Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 434.221
Filtrar
1.
World J Microbiol Biotechnol ; 38(1): 18, 2022 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-34977979

RESUMO

Lantibiotics are a promising class of natural antimicrobial peptides. Lichenicidin is a two-peptide lantibiotic in which two mature peptides act synergistically to exhibit full bioactivity. Considering the two-peptide lantibiotics described so far, only cytolysin has been deeply characterized in terms of toxicity towards eukaryotic cells and it was found to be hemolytic and cytotoxic. This work aimed to improve the production of lichenicidin in vivo and characterize its antibacterial activity and toxicity against human cells. Peptides were purified and minimal inhibitory concentration (MIC) was determined against several strains; a time-kill assay was performed with Staphylococcus aureus. The hemolytic effect of lichenicidin was evaluated on blood samples from healthy donors and its toxicity towards human fibroblasts. The quantity of purified peptides was 1 mg/l Bliα and 0.4 mg/l Bliß. MIC for methicillin-sensitive and resistant S. aureus (MSSA and MRSA) strains were 16-32 µg/ml and 64-128 µg/ml, respectively. At the MIC, lichenicidin took less than 3 h to eliminate MSSA, indicating a strong bactericidal effect. It induces cell lysis at the highest concentration, an effect that might be potentiated by Bliß. Lichenicidin was not cytotoxic to human erythrocytes and fibroblasts. In this work, we evaluated the therapeutic potential of lichenicidin as a possible antimicrobial alternative.


Assuntos
Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Bacteriocinas/farmacologia , Fibroblastos/efeitos dos fármacos , Peptídeos/farmacologia , Sequência de Aminoácidos , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Bacteriocinas/química , Bacteriocinas/isolamento & purificação , Linhagem Celular , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Hemólise , Humanos , Testes de Sensibilidade Microbiana
2.
EMBO J ; 41(1): e108843, 2022 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-34981518

RESUMO

Primary cilia are antenna-like organelles required for signalling transduction. How cilia structure is mechanistically maintained at steady-state to promote signalling is largely unknown. Here, we define that mammalian primary cilia axonemes are formed by proximal segment (PS) and distal segment (DS) delineated by tubulin polyglutamylation-rich and -poor regions, respectively. The analysis of proximal/distal segmentation indicated that perturbations leading to cilia over-elongation influenced PS or DS length with a different impact on cilia behaviour. We identified septins as novel repressors of DS growth. We show that septins control the localisation of MKS3 and CEP290 required for a functional transition zone (TZ), and the cilia tip accumulation of the microtubule-capping kinesin KIF7, a cilia-growth inhibitor. Live-cell imaging and analysis of sonic-hedgehog (SHH) signalling activation established that DS over-extension increased cilia ectocytosis events and decreased SHH activation. Our data underlines the importance of understanding cilia segmentation for length control and cilia-dependent signalling.


Assuntos
Cílios/metabolismo , Septinas/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Microtúbulos/metabolismo , Epitélio Pigmentado da Retina/citologia , Transdução de Sinais
3.
In Vivo ; 36(1): 94-102, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34972704

RESUMO

BACKGROUND/AIM: The promoter region of the telomerase reverse transcriptase (TERT) gene is a regulatory element capable of affecting TERT expression, telomerase activity, and telomerase length. Mutations within the TERT promoter region are the most common mutations in many cancers. In this study, we characterized the TERT promoter mutation status in hepatobiliary, pancreatic, and gastrointestinal cancer cell lines. MATERIALS AND METHODS: TERT promoter mutation status was assessed by digital PCR in 12 liver cancer, 5 cholangiocarcinoma (CCA), 12 pancreatic cancer, 17 gastrointestinal cancer, and 3 healthy control cell lines. RESULTS: The C228T promoter mutation was detected in 9 liver cancer lines, and the C250T TERT mutation was detected in 1 oesophageal squamous cell carcinoma line. CONCLUSION: The C228T promoter mutation is specific to liver cancer cell lines among various gastrointestinal cancer cell lines. These data will contribute to future research on the tumorigenic mechanisms and clinical use of digital PCR to detect mutations.


Assuntos
Neoplasias Gastrointestinais , Neoplasias Hepáticas , Telomerase , Linhagem Celular , Neoplasias Gastrointestinais/genética , Humanos , Neoplasias Hepáticas/genética , Mutação , Regiões Promotoras Genéticas , Telomerase/genética , Telomerase/metabolismo
4.
Nihon Yakurigaku Zasshi ; 157(1): 76-84, 2022.
Artigo em Japonês | MEDLINE | ID: mdl-34980816

RESUMO

Follitropin Delta (Rekovellle Subcutaneous Injection 12 µg/ 36 µg/72 µg Pen) is a recombinant human follicle-stimulating hormone (rFSH) developed by Ferring Pharmaceuticals Co., Ltd. Because human follicle-stimulating hormone (FSH) gene is incorporated into a human-derived cell line (human embryonic retinoblastoma: PER.C6), the Follitropin Delta is produced with having α2.3 and α2.6 linked sialic acid sugar chain which is similar to natural human FSH. Containing these two types of sialic acids linkage, similar blood dynamics with natural FSH can be expected due to the reduction of hepatic clearance. Furthermore, an individual dose algorithm defined by patient blood anti-Müllerian hormone (AMH) level and body weight can be expected to obtain optimal follicle development and reduce the safety risk. In the phase II studies, efficacy and safety of Follitropin Delta are confirmed in a dose-dependent manner, and it is confirmed the individualized dose algorism for non-Japanese is also applicable for Japanese women by the population pharmacokinetic/pharmacodynamic analysis. In the phase III studies the non-inferiority of Follitropin Delta to Follitropin Alfa or Beta is confirmed in ongoing pregnancy rate and the number of oocytes retrievable. In addition, the number of subjects who developed total ovarian hyperstimulation syndrome and/or who underwent prophylactic intervention in the Follitropin Delta was significantly lower than comparators. In conclusion, the clinical benefits of individualized doses of Follitropin Delta were confirmed in infertile women undergoing controlled ovarian stimulation (COS) in assisted reproductive technology (ART), and we propose that Follitropin Delta may provide new options to patients and real clinical settings.


Assuntos
Hormônio Foliculoestimulante Humano , Infertilidade Feminina , Linhagem Celular , DNA Recombinante , Feminino , Humanos , Indução da Ovulação , Gravidez , Proteínas Recombinantes , Tecnologia , Resultado do Tratamento
5.
BMC Genomics ; 23(1): 5, 2022 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-34983375

RESUMO

BACKGROUND: Aspergillus flavus, one of the causative agents of human fungal keratitis, can be phagocytosed by human corneal epithelial (HCE) cells and the conidia containing phagosomes mature into phagolysosomes. But the immunological responses of human corneal epithelial cells interacting with A. flavus are not clear. In this study, we report the expression of immune response related genes of HCE cells exposed to A. flavus spores using targeted transcriptomics. METHODS: Human corneal epithelial cell line and primary cultures were grown in a six-well plate and used for coculture experiments. Internalization of the conidia was confirmed by immunofluorescence microscopy of the colocalized endosomal markers CD71 and LAMP1. Total RNA was isolated, and the quantity and quality of the isolated RNA were assessed using Qubit and Bioanalyzer. NanoString nCounter platform was used for the analysis of mRNA abundance using the Human Immunology panel. R-package and nSolver software were used for data analysis. KEGG and FunRich 3.1.3 tools were used to analyze the differentially expressed genes. RESULTS: Different morphotypes of conidia were observed after 6 h of coculture with human corneal epithelial cells and found to be internalized by epithelial cells. NanoString profiling showed more than 20 differentially expressed genes in immortalized human corneal epithelial cell line and more than ten differentially expressed genes in primary corneal epithelial cells. Distinct set of genes were altered in their expression in cell line and primary corneal epithelial cells. KEGG pathway analysis revealed that genes associated with TNF signaling, NF-KB signaling, and Th17 signaling were up-regulated, and genes associated with chemokine signaling and B cell receptor signaling were down regulated. FunRich pathway analysis showed that pathways such as CDC42 signaling, PI3K signaling, and Arf6 trafficking events were activated by the clinical isolates CI1123 and CI1698 in both type of cells. CONCLUSIONS: Combining the transcript analysis data from cell lines and primary cultures, we showed the up regulation of immune defense genes in A. flavus infected cells. At the same time, chemokine signaling and B cell signaling pathways are downregulated. The variability in the expression levels in the immortalized cell line and the primary cultures is likely due to the variable epigenetic reprogramming in the immortalized cells and primary cultures in the absence of any changes in the genome. It highlights the importance of using both cell types in host-pathogen interaction studies.


Assuntos
Aspergillus flavus , Células Epiteliais/imunologia , Regulação da Expressão Gênica/imunologia , Aspergillus flavus/genética , Linhagem Celular , Quimiocinas/imunologia , Córnea/citologia , Córnea/microbiologia , Células Epiteliais/microbiologia , Humanos , Imunidade , Transdução de Sinais , Esporos Fúngicos
6.
In Vivo ; 36(1): 180-188, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34972713

RESUMO

BACKGROUND/AIM: The role of irisin, the extracellular part of fibronectin type III domain containing 5 (FNDC5), in colorectal cancer (CRC) is unclear. The aim of this study was to investigate immunohistochemical (IHC) expression level of irisin and correlations with clinicopathological factors in patients with CRC. MATERIALS AND METHODS: We collected 222 archived CRC samples and 26 control samples from autopsies conducted at the Department of Forensic Medicine. They were used to perform IHC reactions detecting irisin, Ki-67, minichromosome maintenance protein complex component 3 (MCM3), and urine diphosphate-galactose ceramide galactosyltransferase (UGT3) expression. The correlations with Ki-67, MCM3, and UGT3 were analyzed. Irisin expression was also evaluated in cancer cell lines by immunofluorescence reaction and western blot. RESULTS: Irisin expression was higher in cancer cells compared to the control tissues (p<0.0001). Irisin expression was significantly higher in stage I than in stage III (p=0.013) and IV CRC (p=0.05). CONCLUSION: The correlation between higher expression of irisin and cancer stages indicates its potential usefulness as a marker in CRC.


Assuntos
Neoplasias Colorretais , Fibronectinas , Linhagem Celular , Neoplasias Colorretais/genética , Fibronectinas/genética , Humanos , Fatores de Transcrição
7.
J Toxicol Environ Health A ; 85(1): 29-42, 2022 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-34445936

RESUMO

The aim of this study was to investigate changes in the intracellular metabolism resulting from cisplatin (CDDP)-induced nephrotoxicity in normal kidney tubular epithelial NRK-52E cells. Cytotoxicity, cell cycle analysis, and apoptotic cell death were all evaluated in NRK-52E cells treated with CDDP. Subsequently, proton nuclear magnetic resonance (1H-NMR) spectroscopy was used to investigate cellular metabolic profiles. CDDP-induced nephrotoxicity was determined in vivo model. Cytotoxicity in the NRK-52E cells significantly rose following treatment with CDDP and these increases were found to be concentration-dependent. Both p53 and Bax protein expression was increased in CDDP-treated NRK-52E cells, correlating with enhanced cellular apoptosis. In addition, a number of metabolites were altered in both media and cell lysates in these cells. In cell lysates, citrate, creatinine, and acetate levels were dramatically reduced following treatment with 20 µM CDDP concentrations, while glutamate level was elevated. Lactate and acetate levels were significantly increased in culture media but citrate concentrations were reduced following high 20 µM CDDP concentrations incubation. In addition, excretion of clusterin, calbindin, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), selenium binding protein 1 (SBP1), and pyruvate kinase M2 (PKM2) into the culture media was significantly increased in CDDP-treated cells while expression of acetyl CoA synthetase 1 (AceCS1) was markedly reduced in these cells. These findings suggest that acetate-dependent metabolic pathway may be a reliable and useful biomarker for detecting CDDP-induced nephrotoxicity. Taken together, data demonstrate that the discovery of novel biomarkers by metabolite profiling in target cells may contribute to the detection of nephrotoxicity and new drug development.


Assuntos
Injúria Renal Aguda/metabolismo , Cisplatino/toxicidade , Acetatos/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/patologia , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Metabolômica , Modelos Biológicos , Ratos
8.
FASEB J ; 36(1): e22089, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34888938

RESUMO

Oxidative stress is emerging as a contributing factor to the homeostasis in cystic diseases. However, the role antioxidant enzymes play in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD) remains elusive. Peroxiredoxin 5 (Prdx5) is an antioxidant enzyme that catalyzes the reduction of H2 O2 and alkyl hydroperoxide and plays an important role in different biological processes. In this study, we show that Prdx5 is downregulated in a PKD mutant mouse model and ADPKD patient kidneys. Knockdown of Prdx5 resulted in the formation of cysts in a three-dimensional mouse inner medullar collecting duct (IMCD) cell Matrigel culture system. The mechanisms of Prdx5 deficiency mediated cyst growth include: (1) induction of oxidative stress as indicated by increased mRNA expression of heme oxygenase-1, an oxidant stress marker; (2) activation of Erk, S6 and mTORC1, which contribute to cystic renal epithelial cell proliferation and cyst growth; (3) abnormal centrosome amplification and multipolar spindle formation which result in genome instability; (4) upregulation of Polo-like kinase 1 (Plk1) and Aurora kinase A, important mitotic kinases involved in cell proliferation and ciliogenesis; (5) impaired formation of primary cilia in mouse IMCD3 and retinal pigment epithelial cells, which could be rescued by inhibiting Plk1 activity; and (6) restraining the effect of Wnt3a and Wnt5a ligands on primary cilia in mouse IMCD3 cells, while regulating the activity of the canonical and non-canonical Wnt signaling in a separate cilia independent mechanism, respectively. Importantly, we found that targeting Plk1 with its inhibitor, volasertib, delayed cyst growth in Pkd1 conditional knockout mouse kidneys. Together, these findings indicate that Prdx5 is an important antioxidant that regulates cyst growth via diverse mechanisms, in particular, the Prdx5-Plk1 axis, and that induction and activation of Prdx5, alone or together with inhibition of Plk1, represent a promising strategy for combatting ADPKD.


Assuntos
Antioxidantes/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cílios/enzimologia , Rim/enzimologia , Peroxirredoxinas/metabolismo , Rim Policístico Autossômico Dominante/enzimologia , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular , Cílios/genética , Estabilidade Enzimática , Humanos , Camundongos , Camundongos Knockout , Estresse Oxidativo , Peroxirredoxinas/genética , Rim Policístico Autossômico Dominante/genética , Proteínas Proto-Oncogênicas/genética
9.
Virology ; 565: 13-21, 2022 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-34626907

RESUMO

Eastern equine encephalitis virus (EEEV), western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV) can cause fatal encephalitis in humans and equids. Some MAbs to the E1 glycoprotein are known to be cross-reactive, weakly neutralizing in vitro but can protect from disease in animal models. We investigated the mechanism of neutralization of VEEV infection by the broadly cross-reactive E1-specific MAb 1A4B-6. 1A4B-6 protected 3-week-old Swiss Webster mice prophylactically from lethal VEEV challenge. Likewise, 1A4B-6 inhibited virus growth in vitro at a pre-attachment step after virions were incubated at 37 °C and inhibited virus-mediated cell fusion. Amino acid residue N100 in the fusion loop of E1 protein was identified as critical for binding. The potential to elicit broadly cross-reactive MAbs with limited virus neutralizing activity in vitro but that can inhibit virus entry and protect animals from infection merits further exploration for vaccine and therapeutic developmental research.


Assuntos
Anticorpos Antivirais/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Vírus da Encefalite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/virologia , Proteínas do Envelope Viral/imunologia , Replicação Viral/efeitos dos fármacos , Alphavirus/imunologia , Infecções por Alphavirus/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Chlorocebus aethiops , Reações Cruzadas , Encefalomielite Equina Venezuelana/terapia , Glicoproteínas/imunologia , Imunoterapia , Camundongos , Ligação Proteica , Células Vero , Proteínas do Envelope Viral/metabolismo , Vírion/imunologia , Vírion/metabolismo
10.
Virology ; 565: 82-95, 2022 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-34768112

RESUMO

Adenovirus (Ad) early region 4 (E4) mutants activate cellular DNA damage responses (DDRs) that include non-homologous end joining (NHEJ) pathways mediated by the DNA repair kinase DNA-PK and its associated factors Ku70/Ku86. NHEJ results in concatenation of the viral linear double-stranded DNA genome and inhibits a productive infection. E4 proteins normally prevent activation of cellular DDRs in wild-type Ad type 5 (Ad5) infections, thereby promoting efficient viral growth. The purpose of this study was to evaluate the factors that govern DNA-PK activation during adenovirus infection. Our data indicate that viral DNA replication promotes DNA-PK activation, which is required for genome concatenation by NHEJ. Although the Mre11/Rad50/Nbs1 (MRN) DDR sensor complex is not required for DNA-PK activation, Mre11 is important for recruitment of the NHEJ factor Ku86 to viral replication centers. Our study addresses the interplay between the DNA-PK and MRN complexes during viral genome concatenation by NHEJ.


Assuntos
Infecções por Adenoviridae/metabolismo , Infecções por Adenoviridae/virologia , Adenoviridae/metabolismo , Reparo do DNA por Junção de Extremidades , Replicação do DNA , DNA Viral/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Hidrolases Anidrido Ácido/metabolismo , Proteínas E4 de Adenovirus/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Chlorocebus aethiops , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Genoma Viral , Células HEK293 , Células HeLa , Humanos , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação
11.
Virology ; 565: 106-116, 2022 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-34773868

RESUMO

Influenza NS1 is a promising anti-influenza target, considering its conserved and druggable structure, and key function in influenza replication and pathogenesis. Notwithstanding, target identification and validation, strengthened by experimental data, are lacking. Here, we further explored our previously designed structure-based antiviral rationale directed to highly conserved druggable NS1 regions across a broad spectrum of influenza A viruses. We aimed to identify NS1-mutated viruses exhibiting a reduced growth phenotype and/or an altered cell apoptosis profile. We found that NS1 mutations Y171A, K175A (consensus druggable pocket 1), W102A (consensus druggable pocket 3), Q121A and G184P (multiple consensus druggable pockets) - located at hot spots amenable for pharmacological modulation - significantly impaired A(H1N1)pdm09 virus replication, in vitro. This is the first time that NS1-K175A, -W102A, and -Q121A mutations are characterized. Our map-and-mutate strategy provides the basis to establish the NS1 as a promising target using a rationale with a higher resilience to resistance development.


Assuntos
Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Influenza Humana/virologia , Infecções por Orthomyxoviridae/virologia , Proteínas não Estruturais Virais/genética , Replicação Viral , Substituição de Aminoácidos , Animais , Apoptose , Linhagem Celular , Cães , Descoberta de Drogas , Células HEK293 , Interações entre Hospedeiro e Microrganismos , Humanos , Influenza Humana/metabolismo , Células Madin Darby de Rim Canino , Mutação , Infecções por Orthomyxoviridae/metabolismo
12.
J Sci Food Agric ; 102(2): 764-773, 2022 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-34227118

RESUMO

BACKGROUND: Oleanolic acid (OA) is a pentacyclic triterpenoid compound that is present at high levels in olive oil and has several promising pharmacological effects, such as liver protection and anti-inflammatory, antioxidant, and anticancer effects. The purpose of the present study was to assess whether OA treatment affects gut health compared to a control condition, including gut microbiota and intestinal epithelial immunity. RESULTS: Illumina MiSeq sequencing (16S rRNA gene) was used to investigate the effect of OA on the microbial community of the intestinal tract, while Illumina HiSeq (RNA-seq) technology was used to investigate the regulatory effect of OA on gene expression in intestinal epithelial cells, which allowed for a comprehensive analysis of the effects of OA on intestinal health. The results showed that the consumption of OA initially controlled weight gain in mice and altered the composition of the gut microbiota. At the phylum level, OA significantly increased the relative abundances of cecum Firmicutes but decreased the abundance of Actinobacteria, and at the genus level it increased the relative abundance of potentially beneficial bacteria such as Oscillibacter and Ruminiclostridium 9. Oleanolic acid treatment also altered the expression of 12 genes involved in the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways of complement and coagulation cascades, hematopoietic cell lineage, and leukocyte transendothelial migration in intestinal epithelial cells to improve gut immunity. CONCLUSION: Intake of OA can contribute beneficial effects by optimizing gut microbiota and altering the immune function of intestinal epithelial cells, potentially to improve intestinal health status. © 2021 Society of Chemical Industry.


Assuntos
Células Epiteliais/imunologia , Microbioma Gastrointestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Ácido Oleanólico/farmacologia , Animais , Bactérias/classificação , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/isolamento & purificação , Linhagem Celular , DNA Bacteriano/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA Ribossômico 16S/genética
13.
Anal Chim Acta ; 1189: 339224, 2022 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-34815036

RESUMO

Psoralen ultraviolet A (PUVA) therapy has thrived as a promising treatment for psoriasis. However, overdose of PUVA treatment will cause side-effects, such as melanoma formation. And these side-effects are often ignored during PUVA therapy. Hence, in situ monitoring therapeutic response of PUVA therapy is important to minimize side-effects. Aberrant expression of tyrosinase (TYR) has been proved to be associated with melanoma, indicating that TYR is a potential target for evaluation of PUVA therapy. Herein, we reported a strategy for in situ monitoring TYR activity during PUVA therapy by using a cell-array chip-based SERS platform. The cell-array chip was used to simulate cell survival environment for cell culture. Capture of single cells and living cell analysis were realized in the isolated microchambers. An enzyme-induced core-shell self-assembly substrate was used to evaluate TYR activity in living cells during PUVA therapy. The gold nanoparticle modified with a SERS reporter, 4-mercaptobenzonitrile (4-MBN), was used as the core. In the presence of oxygen and TYR, hydroxylation of l-tyrosine occurred, leading to the reduction of silver ion on the surface of gold cores. The growth of silver shells was accompanied by the increased SERS intensity of the reporter, which is related directly to TYR activity. The detection limit for TYR activity is 0.45 U/mL. Upregulation of TYR activity was successfully monitored after PUVA therapy. Notably, real-time and in situ information of therapeutic response can be obtained through monitoring PUVA therapy by using a cell-array chip-based SERS platform, which has great potential to guide the clinical application of PUVA therapy.


Assuntos
Ouro , Nanopartículas Metálicas , Terapia PUVA , Animais , Linhagem Celular , Camundongos , Prata , Análise Espectral Raman
14.
Exp Neurol ; 347: 113902, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34699789

RESUMO

Immunometabolic changes have been shown to be a key factor in determining the immune cell response in disease models. The immunometabolite, itaconate, is produced by aconitate decarboxylase 1 (Acod1) and has been shown to inhibit inflammatory signaling in macrophages. In this study, we explore the role of Acod1 and itaconate in cerebral ischemia/reperfusion injury. We assessed the effect of global Acod1 knockout (Acod1KO, loss of endogenous itaconate) in a transient ischemia/reperfusion occlusion stroke model. Mice received a transient 90-min middle cerebral artery occlusion followed with 24-h of reperfusion. Stroke lesion volume was measured by MRI analysis and brain tissues were collected for mRNA gene expression analysis. Acod1KO mice showed significant increases in lesion volume compared to control mice, however no differences in pro-inflammatory mRNA levels were observed. Cell specific knockout of Acod1 in myeloid cells (LysM-Cre), microglia cells (CX3CR1, Cre-ERT2) and Endothelial cells (Cdh5(PAC), Cre-ERT2) did not reproduce lesion volume changes seen in global Acod1KO, indicating that circulating myeloid cells, resident microglia and endothelial cell populations are not the primary contributors to the observed phenotype. These effects however do not appear to be driven by changes in inflammatory gene regulation. These data suggests that endogenous Acod1 is protective in cerebral ischemia/reperfusion injury.


Assuntos
Isquemia Encefálica/enzimologia , Isquemia Encefálica/prevenção & controle , Carboxiliases/deficiência , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Isquemia Encefálica/genética , Carboxiliases/genética , Linhagem Celular , Fluxometria por Laser-Doppler/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão/genética
15.
J Environ Sci (China) ; 111: 429-441, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34949371

RESUMO

Airborne fine particulate matter (PM2.5) is known to cause respiratory inflammation such as chronic obstructive pulmonary disease and lung fibrosis. NLRP3 inflammasome activation has been implicated in these diseases; however, due to the complexity in PM2.5 compositions, it is difficult to differentiate the roles of the components in triggering this pathway. We collected eight real-life PM2.5 samples for a comparative analysis of their effects on NLRP3 inflammasome activation and lung fibrosis. In vitro assays showed that although the PM2.5 particles did not induce significant cytotoxicity at the dose range of 12.5 to 100 µg/mL, they induced potent TNF-α and IL-1ß production in PMA differentiated THP-1 human macrophages and TGF-ß1 production in BEAS-2B human bronchial epithelial cells. At the dose of 100 µg/mL, PM2.5 induced NLRP3 inflammasome activation by inducing lysosomal damage and cathepsin B release, leading to IL-1ß production. This was confirmed by using NLRP3- and ASC-deficient cells as well as a cathepsin B inhibitor, ca-074 ME. Administration of PM2.5 via oropharyngeal aspiration at 2 mg/kg induced significant TGF-ß1 production in the bronchoalveolar lavage fluid and collagen deposition in the lung at 21 days post-exposure, suggesting PM2.5 has the potential to induce pulmonary fibrosis. The ranking of in vitro IL-1ß production correlates well with the in vivo total cell count, TGF-ß1 production, and collagen deposition. In summary, we demonstrate that the PM2.5 is capable of inducing NLRP3 inflammasome activation, which triggers a series of cellular responses in the lung to induce fibrosis.


Assuntos
Poluição do Ar , Proteína 3 que Contém Domínio de Pirina da Família NLR , Material Particulado , Poluição do Ar/efeitos adversos , Linhagem Celular , Fibrose , Humanos , Inflamassomos , Interleucina-1beta , Pulmão , Material Particulado/toxicidade , Células THP-1
16.
Mol Carcinog ; 61(1): 99-110, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34727382

RESUMO

E-cadherin plays a central role in the stability of epithelial tissues by facilitating cell-cell adhesion. Loss of E-cadherin expression is a hallmark of epithelial-mesenchymal transition (EMT), a major event in the pathogenesis of several lung diseases. Our earlier studies showed that nickel, a ubiquitous environmental toxicant, induced EMT by persistently downregulating E-cadherin expression in human lung epithelial cells and that the EMT remained irreversible postexposure. However, the molecular basis of persistent E-cadherin downregulation by nickel exposure is not understood. Here, our studies show that the binding of transcription factor Sp1 to the promoter of E-cadherin encoding gene, CDH1, is essential for its expression. Nickel exposure caused a loss of Sp1 binding at the CDH1 promoter, resulting in its downregulation and EMT induction. Loss of Sp1 binding at the CDH1 promoter was associated with an increase in the binding of ZEB1 adjacent to the Sp1 binding site. ZEB1, an EMT master regulator persistently upregulated by nickel exposure, is a negative regulator of CDH1. CRISPR-Cas9-mediated knockout of ZEB1 restored Sp1 binding at the CDH1 promoter. Furthermore, ZEB1 knockout rescued E-cadherin expression and re-established the epithelial phenotype. Since EMT is associated with a number of nickel-exposure-associated chronic inflammatory lung diseases including asthma, fibrosis and cancer and metastasis, our findings provide new insights into the mechanisms associated with nickel pathogenesis.


Assuntos
Antígenos CD/genética , Caderinas/genética , Pulmão/citologia , Níquel/toxicidade , Fator de Transcrição Sp1/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Linhagem Celular , Regulação para Baixo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Técnicas de Inativação de Genes , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Células MCF-7 , Regiões Promotoras Genéticas
17.
Methods Mol Biol ; 2343: 259-270, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34473329

RESUMO

Type-2 diabetes, characterized by hyperglycemia causing various symptoms of metabolic disorders in the heart, kidneys, and brain, has many underlying molecular mechanisms leading to functional insufficiency of these organs. We describe protocols wherein we have optimized conditions for maintenance of hyperglycemic H9c2 cell lines and design to assess the effect of a water-soluble vitamin, Trolox, on the apoptotic pathway. Primarily, the design provides researchers to analyze apoptosis by flow cytometry.


Assuntos
Apoptose , Citometria de Fluxo , Hiperglicemia , Miócitos Cardíacos , Linhagem Celular , Humanos , Miocárdio/citologia
18.
Gene ; 808: 145968, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34530090

RESUMO

Resveratrol (Res) is a polyphenol with a variety of biological activities. However, whether Res can prevent myocardial ischemia-reperfusion (I/R) injury is not yet known. This study aimed to investigate the protective effect of Res on myocardial I/R injury and to explore its potential mechanism. H9c2 cells were used for the in vitro experiments and oxygen-glucose deprivation/reoxygenation (OGD/R) model was established. Rats were ligated and perfused by the left anterior descending branch with or without Res (50 mg/kg·bw) for 14 days.The higher level of oxidative stress and Fe2+ content was observed in OGD/R-induced H9c2 cells than that of normal cells. OGD/R-induced H9c2 cells showed increased ferroptosis, mainly by reducing the expression of glutathione peroxidase 4 (GPX4) and ferritin heavy chain 1 (FTH1), but enhancing the expression of transferrin receptor 1 (TfR1). Both in vivo and in vitro experiments indicated that Res reduced the level of oxidative stress and Fe2 + content. In addition, Res inhibited ferroptosis, decreased TfR1 expression, and increased the expressions of FTH1 and GPX4 in OGD/R-induced H9c2 cells and I/R rats. Moreover, we found that Res inhibited ferroptosis by the regulation of ubiquity specific peptidase 19 (USP19)-Beclin1 autophagy. Res protects against myocardial I/R injury via reducing oxidative stress and attenuating ferroptosis. Res could be a potential agent to the prevention of myocardial I/R injury.


Assuntos
Ferroptose/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Resveratrol/farmacologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ferroptose/fisiologia , Glucose/metabolismo , Coração/efeitos dos fármacos , Masculino , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Oxigênio/metabolismo , Substâncias Protetoras/farmacologia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Resveratrol/metabolismo , Transdução de Sinais/efeitos dos fármacos
19.
Methods Mol Biol ; 2383: 529-545, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34766311

RESUMO

The efficacy of transfection reagents and nanoparticles is often assessed by measuring levels of expressed reporter protein. Fluorescence and luminescence based assays provide sensitive, quantifiable and repeatable approaches. The genes expressing reporter protein can be integrated into the cells to create stable reporter cell lines or can be expressed from a transfected plasmid. Green fluorescent protein, luciferase, and secreted alkaline phosphatase are well-established reporters with versatile applications. Monitoring changes in live cells during and after transfection offer opportunities to reveal related mechanisms, efficacy, and bottlenecks of transfection.In this chapter, we describe the experimental setup and considerations for in vitro screening of delivery vectors. This can further be extended to measurements in reporter cell lines.


Assuntos
Técnicas de Cultura de Células , Mamíferos , Animais , Linhagem Celular , Genes Reporter , Plasmídeos/genética , Transfecção
20.
J Med Virol ; 94(1): 399-403, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34460119

RESUMO

Vaccination generates a neutralizing immune response against SARS-CoV-2. The genomic surveillance is showing the emergence of variants with mutations in spike, the main target of neutralizing antibodies. To understand the impact of these variants, we report the neutralization potency against alpha, gamma, and D614G SARS-CoV-2 variants in 44 individuals that received two doses of CoronaVac vaccine, an inactivated SARS-CoV-2 vaccine. Plasma samples collected at 60 days after the second dose of CoronaVac were analyzed by the reduction of cytopathic effect in Vero E6 cells with the three infectious variants of SARS-CoV-2. Plasma showed lower neutralization with alpha (geometric mean titer [GMT] = 18.5) and gamma (GMT = 10.0) variants than with D614G (GMT = 75.1) variant. Efficient neutralization against the alpha and gamma variants was not detected in 31.8% and 59.1% of plasma, respectively. These findings suggest the alpha and gamma variants could escape from neutralization by antibodies elicited by vaccination. Robust genomic and biological surveillance of viral variants could help to develop effective strategies for the control of SARS-CoV-2.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Adulto , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Linhagem Celular , Chlorocebus aethiops , Feminino , Humanos , Evasão da Resposta Imune/imunologia , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinação , Vacinas de Produtos Inativados/imunologia , Células Vero , Adulto Jovem
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...