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1.
Food Chem ; 305: 125429, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31505415

RESUMO

A simple and rapid magnetic solid-phase extraction (MSPE) method using PEGylated multi-walled carbon nanotubes magnetic nanoparticles (PEG-MWCNTs-MNP) as absorbents is proposed for isolation and enrichment of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), aflatoxin M1 (AFM1), aflatoxin M2 (AFM2), ochratoxin A (OTA), zearalenone (ZEA), zearalanone (ZAN), α-zeralanol (α-ZAL), ß-zeralanol (ß-ZAL), α-zeralenol (α-ZOL), and ß-zeralenol (ß-ZOL) from liquid milk. Combined with ultra-high performance liquid chromatography Q-Exactive high resolution mass spectrometry, simultaneous qualification of these mycotoxins was achieved with sensitivity and specificity. The proposed method showed a good linearity (R2 ≥ 0.995), high sensitivity (limit of detection in the range of 0.005-0.050 µg/kg and limit of quantification in the range of 0.015-0.150 µg/kg), adequate recovery (81.8-106.4%), and good repeatability (intra-day precision in the range of 2.1-8.5% and inter-day precision in the range of 3.9-11.7%). It has been successfully applied to the determination of 13 mycotoxins in real liquid milk samples.

2.
J Vis Exp ; (149)2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31403631

RESUMO

In plant mitochondria, some steady-state transcripts have 5' triphosphate derived from transcription initiation (primary transcripts), while the others contain 5' monophosphate generated post-transcriptionally (processed transcripts). To discriminate between the two types of transcripts, several strategies have been developed, and most of them depend on presence/absence of 5' triphosphate. However, the triphosphate at primary 5' termini is unstable, and it hinders a clear discrimination of the two types of transcripts. To systematically differentiate and map the primary and processed transcripts stably accumulated in maize mitochondrion, we have developed a circular RT-PCR (cRT-PCR)-based strategy by combining cRT-PCR, RNA 5' polyphoshpatase treatment, quantitative RT-PCR (RT-qPCR), and Northern blot. As an improvement, this strategy includes an RNA normalization step to minimize the influence of unstable 5' triphosphate. In this protocol, the enriched mitochondrial RNA is pre-treated by RNA 5' polyphosphatase, which converts 5' triphsophate to monophosphate. After circularization and reverse transcription, the two cDNAs derived from 5' polyphosphatase-treated and non-treated RNAs are normalized by maize 26S mature rRNA, which has a processed 5' end and is insensitive to 5' polyphosphatase. After normalization, the primary and processed transcripts are discriminated by comparing cRT-PCR and RT-qPCR products obtained from the treated and non-treated RNAs. The transcript termini are determined by cloning and sequencing of the cRT-PCR products, and then verified by Northern blot. By using this strategy, most steady-state transcripts in maize mitochondrion have been determined. Due to the complicated transcript pattern of some mitochondrial genes, a few steady-state transcripts were not differentiated and/or mapped, though they were detected in a Northern blot. We are not sure whether this strategy is suitable to discriminate and map the steady-state transcripts in other plant mitochondria or in plastids.

3.
Pestic Biochem Physiol ; 158: 47-53, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31378360

RESUMO

Buprofezin is a chitin synthesis inhibitor that is very effective against Homopteran pests, such as the white-backed planthopper (WBPH), S. furcifera (Horvath). In the present study, resistance selection, cross-resistance and mechanisms of buprofezin resistance were investigated in this planthopper species. However, the mechanism associated with resistance to growth regulator insecticides (IGRs) remains largely unknown. A resistant strain (Bup-R) with a resistance level (22-fold) to buprofezin was developed through continuous selection for 47 generations from a laboratory susceptible strain (Bup-S). The results showed that the Bup-R exhibited no cross-resistance to other tested insecticides. Synergism tests showed that piperonyl butoxide (PBO) (SR = 3.9-fold) and diethyl maleate (DEM) (SR = 1.8-fold) had synergistic effects on buprofezin toxicity in the resistant strain (F47). Enzyme activity results revealed an approximate 5.7-fold difference in cytochrome P450 monooxygenase and a 2-fold difference in glutathione S-transferase (GST) between the resistant and susceptible strains, suggesting that the increased activity of these two enzymes is likely the main detoxification mechanism involved in resistance to buprofezin in this species. Furthermore, the mRNA expression levels of cytochrome P450 (CYP) and GST genes by quantitative real-time PCR results indicated that sixteen P450 and one GST gene were significantly overexpressed in the Bup-R strain, among which thirteen P450 genes and one GST gene were >2-fold higher than in the Bup-S strain. The present study increases our knowledge of the buprofezin resistance mechanism in S. furcifera and provides a useful reference for integrated pest management (IPM) strategies.


Assuntos
Hemípteros/efeitos dos fármacos , Inseticidas/farmacologia , Tiadiazinas/farmacologia , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Hemípteros/metabolismo , Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Maleatos/metabolismo , Butóxido de Piperonila/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
4.
Neurobiol Dis ; 132: 104585, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31445164

RESUMO

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by maternal mutation and paternal imprinting of the gene encoding UBE3A, an E3 ubiquitin ligase. Although several potential target proteins of UBE3A have been reported, how these proteins regulate neuronal development remains unclear. We performed a large-scale quantitative proteomic analysis using stable-isotope labeling of amino acids in mammals (SILAM) in mice with maternal Ube3a mutation. We identified huntingtin (Htt)-associated protein (HAP1), a protein that is involved in Huntington's disease (HD), as a new target of UBE3A. We demonstrate that HAP1 regulates autophagy at the initiation stage by promoting PtdIns3K complex formation and enhancing its activity. HAP1 also co-localized with MAP1LC3 (LC3) and other proteins involved in autophagosome expansion. As a result, HAP1 increased autophagy flux. Strikingly, knocking down of HAP1 alleviated aberrant autophagy in primary neurons from AS mice. Concordantly, treatment of AS neurons with an autophagy inhibitor alleviated the reduction in density of dendritic spines. Furthermore, autophagy inhibition in AS mice partially alleviated a social interaction deficit as shown in open field test. Thus, our results identify HAP1 as an in vivo UBE3A target that contributes to deregulated autophagy and synaptic dysfunction in the central nervous system of AS mouse.

5.
Pestic Biochem Physiol ; 157: 26-32, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31153474

RESUMO

Nitenpyram is very effective in controlling Nilaparvata lugens (brown planthopper, BPH), and its resistance has been reported in field populations; however, the resistance mechanism remains unclear. In the present study, cross-resistance and resistance mechanisms in nitenpyram-resistant BPH were investigated. A resistant strain (NR) with a high resistance level (164.18-fold) to nitenpyram was evolved through successive selection for 42 generations from a laboratory susceptible strain (NS). The bioassay results showed that the NR exhibited cross-resistance to imidacloprid (37.46-fold), thiamethoxam (71.66-fold), clothianidin (149.17-fold), dinotefuran (98.13-fold), sulfoxaflor (47.24-fold), cycloxaprid (9.33-fold), etofenprox (10.51-fold) and isoprocarb (9.97-fold) but not to triflumezopyrim, chlorpyrifos and buprofezin. The NR showed a 3.21-fold increase in cytochrome P450 monooxygenase (P450) activity compared to that in the NS, while resistance was also synergized (4.03-fold) with the inhibitor piperonyl butoxide (PBO), suggesting a role of P450. Furthermore, the mRNA expression levels of cytochrome P450 (CYP) genes by quantitative real-time PCR results indicated that twelve P450 genes were significantly overexpressed in the NR strain, especially CYP6ER1 (203.22-fold). RNA interference (RNAi) suppression of CYP6ER1 through injection of dsCYP6ER1 led to significant susceptibility in the NR strain. The current study expands our understanding of the nitenpyram resistance mechanism in N. lugens, provides an important reference for integrated pest management (IPM), and enriches the theoretical system of insect toxicology.


Assuntos
Hemípteros/efeitos dos fármacos , Neonicotinoides/farmacologia , Animais , Carbamatos/farmacologia , Guanidinas/farmacologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Resistência a Inseticidas , Nitrocompostos/farmacologia , Piretrinas/farmacologia , Piridinas/farmacologia , Pirimidinonas/farmacologia , Interferência de RNA , Tiazóis/farmacologia
6.
Autophagy ; : 1-17, 2019 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-31204559

RESUMO

Mutations in the macroautophagy/autophagy gene WDR45 cause ß-propeller protein-associated neurodegeneration (BPAN); however the molecular and cellular mechanism of the disease process is largely unknown. Here we generated constitutive wdr45 knockout (KO) mice that displayed cognitive impairments, abnormal synaptic transmission and lesions in several brain regions. Immunohistochemistry analysis showed loss of neurons in prefrontal cortex and basal ganglion in aged mice, and increased apoptosis in prefrontal cortex, recapitulating a hallmark of neurodegeneration. Quantitative proteomic analysis showed accumulation of endoplasmic reticulum (ER) proteins in KO mouse. At the cellular level, accumulation of ER proteins due to WDR45 deficiency resulted in increased ER stress and impaired ER quality control. The unfolded protein response (UPR) was elevated through ERN1/IRE1 or EIF2AK3/PERK pathway, and eventually led to neuronal apoptosis. Suppression of ER stress or activation of autophagy through MTOR inhibition alleviated cell death. Thus, the loss of WDR45 cripples macroautophagy machinery in neurons and leads to impairment in organelle autophagy, which provides a mechanistic understanding of cause of BPAN and a potential therapeutic strategy to treat this genetic disorder. Abbreviations: 7-ADD: 7-aminoactinomycin D; ASD: autistic spectrum disorder; ATF6: activating transcription factor 6; ATG: autophagy-related; BafA1: bafilomycin A1; BCAP31: B cell receptor associated protein 31; BPAN: ß-propeller protein-associated neurodegeneration; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CDIPT: CDP-diacylglycerol-inositol 3-phosphatidyltransferase (phosphatidylinositol synthase); DDIT3/CHOP: DNA-damage inducible transcript 3; EIF2A: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; GFP: green fluorescent protein; HIP: hippocampus; HSPA5/GRP78: heat shock protein family A (HSP70) member 5; KO: knockout; LAMP1: lysosomal-associated membrane 1; mEPSCs: miniature excitatory postsynaptic currents; MG132: N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal; MIB: mid-brain; MTOR: mechanistic target of rapamycin kinase; PCR: polymerase chain reaction; PFA: paraformaldehyde; PFC: prefrontal cortex; PRM: parallel reaction monitoring; RBFOX3/NEUN: RNA binding protein, fox-1 homolog [C. elegans] 3; RTN3: reticulon 3; SEC22B: SEC22 homolog B, vesicle trafficking protein; SEC61B: SEC61 translocon beta subunit; SEM: standard error of the mean; SNR: substantia nigra; SQSTM1/p62: sequestosome 1; TH: tyrosine hydroxylase; Tm: tunicamycin; TMT: tandem mass tag; TUDCA: tauroursodeoxycholic acid; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling; UPR: unfolded protein response; WDR45: WD repeat domain 45; WT: wild type; XBP1: X-box binding protein 1.

7.
Mol Cancer ; 18(1): 98, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118036

RESUMO

Cancer-associated chromosomal translocations are reported to generate oncogenic circular RNA (circRNA), contributing to tumorigenesis. The fusion gene SLC34A2-ROS1 (solute carrier family 34 member 2 and ROS proto-oncogene 1) plays an important role in non-small cell lung cancer (NSCLC) progression. However, whether SLC34A2-ROS1 gene can produce circRNA remains unknown. Here, we identified two novel circRNAs (F-circSR1 and F-circSR2) generated from SLC34A2-ROS1 fusion gene, while F-circSR1 has higher expression than F-circSR2. Functional studies through gain- and loss-of-function strategies showed that both F-circSRs promote cell migration in lung cancer cells, whereas they have little effect on cell proliferation. Using the minigene GFP reporter assay, we verified that the flanking complementary sequences with canonical splicing sites are essential for F-circSR biogenesis. Therefore, our findings demonstrate the oncogenic role of F-circSR in NSCLC and highlight its therapeutic potential.

8.
J Econ Entomol ; 112(4): 1866-1874, 2019 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-31081902

RESUMO

In this study, the sensitivity of 20 field populations of Chilo suppressalis (Walker) from five provinces in China to seven insecticides was evaluated during 2016-2018. The results indicated that 20 field populations of C. suppressalis had evolved moderate to high levels of resistance to triazophos (RR 64.5-461.3) and chlorpyrifos (RR 10.1-125.0). Furthermore, C. suppressalis exhibited low to moderate levels of resistance to abamectin (RR 6.5-76.5) and decreased susceptibility to cyantraniliprole (RR 1.0-34.0). The population collected from Nanchang in Jiangxi Province (JXNC) showed high resistance to chlorantraniliprole (RR 148.3-294.3), and other geographical populations remained susceptible to moderate levels of resistance (RR 1.0-37.5). In contrast, C. suppressalis remained susceptible to low levels of resistance to spinetoram (RR 1.0-6.7) and spinosad (RR 1.0-4.6). Significant correlations were found between the Log LC50 values of chlorantraniliprole and cyantraniliprole, chlorpyrifos and triazophos, as well as cyantraniliprole and chlorpyrifos and triazophos. Similarly, significant correlations were found among abamectin, chlorpyrifos, and triazophos. In addition, a significant correlation was also observed between the activity of the detoxification enzymes and the log LC50 values of chlorantraniliprole, cyantraniliprole, abamectin, chlorpyrifos, and triazophos. The findings provide an important reference for implementing effective resistance management strategies and the development of new insecticides in insect pest control.

9.
Pest Manag Sci ; 75(11): 2981-2988, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30884104

RESUMO

BACKGROUND: Sulfoxaflor has been considered as a new tool for Nilaparvata lugens control in the field. In this study, a laboratory-selected resistant strain and a susceptible strain were used to evaluate the inheritance and fitness costs of sulfoxaflor resistance in N. lugens. RESULTS: The resistant strain (SFX-SEL) showed 123.63-fold resistance compared with the susceptible strain (SS). Progenies of reciprocal crosses (F1 RS and F1 SR) showed similar concentration-mortality responses (LC50 ) to sulfoxaflor and also exhibited a similar degree of dominance; -0.16 for F1 RS and -0.26 for F1 SR. Significant differences between the observed and expected mortalities of F2 individuals suggested that sulfoxaflor resistance is associated with multiple genes. The resistant strain had a relative fitness of 0.75 with substantially decreased female adult period, oviposition days, total fecundity, egg hatchability and female adult survival rate, and prolonged pre-adult period and total pre-oviposition period. CONCLUSION: Sulfoxaflor resistance in N. lugens was inherited as autosomal, incompletely recessive and multigene. The development of resistance may have a significant fitness cost for the resistant population. Current research provides valuable information for researchers to establish management strategies to delay the development of sulfoxaflor resistance and control N. lugens sustainably in the field. © 2019 Society of Chemical Industry.

10.
Pestic Biochem Physiol ; 154: 39-45, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30765055

RESUMO

The brown planthopper, Nilaparvata lugens (Stål), is one of the most economically important rice pests in Asia and has become resistant to various kinds of insecticides, including neonicotinoid insecticides. In this study, an N. lugens clothianidin-resistant (CLR) strain and a susceptible (CLS) strain were established, and the potential resistance mechanisms of N. lugens to clothianidin were elucidated. The cross-resistance studies showed that the clothianidin-resistant strain exhibited cross-resistance to most neonicotinoid insecticides, especially nitenpyram (99.19-fold) and dinotefuran (77.68-fold), while there was no cross-resistance to chlorpyrifos (1.79-fold). The synergism assays and the activities of the detoxification enzymes were performed, and we found that a cytochrome P450 conferred the clothianidin resistance. Two P450 genes (CYP6ER1 and CYP6AY1) were found to be significantly overexpressed in the CLR strain compared with the CLS strain based on qRT-PCR. In addition, the knockdown of CYP6ER1 by RNA interference dramatically increased the toxicity of clothianidin against N. lugens. These data demonstrated that the overexpression of CYP6ER1 could contribute to clothianidin resistance in N. lugens. Our findings will help to improve the design of effective resistance management strategies to control brown planthoppers.


Assuntos
Família 6 do Citocromo P450/genética , Guanidinas/toxicidade , Hemípteros/efeitos dos fármacos , Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Inseticidas/toxicidade , Neonicotinoides/toxicidade , Ninfa/efeitos dos fármacos , Tiazóis/toxicidade , Animais , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hemípteros/fisiologia , Ninfa/fisiologia
11.
J Sep Sci ; 42(6): 1289-1298, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30653844

RESUMO

In this work, monoamine oxidase B was immobilised onto magnetic nanoparticles to prepare a new type of affinity solid-phase extraction adsorbent, which was used to extract the possible anti-neurodegenerative components from the Lonicera japonica flower extracts. Coupled with high-performance liquid chromatography with mass spectrometry, two monoamine oxidase B ligands were fished-out and identified as isochlorogenic acid A and isochlorogenic acid C, which were found to be inhibitors of the enzyme for the first time, with similar half maximal inhibitory concentration values of 29.05 ± 0.49 and 29.77 ± 1.03 µM, respectively. Furthermore, equilibrium-dialysis dissociation assay of enzyme-inhibitor complex showed that both compounds have reversible binding patterns to monoamine oxidase B, and kinetic analysis demonstrated that they were mixed-type inhibitors for monoamine oxidase B, with Ki and Kis values of 9.55 and 37.24 µM for isochlorogenic acid A, 9.53 and 35.50 µM for isochlorogenic acid C, respectively. The results indicated that isochlorogenic acid A and isochlorogenic acid C were the major active components responsible for the anti-degenerative activity of the flowers of L. japonica, while magnetic nanoparticles immobilised monoamine oxidase B could serve as an efficient solid-phase extraction adsorbent to specifically extract monoamine oxidase B inhibitors from complex herbal extracts.


Assuntos
Lonicera/química , Nanopartículas de Magnetita/química , Monoaminoxidase/química , Fármacos Neuroprotetores/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Flores/química , Ligantes , Lonicera/metabolismo , Monoaminoxidase/metabolismo , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/metabolismo , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Extração em Fase Sólida
12.
RNA Biol ; 16(1): 104-117, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30585757

RESUMO

In plant mitochondria, some steady-state transcripts contain primary 5' ends derived from transcription initiation, while the others have processed 5' termini generated by post-transcriptional processing. Differentiation and mapping of the primary and processed transcripts are important for unraveling the molecular mechanism(s) underlying transcription and transcript end maturation. However, previous efforts to systematically differentiate these two types of transcripts in plant mitochondria failed. At present, it is considered that the majority of mature mRNAs may have processed 5' ends in Arabidopsis. Here, by combination of circular RT-PCR, quantitative RT-PCR, RNA 5'-polyphosphatase treatment and Northern blot, we successfully discriminated and mapped the primary and processed transcripts in maize mitochondria. Among the thirty-five mature and eight precursor RNAs analyzed in this study, about one half (21/43) were found to have multiple isoforms. In total, seventy-seven steady-state transcripts were determined, and forty-seven of them had primary 5' ends. Most transcription initiation sites (126/167) were downstream of a crTA-motif. These data suggested a major contribution of transcription initiation to 5'-end formation of steady-state transcripts in maize mitochondria. Moreover, the mapping results revealed that mature RNA termini had largely been formed before trans-splicing, and C→U RNA editing was accompanied with trans-splicing and transcript end formation in maize mitochondria.


Assuntos
Regulação da Expressão Gênica de Plantas , Mitocôndrias/genética , Transcrição Genética , Zea mays/genética , Regiões 5' não Traduzidas , Mitocôndrias/metabolismo , Conformação de Ácido Nucleico , Edição de RNA , Processamento Pós-Transcricional do RNA , RNA Mensageiro/química , RNA Mensageiro/genética , Iniciação da Transcrição Genética , Zea mays/metabolismo
13.
Pest Manag Sci ; 2018 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-30488546

RESUMO

BACKGROUND: Sulfoxaflor is a new insecticide for controlling Nilaparvata lugens in the field. The present study was conducted to investigate the risk of resistance development, the cross-resistance spectrum and the resistance mechanisms of sulfoxaflor resistance in N. lugens. RESULTS: A sulfoxaflor-resistant strain was obtained in the laboratory by successive selection with sulfoxaflor for 39 generations from a field population. Sulfoxaflor-resistant populations showed significant levels of cross-resistance to dinotefuran, nitenpyram, thiamethoxam, clothianidin, imidacloprid, and cycloxaprid. However, they exhibited only minor or no cross-resistance to isoprocarb, etofenprox, chlorpyrifos, triflumezopyrim, and buprofezin. Sulfoxaflor was synergized by the inhibitor piperonyl butoxide (PBO) in SFX-SEL with 2.69-fold relative synergistic ratios compared with UNSEL. Compared to UNSEL, the P450 enzyme activity of SFX-SEL was increased 3.50 times, and eight P450 genes were upregulated more than 2.0-fold in SFX-SEL. RNAi reduced the expression of CYP6ER1 (36.87-fold change) and significantly enhanced the susceptibility of SFX-SEL to sulfoxaflor. CONCLUSION: Resistance development and cross-resistance risk of sulfoxaflor-resistance in N. lugens is evident. The enhanced detoxification of P450 enzymes caused by upregulation of several P450 genes is considered to be the metabolic resistance mechanism. These results suggest that CYP6ER1 might play an important role in sulfoxaflor resistance in N. lugens. This article is protected by copyright. All rights reserved.

14.
J Sep Sci ; 41(19): 3772-3781, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30152917

RESUMO

A microchip capillary electrophoresis coupled with laser induced fluorescence detection method for the fast determination of aloin was developed and comprehensively applied for the quantification of aloin A and B present in seven aloe plant species, 42 aloin-containing crude drugs, ten aloe pharmaceutical preparations, and four aloe gel-containing functional foods. The excitation and emission wavelengths for detection of both aloins were set at 473 and 520 nm, respectively. Sample analysis on a 35 mm length of glass microchip channel was completed within 40 s. An interference study indicated that the other main anthraquinones present in the samples did not interrupt with the target aloins detection, demonstrating the good selectivity of this method. It is demonstrated that this method is fast, facile, and specific for determination of aloin A and B from matrix samples which can be applied to the quality control of a wide varieties of aloe species and aloe-derived products.

15.
J Chromatogr A ; 1569: 222-228, 2018 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-30037541

RESUMO

An aptamer-based microchip capillary electrophoresis coupled with laser induced fluorescence (MCE-LIF) detection method for fast determination of Aflatoxin B1 (AFB1) and Ochratoxin A (OTA) was developed. Aptamers that are specific to these two mycotoxins were first hybridized with their aptamer complementary oligonucleotides. The double strand DNA that comes in contact with mycotoxin-containing environment would be unwound into separate aptamer-mycotoxin complex and aptamer complementary single strand. Different types of oligonucleotides can be separated in MCE and detected under the aid of fluorescent dye SYBR gold in LIF detection unit. Under the optimal conditions, on-chip aptamer-mycotoxin conjugates analysis was achieved within 3 min with extremely low LODs (0.026 ng/mL for AFB1 and 0.021 ng/mL for OTA). Specificity study indicated that other major mycotoxins would not cross-react with these two aptamers, demonstrating the good selectivity of the proposed method. Quantification of trace AFB1 and OTA in real food samples was carried out and satisfactory recoveries were obtained. It is demonstrated that this method is fast, facile and specific for Simultaneous determination of trace AFB1 and OTA from foodstuffs.


Assuntos
Aflatoxina B1/análise , Aptâmeros de Nucleotídeos/química , Eletroforese Capilar/métodos , Eletroforese em Microchip/métodos , Análise de Alimentos/métodos , Ocratoxinas/análise , Fluorescência , Corantes Fluorescentes/química , Limite de Detecção , Compostos Orgânicos/química
16.
Cell Death Differ ; 25(5): 904-917, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29234155

RESUMO

PINK1 mutations that disrupt its kinase activity cause autosomal recessive early onset Parkinson's disease (PD). Although research in recent years has elucidated a PINK1-Parkin pathway of mitophagy activation that requires PINK1 kinase activity, mitophagy-independent functions of PINK1 and their possible roles in PD pathogenesis have been proposed. Using an unbiased quantitative mass spectrometry approach to analyze the phosphoproteome in primary neurons from wild type and Pink1 knockout mice after mitochondrial depolarization, we uncovered PINK1-regulated phosphorylation sites, which involve coordinated activation of multiple signaling pathways that control cellular response to stress. We further identified the pro-apoptotic protein BAD as a potential mitochondrial substrate of PINK1 both in vitro and in vivo, and found that cells more susceptible to a12poptosis induced by mitochondrial damage can be rescued by phosphorylation mimic BAD. Our results thus suggest that PINK1 kinase activity is important for pro-apoptotic protein function in regulation of cell death.

17.
Sci Rep ; 7(1): 16913, 2017 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-29209084

RESUMO

In the present study, the effects of sublethal concentrations of buprofezin on life-table traits of S. furcifera were evaluated for two consecutive generations (F0 and F1). Our results exhibited that the fecundity, life span (longevity) and hatchability of the F0 and F1 generations were significantly decreased at LC30 compared to the control. However, copulation was not significantly affected for the F0 or F1 generations at sublethal concentrations. The female life span was affected negatively at both treatments in F0 and at LC30 in F1, compared to the control. Furthermore, significant effects of the sublethal concentrations were found on the developmental rate of all instars except the 3rd instar of F1. However, the pre-adult period, total pre-oviposition period (TPOP) and adult pre-oviposition period (APOP) significantly increased in F1 individuals at LC30 and LC10 compared to the control. Our findings revealed that demographic characters (survival rate, intrinsic rate of increase (ri), finite rate of increase (λ), net reproductive rate (R 0), and gross reproductive rate (GRR)) of the F1 generation (from F0 parents) significantly decreased compared to the untreated group; however, the generation time (T) increased at LC10. Therefore, the results suggested that buprofezin could adversely affect individuals in the successive generation.

18.
Anal Chim Acta ; 995: 99-105, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-29126486

RESUMO

Tyrosinase (TYR) is a key enzyme in melanin biosynthesis and its activity is an important biomarker for dermatological disorders, such as vitiligo, melanoma and actinic damages. Sensitive assay for TYR activity is significant for basic and clinical research. In this work, a facile fluorescent assay for TYR activity based on dopamine functionalized carbon quantum dots (CQDs-Dopa) has been developed. Dopamine (Dopa) was covalently bond to CQDs through a simple one-pot hydrothermal method, and the prepared CQDs-Dopa exhibited a fluorescence emission at 499 nm under exciting wavelength at 310 nm with a quantum yield of approximately 2.1%. When TYR was mixed with CODs-Dopa, the dopamine moiety in CQDs-Dopa conjugate was oxidized to O-dopaquinone, and an intra-particle photo-induced electron transfer (PET) process consequently occurred between CQDs and O-dopaquinone to quench the fluorescence of CQDs-Dopa. TYR activity can be determined based on the fluorescence quenching degree of CQDs-Dopa. This assay covered two broad linear ranges: 44.4-711.1 U L-1 and 711.1-2925.4 U L-1, with detection limit of 17.7 U L-1. The proposed fluorescent assay was applied to TYR activity measurement in human serum samples. It showed promising potential for TYR activity assay in clinical applications.


Assuntos
Técnicas Biossensoriais , Carbono , Dopamina , Monofenol Mono-Oxigenase/análise , Pontos Quânticos , Humanos , Monofenol Mono-Oxigenase/sangue
19.
J Sep Sci ; 40(20): 4022-4031, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28782879

RESUMO

A new, rapid, green, and cost-effective magnetic solid-phase extraction of ochratoxin A from red wine samples was developed using polydopamine-coated magnetic multi-walled carbon nanotubes as the absorbent. The polydopamine-coated magnetic multi-walled carbon nanotubes were fabricated with magnetic multi-walled carbon nanotubes and dopamine by an in situ oxidative self-polymerization approach. Transmission electron microscopy, dynamic light scattering, X-ray photoelectron spectroscopy and vibrating sample magnetometry were used to characterize the absorbents. Ochratoxin A was quantified with high-performance liquid chromatography coupled with fluorescence detection, with excitation and emission wavelengths of 338 and 455 nm, respectively. The conditions affecting the magnetic solid-phase extraction procedure, such as pH, extraction solution, extraction time, absorbent amount, desorption solution and desorption time were investigated to obtain the optimal extraction conditions. Under the optimized conditions, the extraction recovery was 91.8-104.5% for ochratoxin A. A linear calibration curve was obtained in the range of 0.1-2.0 ng/mL. The limit of detection was 0.07 ng/mL, and the limit of quantitation was 0.21 ng/mL. The recoveries of ochratoxin A for spiked red wine sample ranged from 95.65 to 100.65% with relative standard deviation less than 8%. The polydopamine-coated magnetic multi-walled carbon nanotubes showed a high affinity toward ochratoxin A, allowing selective extraction and quantification of ochratoxin A from complex sample matrixes.


Assuntos
Dopamina , Contaminação de Alimentos/análise , Nanotubos de Carbono , Ocratoxinas/isolamento & purificação , Vinho/análise , Extração em Fase Sólida
20.
J Microbiol Biotechnol ; 27(9): 1701-1710, 2017 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-28704902

RESUMO

Classical swine fever virus (CSFV) is the etiologic agent of classical swine fever, a highly contagious disease that causes significant economic losses to the swine industry. The lapinized C-strain, a widely used vaccine strain against CSFV, has low growth efficiency in cell culture, which limits the productivity in the vaccine industry. In this study, a recombinant virus derived from C-strain was constructed and subjected to continuous passaging in PK-15 cells with the goal of acquiring a high progeny virus yield. A cell-adapted virus variant, RecCpp80, had nearly 1,000-fold higher titer than its parent C-strain but lost the ability to induce fever in rabbits. Sequence analysis of cell-adapted RecC variants indicated that at least six nucleotide changes were fixed in RecCpp80. Further adaption of RecCpp80 variant in swine testicle cells led to a higher virus yield without additional mutations. Introduction of each of these residues into the wild-type RecC backbone showed that one mutation, M979R (T3310G), located in the C-terminal region of E2 might be closely related to the cell-adapted phenotype. Rabbit inoculation revealed that RecCpp80+10 failed to induce fever in rabbits, whereas RecCpp40+10 caused a fever response similar to the commercial C-strain vaccine. In conclusion, the C-strain can be adapted to cell culture by introducing specific mutations in its E2 protein. The mutations in RecCpp80 that led to the loss of fever response in rabbits require further investigation. Continuous passaging of the C-strain-based recombinant viruses in PK-15 cells could enhance its in vitro adaption. The non-synonymous mutations at 3310 and 3531 might play major roles in the enhanced capacity of general virus reproduction. Such findings may help design a modified C-strain for improved productivity of commercial vaccines at reduced production cost.


Assuntos
Vírus da Febre Suína Clássica/crescimento & desenvolvimento , Vírus da Febre Suína Clássica/genética , Cultura de Vírus/métodos , Replicação Viral/fisiologia , Animais , Células Cultivadas , Peste Suína Clássica , Vírus da Febre Suína Clássica/fisiologia , Cinética , Coelhos , Inoculações Seriadas , Suínos
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