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1.
Nat Commun ; 11(1): 5079, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033234

RESUMO

Tumor heterogeneity and lack of knowledge about resistant cell states remain a barrier to targeted cancer therapies. Basal cell carcinomas (BCCs) depend on Hedgehog (Hh)/Gli signaling, but can develop mechanisms of Smoothened (SMO) inhibitor resistance. We previously identified a nuclear myocardin-related transcription factor (nMRTF) resistance pathway that amplifies noncanonical Gli1 activity, but characteristics and drivers of the nMRTF cell state remain unknown. Here, we use single cell RNA-sequencing of patient tumors to identify three prognostic surface markers (LYPD3, TACSTD2, and LY6D) which correlate with nMRTF and resistance to SMO inhibitors. The nMRTF cell state resembles transit-amplifying cells of the hair follicle matrix, with AP-1 and TGFß cooperativity driving nMRTF activation. JNK/AP-1 signaling commissions chromatin accessibility and Smad3 DNA binding leading to a transcriptional program of RhoGEFs that facilitate nMRTF activity. Importantly, small molecule AP-1 inhibitors selectively target LYPD3+/TACSTD2+/LY6D+ nMRTF human BCCs ex vivo, opening an avenue for improving combinatorial therapies.


Assuntos
Carcinoma Basocelular/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/metabolismo , DNA de Neoplasias/metabolismo , Resistencia a Medicamentos Antineoplásicos , Matriz Extracelular/metabolismo , Ontologia Genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Folículo Piloso/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Proteína Smad3/metabolismo , Transativadores/metabolismo , Regulação para Cima
2.
PLoS One ; 15(9): e0237851, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32877414

RESUMO

This study examined the antibacterial effect of protoporphyrin IX-ethylenediamine derivative (PPIX-ED)-mediated photodynamic antimicrobial chemotherapy (PPIX-ED-PACT) against Pseudomonas aeruginosa in vitro and in vivo. PPIX-ED potently inhibited the growth of Pseudomonas aeruginosa by inducing reactive oxygen species production via photoactivation. Atomic force microscopy revealed that PPIX-ED-PACT induced the leakage of bacterial content by degrading the bacterial membrane and wall. As revealed using acridine orange/ethidium bromide staining, PPIX-ED-PACT altered the permeability of the bacterial membrane. In addition, the antibacterial effect of PPIX-ED-PACT was demonstrated in an in vivo model of P. aeruginosa-infected wounds. PPIX-ED (100 µM) decreased the number of P. aeruginosa colony-forming units by 4.2 log10. Moreover, histological analysis illustrated that the wound healing rate was 98% on day 14 after treatment, which was 10% higher than that in the control group. According to the present findings, PPIX-ED-PACT can effectively inhibit the growth of P. aeruginosa in vitro and in vivo.


Assuntos
Antibacterianos/uso terapêutico , Fotoquimioterapia , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/fisiologia , Infecção dos Ferimentos/tratamento farmacológico , Infecção dos Ferimentos/microbiologia , Animais , Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/efeitos da radiação , Etilenodiaminas/química , Etilenodiaminas/farmacologia , Etilenodiaminas/uso terapêutico , Feminino , Luz , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana/efeitos dos fármacos , Modelos Biológicos , Células NIH 3T3 , Fotodegradação , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Protoporfirinas/química , Protoporfirinas/farmacologia , Protoporfirinas/uso terapêutico , Pseudomonas aeruginosa/efeitos da radiação , Cicatrização/efeitos dos fármacos
3.
Nat Commun ; 11(1): 4845, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973148

RESUMO

Herpesviruses encode conserved protein kinases (CHPKs) to stimulate phosphorylation-sensitive processes during infection. How CHPKs bind to cellular factors and how this impacts their regulatory functions is poorly understood. Here, we use quantitative proteomics to determine cellular interaction partners of human herpesvirus (HHV) CHPKs. We find that CHPKs can target key regulators of transcription and replication. The interaction with Cyclin A and associated factors is identified as a signature of ß-herpesvirus kinases. Cyclin A is recruited via RXL motifs that overlap with nuclear localization signals (NLS) in the non-catalytic N termini. This architecture is conserved in HHV6, HHV7 and rodent cytomegaloviruses. Cyclin A binding competes with NLS function, enabling dynamic changes in CHPK localization and substrate phosphorylation. The cytomegalovirus kinase M97 sequesters Cyclin A in the cytosol, which is essential for viral inhibition of cellular replication. Our data highlight a fine-tuned and physiologically important interplay between a cellular cyclin and viral kinases.


Assuntos
Replicação do DNA/fisiologia , Infecções por Herpesviridae/metabolismo , Herpesviridae/metabolismo , Proteínas Quinases/metabolismo , Animais , Ciclina A/genética , Ciclina A/metabolismo , Citomegalovirus/genética , DNA/metabolismo , Células HEK293 , Herpesviridae/enzimologia , Herpesviridae/genética , Infecções por Herpesviridae/virologia , Humanos , Camundongos , Células NIH 3T3 , Sinais de Localização Nuclear/metabolismo , Fosforilação , Mapas de Interação de Proteínas , Proteínas Virais/genética , Proteínas Virais/metabolismo
4.
Nat Commun ; 11(1): 4482, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901011

RESUMO

Intracellular trafficking governs receptor signaling, pathogenesis, immune responses and fate of nanomedicines. These processes are typically tracked by observing colocalization of fluorescent markers using confocal microscopy. However, this method is low throughput, limited by the resolution of microscopy, and can miss fleeting interactions. To address this, we developed a localization sensor composed of a quenched SNAP-tag substrate (SNAPSwitch) that can be conjugated to biomolecules using click chemistry. SNAPSwitch enables quantitative detection of trafficking to locations of interest within live cells using flow cytometry. Using SNAPSwitch, we followed the trafficking of DNA complexes from endosomes into the cytosol and nucleus. We show that antibodies against the transferrin or hyaluronan receptor are initially sorted into different compartments following endocytosis. In addition, we can resolve which side of the cellular membrane material was located. These results demonstrate SNAPSwitch is a high-throughput and broadly applicable tool to quantitatively track localization of materials in cells.


Assuntos
DNA/metabolismo , Sondas Moleculares/química , Nanopartículas/metabolismo , Proteínas/metabolismo , Animais , Transporte Biológico Ativo , Técnicas Biossensoriais/métodos , Química Click , Citometria de Fluxo , Corantes Fluorescentes , Células HEK293 , Humanos , Camundongos , Microscopia Confocal , Técnicas de Sonda Molecular , Sondas Moleculares/metabolismo , Células NIH 3T3
5.
Int J Nanomedicine ; 15: 6433-6449, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922010

RESUMO

Background: Electrospun nanofibers based on Colocasia esculenta tuber (CET) protein are considered as a promising material for wound dressing applications. However, the use of these nanofibers in aqueous conditions has poor stability. The present study was performed to obtain insights into the crosslinked electrospun CET's protein-chitosan (CS)-poly(ethylene oxide) (PEO) nanofibers and to evaluate their potential for wound dressing applications. Methods: The electrospun nanofibers were crosslinked with glutaraldehyde (GA) vapor and heat treatment (HT) to enhance their physicochemical stability. The crosslinked nanofibers were characterized by protein profiles, morphology structures, thermal behavior, mechanical properties, and degradation behavior. Furthermore, the antibacterial properties and cytocompatibility were analyzed by antibacterial assessment and cell proliferation. Results: The protein profiles of the electrospun CET's protein-CS-PEO nanofibers before and after HT crosslinking contained one major bioactive protein with a molecular weight of 14.4 kDa. Scanning electron microscopy images of the crosslinked nanofibers indicated preservation of the structure after immersion in phosphate buffered saline. The crosslinked nanofibers resulted in higher ultimate tensile strength and lower ultimate strain compared to the non-crosslinked nanofibers. GA vapor crosslinking showed higher water stability compared to HT crosslinking. The in vitro antibacterial activity of the crosslinked nanofibers showed a stronger bacteriostatic effect on Staphylococcus aureus than on Escherichia coli. Human skin fibroblast cell proliferation on crosslinked GA vapor and HT nanofibers with 1% (w/v) CS and 2% (w/v) CET's protein demonstrated the highest among all the other crosslinked nanofibers after seven days of cell culture. Cell proliferation and cell morphology results revealed that introducing higher CET's protein concentration on crosslinked nanofibers could increase cell proliferation of the crosslinked nanofibers. Conclusion: These results are promising for the potential use of the crosslinked electrospun CET's protein-CS-PEO nanofibers as bioactive wound dressing materials.


Assuntos
Antibacterianos/farmacologia , Quitosana/química , Colocasia/química , Reagentes para Ligações Cruzadas/química , Nanofibras/química , Proteínas de Plantas/química , Tubérculos/química , Polietilenoglicóis/química , Animais , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Células NIH 3T3 , Nanofibras/ultraestrutura , Proteínas de Plantas/ultraestrutura , Staphylococcus aureus/efeitos dos fármacos , Estresse Mecânico , Temperatura
6.
J Environ Pathol Toxicol Oncol ; 39(2): 191-199, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32749127

RESUMO

We have proven that naringin, a phytonutrient, diminishes oxidative damage and inflammatory responses by modulating PPAR-γ expressions in ultraviolet-B radiation (UVB)-induced NIH-3T3 cells. However, the role of naringin against DNA damage, photoaging, and apoptosis in NIH-3T3 cells has yet to be studied, necessitating investigation. We show that Naringin pretreatment significantly reduces UVB-induced alkaline DNA damage and potentially modulates NER gene (XPC, TFIIH, XPE, ERCC1, and GAPDH) expression, thereby augmenting DNA repair. We determined experimentally that naringin pretreatment prevents UVB-induced nuclear fragmentation in NIH-3T3 cells, as well as altering UVB-induced apoptotic marker (Bax, BCl-2, Caspase-9, and Caspase-3) expression in them. In addition, naringin pretreatment inhibits UVB-stimulated matrix metalloproteinase (MMP-2, MMP-9 and MMP-13) expression in these 3T3 cells. Therefore, we report that naringin can effectively avert UVB-mediated DNA damage, photoaging, and apoptosis in NIH-3T3 cells.


Assuntos
Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Flavanonas/farmacologia , Raios Ultravioleta/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Apoptose/efeitos da radiação , Reparo do DNA/efeitos da radiação , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Células NIH 3T3 , Protetores contra Radiação/farmacologia , Envelhecimento da Pele/efeitos dos fármacos
7.
PLoS Biol ; 18(8): e3000774, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32745097

RESUMO

The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVE's proline-rich domain is polyphosphorylated after the complex is activated. Blocking Scar/WAVE activation stops phosphorylation in both Dictyostelium and mammalian cells, implying that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether they are initiated. Unexpectedly, phosphorylation is not promoted by chemotactic signaling but is greatly stimulated by cell:substrate adhesion and diminished when cells deadhere. Phosphorylation-deficient or phosphomimetic Scar/WAVE mutants are both normally functional and rescue the phenotype of knockout cells, demonstrating that phosphorylation is dispensable for activation and actin regulation. However, pseudopods and patches of phosphorylation-deficient Scar/WAVE last substantially longer in mutants, altering the dynamics and size of pseudopods and lamellipods and thus changing migration speed. Scar/WAVE phosphorylation does not require ERK2 in Dictyostelium or mammalian cells. However, the MAPKKK homologue SepA contributes substantially-sepA mutants have less steady-state phosphorylation, which does not increase in response to adhesion. The mutants also behave similarly to cells expressing phosphorylation-deficient Scar, with longer-lived pseudopods and patches of Scar recruitment. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVE's activity and that phosphorylation acts as a pseudopod timer by promoting Scar/WAVE turnover.


Assuntos
Dictyostelium/genética , MAP Quinase Quinase Quinase 3/genética , Proteínas de Protozoários/genética , Pseudópodes/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Animais , Sistemas CRISPR-Cas , Adesão Celular , Linhagem Celular Tumoral , Quimiotaxia/genética , Dictyostelium/metabolismo , Dictyostelium/ultraestrutura , Edição de Genes/métodos , Regulação da Expressão Gênica , MAP Quinase Quinase Quinase 3/metabolismo , Melanócitos/metabolismo , Melanócitos/ultraestrutura , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Mutação , Células NIH 3T3 , Fenótipo , Fosforilação , Ploidias , Proteínas de Protozoários/metabolismo , Pseudópodes/genética , Pseudópodes/ultraestrutura , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo
8.
PLoS Biol ; 18(8): e3000792, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32745129

RESUMO

A ubiquitous feature of the circadian clock across life forms is its organization as a network of cellular oscillators, with individual cellular oscillators within the network often exhibiting considerable heterogeneity in their intrinsic periods. The interaction of coupling and heterogeneity in circadian clock networks is hypothesized to influence clock's entrainability, but our knowledge of mechanisms governing period heterogeneity within circadian clock networks remains largely elusive. In this study, we aimed to explore the principles that underlie intercellular period variation in circadian clock networks (clonal period heterogeneity). To this end, we employed a laboratory selection approach and derived a panel of 25 clonal cell populations exhibiting circadian periods ranging from 22 to 28 h. We report that a single parent clone can produce progeny clones with a wide distribution of circadian periods, and this heterogeneity, in addition to being stochastically driven, has a heritable component. By quantifying the expression of 20 circadian clock and clock-associated genes across our clone panel, we found that inheritance of expression patterns in at least three clock genes might govern clonal period heterogeneity in circadian clock networks. Furthermore, we provide evidence suggesting that heritable epigenetic variation in gene expression regulation might underlie period heterogeneity.


Assuntos
Proteínas CLOCK/genética , Relógios Circadianos/genética , Ritmo Circadiano/genética , Epigênese Genética , Redes Reguladoras de Genes , Animais , Proteínas CLOCK/metabolismo , Linhagem Celular Tumoral , Células Clonais , Perfilação da Expressão Gênica , Genes Reporter , Heterogeneidade Genética , Humanos , Padrões de Herança , Luciferases/genética , Luciferases/metabolismo , Camundongos , Células NIH 3T3 , Osteoblastos/citologia , Osteoblastos/metabolismo , Processos Estocásticos
9.
Nat Commun ; 11(1): 4061, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792541

RESUMO

Adhesions are fibrotic scars that form between abdominal organs following surgery or infection, and may cause bowel obstruction, chronic pain, or infertility. Our understanding of adhesion biology is limited, which explains the paucity of anti-adhesion treatments. Here we present a systematic analysis of mouse and human adhesion tissues. First, we show that adhesions derive primarily from the visceral peritoneum, consistent with our clinical experience that adhesions form primarily following laparotomy rather than laparoscopy. Second, adhesions are formed by poly-clonal proliferating tissue-resident fibroblasts. Third, using single cell RNA-sequencing, we identify heterogeneity among adhesion fibroblasts, which is more pronounced at early timepoints. Fourth, JUN promotes adhesion formation and results in upregulation of PDGFRA expression. With JUN suppression, adhesion formation is diminished. Our findings support JUN as a therapeutic target to prevent adhesions. An anti-JUN therapy that could be applied intra-operatively to prevent adhesion formation could dramatically improve the lives of surgical patients.


Assuntos
Aderências Teciduais/metabolismo , Aderências Teciduais/patologia , Animais , Benzofenonas/farmacologia , Sistemas CRISPR-Cas , Células Cultivadas , Doxiciclina/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Imunofluorescência , Gastroenteropatias/metabolismo , Gastroenteropatias/patologia , Humanos , Imuno-Histoquímica , Isoxazóis/farmacologia , Lipossomos/metabolismo , Camundongos , Células NIH 3T3 , Parabiose , RNA Mensageiro/metabolismo , Tamoxifeno/farmacologia
10.
Int J Nanomedicine ; 15: 5017-5026, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764933

RESUMO

Background: Molecular imaging is of great benefit to early disease diagnosis and timely treatment. One of the most striking innovations is the development of multimodal molecular imaging technology, which integrates two or more imaging modalities, largely in view of making the best of the advantages of each modality while overcoming their respective shortcomings. Hence, engineering a versatile and easily prepared nanomaterial with integrating multimodal molecular imaging function holds great promise, but is still a great challenge. Materials and Methods: We firstly designed and synthesized a BDT-DPP conjugated polymer and then noncovalent self-assembly with phospholipid-polyethylene glycol endowed BDT-DPP with water solubility and biocompatibility. Followed by [Cu] labeling, the acquired multifunctional nanoparticles (NPs) were studied in detail for the photophysical property. The cytotoxicity and biocompatibility of DPP-BDT NPs were examined through MTT assay and H&E stained analysis. In addition, we investigated the accumulation of the NPs in HepG2 tumor models by positron emission tomography (PET) and photoacoustic (PA) dual-mode imaging. Results and Discussion: The DPP-BDT NPs exhibited excellent optical stability, strong near-infrared (NIR) light absorption as well as fine biocompatibility. After tail vein injection into the living mice, the PA signals in the neoplastic tissues were gradually increased and reached to the maximum at the 4-h post-injection, which was consistent with the PET analysis. Such strong PA and PET signals were attributed to the efficient NPs accumulation resulting from the enhanced permeability and retention (EPR) effect. Conclusion: The biocompatible DPP-BDT NPs demonstrated to be strong NIR absorption property and PAI sensitivity. Besides, these novel DPP-BDT NPs can act not only as a PA imaging contrast agent but also as an imaging agent for PET.


Assuntos
Cetonas/química , Nanopartículas/química , Neoplasias Experimentais/diagnóstico por imagem , Técnicas Fotoacústicas/métodos , Tomografia por Emissão de Pósitrons/métodos , Pirróis/química , Animais , Meios de Contraste/química , Radioisótopos de Cobre/administração & dosagem , Radioisótopos de Cobre/farmacocinética , Feminino , Células Hep G2 , Humanos , Injeções , Camundongos , Camundongos Nus , Imagem Multimodal/métodos , Células NIH 3T3 , Nanopartículas/administração & dosagem , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Polímeros/química , Compostos Radiofarmacêuticos/administração & dosagem , Compostos Radiofarmacêuticos/química , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Nat Commun ; 11(1): 4044, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792536

RESUMO

A growing number of optogenetic tools have been developed to reversibly control binding between two engineered protein domains. In contrast, relatively few tools confer light-switchable binding to a generic target protein of interest. Such a capability would offer substantial advantages, enabling photoswitchable binding to endogenous target proteins in cells or light-based protein purification in vitro. Here, we report the development of opto-nanobodies (OptoNBs), a versatile class of chimeric photoswitchable proteins whose binding to proteins of interest can be enhanced or inhibited upon blue light illumination. We find that OptoNBs are suitable for a range of applications including reversibly binding to endogenous intracellular targets, modulating signaling pathway activity, and controlling binding to purified protein targets in vitro. This work represents a step towards programmable photoswitchable regulation of a wide variety of target proteins.


Assuntos
Optogenética/métodos , Biologia Sintética/métodos , Animais , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Ligação Proteica , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologia
12.
Nat Commun ; 11(1): 3455, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32661245

RESUMO

CRISPR-based genetic screening has revolutionized cancer drug target discovery, yet reliable, multiplex gene editing to reveal synergies between gene targets remains a major challenge. Here, we present a simple and robust CRISPR-Cas12a-based approach for combinatorial genetic screening in cancer cells. By engineering the CRISPR-AsCas12a system with key modifications to the Cas protein and its CRISPR RNA (crRNA), we can achieve high efficiency combinatorial genetic screening. We demonstrate the performance of our optimized AsCas12a (opAsCas12a) through double knockout screening against epigenetic regulators. This screen reveals synthetic sick interactions between Brd9&Jmjd6, Kat6a&Jmjd6, and Brpf1&Jmjd6 in leukemia cells.


Assuntos
Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleases/genética , Edição de Genes , Regulação Leucêmica da Expressão Gênica , Leucemia/genética , Animais , Proliferação de Células , Epigênese Genética , Biblioteca Gênica , Engenharia Genética , Genoma Humano , Células HEK293 , Humanos , Células K562 , Camundongos , Células NIH 3T3 , Domínios Proteicos , RNA Guia/genética
13.
Nat Commun ; 11(1): 3698, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32703943

RESUMO

Intellectual disability (ID) is a heterogeneous clinical entity and includes an excess of males who harbor variants on the X-chromosome (XLID). We report rare FAM50A missense variants in the original Armfield XLID syndrome family localized in Xq28 and four additional unrelated males with overlapping features. Our fam50a knockout (KO) zebrafish model exhibits abnormal neurogenesis and craniofacial patterning, and in vivo complementation assays indicate that the patient-derived variants are hypomorphic. RNA sequencing analysis from fam50a KO zebrafish show dysregulation of the transcriptome, with augmented spliceosome mRNAs and depletion of transcripts involved in neurodevelopment. Zebrafish RNA-seq datasets show a preponderance of 3' alternative splicing events in fam50a KO, suggesting a role in the spliceosome C complex. These data are supported with transcriptomic signatures from cell lines derived from affected individuals and FAM50A protein-protein interaction data. In sum, Armfield XLID syndrome is a spliceosomopathy associated with aberrant mRNA processing during development.


Assuntos
Proteínas de Ligação a DNA/genética , Deficiência Intelectual/genética , Retardo Mental Ligado ao Cromossomo X/genética , Mutação/genética , Proteínas de Ligação a RNA/genética , Spliceossomos/metabolismo , Proteínas de Peixe-Zebra/genética , Adulto , Animais , Núcleo Celular/metabolismo , Criança , Pré-Escolar , Família , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Camundongos , Mutação de Sentido Incorreto/genética , Células NIH 3T3 , Linhagem , Fenótipo , Transporte Proteico , Processamento de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/genética , Síndrome , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
14.
Life Sci ; 257: 118062, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32652138

RESUMO

AIMS: In this study, for the first time, the effect of quercetin (Q) on the characteristic properties, antimicrobial activity, and cell viability of polycaprolactone (PCL)/graphene oxide (GO) electrospun scaffold was investigated. MAIN METHODS: Quercetin loaded graphene oxide nanoparticles have been incorporated into the poly-caprolactone solution, and their mixture has been electrospun to be applied as a nanofibrous scaffold for wound dressing and tissue engineering applications. The properties of scaffolds, like their morphology, tensile strength, hydrophilicity, and in vitro biological performance, are investigated. KEY FINDINGS: The SEM micrographs reveal the uniform bead-free nanofibers with smooth structures have been successfully fabricated via the electrospinning procedure. The overall average of cell viability of NIH/3 T3 fibroblast cells on scaffolds is 95% that means the scaffolds have no toxicity, and FESEM shows cells attach and proliferate on scaffolds. Moreover, among all the fabricated scaffolds, the maximum release of quercetin belongs to PCL/GO/Q 0.5 with about 70% after 15 days, and this scaffold reduces bacterial growth by about 50% after 12 h shows the excellent effect of GO/Q on the antibacterial activity of PCL nanofibers. SIGNIFICANCE: The results confirm that more than 1% of GO has some cytotoxicity, which limits its concentration; therefore, a second antibacterial agent is essential to improve the antibacterial activity of PCL/GO scaffold, and quercetin shows that it is an excellent candidate for this purpose.


Assuntos
Grafite/farmacologia , Poliésteres/química , Quercetina/farmacologia , Tecidos Suporte , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Bandagens , Sobrevivência Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Grafite/administração & dosagem , Grafite/toxicidade , Camundongos , Células NIH 3T3 , Nanofibras , Quercetina/administração & dosagem , Engenharia Tecidual
15.
Anticancer Res ; 40(8): 4425-4444, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727773

RESUMO

BACKGROUND/AIM: Patients with metastasized melanoma have limited treatment options and poor diagnosis. Therefore, the development of treatments requires a new therapeutic approach, of which gene therapy using rAAV vectors can be proposed. The aim of the study was to examine the efficiency of the rAAV vector to transduce mouse melanoma cells both in vitro and in vivo. MATERIALS AND METHODS: Different rAAV serotypes encoding GFP under the control of both chicken beta-actin and cytomegalovirus promoters were used in the experiments. Intranasal, intraperitoneal, intravenous and intratumoral pathways of administration of rAAV vectors were tested using quantitative-PCR and immunohistochemical staining. RESULTS: The highest transduction efficiency in metastatic cells in vivo was observed 7 days after intranasal administration of a 1010 gc/0.03 ml dose of rAAV/DJ-CAG. CONCLUSION: Melanoma gene therapy based on rAAV vectors is a possible treatment option.


Assuntos
Dependovirus/genética , Proteínas de Fluorescência Verde/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Melanoma Experimental/genética , Administração Intranasal , Animais , Linhagem Celular Tumoral , Feminino , Terapia Genética , Vetores Genéticos/administração & dosagem , Neoplasias Pulmonares/terapia , Masculino , Melanoma Experimental/terapia , Camundongos , Células NIH 3T3 , Regiões Promotoras Genéticas , Transdução Genética , Resultado do Tratamento
16.
Int J Nanomedicine ; 15: 3319-3331, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32494132

RESUMO

Background: It is of great significance to develop intelligent co-delivery systems for cancer chemotherapy with improved therapeutic efficacy and few side-effects. Materials and Methods: Here, we reported a co-delivery system based on pH-sensitive polyprodrug micelles for simultaneous delivery of doxorubicin (DOX) and paclitaxel (PTX) as a combination chemotherapy with pH-triggered drug release profiles. The physicochemical properties, drug release profiles and mechanism, and cytotoxicity of PTX/DOX-PMs have been thoroughly investigated. Results and Discussion: The pH-sensitive polyprodrug was used as nanocarrier, and PTX was encapsulated into the micelles with high drug-loading content (25.6%). The critical micelle concentration (CMC) was about 3.16 mg/L, indicating the system could form the micelles at low concentration. The particle size of PTX/DOX-PMs was 110.5 nm, and increased to approximately 140 nm after incubation for 5 days which showed that the PTX/DOX-PMs had high serum stability. With decrease in pH value, the particle size first increased, and thenwas no longer detectable. Similar change trend was observed for CMC values. The zetapotential increased sharply with decrease in pH. These results demonstrated the pHsensitivity of PTX/DOX-PMs. In vitro drug release experiments and study on release mechanism showed that the drug release rate and accumulative release for PTX and DOX were dependent on the pH, showing the pH-triggered drug release profiles. Cytotoxicity assay displayed that the block copolymer showed negligible cytotoxicity, while the PTX/DOX-PMs possessed high cytotoxic effect against several tumor cell lines compared with free drugs and control. Conclusion: All the results demonstrated that the co-delivery system based on pH-sensitive polyprodrug could be a potent nanomedicine for combination cancer chemotherapy. In addition, construction based on polyprodrug and chemical drug could be a useful method to prepare multifunctional nanomedicine.


Assuntos
Doxorrubicina/uso terapêutico , Sistemas de Liberação de Medicamentos , Micelas , Neoplasias/tratamento farmacológico , Paclitaxel/uso terapêutico , Pró-Fármacos/farmacologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Liberação Controlada de Fármacos , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Células NIH 3T3 , Neoplasias/patologia , Paclitaxel/farmacologia , Tamanho da Partícula , Polímeros/química , Eletricidade Estática
17.
Nat Commun ; 11(1): 2977, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32532995

RESUMO

Independent scientific achievements have led to the discovery of aberrant splicing patterns in oncogenesis, while more recent advances have uncovered novel gene fusions involving neurotrophic tyrosine receptor kinases (NTRKs) in gliomas. The exploration of NTRK splice variants in normal and neoplastic brain provides an intersection of these two rapidly evolving fields. Tropomyosin receptor kinase B (TrkB), encoded NTRK2, is known for critical roles in neuronal survival, differentiation, molecular properties associated with memory, and exhibits intricate splicing patterns and post-translational modifications. Here, we show a role for a truncated NTRK2 splice variant, TrkB.T1, in human glioma. TrkB.T1 enhances PDGF-driven gliomas in vivo, augments PDGF-induced Akt and STAT3 signaling in vitro, while next generation sequencing broadly implicates TrkB.T1 in the PI3K signaling cascades in a ligand-independent fashion. These TrkB.T1 findings highlight the importance of expanding upon whole gene and gene fusion analyses to include splice variants in basic and translational neuro-oncology research.


Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Glicoproteínas de Membrana/genética , Oncogenes/genética , Isoformas de RNA/genética , Processamento de RNA , Receptor trkB/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Carcinogênese/genética , Células Cultivadas , Perfilação da Expressão Gênica , Ontologia Genética , Glioma/metabolismo , Glioma/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Células NIH 3T3 , Células-Tronco Neurais/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de RNA/metabolismo , Receptor trkB/metabolismo , Transdução de Sinais/genética
18.
Mol Cell ; 79(4): 561-574.e5, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32589966

RESUMO

Translation regulation occurs largely during the initiation phase. Here, we develop selective 40S footprinting to visualize initiating 40S ribosomes on endogenous mRNAs in vivo. This reveals the positions on mRNAs where initiation factors join the ribosome to act and where they leave. We discover that in most human cells, most scanning ribosomes remain attached to the 5' cap. Consequently, only one ribosome scans a 5' UTR at a time, and 5' UTR length affects translation efficiency. We discover that eukaryotic initiation factor 3B (eIF3B,) eIF4G1, and eIF4E remain bound to 80S ribosomes as they begin translating, with a decay half-length of ∼12 codons. Hence, ribosomes retain these initiation factors while translating short upstream open reading frames (uORFs), providing an explanation for how ribosomes can reinitiate translation after uORFs in humans. This method will be of use for studying translation initiation mechanisms in vivo.


Assuntos
Regiões 5' não Traduzidas , Pegada de DNA/métodos , Iniciação Traducional da Cadeia Peptídica , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Animais , Códon de Iniciação , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4G em Eucariotos/genética , Fator de Iniciação 4G em Eucariotos/metabolismo , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Fases de Leitura Aberta , RNA Mensageiro/genética , RNA de Transferência de Metionina/genética , Subunidades Ribossômicas/genética , Subunidades Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética
19.
Nat Immunol ; 21(7): 802-815, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32541832

RESUMO

Microglia and central nervous system (CNS)-associated macrophages (CAMs), such as perivascular and meningeal macrophages, are implicated in virtually all diseases of the CNS. However, little is known about their cell-type-specific roles in the absence of suitable tools that would allow for functional discrimination between the ontogenetically closely related microglia and CAMs. To develop a new microglia gene targeting model, we first applied massively parallel single-cell analyses to compare microglia and CAM signatures during homeostasis and disease and identified hexosaminidase subunit beta (Hexb) as a stably expressed microglia core gene, whereas other microglia core genes were substantially downregulated during pathologies. Next, we generated HexbtdTomato mice to stably monitor microglia behavior in vivo. Finally, the Hexb locus was employed for tamoxifen-inducible Cre-mediated gene manipulation in microglia and for fate mapping of microglia but not CAMs. In sum, we provide valuable new genetic tools to specifically study microglia functions in the CNS.


Assuntos
Encéfalo/patologia , Encefalomielite Autoimune Experimental/patologia , Traumatismos do Nervo Facial/patologia , Microglia/metabolismo , Cadeia beta da beta-Hexosaminidase/metabolismo , Animais , Encéfalo/citologia , Encéfalo/imunologia , Sistemas CRISPR-Cas/genética , Encefalomielite Autoimune Experimental/imunologia , Traumatismos do Nervo Facial/imunologia , Técnicas de Introdução de Genes , Genes Reporter/genética , Loci Gênicos/genética , Humanos , Microscopia Intravital , Substâncias Luminescentes/química , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Microglia/imunologia , Células NIH 3T3 , RNA-Seq , Análise de Célula Única , Transfecção , Cadeia beta da beta-Hexosaminidase/genética
20.
Proc Natl Acad Sci U S A ; 117(19): 10131-10141, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32350144

RESUMO

Over the course of the aging process, fibroblasts lose contractility, leading to reduced connective-tissue stiffness. A promising therapeutic avenue for functional rejuvenation of connective tissue is reprogrammed fibroblast replacement, although major hurdles still remain. Toward this, we recently demonstrated that the laterally confined growth of fibroblasts on micropatterned substrates induces stem-cell-like spheroids. In this study, we embedded these partially reprogrammed spheroids in collagen-I matrices of varying densities, mimicking different three-dimensional (3D) tissue constraints. In response to such matrix constraints, these spheroids regained their fibroblastic properties and sprouted to form 3D connective-tissue networks. Interestingly, we found that these differentiated fibroblasts exhibit reduced DNA damage, enhanced cytoskeletal gene expression, and actomyosin contractility. In addition, the rejuvenated fibroblasts show increased matrix protein (fibronectin and laminin) deposition and collagen remodeling compared to the parental fibroblast tissue network. Furthermore, we show that the partially reprogrammed cells have comparatively open chromatin compaction states and may be more poised to redifferentiate into contractile fibroblasts in 3D-collagen matrix. Collectively, our results highlight efficient fibroblast rejuvenation through laterally confined reprogramming, which has important implications in regenerative medicine.


Assuntos
Diferenciação Celular , Reprogramação Celular , Fibroblastos/citologia , Medicina Regenerativa , Rejuvenescimento/fisiologia , Idoso , Animais , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Humanos , Laminina/metabolismo , Camundongos , Células NIH 3T3 , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo
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