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1.
Cell ; 186(7): 1448-1464.e20, 2023 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-37001504

RESUMO

Neutrophils accumulate in solid tumors, and their abundance correlates with poor prognosis. Neutrophils are not homogeneous, however, and could play different roles in cancer therapy. Here, we investigate the role of neutrophils in immunotherapy, leading to tumor control. We show that successful therapies acutely expanded tumor neutrophil numbers. This expansion could be attributed to a Sellhi state rather than to other neutrophils that accelerate tumor progression. Therapy-elicited neutrophils acquired an interferon gene signature, also seen in human patients, and appeared essential for successful therapy, as loss of the interferon-responsive transcription factor IRF1 in neutrophils led to failure of immunotherapy. The neutrophil response depended on key components of anti-tumor immunity, including BATF3-dependent DCs, IL-12, and IFNγ. In addition, we found that a therapy-elicited systemic neutrophil response positively correlated with disease outcome in lung cancer patients. Thus, we establish a crucial role of a neutrophil state in mediating effective cancer therapy.


Assuntos
Neoplasias Pulmonares , Neutrófilos , Humanos , Neoplasias Pulmonares/genética , Transdução de Sinais/genética , Imunoterapia , Interferons
2.
Mol Cell ; 69(4): 622-635.e6, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29429924

RESUMO

TIA1 and TIAL1 encode a family of U-rich element mRNA-binding proteins ubiquitously expressed and conserved in metazoans. Using PAR-CLIP, we determined that both proteins bind target sites with identical specificity in 3' UTRs and introns proximal to 5' as well as 3' splice sites. Double knockout (DKO) of TIA1 and TIAL1 increased target mRNA abundance proportional to the number of binding sites and also caused accumulation of aberrantly spliced mRNAs, most of which are subject to nonsense-mediated decay. Loss of PRKRA by mis-splicing triggered the activation of the double-stranded RNA (dsRNA)-activated protein kinase EIF2AK2/PKR and stress granule formation. Ectopic expression of PRKRA cDNA or knockout of EIF2AK2 in DKO cells rescued this phenotype. Perturbation of maturation and/or stability of additional targets further compromised cell cycle progression. Our study reveals the essential contributions of the TIA1 protein family to the fidelity of mRNA maturation, translation, and RNA-stress-sensing pathways in human cells.


Assuntos
Ciclo Celular , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Estresse Fisiológico , Antígeno-1 Intracelular de Células T/metabolismo , eIF-2 Quinase/metabolismo , Sistemas CRISPR-Cas , Grânulos Citoplasmáticos/metabolismo , Células HEK293 , Humanos , Sítios de Splice de RNA , Splicing de RNA , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Sequências Reguladoras de Ácido Ribonucleico , Antígeno-1 Intracelular de Células T/antagonistas & inibidores , Antígeno-1 Intracelular de Células T/genética , Uridina/metabolismo , eIF-2 Quinase/genética
3.
Blood ; 142(7): 658-674, 2023 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-37267513

RESUMO

Myeloid cell heterogeneity is known, but whether it is cell-intrinsic or environmentally-directed remains unclear. Here, an inducible/reversible system pausing myeloid differentiation allowed the definition of clone-specific functions that clustered monocytes into subsets with distinctive molecular features. These subsets were orthogonal to the classical/nonclassical categorization and had inherent, restricted characteristics that did not shift under homeostasis, after irradiation, or with infectious stress. Rather, their functional fate was constrained by chromatin accessibility established at or before the granulocyte-monocyte or monocyte-dendritic progenitor level. Subsets of primary monocytes had differential ability to control distinct infectious agents in vivo. Therefore, monocytes are a heterogeneous population of functionally restricted subtypes defined by the epigenome of their progenitors that are differentially selected by physiologic challenges with limited plasticity to transition from one subset to another.


Assuntos
Granulócitos , Monócitos , Células Progenitoras Mieloides , Epigenoma , Epigênese Genética , Diferenciação Celular/genética
4.
Proc Natl Acad Sci U S A ; 114(3): 492-497, 2017 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-28034921

RESUMO

Tumor microvasculature tends to be malformed, more permeable, and more tortuous than vessels in healthy tissue, effects that have been largely attributed to up-regulated VEGF expression. However, tumor tissue tends to stiffen during solid tumor progression, and tissue stiffness is known to alter cell behaviors including proliferation, migration, and cell-cell adhesion, which are all requisite for angiogenesis. Using in vitro, in vivo, and ex ovo models, we investigated the effects of matrix stiffness on vessel growth and integrity during angiogenesis. Our data indicate that angiogenic outgrowth, invasion, and neovessel branching increase with matrix cross-linking. These effects are caused by increased matrix stiffness independent of matrix density, because increased matrix density results in decreased angiogenesis. Notably, matrix stiffness up-regulates matrix metalloproteinase (MMP) activity, and inhibiting MMPs significantly reduces angiogenic outgrowth in stiffer cross-linked gels. To investigate the functional significance of altered endothelial cell behavior in response to matrix stiffness, we measured endothelial cell barrier function on substrates mimicking the stiffness of healthy and tumor tissue. Our data indicate that barrier function is impaired and the localization of vascular endothelial cadherin is altered as function of matrix stiffness. These results demonstrate that matrix stiffness, separately from matrix density, can alter vascular growth and integrity, mimicking the changes that exist in tumor vasculature. These data suggest that therapeutically targeting tumor stiffness or the endothelial cell response to tumor stiffening may help restore vessel structure, minimize metastasis, and aid in drug delivery.


Assuntos
Matriz Extracelular/fisiologia , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/fisiopatologia , Microvasos/fisiopatologia , Animais , Fenômenos Biomecânicos , Bovinos , Células Cultivadas , Embrião de Galinha , Colágeno/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Neoplasias Mamárias Experimentais/patologia , Metaloproteinases da Matriz/metabolismo , Camundongos , Microvasos/patologia , Invasividade Neoplásica/patologia , Invasividade Neoplásica/fisiopatologia , Neovascularização Patológica/patologia , Neovascularização Patológica/fisiopatologia , Fenótipo , Microambiente Tumoral/fisiologia , Rigidez Vascular/fisiologia
5.
bioRxiv ; 2024 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-39185194

RESUMO

The fixation and permeabilization of cells are essential for labeling intracellular biomarkers in flow cytometry. However, these chemical treatments often alter fragile targets, such as cell surface and fluorescent proteins, and can destroy chemically-sensitive fluorescent labels. This reduces measurement accuracy and introduces compromises into sample workflows, leading to losses in data quality. Here, we demonstrate a novel multi-pass flow cytometry approach to address this long-standing problem. Our technique utilizes individual cell barcoding with laser particles, enabling sequential analysis of the same cells with single-cell resolution maintained. Chemically-fragile protein markers and their fluorochrome conjugates are measured prior to destructive sample processing and adjoined to subsequent measurements of intracellular markers after fixation and permeabilization. We demonstrate the effectiveness of our technique in accurately measuring intracellular fluorescent proteins and methanol-sensitive antigens and fluorophores, along with various surface and intracellular markers. This approach significantly enhances assay flexibility, enabling accurate and comprehensive cell analysis without the constraints of conventional one-time measurement flow cytometry. This innovation paves new avenues in flow cytometry for a wide range of applications in immuno-oncology, stem cell research, and cell biology.

6.
Blood Adv ; 7(21): 6608-6623, 2023 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-37450380

RESUMO

Myelodysplastic syndromes (MDSs) are a heterogenous group of diseases affecting the hematopoietic stem cell that are curable only by stem cell transplantation. Both hematopoietic cell intrinsic changes and extrinsic signals from the bone marrow (BM) niche seem to ultimately lead to MDS. Animal models of MDS indicate that alterations in specific mesenchymal progenitor subsets in the BM microenvironment can induce or select for abnormal hematopoietic cells. Here, we identify a subset of human BM mesenchymal cells marked by the expression of CD271, CD146, and CD106. This subset of human mesenchymal cells is comparable with mouse mesenchymal cells that, when perturbed, result in an MDS-like syndrome. Its transcriptional analysis identified Osteopontin (SPP1) as the most overexpressed gene. Selective depletion of Spp1 in the microenvironment of the mouse MDS model, Vav-driven Nup98-HoxD13, resulted in an accelerated progression as demonstrated by increased chimerism, higher mutant myeloid cell burden, and a more pronounced anemia when compared with that in wild-type microenvironment controls. These data indicate that molecular perturbations can occur in specific BM mesenchymal subsets of patients with MDS. However, the niche adaptations to dysplastic clones include Spp1 overexpression that can constrain disease fitness and potentially progression. Therefore, niche changes with malignant disease can also serve to protect the host.


Assuntos
Medula Óssea , Síndromes Mielodisplásicas , Humanos , Camundongos , Animais , Medula Óssea/patologia , Síndromes Mielodisplásicas/genética , Células-Tronco Hematopoéticas/metabolismo , Células da Medula Óssea/metabolismo , Modelos Animais de Doenças , Progressão da Doença
7.
J Microbiol Biol Educ ; 23(1)2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35340450

RESUMO

Since March 2020, in-person science competitions have been cancelled or moved to a virtual space. This reality has encouraged teachers and students to find alternative ways to disseminate student research and participate in a scientific community. Participating in the peer review and publication of one's research offers one such alternative. The Journal of Emerging Investigators (JEI) is a free, online, peer-reviewed science journal specifically for middle school and high school students. JEI provides students the opportunity to engage with professional scientists through the peer review process and share their research with a broad audience, all on a remote platform. This article describes resources that are freely available to help teachers navigate the peer review and publication processes and guide their students through the successful completion of submission and publication of their research papers. Overall, students perceive the experience as attainable and found the JEI resources useful in completing their papers. Importantly, students expressed that the experience of publication increased their confidence and interest in STEM.

8.
Nat Commun ; 12(1): 245, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431855

RESUMO

Acute myeloid leukemia (AML) is a high remission, high relapse fatal blood cancer. Although mTORC1 is a master regulator of cell proliferation and survival, its inhibitors have not performed well as AML treatments. To uncover the dynamics of mTORC1 activity in vivo, fluorescent probes are developed to track single cell proliferation, apoptosis and mTORC1 activity of AML cells in the bone marrow of live animals and to quantify these activities in the context of microanatomical localization and intra-tumoral heterogeneity. When chemotherapy drugs commonly used clinically are given to mice with AML, apoptosis is rapid, diffuse and not preferentially restricted to anatomic sites. Dynamic measurement of mTORC1 activity indicated a decline in mTORC1 activity with AML progression. However, at the time of maximal chemotherapy response, mTORC1 signaling is high and positively correlated with a leukemia stemness transcriptional profile. Cell barcoding reveals the induction of mTORC1 activity rather than selection of mTORC1 high cells and timed inhibition of mTORC1 improved the killing of AML cells. These data define the real-time dynamics of AML and the mTORC1 pathway in association with AML growth, response to and relapse after chemotherapy. They provide guidance for timed intervention with pathway-specific inhibitors.


Assuntos
Leucemia Mieloide Aguda/tratamento farmacológico , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Modelos Biológicos , Células NIH 3T3 , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Transcriptoma/genética , Resultado do Tratamento
9.
Cell Stem Cell ; 28(12): 2090-2103.e9, 2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34551362

RESUMO

Extracellular vesicles (EVs) transfer complex biologic material between cells. However, the role of this process in vivo is poorly defined. Here, we demonstrate that osteoblastic cells in the bone marrow (BM) niche elaborate extracellular vesicles that are taken up by hematopoietic progenitor cells in vivo. Genotoxic or infectious stress rapidly increased stromal-derived extracellular vesicle transfer to granulocyte-monocyte progenitors. The extracellular vesicles contained processed tRNAs (tiRNAs) known to modulate protein translation. 5'-ti-Pro-CGG-1 was preferentially abundant in osteoblast-derived extracellular vesicles and, when transferred to granulocyte-monocyte progenitors, increased protein translation, cell proliferation, and myeloid differentiation. Upregulating EV transfer improved hematopoietic recovery from genotoxic injury and survival from fungal sepsis. Therefore, EV-mediated tiRNA transfer provides a stress-modulated signaling axis in the BM niche distinct from conventional cytokine-driven stress responses.


Assuntos
Vesículas Extracelulares , Células-Tronco Hematopoéticas , Medula Óssea , Células da Medula Óssea , Hematopoese
10.
Curr Protoc Cytom ; 87(1): e50, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30335223

RESUMO

Maintenance of hematopoietic stem cell (HSC) quiescence is critical for self-renewal and differentiation into mature lineages. Therefore, the ability to reliably detect abnormal HSC cycling is essential for experiments that seek to investigate abnormalities of HSC function. The ability to reproducibly evaluate cell cycle status in a rare cell subset requires careful optimization of multiple parameters during cell preparation and sample processing. Here, we describe a method where data acquisition parameters and fluorochrome combination for long-term HSC staining have been specifically designed for concurrent use with DAPI and Ki-67 antibodies. © 2018 by John Wiley & Sons, Inc.


Assuntos
Ciclo Celular , Citometria de Fluxo/métodos , Células-Tronco Hematopoéticas/citologia , Animais , Células da Medula Óssea/citologia , Cor , Indóis/metabolismo , Camundongos , Processamento de Sinais Assistido por Computador
11.
PLoS One ; 10(9): e0139167, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26402367

RESUMO

The porphyrin compound, TMPyP4 (5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)porphine), is widely used as a photosensitizer and a modulator of nucleic acid secondary structure stability. Our group recently showed in cultured cells and forebrain slice cultures that this compound can also down regulate expression of Tyrosine hydroxylase (Th), which encodes the rate-limiting enzyme in catecholamine biosynthesis, by stabilizing DNA secondary structures in the Th proximal promoter. The current study sought to establish whether treatment with TMPyP4 could modify mouse Th expression levels in vivo. Intraperitoneal administration of low TMPyP4 doses (10mg/kg), similar to those used for photosensitization, did not significantly reduce Th transcript levels in several catecholaminergic regions. Administration of a high dose (40 mg/kg), similar to those used for tumor xenograph reduction, unexpectedly induced flaccid paralysis in an age and sex-dependent manner. In vitro analyses revealed that TMPyP4, but not putative metabolites, inhibited Acetylcholinesterase activity and pre-treatment of TMPyP4 with Hemeoxygenase-2 (HO-2) rescued Acetylcholinesterase function. Age-dependent differences in HO-2 expression levels may account for some of the variable in vivo effects of high TMPyP4 doses. Together, these studies indicate that only low doses of TMPyP4, such as those typically used for photosensitization, are well tolerated in vivo. Thus, despite its widespread use in vitro, TMPyP4 is not ideal for modifying neuronal gene expression in vivo by manipulating nucleic acid secondary structure stability, which highlights the need to identify more clinically suitable compounds that can modulate nucleic acid secondary structure and gene expression.


Assuntos
Inibidores da Colinesterase/farmacologia , Conformação de Ácido Nucleico/efeitos dos fármacos , Porfirinas/farmacologia , Envelhecimento/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Inibidores da Colinesterase/química , Relação Dose-Resposta a Droga , Feminino , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Camundongos Transgênicos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Porfirinas/química , Tirosina 3-Mono-Oxigenase/metabolismo
12.
Biofouling ; 19(1): 19-35, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14618686

RESUMO

Acoustic pulses generated by an electrical discharge (pulsed acoustics) were investigated as a means for biofouling control in two test formats, viz. a 5/8" outside diameter titanium tube and a mockup heat exchanger. The pulsed acoustic device, when operated at 17 kV, demonstrated 95% inhibition of microfouling over a 20 ft length of titanium tube over a 4-week period, comparable to chlorination in combination with a high-velocity flush. The pulsed acoustic device inhibited microfouling over a downstream distance of 15 ft, therefore, a single pulsed acoustic device is theoretically capable of protecting at least 30 ft of tube from microfouling (15 ft on either side of the device). A correlation between acoustic intensity in the frequency range 0.01-1 MHz and the level of biofouling inhibition was observed. The threshold acoustic intensity for microfouling inhibition was determined for this frequency range. It was also observed that the orientation of the device is critical to obtaining microfouling inhibition.


Assuntos
Acústica/instrumentação , Biofilmes/crescimento & desenvolvimento , Estimulação Elétrica , Florida , Indústrias , Tecnologia/instrumentação
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