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1.
Medicine (Baltimore) ; 100(24): e26271, 2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34128858

RESUMO

BACKGROUND: Thymic epithelial tumors (TETs), originating from the thymic epithelial cells, are the most common primary neoplasms of the anterior mediastinum. Emerging evidence demonstrated that the competing endogenous RNAs (ceRNAs) exerted a crucial effect on tumor development. Hence, it is urgent to understand the regulatory mechanism of ceRNAs in TETs and its impact on tumor prognosis. METHODS: TETs datasets were harvested from the UCSC Xena as the training cohort, followed by differentially expressed mRNAs (DEmRNAs), lncRNAs (DElncRNAs), and miRNAs (DEmiRNAs) at different pathologic type (A, AB, B, and TC) identified via DESeq2 package. clusterProfiler package was utilized to carry out gene ontology and Kyoto encyclopedia of genes and genomes functional analysis on the DEmRNAs. Subsequently, the lncRNA-miRNA-mRNA regulatory network was constructed to screen the key DEmRNAs. After the key DEmRNAs were verified in the external cohort from Gene Expression Omnibus database, their associated-ceRNAs modules were used to perform the K-M and Cox regression analysis to build a prognostic significance for TETs. Lastly, the feasibility of the prognostic significance was validated by receiver operating characteristic (ROC) curves and the area under the curve. RESULTS: Finally, a total of 463 DEmRNAs, 87 DElncRNAs, and 20 DEmiRNAs were obtained from the intersection of differentially expressed genes in different pathological types of TETs. Functional enrichment analysis showed that the DEmRNAs were closely related to cell proliferation and tumor development. After lncRNA-miRNA-mRNA network construction and external cohort validation, a total of 4 DEmRNAs DOCK11, MCAM, MYO10, and WASF3 were identified and their associated-ceRNA modules were significantly associated with prognosis, which contained 3 lncRNAs (lncRNA LINC00665, lncRNA NR2F1-AS1, and lncRNA RP11-285A1.1), 4 mRNAs (DOCK11, MCAM, MYO10, and WASF3), and 4 miRNAs (hsa-mir-143, hsa-mir-141, hsa-mir-140, and hsa-mir-3199). Meanwhile, ROC curves verified the accuracy of prediction ability of the screened ceRNA modules for prognosis of TETs. CONCLUSION: Our study revealed that ceRNAs modules might exert a crucial role in the progression of TETs. The mRNA associated-ceRNA modules could effectively predict the prognosis of TETs, which might be the potential prognostic and therapeutic markers for TETs patients.


Assuntos
Biologia Computacional/estatística & dados numéricos , MicroRNAs/análise , Neoplasias Epiteliais e Glandulares/genética , RNA Longo não Codificante/análise , RNA Mensageiro/análise , Neoplasias do Timo/genética , Biomarcadores Tumorais/genética , Estudos de Coortes , Biologia Computacional/métodos , Conjuntos de Dados como Assunto , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/genética , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Valor Preditivo dos Testes , Prognóstico , Modelos de Riscos Proporcionais , Curva ROC
2.
Carbohydr Polym ; 268: 118259, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34127229

RESUMO

Nitrocellulose (NC) membrane can have value-added applications for lateral flow assay (LFA)-based diagnostic tools, which has great potential for the detection of pathogens, such as COVID-19, in different environments. However, poor sensitivity of the NC membrane based LFA limits its further application in many cases. Herein, we developed a facile method for LFA sensitivity enhancement, by incorporating two-sugar barrier into LFAs: one between the conjugation pad and the test line, and the other between the test line and the control line. ORF1ab nucleic acid of COVID-19 was used as the model target to demonstrate the concept on the HF120 membrane. Results show that at optimum conditions, the two sugar barrier LFAs have a detection limit of 0.5 nM, which is compared to that of 2.5 nM for the control LFA, achieving a 5-fold sensitivity increase. This low cost, easy-to-fabricate and easy-to-integrate LFA method may have potential applications in other cellulose paper-based platforms.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Colódio/química , RNA Mensageiro/análise , Açúcares/química , Proteínas Virais/genética , Teste de Ácido Nucleico para COVID-19/instrumentação , DNA/química , Sondas de DNA/química , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Poliproteínas/genética , SARS-CoV-2/química , Sensibilidade e Especificidade
3.
Toxins (Basel) ; 13(6)2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073248

RESUMO

Plant materials can be contaminated with Fusarium mycotoxins and their derivatives, whose toxic effects on humans and animals may remain subclinical. Zearalenone (ZEN), a low-molecular-weight compound, is produced by molds in crop plants as a secondary metabolite. The objective of this study will be to analyze the in vivo correlations between very low monotonic doses of ZEN (5, 10, and 15 µg ZEN/kg body weight-BW for 42 days) and the carryover of this mycotoxin and its selected metabolites from the intestinal contents to the intestinal walls, the mRNA expression of estrogen receptor alfa (ERα) and estrogen receptor beta (ERß) genes, and the mRNA expression of genes modulating selected colon enzymes (CYP1A1 and GSTP1) in the intestinal mucosa of pre-pubertal gilts. An in vivo experiment will be performed on 60 clinically healthy animals with initial BW of 14.5 ± 2 kg. The gilts will be randomly divided into a control group (group C, n = 15) and three experimental groups (group ZEN5, group ZEN10, and group ZEN15; n = 15). Group ZEN5 will be administered per os 5 µg ZEN/kg BW (MABEL), group ZEN10-10 µg ZEN/kg BW (NOAEL), and group ZEN15-15 µg ZEN/kg BW (low LOAEL). In each group, five animals will be euthanized on analytical dates 1 (exposure day 7), 2 (exposure day 21), and 3 (exposure day 42). Samples for in vitro analyses will be collected from an intestinal segment resected from the following regions: the third (horizontal) part of the duodenum, jejunum, ileum, cecum, ascending colon, transverse colon, and descending colon. The experimental material will be collected under special conditions, and it will be transported to specialist laboratories where samples will be obtained for further analyses.


Assuntos
Mucosa Intestinal/efeitos dos fármacos , Receptores de Estrogênio/genética , Zearalenona/toxicidade , Animais , Citocromo P-450 CYP1A1/genética , Feminino , Glutationa Transferase/genética , Mucosa Intestinal/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Suínos
4.
Carbohydr Polym ; 268: 118259, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: covidwho-1242891

RESUMO

Nitrocellulose (NC) membrane can have value-added applications for lateral flow assay (LFA)-based diagnostic tools, which has great potential for the detection of pathogens, such as COVID-19, in different environments. However, poor sensitivity of the NC membrane based LFA limits its further application in many cases. Herein, we developed a facile method for LFA sensitivity enhancement, by incorporating two-sugar barrier into LFAs: one between the conjugation pad and the test line, and the other between the test line and the control line. ORF1ab nucleic acid of COVID-19 was used as the model target to demonstrate the concept on the HF120 membrane. Results show that at optimum conditions, the two sugar barrier LFAs have a detection limit of 0.5 nM, which is compared to that of 2.5 nM for the control LFA, achieving a 5-fold sensitivity increase. This low cost, easy-to-fabricate and easy-to-integrate LFA method may have potential applications in other cellulose paper-based platforms.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Colódio/química , RNA Mensageiro/análise , Açúcares/química , Proteínas Virais/genética , Teste de Ácido Nucleico para COVID-19/instrumentação , DNA/química , Sondas de DNA/química , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Poliproteínas/genética , SARS-CoV-2/química , Sensibilidade e Especificidade
5.
J Am Chem Soc ; 143(14): 5413-5424, 2021 04 14.
Artigo em Inglês | MEDLINE | ID: covidwho-1164792

RESUMO

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.


Assuntos
Fluorescência , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Imagem Molecular , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , COVID-19/tratamento farmacológico , Linhagem Celular Tumoral , Citosina/análogos & derivados , Citosina/análise , Citosina/síntese química , Citosina/química , Corantes Fluorescentes/síntese química , Proteínas de Fluorescência Verde/metabolismo , Histonas/metabolismo , Humanos , Estrutura Molecular , RNA Mensageiro/química , RNA Mensageiro/uso terapêutico , Espectrometria de Fluorescência
6.
Nutrients ; 13(4)2021 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-33920401

RESUMO

Diabetes is a disease with an inflammatory component that courses with an anemic state. Vanadium (V) is an antidiabetic agent that acts by stimulating insulin signaling. Hepcidin blocks the intestinal absorption of iron and the release of iron from its deposits. We aim to investigate the effect of V on hepcidin mRNA expression and its consequences on the hematological parameters in streptozotocin-induced diabetic Wistar rats. Control healthy rats, diabetic rats, and diabetic rats treated with 1 mgV/day were examined for five weeks. The mineral levels were measured in diet and serum samples. Hepcidin expression was quantified in liver samples. Inflammatory and hematological parameters were determined in serum or whole blood samples. The inflammatory status was higher in diabetic than in control rats, whereas the hematological parameters were lower in the diabetic rats than in the control rats. Hepcidin mRNA expression was significantly lower in the V-treated diabetic rats than in control and untreated diabetic rats. The inflammatory status remained at a similar level as the untreated diabetic group. However, the hematological profile improved after the V-treatment, reaching similar levels to those found in the control group. Serum iron level was higher in V-treated than in untreated diabetic rats. We conclude that V reduces gene expression of hepcidin in diabetic rats, improving the anemic state caused by diabetes.


Assuntos
Anemia/tratamento farmacológico , Diabetes Mellitus Experimental/tratamento farmacológico , Hepcidinas/genética , Hipoglicemiantes/administração & dosagem , Vanádio/administração & dosagem , Anemia/sangue , Anemia/diagnóstico , Anemia/etiologia , Animais , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Regulação da Expressão Gênica/efeitos dos fármacos , Hepcidinas/metabolismo , Humanos , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Ferro/sangue , Ferro/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ratos , Estreptozocina/administração & dosagem , Estreptozocina/toxicidade
7.
J Am Chem Soc ; 143(14): 5413-5424, 2021 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-33797236

RESUMO

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.


Assuntos
Fluorescência , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Imagem Molecular , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , COVID-19/tratamento farmacológico , Linhagem Celular Tumoral , Citosina/análogos & derivados , Citosina/análise , Citosina/síntese química , Citosina/química , Corantes Fluorescentes/síntese química , Proteínas de Fluorescência Verde/metabolismo , Histonas/metabolismo , Humanos , Estrutura Molecular , RNA Mensageiro/química , RNA Mensageiro/uso terapêutico , Espectrometria de Fluorescência
8.
Methods Mol Biol ; 2300: 203-237, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33792882

RESUMO

For a long time, artificial RNAs have been developed by in vitro selection methodologies like Systematic Evolution of Ligands by EXponential enrichment (SELEX). Yet, even though this technology is extremely powerful to isolate specific and high-affinity binders, it is less suited for the isolation of RNAs optimized for more complex functions such as fluorescence emission or multiple-turnover catalysis. Whereas such RNAs should ideally be developed by screening approaches, conventional microtiter plate assays become rapidly cost-prohibitive. However, the advent of droplet-based microfluidics recently enabled us to devise microfluidic-assisted In Vitro Compartmentalization (µIVC), a strongly miniaturized and highly parallelized screening technology allowing to functionally screen millions of mutants in a single day while using a very low amount of reagent. Used in combination with high-throughput sequencing, the resulting µIVC-seq pipeline described in this chapter now allows rapid and semiautomated screening to be performed at low cost and in an ultrahigh-throughput regime.


Assuntos
Microfluídica/métodos , RNA Mensageiro/análise , Análise de Sequência de RNA/métodos , Biologia Computacional/métodos , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Miniaturização , Mutação
9.
Methods Mol Biol ; 2284: 135-145, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33835441

RESUMO

RNA-sequencing (RNA-seq) is a powerful technology for transcriptome profiling. While most RNA-seq projects focus on gene-level quantification and analysis, there is growing evidence that most mammalian genes are alternatively spliced to generate different isoforms that can be subsequently translated to protein molecules with diverse or even opposing biological functions. Quantifying the expression levels of these isoforms is key to understanding the genes biological functions in healthy tissues and the progression of diseases. Among open source tools developed for isoform quantification, Salmon, Kallisto, and RSEM are recommended based upon previous systematic evaluation of these tools using both experimental and simulated RNA-seq datasets. However, isoform quantification in practical RNA-seq data analysis needs to deal with many QC issues, such as the abundance of rRNAs in mRNA-seq, the efficiency of globin RNA depletion in whole blood samples, and potential sample swapping. To overcome these practical challenges, QuickIsoSeq was developed for large-scale RNA-seq isoform quantification along with QC. In this chapter, we describe the pipeline and detailed the steps required to deploy and use it to analyze RNA-seq datasets in practice. The QuickIsoSeq package can be downloaded from https://github.com/shanrongzhao/QuickIsoSeq.


Assuntos
Isoformas de Proteínas/genética , RNA-Seq/métodos , RNA/genética , Algoritmos , Animais , Sequência de Bases , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Técnicas Genéticas , Humanos , Especificidade de Órgãos/genética , Isoformas de Proteínas/análise , RNA/análise , RNA/química , RNA Mensageiro/análise , RNA Mensageiro/química , RNA Mensageiro/genética , Software
10.
Methods Mol Biol ; 2284: 519-529, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33835461

RESUMO

N6-Methyladenosine (m6A) is the most prevalent posttranscriptional modification in eukaryotes and plays a pivotal role in various biological processes, such as splicing, RNA degradation, and RNA-protein interaction. Accurately identification of the location of m6A is essential for related downstream studies. In this chapter, we introduce a prediction framework WHISTLE, which enables us to acquire so far the most accurate map of the transcriptome-wide human m6A RNA-methylation sites (with an average AUC: 0.948 and 0.880 under the full transcript or mature messenger RNA models, respectively, when tested on independent datasets). Besides, each individual m6A site was also functionally annotated according to the "guilt-by-association" principle by integrating RNA methylation data, gene expression data and protein-protein interaction data. A web server was constructed for conveniently querying the predicted RNA methylation sites and their putative biological functions. The website supports the query by genes, by GO function, table view, and the download of all the functionally annotated map of predicted map of human m6A epitranscriptome. The WHISTLE web server is freely available at: www.xjtlu.edu.cn/biologicalsciences/whistle and http://whistle-epitranscriptome.com .


Assuntos
Adenosina/análogos & derivados , Processamento Pós-Transcricional do RNA , Análise de Sequência de RNA/métodos , Adenosina/metabolismo , Algoritmos , Sítios de Ligação/genética , Biologia Computacional/métodos , Bases de Dados Genéticas , Conjuntos de Dados como Assunto , Perfilação da Expressão Gênica/métodos , Humanos , Internet , RNA Mensageiro/análise , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Software , Transcriptoma
11.
Methods Mol Biol ; 2284: 531-541, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33835462

RESUMO

RNA-seq using long-read sequencing, such as nanopore and SMRT (Single Molecule, Real-Time) sequencing, enabled the identification of the full-length structure of RNA molecules. Several tools for long-read RNA-seq were developed recently. In this section, we introduce an analytical pipeline of long-read RNA-seq for isoform identification and the estimation of expression levels using minimap2, TranscriptClean, and TALON. We applied this pipeline to the public direct RNA-seq data of the HAP1 and HEK293 cell lines to identify transcript isoforms which can be detected only using long-read RNA-seq data.


Assuntos
RNA Mensageiro/análise , RNA-Seq/métodos , Processamento Alternativo/genética , Linhagem Celular , Perfilação da Expressão Gênica/métodos , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Isoformas de Proteínas/genética , Análise de Sequência de RNA/métodos , Transcriptoma/genética
12.
Methods Mol Biol ; 2284: 543-567, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33835463

RESUMO

The poly(A) tail is a homopolymeric stretch of adenosine at the 3'-end of mature RNA transcripts and its length plays an important role in nuclear export, stability, and translational regulation of mRNA. Existing techniques for genome-wide estimation of poly(A) tail length are based on short-read sequencing. These methods are limited because they sequence a synthetic DNA copy of mRNA instead of the native transcripts. Furthermore, they can identify only a short segment of the transcript proximal to the poly(A) tail which makes it difficult to assign the measured poly(A) length uniquely to a single transcript isoform. With the introduction of native RNA sequencing by Oxford Nanopore Technologies, it is now possible to sequence full-length native RNA. A single long read contains both the transcript and the associated poly(A) tail, thereby making transcriptome-wide isoform-specific poly(A) tail length assessment feasible. We developed tailfindr-an R-based package for estimating poly(A) tail length from Oxford Nanopore sequencing data. In this chapter, we describe in detail the pipeline for transcript isoform-specific poly(A) tail profiling based on native RNA Nanopore sequencing-from library preparation to downstream data analysis with tailfindr.


Assuntos
Sequenciamento por Nanoporos/métodos , Poli A/análise , RNA/análise , Análise de Sequência de RNA/métodos , Animais , Estudos de Viabilidade , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Poli A/genética , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , RNA/química , RNA/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/análise , RNA Mensageiro/genética , Transcriptoma , Peixe-Zebra/genética
13.
Biochem Biophys Res Commun ; 551: 155-160, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33740622

RESUMO

OBJECTIVES: Clinically amyopathic dermatomyositis (CADM) is a subtype of dermatomyositis (DM) characterized by low-grade or absent muscle inflammation but frequent and rapidly progressive interstitial lung disease (RP-ILD) and skin ulcers with anti-melanoma differentiation-associated gene 5 (anti-MDA5) autoantibodies. Basic leucine zipper transcription factor ATF-like 2 (BATF2) is thought to function as an inhibitor of tumours and inflammation. Here, we aimed to investigate the roles of BATF2 in Th cell differentiation of CADM with an anti-MDA5 autoantibody (anti-MDA5+ CADM). METHODS: Naive CD4+ T cells from human peripheral blood mononuclear cells (PBMCs) of healthy controls (HCs) were isolated and then cultured with IL-12, TGF-ß or TGF-ß plus IL-6 following anti-CD3 and anti-CD28 stimulations. The expression of BATF2 was measured by real-time PCR. The percentages of Th1, Th17 and Treg CD4+ T cells were detected by flow cytometry. BATF2 knockdown of CD4+ T cells was performed using small interfering RNAs (siRNAs). RESULTS: The expression of BATF2 in PBMCs was higher in anti-MDA5+ CADM patients than in healthy controls. The BATF2 mRNA expression was increased under Th1 and Treg polarization but decreased under Th17 polarization. Th17 cell activation-associated genes were possibly increased while Th1 and Treg cell differentiation-associated genes were inhibited by posttranscriptional gene silencing of BATF2 in CD4+ T cells. CONCLUSIONS: BATF2 promoted Th1 and Treg cell differentiation but suppressed Th17 cell activation in anti-MDA5+ CADM.


Assuntos
Autoanticorpos/imunologia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linfócitos T CD4-Positivos/imunologia , Dermatomiosite/imunologia , Dermatomiosite/metabolismo , Imunidade Celular , Helicase IFIH1 Induzida por Interferon/imunologia , Proteínas Supressoras de Tumor/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Feminino , Humanos , Masculino , RNA Mensageiro/análise , RNA Mensageiro/genética , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Células Th1/citologia , Células Th1/imunologia , Células Th17/citologia , Células Th17/imunologia , Proteínas Supressoras de Tumor/genética , Regulação para Cima
14.
Biochem Biophys Res Commun ; 551: 161-167, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33740623

RESUMO

Physiological oxygen concentration (physioxia) ranges from 1 to 8% in human tissues while many researchers cultivate mammalian cells under an atmospheric concentration of 21% (hyperoxia). Oxygen is one of the significant gases which functions in human cells including energy production in mitochondria, metabolism in peroxidase, and transcription of various genes in company with HIF (Hypoxia-inducible factors) in the nucleus. Thus, mammalian cell culture should be deliberated on the oxygen concentration to mimic in vivo physiology. Here, we studied if the cultivation of human skin cells under physiological conditions could affect skin significant genes in barrier functions and dermal matrix formation. We further examined that some representative active ingredients in dermatology such as glycolic acid, gluconolactone, and salicylic acid work in different ways depending on the oxygen concentration. Taken together, we present the importance of oxygen concentration in skin cell culture for proper screening of novel ingredients as well as the mechanistic study of skin cell regulation.


Assuntos
Hidroxiácidos/farmacologia , Oxigênio/farmacologia , Pele , Linhagem Celular , Colágeno Tipo I/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Gluconatos/metabolismo , Glicolatos/metabolismo , Humanos , Queratina-1/genética , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Lactonas/metabolismo , Metaloproteinase 1 da Matriz/genética , Oxigênio/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Proteínas S100/genética , Ácido Salicílico/metabolismo , Pele/citologia , Pele/efeitos dos fármacos , Pele/metabolismo
15.
Cancer Med ; 10(7): 2300-2309, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33675149

RESUMO

The present study aimed to establish a novel isolation strategy for circulating tumor cells (CTCs) using a microcavity array (MCA) system and to evaluate the clinical significance of CTCs in hepatocellular carcinoma (HCC). We examined recovery rates of HCC cell lines spiked into whole blood in MCA assay. Circulating tumor cells were isolated from peripheral blood samples (3 mL) of 7 healthy donors (HD), 14 patients with liver cirrhosis (LC), and 31 patients with HCC using the MCA system. Additionally, we investigated the mRNA expression of liver-specific genes in isolated CTCs using qPCR. The recovery rates were 65.1% (HepG2), 76.7% (HuH7), and 99.0% (PLC/PRF/5). In HD and patients with LC and HCC, the CTC positivity rate (CTCs ≥10) and average CTC number were as follows: HD 0% and 0.1, LC 14.3% and 5.3, HCC 54.8% and 47.6, respectively. The CTC positivity rate in HCC was significantly higher than that in LC (p < 0.05). The number of CTCs was significantly higher in metastatic HCC (102.2 ± 160.6) than in localized HCC (8.2 ± 7.7) (p < 0.05). The expression of AFP, glypican-3, EpCAM, and albumin (ALB) genes was detected in isolated CTCs. The positive CTCs (CTCs ≥10) significantly reduced the cumulative survival in patients with HCC (p = 0.025), especially in localized patients with HCC (p = 0.046). The newly developed MCA system has the potential to isolate CTCs from HCC with high sensitivity, and mRNA expression could be measured from CTCs. Identification of positive CTCs can help predict clinical outcome of patients with HCC. Thus, analysis of CTCs in patients with HCC may provide important information as a novel biomarker in disease progression.


Assuntos
Carcinoma Hepatocelular/sangue , Separação Celular/métodos , Neoplasias Hepáticas/sangue , Células Neoplásicas Circulantes , Adulto , Idoso , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Contagem de Células , Molécula de Adesão da Célula Epitelial/genética , Feminino , Glipicanas/genética , Humanos , Cirrose Hepática/sangue , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade , Albumina Sérica Humana/genética , alfa-Fetoproteínas/genética
16.
Kidney Blood Press Res ; 46(2): 245-249, 2021.
Artigo em Inglês | MEDLINE | ID: covidwho-1146805

RESUMO

BACKGROUND: Preclinical studies suggested that pharmacological inhibition of the renin-angiotensin-aldosterone system (RAAS) by ACE inhibitors (ACEis) or angiotensin II receptor blockers (ARBs) may increase local angiotensin-converting enzyme 2 (ACE2) expression. METHODS: In this study, we evaluated the effect of ACEi or ARB treatment on expression of ACE2, ACE, and AGTR1 in 3-month protocol kidney allograft biopsies of stable patients using RT-qPCR (n = 48). Protein ACE2 expression was assessed using immunohistochemistry from paraffin sections. RESULTS: The therapy with RAAS blockers was not associated with increased ACE2, ACE, or ATGR1 expression in kidney allografts and also ACE2 protein immunohistochemistry did not reveal differences among groups. CONCLUSIONS: ACEis or ARBs in kidney transplant recipients do not affect local ACE2 expression. This observation supports long-term RAAS treatment in kidney transplant recipients, despite acute complications such as COVID-19 where ACE2 serves as the entry protein for infection.


Assuntos
Aloenxertos/efeitos dos fármacos , Antagonistas de Receptores de Angiotensina/uso terapêutico , Enzima de Conversão de Angiotensina 2/genética , Anti-Hipertensivos/uso terapêutico , Expressão Gênica/efeitos dos fármacos , Rim/efeitos dos fármacos , Adulto , Idoso , Aloenxertos/metabolismo , Antagonistas de Receptores de Angiotensina/farmacologia , Enzima de Conversão de Angiotensina 2/análise , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Anti-Hipertensivos/farmacologia , COVID-19/complicações , COVID-19/genética , Feminino , Humanos , Rim/metabolismo , Transplante de Rim , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , RNA Mensageiro/genética , Sistema Renina-Angiotensina/efeitos dos fármacos
17.
J Enzyme Inhib Med Chem ; 36(1): 885-894, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33752574

RESUMO

Here we investigated the effects of different levels of royal jelly in zebrafish (Danio rerio) diets [0.0% (D1); 0.1% (D2); 0.4% (D3); 1.6% (D4) vs 6.4% (D5)] on the activity and expression profiles of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase and glutathione S-transferase. Muscle, liver and kidney tissue samples were obtained from fish fed during 8 weeks. In these tissues, enzyme activity was determined by means of spectrophotometer and gene expression by quantitative real-time PCR. mRNA levels of the enzymes were elevated in almost all diet groups compared to the control (D1). It was determined that enzyme activities were also increased in general by supplementation of royal jelly although some decreases were also observed. However, the significant correlation between gene expression and enzyme activity was not observed in all tissues. It was concluded that main regulation occurs with post-translational modifications although effects at transcriptomic level demonstrated a snap variation.


Assuntos
Catalase/genética , Ácidos Graxos/farmacologia , Glutationa Peroxidase/genética , Glutationa Redutase/genética , Glutationa Transferase/genética , Estresse Oxidativo/efeitos dos fármacos , Superóxido Dismutase/genética , Peixe-Zebra , Animais , Catalase/análise , Catalase/metabolismo , Dieta , Ácidos Graxos/administração & dosagem , Perfilação da Expressão Gênica , Glutationa Peroxidase/análise , Glutationa Peroxidase/metabolismo , Glutationa Redutase/análise , Glutationa Redutase/metabolismo , Glutationa Transferase/análise , Glutationa Transferase/metabolismo , Estresse Oxidativo/genética , Processamento de Proteína Pós-Traducional , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Espectrofotometria , Superóxido Dismutase/análise , Superóxido Dismutase/metabolismo
18.
J Dairy Res ; 88(1): 73-77, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33663628

RESUMO

Our objective was to determine the influence of chronic coagulase-positive staphylococci (CoPS) or coagulase-negative staphylococci (CoNS) infection on the mRNA and protein levels of two main milk proteins responsible for cheese curd quantity and quality, alpha-S1-casein (CSN1S1) and kappa-casein (CSN3). Measurements were made in cow mammary parenchyma with a prevalence of secretory tissue (MGST). Samples of MGST were collected from the separate quarters and divided into CoPS, CoNS and bacteria-free (H) groups according to the microbiological status of the quarter milk. No differences in CSN1S1 and CSN3 mRNA level were found between groups, however, CSN1S1 protein level was significantly higher in the H group than the CoNS group, and CSN3 protein level was significantly higher in H than CoPS group. Hence, while the CSN1S1 and CSN3 genes appear to be constitutively expressed at the mRNA level in dairy cow MGST during mastitis, CoNS infection negatively affected CSN1S1 protein level, and CoPS infection negatively affected CSN3 protein level. The lack of change at the mRNA level suggests that staphylococcal infection may affect the post-transcriptional or post-translational modifications.


Assuntos
Caseínas/análise , Caseínas/genética , Glândulas Mamárias Animais/química , Mastite Bovina/metabolismo , RNA Mensageiro/análise , Infecções Estafilocócicas/veterinária , Animais , Bovinos , Indústria de Laticínios , Feminino , Expressão Gênica , Glândulas Mamárias Animais/metabolismo , Leite/microbiologia , Infecções Estafilocócicas/metabolismo
19.
Nutrients ; 13(3)2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33670988

RESUMO

Obesity is associated with an impaired balance of CD4+ T cell subsets. Both vitamin D and obesity have been reported to affect the mTOR pathway. In this study, we investigated the effects of vitamin D on CD4+ T cell subsets and the mTOR pathway. Ten-week-old male C57BL/6 mice were divided into four groups and fed diets with different fat (control or high-fat diets: CON or HFD) and vitamin D contents (vitamin D control or supplemented diets: vDC or vDS) for 12 weeks. T cells purified by negative selection were stimulated with anti-CD3/anti-CD28 mAbs and cultured for 48 h. The percentage of CD4+IL-17+ T cells was higher in the vDS than vDC groups. The CD4+CD25+Foxp3+ T cells percentage was higher in HFD than CON groups. The phospho-p70S6K/total-p70S6K ratio was lower in vDS than vDC, but the phospho-AKT/total-AKT ratio was higher in vDS than vDC groups. Hif1α mRNA levels were lower in vDS than vDC groups. These findings suggest HIF1α plays an important role in vitamin-D-mediated regulation of glucose metabolism in T cells, and dietary vitamin D supplementation may contribute to the maintenance of immune homeostasis by regulating the mTOR pathway in T cells.


Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Dieta Hiperlipídica , Obesidade/imunologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Vitamina D/administração & dosagem , Animais , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Suplementos Nutricionais , Fatores de Transcrição Forkhead/análise , Expressão Gênica/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Interferon gama/biossíntese , Interleucina-17/análise , Interleucina-4/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/metabolismo , RNA Mensageiro/análise , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Serina-Treonina Quinases TOR/genética , Vitamina D/análogos & derivados , Vitamina D/sangue , Vitamina D/metabolismo
20.
Nutrients ; 13(3)2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33671116

RESUMO

Ellagic acid, a natural substance found in various fruits and nuts, was previously shown to exhibit beneficial effects towards metabolic syndrome. In this study, using a genetic rat model of metabolic syndrome, we aimed to further specify metabolic and transcriptomic responses to ellagic acid treatment. Adult male rats of the SHR-Zbtb16Lx/k.o. strain were fed a high-fat diet accompanied by daily intragastric gavage of ellagic acid (50 mg/kg body weight; high-fat diet-ellagic acid (HFD-EA) rats) or vehicle only (high-fat diet-control (HFD-CTL) rats). Morphometric and metabolic parameters, along with transcriptomic profile of liver and brown and epididymal adipose tissues, were assessed. HFD-EA rats showed higher relative weight of brown adipose tissue (BAT) and decreased weight of epididymal adipose tissue, although no change in total body weight was observed. Glucose area under the curve, serum insulin, and cholesterol levels, as well as the level of oxidative stress, were significantly lower in HFD-EA rats. The most differentially expressed transcripts reflecting the shift induced by ellagic acid were detected in BAT, showing downregulation of BAT activation markers Dio2 and Nr4a1 and upregulation of insulin-sensitizing gene Pla2g2a. Ellagic acid may provide a useful nutritional supplement to ameliorate features of metabolic syndrome, possibly by suppressing oxidative stress and its effects on brown adipose tissue.


Assuntos
Ácido Elágico/administração & dosagem , Síndrome Metabólica/metabolismo , Síndrome Metabólica/prevenção & controle , Transcriptoma/efeitos dos fármacos , Tecido Adiposo/química , Tecido Adiposo Marrom/química , Animais , Biomarcadores/análise , Glicemia/análise , Dieta Hiperlipídica , Epididimo , Fígado/química , Masculino , Síndrome Metabólica/genética , Estresse Oxidativo/efeitos dos fármacos , RNA/análise , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos SHR
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