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1.
An Acad Bras Cienc ; 93(1): e20180534, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33787681

RESUMO

This study evaluate growth, gas exchange, solute accumulation and activity of antioxidant enzymes in dwarf cashew clones subjected to salinity. Shoot dry mass reduced 26.8% (CCP06) and 41.2% (BRS189) at 16 dS m-1, concerning control. For net photosynthesis, CCP06 and BRS189 presented 69.8% and 34.7% of reduction, respectively. Na+ and Cl- contents increased in leaves and roots, in both clones, although CCP06 leaves presented Na+ concentrations lower than those of BRS189, the first one was the clone that the most accumulated such toxic ion, whereas K+ content remained almost unchanged for both clones. Soluble N-amino was the organic solute that more varied with salinity in cashew seedlings. Salt stress increased the activity of superoxide dismutase in both clones, mainly 16 dS m-1 treatment. Additionally, salinity promoted increases in ascorbate and guaiacol peroxidase activities, and the last enzyme was the main involved in H2O2 removal. Despite the reductions in growth and gas exchange, dwarf cashew seedlings of both clones presented an osmotic adjustment mechanism, and an efficient enzymatic antioxidant system that were able to attenuate the salt and oxidative stress, respectively. Our research suggested that BRS189 clone is more tolerant to salt stress than CCP06.


Assuntos
Anacardium , Antioxidantes , Células Clonais , Peróxido de Hidrogênio , Folhas de Planta , Salinidade
2.
Science ; 371(6535): 1249-1253, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33737485

RESUMO

Although cell lineage information is fundamental to understanding organismal development, very little direct information is available for humans. We performed high-depth (250×) whole-genome sequencing of multiple tissues from three individuals to identify hundreds of somatic single-nucleotide variants (sSNVs). Using these variants as "endogenous barcodes" in single cells, we reconstructed early embryonic cell divisions. Targeted sequencing of clonal sSNVs in different organs (about 25,000×) and in more than 1000 cortical single cells, as well as single-nucleus RNA sequencing and single-nucleus assay for transposase-accessible chromatin sequencing of ~100,000 cortical single cells, demonstrated asymmetric contributions of early progenitors to extraembryonic tissues, distinct germ layers, and organs. Our data suggest onset of gastrulation at an effective progenitor pool of about 170 cells and about 50 to 100 founders for the forebrain. Thus, mosaic mutations provide a permanent record of human embryonic development at very high resolution.


Assuntos
Linhagem da Célula , Gastrulação , Mutação , Células-Tronco Neurais/citologia , Prosencéfalo/citologia , Adolescente , Adulto , Divisão Celular , Células Clonais/citologia , Desenvolvimento Embrionário/genética , Feminino , Gástrula/citologia , Variação Genética , Camadas Germinativas/citologia , Humanos , Masculino , Neurônios/citologia , Organogênese , Polimorfismo de Nucleotídeo Único , Prosencéfalo/embriologia , Análise de Célula Única , Sequenciamento Completo do Genoma
3.
Nat Commun ; 12(1): 1751, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741915

RESUMO

Malignant Pleural Mesothelioma (MPM) is typically diagnosed 20-50 years after exposure to asbestos and evolves along an unknown evolutionary trajectory. To elucidate this path, we conducted multi-regional exome sequencing of 90 tumour samples from 22 MPMs acquired at surgery. Here we show that exomic intratumour heterogeneity varies widely across the cohort. Phylogenetic tree topology ranges from linear to highly branched, reflecting a steep gradient of genomic instability. Using transfer learning, we detect repeated evolution, resolving 5 clusters that are prognostic, with temporally ordered clonal drivers. BAP1/-3p21 and FBXW7/-chr4 events are always early clonal. In contrast, NF2/-22q events, leading to Hippo pathway inactivation are predominantly late clonal, positively selected, and when subclonal, exhibit parallel evolution indicating an evolutionary constraint. Very late somatic alteration of NF2/22q occurred in one patient 12 years after surgery. Clonal architecture and evolutionary clusters dictate MPM inflammation and immune evasion. These results reveal potentially drugable evolutionary bottlenecking in MPM, and an impact of clonal architecture on shaping the immune landscape, with potential to dictate the clinical response to immune checkpoint inhibition.


Assuntos
Deleção Cromossômica , Neoplasias Pulmonares/genética , Mesotelioma/genética , Mutação , Neoplasias Pleurais/genética , Proteínas Supressoras de Tumor/genética , Células Clonais/metabolismo , Células Clonais/patologia , Análise por Conglomerados , Estudos de Coortes , Humanos , Estimativa de Kaplan-Meier , Prognóstico , Microambiente Tumoral/genética , Proteínas Supressoras de Tumor/classificação , Sequenciamento Completo do Exoma/métodos
4.
Nat Commun ; 12(1): 1366, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33649320

RESUMO

Cancer stem cells drive disease progression and relapse in many types of cancer. Despite this, a thorough characterization of these cells remains elusive and with it the ability to eradicate cancer at its source. In acute myeloid leukemia (AML), leukemic stem cells (LSCs) underlie mortality but are difficult to isolate due to their low abundance and high similarity to healthy hematopoietic stem cells (HSCs). Here, we demonstrate that LSCs, HSCs, and pre-leukemic stem cells can be identified and molecularly profiled by combining single-cell transcriptomics with lineage tracing using both nuclear and mitochondrial somatic variants. While mutational status discriminates between healthy and cancerous cells, gene expression distinguishes stem cells and progenitor cell populations. Our approach enables the identification of LSC-specific gene expression programs and the characterization of differentiation blocks induced by leukemic mutations. Taken together, we demonstrate the power of single-cell multi-omic approaches in characterizing cancer stem cells.


Assuntos
Células Clonais/patologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Análise de Célula Única , Transcriptoma/genética , Biomarcadores Tumorais/genética , Medula Óssea/patologia , Diferenciação Celular , Regulação Leucêmica da Expressão Gênica , Genoma , Células-Tronco Hematopoéticas/patologia , Humanos , Células K562 , Mitocôndrias/genética , Mutação/genética
5.
Nat Commun ; 12(1): 1035, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33589603

RESUMO

Stochastic asynchronous replication timing (AS-RT) is a phenomenon in which the time of replication of each allele is different, and the identity of the early allele varies between cells. By taking advantage of stable clonal pre-B cell populations derived from C57BL6/Castaneous mice, we have mapped the genome-wide AS-RT loci, independently of genetic differences. These regions are characterized by differential chromatin accessibility, mono-allelic expression and include new gene families involved in specifying cell identity. By combining population level mapping with single cell FISH, our data reveal the existence of a novel regulatory program that coordinates a fixed relationship between AS-RT regions on any given chromosome, with some loci set to replicate in a parallel and others set in the anti-parallel orientation. Our results show that AS-RT is a highly regulated epigenetic mark established during early embryogenesis that may be used for facilitating the programming of mono-allelic choice throughout development.


Assuntos
Células da Medula Óssea/metabolismo , Cromatina/química , Período de Replicação do DNA , Epigênese Genética , Genoma , Células Precursoras de Linfócitos B/metabolismo , Alelos , Animais , Células da Medula Óssea/citologia , Cromatina/metabolismo , Cromatina/ultraestrutura , Células Clonais , Cruzamentos Genéticos , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Feminino , Loci Gênicos , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Precursoras de Linfócitos B/citologia
6.
Nat Commun ; 12(1): 1047, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594075

RESUMO

Despite the success of checkpoint blockade in some cancer patients, there is an unmet need to improve outcomes. Targeting alternative pathways, such as costimulatory molecules (e.g. OX40, GITR, and 4-1BB), can enhance T cell immunity in tumor-bearing hosts. Here we describe the results from a phase Ib clinical trial (NCT02274155) in which 17 patients with locally advanced head and neck squamous cell carcinoma (HNSCC) received a murine anti-human OX40 agonist antibody (MEDI6469) prior to definitive surgical resection. The primary endpoint was to determine safety and feasibility of the anti-OX40 neoadjuvant treatment. The secondary objective was to assess the effect of anti-OX40 on lymphocyte subsets in the tumor and blood. Neoadjuvant anti-OX40 was well tolerated and did not delay surgery, thus meeting the primary endpoint. Peripheral blood phenotyping data show increases in CD4+ and CD8+ T cell proliferation two weeks after anti-OX40 administration. Comparison of tumor biopsies before and after treatment reveals an increase of activated, conventional CD4+ tumor-infiltrating lymphocytes (TIL) in most patients and higher clonality by TCRß sequencing. Analyses of CD8+ TIL show increases in tumor-antigen reactive, proliferating CD103+ CD39+ cells in 25% of patients with evaluable tumor tissue (N = 4/16), all of whom remain disease-free. These data provide evidence that anti-OX40 prior to surgery is safe and can increase activation and proliferation of CD4+ and CD8+ T cells in blood and tumor. Our work suggests that increases in the tumor-reactive CD103+ CD39+ CD8+ TIL could serve as a potential biomarker of anti-OX40 clinical activity.


Assuntos
Epitopos/imunologia , Terapia Neoadjuvante , Receptores OX40/antagonistas & inibidores , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Biópsia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Células Clonais , Intervalo Livre de Doença , Papillomavirus Humano 16/fisiologia , Humanos , Estimativa de Kaplan-Meier , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Terapia Neoadjuvante/efeitos adversos , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores OX40/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/sangue , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Células Estromais/metabolismo
7.
Sci Immunol ; 6(56)2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33622974

RESUMO

Hyperinflammation contributes to lung injury and subsequent acute respiratory distress syndrome (ARDS) with high mortality in patients with severe coronavirus disease 2019 (COVID-19). To understand the underlying mechanisms involved in lung pathology, we investigated the role of the lung-specific immune response. We profiled immune cells in bronchoalveolar lavage fluid and blood collected from COVID-19 patients with severe disease and bacterial pneumonia patients not associated with viral infection. By tracking T cell clones across tissues, we identified clonally expanded tissue-resident memory-like Th17 cells (Trm17 cells) in the lungs even after viral clearance. These Trm17 cells were characterized by a a potentially pathogenic cytokine expression profile of IL17A and CSF2 (GM-CSF). Interactome analysis suggests that Trm17 cells can interact with lung macrophages and cytotoxic CD8+ T cells, which have been associated with disease severity and lung damage. High IL-17A and GM-CSF protein levels in the serum of COVID-19 patients were associated with a more severe clinical course. Collectively, our study suggests that pulmonary Trm17 cells are one potential orchestrator of the hyperinflammation in severe COVID-19.


Assuntos
/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Memória Imunológica , Pulmão/imunologia , Células Th17/metabolismo , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , /patologia , Células Clonais , Humanos , Inflamação/etiologia , Inflamação/imunologia , Pulmão/patologia , Células Mieloides , Pneumonia Bacteriana/imunologia , Células Th17/imunologia
8.
Pathologe ; 42(2): 241-251, 2021 Mar.
Artigo em Alemão | MEDLINE | ID: mdl-33575888

RESUMO

Malignant lymphomas are derived from a common progenitor cell with a unique rearrangement of immunoglobulin or T­cell receptor genes. Polymerase chain reaction (PCR)-based analyses allow detection of the clone and are an important adjunct for the diagnosis of difficult lymphoproliferations, e.g. for the discrimination of reactive versus malignant lesions. Further applications are detection of disease dissemination and evaluation of the clonal relationship of two lymphomas. However, clonality analysis is not a stand-alone test and must always be considered in context with clinical, histological and immunophenotypic data. For the correct use of clonality analysis, comprehensive knowledge of the biological basis, technical requirements and interpretation are needed in order to avoid incorrect conclusions.


Assuntos
Linfoma , Células Clonais , Humanos , Linfoma/genética , Reação em Cadeia da Polimerase
9.
Nat Commun ; 12(1): 865, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33558546

RESUMO

Chimeric antigen receptor T cell (CAR-T) targeting the CD19 antigen represents an innovative therapeutic approach to improve the outcome of relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). Yet, despite a high initial remission rate, CAR-T therapy ultimately fails for some patients. Notably, around half of relapsing patients develop CD19 negative (CD19neg) B-ALL allowing leukemic cells to evade CD19-targeted therapy. Herein, we investigate leukemic cells of a relapsing B-ALL patient, at two-time points: before (T1) and after (T2) anti-CD19 CAR-T treatment. We show that at T2, the B-ALL relapse is CD19 negative due to the expression of a non-functional CD19 transcript retaining intron 2. Then, using single-cell RNA sequencing (scRNAseq) approach, we demonstrate that CD19neg leukemic cells were present before CAR-T cell therapy and thus that the relapse results from the selection of these rare CD19neg B-ALL clones. In conclusion, our study shows that scRNAseq profiling can reveal pre-existing CD19neg subclones, raising the possibility to assess the risk of targeted therapy failure.


Assuntos
Antígenos CD19/metabolismo , Imunoterapia Adotiva , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Análise de Célula Única , Criança , Células Clonais , Humanos , Recidiva
10.
Nat Commun ; 12(1): 863, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33558489

RESUMO

A concept of polyclonal metastasis has recently been proposed, wherein tumor cell clusters break off from the primary site and are disseminated. However, the involvement of driver mutations in such polyclonal mechanism is not fully understood. Here, we show that non-metastatic AP cells metastasize to the liver with metastatic AKTP cells after co-transplantation to the spleen. Furthermore, AKTP cell depletion after the development of metastases results in the continuous proliferation of the remaining AP cells, indicating a role of AKTP cells in the early step of polyclonal metastasis. Importantly, AKTP cells, but not AP cells, induce fibrotic niche generation when arrested in the sinusoid, and such fibrotic microenvironment promotes the colonization of AP cells. These results indicate that non-metastatic cells can metastasize via the polyclonal metastasis mechanism using the fibrotic niche induced by malignant cells. Thus, targeting the fibrotic niche is an effective strategy for halting polyclonal metastasis.


Assuntos
Metástase Neoplásica/patologia , Neoplasias/genética , Neoplasias/patologia , Animais , Agregação Celular , Proliferação de Células , Células Clonais , Fibrose , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Fígado/irrigação sanguínea , Fígado/patologia , Camundongos Endogâmicos NOD , Organoides/patologia , Fenótipo , Baço/transplante , Fator de Crescimento Transformador beta/farmacologia
11.
Mol Cell ; 81(5): 1013-1026.e11, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33548202

RESUMO

In response to stress, human cells coordinately downregulate transcription and translation of housekeeping genes. To downregulate transcription, the negative elongation factor (NELF) is recruited to gene promoters impairing RNA polymerase II elongation. Here we report that NELF rapidly forms nuclear condensates upon stress in human cells. Condensate formation requires NELF dephosphorylation and SUMOylation induced by stress. The intrinsically disordered region (IDR) in NELFA is necessary for nuclear NELF condensation and can be functionally replaced by the IDR of FUS or EWSR1 protein. We find that biomolecular condensation facilitates enhanced recruitment of NELF to promoters upon stress to drive transcriptional downregulation. Importantly, NELF condensation is required for cellular viability under stressful conditions. We propose that stress-induced NELF condensates reported here are nuclear counterparts of cytosolic stress granules. These two stress-inducible condensates may drive the coordinated downregulation of transcription and translation, likely forming a critical node of the stress survival strategy.


Assuntos
Resposta ao Choque Térmico/genética , Proteínas Intrinsicamente Desordenadas/genética , Processamento de Proteína Pós-Traducional , RNA Polimerase II/genética , Transcrição Genética , Fatores de Elongação da Transcrição/genética , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Cromatina/química , Cromatina/metabolismo , Células Clonais , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Genes Reporter , Células HEK293 , Células HeLa , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fosforilação , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Transdução de Sinais , Estresse Fisiológico , Sumoilação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo
12.
Nat Commun ; 12(1): 1119, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33602930

RESUMO

Regulatory CD4+ T cells (Treg) prevent tumor clearance by conventional T cells (Tconv) comprising a major obstacle of cancer immune-surveillance. Hitherto, the mechanisms of Treg repertoire formation in human cancers remain largely unclear. Here, we analyze Treg clonal origin in breast cancer patients using T-Cell Receptor and single-cell transcriptome sequencing. While Treg in peripheral blood and breast tumors are clonally distinct, Tconv clones, including tumor-antigen reactive effectors (Teff), are detected in both compartments. Tumor-infiltrating CD4+ cells accumulate into distinct transcriptome clusters, including early activated Tconv, uncommitted Teff, Th1 Teff, suppressive Treg and pro-tumorigenic Treg. Trajectory analysis suggests early activated Tconv differentiation either into Th1 Teff or into suppressive and pro-tumorigenic Treg. Importantly, Tconv, activated Tconv and Treg share highly-expanded clones contributing up to 65% of intratumoral Treg. Here we show that Treg in human breast cancer may considerably stem from antigen-experienced Tconv converting into secondary induced Treg through intratumoral activation.


Assuntos
Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linfócitos T Reguladores/imunologia , Antígenos de Neoplasias/metabolismo , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células , Células Clonais , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Ativação Linfocitária/imunologia , Estadiamento de Neoplasias , Receptores de Antígenos de Linfócitos T/imunologia , Análise de Célula Única , Células Th1/imunologia , Transcriptoma/genética
13.
Am J Hematol ; 96(3): 379-394, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33428785

RESUMO

DISEASE OVERVIEW: Ring sideroblasts (RS) are erythroid precursors with abnormal perinuclear mitochondrial iron accumulation. Two myeloid neoplasms defined by the presence of RS, include myelodysplastic syndromes with RS (MDS-RS) and MDS/myeloproliferative neoplasm with RS and thrombocytosis (MDS/MPN-RS-T). DIAGNOSIS: MDS-RS is a lower risk MDS, with single or multilineage dysplasia (MDS-RS-SLD/MLD), <5% bone marrow (BM) blasts, <1% peripheral blood blasts and ≥15% BM RS (≥5% in the presence of SF3B1 mutations). MDS/MPN-RS-T, now a formal entity in the MDS/MPN overlap syndromes, has diagnostic features of MDS-RS-SLD, along with a platelet count ≥450 × 109 /L and large atypical megakaryocytes. MUTATIONS AND KARYOTYPE: Mutations in SF3B1 are seen in ≥80% of patients with MDS-RS-SLD and MDS/MPN-RS-T, and strongly correlate with the presence of BM RS; MDS/MPN-RS-T patients also demonstrate JAK2V617F (50%), DNMT3A, TET2 and ASXL1 mutations. Cytogenetic abnormalities are uncommon in both. RISK STRATIFICATION: Most patients with MDS-RS-SLD are stratified into lower risk groups by the revised-IPSS. Disease outcome in MDS/MPN-RS-T is better than that of MDS-RS-SLD, but worse than that of essential thrombocythemia (MPN). Both diseases are associated with a low risk of leukemic transformation. TREATMENT: Anemia and iron overload are complications seen in both and are managed similar to lower risk MDS and MPN. Luspatercept, a first-in-class erythroid maturation agent is now approved for the management of anemia in patients with MDS-RS and MDS/MPN-RS-T. Aspirin therapy is reasonable in MDS/MPN-RS-T, especially in the presence of JAK2V617F, but the value of platelet-lowering drugs remains to be defined.


Assuntos
Anemia Sideroblástica , Doenças Mieloproliferativas-Mielodisplásicas , Aloenxertos , Anemia Sideroblástica/diagnóstico , Anemia Sideroblástica/etiologia , Anemia Sideroblástica/patologia , Anemia Sideroblástica/terapia , Medula Óssea/patologia , Linhagem da Célula , Células Clonais/patologia , Terapia Combinada , Metilação de DNA/efeitos dos fármacos , Gerenciamento Clínico , Eritroblastos/ultraestrutura , Ferritinas/análise , Hematínicos/uso terapêutico , Transplante de Células-Tronco Hematopoéticas , Humanos , Quelantes de Ferro/uso terapêutico , Mitocôndrias/química , Mutação , Doenças Mieloproliferativas-Mielodisplásicas/diagnóstico , Doenças Mieloproliferativas-Mielodisplásicas/genética , Doenças Mieloproliferativas-Mielodisplásicas/terapia , Fosfoproteínas/genética , Prognóstico , Fatores de Processamento de RNA/genética , Medição de Risco , Trombocitose/diagnóstico , Trombocitose/terapia
14.
Science ; 371(6532)2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33479121

RESUMO

Detailed phylogenies of tumor populations can recount the history and chronology of critical events during cancer progression, such as metastatic dissemination. We applied a Cas9-based, single-cell lineage tracer to study the rates, routes, and drivers of metastasis in a lung cancer xenograft mouse model. We report deeply resolved phylogenies for tens of thousands of cancer cells traced over months of growth and dissemination. This revealed stark heterogeneity in metastatic capacity, arising from preexisting and heritable differences in gene expression. We demonstrate that these identified genes can drive invasiveness and uncovered an unanticipated suppressive role for KRT17 We also show that metastases disseminated via multidirectional tissue routes and complex seeding topologies. Overall, we demonstrate the power of tracing cancer progression at subclonal resolution and vast scale.


Assuntos
Neoplasias Pulmonares/patologia , Metástase Neoplásica , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Linhagem da Célula , Células Clonais , Regulação Neoplásica da Expressão Gênica , Humanos , Queratina-17/genética , Neoplasias Pulmonares/genética , Camundongos , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Inoculação de Neoplasia , Transplante de Neoplasias , Fenótipo , RNA-Seq , Análise de Célula Única , Transcriptoma , Transplante Heterólogo
15.
Nature ; 590(7845): 344-350, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33505024

RESUMO

Identifying the relationships between chromosome structures, nuclear bodies, chromatin states and gene expression is an overarching goal of nuclear-organization studies1-4. Because individual cells appear to be highly variable at all these levels5, it is essential to map different modalities in the same cells. Here we report the imaging of 3,660 chromosomal loci in single mouse embryonic stem (ES) cells using DNA seqFISH+, along with 17 chromatin marks and subnuclear structures by sequential immunofluorescence and the expression profile of 70 RNAs. Many loci were invariably associated with immunofluorescence marks in single mouse ES cells. These loci form 'fixed points' in the nuclear organizations of single cells and often appear on the surfaces of nuclear bodies and zones defined by combinatorial chromatin marks. Furthermore, highly expressed genes appear to be pre-positioned to active nuclear zones, independent of bursting dynamics in single cells. Our analysis also uncovered several distinct mouse ES cell subpopulations with characteristic combinatorial chromatin states. Using clonal analysis, we show that the global levels of some chromatin marks, such as H3 trimethylation at lysine 27 (H3K27me3) and macroH2A1 (mH2A1), are heritable over at least 3-4 generations, whereas other marks fluctuate on a faster time scale. This seqFISH+-based spatial multimodal approach can be used to explore nuclear organization and cell states in diverse biological systems.


Assuntos
Compartimento Celular/genética , Núcleo Celular/genética , Genômica/métodos , Células-Tronco Embrionárias Murinas/citologia , Análise de Célula Única/métodos , Transcriptoma/genética , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Cromossomos de Mamíferos/genética , Células Clonais/citologia , Imunofluorescência , Marcadores Genéticos , Histonas/metabolismo , Lisina/metabolismo , Masculino , Camundongos , Fatores de Tempo
16.
Nat Commun ; 12(1): 472, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33473139

RESUMO

Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a methodology that utilizes barcoded AAV6 donor template libraries, carrying in-frame silent mutations or semi-randomized nucleotides outside the coding region, to track the in vivo lineage contribution of gene-targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in mice. We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.


Assuntos
Alelos , Células Clonais , Marcação de Genes/métodos , Células-Tronco Hematopoéticas , Recombinação Genética , Animais , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Edição de Genes/métodos , Terapia Genética/métodos , Humanos , Camundongos , Mutação , Reparo Gênico Alvo-Dirigido/métodos
17.
Anticancer Res ; 41(2): 1035-1040, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33517312

RESUMO

BACKGROUND/AIM: The definition of multiple oral cancers is based on the distances between the tumors. However, it is not possible to accurately predict tumor origins based only on clinical criteria. PATIENTS AND METHODS: We performed whole-exome sequencing (WES) to analyze the genetic alterations in five tumors of two patients who underwent surgery in our hospital. RESULTS: In case 1, the distances between tumors on the right mandibular gingiva and buccal mucosa were more than 15 mm, leading to a clinical diagnosis of multiple primary tumors. WES revealed common mutations between tumors, suggesting that the tumors were derived from the same clone. In contrast, in case 2, the distance between tumors on the right side of the tongue was only 10 mm, but the tumors were diagnosed as double primary tumors because their mutations were completely different. CONCLUSION: WES, rather than the available clinical criteria, can clarify the clonal origins of multiple oral cancers.


Assuntos
Células Clonais/patologia , Neoplasias Bucais/diagnóstico , Mutação , Neoplasias Primárias Múltiplas/diagnóstico , Sequenciamento Completo do Exoma/métodos , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/genética , Neoplasias Bucais/cirurgia , Metástase Neoplásica , Neoplasias Primárias Múltiplas/genética , Neoplasias Primárias Múltiplas/cirurgia , Especificidade da Espécie
18.
Kidney Int ; 99(2): 303-305, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33509349

RESUMO

Immunotactoid glomerulopathy (ITG) is a rare disease diagnosed by kidney biopsy showing characteristic microtubules, often in parallel arrays, in glomeruli on electron microscopy. Most cases are caused by lymphoproliferative disorders that produce monoclonal immunoglobulins that cause kidney damage, but these disorders do not meet criteria for overt malignancy. The published literature on ITG is limited. In this issue of Kidney International, 2 manuscripts provide significant insight into the clinical presentation, pathology, and treatment of ITG.


Assuntos
Glomerulonefrite , Nefropatias , Células Clonais , Estudos de Coortes , Humanos , Nefropatias/diagnóstico , Nefropatias/terapia , Glomérulos Renais
19.
Nucleic Acids Res ; 49(2): 1173-1198, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33398349

RESUMO

RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by 'tiptoeing' over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of DNA knockout and knock-in procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.


Assuntos
Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Desoxirribonuclease I/metabolismo , Edição de Genes/métodos , Proteínas de Bactérias/genética , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Células Clonais , Desoxirribonuclease I/genética , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Genes Reporter , Técnicas de Genotipagem , Células HEK293 , Células HeLa , Heterocromatina/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas , Polimorfismo Genético , RNA Guia/genética , Proteínas Recombinantes/metabolismo , Streptococcus pyogenes/enzimologia , Especificidade por Substrato , Transfecção
20.
Nat Commun ; 12(1): 293, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436579

RESUMO

Smoldering myeloma (SMM) is associated with a high-risk of progression to myeloma (MM). We report the results of a study of 82 patients with both targeted sequencing that included a capture of the immunoglobulin and MYC regions. By comparing these results to newly diagnosed myeloma (MM) we show fewer NRAS and FAM46C mutations together with fewer adverse translocations, del(1p), del(14q), del(16q), and del(17p) in SMM consistent with their role as drivers of the transition to MM. KRAS mutations are associated with a shorter time to progression (HR 3.5 (1.5-8.1), p = 0.001). In an analysis of change in clonal structure over time we studied 53 samples from nine patients at multiple time points. Branching evolutionary patterns, novel mutations, biallelic hits in crucial tumour suppressor genes, and segmental copy number changes are key mechanisms underlying the transition to MM, which can precede progression and be used to guide early intervention strategies.


Assuntos
Evolução Molecular , Mieloma Múltiplo Latente/genética , Desaminases APOBEC/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Biomarcadores Tumorais/metabolismo , Células Clonais , Variações do Número de Cópias de DNA/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Genoma Humano , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Mutação/genética , Taxa de Mutação , Intervalo Livre de Progressão , Proteínas Proto-Oncogênicas p21(ras)/genética , Mieloma Múltiplo Latente/diagnóstico , Fatores de Tempo , Translocação Genética
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