Molecular Diversity and Evolutionary Relatedness of Paulownia Witches'-Broom Phytoplasma in Different Geographical Distributions in China.

To reveal the distribution and transmission pathway of Paulownia witches'-broom (PaWB) disease, which is caused by phytoplasmas related to genetic variation, and the adaptability to the hosts and environments of the pathogenic population in different geographical regions in China, in this study, we used ten housekeeping gene fragments, including rp, fusA, secY, tuf, secA, dnaK, rpoB, pyrG, gyrB, and ipt, for multilocus sequence typing (MLST). A total of 142 PaWB phytoplasma strains were collected from 18 provinces or municipalities. The results showed that the genetic diversity was comparatively higher among the PaWB phytoplasma strains, and substantially different from that of the other 16SrI subgroup strains. The number of gene variation sites for different housekeeping genes in the PaWB phytoplasma strains ranged from 1 to 14 SNPs. Among them, rpoB (1.47%) and dnaK (1.12%) had higher genetic variation, and rp (0.20%) had the least genetic variation. The tuf and rpoB genes showed the fixation of positively selected beneficial mutations in the PaWB phytoplasma populations, and all housekeeping genes except tuf followed the neutral evolutionary model. We found an absence of recombination among PaWB phytoplasma sequence types (STs) for each housekeeping gene except dnaK, and no evidence for such recombination events for concatenated sequences of PaWB phytoplasma strains. The 22 sequence types were identified among the concatenated sequences of seven housekeeping genes (rp, fusA, secY, secA, tuf, dnaK, and rpoB) from 105 representative strains. We analyzed all 22 STs by goeBURST algorithm, forming two clonal complexes (CCs) and three singletons. Among them, ST1, as the primary founder of CC1, had the widest geographical distribution, accounting for 72.38% of all strains, with a high frequency of shared sequence type. The results of phylogenetic analysis of the concatenated sequences further revealed that the 105 strains were clustered into two representative lineages of PaWB phytoplasma, with obvious geographical differentiation. The ST1 strains of highly homogeneous lineage-1 were a widespread and predominant population in diseased areas. Lineage-2 contained strains from Jiangxi, Fujian, and Shaanxi provinces, highlighting the close genetic relatedness of the strains in these regions, which was also consistent with the results of most single-gene phylogenetic analysis of each gene. We also found that the variability in the northwest China population was higher than in other geographical populations; the range of genetic differentiation between the south of the Yangtze River population and the Huang-huai-hai Plain (or southwest China) population was relatively large. The achieved diversity and evolution data, as well as the MLST technique, are helpful for epidemiological studies and guiding PaWB disease control decisions.

Deficiency of transmembrane AMPA receptor regulatory protein γ-8 leads to attention-deficit hyperactivity disorder-like behavior in mice.

Attention-deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder prevalent in school-age children. At present, however, its etiologies and risk factors are unknown. Transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor regulatory protein γ-8 (TARP γ-8, also known as calcium voltage-gated channel auxiliary subunit gamma 8 (CACNG8)) is an auxiliary AMPA receptor (AMPAR) subunit. Here, we report an association between TARP γ-8 and ADHD, whereby adolescent TARP γ-8 knockout (KO) mice exhibited ADHD-like behaviors, including hyperactivity, impulsivity, anxiety, impaired cognition, and memory deficits. Human single-nucleotide polymorphism (SNP) analysis also revealed strong associations between intronic alleles in CACNG8 genes and ADHD susceptibility. In addition, synaptosomal proteomic analysis revealed dysfunction of the AMPA glutamate receptor complex in the hippocampi of TARP γ-8 KO mice. Proteomic analysis also revealed dysregulation of dopaminergic and glutamatergic transmissions in the prefrontal cortices of TARP γ-8 KO mice. Methylphenidate (MPH), which is commonly used to treat ADHD, significantly rescued the major behavioral deficits and abnormal synaptosomal proteins in TARP γ-8 KO mice. Notably, MPH significantly reversed the up-regulation of Grik2 and Slc6a3 in the prefrontal cortex. MPH also significantly improved synaptic AMPAR complex function by up-regulating other AMPAR auxiliary proteins in hippocampal synaptosomes. Taken together, our results suggest that TARP γ-8 is involved in the development of ADHD in humans. This study provides a useful alternative animal model with ADHD-like phenotypes related to TARP γ-8 deficiency, which has great potential for the development of new therapies.

Catalytic Asymmetric Synthesis of Tetrahydrofuran Spirooxindoles via a Dinuclear Zinc Catalyst.

An asymmetric Michael/hemiketalization and Fridel-Crafts reaction has been reported through a one-pot reaction. A number of structurally novel tetrahydrofuran spirooxindoles are synthesized in the presence of a 10 mol % dinuclear zinc catalyst with diastereomer ratios (dr) of 3:1-13:1 and an enantiomeric excess (ee) of 75-99%. The reaction can be performed on a gram scale without impacting its efficiency. The absolute configuration of products is confirmed by X-ray single crystal structure analysis, and a possible mechanism is proposed.

Screening and identification of potential tyrosinase inhibitors from Semen Oroxyli extract by ultrafiltration LC-MS and in silico molecular docking.

There is an increasing interest in screening and developing natural tyrosinase inhibitors widely applied in medicinal and cosmetic products, as well as in the food industry. In this study, an approach by ultrafiltration LC-MS and molecular docking was used to screen and identify tyrosinase inhibitors from Semen Oroxyli extract. The samples were first incubated with the tyrosinase to select the optimal binding conditions including tyrosinase concentration, incubation time and the molecular weight of ultrafiltration membrane. By comparison of the chromatographic profiles of the extracts after ultrafiltration with activated and inactivated tyrosinase, the potential inhibitors were obtained and then identified by LC-MS. The relative binding affinities of the potential inhibitors were also calculated based on the decrease of peak areas of those. As a result, seven compounds were fished out as tyrosinase inhibitors by this assay. Among them, oroxin A and baicalein showed higher tyrosinase inhibitory than resveratrol as positive drug, and their binding mode with enzyme was further verified via the molecular docking analysis. The test results showed that the proposed method was a simple, rapid, effective, and reliable method for the discovery of natural bioactive compounds, and it can be extended to screen other bioactive compounds from traditional Chinese medicines.

[Quantification of (-)doxazosin at very low concentration in rat plasma samples by SPE-LC-MS/MS].

Previous publications showed that the value of LLOQ (lowest limit of quantification) for doxazosin and its enantiomers in biological samples were above 0.1 ng·m L(-1). The present study was designed to establish a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification at very low concentration of (-)doxazosin in rat plasma after intravenous administration of (-)doxazosin（3.0 mg·kg(-1)）. The plasma samples containing prazosin as an internal standard were extracted by solid-phase extraction (SPE) and separated on Acquity BEH C(18) (50 mm × 2.1 mm, 1.7 µm) column under alkaline conditions of the mobile phase. (-)Doxazosin was monitored under positive ionization condition by multiple reaction monitoring (MRM) with an ESI source. The good linear range of (-)doxazosin varied from 10.4 pg·m L(-1) to 13 ng·m L(-1)（r = 0.992 2), and the lowest limit of quantification was 10.4 pg·m L(-1). An assessment of the matrix effect using post-extraction spiking method and post-column infusion method demonstrated that co-eluting matrix components did not significantly influenced the ionization of (-)doxazosin and prazosin (IS). Using the new method, we accurately measured (-)doxazosin concentration at 48 h after intravenous administration in the rats, and the concentration was 0.034 4 ± 0.010 2 ng·m L(-1). The method is specific, sensitive, and suitable for determining (-)doxazosin at very low concentration in rat plasma samples.

[An overview of effects of traditional medicine on pharmacokinetics of western medicine].

Traditional medicine (herb medicine) began to prevail again over last two decades, and it is about 70% of the world population taking herb medicine as supplement or alternative medicine according to a recent survey. The consumption of herb medicine increased exponentially in Canada, Australia and Europe during last 10 years. Since concomitant administration of herbal and western medicine has become a trend, it requires paying close attention to the problem. Herb-drug interactions have been extensively investigated worldwide, and there is an increasing concern about the clinical herb-drug interaction. In this review we introduced the current progress in the herb-drug interactions including evidence-based clinical studies and establishment of levels of evidence for herb-drug interaction; and in the related mechanisms including the induction and inhibition of metabolic enzymes, inhibition and induction of transport and efflux proteins, alteration of gastrointestinal functions, and alteration in renal elimination. We also analyzed both the achievements and the challenges faced in the concomitant administration of traditional Chinese medicine and western medicine.

(-)Doxazosin is a necessary component for the hypotensive effect of (±)doxazosin during long-term administration in conscious rats.

AIM: Doxazosin is a racemic mixture of (-)doxazosin and (+)doxazosin that is currently used as an add-on therapy for hypertension. In this study we investigated the contribution of each enantiomer to the hypotensive action of long-term administration of (±)doxazosin in conscious rats. METHODS: Blood pressure of conscious SD rats was measured using a volume pressure recording system. The rats were orally administered (-)doxazosin, (+)doxazosin, or (±)doxazosin (8 mg·kg(-1)·d(-1)) for 12 weeks. Plasma concentrations of the agents were analyzed with HPLC. The effect of the agents on α1-adrenoceptor was examined in isolated rat caudal artery preparations. RESULTS: Treatment of conscious rats with a single dose of (±)doxazosin (8 mg/kg) did not affected DBP and MBP, but significantly decreased SBP by 11.9% 4 h after the administration. Long-term treatment of conscious rats with (±)doxazosin significantly decreased SBP, DBP and MBP with a maximal decrease of SBP by 29.3% 8 h after the last administration. The rank order of the hypotensive actions caused by long-term treatment in conscious rats was (±)doxazosin>(+)doxazosin>>(-)doxazosin. However, the pKB values for inhibiting NA-induced contraction of isolated rat caudal artery were (+)doxazosin (8.995)>(±)doxazosin (8.694)>(-)doxazosin (8.032). The plasma concentrations of (-)doxazosin, (+)doxazosin, and (±)doxazosin were 18.26±3.55, 177.11±20.66, and 113.18±13.21 ng/mL, respectively, 8 h after the last administration of these agents. CONCLUSION: Long-term treatment with (±)doxazosin produces potent hypotensive action in conscious rats that seems to result from synergic interaction of the two enantiomers.

Stereoselective binding of doxazosin enantiomers to plasma proteins from rats, dogs and humans in vitro.

AIM: (±)Doxazosin is a long-lasting inhibitor of α1-adrenoceptors that is widely used to treat benign prostatic hyperplasia and lower urinary tract symptoms. In this study we investigated the stereoselective binding of doxazosin enantiomers to the plasma proteins of rats, dogs and humans in vitro. METHODS: Human, dog and rat plasma were prepared. Equilibrium dialysis was used to determine the plasma protein binding of each enantiomer in vitro. Chiral HPLC with fluorescence detection was used to measure the drug concentrations on each side of the dialysis membrane bag. RESULTS: Both the enantiomers were highly bound to the plasma proteins of rats, dogs and humans [(-)doxazosin: 89.4%-94.3%; (+)doxazosin: 90.9%-95.4%]. (+)Doxazosin exhibited significantly higher protein binding capacities than (-)doxazosin in all the three species, and the difference in the bound concentration (Cb) between the two enantiomers was enhanced as their concentrations were increased. Although the percentage of the plasma protein binding in the dog plasma was significantly lower than that in the human plasma at 400 and 800 ng/mL, the corrected percentage of plasma protein binding was dog>human>rat. CONCLUSION: (-)Doxazosin and (+)doxazosin show stereoselective plasma protein binding with a significant species difference among rats, dogs and humans.

[Determination of doxazosin enantiomers in rat plasma and investigation of their chiral inversion].

The study is to establish an HPLC method using fluorescence detector for the determination of doxazosin enantiomers and investigate their chiral inversion in vitro and in vivo. Ultron ES-OVM was taken as the chiral chromatographic column, and the column temperature was 30 degrees C. Isocratic elution using a mobile phase of phosphate buffer-acetonitrile (85 : 15, v/v) at a flow rate of 0.8 mL x min(-1) was done. The fluorescence detection was set at lambda(Ex) = 255 nm and lambda(Em) = 385 nm. Prazosin was used as the internal standard. (-) Doxazosin or (+) doxazosin added into rat plasma in vitro was determined after incubating in 37 degrees C water bath for 2, 5 and 10 days. (-) Doxazosin or (+) doxazosin was administered orally to the rats for one months. Plasma samples were taken at 8 h after the last administration. A good linear relationship was achieved when the concentration of doxazosin enantiomers was within the range of 4 - 2 000 ng x mL(-1). The average recovery for (-) doxazosin was 99.5% with RSD 3.6%, and for (+) doxazosin was 99.3% with RSD 4.3%. Chiral inversion was observed neither in vitro nor in vivo studies. The method is selective, accurate and reproducible, which is suitable for the detection of doxazosin enantiomers in rat plasma. The in vitro and in vivo studies indicate that chiral inversion occurs uneasily between (-) doxazosin and (+) doxazosin in the rat.