A competitive immunoassay for detecting triazophos based on fluorescent catalytic hairpin self-assembly.

A rapid detection method is introduced for residual trace levels of triazophos in water and agricultural products using an immunoassay based on catalytic hairpin self-assembly (CHA). The gold nanoparticle (AuNPs) surface was modified with triazophos antibody and sulfhydryl bio-barcode, and an immune competition reaction system was established between triazophos and its ovalbumin-hapten (OVA-hapten). The bio-barcode served as a catalyst to continuously induce the CHA reaction to achieve the dual signal amplification. The method does not rely on the participation of enzymes, and the addition of fluorescent materials in the last step avoids interfering factors, such as a fluorescence burst. The emitted fluorescence was detected at 489/521 nm excitation/emission wavelengths. The detection range of the developed method was 0.01-50 ng/mL for triazophos, and the limit of detection (LOD) was 0.0048 ng/mL. The developed method correlates well with the results obtained by LC-MS/MS, with satisfactory recovery and sensitivity. In sum, the designed method is reliable and provides a new approach to detect pesticide residues rapidly and quantitatively.

Dissipation, occurrence, and risk assessment of 12 pesticides in Dendrobium officinale Kimura et Migo.

The residual behaviors and dietary risk probability of 12 pesticides in Dendrobium officinale Kimura et Migo cultivated at two representative locations under green house conditions were investigated using liquid chromatography-tandem mass spectrometry. Field trials showed that the half-lives of 12 pesticides ranged from 0.9 to 14.4 days in fresh D. officinale stems. Based on maximum residue levels (MRLs), the ultimate residues of imidacloprid, dimethomorph, metalaxyl, tebuconazole, and cyazofamid at a pre-harvest interval (PHI) of 28 days were within acceptable limits. For abamectin, indoxacarb, and difenoconazole, 35-day PHIs were needed. The PHIs of trifloxystrobin and fluopyram were 42 days, the time required for their residues to be reduced to an MRL of 4 mg/kg. The chronic and acute risk quotients of target pesticides at PHIs of 28-42 days were below 5.929% and 0.532%, respectively, showing that the evaluated D. officinale exhibited an acceptably low dietary risk to the general population.

Significantly increased amino acid accumulation in a novel albino branch of the tea plant (Camellia sinensis).

MAIN CONCLUSION: A normal tea plant with one albino branch was discovered. RNA sequencing, albinism phenotype and ultrastructural observations provided a valuable understanding of the albino mechanism in tea plants. Tea plants with a specific color (white or yellow) have been studied extensively. A normal tea plant (Camellia sinensis cv. quntizhong) with one albino branch was discovered in a local tea plantation in Huangshan, Anhui, China. The pure albino leaves on this special branch had accumulated a fairly high content of amino acids, especially theanine (45.31 mg/g DW), and had a low concentration of polyphenols and an extremely low chlorophyll (Chl) content compared with control leaves. Ultrastructural observation of an albino leaf revealed no chloroplasts, whereas it was viable in the control leaf. RNA sequencing and differentially expressed gene (DEG) analysis were performed on the albino leaves and on control leaves from a normal green branch. The related genes involved in theanine and polyphenol biosynthesis were also investigated in this study. DEG expression patterns in Chl biosynthesis or degradation, carotenoid biosynthesis or degradation, chloroplast development, and biosynthesis were influenced in the albino leaves. Chloroplast deletion in albino leaves had probably destroyed the balance of carbon and nitrogen metabolism, leading to a high accumulation of free amino acids and a low concentration of polyphenols in the albino leaves. The obtained results can provide insight into the mechanism underlying this special albino branch phenotype, and are a valuable contribution toward understanding the albino mechanism in tea plants.

Parthenolide induces autophagy via the depletion of 4E-BP1.

Parthenolide (PTL) is a sesquiterpene lactone isolated from feverfew and exhibits potent antitumor activity against various cancers. Many studies indicate that PTL treatment leads to apoptosis, however, the mechanism has not been defined. Here, we observed that cells underwent autophagy shortly after PTL treatment. Inhibition of autophagy by knocking out autophagy associated gene atg5 blocked PTL-induced apoptosis. Surprisingly, PTL decreased the level of translation initiation factor eIF4E binding protein 1 (4E-BP1) in correlation with autophagy. Ectopic expression or shRNA knockdown of 4E-BP1 further verified the effect of 4E-BP1 on PTL-induced autophagy. Meanwhile, PTL elevated the cellular reactive oxygen species (ROS) which located upstream of the depletion of 4E-BP1, and contributed to the consequent autophagy. This study revealed 4E-BP1 as a trigger for PTL-induced autophagy and may lead to therapeutic strategy to enhance the efficacy of anticancer drugs.

Identifying candidate genes and biological pathways in muscle development through multi-tissue transcriptome comparisons between male and female geese.

Males and females have long shown disparities in body weight and height; yet, the underlying mechanisms influencing growth and development remain unclear. Male and female Zhedong White Geese (ZDW) geese have long been selected for large body size and egg production, respectively. This led to a large difference in body weight between males and females, making them a unique model for studying the effects of sex on growth and development. This study aimed to elucidate these mechanisms by comparing the transcriptomes of muscle and pituitary tissues in male and female ZDW geese to identify the critical genes responsible for the effects of sex on growth performance. Our analysis revealed 1101 differentially expressed genes (DEGs) in leg musculature (507 upregulated, 594 downregulated), 773 DEGs in breast musculature (311 upregulated, 462 downregulated), and 517 DEGs in the pituitary gland (281 upregulated, 236 downregulated) between male and female geese. These DEGs were significantly enriched in gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with endocrine metabolism (e.g., hormonal activities), muscle formation (e.g., sarcomere and myofibril), and bone formation (e.g., bone morphogenesis and cartilage formation). The upregulated genes in males were enriched in KEGG pathways involving nutrient digestion and absorption (vitamin and protein), as well as the secretion of digestive juices (gastric acid and bile). Through protein-protein interaction analyses, we also observed high-density gene networks related to muscle fiber development, calcium ion metabolism, mitochondrial respiratory chain, and bone development. Therefore, our multi-tissue transcriptome analysis provides a deeper understanding of the complex and systematic gender-driven effects on growth and development in geese. IGF1, GHRHR, and NCAPG-LCORL and pathways related to myogenesis might play vital roles in gender differences before hormones exert their effect.

Cost-effective and sensitive indicator-displacement array (IDA) assay for quality monitoring of black tea fermentation.

Herein, a new indicator-displacement array (IDA) sensor was developed for the quality evaluation of black tea fermentation. On the principle of the reversible covalent binding of phenylboronic acid and catechol, phenylboronic acids were selected as acceptors for targeted binding to polyphenols. Pyrocatechol violet and alizarin red S were used as indicators of the reaction. The IDA sensors have sensitive differential responses to fermented tea samples, achieving an assessment of the fermentation degree with accuracies of 80.39-88.00% by support vector machine (SVM). In addition, the key polyphenol components of the fermentation process were accurately predicted by the IDA and SVM regression with ratio of prediction to deviation values of 1.55-1.72, 2.03-2.21, and 2.03-2.08 for total polyphenols, total catechins, and epigallocatechin-3-O-gallate, respectively. In conclusion, the developed IDA sensor is capable of the in-situ quality monitoring of black tea fermentation, with the advantages being cost-effectiveness, sensitivity, and rapidity.

Metabolite analysis and sensory evaluation reveal the effect of roasting on the characteristic flavor of large-leaf yellow tea.

Roasting is essential for processing large-leaf yellow tea (LYT). However, the effect of the roasting on the metabolic and sensory profiles of LYT remains unknown. Herein, the metabolomics and sensory quality of LYT at five roasting degrees were evaluated by liquid/gas chromatography mass spectrometry and quantitative descriptive analysis. A higher degree of roasting resulted in a significantly stronger crispy rice, fried rice, and smoky-burnt aroma (p < 0.05), which is closely associated with heterocyclic compound accumulation (concentrations: 6.47 ± 0.27 - 1065.00 ± 5.58 µg/g). Amino acids, catechins, flavonoid glycosides and N-ethyl-2-pyrrolidone-substituted flavan-3-ol varied with roasting degree. The enhancement of crispy-rice and burnt flavor coupled with the reduction of bitterness and astringency. Correlations analysis revealed the essential compounds responsible for roasting degree, including 2,3-diethyl-5-methylpyrazine, hexanal, isoleucine, N-ethyl-2-pyrrolidone-substituted flavan-3-ol (EPSF), and others. These findings provide a theoretical basis for improving the specific flavors of LYT.

Effects of Microbial Action and Moist-Heat Action on the Nonvolatile Components of Pu-Erh Tea, as Revealed by Metabolomics.

Microbial action and moist-heat action are crucial factors that influence the piling fermentation (PF) of Pu-erh tea. However, their effects on the quality of Pu-erh tea remain unclear. In this study, the effects of spontaneous PF (SPPF) and sterile PF (STPF) on the chemical profile of Pu-erh tea were investigated for the first time, and sun-dried green tea was used as a raw material to determine the factors contributing to the unique quality of Pu-erh tea. The results indicated that the SPPF-processed samples had a stale and mellow taste, whereas the STPF-processed samples had a sweet and mellow taste. Through metabolomics-based analysis, 21 potential markers of microbial action (including kaempferol, quercetin, and dulcitol) and 10 potential markers of moist-heat action (including ellagic acid, ß-glucogallin, and ascorbic acid) were screened among 186 differential metabolites. Correlation analysis with taste revealed that metabolites upregulated by moist-heat and microbial action were the main factors contributing to the staler mellow taste of the SPPF-processed samples and the sweeter mellow taste of the STPF-processed samples. Kaempferol, quercetin, and ellagic acid were the main active substances formed under microbial action. This study provides new knowledge regarding the quality formation mechanism of Pu-erh tea.

Evaluation of the effects of solar withering on nonvolatile compounds in white tea through metabolomics and transcriptomics.

The mechanism through which solar withering (SW) affects the quality of white tea is unclear. To address this gap in the literature, in this study, we used metabolomics and transcriptomics to investigate the effect of SW on the quality of WT. WT that underwent SW was slightly more bitter and astringent than WT that underwent natural withering (control group). Specifically, SW considerably increased the concentration of astringent flavonoids and flavone glycosides in WT. This increase was mainly attributed to the upregulated expression of key genes in the shikimic acid, phenylpropanoid, and flavonoid biosynthesis pathways, such as shikimate kinase, chalcone synthase, and flavonol synthase. In addition, SW experienced considerable heat and light stress. The levels of glycerophosphatidylcholine and carbohydrates increased in response to the stress, which also affected the taste of WT. The results of this study indicate the mechanism through which SW affects the quality of WT.

Effects of Three Different Withering Treatments on the Aroma of White Tea.

White tea (WT) is a slightly fermented tea, and withering is a critical step in its processing. The withering treatment can affect white tea's aroma; different treatments' effects were investigated in this study. White tea was withered indoors (IWT), in a withering-tank (WWT), or under sunlight (SWT). Quantitative descriptive analysis (QDA) results showed that SWT had a more obvious flower aroma, and WWT had a more pronounced grassy aroma. Volatile compounds were extracted and subsequently detected with solvent-assisted flavor evaporation (SAFE) and headspace solid-phase microextraction (HS-SPME) combined in addition to gas chromatography-mass spectrometry (GC-MS). A total of 202 volatile compounds were detected; 35 of these aroma-active compounds met flavor dilution (FD) factor ≥ 4 or odor activity value (OAV) ≥ 1. The nine key potent odorants for which both conditions were met were dimethyl sulfide, 2-methyl-butanal, 1-penten-3-one, hexanal, (Z)-4-heptenal, ß-Myrcene, linalool, geraniol, and trans-ß-ionone. These results were used with QDA to reveal that SWT had a stronger floral aroma mainly due to an increase of geraniol and linalool. Moreover, WWT had a stronger grassy aroma mainly due to increased hexanal. The results could be used to select processing methods for producing white tea with a superior aroma.

A highly sensitive bio-barcode immunoassay for multi-residue detection of organophosphate pesticides based on fluorescence anti-quenching.

Balancing the risks and benefits of organophosphate pesticides (OPs) on human and environmental health relies partly on their accurate measurement. A highly sensitive fluorescence anti-quenching multi-residue bio-barcode immunoassay was developed to detect OPs (triazophos, parathion, and chlorpyrifos) in apples, turnips, cabbages, and rice. Gold nanoparticles were functionalized with monoclonal antibodies against the tested OPs. DNA oligonucleotides were complementarily hybridized with an RNA fluorescent label for signal amplification. The detection signals were generated by DNA-RNA hybridization and ribonuclease H dissociation of the fluorophore. The resulting fluorescence signal enables multiplexed quantification of triazophos, parathion, and chlorpyrifos residues over the concentration range of 0.01-25, 0.01-50, and 0.1-50 ng/mL with limits of detection of 0.014, 0.011, and 0.126 ng/mL, respectively. The mean recovery ranged between 80.3% and 110.8% with relative standard deviations of 7.3%-17.6%, which correlate well with results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proposed bio-barcode immunoassay is stable, reproducible and reliable, and is able to detect low residual levels of multi-residue OPs in agricultural products.

Molecular and Biochemical Characterization of Jasmonic Acid Carboxyl Methyltransferase Involved in Aroma Compound Production of Methyl Jasmonate during Black Tea Processing.

Methyl jasmonate (MeJA), a volatile organic compound, is a principal flowery aromatic compound in tea. During the processing of black tea, MeJA is produced by jasmonic acid carboxyl methyltransferase (JMT) of the jasmonic acid (JA) substrate, forming a specific floral fragrance. CsJMT was cloned from tea leaves; the three-dimensional structure of CsJMT was predicted. Enzyme activity was identified, and protein purification was investigated. Site-directed deletions revealed that N-10, S-22, and Q-25 residues in the beginning amino acids played a key functional role in enzyme activity. The expression patterns of CsJMT in tea organs differed; the highest expression of CsJMT was observed in the fermentation process of black tea. These results aid in further understanding the synthesis of MeJA during black tea processing, which is crucial for improving black tea quality using specific fragrances and could be applied to the aromatic compound regulation and tea breeding improvement in further studies.

Enhanced Bio-Barcode Immunoassay Using Droplet Digital PCR for Multiplex Detection of Organophosphate Pesticides.

A bio-barcode immunoassay based on droplet digital polymerase chain reaction (ddPCR) was developed to simultaneously quantify triazophos, parathion, and chlorpyrifos in apple, cucumber, cabbage, and pear. Three gold nanoparticle (AuNP) probes and magnetic nanoparticle (MNP) probes were prepared, binding through their antibodies with the three pesticides in the same tube. Three groups of primers, probes, templates, and three antibodies were designed to ensure the specificity of the method. Under the optimal conditions, the detection limits (expressed as IC10) of triazophos, parathion, and chlorpyrifos were 0.22, 0.45, and 4.49 ng mL-1, respectively. The linear ranges were 0.01-20, 0.1-100, and 0.1-500 ng mL-1, and the correlation coefficients (R2) were 0.9661, 0.9834, and 0.9612, respectively. The recoveries and relative standard deviations (RSDs) were in the ranges of 75.5-98.9 and 8.3-16.7%. This study provides the first insights into the ddPCR for the determination of organophosphate pesticides. It also laid the foundation for high-throughput detection of other small molecules.

A visual bio-barcode immunoassay for sensitive detection of triazophos based on biochip silver staining signal amplification.

Herein, a novel visual method for detecting triazophos based on competitive bio-barcode immunoassay was described. The competitive immunoassay was established by gold nanoparticles (AuNPs), magnetic microparticle (MMPs) and triazophos, combined with biochip hybridization system to detect the residual of triazophos in water and apple. Because AuNPs carried many bio-barcodes, which hybridized with labeled DNA on the biochip, catalyzed signal amplification using silver staining was detected by grayscale values as well as the naked eye. Notably, the grayscale values decreases with increasing the concentrations of triazophos, and the color change weakened gradually. The detection range was in between 0.05 and 10 ng/mL and the minimum detection limit was set at 0.04 ng/mL. Percent recovery calculated from spiked water and apple samples ranged between 55.4 and 107.8% with relative standard deviations (RSDs) of 12.4-24.9%. It has therefore been shown that this protocol provides a new insight for rapid detection of small molecule pesticides in various matrices.

A sensitive bio-barcode immunoassay based on bimetallic Au@Pt nanozyme for detection of organophosphate pesticides in various agro-products.

Organophosphate pesticides (OPs) are often used as insecticides and acaricides in agriculture, thus improving yields. OP residues may pose a serious threat, duetoinhibitionof the enzymeacetylcholinesterase(AChE). Therefore, a competitive bio-barcode immunoassay was designed for simultaneous quantification of organophosphate pesticide residues using AuNP signal amplification technology and Au@Pt catalysis. The AuNP probes were labelled with antibodies and corresponding bio-barcodes (ssDNAs), MNP probes coated with ovalbumin pesticide haptens and Au@Pt probes functionalized with the complementary ssDNAs were then prepared. Subsequently, pesticides competed with MNP probes to bind the AuNP probes. The recoveries of the developed assay were ranged from 71.26 to 117.47% with RSDs from 2.52 to 14.52%. The LODs were 9.88, 3.91, and 1.47 ng·kg-1, for parathion, triazophos, and chlorpyrifos, respectively. The assay was closely correlated with the data obtained from LC-MS/MS. Therefore, the developed method has the potential to be used as an alternative approach for detection of multiple pesticides.

A highly sensitive bio-barcode immunoassay for multi-residue detection of organophosphate pesticides based on fluorescence anti-quenching / 药物分析学报

Balancing the risks and benefits of organophosphate pesticides(OPs)on human and environmental health relies partly on their accurate measurement.A highly sensitive fluorescence anti-quenching multi-residue bio-barcode immunoassay was developed to detect OPs(triazophos,parathion,and chlorpyrifos)in apples,turnips,cabbages,and rice.Gold nanoparticles were functionalized with monoclonal antibodies against the tested OPs.DNA oligonucleotides were complementarily hybridized with an RNA fluorescent label for signal amplification.The detection signals were generated by DNA-RNA hybridization and ribonuclease H dissociation of the fluorophore.The resulting fluorescence signal en-ables multiplexed quantification of triazophos,parathion,and chlorpyrifos residues over the concen-tration range of 0.01-25,0.01-50,and 0.1-50 ng/mL with limits of detection of 0.014,0.011,and 0.126 ng/mL,respectively.The mean recovery ranged between 80.3％and 110.8％with relative standard deviations of 7.3％-17.6％,which correlate well with results obtained by liquid chromatography-tandem mass spectrometry(LC-MS/MS).The proposed bio-barcode immunoassay is stable,reproducible and reliable,and is able to detect low residual levels of multi-residue OPs in agricultural products.

Vav1 increases Bcl-2 expression by selective activation of Rac2-Akt in leukemia T cells.

Vav proteins are guanine nucleotide exchange factors (GEFs) that activate a group of small G proteins (GTPases). Vav1 is predominantly expressed in hematopoietic cells, whereas Vav2 and Vav3 are ubiquitously distributed in almost all human tissues. All three Vav proteins contain conserved structural motifs and associate with a variety of cellular activities including proliferation, migration, and survival. Previous observation with Jurkat leukemia T cells showed that Vav1 possessed anti-apoptotic activity by enhancing Bcl-2 transcription. However the mechanism has not been unveiled. Here, we explored the effectors of Vav1 in promoting Bcl-2 expression in Jurkat cells and revealed that Rac2-Akt was specifically evoked by the expression of Vav1, but not Vav2 or Vav3. Although all three Vav isoforms existed in Jurkat cells, Rac2 was distinguishably activated by Vav1 and that led to enhanced Bcl-2 expression and cell survival. Akt was modulated downstream of Vav1-Rac2, and the activation of Akt was indispensable in the enhanced transcription of Bcl-2. Intriguingly, neither Vav2 nor Vav3 was able to activate Rac2-Akt pathway as determined by gene silencing approach. Our data illustrated a unique role of Vav1 in T leukemia survival by selectively triggering Rac2-Akt axis and elevating the expression of anti-apoptotic Bcl-2.