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Dried roots of Zanthoxylum nitidum (2n=2x=70, family Rutaceae) was referred as "Liang-Mian-Zhen" in Chinese, acting as a valuable species due to its notable pharmacological activities. Herein, we combined PacBio HiFi data together with Hi-C mapping technology to construct a chromosome-scale reference genome assembly for Z. nitidum. The assembly reached a length of 2.24 Gb, successfully anchoring 99.31% of sequences onto 35 pseudo-chromosomes. Among these, 26 chromosomes achieved telomere-to-telomere assembly, and 11 chromosomes were gap-free. The contigs N50 and scaffolds N50 reached 57.61 Mb and 78.00 Mb, respectively. Transposable elements comprised 81.44% of the Z. nitidum genome, and over 78% of them were long-terminal repeat retrotransposon elements. Furthermore, 32,737 protein-coding genes were identified and 99.38% of all were functionally annotated. The completeness of the genome assembly and final gene sets reached 97.83% and 96.47% based on Benchmarking Universal Single-Copy Orthologs (BUSCO), respectively. Taken together, our results provided a high-quality chromosome-level assembly of Z. nitidum genome and will be a valuable resource that will facilitate breeding varieties with higher alkaloids content. [ABSTRACT FROM AUTHOR]

Anthracnose caused by Colletotrichum gloeosporioides critically threatens the growth and commercial cultivation of Sarcandra glabra. However, the defence responses and underlying mechanisms remain unclear. Herein, we aimed to investigate the molecular reprogramming in S. glabra leaves infected with C. gloeosporioides. Leaf tissues at 0, 24 and 48 h post-inoculation (hpi) were analysed by combining RNA sequencing and Tandem Mass Tag-based liquid chromatography with tandem mass spectrometry. In total, 18 441 and 25 691 differentially expressed genes were identified at 24 and 48 hpi compared to 0 hpi (uninoculated control), respectively. In addition, 1240 and 1570 differentially abundant proteins were discovered at 24 and 48 hpi compared to 0 hpi, respectively. Correlation analysis revealed that transcription and translation levels were highly consistent regarding repeatability and expression. Analyses using databases KEGG and iPATH revealed tricitric acid cycle, glycolysis/gluconeogenesis and phenylpropanoid biosynthesis were induced, whereas photosynthesis and tryptophan were suppressed. Enzymatic activity assay results were consistent with the upregulation of defence-related enzymes including superoxide dismutases, catalases, peroxidases and chitinases. The transcriptome expression results were additionally validated by quantitative real-time polymerase chain reaction analyses. This study provides insights into the molecular reprogramming in S. glabra leaves during infection, which lay a foundation for investigating the mechanisms of host- Colletotrichum interactions and breeding disease-resistant plants. Anthracnose caused by Colletotrichum gloeosporioides critically threatens the growth and commercial cultivation of the medicinal plant Sarcandra glabra. We explored the molecular changes during the early stages of this infection through systems biology. This study provides insights into the molecular reprogramming in S. glabra leaves during infection, which lay a foundation for investigating the mechanisms of host -Colletotrichum interactions and breeding disease-resistant plants. [ABSTRACT FROM AUTHOR]

The safety of medicinal herbs is critical to human health, and the monitoring of pesticide residues in medicinal herbs is necessary. In the present study, a direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed for the determination of imidacloprid in medicinal herbs. Using the optimal conditions, the 50% inhibiting concentration and detection range (20–80% inhibiting concentration) of the established dc-ELISA were 0.08 and 0.03–0.29 ng/mL, respectively. Except for clothianidin (4.64%) and acetamiprid (2.52%), the cross-reactivity with analyte analogs was negligible. In purple perilla, cordate houttuynia, water lily, bitter melon, and mung bean, the average imidacloprid recoveries were from 62.97 to 134.09%, with relative standard deviations between 0.21 and 14.58%. Furthermore, the immunoassay results correlated well with spiked samples analyzed by high-performance liquid chromatography – tandem mass spectrometry (HPLC-MS/MS). The established immunoassay is rapid, sensitive, and specific and suitable to monitor imidacloprid residues in medicinal herbs. [ABSTRACT FROM AUTHOR]

Sodium saccharin is a common artificial sweetener. However, due to its possible carcinogenic effects and causing metabolic disorders, many countries have strictly regulated its use in food. In the study, we prepared a specific monoclonal antibody (mAb 2H11) using the new hapten (6‐carboxylsaccharin) and developed a direct competitive enzyme‐linked immunosorbent assay (dcELISA) for the screening of sodium saccharin residue in food. The half‐maximum inhibition concentration (IC50) and working range (IC20–IC80, the concentrations causing 20% and 80% inhibition by sodium saccharin) were 32.5 and 6.47 to 164 ng/mL, which was 6.5 times more sensitive than the previously reported immunoassay. The average recoveries of sodium saccharin in spiked food samples detected by dcELISA ranged from 82.1% to 117%. Among 70 food samples bought in the physical stores and online, sodium saccharin residues were only detected in four samples purchased online (one canned pineapple, two winter jujube, and one kimchi). The content measured by dcELISA agreed well with those determined by ultra‐performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS). The developed dcELISA was proved to be a sensitive and accurate method for determining sodium saccharin in food. Practical Application: Quantitation of sodium saccharin residue in food is very necessary and important for consumers and regulatory agencies. [ABSTRACT FROM AUTHOR]

In May 2019, leaf spots were found on Amomum villosum in Guangxi Province, China. Light yellow-green spots first appeared at the tips or edges of leaves. A fungus was isolated from the leaves and identified as Colletotrichum fructicola based on morphological characteristics and molecular identification using the DNA sequences from ITS, TUB2, ACT, and GAPDH sequences. Similar leaf spots developed after leaves were inoculated with the isolate. This is the first report that C. fructicola causes leaf spots on A. villosum. [ABSTRACT FROM AUTHOR]

Carbofuran residue in vegetables is a concern to human health. Direct competitive enzyme‐linked immunosorbent assay (dcELISA) and dipstick immunoassay were developed in the present study. The dcELISA showed a 50% inhibition concentration (IC50) and working range of 1.3 and 0.2 to 7.5 ng/mL, respectively, while the cutoff value of dipstick immunoassay was 20 ng/mL. Applying the two immunoassays, we achieved the goal of rapid screening of carbofuran residue in commercial vegetables with a simple sample processing method. Among 46 leek, 39 potato, and 39 sweet potato samples, carbofuran residue was detected in 22% of the leek samples, and two samples exceeded the maximum residue limit of China (0.02 mg/kg). In addition, carbofuran residue was found at less than 2.5 ng/g in one potato and one sweet potato samples. The residual level of carbofuran measured by immunoassays agreed well with those determined by ultra‐performance liquid chromatography tandem mass spectrometry. To ensure food safety and human health, it is greatly necessary and meaningful to monitor carbofuran residue in commercial vegetables. Practical Application: Rapid monitoring of carbofuran residue in vegetables is very necessary and important for consumers, regulatory agencies, and food industry. [ABSTRACT FROM AUTHOR]

Dihydroartemisinin (DHA) and piperaquine (PPQ) are two drugs used in an artemisinin‐based combination therapy (ACT). The circulation of counterfeit antimalarial drugs demands the development of simple, point‐of‐care (POC) tests for monitoring drug quality. Here we aimed to design an antibody‐based lateral flow dipstick assay for simultaneous quality control of DHA and PPQ. To obtain a monoclonal antibody (mAb) for PPQ, one structural unit of the symmetric PPQ molecule was used to derive a carboxylic acid for linkage to a carrier protein as immunogen. Screening of hybridoma cells identified an mAb 4D112B2 that reacted with the PPQ‐based immunogen. A highly‐sensitive icELISA was designed based on this mAb, which showed 50% inhibition concentration of PPQ at 1.66 ng/mL and a working range of 0.35 – 7.40 ng/mL. The mAb showed 10.2, 15.9 and 30.4% cross reactivity to hydroxychloroquine sulfate, chloroquine and amodiaquine, respectively. No cross reactivity was observed to lumefantrine, mefloquine artemisinin and its derivatives. Using our previous DHA dipstick design, a lateral flow dipstick for simultaneous analysis of PPQ and DHA was developed. The indicator ranges for PPQ and DHA were 2 – 5 μg/mL and 250 – 500 ng/mL, respectively. The dipstick was used to semi‐quantitatively analyze PPQ and DHA content in commercial ACT drugs, which produced agreeable results to those determined by high‐performance liquid chromatography. This combination dipstick makes it a potential POC device for quality control of the two active ingredients in a commonly used ACT. [ABSTRACT FROM AUTHOR]

Folates (vitamin B9) are essential for all organisms as cofactors for one-carbon metabolism. However, measurement of folates is technically complicated and time-consuming. In this study, we developed a dipstick immunoassay using a folate-specific monoclonal antibody (mAb), allowing rapid and low-cost detection of folates. The indicator range of the dipstick for 5-formylterahydrofolate (5-CHO-THF), 5-methyltetrahydrofolate (5-CH-THF) and their polyglutamyl forms was 100-200 ng mL; moreover, no cross-reactivity was observed with tetrahydrofolate (THF) or 5,10-methenyltetrahydrofolate (5,10-CH=THF) at 500 ng mL, or with the folate precursors pterin-6-COOH, p-aminobenzoate (pABA), and L-glutamate, or with the folate analogues methotrexate and 10-formyltetrahydrofolate (10-CHO-THF) at up to 1000 ng mL. The dipstick immunoassay was tested in maize seeds; the results classified the seeds into those with low, moderate, and high levels of folates, and were in agreement with those of liquid chromatography-mass spectrometry. Thus, we conclude that the dipstick assay will provide a versatile tool to facilitate large-scale screening of maize rich in folates. [ABSTRACT FROM AUTHOR]

Maize is one of the most important staple food crops worldwide. 5-Formyltetrahydrofolate (5-F-THF), the most dominant and stable folate derivative in maize kernels, is important to human health and acts key roles in several metabolic pathways and biological processes. To assist investigating maize germplasm and subsequent breeding of maize varieties containing high levels of 5-F-THF, a cost-effective and high-throughput methodology for 5-F-THF detection is required. In the present study, monoclonal antibodies (mAbs) against 5-F-THF were raised in mice, followed by establishment of an indirect competitive enzyme-linked immunosorbent assay (icELISA). The icELISA showed an IC of 35.10 ng/mL and a working range of 7.90-263.89 ng/mL for 5-F-THF. The analytical recovery for 5-F-THF in the maize kernels with icELISA was 66-111 %, and that with liquid chromatography-mass spectrometry (LC-MS) was 57-114 %, indicating a good consistency between these two methods (Slope, 0.92; R , 0.98). We conclude that the sensitive and versatile icELISA can be used for high-throughput detection of 5-F-THF in maize kernels. [ABSTRACT FROM AUTHOR]

Artesunate is a frontline antimalarial drug for treating Plasmodium falciparum malaria. To produce specific antibodies to artesunate, the carboxyl group of artesunate was directly conjugated to carrier protein as the immunogen. A specific monoclonal antibody (mAb) 3D2G against artesunate was obtained by high-throughput screening of positive hybridoma clones. This monoclonal antibody had 4.0, 0.5, and 0.9 % cross reactivities with artemisinin, dihydroartemisinin, and artemether, respectively. A dipstick immunoassay was developed, and the indicator range for artesunate was 1000-2000 ng mL. No interference was observed with artemisinin, dihydroartemisinin, artemether, and other commonly used antimalarial drugs for up to 20,000 ng mL. The dipsticks were used for determination of artesunate contents in commercial drugs, and the results were agreeable with those determined by high-performance liquid chromatography. This dipstick, with its specificity and sensitivity for artesunate and simplicity to use, makes it a potential point-of-care device for rapid quality evaluation of artesunate-containing antimalarial drugs. [ABSTRACT FROM AUTHOR]

In the present research, we studied the binding characterization of a single chain fragment variable (scFv) antibody S12B8 with abscisic acid (ABA) and conjugated ABA by competitive enzyme-linked immunosorbent assay (ELISA) and in silico molecular docking. The nucleotide sequences of variable light (VL) and variable heavy (VH) were obtained from hybridoma cells (F12B8), which secrete monoclonal antibodies recognizing ABA and conjugated ABA. And then the genes of VL and VH were constructed to a scFvS12B8 with a nucleotide sequence of a flexible linker (G4S)3 by splicing overlap extension PCR (SOE-PCR). The fragment of S12B8 was cloned into pMAL-c2x with a maltose binding protein tag and expressed in Escherichia coli with a calculated molecular mass of 68 kDa. The results of ELISA showed dose-dependent inhibition of the purified recombinant protein S12B8 by ABA, (ABA-ME) and ABA glucose ester tetra acetyl (ABAGE tetra acetyl). The detection limits (10% inhibition) of ABA, ABA-ME and ABAGE tetra acetyl were 19.71, 8.06 and 3.95 μg/ml, respectively. On the other hand, we investigated the interactions between S12B8 and ligands by homology modeling using RosettaAntibody and molecular docking by Discovery studio 2.5 (DS 2.5). The binding energy of ABA, ABA-ME and ABAGE when fused to the S12B8 antibody was .37.1, .69.6 and .112.0 kcal/mol, respectively, which agreed well with our competitive ELSIA results. [ABSTRACT FROM AUTHOR]

Thidiazuron (TDZ) is a synthetic plant growth regulator, which is widely used in agriculture and tissue culture. A sensitive and specific monoclonal antibody (mAb3D6F5B2) was obtained. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed with mAb3D6F5B2. The established icELISA possessed a 50 % inhibition concentration (IC) of 0.61 ng/mL and a quantitative range of 0.15-2.49 ng/mL. The recoveries of TDZ fortified in water and soil samples were 76-126 %. The residual level of TDZ in soil samples detected by icELISA was confirmed with high performance liquid chromatography-mass spectrometry (LC/MS). The correlation coefficient ( R) between the two assays was 0.9898, demonstrating this icELISA is suitable to detect TDZ residues in environmental and agricultural samples. [ABSTRACT FROM AUTHOR]

The distribution of metallic ions in plant tissues is associated with their toxicity and is important for understanding mechanisms of toxicity tolerance. A quantitative histochemical method can help advance knowledge of cellular and subcellular localization and distribution of heavy metals in plant tissues. An immunohistochemical (IHC) imaging method for cadmium ions (Cd2+) was developed for the first time for the wheat Triticum aestivum grown in Cd2+-fortified soils. Also, 1-(4-Isothiocyanobenzyl)-ethylenediamine-N,N,N,N-tetraacetic acid (ITCB-EDTA) was used to chelate the mobile Cd2+. The ITCB-EDTA/Cd2+ complex was fixed with proteins in situ via the isothiocyano group. A new Cd2+-EDTA specific monoclonal antibody, 4F3B6D9A1, was used to locate the Cd2+-EDTA protein complex. After staining, the fluorescence intensities of sections of Cd2+-positive roots were compared with those of Cd2+-negative roots under a laser confocal scanning microscope, and the location of colloidal gold particles was determined with a transmission electron microscope. The results enable quantification of the Cd2+ content in plant tissues and illustrate Cd2+ translocation and cellular and subcellular responses of T. aestivum to Cd2+ stress. Compared to the conventional metal-S coprecipitation histochemical method, this new IHC method is quantitative, more specific and has less background interference. The subcellular location of Cd2+ was also confirmed with energy-dispersive X-ray microanalysis. The IHC method is suitable for locating and quantifying Cd2+ in plant tissues and can be extended to other heavy metallic ions. [ABSTRACT FROM AUTHOR]

Artemether is one of the artemisinin derivatives that are active ingredients in antimalarial drugs. Counterfeit and substandard antimalarial drugs have become a serious problem, which demands reliable analytical tools and implementation of strict regulation of drug quality. Structural similarity among artemisinin analogs is a challenge to develop immunoassays that are specific to artemisinin derivatives. To produce specific antibodies to artemether, we used microbial fermentation of artemether to obtain 9-hydroxyartemether, which was subsequently used to prepare a 9-O-succinylartemether hapten for conjugation with ovalbumin as the immunogen. A monoclonal antibody (mAb), designated as 2G12E1, was produced with high specificity to artemether. 2G12E1 showed low cross reactivities to dihydroartemisinin, artemisinin, artesunate and other major antimalarial drugs. An indirect competitive enzyme linked immunosorbent assay (icELISA) developed showed a concentration causing 50% of inhibition for artemether as 3.7 ng mL−1 and a working range of 0.7–19 ng mL−1. The icELISA was applied for determination of artemether content in different commercial drugs and the results were comparable to those determined by high-performance liquid chromatography analysis. In comparison with reported broad cross activity of anti-artemisinin mAbs, the most notable advantage of the 2G12E1-based ELISA is its high specificity to artemether only. [ABSTRACT FROM AUTHOR]

Cadmium is classified as a probable human carcinogen. A monoclonal antibody (mAb) against Cd2+-ethylene-diamine-N,N,N′,N′-tetraacetic acid (EDTA) complex was generated by immunising mice with the conjugate of Cd2+-EDTA-aminobenzyl-ovalbumin. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed and applied for the analysis of Cd2+ in water, soil and rape samples. The half-maximum inhibition concentration and detection range were 2.59 ng/ml and 0.20–40 ng/ml, respectively. The assay cross-reactivities with non-target metal-EDTA complexes were all below 1%, except Mn2+ (2.9%) and Hg2+ (1.6%). The average recoveries of Cd2+ in tap water, Jingmi Canal, Xiaoqing River (at concentrations of 2–20 ng/ml), soil (0.25–10 ug/g) and rape (0.1–1.2 ug/g) samples were 103.0%, 101.0%, 103.0%, 82.4% and 93.7%, respectively. Concentrations of Cd2+ measured by this immunoassay agreed well with those determined by graphite furnace atomic absorption spectroscopy (GFAAS) (k=1.1225, r 2=0.9873).The established icELISA was a quick, reliable and sensitive method to detect Cd2+ in environmental and vegetable samples. [ABSTRACT FROM PUBLISHER]

Flubendiamide (FD), the first commercial phthalic acid diamide that targets insect ryanodine receptor (RyRs), has played an important role in pest management. With its extensive worldwide application, a rapid and convenient method to detect its existence in the environment is necessary. In this study, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed to analyse FD residue on environmental and food samples. The established icELISA showed a half maximal inhibition concentration (IC50) of 17.25 µg L−1, with a working range of 4.06-103.59 µg L−1 for FD, and showed no cross-reactivity with chlorantraniliprole, cyantraniliprole, and several FD analogues. Average FD recoveries from spinach, tap water, and soil samples were 89.3-112.3%, 93.0-102.1%, and 86.9-97.6%, respectively. Meanwhile, FD detection results of icELISA were compared with those of ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The comparable results verified that icELISA was suitable for rapid detection of FD residue in environmental and agricultural samples. [ABSTRACT FROM AUTHOR]

Background: Mepiquat chloride (MC) is a plant growth regulator widely used in cotton (Gossypium hirsutum L.) production to suppress excessive vegetative growth, increase root growth and avoid yield losses. To increase root growth, cotton seeds were treated with MC to increase the number of lateral root (LRs) and improve drought resistance. An increased indole-3-acetic acid (IAA) pool appeared to correlate with LR growth, and the principal source of IAA in germinating seeds is IAA conjugates. Here, the role of IAA homeostasis and signaling was investigated in cotton seedlings treated with MC. Results: In the present research, MC significantly increased endogenous IAA levels in the roots, which promoted lateral root initiation (LRI) by upregulating GhARF7/19 and GhLBD18s and subsequently increasing LR quantity and elongation. The levels of IAA-amide conjugates significantly decreased in MC-treated seedlings compared with untreated control seedlings. Sixteen members of the cotton IAA amidohydrolase (IAH) gene family were identified, of which GhIAR3a, GhIAR3b, GhILR1, GhILL3 and GhILL6 were expressed during cotton seed germination. Compared with those in untreated control seedlings, the expression levels of GhIAR3a, GhIAR3b, GhILR1 and GhILL6 in the MC-treated seedlings were markedly elevated. The GhIAR3a/b and GhILR1 genes were cloned and expressed in Escherichia coli; these recombinant proteins exhibited hydrolytic activity that could cleave IAA-phenyalanine (Phe), IAA-methionine (Met), IAA-glycine (Gly) and IAA-leucine (Leu) in vitro, while only GhIAR3a hydrolyzed IAA-alanine (Ala) efficiently. The content of GhIAR3a, as detected via an established sandwich enzyme-linked immunosorbent assay (ELISA), increased in the MC-treated seedlings compared with the untreated control seedlings. In addition, the Arabidopsis iar3 mutant was less responsive to MC-induced LR growth than was wild type. Conclusions: These findings suggested that MC application could mediate IAA homeostasis via increased IAA levels from IAA-amide conjugate hydrolysis by accelerating IAH gene expression, which might promote LRI and increase the LR quantity and elongation. [ABSTRACT FROM AUTHOR]