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A simple, rapid, sensitive, selective, and environmentally friendly method, based on in situ derivatization ultrasound-assisted dispersive liquid-liquid microextraction (in situ DUADLLME) coupled with ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) using multiple reaction monitoring (MRM) mode has been developed for the simultaneous determination of food-related biogenic amines and amino acids. A new mass-spectrometry-sensitive derivatization reagent 4'-carbonyl chloride rosamine (CCR) was designed, synthesized, and first reported. Parameters and conditions of in situ DUADLLME and UHPLC-MS/MS were optimized in detail. Under the optimized conditions, the in situ DUADLLME was completed speedily (within 1 min) with high derivatization efficiencies (≥98.5%). With the cleanup and concentration of microextraction step, good analytical performance was obtained for the analytes. The results showed that this method was accurate and practical for quantification of biogenic amines and amino acids in common food samples (red wine, beer, wine, cheese, sausage, and fish).

To study the functions of the early auxin-responsive genes CsGH3.1 and CsGH3.6 in citrus resistance against canker disease, we cloned CsGH3.1 and CsGH3.6 in ‘Newhall’ Navel Orange ( Citrus sinensis Osbeck). They are 1 797 bp and 1 887 bp and encode 598 and 629 amino acids, respectively. In vitro mature leaves from susceptible ‘Newhall’ and resistant Calamondin ( C. madurensis ) were inoculated by a Xanthomonas axonopodis pv. citri ( Xac ) bacterial suspension, and expression of CsGH3.1 and CsGH3.6 in the two varieties were analyzed using quantitative real-time PCR (qRT-PCR). ‘Newhall’ leaves were treated with different hormones for 3 days, inoculated by Xac bacterial suspension, and then the symptoms in these leaves were investigated. We used qRT-PCR to analyze the effect of different hormones on CsGH3.1 and CsGH3.6 expression in ‘Newhall’ leaves. The expression levels of both CsGH3.1 and CsGH3.6 were significantly induced by Xac in ‘Newhall’ leaves, compared with levels in Calamondin leaves. 1-naphthy acetic acid (NAA) increased the hypertrophy of infection sites in ‘Newhall’ leaves, while naphthyl-phthalamic acid (NPA) had no visible effect on lesion development . NAA hormone greatly improved expression of CsGH3.1 in ‘Newhall’, but not CsGH3.6 . These results indicate that the auxin primary-response gene CsGH3.1 plays an important role in citrus susceptibility to Xac .

A simple, rapid, sensitive and environmentally friendly ultrasound-assisted in situ derivatization dispersive liquid–liquid microextraction (in situ UA-DDLLME) method followed by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has been developed for the simultaneous determination of amino acid neurotransmitters (AANTs) and monoamine neurotransmitters (MANTs) in rat brain microdialysates and baicalein in blood microdialysates of Parkinson's disease (PD) rats with in vivo multiple sites microdialysis in this work. A novel MS sensitive derivatization reagent 4′-carbonyl chloride rosamine (CCR) with a permanent positive charged moiety, has been designed, synthesized and reported for simultaneous labelling of amino and hydroxyl groups in neurotransmitters. Under the selected conditions, the derivatization and microextraction of analytes were simultaneously completed within 1.0 min. Good linearity for each analyte (R > 0.992) was observed with a low limit of detection (LOD, S/N > 3). Taken together, in situ UA-DDLLME coupled with UHPLC-MS/MS analysis was demonstrated to be a promising method for sensitive, accurate and simultaneous monitoring of AANTs and MANTs in rat brain and baicalein in rat blood. This method would be expected to be highly useful in PD clinical diagnostics and monitoring the efficacy of treatments.

This work presents a new triptycene-based stationary phase (TP-PEG) combining the three-dimensional (3D) triptycene (TP) framework with polyethylene glycol (PEG) moieties for gas chromatographic (GC) separations. Its statically coated capillary column showed high column efficiency of 5263 plates/m determined by naphthalene at 120 °C. Its Rohrschneider-McReynolds constants and Abraham solvation system constants were measured to characterize its polarity and molecular interactions with analytes of different types. As evidenced, the TP-PEG column showed high-resolution performance for the isomers of anilines, phenols, halobenzenes and alkanes with distinct advantages over the PEG columns, particularly those critical isomers such as 3,5-/2,3-xylidine (R = 2.94), m-/p-chlorotoluene (R = 1.92), p-/m-cresol (R = 1.89), 2,2-dimethylbutane/2-methylpentane (R = 1.51), 2,2,3-trimethylbutane /2,3-dimethyl pentane (R = 1.74) and 2,3-dimethylpentane/n-heptane (R = 1.92). In addition, it exhibited good column repeatability and reproducibility with the relative standard deviation (RSD) values of 0.02%–0.09% for run-to-run, 0.13%–0.22% for day-to-day and 2.7%–4.1% for column-to-column, respectively, and a wide operational temperature range (30 °C–280 °C) . Its application to GC–MS analysis of the essential oil of Osmanthus fragrans has proven its good potential for practical analysis of complex samples.

Synchronous measurement of brain neurotransmitters and blood drug metabolism by dual sites in vivo microdialysis can provide very important information for several neurological disorders such as post-stroke depression (PSD). In this work, d0/d3-6-N-methyl-rhodamine 6G-N-hydroxysuccinimidyl formate (d0/d3-MRSF) were firstly designed, synthesized and used as stable isotope labeling derivatization reagents for the determination of multiple neurotransmitters by ultra-high performance liquid chromatography-tandem mass spectrometry. The light d0-MRSF was used to label neurotransmitters in real samples and the heavy d3-MRSF was used to label standards, which served as internal standards for quantification to minimize matrix effect. The d0/d3-MRSF-neurotransmitter derivatives were easily adsorbed onto the surface of Fe3O4@graphene oxide. Magnetic dispersive solid phase extraction followed with stable isotope labeling derivatization was developed for efficient sample pretreatment. The permanent positively charged moiety of d0/d3-MRSF significantly enhanced the ionization efficiency of neurotransmitters. The comparison results indicated that the synthesized MRSF derivatization method can bring better detection sensitivity than the commercial dansyl chloride method about tens to hundreds of times on the same instrumentation. Specific and regular product ions of m/z 413.3 and 416.3, m/z 429.4 and 432.4 were observed for d0-and d3-MRSF labeled neurotransmitters, respectively. The limits of detection were in the range of 2.0–40.0 pM. The accuracies ranged from 92.3% to 108.2% with the intra- and inter-day precisions in the range of 2.2–12.8%. Synchronous and dynamic determination of concentrations changes of brain neurotransmitters and blood gastrodin of PSD rats was achieved. The results indicated that gastrodin had analogous regulating effect of brain neurotransmitter, which like the clinical medicine fluoxetine of PSD. Taken together, the developed method was demonstrated to be promising for sensitive, accurate and simultaneous determination of multiple neurotransmitters in trace biofluids.

Marker-free transgenic plants alleviate concerns regarding the biosafety of genetically modified organisms and promote their commercialization. In this study, a transformation vector pLI35SAAT, harboring a Cre/loxP-mediated recombination system combined with the isopentenyl transferase (ipt) selectable marker gene and an anti-bacterial peptide gene AATCB, was used to produce marker-free transgenic ‘Tarocco’ blood orange (Citrus sinensis Osbeck) with enhanced resistance to citrus canker. Using ipt positive selection, a transformation efficiency of 21.4 % was achieved. When the DNA between two loxP sites was excised, phenotypically normal shoots gradually appeared from 74.8 % of the transgenic ipt shoots. Their marker-free transgenic nature was confirmed using PCR and sequencing analyses. In vitro evaluations of citrus canker disease resistance revealed that marker-free transgenic plants exhibited an enhanced resistance to Xanthomonas axonopodis pv. citri. The marker-free transgenic plants appeared phenotypically normal under greenhouse conditions. Thus, marker-free transgenic citrus plants with targeted traits can be efficiently produced using a Cre/loxP-mediated recombination system combined with ipt positive selection.

To improve the resistance of citrus to canker disease caused by Xanthomonas axonopodis pv. citri (Xac), it is important to identify gene promoters that are specifically induced by pathogen infection. Here, we evaluated the functionality of PPP1 and hsr203J (Nicotiana tabacum L.) and gst1 (potato) pathogen-inducible promoters to drive expression of the β-glucuronidase (GUS) reporter gene in transgenic citrus. The activities of these promoters in response to the Xac pathogen and wounding were determined quantitatively and qualitatively by fluorometric and histochemical GUS assays, and compared with that of the cauliflower mosaic virus (CaMV) 35S promoter. In citrus, the hsr203J promoter from tobacco was hardly activated by the Xac pathogen or wounding, whereas the PPP1 and gst1 promoters were rapidly and efficiently activated by both inducers. There was very little visible background expression from the PPP1 promoter, but a high level of background expression from the gst1 promoter. Because of its low background expression, the PPP1 promoter was more responsive to Xac and wounding than was the gst1 promoter. However, the gst1 promoter was more rapidly activated than the PPP1 promoter by Xac and wounding. The inducible activity of the two promoters was restricted to infection sites. Taken together, our results showed that the PPP1 promoter was the most efficient promoter among those evaluated in this study. Its strong responsiveness to Xac and wounding suggests that it would be a good candidate for expression of antibacterial transgenes specifically at infection sites to improve canker disease resistance in citrus.

The auxin early response gene Gretchen Hagen3 (GH3) plays dual roles in plant development and responses to biotic or abiotic stress. It functions in regulating hormone homeostasis through the conjugation of free auxin to amino acids. In citrus, GH3.1 and GH3.1L play important roles in responding to Xanthomonas citri subsp. citri (Xcc). Here, in Wanjingcheng orange (Citrus sinensis Osbeck), the overexpression of CsGH3.1 and CsGH3.1L caused increased branching and drooping dwarfism, as well as smaller, thinner and upward curling leaves compared with wild-type. Hormone determinations showed that overexpressing CsGH3.1 and CsGH3.1L decreased the free auxin contents and accelerated the Xcc-induced decline of free auxin levels in transgenic plants. A resistance analysis showed that transgenic plants had reduced susceptibility to citrus canker, and a transcriptomic analysis revealed that hormone signal transduction-related pathways were significantly affected by the overexpression of CsGH3.1 and CsGH3.1L. A MapMan analysis further showed that overexpressing either of these two genes significantly downregulated the expression levels of the annotated auxin/indole-3-acetic acid family genes and significantly upregulated biotic stress-related functions and pathways. Salicylic acid, jasmonic acid, abscisic acid, ethylene and zeatin levels in transgenic plants displayed obvious changes compared with wild-type. In particular, the salicylic acid and ethylene levels involved in plant resistance responses markedly increased in transgenic plants. Thus, the overexpression of CsGH3.1 and CsGH3.1L reduces plant susceptibility to citrus canker by repressing auxin signaling and enhancing defense responses. Our study demonstrates auxin homeostasis' potential in engineering disease resistance in citrus.

Neurotransmitters (NTs) may play an important role in neurodegenerative disorders such as Alzheimer's disease (AD). In order to investigate the potential links, a new simple, fast, accurate and sensitive analytical method, based on in situ ultrasound-assisted derivatization dispersive liquid-liquid microextraction (in situ UA-DDLLME) coupled with ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS), has been developed and validated. The quantitation of amino acid neurotransmitters (AANTs) and monoamine neurotransmitters (MANTs) in urine of AD rats were performed in this work. The in situ UA-DDLLME procedure involved the rapid injection of the mixture of low toxic 4-bromoanisole (extractant) and acetonitrile (dispersant), which containing the new designed and synthesized 4'-carbonyl chloride rosamine (CCR) as derivatization reagent, into the aqueous phase of real sample and buffer. Under the selected conditions, the derivatization and microextraction of analytes were simultaneously completed within 1min. Good linearity for each analyte (R>0.992) was observed with low limit of detections (LODs, S/N>3). Moreover, the proposed method was compared with direct detection or other reported methods, and the results showed that low matrix effects and good recoveries results were obtained in this work. Taken together, in situ UA-DDLLME coupled with UHPLC-MS/MS analysis was demonstrated to be a good method for sensitive, accurate and simultaneous monitoring of AANTs and MANTs. This method would be expected to be highly useful in AD diseases' clinical diagnostics and may have potential value in monitoring the efficacy of treatment.

This paper, for the first time, reported a simple, rapid, sensitive and environmental friendly ultrasonic-assisted in situ derivatization-dispersive liquid-liquid microextraction (in situ UAD-DLLME) method followed by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for the simultaneous determination of dopamine (DA), serotonin (5-HT) and their biosynthesis precursors and metabolites in rat brain microdialysates. In this work, a commercial reagent, Lissamine rhodamine B sulfonylchloride (LRSC), was proposed as a derivatization reagent. The ionization efficiency of neurotransmitters was greatly enhanced through the introduction of a permanent charged moiety of LRSC into their derivatives during electrospray ionization MS (ESI-MS) analysis. Parameters of in situ UAD-DLLME and UHPLC-MS/MS conditions were all optimized in detail. The optimum conditions of in situ UAD-DLLME were found to be as follows: a mixture of 150μL of acetonitrile (dispersant) containing LRSC (derivatization reagents) and 50μL of low toxic bromobenzene (extractant) was rapidly injected into an aqueous sample containing 30μL of microdialysate and 800μL of NaHCO3-Na2CO3 buffer solution (pH 10.5) at 37°C. After ultrasonication for 3min and centrifuging for 2min, the sedimented phase was conveniently injected for UHPLC-MS/MS analysis. Under the optimized conditions, good linearity was observed with the limits of detection (LODs, S/N>3) and limits of quantification (LOQs, S/N>10) in the range of 0.002-0.008 and 0.015-0.040nmol/L, respectively. Meanwhile, it also brought good results of precision (3.2-13.0%, peak area RSDs %), accuracy (86.4-112%), recovery (73.9-105%), matrix effect (86.2-105%), and stability (3.1-8.8%, peak area RSDs %). The developed method was successfully applied for the simultaneous determination of multiple neurotransmitters, their precursors and metabolites in brain microdialysates of normal and L-DOPA induced dyskinesias (LID) rats.

Simultaneous monitoring of several neurotransmitters (NTs) linked to Parkinson's disease (PD) has important scientific significance for PD related pathology, pharmacology and drug screening. A new simple, fast and sensitive analytical method, based on in situ derivatization-ultrasound-assisted dispersive liquid-liquid microextraction (in situ DUADLLME) in a single step, has been proposed for the quantitative determination of catecholamines and their biosynthesis precursors and metabolites in rat brain microdialysates. The method involved the rapid injection of the mixture of low toxic bromobenzene (extractant) and acetonitrile (dispersant), which containing commercial Lissamine rhodamine B sulfonyl chloride (LRSC) as derivatization reagent, into the aqueous phase of sample and buffer, and the following in situ DUADLLME procedure. After centrifugation, 50μL of the sedimented phase (bromobenzene) was directly injected for ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) detection in multiple reaction monitoring (MRM) mode. This interesting combination brought the advantages of speediness, simpleness, low matrix effects and high sensitivity in an effective way. Parameters of in situ DUADLLME and UHPLC-MS/MS conditions were all optimized in detail. The optimum conditions of in situ DUADLLME were found to be 30μL of microdialysates, 150μL of acetonitrile containing LRSC, 50μL of bromobenzene and 800μL of NaHCO3-Na2CO3 buffer (pH 10.5) for 3.0min at 37°C. Under the optimized conditions, good linearity was observed with LODs (S/N>3) and LOQs (S/N>10) of LRSC derivatized-NTs in the range of 0.002-0.004 and 0.007-0.015 nmol/L, respectively. It also brought good precision (3.2-12.8%, peak area CVs%), accuracy (94.2-108.6%), recovery (94.5-105.5%) and stability (3.8-8.1%, peak area CVs%) results. Moreover, LRSC derivatization significantly improved chromatographic resolution and MS detection sensitivity of NTs when compared with the reported studies through the introduction of a permanent charged moiety from LRSC into NTs. Taken together, this in situ DUADLLME method was successfully applied for the simultaneous determination of six NTs in biological samples.

Transgenic plants of Jincheng orange (Citrus sinensis Osbeck) and Newhall navel orange (C. sinensis Osbeck) containing antibacterial peptide genes Shiva A and Cecropin B were successfully obtained by a novel Agrobacterium tumefaciens-mediated transformation of the mature axillary buds. PCR and Southern blot analysis of the transgenic plants verified that the Shiva A and Cecropin B genes were integrated into the citrus genome. The transgenic plants began to blossom and bear fruit in the 2nd year after grafting on trifoliate orange (Poncirus trifoliata Raf) rootstock in greenhouse. Water-soluble extracts from transgenic citrus leaves exhibit in vitro suppressive effects on Xanthomonas axonopodis pv. citri, suggesting that the expressed products of Shiva A and Cecropin B in citrus retain their native antibacterial activities. Artificial inoculation in greenhouse and open field further indicates significantly increased resistance of transgenic plants to X. axonopodis pv. citri when compared with non-transgenic lines. No significant difference was found in the content of total soluble solids, total acidity, reduced sugar content and other fruit characteristics between transgenic and non-transgenic plants. In this present study, 11 transgenic lines were obtained from 40 transgenic lines, showing enhanced resistance to citrus canker disease.

This paper, for the first time, reported a speedy hyphenated technique of low toxic dual ultrasonic-assisted dispersive liquid-liquid microextraction (dual-UADLLME) coupled with microwave-assisted derivatization (MAD) for the simultaneous determination of 20(S)-protopanaxadiol (PPD) and 20(S)-protopanaxatriol (PPT). The developed method was based on ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detection using multiple-reaction monitoring (MRM) mode. A mass spectrometry sensitizing reagent, 4'-carboxy-substituted rosamine (CSR) with high reaction activity and ionization efficiency was synthesized and firstly used as derivatization reagent. Parameters of dual-UADLLME, MAD and UHPLC-MS/MS conditions were all optimized in detail. Low toxic brominated solvents were used as extractant instead of traditional chlorinated solvents. Satisfactory linearity, recovery, repeatability, accuracy and precision, absence of matrix effect and extremely low limits of detection (LODs, 0.010 and 0.015ng/mL for PPD and PPT, respectively) were achieved. The main advantages were rapid, sensitive and environmentally friendly, and exhibited high selectivity, accuracy and good matrix effect results. The proposed method was successfully applied to pharmacokinetics of PPD and PPT in rat plasma.

Objective To discuss the mechanism of Yiqiyangyin recipe in rats with adriamycin (ADR)-induced nephropathy. Methods We randomly divided 30 Sprague-Dawley rats into 5 groups: control, model, glucocorticoid, Chinese herb, and Chinese herb plus glucocorticoid groups. The 24-h urine volume was collected on days 7, 14, 21, and 28 after ADR injection, and all rats were killed on day 28. We measured the 24-h levels of urinary protein, serum cholesterol, and serum triglycerides, and renal function of all rats by using routine biochemical methods. Pathological changes in the rat kidneys were observed under light and electron microscopes. Heparanase (HPA) mRNA expression levels were measured using real-time fluorescence-quantitative polymerase chain reaction. Urine levels of HPA in all groups were measured using enzyme-linked immunosorbent assay. The expression of nephrin was detected by immunohistochemical staining and quantitatively analyzed using Motic image analysis 3.2 software. Results The levels of urinary protein and serum triglycerides and cholesterol in rats with ADR-induced nephropathy increased on day 14. The serum albumin levels simultaneously decreased. All the changes reached the peak on day 28. Examination under the light microscope showed inflammatory cells and slight fibroplasia in the renal interstitium in the model group, but fewer inflammatory cells were observed in the intervention groups than those in the model group. Examination under the electron microscope showed extensive fusion of foot processes in ADR rats. HPA mRNA expression was higher in the model group than that in the control group. The HPA mRNA levels in the intervention groups, especially in the Chinese herb group, and Chinese herb plus glucocorticoid group were significantly lower than the level in the model group. The HPA expression levels correlated significantly with the proteinuria level. No significant difference was found in the HPA level in urine between the intervention groups and the model group, whereas the model group had a higher urinary HPA level than the control group. Nephrin mRNA expression levels in the model group were higher than those in the control group. Nephrin mRNA expression levels were significantly lower in the intervention groups than that in the model group, especially the Chinese herb plus glucocorticoid group. Compared with the control group, the model group showed increased nephrin expression in the kidney. Nephrin levels in the other groups, especially in the Chinese herb plus glucocorticoid group, were significantly lower than that in the model group. The nephrin levels in the kidney were negatively correlated with the proteinuria level. Conclusion Yiqiyangyin recipe could attenuate foot process injury particularly in combination with a steroid reduce the development of proteinuria possibly by inhibition of HPA in the kidney, and regulate the expression of nephrin in rats with ADR-induced nephropathy. Our study showed that treatment with Yiqiyangyin recipe plus glucocorticoid was better than a singular intervention, and we explored the pharmacological mechanism of this combination by biochemical and molecular biological analysis to provide a basis for clinical application.

A highly efficient Cre-mediated deletion system, offering a good alternative for producing marker-free transgenic plants that will relieve public concerns regarding GMOs, was first developed in citrus. The presence of marker genes in genetically modified crops raises public concerns regarding their safety. The removal of marker genes can prevent the risk of their flow into the environment and hasten the public’s acceptance of transgenic products. In this study, a new construct based on the Cre/loxP site-recombination system was designed to delete marker genes from transgenic citrus. In the construct, the selectable marker gene isopentenyltransferase gene (ipt) from Agrobacterium tumefaciens and the Cre recombinase gene were flanked by two loxP recognition sites in the direct orientation. The green fluorescent protein (gfp) reporter gene for monitoring the transformation of foreign genes was located outside of the loxP sequences. Transformation and deletion efficiencies of the vector were investigated using nopaline synthase gene (NosP) and CaMV 35S promoters to drive expression of Cre. Analysis of GFP activity showed that 28.1 and 13.6 % transformation efficiencies could be obtained by NosP- and CaMV 35S-driven deletions, respectively. Molecular analysis demonstrated that 100 % deletion efficiency was observed in the transgenic plants. The complete excision of the marker gene was found in all deletion events driven by NosP and in 81.8 % of deletion events driven by CaMV 35S. The results showed that Cre/loxP-mediated excision was highly efficient and precise in citrus. This approach provides a reliable strategy for auto-deletion of selectable marker genes from transgenic citrus to produce marker-free transgenic plants.