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In recent decades, infections caused by the opportunistic fungus Candida albicans have increased, especially in patients with immunodeficiency. In this study, we investigated the mechanism of action of sanguinarine (SAN) against C. albicans both in vitro and in vivo. SAN exhibited antifungal activity against C. albicans clinical isolates, with MICs in the range of 112.8-150.5 µM. Furthermore, scanning electron and transmission electron microscopy showed that SAN induced morphological changes as well as structure disruption in C. albicans cells, including masses of cellular debris, ruptured cell walls, and membrane deformation. Flow cytometry revealed that SAN could lead to cell membrane damage, and ergosterol content analysis indicated that SAN could cause ergosterol content reduction exceeding 90%. Further, we validated the efficacy of SAN against candidiasis caused by C. albicans in a murine model, and SAN significantly improved survival and reduced weight loss compared to vehicle. The treatment of 1.5 and 2.5 mg/kg/d SAN obviously reduced the fungal burden in the kidney. In addition, histopathological examination indicated that no fungal cells were observed in lung and kidney tissues after SAN treatment. Hence, this study suggests that SAN is a promising plant-derived compound for the development of an effective anticandidal agent.
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In this paper, a new amphiphilic mono-6-ß-cyclodextrin octadecylimine (6-ß-CD-N-ODMA) copolymer was synthesized based on ß-cyclodextrin and octadecylamine, which can self-assemble to form polymeric micelles. Drug-loaded micelles (a new drug carrier) were obtained using 6-ß-CD-N-ODMA and paclitaxel (PTX) by the dialysis method. Orthogonal experiments were used to optimize the preparation method of the drug-loaded micelles. The drug-loading content of the carrier prepared by the optimized method was 1.97%. The physicochemical properties of blank micelles and drug-loaded micelles were evaluated by the fluorescence probe method, infrared spectra, dynamic light scattering, and scanning electron microscopy. The release properties of the carrier were investigated. The carrier has good pH sensitivity and the cumulative release rate after 96 h was 88% in PBS (pH = 5.0). The Ritger-Peppas equation is the optimal model for PTX released at pH 5.0, implying that the hydrolysis effect of 6-ß-CD-N-ODMA is the main reason for PTX release. The results indicate that the developed carrier can increase the solubility of PTX and possess potential for increased clinical efficacy of PTX.
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We report the discovery of a facile peptide macrocyclization and stapling strategy based on a fluorine thiol displacement reaction (FTDR), which renders a class of peptide analogues with enhanced stability, affinity, cellular uptake, and inhibition of cancer cells. This approach enabled selective modification of the orthogonal fluoroacetamide side chains in unprotected peptides in the presence of intrinsic cysteines. The identified benzenedimethanethiol linker greatly promoted the alpha helicity of a variety of peptide substrates, as corroborated by molecular dynamics simulations. The cellular uptake of benzenedimethanethiol stapled peptides appeared to be universally enhanced compared to the classic ring-closing metathesis (RCM) stapled peptides. Pilot mechanism studies suggested that the uptake of FTDR-stapled peptides may involve multiple endocytosis pathways in a distinct pattern in comparison to peptides stapled by RCM. Consistent with the improved cell permeability, the FTDR-stapled lead Axin and p53 peptide analogues demonstrated enhanced inhibition of cancer cells over the RCM-stapled analogues and the unstapled peptides.
Assuntos
Flúor/química , Compostos Macrocíclicos/química , Peptídeos/química , Compostos de Sulfidrila/química , Sequência de Aminoácidos , Proteína Axina/química , Permeabilidade da Membrana Celular , Peptídeos Penetradores de Células/química , Reagentes de Ligações Cruzadas/química , Ciclização , Células HEK293 , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Dinâmica Molecular , Termodinâmica , Proteína Supressora de Tumor p53/químicaRESUMO
The close relationships of miRNAs with human diseases highlight the urgent needs for miRNA detection. However, the accurate detection of a target miRNA in mixed miRNAs of high sequence homology presents a great challenge. Herein, a novel method called target-protection rolling circle amplification (TP-RCA) is proposed for this purpose. The protective probe is designed so that it can form a fully complementary duplex with the target miRNA and can also mismatch duplexes with other nontarget miRNAs. These duplexes are treated with a single strand-specific nuclease. Consequently, only the target miRNA in a perfect-match duplex can resist the cleavage of nuclease, whereas the nontarget miRNAs in mismatched duplexes will be digested completely. The protected target miRNA can be detected using RCA reactions. MicroRNA let-7 family members (let-7a-let-7f) and nuclease CEL I were used as proof-of-concept models to evaluate the feasibility of the TP-RCA method under different experimental conditions. The experimental results show that the TP-RCA method can unambiguously detect the target let-7 species in mixtures of let-7 family members even though they may differ by only a single nucleotide. This TP-RCA method significantly improves the detection specificity of miRNAs.
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PURPOSE: Paclitaxel resistance in ovarian cancer has become an urgent clinical problem. This study investigated the regulatory effects of RAB17 on the non-coding RNA network of the paclitaxel-resistant ovarian cancer cell A2780/PTX. METHODS: Microarray analysis was used to identify differentially expressed genes in paclitaxel-resistant cell A2780/PTX compared to the parent paclitaxel-sensitive cell A2780. Quantitative real-time PCR and Western blot were used to measure the expression of related mRNAs and proteins. The CCK8 assay was used to determine cell survival ratios and drug resistance indices in ovarian cancer cells. The clone forming assay was used to analyze the cell clone proliferation. Flow cytometry was used to analyze the cell cycle. Dual-luciferase reporter gene assays evaluated the relationship between the genes. RESULTS: RAB17 is highly expressed in A2780/PTX cells. RAB17 knockdown increased the cell sensitivity to paclitaxel, inhibited proliferation, and caused cell cycle arrest in the G1 phase in A2780/PTX. Western blot confirmed that RAB17 influenced cell behavior by activating the CDK6/RB signaling pathway. Bioinformatics analyses identified RAB17 as a new target by the microRNA miR-370-3p, and the latter was predicted to interact with circular RNA hsa_circ_0000714. Hsa_circ_0000714 indeed acted as a miRNA sponge for miR-370-3p allowing its regulation of RAB17 expression. This regulation was accomplished through the CDK6/RB signaling pathway. CONCLUSION: Hsa_circ_0000714 acts as a sponge for miR-370-3p, and regulates RAB17 expression through the CDK6/RB signaling pathway, which plays a role in the malignant progression of the paclitaxel-resistant ovarian cancer cell A2780/PTX.
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Transcription factor (TF)-based metabolite detection mainly depends on TF-regulated gene expression in cells. From TF activation to gene transcription and translation, the signal travels a relatively long way before it is received. Here, we propose a TF-splinting duplex DNA nanoswitch to detect metabolites. We show its feasibility using tryptophan repressor (TrpR) to detect l-tryptophan as a model. The assay has been optimized and characterized after obtaining a proof of concept, and the detection of l-tryptophan in complex biological samples is feasible. Unlike an equivalent gene expression approach, the whole process is a single-step, enzyme-free, and signal-on method. It can be completed within 20 min. This proposed TF-splinting duplex has the potential to be applied to the quick and convenient detection of other metabolites or even TFs.
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Proteínas de Bactérias/química , DNA/química , Proteínas Repressoras/química , Triptofano/análise , Proteínas de Bactérias/genética , Humanos , Proteínas Repressoras/genética , Triptofano/sangue , Triptofano/urinaRESUMO
Seedpod, the nonedible portion of lotus (Nelumbo nucifera Gaertn.), was reported to be rich in polyphenols. The objective of this study was to investigate the major bioactive polyphenols of the lotus seedpods. The total polyphenol content (TPC) from ethanol extract of lotus seedpod (PELS) was found to be 34.23 µg gallic acid equivalents (GAE)/mg extract. Four polyphenolic compounds were identified in the PELS, comprised of one flavan-3-ol (catechin) and three flavonoids (kaemferol, quercetin and hyperoside). In vitro antioxidant and antiproliferative properties of the PELS were evaluated. PELS exhibited 89.38%, 99.82%, 68.25%, and 95.82% scavenging activities against 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide, hydroxyl, and 2,2'azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals, respectively, at 1.6 mg/ml. The Fe3+ reducing power of PELS was 0.605 at 0.32 mg/ml, which is comparable to glutathione (GSH). The PELS showed 31.79% metal chelating capacity and 87.79% inhibition of linoleic acid auto-oxidation at 1.6 mg/ml. PELS showed cytotoxicity toward HepG2 and LNcap cell lines in vitro with IC50 values at 44.59 and 11.50 µg/ml, respectively. The findings of this study provide evidences that the inedible lotus seedpod could be a source for natural antioxidants and anticancer agents.
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Nicking endonucleases (NEs) become increasingly attractive for their promising applications in isothermal amplification. Unfortunately, in comparison with their applications, their catalytic mechanism studies have relatively lagged behind due to a paucity of crystal structure information. Nt.BstNBI is one of those widely used NEs. However, many aspects of its catalytic mechanism still remained to be explored. Herein, we employed only rolling circle amplification (RCA) assay as a major analytic tool and succeeded in identifying the potential binding positions and regions of the DNA substrate based on locked nucleic acid modification, DNA duplex length of substrate, and substrate mismatch designs. Based on these data, we, for the first time, revealed that Nt.BstNBI was likely to recognize six adjacent positions of the recognition sequence (G1rt, A2rt, G3rt, A2rb, C3rb, and T4rb) in the major groove and hold three positions of the cleavage sequence (N3ct, N4ct, and N7cb) in the minor groove of DNA duplex for nicking. Moreover, this work also demonstrated the unexpected efficiency of RCA to study the macromolecular interaction for certain kind of nucleases in an easy and high-throughput way.
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Quebras de DNA de Cadeia Simples , Replicação do DNA , DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , CatáliseRESUMO
The central loop of G-quadruplex molecular beacons is a key element to sense target DNA or RNA sequences. In this study, circular dichroism spectroscopy (CD), thermal difference spectrum (TDS), non-denatured non-denaturing gel electrophoresis, and thermal stability analysis were used to investigate the effect of the central loop length on G-quadruplex features. Two series of G-quadruplexes, AG3TTAG3-(TTA)n-G3TTAG3T (n = 1-8) (named TTA series) and AG3TTTG3-(TTA)n-G3TTTG3T (n = 1-8) (named TTT series) were examined in K+ and Na+ solutions, respectively. CD and TDS spectral data indicated that TTA series adopted an antiparallel G-quadruplex structure in Na+ solution and a hybrid G-quadruplex structure in K+ solution respectively. TTT series exhibited a hybrid G-quadruplex structure in both Na+ and K+ solutions. UV melting curves indicated that the stability of G-quadruplex in both series was reduced by the elongation of central loop. Thermal stability analysis concluded that the G-quadruplex destabilization with long central loop is an entropy-driven process due to more flexible and longer central loops.
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DNA/química , Quadruplex G , Metais/química , Termodinâmica , Dicroísmo Circular , Estrutura Molecular , Conformação de Ácido Nucleico , Raios UltravioletaRESUMO
There is a great demand worldwide for bone-related implant materials. The drawbacks of chronic infections and poor bone healing of current implant materials have limited their clinical applications. Functionalizing the implant surfaces with antibacterial and osteogenic films on implant materials provides new opportunities for fabricating novel implant materials. In the present study, an ultrathin (GO/Lys)8 film of several tens of nanometers was fabricated using a layer-by-layer (LBL) technique with alternative deposition of graphene oxide (GO) and lysozyme (Lys). The deposition of the (GO/Lys) n film exhibited a successive growth as supported by ellipsometry, UV-vis, and Fourier transform infrared data, and the physical properties (morphology, roughness, and stiffness) of this film were characterized with an atomic force microscope. The ultrathin films exhibited a great effect on bacterium sterilization of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli and enhanced osteogenic differentiation efficiency, showing the potential application in bone implant coatings. We believe that this LBL assembling strategy will pave the way for fabricating dual-functional surfaces and guide the design of the implanted surfaces in the future.
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Antibacterianos , Polpa Dentária/metabolismo , Escherichia coli/crescimento & desenvolvimento , Grafite , Membranas Artificiais , Osteogênese/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Células-Tronco/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Polpa Dentária/citologia , Grafite/química , Grafite/farmacologia , Humanos , Muramidase/química , Muramidase/farmacologia , Células-Tronco/citologiaRESUMO
The rapid ascension of immune checkpoint blockade treatments has placed an emphasis on the need for viable, robust, and noninvasive imaging methods for immune checkpoint proteins, which could be of diagnostic value. Immunoconjugate-based positron emission tomography (immuno-PET) allows for sensitive and quantitative imaging of target levels and has promising potential for the noninvasive evaluation of immune checkpoint proteins. However, the advancement of immuno-PET is currently limited by available imaging tools, which heavily rely on full-length IgGs with Fc-mediated effects and are heterogeneous mixtures upon random conjugation with chelators for imaging. Herein, we have developed a site-specific αPD-L1 Fab conjugate with the chelator 1,4,7-triazacyclononane- N, N', Nâ³-triacetic acid (NOTA), enabling radiolabeling for PET imaging, using the amber suppression-mediated genetic incorporation of unnatural amino acid (UAA), p-azidophenylalanine. This Fab conjugate is homogeneous and demonstrated tight binding toward the PD-L1 antigen in vitro. The radiolabeled version, 64Cu-NOTA-αPD-L1, has been employed in PET imaging to allow for effective visualization and mapping of the biodistribution of PD-L1 in two normal mouse models, including the capturing of different PD-L1 expression levels in the spleens of the different mouse types. Follow-up in vivo blocking studies and ex vivo fluorescent staining further validated specific tissue uptakes of the imaging agent. This approach illustrates the utility of UAA-based site-specific Fab conjugation as a general strategy for making sensitive PET imaging probes, which could facilitate the elucidation of the roles of a wide variety of immune checkpoint proteins in immunotherapy.
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Antígeno B7-H1/metabolismo , Sítios de Ligação de Anticorpos , Imunoconjugados/farmacocinética , Tomografia por Emissão de Pósitrons/métodos , Traçadores Radioativos , Animais , Azidas/química , Antígeno B7-H1/imunologia , Quelantes/química , Simulação por Computador , Radioisótopos de Cobre/química , Compostos Heterocíclicos com 1 Anel/química , Imunoconjugados/imunologia , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Mutação , Fenilalanina/análogos & derivados , Fenilalanina/química , Compostos Radiofarmacêuticos/farmacocinética , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Baço/metabolismo , Distribuição TecidualRESUMO
Background: The problem of adulterated meat has become one of the greatest food safety issues in the world. It is reported that the meat used for adulteration includes fox meat, raccoon meat, mink meat, mouse meat, and so on. Although this kind of meat is edible in some areas, such meat is potentially harmful to human health because it is easy to carry bacteria, viruses, and harmful substances. The harm of mouse meat is most frightening. Therefore, it is urgent to develop a fast, accurate, and simple method to effectively identify mouse meat. Methods: In the present study, a new method of isothermal amplification based on the 16S ribosomal RNA gene of the mitochondrial DNA of the mouse was developed. The method is meant to improve the loop-mediated isothermal amplification (LAMP), separating the forward inner primers and backward inner primers, greatly reducing the nonspecific amplification of the method. Results: We have successfully obtained a set of best primers. The developed system allowed for the detection of 0.5% mouse meat from meat mixture effectively and specifically. The best ratio of the primers (F3: F2: F1: RF) was 1:2:2:8, and the optimum concentration of DNA template was 0.35 ng/µL. Conclusions: The assay has great specificity and sensitivity for detecting mouse meat and could provide specific positive results within 1 h. Highlights: We found a new approach of isothermal amplification to detect mouse source components. The LOD is determined to be 0.5 mg/mg. This new method is easy to perform and is able to provide rapid results in the specific detection of mouse meat sources.
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Contaminação de Alimentos/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Ribossômico 16S/análise , Carne Vermelha/análise , Animais , Sequência de Bases , Caniformia , Bovinos , Galinhas , Patos , Equidae , Limite de Detecção , Camundongos , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S/genética , Ovinos , SuínosRESUMO
Our earliest phytochemical separation of Miliusa sinensis aided us in the isolation of a class of unique miliusanes, which were demonstrated as anticancer lead molecules. In the present study, we isolated 19 miliusanes (1-19), including 11 novel ones (5 and 10-19) from another Miliusa plant ( M. balansae), and synthesized additional derivatives to elucidate the structure-activity relationship of miliusanes. When extrapolated to various carcinoma xenograft mouse models, miliusol (1) and its derivatives 20, 26, and 27 (7.5-40 mg/kg) were demonstrated with tumor inhibitory efficacy comparable or even superior to the mainstay chemotherapeutics paclitaxel or fluorouracil. To gain a molecular insight into their anticancer mechanism, 1-3 (GI50 0.03-4.79) were administered to a wide spectrum of human cancer cell lines, including those with specific drug resistance. We further revealed that the antiproliferative properties of miliusanes in carcinoma cells were highly associated with the p21-dependent induction of cellular senescence.
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Annonaceae/química , Antineoplásicos/farmacologia , Senescência Celular/efeitos dos fármacos , Cicloexanonas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Cicloexanóis/síntese química , Cicloexanóis/isolamento & purificação , Cicloexanóis/farmacologia , Cicloexanonas/síntese química , Cicloexanonas/isolamento & purificação , Feminino , Humanos , Masculino , Camundongos Endogâmicos BALB C , Estrutura Molecular , Transdução de Sinais/efeitos dos fármacos , Compostos de Espiro/síntese química , Compostos de Espiro/isolamento & purificação , Compostos de Espiro/farmacologia , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Background: The fraudulent substitution of cheap and low-quality meat for expensive and good-quality meats to gain profit is a common practice in industries worldwide. Adulteration of fox, raccoon, or mink in commercial beef and mutton meat in the supermarket has become a serious problem. Objective: To ensure the meat quality and safety, we have developed a multiplex PCR method to identify the fox, mink, and raccoon components adulterated in beef or mutton with very low detection limits. Methods: PCR primers were designed and tested by examining the size of PCR product, the nuclease digestion products, and DNA sequencing. Results: After primer interference tests, we have established a double PCR method that can clearly identify fox, mink, or raccoon components in beef meat and mutton meat at the 1% (w/w) level. Triplex PCR and quadruple PCR have been also developed, which are able to identify any two types of components or three mixed components in beef meat unambiguously. Conclusions: We have developed multiplex PCR systems. The duplex PCR systems can identify one component (fox, raccoon, or mink) adulterated in beef meat or mutton meat without question, and triplex PCR and quadruple PCR can discriminate two components and three components adulterated in beef meat. Highlights: These methods are convenient, low-cost, highly specific and reliable, and of a great value for meat quality control and food safety quarantine.
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Contaminação de Alimentos/análise , Reação em Cadeia da Polimerase Multiplex/métodos , Carne Vermelha/análise , Animais , Bovinos , DNA/análise , Raposas/genética , Vison/genética , Guaxinins/genética , OvinosRESUMO
The aim of this study is to investigate the protective effect and underlying molecular mechanisms of isobavachalcone on Sephadex-induced lung injury in rats. The result showed isobavachalcone inhibited massive granulomas, decreased inflammatory cells infiltration and oxidative stress markers level, but it can increase antioxidant enzymes level. The ELISA assay exhibited isobavachalcone decreased TNF-α production in BALF and lung tissue. Western blotting analysis showed isobavachalcone can inhibit NF-κB pathway that may be mediated by upregulation of A20. Furthermore, we also found isobavachalcone can activate NRF2/HO-1 pathway and inhibit adhesion molecule expression. Taken together, the present results suggested that isobavachalcone can attenuate Sephadex-induced lung injury that may be related to inhibition of NF-κB mediated by upregulation of A20 and activation of NRF2/HO-1 signaling pathway.
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Lesão Pulmonar Aguda/metabolismo , Chalconas/uso terapêutico , Proteínas de Ligação a DNA/metabolismo , Dextranos/toxicidade , Heme Oxigenase-1/metabolismo , Proteínas de Membrana/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Animais , Chalconas/farmacologia , Proteínas de Ligação a DNA/agonistas , Relação Dose-Resposta a Droga , Masculino , Proteínas de Membrana/agonistas , Fator 2 Relacionado a NF-E2/agonistas , Ratos , Ratos Sprague-Dawley , Proteína 3 Induzida por Fator de Necrose Tumoral alfaRESUMO
Dietary phenols are antioxidants with diverse physiological functions that are beneficial for human health. The objective of this research work was to investigate antioxidant activity of p-coumaric acid (p-CA) using four in vitro methods, the protective effects against oxidative stress in PC12 cells, and hypolipidemic effects on High fat-diet (HFD) mice model. The p-CA exhibited moderate antioxidant activity in the selected in vitro assay. The highest chelating activity of p-CA at 50 µg/mL was found to be 52.22%. Pretreatment with p-CA significantly enhanced cell viability of PC12 cell and suppressed AAPH-induced intracellular ROS generation and AAPH-induced LDH release. The hypolipidemic effects of p-CA (100 mg/kg BW) was directly linked to the increased expression of nuclear factor erythroid 2-related factor (Nrf2) by 2.0-fold, Glutathione peroxidase (Gpx) by 3.8-fold, Superoxide dismutase (SOD-1) by 1.6-fold, Heme oxygenase (HO-1) by 1.72-fold and NAD(P)H Quinone Dehydrogenase 1 (NQO-1) by 1.5-fold compared with HFD group. In addition to these effects, p-CA decreased total cholesterol and atherosclerosis index levels, and increased catalase (CAT) level in serum, total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px) levels in liver as compared HFD group. Administration of p-CA also promoted the recovery of hyperlipidemia steatohepatitis in mice by ameliorating lipid peroxidation. These results suggested that p-CA is a potent antioxidant with potential therapeutic efficacy for treating hyperlipidemia symptoms.
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Antioxidantes/uso terapêutico , Hiperlipidemias/prevenção & controle , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Propionatos/uso terapêutico , Animais , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Ácidos Cumáricos , Dieta Hiperlipídica/efeitos adversos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Sequestradores de Radicais Livres/farmacologia , Sequestradores de Radicais Livres/uso terapêutico , Hiperlipidemias/metabolismo , Peroxidação de Lipídeos/fisiologia , Masculino , Camundongos , Estresse Oxidativo/fisiologia , Células PC12 , Propionatos/farmacologia , Ratos , Resultado do TratamentoRESUMO
Loop-mediated isothermal amplification (LAMP) has been developed as a versatile method for nucleic acid analysis in many applications. However, non-specific LAMP leading to false-positive outcomes has been observed frequently. To solve this problem, we selected six molecules as the additives for evaluating their effects on the improvement of the LAMP specificity. Experimental results show that bovine serum albumin (BSA) and DL-dithiothreitol (DTT) have negative effects on the LAMP specificity; dimethyl sulfoxide (DMSO), tetramethylene sulfoxide (TMSO), and glycerol could inhibit the non-specific LAMP moderately. Surprisingly, pullulan shows an ability to inhibit the non-specific amplification of LAMP significantly without affecting the sample amplification of LAMP, and this inhibitory effect is concentration dependent. Thus, pullulan could be considered as the most promising additive to improve the amplification specificity in the LAMP-based detection and analysis of nucleic acids.
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Glucanos/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/análise , Animais , Sequência de Bases , Bovinos , Dimetil Sulfóxido/química , Ditiotreitol/química , Glicerol/química , Indicadores e Reagentes/química , Soroalbumina Bovina/química , Sulfóxidos/química , Tiofenos/químicaRESUMO
OBJECTIVE: To explore the inhibition of the proliferation of vulvar squamous cell carcinoma (VSCC) by astragaloside IV. METHODS: MTT examined the cell proliferation of VSCC. Flow cytometry analyzed cell cycle and apoptosis. Western blot assay detected the expression of some relevant proteins. RESULTS: AS-IV reduced the proliferation of SW962 cells in a concentration- and time-dependent manner, induced cell-cycle arresting in G0/G1 phase, as demonstrated by the up-regulation of P53 and P21 expression, and the down-regulation of cyclin D1 expression. AS-IV enhanced the expression of Bax and cleaved-caspase 3, and suppressed Bcl-2 and Bcl-xl expression, which resulted in apoptosis increased. Furthermore, the expression of Beclin-1 and LC3-B was upregulated and that of P62 was downregulated, which suggested that AS-IV could increase the incidence of autophagy in SW962 cells. After inhibiting autophagy by 3-methyladenine (3-MA), cell apoptosis decreased upon AS-IV treatment. Similarly, TGF-ß1 stimulated SW962 cells, cell proliferation enhanced, and the expression of TGF-ßRII and Smad4 was decreased. Furthermore, the expression of proteins that promote apoptosis and autophagy decreased. After AS-IV treatment, the expression levels of the above proteins exhibited the opposite effect. CONCLUSION: AS-IV inhibits cell proliferation and induces apoptosis and autophagy through the TGF-ß/Smad signaling pathway in VSCC.
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Carcinoma de Células Escamosas/tratamento farmacológico , Saponinas/farmacologia , Proteínas Smad/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Triterpenos/farmacologia , Neoplasias Vulvares/tratamento farmacológico , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Saponinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Triterpenos/uso terapêutico , Neoplasias Vulvares/patologiaRESUMO
MicroRNAs (miRNAs) diagnostics can be useful for diagnosing or confirming miRNA abundance and are used in screening tests and to assess changes in miRNAs in vivo. At present, the use of traditional nucleic acid amplification assays to detect miRNAs has been limited in laboratory environment because of the time, equipment, and technical expertise required to perform these assays. A specialized, rapid affordable miRNA detection system is necessary when there are limited resources or point-of-care testing needs. We designed a portable and affordable fluorescence-based miRNA detection system based on isothermal signal amplification technology, using SYBR Green II as a fluorescent dye. To reduce costs, we chose LED as a light source and designed the corresponding optical path for LED. The portable detection system shows results consistent with those by real-time PCR, and can be used to detect miR-183 with a limit of detection of approximately 2 fmol. We used the system to detect miR-183 in tissues and blood from patients with hepatocellular carcinoma (HCC). The results from the portable detection device were compared with those from clinical trials and indicated that the miR-183 fluorescence signal could successfully identify HCC and provide information related to cancer progression.
Assuntos
Carcinoma Hepatocelular , Fluorescência , Neoplasias Hepáticas , MicroRNAs , Compostos Orgânicos/química , RNA Neoplásico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adulto , Idoso , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Feminino , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , RNA Neoplásico/genética , RNA Neoplásico/metabolismoRESUMO
ERp57 has been identified to be associated with the chemoresistance of human ovarian cancer. However, its biological roles in the chemoresistance phenotype remain unclear. In the present study, the association of ERp57 with paclitaxelresistant cellular behavior was investigated and the sensitivity enhancement of chemoresistant human ovarian cancer cells to paclitaxel was examined using ERp57small interfering (si)RNA silencing. Cell viability, cell proliferation, cell apoptosis and cell migration were detected using an MTT assay, clonogenic assay, flow cytometry analysis and transwell assay. Furthermore, mRNA expression levels of ERp57 and protein expression levels of ERp57, STAT3, phosphorylated STAT3, PCNA, nucelolin, TUBB3, P-gp, vimentin, Bcl-2, Bax, Bcl-xl, p53, MMP1, MMP2 and MMP9 of paclitaxel-sensitive human SKOV3 ovarian cancer cells were compared with paclitaxel-resistant counterpart SKOV3/tax using the real-time PCR and western blot analysis. ERp57 was highly expressed in the paclitaxelresistant SKOV3/tax cells, and experimental results concluded that the paclitaxelresistance phenotype was due primarily to the activation of the STAT3 signaling pathway. ERp57 overexpression by lentiviral particle infection decreased the sensitivity of SKOV3 cells to paclitaxel. Furthermore, ERp57siRNA silencing restored paclitaxel sensitivity of SKOV3/tax cells. Notably, the IC50 value of ERp57siRNA silenced SKOV3/tax cells was reduced to the original level and colony survival was significantly decreased in comparison with that of SKOV3/tax cells. Additionally, cotreatment of ERp57siRNA silencing and paclitaxel could inhibit the STAT3 signaling pathway and downregulate the expression levels of downstream proteins. Notably, ERp57siRNA and 100 nM paclitaxel cotreatment downregulated Bcl2, Bclxl, MMP2, MMP9, TUBB3 and Pgp expression levels and upregulated the expression of Bax protein. Furthermore, cotreatment promoted change of the isoform of p53 to p53/p47. Bioinformatics analyses supported the experimental observations that ERp57 was associated with drug resistance in ovarian cancer. The present study implies that ERp57 is a potential therapeutic target for the treatment of paclitaxelresistant human ovarian cancer.