele encoding the β-catenin destruction complex protein Axin1 resulted in a further increase in intestinal proliferation. Coexpression of CagA with a null allele of the key β-catenin transcriptional cofactor Tcf4 restored intestinal proliferation to wild-type levels. These results provide in vivo evidence of Wnt pathway activation by CagA downstream of or in parallel to the β-catenin destruction complex and upstream of Tcf4. Long-term transgenic expression of wild-type CagA, but not the phosphorylation-resistant form, resulted in significant hyperplasia of the adult intestinal epithelium. We further utilized this model to demonstrate that oncogenic cooperation between CagA and a loss-of-function allele of p53 is sufficient to induce high rates of intestinal small cell carcinoma and adenocarcinoma, establishing the utility of our transgenic zebrafish model in the study of CagA-associated gastrointestinal cancers.
- Published
- 2013
- Full Text
- View/download PDF
28. β-Catenin-driven cancers require a YAP1 transcriptional complex for survival and tumorigenesis.
- Author
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Rosenbluh J, Nijhawan D, Cox AG, Li X, Neal JT, Schafer EJ, Zack TI, Wang X, Tsherniak A, Schinzel AC, Shao DD, Schumacher SE, Weir BA, Vazquez F, Cowley GS, Root DE, Mesirov JP, Beroukhim R, Kuo CJ, Goessling W, and Hahn WC
- Subjects
- Animals, Cell Line, Tumor, Colon embryology, Colon metabolism, Colonic Neoplasms pathology, Humans, Inhibitor of Apoptosis Proteins genetics, Mice, Mice, Nude, Proto-Oncogene Proteins c-yes antagonists & inhibitors, Proto-Oncogene Proteins c-yes metabolism, Survivin, Transcription Factors, Transcription, Genetic, YAP-Signaling Proteins, Zebrafish embryology, bcl-X Protein genetics, src-Family Kinases antagonists & inhibitors, Adaptor Proteins, Signal Transducing metabolism, Cell Transformation, Neoplastic, Colonic Neoplasms metabolism, Phosphoproteins metabolism, T-Box Domain Proteins metabolism, beta Catenin metabolism
- Abstract
Wnt/β-catenin signaling plays a key role in the pathogenesis of colon and other cancers; emerging evidence indicates that oncogenic β-catenin regulates several biological processes essential for cancer initiation and progression. To decipher the role of β-catenin in transformation, we classified β-catenin activity in 85 cancer cell lines in which we performed genome-scale loss-of-function screens and found that β-catenin active cancers are dependent on a signaling pathway involving the transcriptional regulator YAP1. Specifically, we found that YAP1 and the transcription factor TBX5 form a complex with β-catenin. Phosphorylation of YAP1 by the tyrosine kinase YES1 leads to localization of this complex to the promoters of antiapoptotic genes, including BCL2L1 and BIRC5. A small-molecule inhibitor of YES1 impeded the proliferation of β-catenin-dependent cancers in both cell lines and animal models. These observations define a β-catenin-YAP1-TBX5 complex essential to the transformation and survival of β-catenin-driven cancers., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
29. Identification of genetic modifiers of CagA-induced epithelial disruption in Drosophila.
- Author
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Reid DW, Muyskens JB, Neal JT, Gaddini GW, Cho LY, Wandler AM, Botham CM, and Guillemin K
- Subjects
- Animals, Animals, Genetically Modified, Disease Models, Animal, Drosophila microbiology, Female, Male, Virulence Factors genetics, Antigens, Bacterial genetics, Antigens, Bacterial metabolism, Bacterial Proteins genetics, Bacterial Proteins metabolism, Epithelial Cells microbiology, Helicobacter pylori pathogenicity, Host-Pathogen Interactions, Virulence Factors metabolism
- Abstract
Helicobacter pylori strains containing the CagA protein are associated with high risk of gastric diseases including atrophic gastritis, peptic ulcers, and gastric cancer. CagA is injected into host cells via a Type IV secretion system where it activates growth factor-like signaling, disrupts cell-cell junctions, and perturbs host cell polarity. Using a transgenic Drosophila model, we have shown that CagA expression disrupts the morphogenesis of epithelial tissues such as the adult eye. Here we describe a genetic screen to identify modifiers of CagA-induced eye defects. We determined that reducing the copy number of genes encoding components of signaling pathways known to be targeted by CagA, such as the epidermal growth factor receptor (EGFR), modified the CagA-induced eye phenotypes. In our screen of just over half the Drosophila genome, we discovered 12 genes that either suppressed or enhanced CagA's disruption of the eye epithelium. Included in this list are genes involved in epithelial integrity, intracellular trafficking, and signal transduction. We investigated the mechanism of one suppressor, encoding the epithelial polarity determinant and junction protein Coracle, which is homologous to the mammalian Protein 4.1. We found that loss of a single copy of coracle improved the organization and integrity of larval retinal epithelia expressing CagA, but did not alter CagA's localization to cell junctions. Loss of a single copy of the coracle antagonist crumbs enhanced CagA-associated disruption of the larval retinal epithelium, whereas overexpression of crumbs suppressed this phenotype. Collectively, these results point to new cellular pathways whose disruption by CagA are likely to contribute to H. pylori-associated disease pathology.
- Published
- 2012
- Full Text
- View/download PDF
30. Epithelial cell proliferation in the developing zebrafish intestine is regulated by the Wnt pathway and microbial signaling via Myd88.
- Author
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Cheesman SE, Neal JT, Mittge E, Seredick BM, and Guillemin K
- Subjects
- Adaptor Proteins, Signal Transducing deficiency, Adaptor Proteins, Signal Transducing genetics, Animals, Axin Protein, Bromodeoxyuridine, Immunohistochemistry, In Situ Hybridization, Intestinal Mucosa cytology, Larva growth & development, Larva microbiology, Microscopy, Confocal, Repressor Proteins deficiency, Repressor Proteins genetics, Signal Transduction physiology, Wnt Proteins metabolism, Aeromonas, Cell Proliferation, Intestinal Mucosa growth & development, Intestinal Mucosa microbiology, Myeloid Differentiation Factor 88 metabolism, Zebrafish growth & development, Zebrafish microbiology, beta Catenin metabolism
- Abstract
Rates of cell proliferation in the vertebrate intestinal epithelium are modulated by intrinsic signaling pathways and extrinsic cues. Here, we report that epithelial cell proliferation in the developing zebrafish intestine is stimulated both by the presence of the resident microbiota and by activation of Wnt signaling. We find that the response to microbial proliferation-promoting signals requires Myd88 but not TNF receptor, implicating host innate immune pathways but not inflammation in the establishment of homeostasis in the developing intestinal epithelium. We show that loss of axin1, a component of the β-catenin destruction complex, results in greater than WT levels of intestinal epithelial cell proliferation. Compared with conventionally reared axin1 mutants, germ-free axin1 mutants exhibit decreased intestinal epithelial cell proliferation, whereas monoassociation with the resident intestinal bacterium Aeromonas veronii results in elevated epithelial cell proliferation. Disruption of β-catenin signaling by deletion of the β-catenin coactivator tcf4 partially decreases the proliferation-promoting capacity of A. veronii. We show that numbers of intestinal epithelial cells with cytoplasmic β-catenin are reduced in the absence of the microbiota in both WT and axin1 mutants and elevated in animals' monoassociated A. veronii. Collectively, these data demonstrate that resident intestinal bacteria enhance the stability of β-catenin in intestinal epithelial cells and promote cell proliferation in the developing vertebrate intestine.
- Published
- 2011
- Full Text
- View/download PDF
31. The Purkinje cell degeneration 5J mutation is a single amino acid insertion that destabilizes Nna1 protein.
- Author
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Chakrabarti L, Neal JT, Miles M, Martinez RA, Smith AC, Sopher BL, and La Spada AR
- Subjects
- Alleles, Amino Acid Sequence, Animals, Aspartic Acid metabolism, Cell Line, Cerebellum metabolism, Cerebellum pathology, GTP-Binding Proteins biosynthesis, Humans, Male, Mice, Molecular Sequence Data, Mutation, Nerve Degeneration genetics, Nerve Degeneration metabolism, RNA, Messenger biosynthesis, Retina metabolism, Retina pathology, Serine-Type D-Ala-D-Ala Carboxypeptidase biosynthesis, Aspartic Acid genetics, GTP-Binding Proteins genetics, GTP-Binding Proteins metabolism, Nerve Degeneration pathology, Purkinje Cells physiology, Serine-Type D-Ala-D-Ala Carboxypeptidase genetics, Serine-Type D-Ala-D-Ala Carboxypeptidase metabolism
- Abstract
In the mouse, Purkinje cell degeneration (pcd) is a recessive mutation characterized by degeneration of cerebellar Purkinje cells, retinal photoreceptors, olfactory bulb mitral neurons, and certain thalamic neurons, and is accompanied by defective spermatogenesis. Previous studies of pcd have led to the identification of Nna1 as the causal gene; however, how loss of Nna1 function results in neurodegeneration remains unresolved. One useful approach for establishing which functional domains of a protein underlie a recessive phenotype has been to determine the genetic basis of the various alleles at the locus of interest. Because none of the pcd alleles analyzed at the time of the identification of Nna1 provided insight into the molecular basis of Nna1 loss-of-function, we obtained a recent pcd remutation--pcd5J, and after determining that its phenotype is comparable to existing pcd severe alleles, we sought its genetic basis by sequencing Nna1. In this article we report that pcd5J results from the insertion of a single GAC triplet encoding an aspartic acid residue at position 775 of Nna1. Although this insertion does not affect Nna1 expression at the RNA level, Nna1pcd-5J protein expression is markedly decreased. Pulse-chase experiments reveal that the aspartic acid insertion dramatically destabilizes Nna1pcd-5J protein, accounting for the observation that pcd5J is a severe allele. The presence of a readily detectable genetic mutation in pcd5J confirms that Nna1 loss-of-function alone underlies the broad pcd phenotype and will facilitate further studies of how Nna1 loss-of-function produces neurodegeneration and defective spermatogenesis in pcd mice.
- Published
- 2006
- Full Text
- View/download PDF
32. The impaired dental practitioner: a dilemma requiring action.
- Author
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Neal JT and Tishk MN
- Subjects
- Ethics, Dental, Humans, Missouri, Dentists, Professional Impairment, Substance-Related Disorders
- Published
- 1990
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