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Porcine epidemic diarrhea virus (PEDV) belongs to the Alphacoronavirus genus within the Coronavirus family, causing severe watery diarrhea in piglets and resulting in significant economic losses. Medium-chain acyl-CoA dehydrogenase (ACADM) is an enzyme participating in lipid metabolism associated with metabolic diseases and pathogen infections. Nonetheless, the precise role of ACADM in regulating PEDV replication remains uncertain. In this study, we identified ACADM as the host binding partner of NSP4 via immunoprecipitation-mass spectrometry analysis. The interaction between ACADM and NSP4 was subsequently corroborated through coimmunoprecipitation and laser confocal microscopy. Following this, a notable upsurge in ACADM expression was observed during PEDV infection. ACADM overexpression effectively inhibited virus replication, whereas ACADM knockdown facilitated virus replication, suggesting ACADM has negative regulation effect on PEDV infection. Furthermore, we demonstrated fatty acid ß-oxidation affected PEDV replication for the first time, inhibition of fatty acid ß-oxidation reduced PEDV replication. ACADM decreased PEDV-induced ß-oxidation to suppress PEDV replication. Mechanistically, ACADM reduced cellular free fatty acid levels and subsequent ß-oxidation by hindering AMPK-mediated lipophagy. In summary, our results reveal that ACADM plays a negative regulatory role in PEDV replication by regulating lipid metabolism. The present study introduces a novel approach for the prevention and control of PEDV infection.
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Proteínas Quinasas Activadas por AMP , Oxidación-Reducción , Virus de la Diarrea Epidémica Porcina , Replicación Viral , Virus de la Diarrea Epidémica Porcina/fisiología , Animales , Chlorocebus aethiops , Células Vero , Proteínas Quinasas Activadas por AMP/metabolismo , Porcinos , Humanos , Acil-CoA Deshidrogenasa/metabolismo , Acil-CoA Deshidrogenasa/genética , Metabolismo de los Lípidos , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/virología , Ácidos Grasos/metabolismo , Células HEK293 , Activación EnzimáticaRESUMEN
PEDV, a single-stranded RNA virus, causes significant economic losses in the pig industry. Sin3-associated protein 18 (SAP18) is known for its role in transcriptional inhibition and RNA splicing. However, research on SAP18's involvement in PEDV infection is limited. Here, we identified an interaction between SAP18 and PEDV nonstructural protein 10 (Nsp10) using immunoprecipitation-mass spectrometry (IP-MS) and confirmed it through immunoprecipitation and laser confocal microscopy. Additionally, PEDV Nsp10 reduced SAP18 protein levels and induced its cytoplasmic accumulation. Overexpressing SAP18 suppressed PEDV replication, meanwhile its knockdown via short interfering RNA (siRNA) enhanced replication. SAP18 overexpression boosted IRF3 and NF-κB P65 phosphorylation, nuclear translocation, and IFN-ß antiviral response. Furthermore, SAP18 upregulated RIG-I expression and facilitated its dephosphorylation, while SAP18 knockdown had the opposite effect. Finally, SAP18 interacted with phosphatase 1 (PP1) catalytic subunit alpha (PPP1CA), promoting PPP1CA-RIG-I interaction during PEDV infection. These findings highlight SAP18's role in activating the type I interferon pathway and inhibiting viral replication by promoting RIG-I dephosphorylation through its interaction with PPP1CA.
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Virus de la Diarrea Epidémica Porcina , Proteínas no Estructurales Virales , Replicación Viral , Animales , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Virus de la Diarrea Epidémica Porcina/fisiología , Virus de la Diarrea Epidémica Porcina/genética , Fosforilación , Porcinos , Línea Celular , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/genética , Chlorocebus aethiopsRESUMEN
INTRODUCTION: Bacterial infection in the context of fracture repair remains a severe complication in trauma surgery and may result in long bone nonunion. Since treatment options for aseptic and infected nonunions vary greatly, diagnostic methods should ideally differentiate between these two entities as accurately as possible. Recently, contrast-enhanced ultrasound (CEUS) has been introduced as a preoperative imaging technique to evaluate hypervascularity at the fracture site as sign of bacterial infection. HYPOTHESIS: Preoperative CEUS predicts results of microbiological evaluation obtained either by culture of tissue samples or by analyzing the sonication fluid following removal and sonication of the implant. PATIENTS AND METHODS: Over the course of 6 months, 26 patients with long bone nonunions were included in this study. Patients' clinical data were evaluated. Tissue samples were collected intraoperatively and examined by standard microbiological techniques. The sonication method was applied to removed implants. Additionally, 1-3 days before surgery, CEUS was performed to determine hypervascularity at the nonunion site as a possible parameter for infection. RESULTS: Culture of tissue samples indicated infection in 50% of cases and implant sonication in 57.7% of cases. However, there was merely a fair agreement (κ=0.231) between these two diagnostic methods. CEUS predicted results of tissue culture reliably (sensitivity 92.3% and specificity 100%), whereas implant sonication showed no significant correlations with results from CEUS. Hypertrophic and atrophic nonunions were evaluated separately to determine possible differences in vascularity. We found that contrast peak enhancement of CEUS was similar in atrophic and hypertrophic nonunions with positive culture of tissue samples. Both differed significantly from culture negative cases (p=0.0016 and 0.0062). Results of implant-sonication positive or negative cases in atrophic and hypertrophic nonunions, however, were less clear and could be misleading. DISCUSSION: We were able to confirm CEUS as a valuable preoperative diagnostic tool that reliably predicts microbiology of tissue culture samples, but not of implant sonication. LEVEL OF EVIDENCE: I; diagnostic study.
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Infecciones Bacterianas , Infecciones Relacionadas con Prótesis , Humanos , Prótesis e Implantes , Infecciones Relacionadas con Prótesis/diagnóstico , Sonicación/métodos , UltrasonografíaRESUMEN
The partitioning and diffusion of solute elements in hot rolling and the effect of the partitioning and diffusion on the ferrite-bainite banding formation after hot rolling in the 20CrMnTi steel were experimentally examined by EPMA (electron probe microanalysis) technology and simulated by DICTRTA and MATLAB software. The austenite grain size related to the hot rolling process and the effect of austenite grain size on the ferrite-bainite banding formation were studied. The results show that experimental steel without banding has the most uniform hardness distribution, which is taken from the edge of the cast slab and 1/4 diameter position of the cast slab, heating at 1100 °C for 2 h and above 1200 °C for 2-4 h during the hot rolling, respectively. Cr, Mn, and Si diffuse and inhomogeneously concentrate in austenite during hot rolling, while C homogeneously concentrates in austenite. After the same hot rolling process, ΔAe3 increases and ferrite-bainite banding intensifies with increasing initial segregation width and segregation coefficient K of solute elements. Under the same initial segregation of solute elements, ΔAe3 drops and ferrite-bainite banding reduces with increasing heating temperature and extension heating time. When ΔAe3 drops below 14 °C, ferrite-bainite banding even disappears. What is more, the austenite grain size increases with increasing heating temperature and extension heating time. When the austenite grain size is above 21 µm, the experimental steel will not appear to have a banded structure after hot rolling.
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Gastric cancer is one of the most fatal diseases around the world. However, the mechanism of the development of gastric cancer is still not clarified. In addition, the anticancer drugs have cytotoxicity with different degrees. AnnexinA5, a member of the annexin family, has a great binding ability with the membrane phospholipid in a calcium dependent manner and is involved in the development of various cancers. This study aims to explore the influence of annexinA5 on human gastric cancer cells and whether it has the potential to be an auxiliary treatment to gastric cancer. In this study, the role of annexinA5 was detected from both the endogenous and the exogenous aspects on the gastric cancer cell lines MGC-803 and MKN-45. The cells were divided into a knockdown group in which RNA interference technique was used to suppress annexinA5 expression and a protein-supplementing group in which annexinA5 protein was added in the culture supernatant. After the suppression ratio of RNA interference was determined and the IC50 of annexinA5 protein was decided respectively, the cells' proliferation was detected by MTT assay, colony formation assay, and the expression of PCNA. FCM assay and PI staining methods were applied to test cell apoptosis and necrosis. To investigate whether ANXA5 influence cell metastasis, wound healing assay and transwell assay were employed. To further detect the mechanism of annexinA5 action, the signal pathway was examined with Western Blot method. When ANXA5 gene was knocked down, cell proliferation and metastasis were promoted, while cell apoptosis was suppressed. On the other hand, after the annexinA5 protein was applied to the gastric cancer cells, cell proliferation and metastasis were inhibited, while cell apoptosis and necrosis were promoted. AnnexinA5 played its role via ERK signal pathway. ANXA5 acted as tumor suppressor gene in the gastric cancer by suppressing ERK signal pathway and has the potentiality to be an auxiliary anticancer agent.
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OBJECTIVE: Numerous meta-analyses have revealed the association between gastro-oesophageal reflux disease (GORD) and a range of diseases; however, the certainty of the evidence remains unclear. This study aimed to summarise and assess the certainty of evidence derived from meta-analyses. METHODS: Embase, PubMed, Web of Science, Cochrane Databases of Systematic Reviews, CNKI and Wangfang databases from their inception to 22 February 2020 were queried for systematic reviews and meta-analyses on the association between GORD and various diseases. The methodological quality of the included studies was assessed using A Measurement Tool to Assess Systematic Reviews 2 (AMSTAR 2), and evidence certainty was evaluated using the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) system. Statistical analysis was conducted using Stata V.15. RESULTS: Ten publications with associations between GORD and different types of diseases were included. There was high heterogeneity (I2 >75%) among seven independent meta-analyses. Evidence for publication bias in two independent meta-analyses was also observed. According to the AMSTAR 2 approach, the methodological quality was high for 20% of meta-analyses, moderate for 10%, low for 40% and critically low for 30%. Based on GRADE approach, the certainty of evidence was high for the association between GORD and higher risk of chronic obstructive pulmonary disease (COPD) exacerbation (OR 5.37; 95% CI 2.71 to 10.64) and higher prevalence of oesophageal adenocarcinoma (OR 4.57; 95% CI 3.89 to 5.36), and it was moderate for the association between GORD and higher chronic rhinosinusitis prevalence (OR 2.16; 95% CI 1.37 to 3.48). CONCLUSION: The association between GORD and a range of diseases was extensively studied, and our findings revealed a high certainty of evidence of the association between GORD and an increased risk of COPD exacerbation as well as increased prevalence of oesophageal adenocarcinoma. Further investigations using systematic reviews and meta-analyses of high methodological quality that include prospective large cohort studies and adjusted confounders are warranted. PROSPERO REGISTRATION NUMBER: CRD42019122264.
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Reflujo Gastroesofágico , Sinusitis , Reflujo Gastroesofágico/epidemiología , Humanos , Prevalencia , Estudios Prospectivos , Revisiones Sistemáticas como AsuntoRESUMEN
PURPOSE: Aseptic implant loosening is still a feared complication in the field of orthopaedics. Presumably, a chronic inflammatory response is induced by wear particles, which leads to osteoclast generation, bone degradation and hence loosening of the implant. Since it has been demonstrated in the literature that most implants are in fact colonized by bacteria, the question arises whether aseptic implant loosening is truly aseptic. The aim of this study was to investigate a possibly enhanced inflammatory response to metal wear particles in the context of subclinical infection. PATIENTS AND METHODS: Tissue samples were collected intra-operatively from patients undergoing implant-exchange surgery due to aseptic loosening. Histopathological analysis was performed, as well as gene expression analysis for the pro-inflammatory cytokine Interleukin-8. By a series of in vitro experiments, the effect of metal wear particles on human monocytes, polymorphonuclear neutrophiles and osteoblasts was investigated. Additionally, minor amounts of lipoteichoic acid (LTA) and the bacterial heat shock protein GroEL were added. RESULTS: Histopathology of tissue samples revealed an accumulation of metal wear particles, as well as a cellular infiltrate consisting predominately of mononuclear cells. Furthermore, high expression of IL-8 could be detected in tissue surrounding the implant. Monocytes and osteoblasts in particular showed an increased release of IL-8 after stimulation with metal wear particles and in particular after stimulation with bacterial components and wear particles together. CONCLUSION: We were able to show that minor amounts of bacterial components and metal wear particles together induce an enhanced inflammatory response in human monocytes and osteoblasts. This effect could significantly contribute to the generation of bone-resorbing osteoclasts and hence implant-loosening.
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BACKGROUND: Spinal infections represent a therapeutic challenge. The often protracted course of the disease is accompanied by pain, which can lead to a chronic pain experience even after the infectious disease has been treated successfully. The aim of this study was to investigate possible risk factors of pain chronification. METHODS: In a prospective study, 14 patients with spinal infections were examined at admission (T1), at discharge from inpatient therapy (T2), and three to eight months postoperatively (T3) byquestionnaires on risk factors for pain chronification and by quantitative sensory testing (QST). RESULTS: In-patient treatment lasted on average 45.3 days (±33.13). The patients complained of pain for 3.43 months (±2.77) prior to inpatient treatment. The visual analogue scale (VAS) for pain (0-10) and the Oswestry Disability Index detected significant improvement in the course of the study. However, patients also reported catastrophic thinking, as well as fear of movement and (re)-injury. CONCLUSION: In summary, our results demonstrate that patients with spinal infections did not suffer from pain chronification, but might benefit from an interdisciplinary therapeutic approach, which emphasizes promoting active pain-coping strategies, as well as addressing fear of movement and catastrophic thinking.
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Following the publication of the above article, a number of errors were identified in the paper, and after having consulted with the editor of Molecular Medicine Reports, a corrigendum was published last year ("[Corrigendum] Differential miRNAomics of the synovial membrane in knee osteoarthritis induced by bilateral anterior cruciate ligament transection in rats." Zhou J, Zhao Y, Wu G, Lin B, Li Z and Liu X. Mol Med Rep 20: 5363, 2019). However, following publication of the above corrigendum, the paper was reexamined by the authors, and additional errors were identified; therefore, the authors are going to retract this paper from the publication. All the authors agree to this retraction, and apologize to the Editor of Molecular Medicine Reports and to the readership for any inconvenience caused. [the original article was published in Molecular Medicine Reports 18: 40514057, 2018; DOI: 10.3892/mmr.2018.9385].
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Following the publication of the above article, an interested reader drew to our attention that, in Fig. 4A, in which the authors had presented a western blot image depicting protein expression of IL18, IL1ß, NLRP3 and ßactin from synovial tissue lysates from osteoarthritic rats, upon close examination of the figure a striking similarity was noted between the bands shown for the IL18 and NLRP3stained Sham group experiments, although the bands appeared in an inverted position relative to each other. Following an enquiry with the authors, they realized that they had included incorrect data for this figure; an amended version of Fig. 4, showing the correct data for NLRP3, is shown opposite. Secondly, the authors have realized that, at various points throughout the paper, two miRNAs were written incorrectly: Specifically, references to an 'miR352' should have appeared as miR532, and 'miR233' should have been written as miR223. This error affected the presentation of Fig. 2; therefore, a revised version of this figure, with the miRNAs correctly labelled as miR532 and miR223 respectively, is also shown opposite. Furthermore, mi233 should have been written as miR223 at the following places in the text: p. 4051, righthand column (RHC), line 10 ("Furthermore, the miR223regulated...); p. 4054, Results section, lefthand column (LHC), third subheading ("miR223 negatively regulates the expression of NLRP3."); and in the concluding paragraph of the Discussion on p. 4056, LHC, miR233 should have been written as miR223 in all five instances where this occurred (lines 4, 7, 8, 9, and 10 of this paragraph). Finally, the second sentence featured in the subsection of the Results section entitled "Expression validation of miRs by RTqPCR" on p. 4054 contained additional errors. This sentence should have appeared as follows (changed text is highlighted in bold): "The expression of miR223, 100, 345, 130, 382, 9a and 183 were upregulated, whereas miR377, 532, 200b were downregulated with a fold change of ≥1.5, similar to the microarray data (Fig. 2). All the authors agree to the contents of this Corrigendum, and apologize to the Editor of Molecular Medicine Reports and to the readership for any inconvenience caused. [the original article was published in Molecular Medicine Reports 18: 40514057, 2018; DOI: 10.3892/mmr.2018.9385].
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BACKGROUND: Hepatocellular carcinoma is one of the most fatal malignancies worldwide with high lethality. However, the exact mechanism of liver tumorigenesis is still unclear. AnnexinA7 (ANXA7) is a Ca2+-binding protein which is involved in membrane organization and dynamics and indicated a role of ANXA7 in cancer. However, the action of ANXA7 in hepatocellular carcinoma and the relative mechanism is still indistinct. OBJECTIVE: To gain more insight into the biological function of ANXA7 and assess its possible influence on proliferation and metastasis capacity of human hepatocellular carcinoma cells with the relative mechanism. METHODS: ANXA7 was down-regulated by RNA interference in both HepG2 and smmc-7721 cells. The decreased cell proliferation was detected by MTT method and colony formation assay. To confirm the result of cell proliferation, Ki-67 and cyclinD1 expression was examined by Western Blot. The increased apoptosis capacity of the cells was detected with cell cytometry and PI staining respectively. Bcl-2 and Bax expression was further investigated by Western blot and the decreased ration of Bcl-2/Bax might explain the increased apoptosis. RESULTS: Cell metastasis showed significantly limited ability which was tested by wound healing assay and Transwell assay. Meanwhile, the key biomarkers of cell metastasis E-cadherin expression increased while MMP-9 decreased. Furthermore, we found that ANXA7 played its role via MAPK/ERK pathway. CONCLUSIONS: ANXA7 might involve in the development of hepatocellular carcinoma and act as an oncogene which might be a potential therapeutic target for treatment.
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Anexina A7/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Metaloproteinasa 9 de la Matriz/genética , Apoptosis/genética , Biomarcadores de Tumor/genética , Cadherinas/genética , Carcinoma Hepatocelular/patología , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Humanos , Neoplasias Hepáticas/patología , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
The differential microRNA (miRNA) omics of the synovial membrane were investigated using a rat model of knee osteoarthritis (KOA) induced by bilateral anterior cruciate ligament transection, which produced pathological biomarkers in KOA. SpragueDawley rats were randomly divided into two groups; Shamoperated and KOAoperated group. The KOA rats were subjected to bilateral anterior cruciate ligament transection. After 6 weeks, total RNA was extracted from the knee joint synovial membrane of the rats and a microRNA (miR) microarray was performed to identify differentially expressed miRs. Subsequently, the obtained differentially expressed miRs were validated by reverse transcriptionquantitative polymerase chain reaction (RTqPCR) analysis. A total of 24 miRs were identified with alterations ≥1.5fold in the synovial membrane in the KOAoperated group compared with the shamoperated group, of which 4 miRs (miR5325p, 200b5p, 3773p and 7595p) were decreased and 20 miRs (miR3823p, 2233p, 1005p, 30d5p, 1835p, 130, 92b3p, 125b3p, 1513p, 1553p, 27a3p, 146b3p, 8855p, 352, 184, 3455p, 30a5p and 9a5p) were increased. Subsequently, RTqPCR was used to validate the expressions of miR223, 100, 345, 130, 382, 377, 352, 200b, 9a and 183, which were upregulated by a fold change of ≥1.5 in synovial membranes of KOA rats compared with shams. Furthermore, in vitro miR223 mimic could suppress the luciferase activity of NACHT, LRR and PYD domainscontaining protein 3 (NLRP3) 3' untranslated region by detecting of dual luciferase reporter vector. Additionally, the expression of NLRP3, interleukin (IL)1ß and IL18 significantly increased in the synovial membrane of KOA rats. A total of 24 different miRs were determined by comparing the miRNAomics in the synovial membrane of the KOA model rats. Furthermore, the miR233regulated NLRP3 inflammasome was implicated in synovial membrane injury, which may be an important mechanism of KOA pathogenesis.
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Lesiones del Ligamento Cruzado Anterior/genética , Ligamento Cruzado Anterior/patología , Perfilación de la Expresión Génica , MicroARNs/genética , Osteoartritis de la Rodilla/genética , Membrana Sinovial/metabolismo , Animales , Lesiones del Ligamento Cruzado Anterior/patología , Inflamasomas/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Masculino , MicroARNs/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Osteoartritis de la Rodilla/patología , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Membrana Sinovial/patologíaRESUMEN
BACKGROUND: Annexin A5 (ANXA5) is a kind of Ca2+-dependent phospholipid binding protein which is involved in cell membrane dynamics and organization. Recent data showed that ANXA5 might involve in tumorigenesis. OBJECTIVE: To explore what role ANXA5 play in human uterine cervical carcinoma. MATERIALS AND METHODS: In this study, a recombined ANXA5 plasmid was constructed and uterine cervical carcinoma cell lines HeLa and SiHa were transfected with it. After ANXA5 overexpression was determined by Western Blot, cell proliferation test was detected by MTT assay and colony formation assay respectively. FACS assay and Hochest33258 staining methods were employed to detect cell apoptosis. To further investigate whether ANXA5 influence cell migration and invasion, wound healing assay and transwell assay were applied. At the same time, the relative mechanism was investigated. RESULTS: When ANXA5 expression increased, cell proliferation was inhibited by regulating the expression of bcl-2 and bax while cell metastasis was suppressed by regulating E-cadherin and MMP-9 expression. CONCLUSION: ANXA5 overexpression in the uterine cervical carcinoma might play important roles in cell proliferation and metastasis of uterine cervical cancer cells and act as an anti-cancer gene in uterine cervical cancer.
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Anexina A5/genética , Cadherinas/genética , Metaloproteinasa 9 de la Matriz/genética , Neoplasias del Cuello Uterino/genética , Apoptosis/genética , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transfección , Neoplasias del Cuello Uterino/patología , Proteína X Asociada a bcl-2/genéticaRESUMEN
In this communication, a concentrated solar light (CSL) annealing strategy is proposed with a Fresnel lens as the concentrator for rapid and effective crystallization of nanomaterials. More interestingly, the CSL can be integrated into photoelectrochemical devices and achieved an unprecedented photocurrent density.
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A method for grafting ethylenediamine to a magnetic graphene oxide composite (EDA-GO@Fe3O4) was developed for Cr(VI) decontamination. The physicochemical properties of EDA-GO@Fe3O4 were characterized using HRTEM, EDS, FT-IR, TG-DSC, and XPS. The effects of pH, sorbent dose, foreign anions, time, Cr(VI) concentration, and temperature on decontamination process were studied. The solution pH can largely affect the decontamination process. The pseudo-second-order model is suitable for being applied to fit the adsorption processes of Cr(VI) with GO@Fe3O4 and EDA-GO@Fe3O4. The intra-particle diffusion is not the rate-controlling step. Isotherm experimental data can be described using the Freundlich model. The effects of multiple factors on the Cr(VI) decontamination was investigated by a 25-1 fractional factorial design (FFD). The adsorption process can significantly be affected by the main effects of A (pH), B (Cr(VI) concentration), and E (Adsorbent dose). The combined factors of AB (pH × Cr(VI) concentration), AE (pH × Adsorbent dose), and BC (Cr(VI) concentration × Temperature) had larger effects than other factors on Cr(VI) removal. These results indicated that EDA-GO@Fe3O4 is a potential and suitable candidate for treatment of heavy metal wastewater.
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Cromo/aislamiento & purificación , Etilenodiaminas/química , Óxido Ferrosoférrico/química , Grafito/química , Adsorción , Concentración de Iones de Hidrógeno , Microscopía Electrónica de Transmisión , TermodinámicaRESUMEN
In this communication, we report the first demonstration of an efficient photoelectrochemical aptasensor based on sputtering Au nanoparticle-modified nanoporous BiVO4 for the excellent sensitive and selective detection of thrombin with a low detection limit of 0.5 pM and a large linear range.
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Bismuto/química , Técnicas Electroquímicas , Oro/química , Nanopartículas del Metal/química , Trombina/análisis , Vanadatos/química , Tamaño de la Partícula , Procesos Fotoquímicos , Porosidad , Propiedades de SuperficieRESUMEN
Sensitive and specific detection of disease-related gene and single nucleotide polymorphism (SNP) is of great importance in cancer diagnosis. Here, a colorimetric and fluorescent approach is described for detection of the p53 gene and SNP in homogeneous solution by using gold nanorods (GNRs) as both colorimetric probe and fluorescence quencher. Hairpin oligonucleotide was utilized as DNA probe to ensure highly sequence-specific detection of target DNA. In the presence of target DNA, the formation of DNA duplex greatly changed the electrostatic interaction between GNR and DNAs, leading to an obvious change in fluorescence and colorimetric response. The detection limit of fluorescent and colorimetric assay is 0.26 pM and 0.3 nM, respectively. Both fluorescence and colorimetric strategies were able to effectively discriminate complementary DNA from single-base mismatched DNA, which is meaningful for cancer diagnosis. More important, target DNA can be detected as low as 10 nM by the naked eye. Furthermore, transmission electron microscopy and fluorescence anisotropy measurements demonstrated that the color change as well as fluorescence quenching is ascribed to the DNA hybridization-induced aggregation of GNRs. Therefore, the assay provided a fast, sensitive, cost-effective, and specific sensing platform for detecting disease-related gene and SNP.
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ADN/química , Fluorescencia , Oro/química , Mutación , Nanotubos/química , Neoplasias/genética , Polimorfismo de Nucleótido Simple , Colorimetría/métodos , ADN/genética , Sondas de ADN/química , Sondas de ADN/genética , Humanos , Neoplasias/diagnósticoRESUMEN
A label-free fluorescent AND logic gate has been developed utilizing ion-tuned configuration conversion of DNA probe with K(+) and Pb(2+) as two inputs. A well-designed hairpin DNA which is composed of a poly-G loop and a GR-5 DNAzyme stem serves as a recognition probe, and an derivative of aloe-emodin (AED) was designed and synthesized as signal probe. In the presence of Pb(2+), the substrate strand of DNAzyme is irreversibly and specifically cleaved at the cleavage site, which made the poly-G loop form G-quadruplex in the presence of a constant concentration of K(+). Such a structural change significantly affects the spectral behaviors of AED, which can be explored to ultra-sensitively detect Pb(2+) with a limit of detection of 22.8pM. By combing the high specificity of hairpin DNA and GR-5 DNAzyme, Pb(2+) can be highly selectively detected even when coexisted with other metal ions. Circular dichroism (CD), UV-vis absorption spectrometry and fluorescence polarization (FP) measurements further verified the reliability and reasonability of the sensing mechanism. Therefore, it provides a simple and label-free approach to detect ions with high sensitivity and specificity, and promises to provide a solid sensing platform for the detection of targets by altering the specific sequence of nucleic acid probe.
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Técnicas Biosensibles/instrumentación , ADN/química , G-Cuádruplex , Plomo/análisis , Procesamiento de Señales Asistido por Computador/instrumentación , Espectrometría de Fluorescencia/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Iones , Plomo/químicaRESUMEN
Specific and homogeneous detection of heavy metal ion is of great importance for both human health care and environmental protection. We reported a highly specific and sensitive assay for fluorescent detection of Pb(2+) based on the difference in quenching ability between deoxyguanosines and G-quartet by using carboxyfluorescein-labeled hairpin DNA (F-hpDNA) as a recognition probe. In the absence of target, the fluorescence of F-hpDNA can be quenched through photoinduced electron transfer from the dye to deoxyguanosines because the formation of hairpin brings deoxyguanosines close to the FAM. In the presence of Pb(2+), the formation of G-quadruplex DNA leads to a significant decrease in fluorescence due to the effective stack of dye on the G-quartet, which obviously intensified the quenching of fluorophore. In comparison with linear DNA probe, hairpin DNA probe greatly improved the specificity, and Pb(2+) can be highly selective detected even when coexisted with other metal ions. The quenching efficiency is linear with the concentration of lead(II) over the range of 0.5-500 nM, with a limit of detection of 0.4 nM. Conformational switch from hairpin to G-quadruplex was verified by CD measurements. Moreover, the application for detection of real samples further demonstrated its reliability. Therefore, it is a selective, simple and sensitive approach for detection of lead ion, as such, it promises to provide a solid foundation for developing universal analytical method for heavy metal ions.