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Pesticides are widely used on tea plants, and pesticide residues are of significant concern to consumers. The National Food Safety Standard Maximum Residue Limits for Pesticides in Food (GB 2763-2021) was recently amended. However, detection methods for pesticides newly added to the list of residues in beverages have not yet been established. For that reason, this study developed a solid-phase extraction (SPE) and gas chromatography-tandem mass spectrometry (GC-MS/MS) method for determining the residues of 12 pesticides, including four newly added, in black and green tea. Sample preparation processes (sample extraction, SPE clean-up, elution solvent, and elution volume) were optimized to monitor these residues reliably. Multiple reaction monitoring (MRM) was used for GC-MS/MS electron impact (EI) mode determination. Finally, satisfactory recoveries (70.7-113.0% for green tea and 72.0-99.1% for black tea) were achieved at three concentrations (10 µg/kg, 20 µg/kg, and 100 µg/kg). The LOQs were 0.04-8.69 µg/kg, and the LODs were 0.01-3.14 µg/kg. This study provides a reliable and sensitive workflow for determining 12 pesticide residues in tea, filling a gap in the newly revised National Standards.
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Camellia sinensis , Residuos de Plaguicidas , Plaguicidas , Residuos de Plaguicidas/análisis , Espectrometría de Masas en Tándem/métodos , Plaguicidas/análisis , Cromatografía de Gases y Espectrometría de Masas/métodos , Extracción en Fase Sólida/métodos , Té/química , Camellia sinensis/químicaRESUMEN
The fibrillar protein deposits of the human islet amyloid polypeptide (hIAPP) in the pancreatic islet of Langerhans are pathological hallmark of type II diabetes. Extensive experimental studies have revealed that the oligomeric formations of the hIAPP are more toxic than the mature fibrils. Exploring the oligomeric conformations in the early aggregation state is valuable for effective therapeutics. In this work, using the all-atom explicit-solvent replica exchange molecular dynamic (REMD) simulations, we investigated the structural features and the assembly mechanisms of the full-length hIAPP trimer in solution. The hIAPP trimer adopted more ß-sheets than a-helix conformations, and three types of ordered conformations including open ß-barrel, single-layer, and double-layer U-shaped ß-sheet structures with five ß-strands were captured in our simulations. A representative single-layer ß-sheet conformation with a CCS value of 1400 Å2 in our simulations matches exactly the experimentally ESI-IMS-MS-derived hIAPP trimer sample. These five ß-strand conformations formed via the ß-hairpin lateral and longitudinal association, respectively, showing two ß-protofibril formation models. To the best of our knowledge, it is the first time to reveal two routes to ß-sheet formation in the hIAPP trimers on the atomic level. The contact probabilities between pairs of the ß-stranded residue show that the hydrophobic interactions between the residues F15 â¼ V17 and A25 â¼ L27 are responsible for the inter- and intra-peptide ß-hairpin formations. All of these results indicate that the ß-sheet formation is the first step in the conformational changes toward pathological aggregation and provides evidence of the ß-sheet assembly mechanism into hIAPP aggregation.
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Diabetes Mellitus Tipo 2 , Humanos , Diabetes Mellitus Tipo 2/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/química , Amiloide/química , Simulación de Dinámica Molecular , Conformación Proteica en Lámina betaRESUMEN
(1) Background: The Hypsizygus marmoreus is a popular edible mushroom in East Asian markets. In a previous study, we reported the proteomic analyses of different developmental stages of H. marmoreus, from primordium to mature fruiting body. However, the growth and protein expression changes from scratching to primordium are unclear. (2) Methods: A label-free LC-MS/MS quantitative proteomic analysis technique was adopted to obtain the protein expression profiles of three groups of samples collected in different growth stages from scratching to the tenth day after scratching. The Pearson's correlation coefficient analysis and principal component analysis were performed to reveal the correlation among samples. The differentially expressed proteins (DEPs) were organized. Gene Ontology (GO) analysis was performed to divide the DEPs into different metabolic processes and pathways. (3) Results: From the 3rd day to the 10th day after scratching, mycelium recovered gradually and formed primordia. Compared with the Rec stage, 218 highly expressed proteins were identified in the Knot stage. Compared with the Pri stage, 217 highly expressed proteins were identified in the Rec stage. Compared with the Pri stage, 53 highly expressed proteins were identified in the Knot stage. A variety of the same highly expressed proteins were identified in these three developmental stages, including: glutathione S-transferase, acetyltransferase, importin, dehydrogenase, heat-shock proteins, ribosomal proteins, methyltransferase, etc. The key pathways in the development of H. marmoreus are metabolic process, catabolic process, oxidoreductase activity and hydrolase activity. DEPs in the Knot or Pri stages compared with the Rec stage were significantly decreased in the metabolic-, catabolic- and carbohydrate-related process; and the oxidoreductase, peptidase, and hydrolase activity, which can serve as targets for selectable molecular breeding in H. marmoreus. A total of 2000 proteins were classified into eight different modules by WGCNA, wherein 490 proteins were classified into the turquoise module. (4) Conclusions: Generally, from the 3rd day to the 10th day after scratching, mycelium recovered gradually and formed primordia. Importin, dehydrogenase, heat-shock proteins, ribosomal proteins, transferases were all highly expressed in these three developmental stages. DEPs in the Rec stage compared with the Knot or Pri stages were significantly enriched in the metabolic-, catabolic- and carbohydrate-related process; and in oxidoreductase, peptidase and hydrolase activities. This research contributes to the understanding of the mechanisms of the development changes before primordium of H. marmoreus.
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Rhynchophylline (RIN) and isorhynchophylline (IRN), two of the representative types of indole alkaloids, showed the unique spiroindole structures produced in Uncaria rhynchophylla. As the bioactive constituent of U. rhynchophylla, IRN has recently drawn extensive attention toward antihypertensive and neuroprotective activities. Despite their medicinal importance and unique chemical structure, the biosynthetic pathways of plant spiroindole alkaloids are still largely unknown. In this study, we used U. rhynchophylla, extensively used in traditional Chinese medicine (TCM), a widely cultivated plant of the Uncaria genus, to investigate the biosynthetic genes and characterize the functional enzymes in the spiroindole alkaloids. We aim to use the transcriptome platform to analyse the tissue-specific gene expression in spiroindole alkaloids-producing tissues, including root, bud, stem bark and leaf. The critical genes involved in the biosynthesis of precursors and scaffold formation of spiroindole alkaloids were discovered and characterized. The datasets from this work provide an essential resource for further investigating metabolic pathways in U. rhynchophylla and facilitate novel functional enzyme characterization and a good biopharming approach to spiroindole alkaloids.
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Alcaloides , Alcaloides Indólicos , Oxindoles , Alcaloides Indólicos/química , Alcaloides/químicaRESUMEN
Detoxification enzymes play significant roles in the interactions between insects and host plants, wherein detoxification-related genes make great contributions. As herbivorous pests, aphids reproduce rapidly due to parthenogenesis. They are good biological materials for studying the mechanisms that allow insect adaptation to host plants. Insect detoxification gene families are associated with insect adaptation to host plants. The Aphidinae is the largest subfamily in the Aphididae with at least 2483 species in 256 genera in 2 tribes: the Macrosiphini (with 3/4 of the species) and the Aphidini. Most aphid pests on crops and ornamental plants are Aphidinae. Members of the Aphidinae occur in nearly every region of the world. The body shape and colour vary significantly. To research the role that detoxification gene families played in the process of aphid adaptation to host evolution, we analyzed the phylogeny and evolution of these detoxification gene families in Aphidinae. In general, the P450/GST/CCE gene families contract, whereas the ABC/UGT families are conserved in Aphidinae species compared to these families in other herbivorous insects. Genus-specific expansions of P450 CYP4, and GST Delta have occurred in the genus Acyrthosiphon. In addition, the evolutionary rates of five detoxification gene families in the evolution process of Aphidinae are different. The comparison of five detoxification gene families among nine Aphidinae species and the estimated relative evolutionary rates provided herein support an understanding of the interaction between and the co-evolution of Aphidinae and plants.
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Áfidos/genética , Coevolución Biológica , Genes de Insecto , Plantas/parasitología , Adaptación Fisiológica , Animales , Áfidos/fisiología , Filogenia , Plantas/genéticaRESUMEN
BACKGROUND: Pleurotus ostreatus is a popular edible mushroom in East Asian markets. Research on the responses of P. ostreatus under different carbon dioxide concentrations is limited. METHODS: Label-free LC-MS/MS quantitative proteomics analysis technique was adopted to obtain the protein expression profiles of P. ostreatus fruiting body pileus collected under different carbon dioxide concentrations. The Pearson correlation coefficient analysis and principal component analysis were performed to reveal the correlation among samples. The differentially expressed proteins (DEPs) were organized. Gene ontology analysis was performed to divide the DEPs into different metabolic processes and pathways. RESULTS: The expansion of stipes was inhibited in the high CO2 group compared with that in the low CO2 group. There were 415 DEPs (131 up- and 284 down-regulated) in P. ostreatus PH11 treated with 1% CO2 concentration compared with P. ostreatus under atmospheric conditions. Proteins related to hydrolase activity, including several amidohydrolases and cell wall synthesis proteins, were highly expressed under high CO2 concentration. Most of the kinases and elongation factors were significantly down-regulated under high CO2 concentration. The results suggest that the metabolic regulation and development processes were inhibited under high CO2 concentrations. In addition, the sexual differentiation process protein Isp4 was inhibited under high CO2 concentrations, indicating that the sexual reproductive process was also inhibited under high CO2 concentrations, which is inconsistent with the small fruiting body pileus under high CO2 concentrations. CONCLUSIONS: This research reports the proteome analysis of commercially relevant edible fungi P. ostreatus under different carbon dioxide concentrations. This study deepens our understanding of the mechanism for CO2-induced morphological change in the P. ostreatus fruiting body, which will facilitate the artificial cultivation of edible mushrooms.
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(1) Background: The white Hypsizygus marmoreus is a popular edible mushroom in East Asia markets. Research on the systematic investigation of the protein expression changes in the cultivation process of this mushroom are few. (2) Methods: Label-free LC-MS/MS quantitative proteomics analysis technique was adopted to obtain the protein expression profiles of six groups of samples collected in different growth stages. A total of 3468 proteins were identified. The UpSetR plot analysis, Pearson correlation coefficient (PCC) analysis, and principal component (PC) analysis were performed to reveal the correlation among the six groups of samples. The differentially expressed proteins (DEPs) were organised by One-way ANOVA test and divided into four clusters. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to divide the DEPs into different metabolic processes and pathways in each cluster. (3) Results: The DEPs in cluster 1 are of the highest abundance in the mycelium and are mainly involved in protein biosynthesis, biosynthesis of cofactors, lipid metabolism, spliceosome, cell cycle regulation, and MAPK signaling pathway. The DEPs in cluster 2 are enriched in the stem and are mainly associated with protein biosynthesis, biosynthesis of cofactors, carbon, and energy metabolism. The DEPs in cluster 3 are highly expressed in the primordia and unmatured fruiting bodies and are related to amino acids metabolism, carbon and carbohydrate metabolism, protein biosynthesis and processing, biosynthesis of cofactors, cell cycle regulation, MAPK signaling pathway, ubiquitin-mediated proteolysis, and proteasome. The DEPs in cluster 4 are of the highest abundance in the cap and are mainly associated with spliceosome, endocytosis, nucleocytoplasmic transport, protein processing, oxidative phosphorylation, biosynthesis of cofactors, amino acids metabolism, and lipid metabolism. (4) Conclusions: This research reports the proteome analysis of different developmental stages during the cultivation of the commercially relevant edible fungi the white H. marmoreus. In the mycelium stage, most of the DEPs are associated with cell proliferation, signal response, and mycelium growth. In the primordia and unmatured fruiting bodies stage, the DEPs are mainly involved in biomass increase, cell proliferation, signal response, and differentiation. In the mature fruiting body stage, the DEPs in the stem are largely associated with cell elongation and increase in biomass, and most of the DEPs in the cap are mainly related to pileus expansion. Several carbohydrate-active enzymes, transcription factors, heat shock proteins, and some DEPs involved in MAPK and cAMP signaling pathways were determined. These proteins might play vital roles in metabolic processes and activities. This research can add value to the understanding of mechanisms concerning mushroom development during commercial production.
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Medication dosing in a critical care environment is a complex task that involves close monitoring of relevant physiologic and laboratory biomarkers and corresponding sequential adjustment of the prescribed dose. Misdosing of medications with narrow therapeutic windows (such as intravenous [IV] heparin) can result in preventable adverse events, decrease quality of care and increase cost. Therefore, a robust recommendation system can help clinicians by providing individualized dosing suggestions or corrections to existing protocols. We present a clinician-in-the-loop framework for adjusting IV heparin dose using deep reinforcement learning (RL). Our main objectives were to learn a new IV heparin dosing policy based on the multi-dimensional features of patients, and evaluate the effectiveness of the learned policy in the presence of other confounding factors that may contribute to heparin-related side effects. The data used in the experiments included 2598 intensive care patients from the publicly available MIMIC database and 2310 patients from the Emory University clinical data warehouse. Experimental results suggested that the distance from RL policy had a statistically significant association with anticoagulant complications $(p< 0.05)$, after adjusting for the effects of confounding factors.