RESUMO
Plant genomes encode many receptor-like kinases (RLKs) that localize to the cell surface and perceive a wide variety of environmental cues to initiate downstream signaling cascades. Whether these RLKs participate in dehydration stress signaling in plants is largely unknown. DROOPY LEAF1 (DPY1), a leucine-rich repeat (LRR)-RLK, was recently shown to regulate plant architecture by orchestrating early brassinosteroid signaling in foxtail millet (Setaria italica). Here, we show that DPY1 is essential for the acclimation of foxtail millet to drought stress. DPY1 can be phosphorylated and activated in response to osmotic stress and is required for more than half of osmotic stress-induced global phosphorylation events, including the phosphorylation of sucrose nonfermenting kinase 2s (SnRK2s), the central kinases involved in osmotic stress. DPY1 acts upstream of STRESS-ACTIVATED PROTEIN KINASE 6 (SAPK6, a subclass I SnRK2) and is required for full SAPK6 activation, thereby allowing regulation of downstream genes to mount a response against drought stress. These signaling events are largely independent of DPY1-mediated brassinosteroid signaling. The DPY1-SAPK6 module is specific to seed plants and is absent in ancestral nonseed plants. Our findings reveal a dehydration stress-activated RLK that plays an indispensable role in osmotic stress signaling and mediates SnRK2 activation at the cell surface.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Setaria (Planta) , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pressão Osmótica/fisiologia , Setaria (Planta)/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Resistência à Seca , Brassinosteroides/metabolismo , Desidratação , Ácido Abscísico/metabolismo , Plantas/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
Maize (Zea mays) smut is a common biotrophic fungal disease caused by Ustilago maydis and leads to low maize yield. Maize resistance to U. maydis is a quantitative trait. However, the molecular mechanism underlying the resistance of maize to U. maydis is poorly understood. Here, we reported that a maize mutant caused by a single gene mutation exhibited defects in both fungal resistance and plant development. maize mutant highly susceptible to U. maydis (mmsu) with a dwarf phenotype forms tumors in the ear. A map-based cloning and allelism test demonstrated that 1 gene encoding a putative arogenate dehydratase/prephenate dehydratase (ADT/PDT) is responsible for the phenotypes of the mmsu and was designated as ZmADT2. Combined transcriptomic and metabolomic analyses revealed that mmsu had substantial differences in multiple metabolic pathways in response to U. maydis infection compared with the wild type. Disruption of ZmADT2 caused damage to the chloroplast ultrastructure and function, metabolic flux redirection, and reduced the amounts of salicylic acid (SA) and lignin, leading to susceptibility to U. maydis and dwarf phenotype. These results suggested that ZmADT2 is required for maintaining metabolic flux, as well as resistance to U. maydis and plant development in maize. Meanwhile, our findings provided insights into the maize response mechanism to U. maydis infection.
Assuntos
Resistência à Doença , Doenças das Plantas , Zea mays , Zea mays/microbiologia , Zea mays/genética , Zea mays/crescimento & desenvolvimento , Doenças das Plantas/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Resistência à Doença/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Hidroliases/genética , Hidroliases/metabolismo , Basidiomycota/fisiologia , Regulação da Expressão Gênica de Plantas , Fenótipo , Mutação/genética , Ácido Salicílico/metabolismo , Ustilago/genéticaRESUMO
Wood formation, which occurs mainly through secondary xylem development, is important not only for supplying raw material for the 'ligno-chemical' industry but also for driving the storage of carbon. However, the complex mechanisms underlying the promotion of xylem formation remain to be elucidated. Here, we found that overexpression of Auxin-Regulated Gene involved in Organ Size (ARGOS) in hybrid poplar 84 K (Populus alba × Populus tremula var. glandulosa) enlarged organ size. In particular, PagARGOS promoted secondary growth of stems with increased xylem formation. To gain further insight into how PagARGOS regulates xylem development, we further carried out yeast two-hybrid screening and identified that the auxin transporter WALLS ARE THIN1 (WAT1) interacts with PagARGOS. Overexpression of PagARGOS up-regulated WAT1, activating a downstream auxin response promoting cambial cell division and xylem differentiation for wood formation. Moreover, overexpressing PagARGOS caused not only higher wood yield but also lower lignin content compared with wild-type controls. PagARGOS is therefore a potential candidate gene for engineering fast-growing and low-lignin trees with improved biomass production.
Assuntos
Regulação da Expressão Gênica de Plantas , Lignina , Proteínas de Plantas , Populus , Madeira , Xilema , Populus/genética , Populus/crescimento & desenvolvimento , Populus/metabolismo , Lignina/metabolismo , Madeira/crescimento & desenvolvimento , Madeira/genética , Madeira/metabolismo , Xilema/metabolismo , Xilema/crescimento & desenvolvimento , Xilema/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Ácidos Indolacéticos/metabolismoRESUMO
Plants delicately regulate endogenous auxin levels through the coordination of transport, biosynthesis, and inactivation, which is crucial for growth and development. While it is well-established that the actin cytoskeleton can regulate auxin levels by affecting polar transport, its potential role in auxin biosynthesis has remained largely unexplored. Using LC-MS/MS-based methods combined with fluorescent auxin marker detection, we observed a significant increase in root auxin levels upon deletion of the actin bundling proteins AtFIM4 and AtFIM5. Fluorescent observation, immunoblotting analysis, and biochemical approaches revealed that AtFIM4 and AtFIM5 affect the protein abundance of the key auxin synthesis enzyme YUC8 in roots. AtFIM4 and AtFIM5 regulate the auxin synthesis enzyme YUC8 at the protein level, with its degradation mediated by the 26S proteasome. This regulation modulates auxin synthesis and endogenous auxin levels in roots, consequently impacting root development. Based on these findings, we propose a molecular pathway centered on the 'actin cytoskeleton-26S proteasome-YUC8-auxin' axis that controls auxin levels. Our findings shed light on a new pathway through which plants regulate auxin synthesis. Moreover, this study illuminates a newfound role of the actin cytoskeleton in regulating plant growth and development, particularly through its involvement in maintaining protein homeostasis via the 26S proteasome.
RESUMO
New psychoactive substances are constantly emerging, among which ketamine analogs with the core structure of 2-amino-2-phenylcyclohexanone have attracted global attention due to their continued involvement in acute intoxications. The monitoring of these substances largely relies on the acquisition of metabolic data. However, the lack of in vitro human metabolism information for these emerging structural analogs presents significant challenges to drug control efforts. To address this challenge, we investigated the first-phase metabolism patterns of four novel ketamine structural analogs of 2-FXE [2-(ethylamino)-2-(2-fluorophenyl) cyclohexan-1-one], 2-MDCK [2-(methylamino)-2-(o-tolyl) cyclohexan-1-one], 3-DMXE [2-(ethylamino)-2-(m-tolyl) cyclohexan-1-one], and 2-DMXE [2-(ethylamino)-2-(o-tolyl) cyclohexan-1-one] utilizing human liver microsomes for the first time. Metabolites were identified using ultra-performance liquid chromatography coupled with high-resolution tandem mass spectrometry. Our findings reveal that N-dealkylation and hydroxylation are the primary metabolic reactions, alongside other notable reactions, including oxidation, reduction, and dehydration. Based on our extensive research, we propose N-dealkylation and hydroxylation metabolites as appropriate analytical markers for monitoring the consumption of these substances.
Assuntos
Ketamina , Microssomos Hepáticos , Humanos , Microssomos Hepáticos/metabolismo , Espectrometria de Massas em Tandem/métodos , Ketamina/metabolismo , Espectrometria de Massa com Cromatografia Líquida , Hidroxilação , Cromatografia Líquida de Alta Pressão/métodosRESUMO
The phytohormone abscisic acid (ABA) regulates ion channel activity and stomatal movement in response to drought stress. Cellular ABA levels change depending on cellular and environmental conditions via modulation of its biosynthesis, catabolism and transport. Although factors involved in ABA biosynthesis and degradation have been studied extensively, how ABA transporters are modulated to fine-tune ABA levels, especially under drought stress, remains elusive. Here, we show that Arabidopsis thaliana SORTING NEXIN 2 (SNX2) proteins play a critical role in endosomal trafficking of the ABA exporter ATP BINDING CASETTE G25 (ABCG25) via direct interaction at endosomes, leading to its degradation in the vacuole. In agreement, snx2a and snx2b mutant plants showed enhanced recycling of GFP-ABCG25 from early endosomes to the plasma membrane and higher accumulation of GFP-ABCG25. Phenotypically, snx2a and snx2b plants were highly sensitive to exogenous ABA and displayed enhanced ABA-mediated inhibition of inward K+ currents and ABA-mediated activation of slow anion currents in guard cells, resulting in an increased tolerance to drought stress. Based on these results, we propose that SNX2 proteins play a crucial role in stomatal movement and tolerance to drought stress by modulating the endosomal trafficking of ABCG25 and thus cellular ABA levels.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Secas , Estômatos de Plantas/fisiologiaRESUMO
MAIN CONCLUSION: A lncRNA MtCIR1 negatively regulates the response to salt stress in Medicago truncatula seed germination by modulating seedling growth and ABA metabolism and signaling by enhancing Na+ accumulation. Increasing evidence suggests that long non-coding RNAs (lncRNAs) are involved in the regulation of plant tolerance to varying abiotic stresses. A large number of lncRNAs that are responsive to abiotic stress have been identified in plants; however, the mechanisms underlying the regulation of plant responses to abiotic stress by lncRNAs are largely unclear. Here, we functionally characterized a salt stress-responsive lncRNA derived from the leguminous model plant M. truncatula, referred to as MtCIR1, by expressing MtCIR1 in Arabidopsis thaliana in which no such homologous sequence was observed. Expression of MtCIR1 rendered seed germination more sensitive to salt stress by enhanced accumulation of abscisic acid (ABA) due to suppressing the expression of the ABA catabolic enzyme CYP707A2. Expression of MtCIR1 also suppressed the expression of genes associated with ABA receptors and signaling. The ABA-responsive gene AtPGIP2 that was involved in degradation of cell wall during seed germination was up-regulated by expressing MtCIR1. On the other hand, expression of MtCIR1 in Arabidopsis thaliana enhanced foliar Na+ accumulation by down-regulating genes encoding Na+ transporters, thus rendering the transgenic plants more sensitive to salt stress. These results demonstrate that the M. truncatula lncRNA MtCIR1 negatively regulates salt stress response by targeting ABA metabolism and signaling during seed germination and foliar Na+ accumulation by affecting Na+ transport under salt stress during seedling growth. These novel findings would advance our knowledge on the regulatory roles of lncRNAs in response of plants to salt stress.
Assuntos
Medicago truncatula , RNA Longo não Codificante , Estresse Salino , Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Germinação/genética , Medicago truncatula/genética , Medicago truncatula/metabolismo , Plantas Geneticamente Modificadas/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Estresse Salino/genética , Plântula , Estresse Fisiológico/genéticaRESUMO
OBJECTIVES: To establish the GC-MS qualitative and quantitative analysis methods for the synthetic cannabinoids, its main matrix and additives in suspicious electronic cigarette (e-cigarette) oil samples. METHODS: The e-cigarette oil samples were analyzed by GC-MS after diluted with methanol. Synthetic cannabinoids, its main matrix and additives in e-cigarette oil samples were qualitatively analyzed by the characteristic fragment ions and retention time. The synthetic cannabinoids were quantitatively analyzed by using the selective ion monitoring mode. RESULTS: The linear range of each compound in GC-MS quantitative method was 0.025-1 mg/mL, the matrix recovery rate was 94%-103%, the intra-day precision relative standard deviations (RSD) was less than 2.5%, and inter-day precision RSD was less than 4.0%. Five indoles or indazole amide synthetic cannabinoids were detected in 25 e-cigarette samples. The main matrixes of e-cigarette samples were propylene glycol and glycerol. Additives such as N,2,3-trimethyl-2-isopropyl butanamide (WS-23), glycerol triacetate and nicotine were detected in some samples. The content range of synthetic cannabinoids in 25 e-cigarette samples was 0.05%-2.74%. CONCLUSIONS: The GC-MS method for synthesizing cannabinoid, matrix and additive in e-cigarette oil samples has good selectivity, high resolution, low detection limit, and can be used for simultaneous qualitative and quantitative analysis of multiple components; The explored fragment ion fragmentation mechanism of the electron bombardment ion source of indole or indoxamide compounds helps to identify such substances or other compounds with similar structures in cases.
Assuntos
Canabinoides , Sistemas Eletrônicos de Liberação de Nicotina , Drogas Ilícitas , Cromatografia Gasosa-Espectrometria de Massas/métodos , Drogas Ilícitas/análise , Indazóis/química , Glicerol/análise , Indóis/química , ÍonsRESUMO
MicroRNAs (miRNAs) play key regulatory roles in seed development and emerge as new key targets for engineering grain size and yield. The Zma-miRNA169 family is highly expressed during maize seed development, but its functional roles in seed development remain elusive. Here, we generated zma-miR169o and ZmNF-YA13 transgenic plants. Phenotypic and genetic analyses were performed on these lines. Seed development and auxins contents were investigated. Overexpression of maize miRNA zma-miR169o increases seed size and weight, whereas the opposite is true when its expression is suppressed. Further studies revealed that zma-miR169 acts by negatively regulating its target gene, a transcription factor ZmNF-YA13 that also plays a key role in determining seed size. We demonstrate that ZmNF-YA13 regulates the expression of the auxin biosynthetic gene ZmYUC1, which modulates auxin levels in the early developing seeds and determines the number of endosperm cells, thereby governing maize seed size and ultimately yield. Overall, our present study has identified zma-miR169o and ZmNF-YA13 that form a functional module regulating auxin accumulation in maize seeds and playing an important role in determining maize seed size and yield, providing a set of novel molecular tools for yield improvement in molecular breeding and genetic engineering.
Assuntos
MicroRNAs , Zea mays , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Sementes/genética , Sementes/metabolismo , Zea mays/metabolismoRESUMO
Sample preparation remains a bottleneck in the rapid and reliable quantification of gibberellins (GAs) for obtaining an insight into the physiological processes mediated by GAs. The challenges arise from not only the extremely low content of GAs in complex plant matrices, but the poor detectability of GAs by mass spectrometry (MS) in negative ion mode. In an effort to solve these urgent difficulties, we present a spatial-resolved analysis method to investigate the distribution of GAs in tiny plant tissues based on a simplified one-pot sample preparation approach coupled with ultrahigh-performance liquid chromatography-tandem MS. By integrating extraction and derivatization into one step, target GAs were effectively extracted from plant materials and simultaneously reacted with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, the sample preparation time was largely shortened, the probability of sample loss was minimized and the detection sensitivity of MS was also greatly improved compared with underivatized GAs. Under optimal conditions, the method was validated from the quantification linearity, limits of detection and limits of quantification in the presence of plant matrices, recoveries, and precision. With the proposed method, 15 endogenous GAs were detected and, among these, 11 GAs could be quantified in 0.50 mg fresh weight (FW) wheat shoot samples, and five GAs were quantified in only 0.15 mg FW developing seed samples of Arabidopsis thaliana. The distribution patterns of GAs along both the non-13-hydroxylation pathway and the early 13-hydroxylation pathway in a single shoot of germinating wheat, rice and maize seeds were finally profiled with a spatial resolution down to approximately 1 mm2 .
Assuntos
Grão Comestível/metabolismo , Giberelinas/metabolismo , Brotos de Planta/metabolismo , Sementes/metabolismo , Arabidopsis , Carbodi-Imidas/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida/métodos , Germinação , Giberelinas/isolamento & purificação , Metilaminas/metabolismo , Oryza/metabolismo , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Poaceae/metabolismo , Temperatura , Fatores de Tempo , Triticum/metabolismoRESUMO
N6 -isopentenyladenosine (i6 A) is an RNA modification found in cytokinins, which regulate plant growth/differentiation, and a subset of tRNAs, where it improves the efficiency and accuracy of translation. The installation and removal of this modification is mediated by prenyltransferases and cytokinin oxidases, and a chemical approach to selective deprenylation of i6 A has not been developed. We show that a selected group of oxoammonium cations function as artificial deprenylases to promote highly selective deprenylation of i6 A in nucleosides, oligonucleotides, and live cells. Importantly, other epigenetic modifications, amino acid residues, and natural products were not affected. Moreover, a significant phenotype difference in the Arabidopsis thaliana shoot and root development was observed with incubation of the cation. These results establish these small organic molecules as direct chemical regulators/artificial deprenylases of i6 A.
Assuntos
Óxidos N-Cíclicos/farmacologia , Citocininas/metabolismo , Isopenteniladenosina/metabolismo , Piperidinas/farmacologia , Prenilação/efeitos dos fármacos , RNA/metabolismo , Arabidopsis/efeitos dos fármacos , Óxidos N-Cíclicos/química , Óxidos N-Cíclicos/toxicidade , Citocininas/química , Epigênese Genética/efeitos dos fármacos , Humanos , Isopenteniladenosina/química , Células MCF-7 , Oligorribonucleotídeos/química , Oligorribonucleotídeos/metabolismo , Piperidinas/química , Piperidinas/toxicidade , Reguladores de Crescimento de Plantas/química , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Brotos de Planta/efeitos dos fármacos , RNA/químicaRESUMO
There remains a methodological bottleneck in the quantification of ultra-trace plant hormones in very tiny plant organs at fresh weights below a milligram. The challenge becomes even more serious in the determination of endogenous gibberellins (GAs), which are a class of compounds that are difficult to separate and detect. Herein, a quantification method using ultra-high-performance liquid chromatography-tandem mass spectrometry was developed, combined with a derivatization technique in which GAs react with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide in ethanol. The method was validated as capable of determining GAs in floral organs (about 80-250 µg) - pistil, stamens, petals, sepals and receptacle - which were dissected from only one flower of Arabidopsis thaliana. Substantially different abundance patterns of GAs were measured from the floral organs at floral stages 13, 14 and 15 along the non-13-hydroxylation pathway and the early 13-hydroxylation pathway in plants. This allows sub-flower-level insights into how GAs affect floral development. The method exhibited excellent limit of detection and limit of quantification down to 5.41 and 18.0 attomole, respectively, and offered a fairly wide linear range from 0.01 to 25 femtomole with linear coefficients above 0.9961. The precision of the method was evaluated with relative standard deviations below 10.6% for intra-day and 11.4% for inter-day assays, and recoveries ranged from 64.0% to 107%.
Assuntos
Arabidopsis/metabolismo , Flores/metabolismo , Giberelinas/metabolismo , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
It remains an issue to directly quantify trace biologically important carboxyl compounds in body fluids. Herein we propose an innovative method to determine α-lipoic acid, 2-(ß-carboxyethyl)-6-hydroxy-2,7,8-trimethylchroman, prostaglandin E2, cholic acid, and chenodeoxycholic acid in saliva. The method consists of two successive steps: fast and direct labeling of the target analytes with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide followed by ultrahigh-performance liquid chromatography-tandem mass spectrometry analysis. The method exhibited a wide linear range from 2.5 to 2500 pg/mL, with linear coefficients greater than 0.9963 and limits of detection and quantification as low as 0.10 and 0.33 pg/mL, respectively. The method precision was evaluated, with relative standard deviations ranging from 2.12% to 10.63% for intraday assays and from 2.98% to 12.88% for interday assays. The recoveries were measured by our spiking saliva samples with standards at three different levels, and ranged from 72.5% to 98.0%. Real applicability was validated by direct quantification of trace target analytes in human saliva, with simple pretreatment, use of a small sample volume, and a short analysis time. Graphical abstract Sequential steps to extract, label, and determine the ultratrace carboxylic acids in saliva. CDCA chenodeoxycholic acid, γ-CEHC 2-(ß-carboxyethyl)-6-hydroxy-2,7,8-trimethylchroman, α-LA α-lipoic acid, PGE2 prostaglandin E2, UHPLC-MS/MS ultrahigh-performance liquid chromatography-tandem mass spectrometry.
Assuntos
Ácidos Carboxílicos/análise , Cromatografia Líquida de Alta Pressão/métodos , Saliva/química , Espectrometria de Massas em Tandem/métodos , Carbodi-Imidas/química , Ácido Quenodesoxicólico/análise , Ácido Cólico/análise , Dinoprostona/análise , Humanos , Limite de Detecção , Metilaminas/química , Reprodutibilidade dos Testes , Ácido Tióctico/análiseRESUMO
Since 2012, several cannabimimetic indazole and indole derivatives with valine amino acid amide residue have emerged in the illicit drug market, and have gradually replaced the old generations of synthetic cannabinoids (SCs) with naphthyl or adamantine groups. Among them, ADB-FUBICA [N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indole-3-carboxamide], AB-FUBICA [N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indole-3-carboxamide], AB-BICA [N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-benzyl-1H-indole-3-carboxamide] and ADB-BICA [N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-benzyl-1H-indole-3-carboxamide] were detected in China recently, but unfortunately no information about their in vitro human metabolism is available. Therefore, biomonitoring studies to screen their consumption lack any information about the potential biomarkers (e.g. metabolites) to target. To bridge this gap, we investigated their phase I metabolism by incubating with human liver microsomes, and the metabolites were identified by ultra-performance liquid chromatography-high resolution-tandem mass spectrometry. Metabolites generated by N-dealkylation and hydroxylation on the 1-amino-alkyl moiety were found to be predominant for all these four substances, and others which underwent hydroxylation, amide hydrolysis and dehydrogenation were also observed in our investigation. Based on our research, we recommend that the N-dealkylation and hydroxylation metabolites are suitable and appropriate analytical markers for monitoring their intake.
Assuntos
Canabinoides/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Indazóis/metabolismo , Microssomos Hepáticos/metabolismo , Espectrometria de Massas em Tandem/métodos , Canabinoides/análise , Canabinoides/química , Humanos , Indazóis/análise , Indazóis/químicaRESUMO
BACKGROUND: Neuregulin-1 (NRG-1) has been shown to act as a neuroprotectant in animal models of nerve agent intoxication and other acute brain injuries. We recently demonstrated that NRG-1 blocked delayed neuronal death in rats intoxicated with the organophosphate (OP) neurotoxin diisopropylflurophosphate (DFP). It has been proposed that inflammatory mediators are involved in the pathogenesis of OP neurotoxin-mediated brain damage. METHODS: We examined the influence of NRG-1 on inflammatory responses in the rat brain following DFP intoxication. Microglial activation was determined by immunohistchemistry using anti-CD11b and anti-ED1 antibodies. Gene expression profiling was performed with brain tissues using Affymetrix gene arrays and analyzed using the Ingenuity Pathway Analysis software. Cytokine mRNA levels following DFP and NRG-1 treatment was validated by real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: DFP administration resulted in microglial activation in multiple brain regions, and this response was suppressed by treatment with NRG-1. Using microarray gene expression profiling, we observed that DFP increased mRNA levels of approximately 1,300 genes in the hippocampus 24 h after administration. NRG-1 treatment suppressed by 50% or more a small fraction of DFP-induced genes, which were primarily associated with inflammatory responses. Real-time RT-PCR confirmed that the mRNAs for pro-inflammatory cytokines interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) were significantly increased following DFP exposure and that NRG-1 significantly attenuated this transcriptional response. In contrast, tumor necrosis factor α (TNFα) transcript levels were unchanged in both DFP and DFP + NRG-1 treated brains relative to controls. CONCLUSION: Neuroprotection by NRG-1 against OP neurotoxicity is associated with the suppression of pro-inflammatory responses in brain microglia. These findings provide new insight regarding the molecular mechanisms involved in the neuroprotective role of NRG-1 in acute brain injuries.
Assuntos
Inibidores da Colinesterase/toxicidade , Inibidores da Colinesterase/uso terapêutico , Encefalite/induzido quimicamente , Isoflurofato/toxicidade , Neuregulina-1/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Animais , Encéfalo/patologia , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Injeções Intra-Arteriais , Masculino , Microglia/efeitos dos fármacos , Microglia/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To evaluate the prophylactic application of antibiotics in oral and maxillofacial surgery and to provide a scientific basis for its reasonable use. METHODS: The use of prophylactic antibiotics in the oral and maxillofacial surgery was conducted in our hospital from January 2011 to August 2013 based on a retrospective survey, and the conditions and affecting factors were analyzed. RESULTS: The utilization rates of prophylactic antibiotics were respectively 98.9%, 61.8%, and 24.6%, showing a downward trend. But the infection rate of surgical site did not significantly increase, and by Fisher's exact test, the difference was not significant (P>0.05). Surgical site infections (SSI) rates did not rise between using and not using prophylactic antibiotics (P>0.05). CONCLUSION: The use of prophylactic antibiotics is greatly influenced by the policy, and along with the decline in antibiotic usage, SSI have not increased significantly.
Assuntos
Antibacterianos/uso terapêutico , Antibioticoprofilaxia/estatística & dados numéricos , Procedimentos Cirúrgicos Bucais , Infecção da Ferida Cirúrgica/prevenção & controle , Humanos , Estudos RetrospectivosRESUMO
Afghanistan and Myanmar are two overwhelming opium production places. In this study, rapid and efficient methods for distinguishing opium from Afghanistan and Myanmar were developed using infrared spectroscopy (IR) coupled with multiple machine learning (ML) methods for the first time. A total of 146 authentic opium samples were analyzed by mid-IR (MIR) and near-IR (NIR), within them 116 were used for model training and 30 were used for model validation. Six ML methods, including partial least squares discriminant analysis (PLS-DA), orthogonal PLS-DA (OPLS-DA), k-nearest neighbour (KNN), support vector machine (SVM), random forest (RF), and artificial neural networks (ANNs) were constructed and compared to get the best classification effect. For MIR data, the average of precision, recall and f1-score for all classification models were 1.0. For NIR data, the average of precision, recall and f1-score for different classification models ranged from 0.90 to 0.94. The comparison results of six ML models for MIR and NIR data showed that MIR was more suitable for opium geography classification. Compared with traditional chromatography and mass spectrometry profiling methods, the advantages of MIR are simple, rapid, cost-effective, and environmentally friendly. The developed IR chemical profiling methodology may find wide application in classification of opium from Afghanistan and Myanmar, and also to differentiate them from opium originating from other opium producing countries. This study presented new insights into the application of IR and ML to rapid drug profiling analysis.
Assuntos
Ópio , Espectroscopia de Luz Próxima ao Infravermelho , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Afeganistão , Mianmar , Espectrofotometria Infravermelho , Análise Discriminante , Análise dos Mínimos Quadrados , Máquina de Vetores de SuporteRESUMO
The effective implementation of drug precursor legislation has driven the innovation and design of new alternative substances. The application of 1,3-dicarbonyl precursors as alternative precursors for the synthesis of 1-phenyl-2-propanone (P2P) and 3,4-methylenedioxyphenyl-2-propanone (MDP2P) has created new challenges to legal control. Their 1,3-dicarbonyl structure allows the precursors to exist as an equilibrium mixture of the tautomeric diketo and keto-enolic forms during the nuclear magnetic resonance (NMR) analysis. In this study, the keto-enol tautomerism of four 1,3-dicarbonyl drug pre-precursors, α-phenylacetoacetamide (APAA), methyl α-phenylacetoacetate (MAPA), ethyl α-phenylacetoacetate (EAPA), and methyl 2-(benzo[d][1,3]dioxol-5-yl)-3-oxobutanoate (MAMDPA) were investigated through NMR. One-dimensional (1D) and 2D NMR were combined to assign signals for the diketo and keto-enolic tautomers. Results showed that the keto-enol tautomerism was solvent-dependent but was also influenced by the substituent present in the molecule. Further, the analysis results indicated that majority of substances existed mainly in the diketo form. The enol-keto equilibrium constant (Keq) was stable in dimethyl sulfoxide-d6 and chloroform-d, while unstable for some compounds in acetone-d6 and deuterated methanol. The presence of impurities in the seized sample may disrupt the equilibrium between keto-enol tautomers in 1,3-dicarbonyl precursors. After the optimization of several key quantitative parameters, a quantitative NMR method for the quantification of 1,3-dicarbonyl drug precursors were also developed to facilitate their quantitative analysis. This is the first study to investigate the keto-enol tautomerism and quantification of 1,3-dicarbonyl drug precursors by NMR, providing a new approach for structure analysis and quantification of new precursor analogues.
RESUMO
In this study, the analytical characterization of three cathinones and one N-pyrrolidinyl-substituted amphetamine derivative is described: 1-([3,4-methylenedioxyphenyl])-2-(propylamino)butan-1-one (N-propyl norbutylone 1), 1-([3,4-methylenedioxyphenyl])-2-(butylamino)butan-1-one (N-butyl norbutylone 2), 2-(benzylamino)-1-phenylheptan-1-one (N-benzyl norheptedrone 3), and 1-(1-[3,4-dimethoxyphenyl]propan-2-yl)pyrrolidine (N-pyrrolidinyl-3,4-DMA 4). The identification was based on ultra-high-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (UHPLC-QTOF-MS), gas chromatography-orbitrap MS (GC-Orbitrap-MS), nuclear magnetic resonance spectroscopy (NMR), and Fourier transform infrared (FT-IR). GC-Orbitrap-MS, with higher mass accuracy, benefit more on the accurate structure elucidation of product ions compared with the low-resolution GC-MS. The collision-induced dissociation (CID) and electron ionization (EI) pathways of these compounds were examined to assist forensic laboratories in elucidating the structure of new psychoactive substances (NPS) with similar structure in their case work. In addition, electron activated dissociation (EAD) was applied to analyze N-benzyl norheptedrone, which showed only one product ion in the CID mode. The result showed that for compound with limited product ions in the CID mode, the EAD mode can give more complementary information for structure elucidation. In addition, quantitative NMR (qNMR) was applied for the quantification of four powdered/crystal and two herbal blend seized samples. To our knowledge, no analytical data about the compounds 3 and 4 have appeared until now, making this the first report on these compounds.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas , Cromatografia Gasosa-Espectrometria de Massas/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Espectrometria de Massas/métodos , Cromatografia Líquida de Alta Pressão/métodos , Espectroscopia de Ressonância Magnética/métodosRESUMO
Due to the diversity and fast evolution of new psychoactive substances (NPS), both public health and safety are threatened around the world. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), which serves as a simple and rapid technique for targeted NPS screening, is challenging with the rapid structural modifications of NPS. To achieve the fast non-targeted screening of NPS, six machine learning (ML) models were constructed to classify eight categories of NPS, including synthetic cannabinoids, synthetic cathinones, phenethylamines, fentanyl analogues, tryptamines, phencyclidine types, benzodiazepines, and "other substances" based on the 1099 IR spectra data items of 362 types of NPS collected by one desktop ATR-FTIR and two portable FTIR spectrometers. All these six ML classification models, including k-nearest neighbor (KNN), support vector machine (SVM), random forest (RF), extra trees (ET), voting, and artificial neural networks (ANNs) were trained through cross validation, and f1-scores of 0.87-1.00 were achieved. In addition, hierarchical cluster analysis (HCA) was performed on 100 synthetic cannabinoids with the most complex structural variation to investigate the structure-spectral property relationship, which leads to a summary of eight synthetic cannabinoid sub-categories with different "linked groups". ML models were also constructed to classify eight synthetic cannabinoid sub-categories. For the first time, this study developed six ML models, which were suitable for both desktop and portable spectrometers, to classify eight categories of NPS and eight synthetic cannabinoids sub-categories. These models can be applied for the fast, accurate, cost-effective, and on-site non-targeted screening of newly emerging NPS with no reference data available.