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1.
J Environ Sci (China) ; 85: 94-106, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31471036

RESUMO

Titanium dioxide nanoparticles (TiO2 NPs) are subjected to various transformation processes (chemical, physical and biological processes) in the environment, potentially affecting their bioavailability and toxic properties. However, the size variation of TiO2 NPs during aging process and subsequent effects in mammalian cells are largely unknown. The aim of this study was to illustrate the adverse effects of TiO2 NPs in different sizes (5, 15 and <100 nm) during aging process on human-hamster hybrid (AL) cells. There was an aging-time dependent enhancement of average hydrodynamic size in TiO2 NPs stock suspensions. The cytotoxicity of fresh TiO2 NPs increased in a size-dependent manner; in contrast, their genotoxicity decreased with the increasing sizes of NPs. No significant toxicity difference was observed in cells exposed to either fresh or 60 day-aged TiO2 NPs. Both Fresh and aged TiO2 NPs efficiently induced mitochondrial dysfunction and activated Caspase-3/7 in a size-dependent manner. Using mitochondrial-DNA deficient (ρ0) AL cells, we further discovered that mitochondrial dysfunction made significant contribution to the size-dependent toxicity induced by TiO2 NPs during the aging process. Taken together, our data indicated that TiO2 NPs could significantly induced the cytotoxicity and genotoxicity in an aging time-independent and size-dependent manner, which were triggered by mitochondrial dysfunction. Our study suggested the necessity to include size as an additional parameter for the cautious monitoring of TiO2 NPs disposal before entering the environment.


Assuntos
Nanopartículas/toxicidade , Titânio/toxicidade , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Dano ao DNA , Humanos , Testes de Toxicidade
2.
J Biomed Nanotechnol ; 15(9): 1867-1880, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31387675

RESUMO

The present study aims to evaluate the effect of the ethyl acetate extract of Cichorium (EAEC) as a novel photosensitizer in photodynamic therapy (PDT) of colorectal carcinoma (CRC) HCT116 and SW620 cells. The absorption and fluorescence spectra of EAEC were measured using a UV-vis spectrophotometer and fluorescence spectrophotometer, respectively. EAEC-induced reactive oxygen species (ROS) production in HCT116 and SW620 cells was detected using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and glutathione/glutathione disulfide (GSH/GSSG). The photo- and dark toxicities of EAEC were estimated using the Cell Counting Kit-8 (CCK-8) assay. Cellular uptake and localization of EAEC were detected by confocal laser fluorescence microscopy. Annexin V-FITC/PI staining, Western blotting and immunofluorescence staining were used to assess apoptosis and autophagy. The antitumor activity of EAEC was confirmed in a xenograft model. Finally, effects on the PERK pathway were verified using qRT-PCR and Western blotting. EAEC displayed absorption and fluorescence emission peaks at 660 nm and 678 nm, respectively. EAEC induced ROS production in CRC cells. Assessment of dark toxicity showed that treatment with EAEC alone induced little cytotoxicity in CRC or normal cells but that EAEC-PDT induced significant photocytotoxicity in CRC cells in a time- and dose-dependent manner. After cellular uptake, EAEC was located in the mitochondria. Treatment with EAEC-PDT reduced xenograft tumor size. Further evaluation suggested that activation of the PERK pathway mediates these effects, as the apoptotic rate and autophagy flux increased markedly after EAEC-PDT. EAEC, a natural photosensitizer extracted from Cichorium, displays potential utility in PDT of CRC by targeting the PERK pathway.


Assuntos
Autofagia , Neoplasias Colorretais , Fotoquimioterapia , Acetatos , Apoptose , Linhagem Celular , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático , Humanos , Fármacos Fotossensibilizantes , Proteínas Quinases , Espécies Reativas de Oxigênio
3.
Biol Res ; 52(1): 44, 2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31426858

RESUMO

BACKGROUND: Free fatty acid receptor 1 (FFAR1) is G-protein coupled receptor predominantly expressed in pancreatic ß-cells that is activated by a variety of free fatty acids (FFAs). Once activated, it promotes glucose-stimulated insulin secretion (GSIS). However, increased levels of FFAs lead to lipotoxicity, inducing loss of ß-cell function. FFAR1 plays a key role in the development of type 2 diabetes (T2D), and previous studies have indicated the importance of developing anti-diabetic therapies against FFAR1, although its role in the regulation of ß-cell function remains unclear. The present study investigated the role of FFAR1 under lipotoxic conditions using palmitic acid (PA). The rat insulinoma 1 clone 832/13 (INS-1 832/13) cell line was used as a model as it physiologically resembles native pancreatic ß-cells. Key players of the insulin signaling pathway, such as mTOR, Akt, IRS-1, and the insulin receptor (INSR1ß), were selected as candidates to be analyzed under lipotoxic conditions. RESULTS: We revealed that PA-induced lipotoxicity affected GSIS in INS-1 cells and negatively modulated the activity of both IRS-1 and Akt. Reduced phosphorylation of both IRS-1 S636/639 and Akt S473 was observed, in addition to decreased expression of both INSR1ß and FFAR1. Moreover, transient knockdown of FFAR1 led to a reduction in IRS-1 mRNA expression and an increase in INSR1ß mRNA. Finally, PA affected localization of FFAR1 from the cytoplasm to the perinucleus. CONCLUSIONS: In conclusion, our study suggests a novel regulatory involvement of FFAR1 in crosstalk with mTOR-Akt and IRS-1 signaling in ß-cells under lipotoxic conditions.


Assuntos
Células Secretoras de Insulina/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Ácido Palmítico/toxicidade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose , Linhagem Celular , Células Secretoras de Insulina/metabolismo , Ratos , Transdução de Sinais
4.
Gene ; 716: 144031, 2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31377314

RESUMO

Circular RNAs (circRNAs), a novel class of widespread and diverse endogenous RNAs, have been identified as critical regulators of various cancers, including hepatocellular carcinoma (HCC). However, the specific roles of circRNAs in HCC are largely unknown. In this study, we identified a novel circRNA, circ-IGF1R, in HCC tumour tissues and cell lines. Circ-IGF1R levels were found to be significantly upregulated in HCC tissues compared with levels in paired peritumoural tissues. The high expression levels of circ-IGF1R in HCC were associated with tumour size. Moreover, knocking down circ-IGF1R with siRNA significantly attenuated cell proliferation and induced cell apoptosis and cell cycle arrest in vitro. Further investigation revealed that PI3K/AKT signalling pathway activation was involved in the oncogenic functions of circ-IGF1R in HCC. Our study suggests that circ-IGF1R may be a potential target for the prevention and treatment of HCC.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , RNA/metabolismo , Apoptose , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinase/antagonistas & inibidores , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Regulação para Cima
5.
Braz Oral Res ; 33: e058, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31432925

RESUMO

Cementum is the mineralized tissue covering the tooth root that functions in tooth attachment and post-eruptive adjustment of tooth position. It has been reported to be highly similar to bone in several respects but remains poorly understood in terms of development and regeneration. Here, we investigate whether cementocytes, the residing cells in cellular cementum, have the potential to be protagonist in cementum homeostasis, responding to endocrine signals and directing local cementum metabolism. Cells from healthy erupted human teeth were isolated using sequential collagenase/EDTA digestions, and maintained in standard cell culture conditions. A cementocyte-like cell line was cloned (HCY-23, for human cementocyte clone 23), which presented a cementocyte compatible gene expression signature, including the expression of dentin matrix protein 1 ( DMP1 ), sclerostin ( SOST ), and E11/gp38/podoplanin ( E11 ). In contrast, these cells did not express the odontoblast/dentin marker dentin sialoprotein ( DSPP ). HCY-23 cells produced mineral-like nodules in vitro under differentiation conditions, and were highly responsive to inorganic phosphate (Pi). Within the limits of the present study, it can be concluded that cementocytes are phosphate-responsive cells, and have the potential do play a key role in periodontal homeostasis and regeneration.


Assuntos
Técnicas de Cultura de Células/métodos , Cemento Dentário/citologia , Adolescente , Adulto , Análise de Variância , Proteínas Morfogenéticas Ósseas/análise , Proteínas Morfogenéticas Ósseas/genética , Linhagem Celular , Cemento Dentário/metabolismo , Proteínas da Matriz Extracelular/análise , Proteínas da Matriz Extracelular/genética , Feminino , Imunofluorescência , Expressão Gênica , Marcadores Genéticos/genética , Humanos , Masculino , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/genética , Dente Molar/citologia , Fosfatos/farmacologia , Fosfoproteínas/análise , Fosfoproteínas/genética , Sialoglicoproteínas/análise , Sialoglicoproteínas/genética , Fatores de Tempo , Adulto Jovem
6.
7.
Cell Physiol Biochem ; 53(2): 413-428, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31415717

RESUMO

BACKGROUND/AIMS: Amyloid plaques, generated during the progression of Alzheimer's disease, cause major neurological deficits due to substantial cell toxicity and death. The underlying cause of plaque generation stems from cleavage of the amyloid precursor protein (APP) by ß-secretase (BACE1). A resulting amyloid-ß (Aß) fragment forms aggregates to produce the main constituent of a plaque. METHODS: Phage display and biopanning techniques were used to identify a 12-mer peptide that had a natural affinity for the BACE1 enzyme. The peptide was translated from phage DNA and synthetically produced. The peptide, at concentrations of 1nM, 10nM and 100nM, was used to confirm binding by direct assay. Non-specific binding to BACE2, renin and cathepsin D was tested by direct binding assay. A BACE1 activity assay was used to determine the peptide effect on cleavage of an APP substrate. Treatment of SY5Y cells with the peptide was used to determine toxicity and prevention of Aß40 and Aß42 production. RESULTS: After identification and synthetic production, the peptide exhibited a strong affinity for BACE1 at nanomolar concentrations in the direct assay. In case of non-specific binding to homologous BACE2, renin and cathepsin D, the peptide showed minor binding but was nullified when in solution with BACE1. The peptide addition to a BACE1 activity assay was able to significantly reduce the amount of substrate cleavage. SY5Y cells, when treated with the peptide, did not show any detrimental morphological changes while being able to reduce the production of natural Aß40 and Aß42. Even under stressed conditions (H2O2 treatment) where the Aß production was higher, the peptide was still able to significantly reduce the effect of BACE1 while not effecting cell viability. CONCLUSION: The identified peptide exhibited strong binding to BACE1 in vitro and was able to reduce production of Aß, suggesting a favourable BACE1 inhibitor for future refining and characterisation.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fragmentos de Peptídeos/antagonistas & inibidores , Peptídeos/farmacologia , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Linhagem Celular , Descoberta de Drogas , Inibidores Enzimáticos/metabolismo , Humanos , Fragmentos de Peptídeos/metabolismo , Peptídeos/metabolismo
8.
Gene ; 715: 144028, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31374326

RESUMO

BACKGROUND: Type 2 diabetes (T2D) is a complex polygenic disease with unclear mechanism. In an attempt to identify novel genes involved in ß-cell function, we harness a bioinformatics method called Loss-of-function tool (LoFtool) gene score. METHODS: RNA-sequencing data from human islets were used to cross-reference genes within the 1st quartile of most intolerant LoFtool score with the 100th most expressed genes in human islets. Out of these genes, GNAS and EEF1A1 genes were selected for further investigation in diabetic islets, metabolic tissues along with their correlation with diabetic phenotypes. The influence of GNAS and EEF1A1 on insulin secretion and ß-cell function were validated in INS-1 cells. RESULTS: A comparatively higher expression level of GNAS and EEF1A1 was observed in human islets than fat, liver and muscle tissues. Furthermore, diabetic islets displayed a reduced expression of GNAS, but not of EEF1A, compared to non-diabetic islets. The expression of GNAS was positively correlated with insulin secretory index, GLP1R, GIPR and inversely correlated with HbA1c. Diabetic human islets displayed a reduced cAMP generation and insulin secretory capacity in response to glucose. Moreover, siRNA silencing of GNAS in INS-1 cells reduced insulin secretion, insulin content, and cAMP production. In addition, the expression of Insulin, PDX1, and MAFA was significantly down-regulated in GNAS-silenced cells. However, cell viability and apoptosis rate were unaffected. CONCLUSION: LoFtool is a powerful tool to identify genes associated with pancreatic islets dysfunction. GNAS is a crucial gene for the ß-cell insulin secretory capacity.


Assuntos
Cromograninas/biossíntese , Subunidades alfa Gs de Proteínas de Ligação ao GTP/biossíntese , Regulação da Expressão Gênica , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Idoso , Animais , Linhagem Celular , Cromograninas/genética , AMP Cíclico/genética , AMP Cíclico/metabolismo , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Humanos , Células Secretoras de Insulina/citologia , Masculino , Pessoa de Meia-Idade , Especificidade de Órgãos , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Ratos
9.
Cell Physiol Biochem ; 53(2): 355-365, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31385664

RESUMO

BACKGROUND/AIMS: NLRP3 inflammasome activation has been reported to be an early mechanism responsible for glomerular inflammation and injury in obese mice. However, the precise mechanism of obesity-induced NLRP3 inflammasome activation remains unknown. The present study explored whether adipokine visfatin mediates obesity-induced NLRP3 inflammasome activation and consequent podocyte injury. METHODS: Inflammasome formation and immunofluorescence expressions were quantified by confocal microscopy. Caspase-activity, IL-1ß production and VEGF concentrations were measured by ELISA. RESULTS: Confocal microscopic analysis showed that visfatin treatment increased the colocalization of Nlrp3 with Asc or Nlrp3 with caspase-1 in podocytes indicating the formation of NLRP3 inflammasomes. This visfatin-induced NLRP3 inflammasome formation was abolished by pretreatment of podocytes with Asc siRNA. Correspondingly, visfatin treatment significantly increased the caspase-1 activity and IL-1ß production in podocytes, which was significantly attenuated by Asc siRNA transfection. Further RT-PCR and confocal microscopic analysis demonstrated that visfatin treatment significantly decreased the podocin expression (podocyte damage). Podocytes pretreatment with Asc siRNA or caspase-1 inhibitor, WEHD attenuated this visfatin-induced podocin reduction. Furthermore, Asc siRNA transfection was found to preserve podocyte morphology by maintaining the distinct arrangement of F-actin fibers normally lost in response to visfatin. It also prevented podocyte dysfunction by restoring visfatin-induced suppression of VEGF production and secretion. CONCLUSION: Visfatin induces NLRP3 inflammasome activation in podocytes and thereby resulting in podocyte injury.


Assuntos
Adipocinas/imunologia , Inflamassomos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Nicotinamida Fosforribosiltransferase/imunologia , Podócitos/imunologia , Animais , Linhagem Celular , Inflamação/imunologia , Inflamação/patologia , Interleucina-1beta/imunologia , Camundongos , Obesidade/imunologia , Obesidade/patologia , Podócitos/citologia , Podócitos/patologia , Fator A de Crescimento do Endotélio Vascular/imunologia
10.
Cell Physiol Biochem ; 53(2): 366-387, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31385665

RESUMO

BACKGROUND/AIMS: The extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase (MAPK) family. Upon stimulation, these kinases translocate from the cytoplasm to the nucleus, where they induce physiological processes such as proliferation and differentiation. The mechanism of translocation of this kinase involves phosphorylation of two Ser residues within a nuclear translocation signal (NTS), which allows binding to importin7 and a subsequent penetration via nuclear pores. However, the regulation of this process and the protein kinases involved are not yet clear. METHODS: To answer this point we developed specific anti phospho-SPS antibody, used this and other antibodies in Western blots and crystalized the phospho-mimetic mutated ERK. RESULTS: Here we show that the phosphorylation of both Ser residues is mediated mainly by casein kinase 2 (CK2) and that active ERK may assist in the phosphorylation of the N-terminal Ser. We also demonstrate that the phosphorylation is dependent on the release of ERK from cytoplasmic anchoring proteins. Crystal structure of the phosphomimetic ERK revealed that the NTS phosphorylation creates an acidic patch in ERK. Our model is that in resting cells ERK is bound to cytoplasmic anchors, which prevent its NTS phosphorylation. Upon stimulation, phosphorylation of the ERK TEY domain releases ERK and allows phosphorylation of its NTS by CK2 and active ERK to generate a negatively charged patch in ERK, binding to importin 7 and nuclear translocation. CONCLUSION: These results provide an important role of CK2 in regulating nuclear ERK activities.


Assuntos
Núcleo Celular/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transporte Ativo do Núcleo Celular , Caseína Quinase II/metabolismo , Linhagem Celular , Humanos , Carioferinas/metabolismo , Fosforilação , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/metabolismo
11.
Pestic Biochem Physiol ; 159: 41-50, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400783

RESUMO

Emerging fungal phytodiseases are a food security threat and novel fungicides are in an urgent need. Herein, a series of isobutyrophenone derivatives were designed and synthesized. The derivatives exhibited excellent fungicidal activities against seven fungi. The structure-activity relationship (SAR) study indicated that the introduction of a bromo group at the position 3 or 5 of the phenyl ring, as well as esterification of the 4-hydroxy with a chloroacetyl group, could substantially increase the antifungal activity and spectrum of the compounds. Among all 23 compounds, 2-bromo-3-hydroxy-4-isobutyryl-6-methylphenyl 2-chloroacetate (12b) showed the highest fungicidal activity against all seven tested fungal pathogens with EC50 values ranging from 1.22 to 39.94 µg/mL and exhibited the most potent inhibition against class II fructose-1,6-bisphosphate aldolase with an IC50 of 3.63 µM. The lead compounds were proven to be safe to NIH3T3/293 T cells and silkworm larvae, and relatively stable under different harsh conditions. Detached fruit tests showed the practical potential of lead compounds for fruit (or plant) protection. Taken together, our results indicated that the isobutyrophenone derivatives could be further optimized and developed as advanced leads for new fungicides.


Assuntos
Antifúngicos/química , Antifúngicos/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Animais , Bombyx/metabolismo , Linhagem Celular , Frutose-Bifosfato Aldolase/genética , Humanos , Larva/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Células NIH 3T3 , Relação Estrutura-Atividade
12.
Chem Commun (Camb) ; 55(57): 8227-8230, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31268107

RESUMO

An original family of multivalent vectors encompassing gemini and facial amphiphilicity, namely cationic Siamese twin surfactants, has been prepared from the disaccharide trehalose; molecular engineering lets us modulate the self-assembling properties and the topology of the nanocomplexes with plasmid DNA for efficient gene delivery in vitro and in vivo.


Assuntos
Nanoestruturas/química , Plasmídeos/química , Tensoativos/química , Transfecção/métodos , Trealose/química , Animais , Linhagem Celular , Cercopithecus aethiops , Humanos , Camundongos , Plasmídeos/metabolismo
13.
Cell Physiol Biochem ; 53(2): 323-336, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31359737

RESUMO

BACKGROUND/AIMS: Vascular calcification represents a huge clinical problem contributing to adverse cardiovascular events, with no effective treatment currently available. Upregulation of hepatocyte growth factor has been linked with vascular calcification, and thus, represent a potential target in the development of a novel therapeutic strategy. Glycomimetics have been shown to interrupt HGF-receptor signalling, therefore this study investigated the effect of novel glycomimetics on osteogenic signalling and vascular calcification in vitro. METHODS: Primary human vascular smooth muscle cells (HVSMCs) were induced by ß-glycerophosphate (ß-GP) and treated with 4 glycomimetic compounds (C1-C4). The effect of ß-GP and C1-C4 on alkaline phosphatase (ALP), osteogenic markers and c-Met/Notch3/HES1 signalling was determined using colorimetric assays, qRT-PCR and western blotting respectively. RESULTS: C1-C4 significantly attenuated ß-GP-induced calcification, as shown by Alizarin Red S staining and calcium content by day 14. In addition, C1-C4 reduced ALP activity and prevented upregulation of the osteogenic markers, BMP-2, Runx2, Msx2 and OPN. Furthermore, ß-GP increased c-Met phosphorylation at day 21, an effect ameliorated by C2 and C4 and the c-Met inhibitor, crizotinib. We next interrogated the effects of the Notch inhibitor DAPT and confirmed an inhibition of ß-GP up-regulated Notch3 protein by C2, DAPT and crizotinib compared to controls. Hes-1 protein upregulation by ß-GP, was also significantly downregulated by C2 and DAPT. GOLD docking analysis identified a potential binding interaction of C1-C4 to HGF which will be investigated further. CONCLUSION: These findings demonstrate that glycomimetics have potent anti-calcification properties acting via HGF/c-Met and Notch signalling.


Assuntos
Músculo Liso Vascular/citologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptor Notch3/metabolismo , Fatores de Transcrição HES-1/metabolismo , Calcificação Vascular/metabolismo , Materiais Biomiméticos/farmacologia , Proteína Morfogenética Óssea 2/metabolismo , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Glicerofosfatos/farmacologia , Proteínas de Homeodomínio/metabolismo , Humanos , Miócitos de Músculo Liso/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo
14.
Genome Biol ; 20(1): 142, 2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31315641

RESUMO

We develop CellSIUS (Cell Subtype Identification from Upregulated gene Sets) to fill a methodology gap for rare cell population identification for scRNA-seq data. CellSIUS outperforms existing algorithms for specificity and selectivity for rare cell types and their transcriptomic signature identification in synthetic and complex biological data. Characterization of a human pluripotent cell differentiation protocol recapitulating deep-layer corticogenesis using CellSIUS reveals unrecognized complexity in human stem cell-derived cellular populations. CellSIUS enables identification of novel rare cell populations and their signature genes providing the means to study those populations in vitro in light of their role in health and disease.


Assuntos
Análise de Célula Única/métodos , Transcriptoma , Algoritmos , Linhagem Celular , Humanos , Neurônios/citologia
15.
Toxicol Lett ; 313: 130-136, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31276767

RESUMO

We previously demonstrated that based on their potency, contact allergens differently modulate Blimp-1/NLRP12 expression in human keratinocytes, with the extreme allergen 2,4-dinitrochlorobenzene (DNCB) more rapidly upregulating Blimp-1, leading to downregulation of NLRP12, and to the production of interleukin-18 (IL-18). The purpose of this study was to further investigate the effects of DNCB and para-phenylenediamine (PPD) on the expression of the proteins of the inflammasome, namely NLRP3, ASC and caspase 1 by western blot analysis; to define the intracellular localization and co-localization of NLRP3 and NLPR12 by immunoprecipitation and immunohistochemistry; and to define the role of NF-κB in Blimp-1 induction by pharmacological inhibition. The human keratinocyte cell line NCTC2544 was used for all experiments. Dose and time course experiments were performed to evaluate the effect of the selected contact allergens on the parameters investigated. Results indicate, that consistent with previous finding, DNCB more rapidly (3 h) induces NLRP3, ASC protein expression and caspase-1 activation compared to PPD. Immunoprecipitation studies show the recruitment of ASC to the inflammasome following exposure to both allergens, while high level of NLRP12 and less ASC protein were found associated in control cells. By immunohistochemistry, we found increased NLRP3 expression following exposure to contact allergens, and observed a nuclear co-localization of the two proteins, indicating the NLRP12 likely acts preventing the cytosolic localization of NLRP3 and inflammasome assembly. Finally, contact allergen-induced Blimp-1 mRNA and protein expression can be completely blocked by inhibiting NF-κB activation, confirming the central role of NF-κB in contact allergen-induced keratinocyte activation.


Assuntos
Alérgenos/toxicidade , Dermatite Alérgica de Contato/etiologia , Dinitroclorobenzeno/toxicidade , Inflamassomos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Queratinócitos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fenilenodiaminas/toxicidade , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 1/metabolismo , Linhagem Celular , Dermatite Alérgica de Contato/genética , Dermatite Alérgica de Contato/metabolismo , Relação Dose-Resposta a Droga , Humanos , Inflamassomos/genética , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fatores de Tempo
16.
Toxicol Lett ; 313: 150-158, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31276768

RESUMO

Ochratoxin A (OTA), one of the most abundant food-contaminating mycotoxins, is a possible carcinogen to humans. We previously demonstrated that long-term (40 weeks) OTA exposure induces the malignant transformation of human gastric epithelium cells (GES-1) in vitro. However, the specific mechanism underlying OTA-induced gastric carcinogenesis is complex. In the present study, we used 2-DE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF MS) combined with bioinformatics and immunoblotting to investigate the differentially expressed proteins between GES-1 and OTA-malignant transformed GES-1 cells (OTA-GES-1T cells) in vitro. We found that four differentially expressed proteins were identified after malignant transformation, including actin, cytoplasmic 1 (ACTB), F-actin-capping protein subunit alpha-1 (CAPZA1), Annexin A3 (ANXA3), thioredoxin peroxidase B from red blood cells (TPx-B) and Fibrinogen beta B (Fibrinogen ß). Among the differentially expressed proteins, the effect of Annexin A3 was analyzed by MTT assay, western blot, cell cycle analysis, wound healing assay, Transwell assay, and colony formation assay in OTA-GES-1T cells. The results showed that inhibition of Annexin A3 by siRNA effectively prevented the proliferation, migration, and invasion abilities of OTA-GES-1T cells. Collectively, the results of this study will guide future research on OTA carcinogenicity.


Assuntos
Anexina A3/metabolismo , Carcinógenos/toxicidade , Transformação Celular Neoplásica/induzido quimicamente , Células Epiteliais/efeitos dos fármacos , Mucosa Gástrica/efeitos dos fármacos , Ocratoxinas/toxicidade , Neoplasias Gástricas/induzido quimicamente , Anexina A3/genética , Western Blotting , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Biologia Computacional , Eletroforese em Gel Bidimensional , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Humanos , Invasividade Neoplásica , Proteômica/métodos , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia
17.
J Agric Food Chem ; 67(31): 8668-8676, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31271028

RESUMO

This study investigated the effect of Chlorella vulgaris (C. vulgaris) on genotoxicity, cytotoxicity, and apoptosis in Caco-2 and HT-29 cells. C. vulgaris significantly induced DNA damage in both cell lines at a concentration of 200 µg dry matter/mL (comet tail intensity CTI: 24.6 ± 4.7% for Caco-2, 16.6 ± 0.9% for HT-29). The application of processing (sonication, ball-milling) did not affect the genotoxicity negatively and lowered the lipid peroxidation in C. vulgaris preparations. C. vulgaris-induced intracellular formation of reactive oxygen species in human cell lines and might be responsible for the genotoxic effect. A solid fraction mainly triggered the observed DNA damage (CTI: 41.5 ± 1.9%), whereas a hydrophilic (CTI: 7.9 ± 1.7%) and lipophilic (CTI: 10.2 ± 2.1%) fraction revealed a significantly lower tail intensity. C. vulgaris significantly induced DNA damage in both cell lines possibly through intracellular formation of reactive oxygen species; however, it was repaired after a 2 h recovery time or was even avoided at lower concentrations. In addition, none of the preparations indicated an adverse effect on cell proliferation or revealed apoptotic activity.


Assuntos
Chlorella vulgaris/química , Dano ao DNA/efeitos dos fármacos , Células Epiteliais/citologia , Mutagênicos/toxicidade , Extratos Vegetais/toxicidade , Apoptose/efeitos dos fármacos , Processos Autotróficos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Chlorella vulgaris/crescimento & desenvolvimento , Chlorella vulgaris/efeitos da radiação , Ensaio Cometa , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Luz , Peroxidação de Lipídeos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
18.
J Agric Food Chem ; 67(31): 8649-8659, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31283213

RESUMO

Spent coffee grounds (SCG) are the most abundant coffee byproduct and are generally discarded as waste. The horticultural use of SCG and SCG compost (SCGC) has become popular due to a growing interest in environmentally friendly measures for waste disposal. Estrogen-like endocrine disrupting chemicals in the soil can be absorbed by plants and subsequently by humans who consume these plants. The objectives of this study are to determine the phytochemical profiles of extracts of SCG and SCGC and to evaluate the estrogen-like activities of SCG, SCGC, and the major coffee phenolic acids, specifically, 5-O-caffeoylquinic acid (CQA), caffeic acid, and ferulic acid. Their inductive effects on estrogen receptor (ER)-mediated gene transcription have been examined in cultured cell lines. CQA was the most abundant phenolic acid in SCG and SCGC and was further examined for its ER-mediated estrogen-like activity using various assays. This is the first study to report the estrogen-like signaling activities of coffee byproducts and their major constituents.


Assuntos
Coffea/química , Hidroxibenzoatos/metabolismo , Fitoestrógenos/metabolismo , Extratos Vegetais/metabolismo , Receptores Estrogênicos/genética , Ativação Transcricional , Resíduos/análise , Animais , Ácidos Cafeicos/análise , Ácidos Cafeicos/metabolismo , Linhagem Celular , Compostagem , Feminino , Genes Reporter , Humanos , Hidroxibenzoatos/química , Fitoestrógenos/química , Extratos Vegetais/química , Ratos , Ratos Sprague-Dawley , Receptores Estrogênicos/metabolismo , Sementes/química
19.
J Agric Food Chem ; 67(33): 9286-9294, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31339733

RESUMO

Natural aryl hydrocarbon (AHR) ligands have been identified in food and herbal medicines, and they may exhibit beneficial activity in humans. In this study, white button (WB) feeding significantly decreased AHR target gene expression in the small intestine of both conventional and germ-free mice. High-performance liquid chromatography (HPLC) fractionation and ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) combined with an AHR-responsive cell-based luciferase gene reporter assay were used to isolate and characterize benzothiazole (BT) derivatives and 6-methylisoquinoline (6-MIQ) as AHR modulators from WB mushrooms. The study showed dose-dependent changes of AHR transformation determined by the cell-based luciferase gene reporter assay and transcription of CYP1A1 in human Caco-2 cells by BT derivatives and 6-MIQ. These findings suggested that WB mushroom contains new classes of natural AHR modulators and demonstrated HPLC fractionation and UHPLC-MS/MS combined with a cell-based luciferase gene reporter assay as a useful approach for isolation and characterization of the previously unidentifed AHR modulators from natural products.


Assuntos
Agaricus/química , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Receptores de Hidrocarboneto Arílico/genética , Animais , Benzotiazóis/química , Benzotiazóis/isolamento & purificação , Benzotiazóis/farmacologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Genes Reporter , Humanos , Isoquinolinas/química , Isoquinolinas/isolamento & purificação , Isoquinolinas/farmacologia , Ligantes , Camundongos , Extratos Vegetais/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Espectrometria de Massas em Tandem , Ativação Transcricional/efeitos dos fármacos , Verduras/química
20.
Chem Biodivers ; 16(8): e1900299, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31287220

RESUMO

The biotransformation of huperzine B (hupB), one of the characteristic bioactive constituents of the medicinal plant Huperzia serrata, by a fungal endophyte of the host plant was studied. One new compound, 8α,15α-epoxyhuperzine B (1), along with two known oxygenated hupB analogs, 16-hydroxyhuperzine B (2) and carinatumin B (3), was isolated and identified. The structures of all the isolates were deduced by spectroscopic methods including NMR, MS, IR, and UV spectra. The known compounds 2 and 3 were obtained from a microbial source for the first time. To the best of our knowledge, it is the first report on the microbial transformation of hupB and would facilitate further structural modification of hupB by chemo-enzymatic method. In the LPS-induced neuro-inflammation injury assay, 8α,15α-epoxyhuperzine B (1) exhibited moderate neuroprotective activity by increasing the viability of U251 cell lines with an EC50 of 40.1 nm.


Assuntos
Alcaloides/química , Huperzia/química , Alcaloides/metabolismo , Alcaloides/farmacologia , Biotransformação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Huperzia/metabolismo , Lipopolissacarídeos/toxicidade , Conformação Molecular , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Plantas Medicinais/química , Plantas Medicinais/metabolismo , Substâncias Protetoras/química , Substâncias Protetoras/farmacologia
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