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1.
Arthritis Res Ther ; 26(1): 12, 2024 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-38173036

RESUMO

BACKGROUND: Low back pain is a leading cause of disability worldwide and is frequently attributed to intervertebral disc (IVD) degeneration. Though the contributions of the adjacent cartilage endplates (CEP) to IVD degeneration are well documented, the phenotype and functions of the resident CEP cells are critically understudied. To better characterize CEP cell phenotype and possible mechanisms of CEP degeneration, bulk and single-cell RNA sequencing of non-degenerated and degenerated CEP cells were performed. METHODS: Human lumbar CEP cells from degenerated (Thompson grade ≥ 4) and non-degenerated (Thompson grade ≤ 2) discs were expanded for bulk (N=4 non-degenerated, N=4 degenerated) and single-cell (N=1 non-degenerated, N=1 degenerated) RNA sequencing. Genes identified from bulk RNA sequencing were categorized by function and their expression in non-degenerated and degenerated CEP cells were compared. A PubMed literature review was also performed to determine which genes were previously identified and studied in the CEP, IVD, and other cartilaginous tissues. For single-cell RNA sequencing, different cell clusters were resolved using unsupervised clustering and functional annotation. Differential gene expression analysis and Gene Ontology, respectively, were used to compare gene expression and functional enrichment between cell clusters, as well as between non-degenerated and degenerated CEP samples. RESULTS: Bulk RNA sequencing revealed 38 genes were significantly upregulated and 15 genes were significantly downregulated in degenerated CEP cells relative to non-degenerated cells (|fold change| ≥ 1.5). Of these, only 2 genes were previously studied in CEP cells, and 31 were previously studied in the IVD and other cartilaginous tissues. Single-cell RNA sequencing revealed 11 unique cell clusters, including multiple chondrocyte and progenitor subpopulations with distinct gene expression and functional profiles. Analysis of genes in the bulk RNA sequencing dataset showed that progenitor cell clusters from both samples were enriched in "non-degenerated" genes but not "degenerated" genes. For both bulk- and single-cell analyses, gene expression and pathway enrichment analyses highlighted several pathways that may regulate CEP degeneration, including transcriptional regulation, translational regulation, intracellular transport, and mitochondrial dysfunction. CONCLUSIONS: This thorough analysis using RNA sequencing methods highlighted numerous differences between non-degenerated and degenerated CEP cells, the phenotypic heterogeneity of CEP cells, and several pathways of interest that may be relevant in CEP degeneration.


Assuntos
Degeneração do Disco Intervertebral , Disco Intervertebral , Humanos , Disco Intervertebral/metabolismo , Cartilagem/metabolismo , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/metabolismo , Condrócitos/metabolismo , Células-Tronco/metabolismo
2.
Nat Cancer ; 4(11): 1592-1609, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37904046

RESUMO

Safely expanding indications for cellular therapies has been challenging given a lack of highly cancer-specific surface markers. Here we explore the hypothesis that tumor cells express cancer-specific surface protein conformations that are invisible to standard target discovery pipelines evaluating gene or protein expression, and these conformations can be identified and immunotherapeutically targeted. We term this strategy integrating cross-linking mass spectrometry with glycoprotein surface capture 'structural surfaceomics'. As a proof of principle, we apply this technology to acute myeloid leukemia (AML), a hematologic malignancy with dismal outcomes and no known optimal immunotherapy target. We identify the activated conformation of integrin ß2 as a structurally defined, widely expressed AML-specific target. We develop and characterize recombinant antibodies to this protein conformation and show that chimeric antigen receptor T cells eliminate AML cells and patient-derived xenografts without notable toxicity toward normal hematopoietic cells. Our findings validate an AML conformation-specific target antigen and demonstrate a tool kit for applying these strategies more broadly.


Assuntos
Leucemia Mieloide Aguda , Receptores de Antígenos Quiméricos , Humanos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T , Integrinas/metabolismo , Imunoterapia Adotiva/métodos , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/genética
3.
Cell ; 186(2): 446-460.e19, 2023 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-36638795

RESUMO

Precise targeting of large transgenes to T cells using homology-directed repair has been transformative for adoptive cell therapies and T cell biology. Delivery of DNA templates via adeno-associated virus (AAV) has greatly improved knockin efficiencies, but the tropism of current AAV serotypes restricts their use to human T cells employed in immunodeficient mouse models. To enable targeted knockins in murine T cells, we evolved Ark313, a synthetic AAV that exhibits high transduction efficiency in murine T cells. We performed a genome-wide knockout screen and identified QA2 as an essential factor for Ark313 infection. We demonstrate that Ark313 can be used for nucleofection-free DNA delivery, CRISPR-Cas9-mediated knockouts, and targeted integration of large transgenes. Ark313 enables preclinical modeling of Trac-targeted CAR-T and transgenic TCR-T cells in immunocompetent models. Efficient gene targeting in murine T cells holds great potential for improved cell therapies and opens avenues in experimental T cell immunology.


Assuntos
Dependovirus , Engenharia Genética , Linfócitos T , Animais , Camundongos , Sistemas CRISPR-Cas/genética , Dependovirus/genética , Marcação de Genes , Engenharia Genética/métodos
4.
Chem Commun (Camb) ; 58(40): 5992-5995, 2022 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-35485326

RESUMO

In this work, we describe the preparation of double-decker cage [1-H6]6+ comprising two binding pockets, each with three ammonium and three amide hydrogen bonding sites. This novel host possesses a high affinity for trapping two molecules of ATP in an allosteric fashion, with both experiments and theory suggesting the synergistic action of charged hydrogen bonds and π-π stacking in the encapsulation.


Assuntos
Trifosfato de Adenosina , Trifosfato de Adenosina/metabolismo , Ligação de Hidrogênio
5.
Angew Chem Int Ed Engl ; 61(12): e202116518, 2022 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-35038355

RESUMO

Supramolecular hosts bind to inorganic anions at a fast rate and select them in proportion with thermodynamic stability of the corresponding [anion⊂host] complexes, forming in a reversible manner. In this study, we describe the action of hexapodal capsule 1 and its remarkable ability to select anions based on a large span of rates by which they enter this host. The thermodynamic affinity of 1 toward eighteen anions extends over eight orders of magnitude (0

Assuntos
Carbonatos , Ânions/química , Cinética , Espectroscopia de Ressonância Magnética , Termodinâmica
6.
Sci Adv ; 7(41): eabh2443, 2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34613780

RESUMO

Cell state transitions control the functional behavior of cancer cells. Epithelial-to-mesenchymal transition (EMT) confers cancer stem cell-like properties, enhanced tumorigenicity and drug resistance to tumor cells, while mesenchymal-epithelial transition (MET) reverses these phenotypes. Using high-throughput chemical library screens, retinoids are found to be potent promoters of MET that inhibit tumorigenicity in basal-like breast cancer. Cell state transitions are defined by reprogramming of lipid metabolism. Retinoids bind cognate nuclear receptors, which target lipid metabolism genes, thereby redirecting fatty acids for ß-oxidation in the mesenchymal cell state towards lipid storage in the epithelial cell state. Disruptions of key metabolic enzymes mediating this flux inhibit MET. Conversely, perturbations to fatty acid oxidation (FAO) rechannel fatty acid flux and promote a more epithelial cell phenotype, blocking EMT-driven breast cancer metastasis in animal models. FAO impinges on the epigenetic control of EMT through acetyl-CoA-dependent regulation of histone acetylation on EMT genes, thus determining cell states.

7.
JAMA Netw Open ; 3(3): e201074, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32181827

RESUMO

Importance: Promoting patient mobility during hospitalization is associated with improved outcomes and reduced risk of hospitalization-associated functional decline. Therefore, accurate measurement of mobility with high-information content data may be key to improved risk prediction models, identification of at-risk patients, and the development of interventions to improve outcomes. Remote monitoring enables measurement of multiple ambulation metrics incorporating both distance and speed. Objective: To evaluate novel ambulation metrics in predicting 30-day readmission rates, discharge location, and length of stay using a real-time location system to continuously monitor the voluntary ambulations of postoperative cardiac surgery patients. Design, Setting, and Participants: This prognostic cohort study of the mobility of 100 patients after cardiac surgery in a progressive care unit at Johns Hopkins Hospital was performed using a real-time location system. Enrollment occurred between August 29, 2016, and April 4, 2018. Data analysis was performed from June 2018 to December 2019. Main Outcomes and Measures: Outcome measures included 30-day readmission, discharge location, and length of stay. Digital records of all voluntary ambulations were created where each ambulation consisted of multiple segments defined by distance and speed. Ambulation profiles consisted of 19 parameters derived from the digital ambulation records. Results: A total of 100 patients (81 men [81%]; mean [SD] age, 63.1 [11.6] years) were evaluated. Distance and speed were recorded for more than 14 000 segments in 840 voluntary ambulations, corresponding to a total of 127.8 km (79.4 miles) using a real-time location system. Patient ambulation profiles were predictive of 30-day readmission (sensitivity, 86.7%; specificity, 88.2%; C statistic, 0.925 [95% CI, 0.836-1.000]), discharge to acute rehabilitation (sensitivity, 84.6%; specificity, 86.4%; C statistic, 0.930 [95% CI, 0.855-1.000]), and length of stay (correlation coefficient, 0.927). Conclusions and Relevance: Remote monitoring provides a high-information content description of mobility, incorporating elements of step count (ambulation distance and related parameters), gait speed (ambulation speed and related parameters), frequency of ambulation, and changes in parameters on successive ambulations. Ambulation profiles incorporating multiple aspects of mobility enables accurate prediction of clinically relevant outcomes.


Assuntos
Reabilitação Cardíaca/estatística & dados numéricos , Procedimentos Cirúrgicos Cardíacos/reabilitação , Análise da Marcha/métodos , Hospitalização/estatística & dados numéricos , Medição de Risco/métodos , Idoso , Feminino , Análise da Marcha/estatística & dados numéricos , Humanos , Tempo de Internação/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Alta do Paciente/estatística & dados numéricos , Readmissão do Paciente/estatística & dados numéricos , Valor Preditivo dos Testes , Prognóstico , Medição de Risco/estatística & dados numéricos , Sensibilidade e Especificidade , Caminhada
8.
J Biol Chem ; 293(49): 18864-18878, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30291141

RESUMO

The inflammasome is a critical molecular complex that activates interleukin-1 driven inflammation in response to pathogen- and danger-associated signals. Germline mutations in the inflammasome sensor NLRP1 cause Mendelian systemic autoimmunity and skin cancer susceptibility, but its endogenous regulation remains less understood. Here we use a proteomics screen to uncover dipeptidyl dipeptidase DPP9 as a novel interacting partner with human NLRP1 and a related inflammasome regulator, CARD8. DPP9 functions as an endogenous inhibitor of NLRP1 inflammasome in diverse primary cell types from human and mice. DPP8/9 inhibition via small molecule drugs and CRISPR/Cas9-mediated genetic deletion specifically activate the human NLRP1 inflammasome, leading to ASC speck formation, pyroptotic cell death, and secretion of cleaved interleukin-1ß. Mechanistically, DPP9 interacts with a unique autoproteolytic domain (Function to Find Domain (FIIND)) found in NLRP1 and CARD8. This scaffolding function of DPP9 and its catalytic activity act synergistically to maintain NLRP1 in its inactive state and repress downstream inflammasome activation. We further identified a single patient-derived germline missense mutation in the NLRP1 FIIND domain that abrogates DPP9 binding, leading to inflammasome hyperactivation seen in the Mendelian autoinflammatory disease Autoinflammation with Arthritis and Dyskeratosis. These results unite recent findings on the regulation of murine Nlrp1b by Dpp8/9 and uncover a new regulatory mechanism for the NLRP1 inflammasome in primary human cells. Our results further suggest that DPP9 could be a multifunctional inflammasome regulator involved in human autoinflammatory diseases.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Inflamassomos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Ácidos Borônicos/farmacologia , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Dipeptídeos/farmacologia , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Mutação em Linhagem Germinativa , Células HEK293 , Humanos , Inflamação/genética , Mutação de Sentido Incorreto , Proteínas NLR , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Domínios Proteicos
9.
Elife ; 72018 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-29784083

RESUMO

Calcium/calmodulin-dependent protein kinase II (CAMK2) plays fundamental roles in synaptic plasticity that underlies learning and memory. Here, we describe a new recessive neurodevelopmental syndrome with global developmental delay, seizures and intellectual disability. Using linkage analysis and exome sequencing, we found that this disease maps to chromosome 5q31.1-q34 and is caused by a biallelic germline mutation in CAMK2A. The missense mutation, p.His477Tyr is located in the CAMK2A association domain that is critical for its function and localization. Biochemically, the p.His477Tyr mutant is defective in self-oligomerization and unable to assemble into the multimeric holoenzyme.In vivo, CAMK2AH477Y failed to rescue neuronal defects in C. elegans lacking unc-43, the ortholog of human CAMK2A. In vitro, neurons derived from patient iPSCs displayed profound synaptic defects. Together, our data demonstrate that a recessive germline mutation in CAMK2A leads to neurodevelopmental defects in humans and suggest that dysfunctional CAMK2 paralogs may contribute to other neurological disorders.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Deficiências do Desenvolvimento/genética , Homozigoto , Deficiência Intelectual/genética , Mutação com Perda de Função , Convulsões/genética , Cromossomos Humanos Par 5 , Consanguinidade , Saúde da Família , Ligação Genética , Humanos , Jordânia , Mutação de Sentido Incorreto , Análise de Sequência de DNA
10.
ACS Appl Mater Interfaces ; 7(9): 5438-43, 2015 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-25658137

RESUMO

HNO has recently been found to possess distinct biological functions from NO. Studying the biological functions of HNO calls for the development of sensitive and selective fluorescent probes. Herein, we designed and synthesized a FRET-based ratiometric probe to detect HNO in living cells. Our studies revealed that the probe is capable of detecting HNO in a rapid and ratiometric manner under physiological conditions. In bioimaging studies, the probe displayed a clear color change from blue to green when treated with HNO.


Assuntos
Corantes Fluorescentes/química , Microscopia de Fluorescência , Óxidos de Nitrogênio/análise , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos
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