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1.
Gene ; 806: 145922, 2022 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-34454032

RESUMO

Gastric cancer (GC)-derived cell lines were generally used in basic cancer research and drug screening. However, it is always concerned about the difference between cultured cells and primary tumor by oncologists. To address this question, we compared differentially expressed genes (DEGs) in primary cancers, healthy tissues, and cell lines both in vitro and in silico. Seven reported genes with decreased expression in GCs by DNA methylation were analyzed in our cohort studies and experimentally validation. Selected datasets from TCGA (The Cancer Genome Atlas), CCLE (The Broad Institute Cancer Cell Line Encyclopedia), and GTEx (The Genotype-Tissue Expression project) were used to represent GCs, GC-derived cell lines, and healthy tissues respectively in the in silico analysis. Thirty gastric tissues together with six cell lines were used for validations. Unexpectedly, we experimentally found that reported cancer-related downregulated genes were only found in cancer cell lines but not in biopsies. The unchanged gene expressions in primary GCs were generally consistent with our cohort study, using information from cancerous (TCGA) and healthy tissues (GETx). Substantial differences were also found between DEGs of cancer tissues (TGCA)/ healthy tissues (GTEx) pair and cell lines (CCLE)/ healthy tissues (GTEx) pair, which confirmed the significant differences between primary cancer and cancer cell lines. Moreover, elevated expression of YWHAQ (14-3-3 δ) and THBS1 were observed in the GC biopsies, which might be potential biomarkers for GC diagnosis, considering the increased YWHAQ and THBS1 associated with poor survival rates in gastric cancer patients. In sum, it is suggested that cautions should be taken when using GC cell lines to study genes that show great differences between cell lines and tissues.


Assuntos
Proteínas 14-3-3/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Neoplasias Gástricas/genética , Trombospondinas/genética , Proteínas 14-3-3/metabolismo , Idoso , Atlas como Assunto , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Metilação de DNA , Conjuntos de Dados como Assunto , Epigênese Genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Cultura Primária de Células , Prognóstico , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Análise de Sobrevida , Trombospondinas/metabolismo , Células Tumorais Cultivadas
2.
Am J Hum Genet ; 108(9): 1611-1630, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34343493

RESUMO

Genome-wide association studies (GWASs) have identified a melanoma-associated locus on chromosome band 7p21.1 with rs117132860 as the lead SNP and a secondary independent signal marked by rs73069846. rs117132860 is also associated with tanning ability and cutaneous squamous cell carcinoma (cSCC). Because ultraviolet radiation (UVR) is a key environmental exposure for all three traits, we investigated the mechanisms by which this locus contributes to melanoma risk, focusing on cellular response to UVR. Fine-mapping of melanoma GWASs identified four independent sets of candidate causal variants. A GWAS region-focused Capture-C study of primary melanocytes identified physical interactions between two causal sets and the promoter of the aryl hydrocarbon receptor (AHR). Subsequent chromatin state annotation, eQTL, and luciferase assays identified rs117132860 as a functional variant and reinforced AHR as a likely causal gene. Because AHR plays critical roles in cellular response to dioxin and UVR, we explored links between this SNP and AHR expression after both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and ultraviolet B (UVB) exposure. Allele-specific AHR binding to rs117132860-G was enhanced following both, consistent with predicted weakened AHR binding to the risk/poor-tanning rs117132860-A allele, and allele-preferential AHR expression driven from the protective rs117132860-G allele was observed following UVB exposure. Small deletions surrounding rs117132860 introduced via CRISPR abrogates AHR binding, reduces melanocyte cell growth, and prolongs growth arrest following UVB exposure. These data suggest AHR is a melanoma susceptibility gene at the 7p21.1 risk locus and rs117132860 is a functional variant within a UVB-responsive element, leading to allelic AHR expression and altering melanocyte growth phenotypes upon exposure.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinoma de Células Escamosas/genética , Cromossomos Humanos Par 7 , Loci Gênicos , Melanócitos/metabolismo , Melanoma/genética , Receptores de Hidrocarboneto Arílico/genética , Neoplasias Cutâneas/genética , Alelos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Cromatina/química , Cromatina/metabolismo , Regulação da Expressão Gênica , Predisposição Genética para Doença , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Melanócitos/efeitos dos fármacos , Melanócitos/patologia , Melanócitos/efeitos da radiação , Melanoma/metabolismo , Melanoma/patologia , Dibenzodioxinas Policloradas/toxicidade , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células , Regiões Promotoras Genéticas , Receptores de Hidrocarboneto Arílico/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Banho de Sol , Raios Ultravioleta/efeitos adversos
3.
J Immunol ; 207(5): 1478-1492, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34389622

RESUMO

Stable, long-term culture of primary B lymphocytes has many potential scientific and medical applications, but remains an elusive feat. A major obstacle to long-term culture is that in vitro mitogens quickly drive B cells to differentiate into short-lived plasma cells (PCs). PC differentiation is governed by opposing teams of transcription factors: Pax5, Bach2, and Bcl6 suppress PC commitment, whereas IFN regulatory factor 4 and Blimp1 promote it. To determine whether transcriptional programming could prolong B cell culture by blocking PC commitment, we generated mouse primary B cells harboring gain- or loss-of-function in the key transcription factors, continuously stimulated these cells with CD154 and IL-21, and determined growth potential and phenotypes in vitro. We found that transgenic expression of Bach2 prohibits PC commitment and endows B cells with extraordinary growth potential in response to external proliferation and survival cues. Long-term Bach2-transgenic B cell lines have genetically stable BCRs [i.e., do not acquire V(D)J mutations], express high levels of MHC class II and molecules for costimulation of T cells, and transduce intracellular signals when incubated with BCR ligands. Silencing the Bach2 transgene in an established transgenic cell line causes the cells to secrete large quantities of Ig. This system has potential applications in mAb production, BCR signaling studies, Ag presentation to T cells, and ex vivo clonal expansion for adoptive cell transfer. Additionally, our results provide insight into molecular control over activated B cell fate and suggest that forced Bach2 expression in vivo may augment germinal center B cell or memory B cell differentiation at the expense of PC commitment.


Assuntos
Linfócitos B/imunologia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Centro Germinativo/imunologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Regulação da Expressão Gênica , Memória Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Fator de Transcrição PAX5/genética , Fator de Transcrição PAX5/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo
4.
Molecules ; 26(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34361702

RESUMO

Neurodegenerative diseases have a complex nature which highlights the need for multitarget ligands to address the complementary pathways involved in these diseases. Over the last decade, many innovative curcumin-based compounds have been designed and synthesized, searching for new derivatives having anti-amyloidogenic, inhibitory of tau formation, as well as anti-neuroinflammation, antioxidative, and AChE inhibitory activities. Regarding our experience studying 3-substituted coumarins with interesting properties for neurodegenerative diseases, our aim was to synthesize a new series of curcumin-coumarin hybrid analogues and evaluate their activity. Most of the 3-(7-phenyl-3,5-dioxohepta-1,6-dien-1-yl)coumarin derivatives 11-18 resulted in moderated inhibitors of hMAO isoforms and AChE and BuChE activity. Some of them are also capable of scavenger the free radical DPPH. Furthermore, compounds 14 and 16 showed neuroprotective activity against H2O2 in SH-SY5Y cell line. Nanoparticles formulation of these derivatives improved this property increasing the neuroprotective activity to the nanomolar range. Results suggest that by modulating the substitution pattern on both coumarin moiety and phenyl ring, ChE and MAO-targeted derivatives or derivatives with activity in cell-based phenotypic assays can be obtained.


Assuntos
Antioxidantes/síntese química , Inibidores da Colinesterase/síntese química , Cumarínicos/síntese química , Curcumina/análogos & derivados , Inibidores da Monoaminoxidase/síntese química , Fármacos Neuroprotetores/síntese química , Acetilcolinesterase/metabolismo , Animais , Antioxidantes/farmacologia , Compostos de Bifenilo/antagonistas & inibidores , Butirilcolinesterase/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidores da Colinesterase/farmacologia , Cumarínicos/farmacologia , Curcumina/farmacologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/farmacologia , Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Córtex Motor/citologia , Córtex Motor/enzimologia , Nanopartículas/química , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Fármacos Neuroprotetores/farmacologia , Picratos/antagonistas & inibidores , Cultura Primária de Células , Ratos , Relação Estrutura-Atividade
5.
Front Immunol ; 12: 655122, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34408743

RESUMO

FOXP3+ regulatory T cells (Tregs) are central for maintaining peripheral tolerance and immune homeostasis. Because of their immunosuppressive characteristics, Tregs are a potential therapeutic target in various diseases such as autoimmunity, transplantation and infectious diseases like COVID-19. Numerous studies are currently exploring the potential of adoptive Treg therapy in different disease settings and novel genome editing techniques like CRISPR/Cas will likely widen possibilities to strengthen its efficacy. However, robust and expeditious protocols for genome editing of human Tregs are limited. Here, we describe a rapid and effective protocol for reaching high genome editing efficiencies in human Tregs without compromising cell integrity, suitable for potential therapeutic applications. By deletion of IL2RA encoding for IL-2 receptor α-chain (CD25) in Tregs, we demonstrated the applicability of the method for downstream functional assays and highlighted the importance for CD25 for in vitro suppressive function of human Tregs. Moreover, deletion of IL6RA (CD126) in human Tregs elicits cytokine unresponsiveness and thus may prevent IL-6-mediated instability of Tregs, making it an attractive target to potentially boost functionality in settings of adoptive Treg therapies to contain overreaching inflammation or autoimmunity. Thus, our rapid and efficient protocol for genome editing in human Tregs may advance possibilities for Treg-based cellular therapies.


Assuntos
Edição de Genes/métodos , Subunidade alfa de Receptor de Interleucina-2/genética , Receptores de Interleucina-6/genética , Linfócitos T Reguladores/metabolismo , Buffy Coat/citologia , Sistemas CRISPR-Cas/genética , Fatores de Transcrição Forkhead/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Voluntários Saudáveis , Humanos , Imunoterapia Adotiva/métodos , Cultura Primária de Células , RNA Guia/genética , Fatores de Tempo
6.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361027

RESUMO

The experimental animal model is still essential in the development of new anticancer drugs. We characterized mouse tumors derived from two-dimensional (2D) monolayer cells or three-dimensional (3D) spheroids to establish an in vivo model with highly standardized conditions. Primary cancer-associated fibroblasts (CAFs) were cultured from head and neck squamous cell carcinoma (HNSCC) tumor tissues and co-injected with monolayer cancer cells or spheroids into the oral mucosa of mice. Mice tumor blood vessels were stained, followed by tissue clearing and 3D Lightsheet fluorescent imaging. We compared the effect of exosomes secreted from 2D or 3D culture conditions on the angiogenesis-related genes in HNSCC cells. Our results showed that both the cells and spheroids co-injected with primary CAFs formed tumors. Interestingly, vasculature was abundantly distributed inside the spheroid-derived but not the monolayer-derived mice tumors. In addition, cisplatin injection more significantly decreased spheroid-derived but not monolayer-derived tumor size in mice. Additionally, exosomes isolated from co-culture media of FaDu spheroid and CAF upregulated angiogenesis-related genes in HNSCC cells as compared to exosomes from FaDu cell and CAF co-culture media under in vitro conditions. The mouse tumor xenograft model derived from 3D spheroids of HNSCC cells with primary CAFs is expected to produce reliable chemotherapy drug screening results given the robust angiogenesis and lack of necrosis inside tumor tissues.


Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias Bucais/patologia , Neovascularização Patológica/patologia , Esferoides Celulares/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinoma de Células Escamosas/metabolismo , Exossomos/metabolismo , Feminino , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Bucais/metabolismo , Neovascularização Patológica/metabolismo , Cultura Primária de Células/métodos , Esferoides Celulares/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto/normas
7.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360874

RESUMO

Osteoarthritis (OA) is still a recalcitrant musculoskeletal disease on account of its complex biochemistry and mechanical stimulations. Apart from stimulation by external mechanical forces, the regulation of intracellular mechanics in chondrocytes has also been linked to OA development. Recently, visfatin has received significant attention because of the clinical finding of the positive correlation between its serum/synovial level and OA progression. However, the precise mechanism involved is still unclear. This study determined the effect of visfatin on intracellular mechanics and catabolism in human primary chondrocytes isolated from patients. The intracellular stiffness of chondrocytes was analyzed by the particle-tracking microrheology method. It was shown that visfatin damages the microtubule and microfilament networks to influence intracellular mechanics to decrease the intracellular elasticity and viscosity via glycogen synthase kinase 3ß (GSK3ß) inactivation induced by p38 signaling. Further, microtubule network destruction in human primary chondrocytes is predominantly responsible for the catabolic effect of visfatin on the cyclooxygenase 2 upregulation. The present study shows a more comprehensive interpretation of OA development induced by visfatin through biochemical and biophysical perspectives. Finally, the role of GSK3ß inactivation, and subsequent regulation of intracellular mechanics, might be considered as theranostic targets for future drug development for OA.


Assuntos
Condrócitos , Citocinas/fisiologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Nicotinamida Fosforribosiltransferase/fisiologia , Osteoartrite , Citoesqueleto de Actina/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Humanos , Microtúbulos/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Cultura Primária de Células
8.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360875

RESUMO

Single prostate stem cells can generate stem and progenitor cells to form prostaspheres in 3D culture. Using a prostasphere-based label retention assay, we recently identified keratin 13 (KRT13)-enriched prostate stem cells at single-cell resolution, distinguishing them from daughter progenitors. Herein, we characterized the epithelial cell lineage hierarchy in prostaspheres using single-cell RNA-seq analysis. Keratin profiling revealed three clusters of label-retaining prostate stem cells; cluster I represents quiescent stem cells (PSCA, CD36, SPINK1, and KRT13/23/80/78/4 enriched), while clusters II and III represent active stem and bipotent progenitor cells (KRT16/17/6 enriched). Gene set enrichment analysis revealed enrichment of stem and cancer-related pathways in cluster I. In non-label-retaining daughter progenitor cells, three clusters were identified; cluster IV represents basal progenitors (KRT5/14/6/16 enriched), while clusters V and VI represent early and late-stage luminal progenitors, respectively (KRT8/18/10 enriched). Furthermore, MetaCore analysis showed enrichment of the "cytoskeleton remodeling-keratin filaments" pathway in cancer stem-like cells from human prostate cancer specimens. Along with common keratins (KRT13/23/80/78/4) in normal stem cells, unique keratins (KRT10/19/6C/16) were enriched in cancer stem-like cells. Clarification of these keratin profiles in human prostate stem cell lineage hierarchy and cancer stem-like cells can facilitate the identification and therapeutic targeting of prostate cancer stem-like cells.


Assuntos
Queratinas/metabolismo , Células-Tronco Neoplásicas , Neoplasias da Próstata , RNA/metabolismo , Adulto , Células Cultivadas , Humanos , Masculino , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Cultura Primária de Células , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Análise de Célula Única , Adulto Jovem
9.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445494

RESUMO

Despite significant advances in treatment of acute coronary syndromes (ACS) many subjects still develop heart failure due to significantly reduced ejection fraction. Currently, there are no commonly available treatment strategies that replace the infarcted/dysfunctional myocardium. Therefore, understanding the mechanisms that control the regeneration of the heart muscle is important. The development of new coronary vessels plays a pivotal role in cardiac regeneration. Employing microarray expression assays and RT-qPCR validation expression pattern of genes in long-term primary cultured cells isolated form the right atrial appendage (RAA) and right atrium (RA) was evaluated. After using DAVID software, it indicated the analysis expression profiles of genes involved in ontological groups such as: "angiogenesis", "blood vessel morphogenesis", "circulatory system development", "regulation of vasculature development", and "vasculature development" associated with the process of creation new blood vessels. The performed transcriptomic comparative analysis between two different compartments of the heart muscle allowed us to indicate the presence of differences in the expression of key transcripts depending on the cell source. Increases in culture intervals significantly increased expression of SFRP2, PRRX1 genes and some other genes involved in inflammatory process, such as: CCL2, IL6, and ROBO1. Moreover, the right atrial appendage gene encoding lysyl oxidase (LOX) showed much higher expression compared to the pre-cultivation state.


Assuntos
Vasos Coronários/crescimento & desenvolvimento , Perfilação da Expressão Gênica/métodos , Desenvolvimento Muscular , Miocárdio/citologia , Animais , Células Cultivadas , Vasos Coronários/química , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Miocárdio/química , Análise de Sequência com Séries de Oligonucleotídeos , Cultura Primária de Células , Suínos , Sequenciamento Completo do Exoma
10.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445513

RESUMO

The activation of signal transducer and activator of transcription 3 (STAT3), as well as up-regulation of cytokines and growth factors to promote STAT3 activation, have been found in the epidermis of psoriatic lesions. Recently, a series of synthetic compounds possessing the Michael acceptor have been reported as STAT3 inhibitors by covalently binding to cysteine of STAT3. We synthesized a Michael acceptor analog, SKSI-0412, and confirmed the binding affinity between STAT3 and SKSI-0412. We hypothesized that the SKSI-0412 can inhibit interleukin (IL)-17A-induced inflammation in keratinocytes. The introduction of IL-17A increased the phosphorylation of STAT3 in keratinocytes, whereas the inactivation of STAT3 by SKSI-0412 reduced IL-17A-induced STAT3 phosphorylation and IκBζ expression. In addition, human ß defensin-2 and S100A7, which are regulated by IκBζ, were significantly decreased with SKSI-0412 administration. We also confirmed that SKSI-0412 regulates cell proliferation, which is the major phenotype of psoriasis. Based on these results, we suggest targeting STAT3 with SKSI-0412 as a novel therapeutic strategy to regulate IL-17A-induced psoriatic inflammation in keratinocytes.


Assuntos
Anti-Inflamatórios/farmacologia , Interleucina-17/efeitos adversos , Queratinócitos/citologia , Fator de Transcrição STAT3/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/química , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Fator de Transcrição STAT3/química , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química
11.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360637

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by increased activation of fibroblasts/myofibroblasts. Previous reports have shown that IPF fibroblasts are resistant to apoptosis, but the mechanisms remain unclear. Since inhibition of the mitochondrial permeability transition pore (mPTP) has been implicated in the resistance to apoptosis, in this study, we analyzed the role of mitochondrial function and the mPTP on the apoptosis resistance of IPF fibroblasts under basal conditions and after mitomycin C-induced apoptosis. We measured the release of cytochrome c, mPTP opening, mitochondrial calcium release, oxygen consumption, mitochondrial membrane potential, ADP/ATP ratio, ATP concentration, and mitochondrial morphology. We found that IPF fibroblasts were resistant to mitomycin C-induced apoptosis and that calcium, a well-established activator of mPTP, is decreased as well as the release of pro-apoptotic proteins such as cytochrome c. Likewise, IPF fibroblasts showed decreased mitochondrial function, while mPTP was less sensitive to ionomycin-induced opening. Although IPF fibroblasts did not present changes in the mitochondrial membrane potential, we found a fragmented mitochondrial network with scarce, thinned, and disordered mitochondria with reduced ATP levels. Our findings demonstrate that IPF fibroblasts are resistant to mitomycin C-induced apoptosis and that altered mPTP opening contributes to this resistance. In addition, IPF fibroblasts show mitochondrial dysfunction evidenced by a decrease in respiratory parameters.


Assuntos
Apoptose , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Mitocôndrias/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Citocromos c/metabolismo , Fibroblastos/patologia , Humanos , Fibrose Pulmonar Idiopática/etiologia , Fibrose Pulmonar Idiopática/patologia , Ionomicina , Mitocôndrias/patologia , Mitomicina , Oxigênio/metabolismo , Cultura Primária de Células
12.
Am J Hum Genet ; 108(9): 1647-1668, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34416157

RESUMO

Interpretation of the function of non-coding risk loci for neuropsychiatric disorders and brain-relevant traits via gene expression and alternative splicing quantitative trait locus (e/sQTL) analyses is generally performed in bulk post-mortem adult tissue. However, genetic risk loci are enriched in regulatory elements active during neocortical differentiation, and regulatory effects of risk variants may be masked by heterogeneity in bulk tissue. Here, we map e/sQTLs, and allele-specific expression in cultured cells representing two major developmental stages, primary human neural progenitors (n = 85) and their sorted neuronal progeny (n = 74), identifying numerous loci not detected in either bulk developing cortical wall or adult cortex. Using colocalization and genetic imputation via transcriptome-wide association, we uncover cell-type-specific regulatory mechanisms underlying risk for brain-relevant traits that are active during neocortical differentiation. Specifically, we identified a progenitor-specific eQTL for CENPW co-localized with common variant associations for cortical surface area and educational attainment.


Assuntos
Proteínas Cromossômicas não Histona/genética , Regulação da Expressão Gênica no Desenvolvimento , Neocórtex/metabolismo , Neurogênese/genética , Neurônios/metabolismo , Locos de Características Quantitativas , Alelos , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Diferenciação Celular , Cromatina/química , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Mapeamento Cromossômico , Escolaridade , Feminino , Feto , Predisposição Genética para Doença , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Masculino , Neocórtex/citologia , Neocórtex/crescimento & desenvolvimento , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neuroticismo , Doença de Parkinson/diagnóstico , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Cultura Primária de Células , Prognóstico , Esquizofrenia/diagnóstico , Esquizofrenia/genética , Esquizofrenia/metabolismo , Transcriptoma
13.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361776

RESUMO

In this study, we examined aqueous extracts of the edible mushrooms Pleurotus ostreatus (oyster mushroom) and Lentinula edodes (shiitake mushroom). Proteome analysis was conducted using LC-Triple TOF-MS and showed the expression of 753 proteins by Pleurotus ostreatus, and 432 proteins by Lentinula edodes. Bioactive peptides: Rab GDP dissociation inhibitor, superoxide dismutase, thioredoxin reductase, serine proteinase and lectin, were identified in both mushrooms. The extracts also included promising bioactive compounds including phenolics, flavonoids, vitamins and amino acids. The extracts showed promising antiviral activities, with a selectivity index (SI) of 4.5 for Pleurotus ostreatus against adenovirus (Ad7), and a slight activity for Lentinula edodes against herpes simplex-II (HSV-2). The extracts were not cytotoxic to normal human peripheral blood mononuclear cells (PBMCs). On the contrary, they showed moderate cytotoxicity against various cancer cell lines. Additionally, antioxidant activity was assessed using DPPH radical scavenging, ABTS radical cation scavenging and ORAC assays. The two extracts showed potential antioxidant activities, with the maximum activity seen for Pleurotus ostreatus (IC50 µg/mL) = 39.46 ± 1.27 for DPPH; 11.22 ± 1.81 for ABTS; and 21.40 ± 2.20 for ORAC assays. This study encourages the use of these mushrooms in medicine in the light of their low cytotoxicity on normal PBMCs vis à vis their antiviral, antitumor and antioxidant capabilities.


Assuntos
Antineoplásicos/química , Antioxidantes/química , Antivirais/química , Proteínas Fúngicas/química , Pleurotus/química , Proteoma/química , Cogumelos Shiitake/química , Aminoácidos/química , Aminoácidos/isolamento & purificação , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Antivirais/isolamento & purificação , Antivirais/farmacologia , Benzotiazóis/antagonistas & inibidores , Compostos de Bifenilo/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Misturas Complexas/química , Flavonoides/química , Flavonoides/isolamento & purificação , Proteínas Fúngicas/classificação , Proteínas Fúngicas/isolamento & purificação , Humanos , Lectinas/química , Lectinas/isolamento & purificação , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Especificidade de Órgãos , Fenóis/química , Fenóis/isolamento & purificação , Picratos/antagonistas & inibidores , Pleurotus/metabolismo , Cultura Primária de Células , Proteoma/classificação , Proteoma/isolamento & purificação , Serina Proteases/química , Serina Proteases/isolamento & purificação , Cogumelos Shiitake/metabolismo , Ácidos Sulfônicos/antagonistas & inibidores , Superóxido Dismutase/química , Superóxido Dismutase/isolamento & purificação , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxina Dissulfeto Redutase/isolamento & purificação , Vitaminas/química , Vitaminas/isolamento & purificação , Água/química
14.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361797

RESUMO

Carpesium divaricatum Sieb. & Zucc., a traditional medicinal plant used as an inflammation-relieving remedy, is a rich source of terpenoids. At least 40 germacrane-type sesquiterpene lactones, representatives of four different structural groups, were isolated from the plant. Cytotoxicity against cancer cells in vitro is the most frequently described biological activity of the compounds. However, little is known about the selectivity of the cytotoxic effect. The anti-inflammatory activity of the germacranolides is also poorly documented. The objective of the present study was to assess the cytotoxic activity of selected C. divaricatum germacranolides-derivatives of 4,5,8,9-tetrahydroxy-3-oxo-germacran-6,12-olide towards cancer and normal cell lines (including cells of different p53 status). Moreover, to assess the anti-inflammatory effect of the compounds, the release of four proinflammatory cytokines/chemokines (IL-1ß, IL-8, TNF-α and CCL2) by lipopolysaccharide-stimulated human neutrophils was measured by ELISA. The investigated sesquiterpene lactones demonstrated nonselective activity towards prostate cancer (Du145 and PC3) and normal prostate epithelial cells (PNT2) as well as against melanoma cells (A375 and HTB140) and keratinocytes (HaCaT). Cytotoxic activity against osteosarcoma cells was independent of their p53 status. In sub-cytotoxic concentrations (0.5-2.5 µM) the studied compounds significantly decreased cytokine/chemokine release by lipopolysaccharide-stimulated human leukocytes.


Assuntos
Anti-Inflamatórios/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Asteraceae/química , Citotoxinas/farmacologia , Sesquiterpenos de Germacrano/farmacologia , Anti-Inflamatórios/química , Anti-Inflamatórios/classificação , Anti-Inflamatórios/isolamento & purificação , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/classificação , Antineoplásicos Fitogênicos/isolamento & purificação , Asteraceae/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Citotoxinas/química , Citotoxinas/classificação , Citotoxinas/isolamento & purificação , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Concentração Inibidora 50 , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Componentes Aéreos da Planta/química , Componentes Aéreos da Planta/metabolismo , Extratos Vegetais/química , Plantas Medicinais , Polônia , Cultura Primária de Células , Sesquiterpenos de Germacrano/química , Sesquiterpenos de Germacrano/classificação , Sesquiterpenos de Germacrano/isolamento & purificação , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia
15.
Viruses ; 13(7)2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34372571

RESUMO

Anti-cancer activity can be improved by engineering immune cells to express chimeric antigen receptors (CARs) that recognize tumor-associated antigens. Retroviral vector gene transfer strategies allow stable and durable transgene expression. Here, we used alpharetroviral vectors to modify NK-92 cells, a natural killer cell line, with a third-generation CAR designed to target the IL-3 receptor subunit alpha (CD123), which is strongly expressed on the surface of acute myeloid leukemia (AML) cells. Alpharetroviral vectors also contained a transgene cassette to allow constitutive expression of human IL-15 for increased NK cell persistence in vivo. The anti-AML activity of CAR-NK-92 cells was tested via in vitro cytotoxicity assays with the CD123+ AML cell line KG-1a and in vivo in a patient-derived xenotransplantation CD123+ AML model. Unmodified NK-92 cells or NK-92 cells modified with a truncated version of the CAR that lacked the signaling domain served as controls. Alpharetroviral vector-modified NK-92 cells stably expressed the transgenes and secreted IL-15. Anti-CD123-CAR-NK-92 cells exhibited enhanced anti-AML activity in vitro and in vivo as compared to control NK-92 cells. Our data (1) shows the importance of IL-15 expression for in vivo persistence of NK-92 cells, (2) supports continued investigation of anti-CD123-CAR-NK cells to target AML, and (3) points towards potential strategies to further improve CAR-NK anti-AML activity.


Assuntos
Imunoterapia Adotiva/métodos , Subunidade alfa de Receptor de Interleucina-3/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Idoso , Alpharetrovirus/genética , Animais , Linhagem Celular Tumoral , Feminino , Terapia Genética , Vetores Genéticos/genética , Humanos , Subunidade alfa de Receptor de Interleucina-3/genética , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Cultura Primária de Células , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Transdução Genética , Transgenes , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Bioengineered ; 12(1): 4407-4419, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34436976

RESUMO

Widespread infection due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has led to a global pandemic. Currently, various approaches are being taken up to develop vaccines and therapeutics to treat SARS-CoV2 infection. Consequently, the S protein has become an important target protein for developing vaccines and therapeutics against SARS-CoV2. However, the highly infective nature of SARS-CoV2 restricts experimentation with the virus to highly secure BSL3 facilities. The availability of fusion-enabled, nonreplicating, and nonbiohazardous mimics of SARS-CoV2 virus fusion, containing the viral S or S and M protein in their native conformation on mammalian cells, can serve as a useful substitute for studying viral fusion for testing various inhibitors of viral fusion. This would avoid the use of the BSL3 facility for fusion studies required to develop therapeutics. In the present study, we have developed SARS-CoV2 virus fusion mimics (SCFMs) using mammalian cells transfected with constructs coding for S or S and M protein. The fusogenic property of the mimic(s) and their interaction with the functional human ACE2 receptors was confirmed experimentally. We have also shown that such mimics can easily be used in an inhibition assay. These mimic(s) can be easily prepared on a large scale, and such SCFMs can serve as an invaluable resource for viral fusion inhibition assays and in vitro screening of antiviral agents, which can be shared/handled between labs/facilities without worrying about any biohazard while working under routine laboratory conditions, avoiding the use of BSL3 laboratory.Abbreviations :SCFM: SARS-CoV2 Virus Fusion Mimic; ACE2: Angiotensin-Converting Enzyme 2; hACE2: Human Angiotensin-Converting enzyme 2; MEF: Mouse Embryonic Fibroblasts; HBSS: Hanks Balanced Salt Solution; FBS: Fetal Bovine Serum.


Assuntos
Anticorpos Neutralizantes/farmacologia , Contenção de Riscos Biológicos/métodos , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Proteínas da Matriz Viral/antagonistas & inibidores , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Chlorocebus aethiops , Embrião de Mamíferos , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células MCF-7 , Camundongos , Mimetismo Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , Ligação Proteica , Receptores Virais/genética , Receptores Virais/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Transfecção , Células Vero , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
17.
Nat Commun ; 12(1): 4171, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234116

RESUMO

Here we report the pharmacologic blockade of voltage-gated sodium ion channels (NaVs) by a synthetic saxitoxin derivative affixed to a photocleavable protecting group. We demonstrate that a functionalized saxitoxin (STX-eac) enables exquisite spatiotemporal control of NaVs to interrupt action potentials in dissociated neurons and nerve fiber bundles. The photo-uncaged inhibitor (STX-ea) is a nanomolar potent, reversible binder of NaVs. We use STX-eac to reveal differential susceptibility of myelinated and unmyelinated axons in the corpus callosum to NaV-dependent alterations in action potential propagation, with unmyelinated axons preferentially showing reduced action potential fidelity under conditions of partial NaV block. These results validate STX-eac as a high precision tool for robust photocontrol of neuronal excitability and action potential generation.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Saxitoxina/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Células CHO , Células Cultivadas , Corpo Caloso/citologia , Corpo Caloso/efeitos dos fármacos , Corpo Caloso/metabolismo , Cricetulus , Embrião de Mamíferos , Feminino , Hipocampo/citologia , Masculino , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Técnicas de Patch-Clamp , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saxitoxina/análogos & derivados , Saxitoxina/efeitos da radiação , Análise de Célula Única , Análise Espaço-Temporal , Raios Ultravioleta , Bloqueadores do Canal de Sódio Disparado por Voltagem/efeitos da radiação
18.
Nat Commun ; 12(1): 4229, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244477

RESUMO

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved.


Assuntos
Citoesqueleto de Actina/metabolismo , Adesão Celular/fisiologia , Mecanotransdução Celular/fisiologia , Citoesqueleto de Actina/ultraestrutura , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Fibroblastos , Técnicas de Silenciamento de Genes , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/fisiologia , Masculino , Camundongos , Camundongos Knockout , Microscopia de Força Atômica , Pinças Ópticas , Paxilina/metabolismo , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Respiração , Organismos Livres de Patógenos Específicos , Talina/genética , Talina/metabolismo
19.
Molecules ; 26(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199417

RESUMO

Blockade of the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction is currently the focus in the field of cancer immunotherapy, and so far, several monoclonal antibodies (mAbs) have achieved encouraging outcomes in cancer treatment. Despite this achievement, mAbs-based therapies are struggling with limitations including poor tissue and tumor penetration, long half-life time, poor oral bioavailability, and expensive production costs, which prompted a shift towards the development of the small-molecule inhibitors of PD-1/PD-L1 pathways. Even though many small-molecule inhibitors targeting PD-1/PD-L1 interaction have been reported, their development lags behind the corresponding mAb, partly due to the challenges of developing drug-like small molecules. Herein, we report the discovery of a series of novel inhibitors targeting PD-1/PD-L1 interaction via structural simplification strategy by using BMS-1058 as a starting point. Among them, compound A9 stands out as the most promising candidate with excellent PD-L1 inhibitory activity (IC50 = 0.93 nM, LE = 0.43) and high binding affinity to hPD-L1 (KD = 3.64 nM, LE = 0.40). Furthermore, A9 can significantly promote the production of IFN-γ in a dose-dependent manner by rescuing PD-L1 mediated T-cell inhibition in Hep3B/OS-8/hPD-L1 and CD3-positive T cells co-culture assay. Taken together, these results suggest that A9 is a promising inhibitor of PD-1/PD-L1 interaction and is worthy for further study.


Assuntos
Antígeno B7-H1/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T/citologia , Antígeno B7-H1/química , Linhagem Celular , Cristalografia por Raios X , Humanos , Interferon gama/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Cultura Primária de Células , Receptor de Morte Celular Programada 1/química , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
20.
Int J Mol Sci ; 22(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299331

RESUMO

BACKGROUND: Due to its prominence in the regulation of metabolism and inflammation, adipose tissue is a major target to investigate alterations in insulin action. This hormone activates PI3K/AKT pathway which is essential for glucose homeostasis, cell differentiation, and proliferation in insulin-sensitive tissues, like adipose tissue. The aim of this work was to evaluate the impact of chronic and intermittent high glucose on the expression of biomolecules of insulin signaling pathway during the differentiation and maturation of human visceral preadipocytes. METHODS: Human visceral preadipocytes (HPA-V) cells were treated with high glucose (30 mM)during the proliferation and/or differentiation and/or maturation stage. The level of mRNA (by Real-Time PCR) and protein (by Elisa tests) expression of IRS1, PI3K, PTEN, AKT2, and GLUT4 was examined after each culture stage. Furthermore, we investigated whether miR-29a-3p, miR-143-3p, miR-152-3p, miR-186-5p, miR-370-3p, and miR-374b-5p may affect the expression of biomolecules of the insulin signaling pathway. RESULTS: Both chronic and intermittent hyperglycemia affects insulin signaling in visceral pre/adipocytes by upregulation of analyzed PI3K/AKT pathway molecules. Both mRNA and protein expression level is more dependent on stage-specific events than the length of the period of high glucose exposure. What is more, miRs expression changes seem to be involved in PI3K/AKT expression regulation in response to hyperglycemic stimulation.


Assuntos
Hiperglicemia/metabolismo , Gordura Intra-Abdominal/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Feminino , Expressão Gênica , Glucose/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Humanos , Hiperglicemia/patologia , Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Resistência à Insulina , Gordura Intra-Abdominal/citologia , Gordura Intra-Abdominal/fisiologia , MicroRNAs/genética , MicroRNAs/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/genética , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais
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