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Oxidative stress is one of the major culprits causing dopaminergic neuron loss in Parkinson's disease (PD). DJ-1 is a protein with multiple actions against oxidative stress, apoptosis, neuroinflammation, etc. DJ-1 expression is decreased in sporadic PD, therefore increasing DJ-1 expression might be beneficial in PD treatment. However, drugs known to upregulate DJ-1 are still lacking. In this study, we identified a novel DJ-1-elevating compound called ChemJ through luciferase assay-based high-throughput compound screening in SH-SY5Y cells and confirmed that ChemJ upregulated DJ-1 in SH-SY5Y cell line and primary cortical neurons. DJ-1 upregulation by ChemJ alleviated MPP+-induced oxidative stress. In exploring the underlying mechanisms, we found that the transcription factor CREB1 bound to DJ-1 promoter and positively regulated its expression under both unstressed and 1-methyl-4-phenylpyridinium-induced oxidative stress conditions and that ChemJ promoted DJ-1 expression via activating PKA/CREB1 pathway in SH-SY5Y cells. Our results demonstrated that ChemJ alleviated the MPP+-induced oxidative stress through a PKA/CREB1-mediated regulation of DJ-1 expression, thus offering a novel and promising avenue for PD treatment.
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Unified visual perception requires integration of bottom-up and top-down inputs in the primary visual cortex (V1), yet the organization of top-down inputs in V1 remains unclear. Here, we used optogenetics-assisted circuit mapping to identify how multiple top-down inputs from higher-order cortical and thalamic areas engage V1 excitatory and inhibitory neurons. Top-down inputs overlap in superficial layers yet segregate in deep layers. Inputs from the medial secondary visual cortex (V2M) and anterior cingulate cortex (ACA) converge on L6 Pyrs, whereas ventrolateral orbitofrontal cortex (ORBvl) and lateral posterior thalamic nucleus (LP) inputs are processed in parallel in Pyr-type-specific subnetworks (PyrâORBvl and PyrâLP) and drive mutual inhibition between them via local interneurons. Our study deepens understanding of the top-down modulation mechanisms of visual processing and establishes that V2M and ACA inputs in L6 employ integrated processing distinct from the parallel processing of LP and ORBvl inputs in L5.
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Optogenética , Corteza Visual Primaria , Animales , Corteza Visual Primaria/fisiología , Masculino , Tálamo/fisiología , Vías Visuales/fisiología , Neuronas/fisiología , Corteza Visual/fisiología , Giro del Cíngulo/fisiología , Interneuronas/fisiología , Percepción Visual/fisiología , Ratones , Femenino , Mapeo EncefálicoRESUMEN
Interhemispheric communication through the corpus callosum is required for both sensory and cognitive processes. Impaired transcallosal inhibition causing interhemispheric imbalance is believed to underlie visuospatial bias after frontoparietal cortical damage, but the synaptic circuits involved remain largely unknown. Here, we show that lesions in the mouse anterior cingulate area (ACA) cause severe visuospatial bias mediated by a transcallosal inhibition loop. In a visual-change-detection task, ACA callosal-projection neurons (CPNs) were more active with contralateral visual field changes than with ipsilateral changes. Unilateral CPN inactivation impaired contralateral change detection but improved ipsilateral detection by altering interhemispheric interaction through callosal projections. CPNs strongly activated contralateral parvalbumin-positive (PV+) neurons, and callosal-input-driven PV+ neurons preferentially inhibited ipsilateral CPNs, thus mediating transcallosal inhibition. Unilateral PV+ neuron activation caused a similar behavioral bias to contralateral CPN activation and ipsilateral CPN inactivation, and bilateral PV+ neuron activation eliminated this bias. Notably, restoring interhemispheric balance by activating contralesional PV+ neurons significantly improved contralesional detection in ACA-lesioned animals. Thus, a frontal transcallosal inhibition loop comprising CPNs and callosal-input-driven PV+ neurons mediates interhemispheric balance in visuospatial processing, and enhancing contralesional transcallosal inhibition restores interhemispheric balance while also reversing lesion-induced bias.
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Inhibición Psicológica , Interneuronas , Animales , Ratones , Neuronas , Cuerpo Calloso , ParvalbúminasRESUMEN
Astrocytes play a crucial role in regulating sleep-wake behavior, and adenosine signaling is generally thought to be involved. Here we show multiple lines of evidence supporting that modulation of the sleep-wake behavior by astrocyte Ca2+ activity could occur without adenosine signaling. In the basal forebrain and the brainstem, two brain regions that are known to be essential for sleep-wake regulation, chemogenetically-induced astrocyte Ca2+ elevation significantly modulated the sleep-wake cycle. Although astrocyte Ca2+ level positively correlated with the amount of extracellular adenosine, as revealed by a genetically encoded adenosine sensor, we found no detectable change in adenosine level after suppressing astrocyte Ca2+ elevation, and transgenic mice lacking one of the major extracellular ATP-adenosine conversion enzymes showed similar extracellular adenosine level and astrocyte Ca2+-induced sleep modulation. Furthermore, astrocyte Ca2+ is dependent primarily on local neuronal activity, causing brain region-specific regulation of the sleep-wake cycle. Thus, neural activity-dependent astrocyte activity could regulate the sleep-wake behavior independent of adenosine signaling.
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DNA methylation has been proposed to regulate plant stress resistance. However, the dynamic changes in DNA methylation in woody plants and their correlations with pathogenic responses are not fully understood. Here, we present single-base maps of the DNA methylomes of mulberry (Morus notabilis) leaves that were subjected to a mock treatment or inoculation with Botrytis cinerea. Compared with the former, the latter showed decreased mCG and mCHG levels and increased mCHH levels. DNA methylation inhibitors reduced resistance gene methylation levels and enhanced mulberry resistance, suggesting that the hypomethylation of resistance genes affects mulberry resistance to B. cinerea. Virus-induced gene silencing of MnMET1 enhanced the expression of mulberry-resistance genes, thereby increasing the plant's resistance to B. cinerea. We also found that MITEs play a dominant role in controlling DNA methylation levels. MITEs appear to be the main sources of 24-nt siRNAs that regulate gene expression through the RNA-directed DNA methylation pathway.
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Amyotrophic lateral sclerosis is a deleterious neurodegenerative disease without effective treatment options. Recent studies have indicated the involvement of the dysregulation of RNA metabolism in the pathogenesis of amyotrophic lateral sclerosis. Among the various RNA regulatory machineries, nonsense-mediated mRNA decay (NMD) is a stress responsive cellular surveillance system that degrades selected mRNA substrates to prevent the translation of defective or harmful proteins. Whether this pathway is affected in neurodegenerative diseases is unclear. Here we report the inhibition of NMD by arginine-rich dipeptide repeats derived from C9orf72 hexanucleotide repeat expansion, the most common cause of familial amyotrophic lateral sclerosis. Bioinformatic analysis of multiple transcriptome profiles revealed significant overlap of upregulated genes in NMD-defective cells with those in the brain tissues, micro-dissected motor neurons, or induced pluripotent stem cell-derived motor neurons specifically from amyotrophic lateral sclerosis patients carrying C9orf72 hexanucleotide repeat expansion, suggesting the suppression of NMD pathway in these patients. Using Drosophila as a model, we have validated that the C9orf72 hexanucleotide repeat expansion products could lead to the accumulation of the NMD substrates and identified arginine-rich dipeptide repeats, including poly glycine-arginine and poly proline-arginine, as the main culprits of NMD inhibition. Furthermore, in human SH-SY5Y neuroblastoma cells and in mouse brains, expression of glycine-arginine with 36 repeats (GR36) was sufficient to cause NMD inhibition. In cells expressing GR36, stress granule accumulation was accompanied by decreased processing body formation, which contributed to the inhibition of NMD. Remarkably, expression of UPF1, a core gene in the NMD pathway, efficiently blocked neurotoxicity caused by arginine-rich dipeptide repeats in both cellular and Drosophila models. Although not as effective as UPF1, expression of another NMD gene UPF2 also ameliorated the degenerative phenotypes in dipeptide repeat-expressing flies, indicating that genetically reactivating the NMD pathway could suppress dipeptide repeat toxicity. Finally, after validating tranilast as an NMD-activating drug, we demonstrated the therapeutic potential of this asthma drug in cellular and Drosophila models of C9orf72 dipeptide repeat neurotoxicity. Therefore, our study has revealed a cellular mechanism whereby arginine-rich C9orf72 dipeptide repeats could inhibit NMD activities by reducing the abundance of processing bodies. Furthermore, our results suggested that activation of the NMD pathway could be a potential therapeutic strategy for amyotrophic lateral sclerosis with defective RNA metabolism.
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Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido/fisiología , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Animales , Animales Modificados Genéticamente , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/uso terapéutico , Línea Celular Tumoral , Dipéptidos/genética , Dipéptidos/metabolismo , Drosophila , Femenino , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Degradación de ARNm Mediada por Codón sin Sentido/efectos de los fármacos , ortoaminobenzoatos/farmacología , ortoaminobenzoatos/uso terapéuticoRESUMEN
BACKGROUND: High levels of harmful pesticide residues in rice can cause undesirable side effects and are a source of great concern to consumers. Reduction of pesticide residues to provide rice security has thus became an urgent problem. RESULTS: In this study, the effects of commercial and home processing on removal of chlorpyrifos and carbosulfan residues from rice, and the formation of metabolites during processing, were studied. The results showed that 3,5,6-trichloro-2-pyridinol (0.87 mg kg-1 ) and carbofuran (0.43 mg kg-1 ) were the predominant components detected in paddy rice. All detected residues were primarily deposited on the rice hull and bran. Washing twice followed by high-pressure cooking was able to further decrease residues in polished rice with the processing factor value <0.25. Following application of pesticides at the recommended rate and twice the recommended rate, with a preharvest interval of 28 days, changes in residues from harvest to dining table based on efficient processing techniques were investigated. The final residues dropped to below maximum residue levels after washing twice followed by high-pressure cooking. CONCLUSION: This simple cooking process thus reduces the risk of dietary exposure, and it is recommended that it is adopted by all consumers. © 2019 Society of Chemical Industry.
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Culinaria/métodos , Oryza/química , Residuos de Plaguicidas/química , Carbamatos/química , Carbofurano/química , Cloropirifos/química , Contaminación de Alimentos/análisis , CinéticaRESUMEN
Stress granule formation is important for stress response in normal cells and could lead to chemotherapy resistance in cancer cells. Aberrant stress granule dynamics are also known to disrupt proteostasis, affect RNA metabolism, and contribute to neuronal cell death. Meanwhile, circadian abnormality is an aging-related risk factor for cancer and neurodegeneration. Whether stress granule dynamics are circadian regulated is entirely unknown. Here we show that the formation of stress granules varied by zeitgeber time in mouse liver. Moreover, altering circadian regulation by silencing the core circadian gene Bmal1 in a cell line expressing an endogenous GFP-tagged G3BP1 significantly increased stress granule dynamics, while the overexpression of Bmal1 decreased them. Surprisingly, increased stress granule dynamics and formation by transient decrease of BMAL1 coincided with increased resistance to stress-induced cell death. The circadian regulation of stress granules was mediated by oscillating eIF2α expression. At zeitgeber time when BMAL1 and eIF2α were at nadir, reduction of unphosphorylated eIF2α could significantly alter the ratio of phosphorylated/total eIF2α and quickly lead to increased formation of stress granules. Therefore, diurnal oscillating eIF2α connects the circadian cue to a cellular stress response mechanism that is vital for both neurodegeneration and cancer.
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Ritmo Circadiano/genética , Factor 2 Eucariótico de Iniciación/fisiología , Estrés Fisiológico , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Factores de Transcripción ARNTL/fisiología , Animales , Resistencia a Antineoplásicos , Factor 2 Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , Masculino , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Circadian rhythms are oscillating physiological and behavioral changes governed by an internal molecular clock, and dysfunctions in circadian rhythms have been associated with ageing and various neurodegenerative diseases. However, the evidence directly connecting the neurodegeneration-associated proteins to circadian control at the molecular level remains sparse. METHODS: Using meta-analysis, synchronized animals and cell lines, cells and tissues from FUS R521C knock-in rats, we examined the role of FUS in circadian gene expression regulation. RESULTS: We found that FUS, an oscillating expressed nuclear protein implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), exerted a novel feedback route to regulate circadian gene expression. Nr1d1-encoded core circadian protein REV-ERBα bound the Fus promoter and regulated the expression of Fus. Meanwhile, FUS was in the same complex as PER/CRY, and repressed the expression of E box-containing core circadian genes, such as Per2, by mediating the promoter occupancy of PSF-HDAC1. Remarkably, a common pathogenic mutant FUS (R521C) showed increased binding to PSF, and caused decreased expression of Per2. CONCLUSIONS: Therefore, we have demonstrated FUS as a modulator of circadian gene expression, and provided novel mechanistic insights into the mutual influence between circadian control and neurodegeneration-associated proteins.
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Huntingtin interaction protein 2 (HIP2) is an E2 ubiquitin-conjugating enzyme associated with neurodegenerative diseases, and HIP2 mRNA has been implicated as a potential blood biomarker for Parkinson's disease (PD). However, it is unclear whether the alteration of HIP2 expression may contribute to the development of PD, and whether the change of HIP2 in blood could reflect its expression in the brain or motor functions in PD patients. In this study, we established a mouse line with HIP2 haploinsufficiency. The reduction of the HIP2 expression led to spontaneous motor function impairment and dopaminergic neuronal loss. Furthermore, HIP2 haploinsufficiency increased the susceptibility of mice to 6-hydroxydopamine (6-OHDA) and caused severe loss of dopaminergic neurons. Interestingly, in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model for PD, we observed concurrent, highly correlated decrease of HIP2 expression in the brain and in the blood. Using blood samples from more than 300 patients, we validated the decreased HIP2 mRNA in PD patients, including de novo patients. Finally, in a 1-year, 20-patient study, we observed reversed blood HIP2 mRNA levels accompanying improved motor and overall daily functions in 75% of the PD patients with instructed Tai Chi training. Therefore, our in vivo studies have indicated HIP2 insufficiency as a contributing factor for PD, and functionally validated blood HIP2 as a useful and reversible biomarker for PD.
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Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Actividad Motora/fisiología , Enfermedad de Parkinson/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/efectos de los fármacos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Oxidopamina/farmacologíaRESUMEN
Mutations in fused in sarcoma (Fus) cause familial amyotrophic lateral sclerosis (ALS) and occasionally frontotemporal dementia. Here we report the establishment and characterization of a novel knockin (KI) rat model expressing a Fus point mutation (R521C) via CRISPR/Cas9. The mutant animals developed adult-onset learning and memory behavioral deficits, with reduced spine density in hippocampal neurons. Remarkably, sleep-wake cycle and circadian abnormalities preceded the onset of cognitive deficit. RNA-seq study further demonstrated altered expression of some key sleep and circadian regulators, such as orexin/hypocretin receptor type 2 and casein kinase 1 epsilon, in the mutant rats. Therefore, we have established a rodent model expressing physiological level of a pathogenic mutant FUS, and we found cognitive impairment as a main behavioral deficit at mid age. Furthermore, we have revealed a new role of FUS in sleep and circadian regulation and demonstrated that functional change in FUS could cause sleep-wake and circadian disturbance as early symptoms.
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Conducta Animal , Trastornos Cronobiológicos/genética , Disfunción Cognitiva/genética , Proteína FUS de Unión a ARN/metabolismo , Sueño/genética , Vigilia/genética , Factores de Edad , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Electroencefalografía , Electromiografía , Embrión de Mamíferos , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Trastornos del Sueño del Ritmo Circadiano/genéticaRESUMEN
The zinc-regulated transporters, iron-regulated transporter-like proteins (ZIPs), the natural resistance and macrophage proteins (NRAMP), the heavy metal ATPases (HMAs) and the metal tolerance or transporter proteins (MTPs) families are involved in cadmium (Cd) uptake, translocation and sequestration in plants. Mulberry (Morus L.), one of the most ecologically and economically important (as a food plant for silkworm production) genera of perennial trees, exhibits excellent potential for remediating Cd-contaminated soils. However, there is no detailed information about the genes involved in Cd2+ transport in mulberry. In this study, we identified 31 genes based on a genome-wide analysis of the Morus notabilis genome database. According to bioinformatics analysis, the four transporter gene families in Morus were distributed in each group of the phylogenetic tree, and the gene exon/intron structure and protein motif structure were similar among members of the same group. Subcellular localization software predicted that these transporters were mainly distributed in the plasma membrane and the vacuolar membrane, with members of the same group exhibiting similar subcellular locations. Most of the gene promoters contained abiotic stress-related cis-elements. The expression patterns of these genes in different organs were determined, and the patterns identified, allowing the categorization of these genes into four groups. Under low or high-Cd2+ concentrations (30 µM or 100 µM, respectively), the transcriptional regulation of the 31 genes in root, stem and leaf tissues of M. alba seedlings differed with regard to tissue and time of peak expression. Heterologous expression of MaNRAMP1, MaHMA3, MaZIP4, and MaIRT1 in Saccharomyces cerevisiae increased the sensitivity of yeast to Cd, suggested that these transporters had Cd transport activity. Subcellular localization experiment showed that the four transporters were localized to the plasma membrane of yeast and tobacco. These results provide the basis for further understanding of the Cd tolerance mechanism in Morus, which can be exploited in Cd phytoremediation.
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Sulfur (S) fertilizer application in rice (Oryza sativa L.) is crucial in determining rice grain productivity and quality. However, little information is available concerning the effect of S supply on cadmium (Cd) uptake and translocation in rice. In this study, both hydroponic and soil experiments were conducted to investigate the influence of S supply on Cd accumulation in rice under two Cd levels (0 and 50⯵M), combined with three S concentrations (0, 2.64 and 5.28â¯mM). The moderate and excessive S supply (2.64 and 5.28â¯mM) tended to increase plant growth, root length, root and shoot dry weights of rice seedlings, and significantly decreased Cd concentrations in rice plants and grains in the absence or presence of Cd. The subcellular distribution and chemical forms of Cd in roots and shoots also varied with S supply levels. The decreased Cd uptake and translocation in rice grains could be ascribed to the enhanced formation of iron (Fe) plaque on the root surfaces and increased Cd chelation and vacuolar sequestration in roots, since Fe, Mn concentrations in Fe plaque, glutathione and phytochelatins contents, as well as phytochelatin synthase (OsPCS) and tonoplast heavy metal ATPase (OsHMA3) expressions in roots significantly increased with increased S supply. This work provides more insight into the mechanisms of Cd uptake and translocation in rice, and will be helpful for developing strategies to reduce rice grain Cd through S fertilizer application in Cd-contaminated soil.
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Cadmio/metabolismo , Hierro/metabolismo , Oryza/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Plantones/metabolismo , Contaminantes del Suelo/metabolismo , Azufre/farmacología , Adenosina Trifosfatasas/metabolismo , Aminoaciltransferasas/metabolismo , Transporte Biológico/efectos de los fármacos , Cadmio/análisis , Grano Comestible/química , Fertilizantes/análisis , Glutatión/metabolismo , Hierro/análisis , Oryza/crecimiento & desarrollo , Fitoquelatinas/metabolismo , Raíces de Plantas/metabolismo , Suelo/química , Contaminantes del Suelo/análisis , Azufre/análisisRESUMEN
A two-dimensional liquid chromatography (2D LC) system was developed to separate proteins from rice leaves, which was extracted by phenol method, followed by the analysis with linear trap quadrupole orbitrap mass spectrometry (LTQ/Orbitrap MS). After proteins were extracted with phenol method, the enzymolytic peptides were separated by offline two-dimensional RP-RP system and detected by LTQ/Orbitrap MS, yielding 2712 proteins. Liquid chromatography separation system (1D LC and 2D LC) and protein extraction methods (phenol method, sodium dodecyl sulfate method (SDS method) and trichloroacetic acid/acetone method (TCA/acetone method)) were compared. Proteins identified by 2D LC were 2712, 2415 and 1914 with the above three extraction methods, respectively. The proteins were 2.7-fold, 2.5-fold and 1.9-fold the number of proteins identified by 1D LC respectively. And in terms of 2D LC, the proteins identified by phenol method were 297 and 798 more than SDS method and TCA/acetone method, respectively. Some proteins with extreme properties, such as very acidic or basic protein and high relative molecular mass proteins, were only identified in phenol method. Furthermore, proteins, which were extracted by different extraction methods and separated by 2D LC, were classified according to biological functions. It was found that protein functions by the three extraction methods were complementary. However, phenol method had the most variety of functions. The method provides technological support for rice proteomics and reference for research techniques of other crop proteomics.
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Cromatografía Liquida , Espectrometría de Masas , Oryza/metabolismo , Proteoma/análisis , Acetona , Fenol , Fenoles , Hojas de la Planta/metabolismo , Proteómica , Dodecil Sulfato de Sodio , Ácido TricloroacéticoRESUMEN
The authors would like to call the reader's attention to the fact that unfortunately during a recent cross-check of the experimental record, they found that the positions of intercept and slope were reversed in Table 1 in the original manuscript. The authors apologize for the mistake.
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A modified Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) sample preparation method, coupled with liquid chromatography-electrospray ionization tandem mass spectrometry, has been developed for the simultaneous analysis of four commonly used sulfonylurea herbicides, ethoxysulfuron, halosulfuron-methyl, mesosulfuron-methyl and orthosulfamuron, in rice, maize, wheat and soybean. Adsorption of the analytes onto the primary-secondary amine used in the clean-up step was avoided by using 1% formic acid in acetonitrile as the extraction solvent to maintain the acidic herbicides in a non-ionized state. Trueness studies were carried out at three levels (2.5, 25 and 250⯵g/kg for ethoxysulfuron and 5, 50 and 500⯵g/kg for the other three analytes). Promising trueness (70.2%-119.8%) was achieved for all herbicides in all matrices after clean-up, with relative standard deviations (RSDr)â¯<â¯18.6%. Satisfactory matrix effects (-19.7% to 14.8%) were also obtained. Good linearity of the calibration curves was achieved, with determination coefficients (r)â¯≥â¯0.9956, when the concentration of ethoxysulfuron was in the range 0.5-50⯵g/L and that of the other three analytes was in the range 1.0-500⯵g/L. The RSDwR for within-laboratory reproducibility was 5.7%. The validated method was successfully used to analyze real samples.
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Cromatografía Liquida , Grano Comestible/química , Análisis de los Alimentos/métodos , Herbicidas/análisis , Espectrometría de Masas en Tándem , Reproducibilidad de los ResultadosRESUMEN
A highly sensitive method was developed for the simultaneous separation and determination of organic and inorganic selenium species in rice by ion-pairing reversed-phase chromatography combined with inductively coupled plasma tandem mass spectrometry. To achieve a good separation of these species, a comparison between anion-exchange chromatography and ion-pairing reversed-phase chromatography was performed. The results indicated that ion-pairing reversed-phase chromatography was more suitable due to better separation and higher sensitivity for all analytes. In this case, a StableBond C18 column proved to be more robust or to have a better resolution than other C18 columns, when 0.5 mM tetrabutylammonium hydroxide and 10 mM ammonium acetate at pH 5.5 were used as the mobile phase. Moreover, an excellent sensitivity was obtained in terms of interferences by means of tandem mass spectrometry in the hydrogen mode. The detection limits were 0.02-0.12 µg/L, and recoveries of five selenium species were 75-114%, with relative standard deviations ≤ 9.4%. This method was successfully applied to the analysis of rice samples. Compared with previous studies, the proposed method not only gave comparable results when used for measuring selenium-enriched rice, but it can provide greater sensitivity for the detection of low concentrations of selenium species in rice.
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Oryza/química , Selenio/análisis , Selenio/aislamiento & purificación , Acetatos/química , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Hidrógeno/química , Concentración de Iones de Hidrógeno , Límite de Detección , Compuestos de Amonio Cuaternario/química , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Análisis Espectral , Espectrometría de Masas en TándemRESUMEN
A novel method has been developed for the direct, sensitive, and rapid detection of bronopol in rice using a simple solid-phase extraction (SPE) procedure followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), with electrospray ionization (ESI). Bronopol was stable under acidic conditions, and an acidic environment was thus needed before sample loading to ensure the stability of bronopol. Rice extracts containing bronopol were pretreated using a hydrophilic-lipophilic balanced (Bond Elut Plexa) cartridge to reduce the matrix effect. An XDB-C18 column (150 mm × 2.1 mm, 3.5 µm) was used for chromatographic separations, with a mobile phase comprising methanol and aqueous ammonium formate (5 mM). The linearity of the method was satisfactory with regression coefficient (R 2) = 0.9992. The limit of quantification was 3.3 µg kg-1. Three spiked levels (25, 125 and 625 µg kg-1) were used to determine the recovery of bronopol, which was found to be 73.3-96.7%, with relative standard deviations (RSD) in the range 1.2-7.9%. The RSD for intra-day precision (n = 7) was 7.6% and the RSD for inter-day precision (n = 15) was 8.3%. The newly developed analytical method was successfully used to quantify bronopol in rice samples.
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Residuos de Medicamentos/análisis , Oryza/química , Glicoles de Propileno/análisis , Extracción en Fase Sólida/métodos , Espectrometría de Masas en Tándem/métodos , Cromatografía Líquida de Alta Presión/métodos , Límite de Detección , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
A method was developed for the determination of pyriminobac-methyl and bispyribac-sodium residues in rice by liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with modified QuEChERS. The samples were extracted with acidified acetonitrile, and then purified by octadecylsilane bonded silica (C18) adsorbents. The analytes were separated on a ZORBAX SB C18 column through a gradient elution using 0.1% (v/v) aqueous formic acid aqueous containing 5 mmol/L ammonium acetate and acetonitrile as mobile phases. Positive electrospray ionization (ESI+) was used. Qualitative work was performed using selected dynamic multiple reaction monitoring (dynamic MRM) mode. Quantization was performed using external standard method. The results showed good linearities of pyriminobac-methyl and bispyribac-sodium with correlation coefficients (r2) not less than 0.996. The limits of detection (LODs) of the method were 0.8 µ g/kg for pyriminobac-methyl, and 3 µ g/kg for bispyribac-sodium. The mean spiked recoveries of pyriminobac-methyl and bispyribac-sodium at three spiked levels were 76.6%-85.6% and 73.0%-86.7%, respectively, and the relative standard deviations (RSDs) of pyriminobac-methyl and bispyribac-sodium were 0.9%-3.4% and 1.2%-5.5%, respectively. This method is simple, rapid, sensitive, and suitable for the simultaneous determination of pyriminobac-methyl and bispyribac-sodium in rice.
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Benzoatos/análisis , Oryza/química , Pirimidinas/análisis , Espectrometría de Masas en Tándem , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , SodioRESUMEN
Autophagy-lysosomal pathway is a cellular protective system to remove aggregated proteins and damaged organelles. Meanwhile, exosome secretion has emerged as a mode to selectively clear the neurotoxic proteins, such as α-synuclein. Mounting evidence suggests that these two cellular processes are coordinated to facilitate the clearance of toxic cellular waste; however the regulators for the transition between these two processes are unclear. Here we show that SCAMP5, a secretory carrier membrane protein significantly induced in the brains of Huntington's disease patients, is quickly and transiently induced by protein stress and autophagic stimulation, and is regulated by the master autophagy transcriptional regulator TFEB. Ironically, SCAMP5 inhibits autophagy flux by blocking the fusion of autophagosomes and lysosomes. Although autophagy is blocked, SCAMP5 does not cause significant protein aggregation in cells. Instead, it promotes the Golgi fragmentation and stimulates the unconventional secretion of the co-localizing α-synuclein via exosome as an exosome component. Therefore, we have identified SCAMP5 as a novel coordinator of autophagy and exosome secretion, which is induced upon protein stress to channel the efficient clearance of toxic proteins via the exosomes rather than autophagy-lysosomal pathway.