Delineating the organization of projection neuron subsets in primary visual cortex with multiple fluorescent rabies virus tracing.

The impressive functions of the brain rely on an extensive connectivity matrix between specific neurons, the architecture of which is frequently characterized by one brain nucleus/region connecting to multiple targets, either via collaterals of the same projection neuron or several, differentially specified neurons. Delineating the fine architecture of projection neuron subsets in a specific brain region could greatly facilitate its circuit, computational, and functional resolution. Here, we developed multiple fluorescent rabies viruses (RV) to delineate the fine organization of corticothalamic projection neuron subsets in the primary visual cortex (V1). By simultaneously retrograde labeling multiple distinct subsets of corticothalamic projection neurons in V1 from their target nuclei in thalamus (dLGN, LP, LD), we observed that V1-dLGN corticothalamic projection neurons were densely concentrated in layer VI, except for several sparsely scattered neurons in layer V, while V1-LP and V1-LD corticothalamic projection neurons were localized to both layers V and VI. Meanwhile, we observed a fraction of V1 corticothalamic projection neurons targeting two thalamic nuclei, which was further confirmed by fMOST whole-brain imaging. The multiple fluorescent RV tracing tools can be extensively applied to resolve the architecture of projection neuron subsets in certain brain regions, with a strong potential to delineate the computational and functional organization of these brain regions.

Photolysis kinetics of cartap and nereistoxin in water and tea beverages under irradiation of simulated sunlight and ultraviolet under laboratory conditions.

Cartap applied widely in agricultural crops and tea plants is readily degraded into nereistoxin, resulting in a longer residual period and higher exposure risk to humans. The photolysis kinetics of cartap and nereistoxin in water and tea beverages was firstly investigated to explore the effect and mechanism of pesticide residue removal. Cartap and nereistoxin could be effectively photolyzed by ultraviolet and their photolysis rate increased with light intensity increasing. The photolysis percentage of cartap and nereistoxin in different solutions under ultraviolet irradiation of 200 W mercury lamp reached 81.8%-100.0% within 6 h. Relative to water solution, the water-soluble components in tea had an inhibition effect on the photodegradation of cartap and nereistoxin. This research provided a reference for the development of effective methods for the removal of cartap and its metabolite in water and tea beverages.

Residue pattern of chlorpyrifos and its metabolite in tea from cultivation to consumption.

BACKGROUND: Chlorpyrifos (CPF) is a broad-spectrum organophosphorus pesticide widely used to control tea geometrid (Ectropis oblique) and tea green leafhoppers (Empoasca pirisuga Matsumura) in tea trees. The major metabolite of CPF in water, plants, and animals is 3,5,6-trichloro-2-pyridinol, which is more toxic than CPF. However, the dissipation pattern of CPF in tea is unknown. RESULTS: An optimized QuEChERS sample preparation method combined with ultra-performance liquid chromatography-tandem mass spectrometry was applied to determine the residues of chlorpyrifos and its metabolite in tea during tea planting and green tea processing. During tea planting, the sum of chlorpyrifos and its metabolite dissipated rapidly with a half-life of 1.93 days for tea shoots. The residues of chlorpyrifos and its metabolite in made green tea were 96.89 and 35.88 µg kg-1 on the seventh day. The values for processing factors of chlorpyrifos and its metabolite were all less than 1, showing that each green tea manufacturing step was responsible for the reduction. The transfer rates of chlorpyrifos and its metabolite from made green tea to its infusion were 0.68-4.62% and 62.93-71.79%, respectively. CONCLUSION: The risk of chlorpyrifos was negligible to human health based on the hazard quotient, which was 7.4%. This study provides information relevant to the reasonable application of chlorpyrifos in tea planting and is potentially helpful for tea exporting and importing countries to establish harmonized maximum residue limits. © 2020 Society of Chemical Industry.

A Dissipation Pattern of Gibberellic Acid and Its Metabolite, Isogibberellic Acid, during Tea Planting, Manufacturing, and Brewing.

As a widely used plant growth regulator, the gibberellic acid (GA3) residue in tea has potential risk for human health. Herein, the degradation of GA3 and its conversion into main metabolites were investigated during tea planting, manufacturing, and brewing using ultrahigh-performance liquid chromatography tandem mass spectrometry. The metabolite iso-GA3 was first discovered during the tea production chain and identified using Q-Exactive Orbitrap mass spectrometry. GA3 dissipated following first-order kinetics in tea shoots with half-lives ranging from 2.46 to 2.74 days. It was degraded into iso-GA3 in tea shoots, which had a longer residual period than GA3. Meanwhile, external application of GA3 could increase the proportion of growth-promoting endogenous phytohormones and lead to rapid growth of tea plants. During tea manufacturing, iso-GA3 was quickly and massively converted from GA3. Fixing (heat at 220-230 °C) played an important role in the dissipation of GA3 and iso-GA3 during green tea manufacturing, but there were high residues of iso-GA3 in black tea. High transfer rates (77.3 to 94.5%) of GA3 and iso-GA3 were observed during tea brewing. These results could provide a practical reference for food safety in tea and other agricultural products and the guidance for scientific application of GA3 in tea planting.

Determination of thirteen acidic phytohormones and their analogues in tea (Camellia sinensis) leaves using ultra high performance liquid chromatography tandem mass spectrometry.

Trace plant hormones play an important role in tea growth, development and quick response to biotic and abiotic stresses. However, lack of a sensitive method limits the research on plant hormone regulation for tea quality and yields. Herein, a highly sensitive method was developed using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for profiling and quantification of 13 acidic phytohormones and their analogues, including auxins, abscisic acid and gibberellins in fresh tea leaves. After optimizing the different C18 columns and mobile phase systematically, an Agilent Eclipse Plus C18 column combined with the mobile phase A (acetonitrile) and B (water) was employed. Target acidic phytohormones were extracted using acidified methanol, and tea matrices were cleaned up by dispersive solid phase adsorbents of polyvinylpolypyrrolidone (PVPP) and graphitized carbon black (GCB) followed by polymer-based mixed-mode cation-exchange solid phase extraction. The method showed good linearity for all 13 analytes with regression coefficients (R2) > 0.998. Satisfactory recoveries of 12 analytes spiked with three levels ranged from 71.8% to 109.9%, while intra-day and inter-day precisions were below 20%. Limits of detection (LODs) and limits of quantitation (LODs) for 12 acidic phytohormones were 0.1-4.2 µg kg-1 and 0.3-13.9 µg kg-1, respectively. Finally, this method was firstly employed to analyze 13 analytes in fresh tea leaves (with the treatment of dormancy, light qualities, exogenous hormones and infestation of pests), highlighting its sufficient capability for rapid analysis of multiclass phytohormones in agriculture field.

Determination of polychlorinated biphenyls in tea using gas chromatography-tandem mass spectrometry combined with dispersive solid phase extraction.

A gas chromatography-tandem mass spectrometry method was developed for simultaneous determination of 38 polychlorinated biphenyls (PCBs) in tea. Sample preparation was based on a dispersive solid phase extraction procedure through an extraction of target compounds. An appropriate amount of polyvinylpolypyrrolidone was directly added in tea extractions to effectively remove polyphenols, and then tea extracts were cleaned up with primary secondary amine, florisil and graphitised carbon black. The method was validated, and linearity with correlation coefficients higher than 0.99 was obtained. Satisfactory recoveries at 2, 10, 50, and 100 µg kg-1 ranged from 71% to 117% with a maximum relative standard deviation of 23%, except for PCB 81, 77, 126 and 169, of which recoveries were in the range of 32%-63%. Limits of quantitation for PCBs were 2 or 10 µg kg-1, which was set as the lowest validated and spiked level meeting the acceptable accuracy and precision.

Dissipation pattern and safety evaluation of cartap and its metabolites during tea planting, tea manufacturing and brewing.

There are few studies for risk assessment of cartap and its metabolites, although cartap is easily transformed into metabolites which could induce higher toxicity. This study aimed to investigate the dissipation pattern of cartap and its metabolites during tea planting, manufacturing and brewing for evaluating the safety of cartap pesticide. Cartap metabolites were identified using Q-Exactive Orbitrap mass spectrometry. Half-lives of cartap in fresh tea leaves ranged from 0.49 to 0.59 days. Cartap decreased rapidly with time, and it was degraded into nereistoxin and cartap monothiol during tea production chain. Cartap monothiol residues dissipated rapidly by 98% in three days during tea planting. Nereistoxin had a longer residual period than cartap and it dominated the total residue in made tea after tea manufacturing. Transfer rates of nereistoxin during tea brewing ranged from 78.24% to 121.56%. Therefore, we suggested sum of cartap and nereistoxin residues as maximum residual limits in tea.

In Vivo Two-photon Calcium Imaging in Dendrites of Rabies Virus-labeled V1 Corticothalamic Neurons.

Monitoring neuronal activity in vivo is critical to understanding the physiological or pathological functions of the brain. Two-photon Ca2+ imaging in vivo using a cranial window and specific neuronal labeling enables real-time, in situ, and long-term imaging of the living brain. Here, we constructed a recombinant rabies virus containing the Ca2+ indicator GCaMP6s along with the fluorescent protein DsRed2 as a baseline reference to ensure GCaMP6s signal reliability. This functional tracer was applied to retrogradely label specific V1-thalamus circuits and detect spontaneous Ca2+ activity in the dendrites of V1 corticothalamic neurons by in vivo two-photon Ca2+ imaging. Notably, we were able to record single-spine spontaneous Ca2+ activity in specific circuits. Distinct spontaneous Ca2+ dynamics in dendrites of V1 corticothalamic neurons were found for different V1-thalamus circuits. Our method can be applied to monitor Ca2+ dynamics in specific input circuits in vivo, and contribute to functional studies of defined neural circuits and the dissection of functional circuit connections.

Simultaneous determination of cartap and its metabolite in tea using hydrophilic interaction chromatography tandem mass spectrometry and the combination of dispersive solid phase extraction and solid phase extraction.

Risk assessment of cartap residue in tea should include the exposure of cartap and its metabolite due to rapid degradation of cartap into nereistoxin. Herein, a reliable method for determination of cartap and nereistoxin in tea was developed by hydrophilic interaction chromatography tandem mass spectrometry. Target compounds were extracted with water containing 1% formic acid and 5 mM ammonium formate. The use of dichloromethane effectively removed caffeine. Tea extracts were cleaned up by dispersive adsorbents of octadecylsilane and strong anion exchanger, then further purified using hydrophilic lipophilic balanced solid phase extraction cartridge. Isotopic internal standard was employed to calibrate the loss of analytes during sample preparation and compensate matrix effects. Method validation illustrated excellent linearity, with correlation coefficients (R2) higher than 0.999. Satisfactory recoveries of target compounds spiked in green tea, black tea and oolong tea ranged from 87.6% to 119.9% with intra- and inter-day precisions below 20%. Limits of quantification of cartap and nereistoxin were 10.0 µg kg-1, and limits of detection were 2.0 µg kg-1 for cartap and 4.0 µg kg-1 for nereistoxin. The developed method was applied to determine cartap and nereistoxin in thirty tea samples.

Enantioselectivity and residue analysis of fipronil in tea (Camellia sinensis) by ultra-performance liquid chromatography Orbitrap mass spectrometry.

This study aimed to determine the residues of fipronil, metabolites, and enantiomers in tea (Camellia sinensis) during tea planting and green tea manufacture. An AD-RH chiral column was used to separate the fipronil enantiomers. During tea planting, the half-lives of the sum of fipronil and metabolites were similar to those of fipronil, which were 2.37 and 3.88 days for tea shoots and mature leaves, respectively. The residues of fipronil and its metabolites increased 2.3-3.6-fold during green tea manufacture. The values for the processing factors of fipronil and metabolites ranged from 1.0 to 2.1. A slightly significant enantioselectivity of (R)- and (S)-fipronil was observed during tea planting and green tea manufacturing. The residue pattern indicated that fipronil should not be applied in tea gardens due to its long persistence. The maximum residual limits of fipronil and metabolites at 2 µg kg-1 were considered optimal.

[Antimicrobial and anti-tumor activities of endophytes from Lycium barbarum of Ningxia].

The antimicrobial activity and cytotoxicity in vitro of the fermentation broth of 10 endophytic strains isolated from Lycium barbarum were determined to screen high activity endophytic strains. Sequences analysis of ITS and 16S rDNA was used for molecular identification of the strains. The results showed that 5 endophytic fungi had no inhibitory activity against the tested pathogens. Endophytic actinomycete strain AL6 had a certain inhibitory effect on 3 kinds of pathogenic fungi, and strain AL5 only had strong inhibitory activity against Staphylococcus aureus. However, the anti-tumor activity of endophytic fungi was significantly higher than that of actinomycetes. Four endophytic fungi strains exhibited the growth inhibition rate of above 50% against at least one of the tested tumor cells when the concentration of fermentation broth was 0.2 g•L⁻¹. Sequences analysis showed that 5 endophytic fungi strains belonged to genus Aspergillus, Penicillium and Emericella, and the 5 endophytic actinomycetes strains belonged to genus Aspergillus, Penicillium and Emericella. Aspergillus strain FL1 had stronger inhibitory activity against A549 and HeLa cells, and the IC50 values were 0.022，0.028 g•L⁻¹, respectively, which was worthy of further study.

[Screening and identification of antioxidant endophytes from Lycium barbarum of Ningxia].

In this paper, 29 endophytes were isolated from different organs and tissues of Lycium barbarum of Ningxia by tablet coating method, 18 of them was fungi, and 11 of them was actinomycetes. The endophytes quantity in the different tissues were leaves > flowers > roots >fruits; The hydroxyl radical scavenging activities of 11 endophytes were investigated by Fenton reaction, and total antioxidant capacities of them were examined by a. total antioxidant capacity test kit; culture features and strain-specific sequence analysis were employed to explore the diversity of the 11 endophytes. The result showed that 5 fungi and 6 actinomycetes that having antioxidant activity could be phylogenetically classified into 3 genera, 3 genera and 3 families, respectively. The total antioxidant capacity and hydroxyl radical scavenging activity of the 11 endophytes showed distinct difference. The antioxidant activity of Aspergillus were stronger, among which total antioxidant capacity of fL1 was (188.5 ± 0.549) U · mL⁻¹ and the IC50 was 0.3 mg · L⁻¹; the IC50 of strain fL1 was 0.42 mg · L⁻¹ and the total antioxidant capacity of fL9 was (113.63 ± 1.021) U · mL⁻¹, all of them were stronger than the positive control Vit C. The experimental results indicated that endophytic fungi of L. barbarum of Ningxia have a great developing and application prospect for the development of antioxidant agent.

Postnatal ontogeny of the transcription factor Lmx1b in the mouse central nervous system.

The expression profile of Lim homeodomain transcription factor Lmx1b in the mouse brain was investigated at different postnatal stages by immunohistochemistry and in situ hybridization. At postnatal day (P) 7, many Lmx1b-expressing neurons were found in the posterior hypothalamic area, supramammillary nucleus, ventral premammillary nucleus, and subthalamic nucleus. In the midbrain, numerous Lmx1b-expressing neurons were present in the substantia nigra pars compacta and ventral tegmental area. In the hindbrain, Lmx1b-expressing neurons were primarily observed in the raphe nuclei, parabrachial nuclei, principal sensory trigeminal nucleus, nucleus of the solitary tract, and laminae I-II of the medullary dorsal horn as well as spinal dorsal horn. Although expression levels diminished as postnatal life progressed, persistent expression throughout the first year of life was observed in many of these regions. In contrast, Lmx1b was present in a few brain regions (e.g., principal sensory trigeminal nucleus) only in early life with expression expiring by P60. Lmx1b was observed in dopaminergic neurons in the midbrain and serotonergic neurons in the hindbrain, as determined by double labeling with specific markers. In addition, we found that Lmx1b-expressing neurons are not GABAergic, and Lmx1b was colocalized with Tlx3 in the parabrachial nuclei, principal sensory trigeminal nucleus, nucleus of the solitary tract. as well as the medullary and spinal dorsal horns, suggesting that Lmx1b-expressing cells in these areas are excitatory neurons. Our data suggest that Lmx1b is involved in the postnatal maturation of certain types of neurons and maintenance of their normal functions in the adult brain.