57 results on '"Neff NF"'
Search Results
2. Genetically corrected RAG2-SCID human hematopoietic stem cells restore V(D)J-recombinase and rescue lymphoid deficiency.
- Author
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Pavel-Dinu M, Gardner CL, Nakauchi Y, Kawai T, Delmonte OM, Palterer B, Bosticardo M, Pala F, Viel S, Malech HL, Ghanim HY, Bode NM, Kurgan GL, Detweiler AM, Vakulskas CA, Neff NF, Sheikali A, Menezes ST, Chrobok J, Hernández González EM, Majeti R, Notarangelo LD, and Porteus MH
- Subjects
- Humans, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Hematopoietic Stem Cells metabolism, Nuclear Proteins, Receptors, Antigen, T-Cell, alpha-beta genetics, VDJ Recombinases, Homeodomain Proteins genetics, Homeodomain Proteins metabolism, Severe Combined Immunodeficiency genetics, Severe Combined Immunodeficiency therapy
- Abstract
Abstract: Recombination-activating genes (RAG1 and RAG2) are critical for lymphoid cell development and function by initiating the variable (V), diversity (D), and joining (J) (V(D)J)-recombination process to generate polyclonal lymphocytes with broad antigen specificity. The clinical manifestations of defective RAG1/2 genes range from immune dysregulation to severe combined immunodeficiencies (SCIDs), causing life-threatening infections and death early in life without hematopoietic cell transplantation (HCT). Despite improvements, haploidentical HCT without myeloablative conditioning carries a high risk of graft failure and incomplete immune reconstitution. The RAG complex is only expressed during the G0-G1 phase of the cell cycle in the early stages of T- and B-cell development, underscoring that a direct gene correction might capture the precise temporal expression of the endogenous gene. Here, we report a feasibility study using the CRISPR/Cas9-based "universal gene-correction" approach for the RAG2 locus in human hematopoietic stem/progenitor cells (HSPCs) from healthy donors and RAG2-SCID patient. V(D)J-recombinase activity was restored after gene correction of RAG2-SCID-derived HSPCs, resulting in the development of T-cell receptor (TCR) αβ and γδ CD3+ cells and single-positive CD4+ and CD8+ lymphocytes. TCR repertoire analysis indicated a normal distribution of CDR3 length and preserved usage of the distal TRAV genes. We confirmed the in vivo rescue of B-cell development with normal immunoglobulin M surface expression and a significant decrease in CD56bright natural killer cells. Together, we provide specificity, toxicity, and efficacy data supporting the development of a gene-correction therapy to benefit RAG2-deficient patients., (Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution.)
- Published
- 2024
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3. Prolonged delays in human microbiota transmission after a controlled antibiotic perturbation.
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Xue KS, Walton SJ, Goldman DA, Morrison ML, Verster AJ, Parrott AB, Yu FB, Neff NF, Rosenberg NA, Ross BD, Petrov DA, Huang KC, Good BH, and Relman DA
- Abstract
Humans constantly encounter new microbes, but few become long-term residents of the adult gut microbiome. Classical theories predict that colonization is determined by the availability of open niches, but it remains unclear whether other ecological barriers limit commensal colonization in natural settings. To disentangle these effects, we used a controlled perturbation with the antibiotic ciprofloxacin to investigate the dynamics of gut microbiome transmission in 22 households of healthy, cohabiting adults. Colonization was rare in three-quarters of antibiotic-taking subjects, whose resident strains rapidly recovered in the week after antibiotics ended. In contrast, the remaining antibiotic-taking subjects exhibited lasting responses, with extensive species losses and transient expansions of potential opportunistic pathogens. These subjects experienced elevated rates of commensal colonization, but only after long delays: many new colonizers underwent sudden, correlated expansions months after the antibiotic perturbation. Furthermore, strains that had previously transmitted between cohabiting partners rarely recolonized after antibiotic disruptions, showing that colonization displays substantial historical contingency. This work demonstrates that there remain substantial ecological barriers to colonization even after major microbiome disruptions, suggesting that dispersal interactions and priority effects limit the pace of community change.
- Published
- 2023
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4. KRAS(G12D) drives lepidic adenocarcinoma through stem-cell reprogramming.
- Author
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Juul NH, Yoon JK, Martinez MC, Rishi N, Kazadaeva YI, Morri M, Neff NF, Trope WL, Shrager JB, Sinha R, and Desai TJ
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- Humans, Extracellular Signal-Regulated MAP Kinases metabolism, Adenocarcinoma of Lung genetics, Adenocarcinoma of Lung pathology, Cellular Reprogramming genetics, Lung Neoplasms genetics, Lung Neoplasms pathology, Proto-Oncogene Proteins p21(ras) genetics, Proto-Oncogene Proteins p21(ras) metabolism, Stem Cells metabolism, Stem Cells pathology
- Abstract
Many cancers originate from stem or progenitor cells hijacked by somatic mutations that drive replication, exemplified by adenomatous transformation of pulmonary alveolar epithelial type II (AT2) cells
1 . Here we demonstrate a different scenario: expression of KRAS(G12D) in differentiated AT1 cells reprograms them slowly and asynchronously back into AT2 stem cells that go on to generate indolent tumours. Like human lepidic adenocarcinoma, the tumour cells slowly spread along alveolar walls in a non-destructive manner and have low ERK activity. We find that AT1 and AT2 cells act as distinct cells of origin and manifest divergent responses to concomitant WNT activation and KRAS(G12D) induction, which accelerates AT2-derived but inhibits AT1-derived adenoma proliferation. Augmentation of ERK activity in KRAS(G12D)-induced AT1 cells increases transformation efficiency, proliferation and progression from lepidic to mixed tumour histology. Overall, we have identified a new cell of origin for lung adenocarcinoma, the AT1 cell, which recapitulates features of human lepidic cancer. In so doing, we also uncover a capacity for oncogenic KRAS to reprogram a differentiated and quiescent cell back into its parent stem cell en route to adenomatous transformation. Our work further reveals that irrespective of a given cancer's current molecular profile and driver oncogene, the cell of origin exerts a pervasive and perduring influence on its subsequent behaviour., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2023
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5. Purification and characterization of human neural stem and progenitor cells.
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Liu DD, He JQ, Sinha R, Eastman AE, Toland AM, Morri M, Neff NF, Vogel H, Uchida N, and Weissman IL
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- Mice, Animals, Humans, Neurons, Cell Differentiation physiology, Neuroglia metabolism, Brain, Astrocytes, Neural Stem Cells metabolism
- Abstract
The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers. CD24
- THY1-/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain. THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+ THY1-/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a transcriptomically distinct THY1hi EGFRhi PDGFRA- bipotent glial progenitor cell (GPC), which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. Our study provides a framework for the functional study of distinct cell types in human neurodevelopment., Competing Interests: Declaration of interests D.D.L., J.Q.H., R.S., N.U., and I.L.W. are listed as inventors on a pending patent related to this work. I.L.W. is a cofounder of Bitterroot Bio, Inc. and Pheast, Inc., neither of which are related to the current study. I.L.W. was an initial cofounder and N.U. a former employee of Stem Cells, Inc., but currently are not consultants or employees of it or its successor., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2023
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6. Intermittent fasting induces rapid hepatocyte proliferation to restore the hepatostat in the mouse liver.
- Author
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Sarkar A, Jin Y, DeFelice BC, Logan CY, Yang Y, Anbarchian T, Wu P, Morri M, Neff NF, Nguyen H, Rulifson E, Fish M, Kaye AG, Martínez Jaimes AM, and Nusse R
- Subjects
- Mice, Animals, Liver, Fasting, Hepatocytes, Cell Proliferation, Liver Regeneration, Intermittent Fasting
- Abstract
Nutrient availability fluctuates in most natural populations, forcing organisms to undergo periods of fasting and re-feeding. It is unknown how dietary changes influence liver homeostasis. Here, we show that a switch from ad libitum feeding to intermittent fasting (IF) promotes rapid hepatocyte proliferation. Mechanistically, IF-induced hepatocyte proliferation is driven by the combined action of systemic FGF15 and localized WNT signaling. Hepatocyte proliferation during periods of fasting and re-feeding re-establishes a constant liver-to-body mass ratio, thus maintaining the hepatostat. This study provides the first example of dietary influence on adult hepatocyte proliferation and challenges the widely held view that liver tissue is mostly quiescent unless chemically or mechanically injured., Competing Interests: AS, YJ, BD, CL, YY, TA, PW, MM, NN, HN, ER, MF, AK, AM No competing interests declared, RN Reviewing editor, eLife, (© 2023, Sarkar et al.)
- Published
- 2023
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7. Concordance of MERFISH spatial transcriptomics with bulk and single-cell RNA sequencing.
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Liu J, Tran V, Vemuri VNP, Byrne A, Borja M, Kim YJ, Agarwal S, Wang R, Awayan K, Murti A, Taychameekiatchai A, Wang B, Emanuel G, He J, Haliburton J, Oliveira Pisco A, and Neff NF
- Subjects
- Mice, Animals, In Situ Hybridization, Fluorescence methods, Gene Expression Profiling methods, RNA-Seq, Single-Cell Analysis methods, Transcriptome genetics
- Abstract
Spatial transcriptomics extends single-cell RNA sequencing (scRNA-seq) by providing spatial context for cell type identification and analysis. Imaging-based spatial technologies such as multiplexed error-robust fluorescence in situ hybridization (MERFISH) can achieve single-cell resolution, directly mapping single-cell identities to spatial positions. MERFISH produces a different data type than scRNA-seq, and a technical comparison between the two modalities is necessary to ascertain how to best integrate them. We performed MERFISH on the mouse liver and kidney and compared the resulting bulk and single-cell RNA statistics with those from the Tabula Muris Senis cell atlas and from two Visium datasets. MERFISH quantitatively reproduced the bulk RNA-seq and scRNA-seq results with improvements in overall dropout rates and sensitivity. Finally, we found that MERFISH independently resolved distinct cell types and spatial structure in both the liver and kidney. Computational integration with the Tabula Muris Senis atlas did not enhance these results. We conclude that MERFISH provides a quantitatively comparable method for single-cell gene expression and can identify cell types without the need for computational integration with scRNA-seq atlases., (© 2022 Liu et al.)
- Published
- 2022
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8. Deconvoluting complex correlates of COVID-19 severity with a multi-omic pandemic tracking strategy.
- Author
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Parikh VN, Ioannidis AG, Jimenez-Morales D, Gorzynski JE, De Jong HN, Liu X, Roque J, Cepeda-Espinoza VP, Osoegawa K, Hughes C, Sutton SC, Youlton N, Joshi R, Amar D, Tanigawa Y, Russo D, Wong J, Lauzon JT, Edelson J, Mas Montserrat D, Kwon Y, Rubinacci S, Delaneau O, Cappello L, Kim J, Shoura MJ, Raja AN, Watson N, Hammond N, Spiteri E, Mallempati KC, Montero-Martín G, Christle J, Kim J, Kirillova A, Seo K, Huang Y, Zhao C, Moreno-Grau S, Hershman SG, Dalton KP, Zhen J, Kamm J, Bhatt KD, Isakova A, Morri M, Ranganath T, Blish CA, Rogers AJ, Nadeau K, Yang S, Blomkalns A, O'Hara R, Neff NF, DeBoever C, Szalma S, Wheeler MT, Gates CM, Farh K, Schroth GP, Febbo P, deSouza F, Cornejo OE, Fernandez-Vina M, Kistler A, Palacios JA, Pinsky BA, Bustamante CD, Rivas MA, and Ashley EA
- Subjects
- Genome, Viral, Genome-Wide Association Study, Humans, SARS-CoV-2 genetics, COVID-19 epidemiology, Pandemics
- Abstract
The SARS-CoV-2 pandemic has differentially impacted populations across race and ethnicity. A multi-omic approach represents a powerful tool to examine risk across multi-ancestry genomes. We leverage a pandemic tracking strategy in which we sequence viral and host genomes and transcriptomes from nasopharyngeal swabs of 1049 individuals (736 SARS-CoV-2 positive and 313 SARS-CoV-2 negative) and integrate them with digital phenotypes from electronic health records from a diverse catchment area in Northern California. Genome-wide association disaggregated by admixture mapping reveals novel COVID-19-severity-associated regions containing previously reported markers of neurologic, pulmonary and viral disease susceptibility. Phylodynamic tracking of consensus viral genomes reveals no association with disease severity or inferred ancestry. Summary data from multiomic investigation reveals metagenomic and HLA associations with severe COVID-19. The wealth of data available from residual nasopharyngeal swabs in combination with clinical data abstracted automatically at scale highlights a powerful strategy for pandemic tracking, and reveals distinct epidemiologic, genetic, and biological associations for those at the highest risk., (© 2022. The Author(s).)
- Published
- 2022
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9. The Tabula Sapiens: A multiple-organ, single-cell transcriptomic atlas of humans.
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Jones RC, Karkanias J, Krasnow MA, Pisco AO, Quake SR, Salzman J, Yosef N, Bulthaup B, Brown P, Harper W, Hemenez M, Ponnusamy R, Salehi A, Sanagavarapu BA, Spallino E, Aaron KA, Concepcion W, Gardner JM, Kelly B, Neidlinger N, Wang Z, Crasta S, Kolluru S, Morri M, Tan SY, Travaglini KJ, Xu C, Alcántara-Hernández M, Almanzar N, Antony J, Beyersdorf B, Burhan D, Calcuttawala K, Carter MM, Chan CKF, Chang CA, Chang S, Colville A, Culver RN, Cvijović I, D'Amato G, Ezran C, Galdos FX, Gillich A, Goodyer WR, Hang Y, Hayashi A, Houshdaran S, Huang X, Irwin JC, Jang S, Juanico JV, Kershner AM, Kim S, Kiss B, Kong W, Kumar ME, Kuo AH, Li B, Loeb GB, Lu WJ, Mantri S, Markovic M, McAlpine PL, de Morree A, Mrouj K, Mukherjee S, Muser T, Neuhöfer P, Nguyen TD, Perez K, Puluca N, Qi Z, Rao P, Raquer-McKay H, Schaum N, Scott B, Seddighzadeh B, Segal J, Sen S, Sikandar S, Spencer SP, Steffes LC, Subramaniam VR, Swarup A, Swift M, Van Treuren W, Trimm E, Veizades S, Vijayakumar S, Vo KC, Vorperian SK, Wang W, Weinstein HNW, Winkler J, Wu TTH, Xie J, Yung AR, Zhang Y, Detweiler AM, Mekonen H, Neff NF, Sit RV, Tan M, Yan J, Bean GR, Charu V, Forgó E, Martin BA, Ozawa MG, Silva O, Toland A, Vemuri VNP, Afik S, Awayan K, Botvinnik OB, Byrne A, Chen M, Dehghannasiri R, Gayoso A, Granados AA, Li Q, Mahmoudabadi G, McGeever A, Olivieri JE, Park M, Ravikumar N, Stanley G, Tan W, Tarashansky AJ, Vanheusden R, Wang P, Wang S, Xing G, Dethlefsen L, Ezran C, Gillich A, Hang Y, Ho PY, Irwin JC, Jang S, Leylek R, Liu S, Maltzman JS, Metzger RJ, Phansalkar R, Sasagawa K, Sinha R, Song H, Swarup A, Trimm E, Veizades S, Wang B, Beachy PA, Clarke MF, Giudice LC, Huang FW, Huang KC, Idoyaga J, Kim SK, Kuo CS, Nguyen P, Rando TA, Red-Horse K, Reiter J, Relman DA, Sonnenburg JL, Wu A, Wu SM, and Wyss-Coray T
- Subjects
- B-Lymphocytes metabolism, Humans, T-Lymphocytes metabolism, Atlases as Topic, Cells metabolism, Organ Specificity genetics, RNA Splicing, Single-Cell Analysis, Transcriptome
- Abstract
Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type-specific RNA splicing was discovered and analyzed across tissues within an individual.
- Published
- 2022
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10. Molecular hallmarks of heterochronic parabiosis at single-cell resolution.
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Pálovics R, Keller A, Schaum N, Tan W, Fehlmann T, Borja M, Kern F, Bonanno L, Calcuttawala K, Webber J, McGeever A, Luo J, Pisco AO, Karkanias J, Neff NF, Darmanis S, Quake SR, and Wyss-Coray T
- Subjects
- Adipocytes, Aging genetics, Electron Transport genetics, Hematopoietic Stem Cells, Hepatocytes, Mesenchymal Stem Cells, Mitochondria, Organ Specificity genetics, RNA-Seq, Rejuvenation, Parabiosis, Single-Cell Analysis
- Abstract
The ability to slow or reverse biological ageing would have major implications for mitigating disease risk and maintaining vitality
1 . Although an increasing number of interventions show promise for rejuvenation2 , their effectiveness on disparate cell types across the body and the molecular pathways susceptible to rejuvenation remain largely unexplored. Here we performed single-cell RNA sequencing on 20 organs to reveal cell-type-specific responses to young and aged blood in heterochronic parabiosis. Adipose mesenchymal stromal cells, haematopoietic stem cells and hepatocytes are among those cell types that are especially responsive. On the pathway level, young blood invokes new gene sets in addition to reversing established ageing patterns, with the global rescue of genes encoding electron transport chain subunits pinpointing a prominent role of mitochondrial function in parabiosis-mediated rejuvenation. We observed an almost universal loss of gene expression with age that is largely mimicked by parabiosis: aged blood reduces global gene expression, and young blood restores it in select cell types. Together, these data lay the groundwork for a systemic understanding of the interplay between blood-borne factors and cellular integrity., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2022
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11. Establishment and characterization of stable, diverse, fecal-derived in vitro microbial communities that model the intestinal microbiota.
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Aranda-Díaz A, Ng KM, Thomsen T, Real-Ramírez I, Dahan D, Dittmar S, Gonzalez CG, Chavez T, Vasquez KS, Nguyen TH, Yu FB, Higginbottom SK, Neff NF, Elias JE, Sonnenburg JL, and Huang KC
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- Animals, Bacteria, Bacteroides, Feces microbiology, Humans, Mice, Gastrointestinal Microbiome, Microbiota
- Abstract
Efforts to probe the role of the gut microbiota in disease would benefit from a system in which patient-derived bacterial communities can be studied at scale. We addressed this by validating a strategy to propagate phylogenetically complex, diverse, stable, and highly reproducible stool-derived communities in vitro. We generated hundreds of in vitro communities cultured from diverse stool samples in various media; certain media generally preserved inoculum composition, and inocula from different subjects yielded source-specific community compositions. Upon colonization of germ-free mice, community composition was maintained, and the host proteome resembled the host from which the community was derived. Treatment with ciprofloxacin in vivo increased susceptibility to Salmonella invasion in vitro, and the in vitro response to ciprofloxacin was predictive of compositional changes observed in vivo, including the resilience and sensitivity of each Bacteroides species. These findings demonstrate that stool-derived in vitro communities can serve as a powerful system for microbiota research., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)
- Published
- 2022
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12. Early prediction of preeclampsia in pregnancy with cell-free RNA.
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Moufarrej MN, Vorperian SK, Wong RJ, Campos AA, Quaintance CC, Sit RV, Tan M, Detweiler AM, Mekonen H, Neff NF, Baruch-Gravett C, Litch JA, Druzin ML, Winn VD, Shaw GM, Stevenson DK, and Quake SR
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- Blood Pressure, Female, Humans, Mothers, Pregnancy, Transcriptome, Cell-Free Nucleic Acids blood, Cell-Free Nucleic Acids genetics, Early Diagnosis, Pre-Eclampsia diagnosis, Pre-Eclampsia genetics, RNA blood, RNA genetics
- Abstract
Liquid biopsies that measure circulating cell-free RNA (cfRNA) offer an opportunity to study the development of pregnancy-related complications in a non-invasive manner and to bridge gaps in clinical care
1-4 . Here we used 404 blood samples from 199 pregnant mothers to identify and validate cfRNA transcriptomic changes that are associated with preeclampsia, a multi-organ syndrome that is the second largest cause of maternal death globally5 . We find that changes in cfRNA gene expression between normotensive and preeclamptic mothers are marked and stable early in gestation, well before the onset of symptoms. These changes are enriched for genes specific to neuromuscular, endothelial and immune cell types and tissues that reflect key aspects of preeclampsia physiology6-9 , suggest new hypotheses for disease progression and correlate with maternal organ health. This enabled the identification and independent validation of a panel of 18 genes that when measured between 5 and 16 weeks of gestation can form the basis of a liquid biopsy test that would identify mothers at risk of preeclampsia long before clinical symptoms manifest themselves. Tests based on these observations could help predict and manage who is at risk for preeclampsia-an important objective for obstetric care10,11 ., (© 2022. The Author(s).)- Published
- 2022
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13. Aged skeletal stem cells generate an inflammatory degenerative niche.
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Ambrosi TH, Marecic O, McArdle A, Sinha R, Gulati GS, Tong X, Wang Y, Steininger HM, Hoover MY, Koepke LS, Murphy MP, Sokol J, Seo EY, Tevlin R, Lopez M, Brewer RE, Mascharak S, Lu L, Ajanaku O, Conley SD, Seita J, Morri M, Neff NF, Sahoo D, Yang F, Weissman IL, Longaker MT, and Chan CKF
- Subjects
- Animals, Bone Morphogenetic Protein 2 metabolism, Bone Regeneration, Cell Lineage, Female, Fracture Healing, Hematopoiesis, Macrophage Colony-Stimulating Factor metabolism, Male, Mice, Myeloid Cells cytology, Osteoclasts cytology, Rejuvenation, Aging pathology, Bone and Bones pathology, Cellular Senescence, Inflammation pathology, Stem Cell Niche, Stem Cells pathology
- Abstract
Loss of skeletal integrity during ageing and disease is associated with an imbalance in the opposing actions of osteoblasts and osteoclasts
1 . Here we show that intrinsic ageing of skeletal stem cells (SSCs)2 in mice alters signalling in the bone marrow niche and skews the differentiation of bone and blood lineages, leading to fragile bones that regenerate poorly. Functionally, aged SSCs have a decreased bone- and cartilage-forming potential but produce more stromal lineages that express high levels of pro-inflammatory and pro-resorptive cytokines. Single-cell RNA-sequencing studies link the functional loss to a diminished transcriptomic diversity of SSCs in aged mice, which thereby contributes to the transformation of the bone marrow niche. Exposure to a youthful circulation through heterochronic parabiosis or systemic reconstitution with young haematopoietic stem cells did not reverse the diminished osteochondrogenic activity of aged SSCs, or improve bone mass or skeletal healing parameters in aged mice. Conversely, the aged SSC lineage promoted osteoclastic activity and myeloid skewing by haematopoietic stem and progenitor cells, suggesting that the ageing of SSCs is a driver of haematopoietic ageing. Deficient bone regeneration in aged mice could only be returned to youthful levels by applying a combinatorial treatment of BMP2 and a CSF1 antagonist locally to fractures, which reactivated aged SSCs and simultaneously ablated the inflammatory, pro-osteoclastic milieu. Our findings provide mechanistic insights into the complex, multifactorial mechanisms that underlie skeletal ageing and offer prospects for rejuvenating the aged skeletal system., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2021
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14. Distinct skeletal stem cell types orchestrate long bone skeletogenesis.
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Ambrosi TH, Sinha R, Steininger HM, Hoover MY, Murphy MP, Koepke LS, Wang Y, Lu WJ, Morri M, Neff NF, Weissman IL, Longaker MT, and Chan CK
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- Adipose Tissue, Animals, Bone Marrow, Bone Marrow Cells, Gene Expression Regulation, Developmental, Hematopoietic Stem Cells metabolism, Male, Mice, Mice, Inbred C57BL, Pericytes, Stem Cell Niche, Transcriptome, Bone Development, Bone and Bones metabolism, Stromal Cells metabolism
- Abstract
Skeletal stem and progenitor cell populations are crucial for bone physiology. Characterization of these cell types remains restricted to heterogenous bulk populations with limited information on whether they are unique or overlap with previously characterized cell types. Here we show, through comprehensive functional and single-cell transcriptomic analyses, that postnatal long bones of mice contain at least two types of bone progenitors with bona fide skeletal stem cell (SSC) characteristics. An early osteochondral SSC (ocSSC) facilitates long bone growth and repair, while a second type, a perivascular SSC (pvSSC), co-emerges with long bone marrow and contributes to shape the hematopoietic stem cell niche and regenerative demand. We establish that pvSSCs, but not ocSSCs, are the origin of bone marrow adipose tissue. Lastly, we also provide insight into residual SSC heterogeneity as well as potential crosstalk between the two spatially distinct cell populations. These findings comprehensively address previously unappreciated shortcomings of SSC research., Competing Interests: TA, RS, HS, MH, MM, LK, YW, WL, MM, NN, IW, ML, CC No competing interests declared, (© 2021, Ambrosi et al.)
- Published
- 2021
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15. Sexual and asexual development: two distinct programs producing the same tunicate.
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Kowarsky M, Anselmi C, Hotta K, Burighel P, Zaniolo G, Caicci F, Rosental B, Neff NF, Ishizuka KJ, Palmeri KJ, Okamoto J, Gordon T, Weissman IL, Quake SR, Manni L, and Voskoboynik A
- Subjects
- Animals, Embryonic Development genetics, Reproduction, Asexual genetics, Sexual Development genetics, Urochordata genetics
- Abstract
Colonial tunicates are the only chordate that possess two distinct developmental pathways to produce an adult body: either sexually through embryogenesis or asexually through a stem cell-mediated renewal termed blastogenesis. Using the colonial tunicate Botryllus schlosseri, we combine transcriptomics and microscopy to build an atlas of the molecular and morphological signatures at each developmental stage for both pathways. The general molecular profiles of these processes are largely distinct. However, the relative timing of organogenesis and ordering of tissue-specific gene expression are conserved. By comparing the developmental pathways of B. schlosseri with other chordates, we identify hundreds of putative transcription factors with conserved temporal expression. Our findings demonstrate that convergent morphology need not imply convergent molecular mechanisms but that it showcases the importance that tissue-specific stem cells and transcription factors play in producing the same mature body through different pathways., Competing Interests: Declarations of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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16. Rapid deployment of SARS-CoV-2 testing: The CLIAHUB.
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Crawford ED, Acosta I, Ahyong V, Anderson EC, Arevalo S, Asarnow D, Axelrod S, Ayscue P, Azimi CS, Azumaya CM, Bachl S, Bachmutsky I, Bhaduri A, Brown JB, Batson J, Behnert A, Boileau RM, Bollam SR, Bonny AR, Booth D, Borja MJB, Brown D, Buie B, Burnett CE, Byrnes LE, Cabral KA, Cabrera JP, Caldera S, Canales G, Castañeda GR, Chan AP, Chang CR, Charles-Orszag A, Cheung C, Chio U, Chow ED, Citron YR, Cohen A, Cohn LB, Chiu C, Cole MA, Conrad DN, Constantino A, Cote A, Crayton-Hall T, Darmanis S, Detweiler AM, Dial RL, Dong S, Duarte EM, Dynerman D, Egger R, Fanton A, Frumm SM, Fu BXH, Garcia VE, Garcia J, Gladkova C, Goldman M, Gomez-Sjoberg R, Gordon MG, Grove JCR, Gupta S, Haddjeri-Hopkins A, Hadley P, Haliburton J, Hao SL, Hartoularos G, Herrera N, Hilberg M, Ho KYE, Hoppe N, Hosseinzadeh S, Howard CJ, Hussmann JA, Hwang E, Ingebrigtsen D, Jackson JR, Jowhar ZM, Kain D, Kim JYS, Kistler A, Kreutzfeld O, Kulsuptrakul J, Kung AF, Langelier C, Laurie MT, Lee L, Leng K, Leon KE, Leonetti MD, Levan SR, Li S, Li AW, Liu J, Lubin HS, Lyden A, Mann J, Mann S, Margulis G, Marquez DM, Marsh BP, Martyn C, McCarthy EE, McGeever A, Merriman AF, Meyer LK, Miller S, Moore MK, Mowery CT, Mukhtar T, Mwakibete LL, Narez N, Neff NF, Osso LA, Oviedo D, Peng S, Phelps M, Phong K, Picard P, Pieper LM, Pincha N, Pisco AO, Pogson A, Pourmal S, Puccinelli RR, Puschnik AS, Rackaityte E, Raghavan P, Raghavan M, Reese J, Replogle JM, Retallack H, Reyes H, Rose D, Rosenberg MF, Sanchez-Guerrero E, Sattler SM, Savy L, See SK, Sellers KK, Serpa PH, Sheehy M, Sheu J, Silas S, Streithorst JA, Strickland J, Stryke D, Sunshine S, Suslow P, Sutanto R, Tamura S, Tan M, Tan J, Tang A, Tato CM, Taylor JC, Tenvooren I, Thompson EM, Thornborrow EC, Tse E, Tung T, Turner ML, Turner VS, Turnham RE, Turocy MJ, Vaidyanathan TV, Vainchtein ID, Vanaerschot M, Vazquez SE, Wandler AM, Wapniarski A, Webber JT, Weinberg ZY, Westbrook A, Wong AW, Wong E, Worthington G, Xie F, Xu A, Yamamoto T, Yang Y, Yarza F, Zaltsman Y, Zheng T, and DeRisi JL
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- Betacoronavirus, COVID-19, COVID-19 Testing, California, Humans, Pandemics, SARS-CoV-2, Workflow, Clinical Laboratory Services supply & distribution, Clinical Laboratory Techniques methods, Coronavirus Infections diagnosis, Pneumonia, Viral diagnosis
- Abstract
Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2020
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17. Recovery of the Gut Microbiota after Antibiotics Depends on Host Diet, Community Context, and Environmental Reservoirs.
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Ng KM, Aranda-Díaz A, Tropini C, Frankel MR, Van Treuren W, O'Loughlin CT, Merrill BD, Yu FB, Pruss KM, Oliveira RA, Higginbottom SK, Neff NF, Fischbach MA, Xavier KB, Sonnenburg JL, and Huang KC
- Published
- 2020
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18. High-throughput SARS-CoV-2 and host genome sequencing from single nasopharyngeal swabs.
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Gorzynski JE, De Jong HN, Amar D, Hughes CR, Ioannidis A, Bierman R, Liu D, Tanigawa Y, Kistler A, Kamm J, Kim J, Cappello L, Neff NF, Rubinacci S, Delaneau O, Shoura MJ, Seo K, Kirillova A, Raja A, Sutton S, Huang C, Sahoo MK, Mallempati KC, Montero-Martin G, Osoegawa K, Jimenez-Morales D, Watson N, Hammond N, Joshi R, Fernandez-Vina M, Christle JW, Wheeler MT, Febbo P, Farh K, Schroth G, Desouza F, Palacios J, Salzman J, Pinsky BA, Rivas MA, Bustamante CD, Ashley EA, and Parikh VN
- Abstract
During COVID19 and other viral pandemics, rapid generation of host and pathogen genomic data is critical to tracking infection and informing therapies. There is an urgent need for efficient approaches to this data generation at scale. We have developed a scalable, high throughput approach to generate high fidelity low pass whole genome and HLA sequencing, viral genomes, and representation of human transcriptome from single nasopharyngeal swabs of COVID19 patients.
- Published
- 2020
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19. Ageing compromises mouse thymus function and remodels epithelial cell differentiation.
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Baran-Gale J, Morgan MD, Maio S, Dhalla F, Calvo-Asensio I, Deadman ME, Handel AE, Maynard A, Chen S, Green F, Sit RV, Neff NF, Darmanis S, Tan W, May AP, Marioni JC, Ponting CP, and Holländer GA
- Subjects
- Animals, Female, Mice, Mice, Inbred C57BL, Single-Cell Analysis, Aging, Cell Differentiation, Epithelial Cells physiology, Thymus Gland physiopathology, Transcriptome physiology
- Abstract
Ageing is characterised by cellular senescence, leading to imbalanced tissue maintenance, cell death and compromised organ function. This is first observed in the thymus, the primary lymphoid organ that generates and selects T cells. However, the molecular and cellular mechanisms underpinning these ageing processes remain unclear. Here, we show that mouse ageing leads to less efficient T cell selection, decreased self-antigen representation and increased T cell receptor repertoire diversity. Using a combination of single-cell RNA-seq and lineage-tracing, we find that progenitor cells are the principal targets of ageing, whereas the function of individual mature thymic epithelial cells is compromised only modestly. Specifically, an early-life precursor cell population, retained in the mouse cortex postnatally, is virtually extinguished at puberty. Concomitantly, a medullary precursor cell quiesces, thereby impairing maintenance of the medullary epithelium. Thus, ageing disrupts thymic progenitor differentiation and impairs the core immunological functions of the thymus., Competing Interests: JB, MM, SM, FD, IC, MD, AH, AM, SC, FG, RS, NN, SD, WT, AM, JM, GH No competing interests declared, CP Reviewing editor, eLife, (© 2020, Baran-Gale et al.)
- Published
- 2020
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20. Ageing hallmarks exhibit organ-specific temporal signatures.
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Schaum N, Lehallier B, Hahn O, Pálovics R, Hosseinzadeh S, Lee SE, Sit R, Lee DP, Losada PM, Zardeneta ME, Fehlmann T, Webber JT, McGeever A, Calcuttawala K, Zhang H, Berdnik D, Mathur V, Tan W, Zee A, Tan M, Pisco AO, Karkanias J, Neff NF, Keller A, Darmanis S, Quake SR, and Wyss-Coray T
- Subjects
- Animals, Blood Proteins analysis, Blood Proteins genetics, Female, Immunoglobulin J-Chains genetics, Immunoglobulin J-Chains metabolism, Male, Mice, Plasma Cells cytology, Plasma Cells metabolism, RNA, Messenger analysis, RNA, Messenger genetics, RNA-Seq, Single-Cell Analysis, T-Lymphocytes cytology, T-Lymphocytes metabolism, Time Factors, Transcriptome, Aging genetics, Aging physiology, Gene Expression Regulation, Organ Specificity genetics
- Abstract
Ageing is the single greatest cause of disease and death worldwide, and understanding the associated processes could vastly improve quality of life. Although major categories of ageing damage have been identified-such as altered intercellular communication, loss of proteostasis and eroded mitochondrial function
1 -these deleterious processes interact with extraordinary complexity within and between organs, and a comprehensive, whole-organism analysis of ageing dynamics has been lacking. Here we performed bulk RNA sequencing of 17 organs and plasma proteomics at 10 ages across the lifespan of Mus musculus, and integrated these findings with data from the accompanying Tabula Muris Senis2 -or 'Mouse Ageing Cell Atlas'-which follows on from the original Tabula Muris3 . We reveal linear and nonlinear shifts in gene expression during ageing, with the associated genes clustered in consistent trajectory groups with coherent biological functions-including extracellular matrix regulation, unfolded protein binding, mitochondrial function, and inflammatory and immune response. Notably, these gene sets show similar expression across tissues, differing only in the amplitude and the age of onset of expression. Widespread activation of immune cells is especially pronounced, and is first detectable in white adipose depots during middle age. Single-cell RNA sequencing confirms the accumulation of T cells and B cells in adipose tissue-including plasma cells that express immunoglobulin J-which also accrue concurrently across diverse organs. Finally, we show how gene expression shifts in distinct tissues are highly correlated with corresponding protein levels in plasma, thus potentially contributing to the ageing of the systemic circulation. Together, these data demonstrate a similar yet asynchronous inter- and intra-organ progression of ageing, providing a foundation from which to track systemic sources of declining health at old age.- Published
- 2020
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21. Recovery of the Gut Microbiota after Antibiotics Depends on Host Diet, Community Context, and Environmental Reservoirs.
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Ng KM, Aranda-Díaz A, Tropini C, Frankel MR, Van Treuren W, O'Loughlin CT, Merrill BD, Yu FB, Pruss KM, Oliveira RA, Higginbottom SK, Neff NF, Fischbach MA, Xavier KB, Sonnenburg JL, and Huang KC
- Subjects
- Animals, Bacteroides classification, Bacteroides isolation & purification, Biodiversity, Ciprofloxacin pharmacology, Diet, Female, Gastrointestinal Tract drug effects, Germ-Free Life, Humans, Male, Mice, Rifaximin pharmacology, Streptomycin pharmacology, Anti-Bacterial Agents pharmacology, Bacterial Load drug effects, Bacteroides growth & development, Gastrointestinal Microbiome drug effects, Gastrointestinal Tract microbiology
- Abstract
Antibiotics alter microbiota composition and increase infection susceptibility. However, the generalizable effects of antibiotics on and the contribution of environmental variables to gut commensals remain unclear. To address this, we tracked microbiota dynamics with high temporal and taxonomic resolution during antibiotic treatment in a controlled murine system by isolating variables such as diet, treatment history, and housing co-inhabitants. Human microbiotas were remarkably resilient and recovered during antibiotic treatment, with transient dominance of resistant Bacteroides and taxa-asymmetric diversity reduction. In certain cases, in vitro sensitivities were not predictive of in vivo responses, underscoring the significance of host and community context. A fiber-deficient diet exacerbated microbiota collapse and delayed recovery. Species replacement through cross housing after ciprofloxacin treatment established resilience to a second treatment. Single housing drastically disrupted recovery, highlighting the importance of environmental reservoirs. Our findings highlight deterministic microbiota adaptations to perturbations and the translational potential for modulating diet, sanitation, and microbiota composition during antibiotics., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
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22. Single Cell Transcriptomes Derived from Human Cervical and Uterine Tissue during Pregnancy.
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Koh W, Wu A, Penland L, Treutlein B, Neff NF, Mantalas GL, Blumenfeld YJ, El-Sayed YY, Stevenson DK, Shaw GM, and Quake SR
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- Adult, Cesarean Section, Female, Humans, Pregnancy, Cervix Uteri metabolism, Placenta Diseases metabolism, Single-Cell Analysis, Transcriptome
- Abstract
This work presents the workflow for generating single cell transcriptomes derived from primary human uterine and cervical tissue obtained during planned cesarean hysterectomies. In total, a catalogue of 310 single cell transcriptomes are obtained, cell types present in these biopsies are inferred, and specific genes defining each of the cellular types present in the tissue are identified. Further validation of the inferred cell identity is also demonstrated via meta-analysis of independent repositories in literature generated by bulk sequenced data of fluorescence-activated cell sorting sorted cells., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2019
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23. FLASH: a next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences.
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Quan J, Langelier C, Kuchta A, Batson J, Teyssier N, Lyden A, Caldera S, McGeever A, Dimitrov B, King R, Wilheim J, Murphy M, Ares LP, Travisano KA, Sit R, Amato R, Mumbengegwi DR, Smith JL, Bennett A, Gosling R, Mourani PM, Calfee CS, Neff NF, Chow ED, Kim PS, Greenhouse B, DeRisi JL, and Crawford ED
- Subjects
- Bacteria classification, Bacteria drug effects, Bacteria genetics, Bacterial Infections diagnosis, Bacterial Infections genetics, Bacterial Infections prevention & control, Drug Resistance, Bacterial genetics, Humans, Metagenomics methods, Reproducibility of Results, Sensitivity and Specificity, Anti-Bacterial Agents pharmacology, CRISPR-Cas Systems, Computational Biology methods, Drug Resistance, Bacterial drug effects, High-Throughput Nucleotide Sequencing methods
- Abstract
The growing prevalence of deadly microbes with resistance to previously life-saving drug therapies is a dire threat to human health. Detection of low abundance pathogen sequences remains a challenge for metagenomic Next Generation Sequencing (NGS). We introduce FLASH (Finding Low Abundance Sequences by Hybridization), a next-generation CRISPR/Cas9 diagnostic method that takes advantage of the efficiency, specificity and flexibility of Cas9 to enrich for a programmed set of sequences. FLASH-NGS achieves up to 5 orders of magnitude of enrichment and sub-attomolar gene detection with minimal background. We provide an open-source software tool (FLASHit) for guide RNA design. Here we applied it to detection of antimicrobial resistance genes in respiratory fluid and dried blood spots, but FLASH-NGS is applicable to all areas that rely on multiplex PCR., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2019
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24. Developmental Heterogeneity of Microglia and Brain Myeloid Cells Revealed by Deep Single-Cell RNA Sequencing.
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Li Q, Cheng Z, Zhou L, Darmanis S, Neff NF, Okamoto J, Gulati G, Bennett ML, Sun LO, Clarke LE, Marschallinger J, Yu G, Quake SR, Wyss-Coray T, and Barres BA
- Subjects
- Algorithms, Animals, Animals, Newborn, Antigens, CD metabolism, Cell Proliferation physiology, Choroid Plexus cytology, Cluster Analysis, Computer Simulation, Embryo, Mammalian, Gene Regulatory Networks physiology, High-Throughput Nucleotide Sequencing, Mice, Mice, Inbred C57BL, Mice, Transgenic, Oligodendroglia physiology, Phagocytosis physiology, Brain cytology, Brain embryology, Brain growth & development, Gene Expression Regulation, Developmental physiology, Microglia physiology, Myeloid Cells physiology, Sequence Analysis, RNA, Transcriptome physiology
- Abstract
Microglia are increasingly recognized for their major contributions during brain development and neurodegenerative disease. It is currently unknown whether these functions are carried out by subsets of microglia during different stages of development and adulthood or within specific brain regions. Here, we performed deep single-cell RNA sequencing (scRNA-seq) of microglia and related myeloid cells sorted from various regions of embryonic, early postnatal, and adult mouse brains. We found that the majority of adult microglia expressing homeostatic genes are remarkably similar in transcriptomes, regardless of brain region. By contrast, early postnatal microglia are more heterogeneous. We discovered a proliferative-region-associated microglia (PAM) subset, mainly found in developing white matter, that shares a characteristic gene signature with degenerative disease-associated microglia (DAM). Such PAM have amoeboid morphology, are metabolically active, and phagocytose newly formed oligodendrocytes. This scRNA-seq atlas will be a valuable resource for dissecting innate immune functions in health and disease., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2019
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25. Complex mammalian-like haematopoietic system found in a colonial chordate.
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Rosental B, Kowarsky M, Seita J, Corey DM, Ishizuka KJ, Palmeri KJ, Chen SY, Sinha R, Okamoto J, Mantalas G, Manni L, Raveh T, Clarke DN, Tsai JM, Newman AM, Neff NF, Nolan GP, Quake SR, Weissman IL, and Voskoboynik A
- Subjects
- Animals, Cell Differentiation, Cell Lineage, Cytotoxicity, Immunologic, Female, Flow Cytometry, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells immunology, Immunity, Cellular, Isoantigens immunology, Male, Mammals anatomy & histology, Myeloid Cells cytology, Myeloid Cells immunology, Phagocytosis immunology, Stem Cell Niche, Transcriptome genetics, Urochordata anatomy & histology, Urochordata genetics, Urochordata immunology, Hematopoiesis, Hematopoietic System cytology, Mammals blood, Phylogeny, Urochordata cytology
- Abstract
Haematopoiesis is an essential process that evolved in multicellular animals. At the heart of this process are haematopoietic stem cells (HSCs), which are multipotent and self-renewing, and generate the entire repertoire of blood and immune cells throughout an animal's life
1 . Although there have been comprehensive studies on self-renewal, differentiation, physiological regulation and niche occupation in vertebrate HSCs, relatively little is known about the evolutionary origin and niches of these cells. Here we describe the haematopoietic system of Botryllus schlosseri, a colonial tunicate that has a vasculature and circulating blood cells, and interesting stem-cell biology and immunity characteristics2-8 . Self-recognition between genetically compatible B. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other3,4,7 . Using flow cytometry, whole-transcriptome sequencing of defined cell populations and diverse functional assays, we identify HSCs, progenitors, immune effector cells and an HSC niche, and demonstrate that self-recognition inhibits allospecific cytotoxic reactions. Our results show that HSC and myeloid lineage immune cells emerged in a common ancestor of tunicates and vertebrates, and also suggest that haematopoietic bone marrow and the B. schlosseri endostyle niche evolved from a common origin.- Published
- 2018
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26. Noninvasive blood tests for fetal development predict gestational age and preterm delivery.
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Ngo TTM, Moufarrej MN, Rasmussen MH, Camunas-Soler J, Pan W, Okamoto J, Neff NF, Liu K, Wong RJ, Downes K, Tibshirani R, Shaw GM, Skotte L, Stevenson DK, Biggio JR, Elovitz MA, Melbye M, and Quake SR
- Subjects
- Adult, Female, Humans, Pilot Projects, Pregnancy, Prenatal Care, Young Adult, Blood Chemical Analysis methods, Cell-Free Nucleic Acids blood, Fetal Development, Fetal Monitoring methods, Gestational Age, Premature Birth blood, Premature Birth diagnosis
- Abstract
Noninvasive blood tests that provide information about fetal development and gestational age could potentially improve prenatal care. Ultrasound, the current gold standard, is not always affordable in low-resource settings and does not predict spontaneous preterm birth, a leading cause of infant death. In a pilot study of 31 healthy pregnant women, we found that measurement of nine cell-free RNA (cfRNA) transcripts in maternal blood predicted gestational age with comparable accuracy to ultrasound but at substantially lower cost. In a related study of 38 women (23 full-term and 15 preterm deliveries), all at elevated risk of delivering preterm, we identified seven cfRNA transcripts that accurately classified women who delivered preterm up to 2 months in advance of labor. These tests hold promise for prenatal care in both the developed and developing worlds, although they require validation in larger, blinded clinical trials., (Copyright © 2018, American Association for the Advancement of Science.)
- Published
- 2018
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27. High-throughput full-length single-cell mRNA-seq of rare cells.
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Ooi CC, Mantalas GL, Koh W, Neff NF, Fuchigami T, Wong DJ, Wilson RJ, Park SM, Gambhir SS, Quake SR, and Wang SX
- Subjects
- Cell Line, Tumor, Gene Expression Profiling, Humans, Lung Neoplasms genetics, Lung Neoplasms pathology, Mutation, High-Throughput Nucleotide Sequencing methods, RNA, Messenger genetics
- Abstract
Single-cell characterization techniques, such as mRNA-seq, have been applied to a diverse range of applications in cancer biology, yielding great insight into mechanisms leading to therapy resistance and tumor clonality. While single-cell techniques can yield a wealth of information, a common bottleneck is the lack of throughput, with many current processing methods being limited to the analysis of small volumes of single cell suspensions with cell densities on the order of 107 per mL. In this work, we present a high-throughput full-length mRNA-seq protocol incorporating a magnetic sifter and magnetic nanoparticle-antibody conjugates for rare cell enrichment, and Smart-seq2 chemistry for sequencing. We evaluate the efficiency and quality of this protocol with a simulated circulating tumor cell system, whereby non-small-cell lung cancer cell lines (NCI-H1650 and NCI-H1975) are spiked into whole blood, before being enriched for single-cell mRNA-seq by EpCAM-functionalized magnetic nanoparticles and the magnetic sifter. We obtain high efficiency (> 90%) capture and release of these simulated rare cells via the magnetic sifter, with reproducible transcriptome data. In addition, while mRNA-seq data is typically only used for gene expression analysis of transcriptomic data, we demonstrate the use of full-length mRNA-seq chemistries like Smart-seq2 to facilitate variant analysis of expressed genes. This enables the use of mRNA-seq data for differentiating cells in a heterogeneous population by both their phenotypic and variant profile. In a simulated heterogeneous mixture of circulating tumor cells in whole blood, we utilize this high-throughput protocol to differentiate these heterogeneous cells by both their phenotype (lung cancer versus white blood cells), and mutational profile (H1650 versus H1975 cells), in a single sequencing run. This high-throughput method can help facilitate single-cell analysis of rare cell populations, such as circulating tumor or endothelial cells, with demonstrably high-quality transcriptomic data.
- Published
- 2017
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28. Numerous uncharacterized and highly divergent microbes which colonize humans are revealed by circulating cell-free DNA.
- Author
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Kowarsky M, Camunas-Soler J, Kertesz M, De Vlaminck I, Koh W, Pan W, Martin L, Neff NF, Okamoto J, Wong RJ, Kharbanda S, El-Sayed Y, Blumenfeld Y, Stevenson DK, Shaw GM, Wolfe ND, and Quake SR
- Subjects
- Genetic Variation, High-Throughput Nucleotide Sequencing methods, Humans, Metagenomics methods, Phylogeny, Cell-Free Nucleic Acids blood, Cell-Free Nucleic Acids genetics, DNA, Bacterial blood, DNA, Bacterial genetics, DNA, Viral blood, DNA, Viral genetics, Microbiota genetics
- Abstract
Blood circulates throughout the human body and contains molecules drawn from virtually every tissue, including the microbes and viruses which colonize the body. Through massive shotgun sequencing of circulating cell-free DNA from the blood, we identified hundreds of new bacteria and viruses which represent previously unidentified members of the human microbiome. Analyzing cumulative sequence data from 1,351 blood samples collected from 188 patients enabled us to assemble 7,190 contiguous regions (contigs) larger than 1 kbp, of which 3,761 are novel with little or no sequence homology in any existing databases. The vast majority of these novel contigs possess coding sequences, and we have validated their existence both by finding their presence in independent experiments and by performing direct PCR amplification. When their nearest neighbors are located in the tree of life, many of the organisms represent entirely novel taxa, showing that microbial diversity within the human body is substantially broader than previously appreciated., Competing Interests: Conflict of interest statement: N.D.W. is an employee of Metabiota and founder of Global Viral; S.R.Q. is a founder of Karius. M. Kertesz is an employee and founder of Karius, but all work was performed while at Stanford and before he joined the company. All other authors declare no conflict of interest.
- Published
- 2017
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29. Early somatic mosaicism is a rare cause of long-QT syndrome.
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Priest JR, Gawad C, Kahlig KM, Yu JK, O'Hara T, Boyle PM, Rajamani S, Clark MJ, Garcia ST, Ceresnak S, Harris J, Boyle S, Dewey FE, Malloy-Walton L, Dunn K, Grove M, Perez MV, Neff NF, Chen R, Maeda K, Dubin A, Belardinelli L, West J, Antolik C, Macaya D, Quertermous T, Trayanova NA, Quake SR, and Ashley EA
- Subjects
- Action Potentials, Arrhythmias, Cardiac complications, Arrhythmias, Cardiac genetics, Arrhythmias, Cardiac physiopathology, Base Sequence, Cardiomyopathy, Dilated complications, Cardiomyopathy, Dilated genetics, Cardiomyopathy, Dilated physiopathology, Computer Simulation, Diffusion, Electrocardiography, Gene Frequency genetics, Genes, Dominant, Genetic Loci, Genotyping Techniques, Heart Conduction System physiopathology, High-Throughput Nucleotide Sequencing, Humans, Infant, Ion Channel Gating genetics, Long QT Syndrome complications, Long QT Syndrome diagnostic imaging, Long QT Syndrome physiopathology, Models, Biological, Mutation genetics, Myocytes, Cardiac metabolism, NAV1.5 Voltage-Gated Sodium Channel genetics, Phenotype, Single-Cell Analysis, Genetic Predisposition to Disease, Long QT Syndrome genetics, Mosaicism
- Abstract
Somatic mosaicism, the occurrence and propagation of genetic variation in cell lineages after fertilization, is increasingly recognized to play a causal role in a variety of human diseases. We investigated the case of life-threatening arrhythmia in a 10-day-old infant with long QT syndrome (LQTS). Rapid genome sequencing suggested a variant in the sodium channel Na
V 1.5 encoded by SCN5A, NM_000335:c.5284G > T predicting p.(V1762L), but read depth was insufficient to be diagnostic. Exome sequencing of the trio confirmed read ratios inconsistent with Mendelian inheritance only in the proband. Genotyping of single circulating leukocytes demonstrated the mutation in the genomes of 8% of patient cells, and RNA sequencing of cardiac tissue from the infant confirmed the expression of the mutant allele at mosaic ratios. Heterologous expression of the mutant channel revealed significantly delayed sodium current with a dominant negative effect. To investigate the mechanism by which mosaicism might cause arrhythmia, we built a finite element simulation model incorporating Purkinje fiber activation. This model confirmed the pathogenic consequences of cardiac cellular mosaicism and, under the presenting conditions of this case, recapitulated 2:1 AV block and arrhythmia. To investigate the extent to which mosaicism might explain undiagnosed arrhythmia, we studied 7,500 affected probands undergoing commercial gene-panel testing. Four individuals with pathogenic variants arising from early somatic mutation events were found. Here we establish cardiac mosaicism as a causal mechanism for LQTS and present methods by which the general phenomenon, likely to be relevant for all genetic diseases, can be detected through single-cell analysis and next-generation sequencing., Competing Interests: At the time of this work K.M.K., S.R., and L.B. were employed by Gilead Sciences. M.J.C., S.T.K.G., S.B., J.W., and R.C. were employed by Personalis, Inc. J.W. and E.A.A. are founders of Personalis, Inc. which offers clinical genetic testing but does not offer Clinical Laboratory Improvement Amendments (CLIA)-certified rapid-turnaround whole-genome sequencing. N.A.T. is a co-founder of CardioSolv LLC.- Published
- 2016
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30. Cellular Taxonomy of the Mouse Striatum as Revealed by Single-Cell RNA-Seq.
- Author
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Gokce O, Stanley GM, Treutlein B, Neff NF, Camp JG, Malenka RC, Rothwell PE, Fuccillo MV, Südhof TC, and Quake SR
- Subjects
- Animals, Astrocytes metabolism, Astrocytes physiology, Behavior, Addictive metabolism, Behavior, Addictive physiopathology, Cell Differentiation physiology, Cognitive Dysfunction metabolism, Cognitive Dysfunction physiopathology, Corpus Striatum metabolism, Ependyma metabolism, Ependyma physiology, Male, Mice, Neurons metabolism, Neurons physiology, Oligodendroglia metabolism, Oligodendroglia physiology, Transcription Factors metabolism, Transcriptome physiology, Corpus Striatum physiology, RNA genetics
- Abstract
The striatum contributes to many cognitive processes and disorders, but its cell types are incompletely characterized. We show that microfluidic and FACS-based single-cell RNA sequencing of mouse striatum provides a well-resolved classification of striatal cell type diversity. Transcriptome analysis revealed ten differentiated, distinct cell types, including neurons, astrocytes, oligodendrocytes, ependymal, immune, and vascular cells, and enabled the discovery of numerous marker genes. Furthermore, we identified two discrete subtypes of medium spiny neurons (MSNs) that have specific markers and that overexpress genes linked to cognitive disorders and addiction. We also describe continuous cellular identities, which increase heterogeneity within discrete cell types. Finally, we identified cell type-specific transcription and splicing factors that shape cellular identities by regulating splicing and expression patterns. Our findings suggest that functional diversity within a complex tissue arises from a small number of discrete cell types, which can exist in a continuous spectrum of functional states., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2016
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31. Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq.
- Author
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Treutlein B, Lee QY, Camp JG, Mall M, Koh W, Shariati SA, Sim S, Neff NF, Skotheim JM, Wernig M, and Quake SR
- Subjects
- Animals, Basic Helix-Loop-Helix Transcription Factors genetics, Basic Helix-Loop-Helix Transcription Factors metabolism, Cell Cycle genetics, Cell Lineage genetics, Cell Transdifferentiation genetics, Embryo, Mammalian cytology, Gene Expression Profiling, Gene Silencing, Homeodomain Proteins metabolism, Mice, Nerve Tissue Proteins metabolism, POU Domain Factors metabolism, Time Factors, Transcription Factors metabolism, Transcriptome genetics, Transgenes genetics, Cellular Reprogramming genetics, Fibroblasts cytology, Fibroblasts metabolism, Neurons cytology, Neurons metabolism, Sequence Analysis, RNA, Single-Cell Analysis
- Abstract
Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states. However, the intermediate stages through which individual cells progress during reprogramming are largely undefined. Here we use single-cell RNA sequencing at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts to induced neuronal cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts, suggesting that the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.
- Published
- 2016
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32. Noninvasive monitoring of infection and rejection after lung transplantation.
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De Vlaminck I, Martin L, Kertesz M, Patel K, Kowarsky M, Strehl C, Cohen G, Luikart H, Neff NF, Okamoto J, Nicolls MR, Cornfield D, Weill D, Valantine H, Khush KK, and Quake SR
- Subjects
- Base Sequence, Cytomegalovirus genetics, Humans, Molecular Sequence Data, Polymorphism, Single Nucleotide genetics, Sequence Analysis, DNA, Species Specificity, Surgical Wound Infection virology, DNA, Viral blood, Graft Rejection diagnosis, Lung Transplantation adverse effects, Postoperative Care methods, Surgical Wound Infection diagnosis
- Abstract
The survival rate following lung transplantation is among the lowest of all solid-organ transplants, and current diagnostic tests often fail to distinguish between infection and rejection, the two primary posttransplant clinical complications. We describe a diagnostic assay that simultaneously monitors for rejection and infection in lung transplant recipients by sequencing of cell-free DNA (cfDNA) in plasma. We determined that the levels of donor-derived cfDNA directly correlate with the results of invasive tests of rejection (area under the curve 0.9). We also analyzed the nonhuman cfDNA as a hypothesis-free approach to test for infections. Cytomegalovirus is most frequently assayed clinically, and the levels of CMV-derived sequences in cfDNA are consistent with clinical results. We furthermore show that hypothesis-free monitoring for pathogens using cfDNA reveals undiagnosed cases of infection, and that certain infectious pathogens such as human herpesvirus (HHV) 6, HHV-7, and adenovirus, which are not often tested clinically, occur with high frequency in this cohort.
- Published
- 2015
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33. Circulating cell-free DNA enables noninvasive diagnosis of heart transplant rejection.
- Author
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De Vlaminck I, Valantine HA, Snyder TM, Strehl C, Cohen G, Luikart H, Neff NF, Okamoto J, Bernstein D, Weisshaar D, Quake SR, and Khush KK
- Subjects
- Early Diagnosis, Humans, Retrospective Studies, DNA blood, Graft Rejection diagnosis, Heart Transplantation
- Abstract
Monitoring allograft health is an important component of posttransplant therapy. Endomyocardial biopsy is the current gold standard for cardiac allograft monitoring but is an expensive and invasive procedure. Proof of principle of a universal, noninvasive diagnostic method based on high-throughput screening of circulating cell-free donor-derived DNA (cfdDNA) was recently demonstrated in a small retrospective cohort. We present the results of a prospective cohort study (65 patients, 565 samples) that tested the utility of cfdDNA in measuring acute rejection after heart transplantation. Circulating cell-free DNA was purified from plasma and sequenced (mean depth, 1.2 giga-base pairs) to quantify the fraction of cfdDNA. Through a comparison with endomyocardial biopsy results, we demonstrate that cfdDNA enables diagnosis of acute rejection after heart transplantation, with an area under the receiver operating characteristic curve of 0.83 and sensitivity and specificity that are comparable to the intrinsic performance of the biopsy itself. This noninvasive genome transplant dynamics approach is a powerful and informative method for routine monitoring of allograft health without incurring the risk, discomfort, and expense of an invasive biopsy., (Copyright © 2014, American Association for the Advancement of Science.)
- Published
- 2014
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34. Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq.
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Treutlein B, Brownfield DG, Wu AR, Neff NF, Mantalas GL, Espinoza FH, Desai TJ, Krasnow MA, and Quake SR
- Subjects
- Animals, Bronchi cytology, Cell Differentiation genetics, Epithelial Cells classification, Female, Genetic Markers, Genome genetics, Genomics, Lung embryology, Mice, Mice, Inbred C57BL, Pulmonary Alveoli cytology, Pulmonary Gas Exchange, Stem Cells cytology, Transcriptome genetics, Cell Lineage genetics, Epithelial Cells cytology, Epithelial Cells metabolism, Lung cytology, Sequence Analysis, RNA methods, Single-Cell Analysis methods
- Abstract
The mammalian lung is a highly branched network in which the distal regions of the bronchial tree transform during development into a densely packed honeycomb of alveolar air sacs that mediate gas exchange. Although this transformation has been studied by marker expression analysis and fate-mapping, the mechanisms that control the progression of lung progenitors along distinct lineages into mature alveolar cell types are still incompletely known, in part because of the limited number of lineage markers and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations. Here we show that single-cell transcriptome analysis circumvents these problems and enables direct measurement of the various cell types and hierarchies in the developing lung. We used microfluidic single-cell RNA sequencing (RNA-seq) on 198 individual cells at four different stages encompassing alveolar differentiation to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We empirically classified cells into distinct groups by using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or the previous purification of cell populations. The results confirmed the basic outlines of the classical model of epithelial cell-type diversity in the distal lung and led to the discovery of many previously unknown cell-type markers, including transcriptional regulators that discriminate between the different populations. We reconstructed the molecular steps during maturation of bipotential progenitors along both alveolar lineages and elucidated the full life cycle of the alveolar type 2 cell lineage. This single-cell genomics approach is applicable to any developing or mature tissue to robustly delineate molecularly distinct cell types, define progenitors and lineage hierarchies, and identify lineage-specific regulatory factors.
- Published
- 2014
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35. Quantitative assessment of single-cell RNA-sequencing methods.
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Wu AR, Neff NF, Kalisky T, Dalerba P, Treutlein B, Rothenberg ME, Mburu FM, Mantalas GL, Sim S, Clarke MF, and Quake SR
- Subjects
- Data Interpretation, Statistical, Electronic Data Processing, Gene Expression Profiling, Gene Expression Regulation, HCT116 Cells, Humans, Microfluidics, Nucleic Acid Amplification Techniques, Polymerase Chain Reaction methods, Reproducibility of Results, Sequence Analysis, DNA, Transcriptome, Sequence Analysis, RNA methods, Single-Cell Analysis methods
- Abstract
Interest in single-cell whole-transcriptome analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. We compared commercially available single-cell RNA amplification methods with both microliter and nanoliter volumes, using sequence from bulk total RNA and multiplexed quantitative PCR as benchmarks to systematically evaluate the sensitivity and accuracy of various single-cell RNA-seq approaches. We show that single-cell RNA-seq can be used to perform accurate quantitative transcriptome measurement in individual cells with a relatively small number of sequencing reads and that sequencing large numbers of single cells can recapitulate bulk transcriptome complexity.
- Published
- 2014
- Full Text
- View/download PDF
36. Temporal response of the human virome to immunosuppression and antiviral therapy.
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De Vlaminck I, Khush KK, Strehl C, Kohli B, Luikart H, Neff NF, Okamoto J, Snyder TM, Cornfield DN, Nicolls MR, Weill D, Bernstein D, Valantine HA, and Quake SR
- Subjects
- Adult, Antibiotic Prophylaxis, Blood microbiology, Child, DNA blood, DNA genetics, Humans, Viruses classification, Antiviral Agents therapeutic use, Blood virology, Heart Transplantation, Immunosuppressive Agents therapeutic use, Lung Transplantation, Viruses isolation & purification
- Abstract
There are few substantive methods to measure the health of the immune system, and the connection between immune strength and the viral component of the microbiome is poorly understood. Organ transplant recipients are treated with posttransplant therapies that combine immunosuppressive and antiviral drugs, offering a window into the effects of immune modulation on the virome. We used sequencing of cell-free DNA in plasma to investigate drug-virome interactions in a cohort of organ transplant recipients (656 samples, 96 patients) and find that antivirals and immunosuppressants strongly affect the structure of the virome in plasma. We observe marked virome compositional dynamics at the onset of the therapy and find that the total viral load increases with immunosuppression, whereas the bacterial component of the microbiome remains largely unaffected. The data provide insight into the relationship between the human virome, the state of the immune system, and the effects of pharmacological treatment and offer a potential application of the virome state to predict immunocompetence., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
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37. Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons.
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Wapinski OL, Vierbuchen T, Qu K, Lee QY, Chanda S, Fuentes DR, Giresi PG, Ng YH, Marro S, Neff NF, Drechsel D, Martynoga B, Castro DS, Webb AE, Südhof TC, Brunet A, Guillemot F, Chang HY, and Wernig M
- Subjects
- Animals, Basic Helix-Loop-Helix Transcription Factors metabolism, Cell Differentiation, Chromatin metabolism, Fibroblasts metabolism, Genome-Wide Association Study, Humans, Mice, Nerve Tissue Proteins metabolism, Neurons metabolism, POU Domain Factors metabolism, Repressor Proteins metabolism, Transcription Factors metabolism, Cellular Reprogramming, Embryo, Mammalian cytology, Fibroblasts cytology, Gene Regulatory Networks, Neurons cytology
- Abstract
Direct lineage reprogramming is a promising approach for human disease modeling and regenerative medicine, with poorly understood mechanisms. Here, we reveal a hierarchical mechanism in the direct conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an "on-target" pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myt1l do not access fibroblast chromatin productively on their own; instead, Ascl1 recruits Brn2 to Ascl1 sites genome wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, we identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, a precise match between pioneer factors and the chromatin context at key target genes is determinative for transdifferentiation to neurons and likely other cell types., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
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38. Identification of a colonial chordate histocompatibility gene.
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Voskoboynik A, Newman AM, Corey DM, Sahoo D, Pushkarev D, Neff NF, Passarelli B, Koh W, Ishizuka KJ, Palmeri KJ, Dimov IK, Keasar C, Fan HC, Mantalas GL, Sinha R, Penland L, Quake SR, and Weissman IL
- Subjects
- Alleles, Animals, Genome, Genotype, Immune Tolerance, Molecular Sequence Data, Sequence Analysis, DNA, Transcriptome, Up-Regulation, Urochordata physiology, Genes, Histocompatibility genetics, Urochordata genetics, Urochordata immunology
- Abstract
Histocompatibility is the basis by which multicellular organisms of the same species distinguish self from nonself. Relatively little is known about the mechanisms underlying histocompatibility reactions in lower organisms. Botryllus schlosseri is a colonial urochordate, a sister group of vertebrates, that exhibits a genetically determined natural transplantation reaction, whereby self-recognition between colonies leads to formation of parabionts with a common vasculature, whereas rejection occurs between incompatible colonies. Using genetically defined lines, whole-transcriptome sequencing, and genomics, we identified a single gene that encodes self-nonself and determines "graft" outcomes in this organism. This gene is significantly up-regulated in colonies poised to undergo fusion and/or rejection, is highly expressed in the vasculature, and is functionally linked to histocompatibility outcomes. These findings establish a platform for advancing the science of allorecognition.
- Published
- 2013
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39. The genome sequence of the colonial chordate, Botryllus schlosseri.
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Voskoboynik A, Neff NF, Sahoo D, Newman AM, Pushkarev D, Koh W, Passarelli B, Fan HC, Mantalas GL, Palmeri KJ, Ishizuka KJ, Gissi C, Griggio F, Ben-Shlomo R, Corey DM, Penland L, White RA 3rd, Weissman IL, and Quake SR
- Subjects
- Animals, Chordata classification, Chordata physiology, Chromosome Mapping, High-Throughput Nucleotide Sequencing, Phylogeny, Reproduction, Chordata genetics, Genome
- Abstract
Botryllus schlosseri is a colonial urochordate that follows the chordate plan of development following sexual reproduction, but invokes a stem cell-mediated budding program during subsequent rounds of asexual reproduction. As urochordates are considered to be the closest living invertebrate relatives of vertebrates, they are ideal subjects for whole genome sequence analyses. Using a novel method for high-throughput sequencing of eukaryotic genomes, we sequenced and assembled 580 Mbp of the B. schlosseri genome. The genome assembly is comprised of nearly 14,000 intron-containing predicted genes, and 13,500 intron-less predicted genes, 40% of which could be confidently parceled into 13 (of 16 haploid) chromosomes. A comparison of homologous genes between B. schlosseri and other diverse taxonomic groups revealed genomic events underlying the evolution of vertebrates and lymphoid-mediated immunity. The B. schlosseri genome is a community resource for studying alternative modes of reproduction, natural transplantation reactions, and stem cell-mediated regeneration. DOI:http://dx.doi.org/10.7554/eLife.00569.001.
- Published
- 2013
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40. Single-cell dissection of transcriptional heterogeneity in human colon tumors.
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Dalerba P, Kalisky T, Sahoo D, Rajendran PS, Rothenberg ME, Leyrat AA, Sim S, Okamoto J, Johnston DM, Qian D, Zabala M, Bueno J, Neff NF, Wang J, Shelton AA, Visser B, Hisamori S, Shimono Y, van de Wetering M, Clevers H, Clarke MF, and Quake SR
- Subjects
- Adenocarcinoma genetics, Adenocarcinoma pathology, Adult, Aged, Aged, 80 and over, Animals, Colonic Neoplasms genetics, Colonic Neoplasms pathology, Disease-Free Survival, Female, Flow Cytometry, HCT116 Cells, Humans, Kaplan-Meier Estimate, Male, Mice, Middle Aged, Neoplasm Staging, Transplantation, Heterologous, Treatment Outcome, Adenocarcinoma metabolism, Cell Differentiation genetics, Cell Lineage genetics, Colonic Neoplasms metabolism, Gene Expression Regulation, Neoplastic, Single-Cell Analysis methods, Transcription, Genetic
- Abstract
Cancer is often viewed as a caricature of normal developmental processes, but the extent to which its cellular heterogeneity truly recapitulates multilineage differentiation processes of normal tissues remains unknown. Here we implement single-cell PCR gene-expression analysis to dissect the cellular composition of primary human normal colon and colon cancer epithelia. We show that human colon cancer tissues contain distinct cell populations whose transcriptional identities mirror those of the different cellular lineages of normal colon. By creating monoclonal tumor xenografts from injection of a single (n = 1) cell, we demonstrate that the transcriptional diversity of cancer tissues is largely explained by in vivo multilineage differentiation and not only by clonal genetic heterogeneity. Finally, we show that the different gene-expression programs linked to multilineage differentiation are strongly associated with patient survival. We develop two-gene classifier systems (KRT20 versus CA1, MS4A12, CD177, SLC26A3) that predict clinical outcomes with hazard ratios superior to those of pathological grade and comparable to those of microarray-derived multigene expression signatures.
- Published
- 2011
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41. Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding.
- Author
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Lu R, Neff NF, Quake SR, and Weissman IL
- Subjects
- Animals, Cell Differentiation genetics, Cell Differentiation radiation effects, Cell Lineage genetics, Cell Lineage radiation effects, Clone Cells, Gene Library, Hematopoietic Stem Cells radiation effects, Lentivirus radiation effects, Mice, Mice, Inbred C57BL, Radiation, Sequence Analysis, DNA, DNA Barcoding, Taxonomic methods, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells virology, High-Throughput Nucleotide Sequencing methods, Lentivirus genetics, Single-Cell Analysis methods
- Abstract
Disentangling cellular heterogeneity is a challenge in many fields, particularly in the stem cell and cancer biology fields. Here we demonstrate how to combine viral genetic barcoding with high-throughput sequencing to track single cells in a heterogeneous population. We use this technique to track the in vivo differentiation of unitary hematopoietic stem cells (HSCs). The results are consistent with single-cell transplantation studies but require two orders of magnitude fewer mice. In addition to its high throughput, the high sensitivity of the technique allows for a direct examination of the clonality of sparse cell populations such as HSCs. We show how these capabilities offer a clonal perspective of the HSC differentiation process. In particular, our data suggest that HSCs do not equally contribute to blood cells after irradiation-mediated transplantation, and that two distinct HSC differentiation patterns co-exist in the same recipient mouse after irradiation. This technique can be applied to any virus-accessible cell type for both in vitro and in vivo processes.
- Published
- 2011
- Full Text
- View/download PDF
42. Clinical assessment incorporating a personal genome.
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Ashley EA, Butte AJ, Wheeler MT, Chen R, Klein TE, Dewey FE, Dudley JT, Ormond KE, Pavlovic A, Morgan AA, Pushkarev D, Neff NF, Hudgins L, Gong L, Hodges LM, Berlin DS, Thorn CF, Sangkuhl K, Hebert JM, Woon M, Sagreiya H, Whaley R, Knowles JW, Chou MF, Thakuria JV, Rosenbaum AM, Zaranek AW, Church GM, Greely HT, Quake SR, and Altman RB
- Subjects
- Adult, Aryl Hydrocarbon Hydroxylases genetics, Carrier Proteins genetics, Cytochrome P-450 CYP2C19, Cytochrome P-450 Enzyme System genetics, Cytochrome P450 Family 4, Death, Sudden, Cardiac, Desmoplakins genetics, Environment, Family Health, Genetic Counseling, Humans, Lipoprotein(a) genetics, Male, Membrane Proteins genetics, Mixed Function Oxygenases genetics, Mutation, Osteoarthritis genetics, Pedigree, Pharmacogenetics, Polymorphism, Single Nucleotide, Risk Assessment, Vitamin K Epoxide Reductases, Genetic Predisposition to Disease genetics, Genetic Testing, Genome, Human, Sequence Analysis, DNA, Vascular Diseases genetics
- Abstract
Background: The cost of genomic information has fallen steeply, but the clinical translation of genetic risk estimates remains unclear. We aimed to undertake an integrated analysis of a complete human genome in a clinical context., Methods: We assessed a patient with a family history of vascular disease and early sudden death. Clinical assessment included analysis of this patient's full genome sequence, risk prediction for coronary artery disease, screening for causes of sudden cardiac death, and genetic counselling. Genetic analysis included the development of novel methods for the integration of whole genome and clinical risk. Disease and risk analysis focused on prediction of genetic risk of variants associated with mendelian disease, recognised drug responses, and pathogenicity for novel variants. We queried disease-specific mutation databases and pharmacogenomics databases to identify genes and mutations with known associations with disease and drug response. We estimated post-test probabilities of disease by applying likelihood ratios derived from integration of multiple common variants to age-appropriate and sex-appropriate pre-test probabilities. We also accounted for gene-environment interactions and conditionally dependent risks., Findings: Analysis of 2.6 million single nucleotide polymorphisms and 752 copy number variations showed increased genetic risk for myocardial infarction, type 2 diabetes, and some cancers. We discovered rare variants in three genes that are clinically associated with sudden cardiac death-TMEM43, DSP, and MYBPC3. A variant in LPA was consistent with a family history of coronary artery disease. The patient had a heterozygous null mutation in CYP2C19 suggesting probable clopidogrel resistance, several variants associated with a positive response to lipid-lowering therapy, and variants in CYP4F2 and VKORC1 that suggest he might have a low initial dosing requirement for warfarin. Many variants of uncertain importance were reported., Interpretation: Although challenges remain, our results suggest that whole-genome sequencing can yield useful and clinically relevant information for individual patients., Funding: National Institute of General Medical Sciences; National Heart, Lung And Blood Institute; National Human Genome Research Institute; Howard Hughes Medical Institute; National Library of Medicine, Lucile Packard Foundation for Children's Health; Hewlett Packard Foundation; Breetwor Family Foundation., (Copyright 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
43. Genomic determination of the glucocorticoid response reveals unexpected mechanisms of gene regulation.
- Author
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Reddy TE, Pauli F, Sprouse RO, Neff NF, Newberry KM, Garabedian MJ, and Myers RM
- Subjects
- Binding Sites, Chromatin Immunoprecipitation, Dexamethasone metabolism, Genome drug effects, Humans, Lung metabolism, Proteins genetics, Proteins metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Sequence Analysis, DNA, Dexamethasone pharmacology, Gene Expression Regulation drug effects, Lung cytology, Lung drug effects, Receptors, Glucocorticoid metabolism
- Abstract
The glucocorticoid steroid hormone cortisol is released by the adrenal glands in response to stress and serves as a messenger in circadian rhythms. Transcriptional responses to this hormonal signal are mediated by the glucocorticoid receptor (GR). We determined GR binding throughout the human genome by using chromatin immunoprecipitation followed by next-generation DNA sequencing, and measured related changes in gene expression with mRNA sequencing in response to the glucocorticoid dexamethasone (DEX). We identified 4392 genomic positions occupied by the GR and 234 genes with significant changes in expression in response to DEX. This genomic census revealed striking differences between gene activation and repression by the GR. While genes activated with DEX treatment have GR bound within a median distance of 11 kb from the transcriptional start site (TSS), the nearest GR binding for genes repressed with DEX treatment is a median of 146 kb from the TSS, suggesting that DEX-mediated repression occurs independently of promoter-proximal GR binding. In addition to the dramatic differences in proximity of GR binding, we found differences in the kinetics of gene expression response for induced and repressed genes, with repression occurring substantially after induction. We also found that the GR can respond to different levels of corticosteroids in a gene-specific manner. For example, low doses of DEX selectively induced PER1, a transcription factor involved in regulating circadian rhythms. Overall, the genome-wide determination and analysis of GR:DNA binding and transcriptional response to hormone reveals new insights into the complexities of gene regulatory activities managed by GR.
- Published
- 2009
- Full Text
- View/download PDF
44. Single-molecule sequencing of an individual human genome.
- Author
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Pushkarev D, Neff NF, and Quake SR
- Subjects
- Computer Simulation, Humans, Polymorphism, Single Nucleotide, Reproducibility of Results, Genome, Human, Genomics methods, Sequence Analysis, DNA methods
- Abstract
Recent advances in high-throughput DNA sequencing technologies have enabled order-of-magnitude improvements in both cost and throughput. Here we report the use of single-molecule methods to sequence an individual human genome. We aligned billions of 24- to 70-bp reads (32 bp average) to approximately 90% of the National Center for Biotechnology Information (NCBI) reference genome, with 28x average coverage. Our results were obtained on one sequencing instrument by a single operator with four data collection runs. Single-molecule sequencing enabled analysis of human genomic information without the need for cloning, amplification or ligation. We determined approximately 2.8 million single nucleotide polymorphisms (SNPs) with a false-positive rate of less than 1% as validated by Sanger sequencing and 99.8% concordance with SNP genotyping arrays. We identified 752 regions of copy number variation by analyzing coverage depth alone and validated 27 of these using digital PCR. This milestone should allow widespread application of genome sequencing to many aspects of genetics and human health, including personal genomics.
- Published
- 2009
- Full Text
- View/download PDF
45. Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver.
- Author
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Brunner AL, Johnson DS, Kim SW, Valouev A, Reddy TE, Neff NF, Anton E, Medina C, Nguyen L, Chiao E, Oyolu CB, Schroth GP, Absher DM, Baker JC, and Myers RM
- Subjects
- Binding Sites, Cell Differentiation genetics, Cell Line, Cells, Cultured, Chromosome Mapping, Cluster Analysis, CpG Islands genetics, Embryonic Stem Cells cytology, Gene Expression Profiling, Genome, Human genetics, Histones metabolism, Humans, Liver cytology, Liver embryology, Lysine metabolism, Methylation, Promoter Regions, Genetic genetics, Sequence Analysis, DNA, DNA Methylation, Embryonic Stem Cells metabolism, Liver metabolism
- Abstract
To investigate the role of DNA methylation during human development, we developed Methyl-seq, a method that assays DNA methylation at more than 90,000 regions throughout the genome. Performing Methyl-seq on human embryonic stem cells (hESCs), their derivatives, and human tissues allowed us to identify several trends during hESC and in vivo liver differentiation. First, differentiation results in DNA methylation changes at a minimal number of assayed regions, both in vitro and in vivo (2%-11%). Second, in vitro hESC differentiation is characterized by both de novo methylation and demethylation, whereas in vivo fetal liver development is characterized predominantly by demethylation. Third, hESC differentiation is uniquely characterized by methylation changes specifically at H3K27me3-occupied regions, bivalent domains, and low density CpG promoters (LCPs), suggesting that these regions are more likely to be involved in transcriptional regulation during hESC differentiation. Although both H3K27me3-occupied domains and LCPs are also regions of high variability in DNA methylation state during human liver development, these regions become highly unmethylated, which is a distinct trend from that observed in hESCs. Taken together, our results indicate that hESC differentiation has a unique DNA methylation signature that may not be indicative of in vivo differentiation.
- Published
- 2009
- Full Text
- View/download PDF
46. Telomere and ribosomal DNA repeats are chromosomal targets of the bloom syndrome DNA helicase.
- Author
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Schawalder J, Paric E, and Neff NF
- Subjects
- Adenosine Triphosphatases chemistry, Adenosine Triphosphatases metabolism, Alleles, Binding Sites, Bloom Syndrome etiology, Bloom Syndrome genetics, Cell Cycle, Cell Line, Cell Nucleolus enzymology, Cell Nucleus Structures enzymology, Chromosomes ultrastructure, DNA Helicases chemistry, DNA Helicases metabolism, DNA Repair, DNA Replication, DNA, Ribosomal analysis, Genetic Variation, Humans, Protein Structure, Tertiary, RecQ Helicases, Recombinant Fusion Proteins analysis, Recombinant Fusion Proteins isolation & purification, Recombination, Genetic, Repetitive Sequences, Nucleic Acid, Telomere enzymology, Adenosine Triphosphatases analysis, Bloom Syndrome enzymology, Chromosomes enzymology, DNA Helicases analysis, DNA, Ribosomal chemistry, Telomere chemistry
- Abstract
Background: Bloom syndrome is one of the most cancer-predisposing disorders and is characterized by genomic instability and a high frequency of sister chromatid exchange. The disorder is caused by loss of function of a 3' to 5' RecQ DNA helicase, BLM. The exact role of BLM in maintaining genomic integrity is not known but the helicase has been found to associate with several DNA repair complexes and some DNA replication foci., Results: Chromatin immunoprecipitation of BLM complexes recovered telomere and ribosomal DNA repeats. The N-terminus of BLM, required for NB localization, is the same as the telomere association domain of BLM. The C-terminus is required for ribosomal DNA localization. BLM localizes primarily to the non-transcribed spacer region of the ribosomal DNA repeat where replication forks initiate. Bloom syndrome cells expressing the deletion alleles lacking the ribosomal DNA and telomere association domains have altered cell cycle populations with increased S or G2/M cells relative to normal., Conclusion: These results identify telomere and ribosomal DNA repeated sequence elements as chromosomal targets for the BLM DNA helicase during the S/G2 phase of the cell cycle. BLM is localized in nuclear bodies when it associates with telomeric repeats in both telomerase positive and negative cells. The BLM DNA helicase participates in genomic stability at ribosomal DNA repeats and telomeres.
- Published
- 2003
- Full Text
- View/download PDF
47. The C-terminal domain of the Bloom syndrome DNA helicase is essential for genomic stability.
- Author
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Yankiwski V, Noonan JP, and Neff NF
- Subjects
- Adenosine Triphosphatases genetics, Alleles, Cell Line, Transformed, Cell Nucleolus enzymology, Cell Nucleus enzymology, Chromosomes ultrastructure, DNA Helicases genetics, DNA Repair, Dose-Response Relationship, Drug, Doxycycline pharmacology, Genome, Green Fluorescent Proteins, Humans, Luminescent Proteins genetics, Mutation, Protein Structure, Tertiary, RecQ Helicases, Recombinant Fusion Proteins metabolism, Transcriptional Activation, Transfection, Adenosine Triphosphatases chemistry, Adenosine Triphosphatases physiology, DNA Helicases chemistry, DNA Helicases physiology, Sister Chromatid Exchange
- Abstract
Background: Bloom syndrome is a rare cancer-prone disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. Bloom syndrome cells have a distinctive high frequency of sister chromatid exchange and quadriradial formation. BLM, the protein altered in BS, is a member of the RecQ DNA helicase family, whose members share an average of 40% identity in the helicase domain and have divergent N-terminal and C-terminal flanking regions of variable lengths. The BLM DNA helicase has been shown to localize to the ND10 (nuclear domain 10) or PML (promyelocytic leukemia) nuclear bodies, where it associates with TOPIIIalpha, and to the nucleolus., Results: This report demonstrates that the N-terminal domain of BLM is responsible for localization of the protein to the nuclear bodies, while the C-terminal domain directs the protein to the nucleolus. Deletions of the N-terminal domain of BLM have little effect on sister chromatid exchange frequency and chromosome stability as compared to helicase and C-terminal mutations which can increase SCE frequency and chromosome abnormalities., Conclusion: The helicase activity and the C-terminal domain of BLM are critical for maintaining genomic stability as measured by the sister chromatid exchange assay. The localization of BLM into the nucleolus by the C-terminal domain appears to be more important to genomic stability than localization in the nuclear bodies.
- Published
- 2001
- Full Text
- View/download PDF
48. Nuclear structure in normal and Bloom syndrome cells.
- Author
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Yankiwski V, Marciniak RA, Guarente L, and Neff NF
- Subjects
- Adolescent, Adult, Bloom Syndrome enzymology, Bloom Syndrome genetics, Cell Line, Cell Nucleolus enzymology, Cell Nucleolus ultrastructure, Cell Nucleus enzymology, Cell Nucleus pathology, Child, Child, Preschool, Consanguinity, Exodeoxyribonucleases, Female, Humans, Jews genetics, Male, Middle Aged, Pedigree, Polymorphism, Restriction Fragment Length, RecQ Helicases, Reference Values, S Phase, Telomere enzymology, Telomere ultrastructure, Werner Syndrome enzymology, Werner Syndrome Helicase, Adenosine Triphosphatases analysis, Bloom Syndrome pathology, Cell Nucleus ultrastructure, DNA Helicases analysis
- Abstract
Bloom syndrome (BS) is a rare cancer-predisposing disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. BLM, the protein altered in BS, is a RecQ DNA helicase. This report shows that BLM is found in the nucleus of normal human cells in the nuclear domain 10 or promyelocytic leukemia nuclear bodies. These structures are punctate depots of proteins disrupted upon viral infection and in certain human malignancies. BLM is found primarily in nuclear domain 10 except during S phase when it colocalizes with the Werner syndrome gene product, WRN, in the nucleolus. BLM colocalizes with a select subset of telomeres in normal cells and with large telomeric clusters seen in simian virus 40-transformed normal fibroblasts. During S phase, BS cells expel micronuclei containing sites of DNA synthesis. BLM is likely to be part of a DNA surveillance mechanism operating during S phase.
- Published
- 2000
- Full Text
- View/download PDF
49. Association of the Bloom syndrome protein with topoisomerase IIIalpha in somatic and meiotic cells.
- Author
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Johnson FB, Lombard DB, Neff NF, Mastrangelo MA, Dewolf W, Ellis NA, Marciniak RA, Yin Y, Jaenisch R, and Guarente L
- Subjects
- Adenosine Triphosphatases genetics, DNA Helicases genetics, DNA Topoisomerases, Type I genetics, Gene Expression Regulation, Humans, RecQ Helicases, Tumor Cells, Cultured, Adenosine Triphosphatases metabolism, DNA Helicases metabolism, DNA Topoisomerases, Type I metabolism, Meiosis
- Abstract
Bloom syndrome (BS) is characterized by genomic instability and cancer susceptibility caused by defects in BLM, a DNA helicase of the RecQ-family (J. German and N. A. Ellis, The Genetic Basis of Human Cancer, pp. 301-316, 1998). RecQ helicases and topoisomerase III proteins interact physically and functionally in yeast (S. Gangloff et al., Mol. Cell. Biol., 14: 8391-8398, 1994) and in Escherichia coli can function together to enable passage of double-stranded DNA (F. G. Harmon et al., Mol. Cell, 3: 611-620, 1999). We demonstrate in somatic and meiotic human cells an association between BLM and topoisomerase IIIalpha. These proteins colocalize in promyelocytic leukemia protein nuclear bodies, and this localization is disrupted in BS cells. Thus, mechanisms by which RecQ helicases and topoisomerase III proteins cooperate to maintain genomic stability in model organisms likely apply to humans.
- Published
- 2000
50. Bloom's syndrome protein, BLM, colocalizes with replication protein A in meiotic prophase nuclei of mammalian spermatocytes.
- Author
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Walpita D, Plug AW, Neff NF, German J, and Ashley T
- Subjects
- Adenosine Triphosphatases analysis, Animals, DNA Helicases analysis, DNA Replication, DNA-Binding Proteins analysis, Humans, Image Processing, Computer-Assisted, Male, Mice, Microscopy, Fluorescence, Prophase, RecQ Helicases, Recombination, Genetic, Replication Protein A, Synaptonemal Complex genetics, Adenosine Triphosphatases metabolism, DNA Helicases metabolism, DNA-Binding Proteins metabolism, Spermatocytes metabolism
- Abstract
Bloom's syndrome (BS) is a rare autosomal recessive disorder of humans characterized by severe pre- and postnatal growth deficiency, immunodeficiency, genomic instability, and a predisposition to a wide variety of neoplasms. The genomic instability is evidenced in BS somatic cells as a high incidence of gaps and breaks, chromatid exchanges, chromosome rearrangements, and locus-specific mutations. BS arises from a mutation in BLM, a gene encoding a protein with homology to the RecQ helicase family. Men with BS are sterile; women have reduced fertility and a shortened reproductive span. The current immunocytological study on mouse spermatocytes shows that the BLM protein is first evident as discrete foci along the synaptonemal complexes (SCs) of homologously synapsed autosomal bivalents in late zygonema of meiotic prophase. BLM foci progressively dissociate from the synapsed autosomal axes during early pachynema and are no longer seen in mid-pachynema. BLM colocalizes with the single-stranded DNA binding replication protein A, which has been shown to be involved in meiotic synapsis. However, there is a temporal delay in the appearance of BLM protein along the SCs relative to replication protein A, suggesting that BLM is required for a late step in processing of a subset of genomic DNA involved in establishment of interhomologue interactions in early meiotic prophase. In late pachynema and into diplonema, BLM is more dispersed in the nucleoplasm, especially over the chromatin most intimately associated with the SCs, suggesting a possible involvement of BLM in resolution of interlocks in preparation for homologous chromosome disjunction during anaphase I.
- Published
- 1999
- Full Text
- View/download PDF
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