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Amitraz is one of the critical acaricides/insecticides for effective control of pest infestation of Varroa destructor mite, a devastating parasite of Apis mellifera, because of its low toxicity to honeybees. Previous assays verified that a typical G protein-coupled receptor, β-adrenergic-like octopamine receptor (Octβ2R), is the unique target of amitraz, but the honeybee Octβ2R resists to amitraz. However, the underlying molecular mechanism of the enhanced sensitivity or toxicity of amitraz to mutated honeybee Octβ2RWe found that amitraz binding stabilized the structure of Octβ2R, particularly the intracellular loop 3 associated with the Octβ2R signaling. Then, it was further demonstrated that both mutations and ligand binding resulted in a more rigid and compact amitraz binding site, as well as the outward movement of the transmembrane helix 6, which was a prerequisite for G protein coupling and activation. Moreover, mutations were found to promote the binding between Octβ2R and amitraz. Finally, community analysis illuminated that mutations and amitraz strengthened the residue-residue communication within the transmembrane domain, which might facilitate the allosteric signal propagation and activation of Octβ2R.Our results unveiled structural determinants of improved sensitivity in the Octβ2R-amitraz complex and may contribute to further structure-based drug design for safer and less toxic selective insecticides. © 2022 Society of Chemical Industry.

The parathyroid hormone type 1 receptor (PTH1R) acts as a canonical class B G protein-coupled receptor, regulating crucial functions including calcium homeostasis and bone formation. The identification and development of PTH1R non-peptide allosteric modulators have obtained widespread attention. It has been found that a negative allosteric modulator (NAM) could inhibit the activation of PTH1R, but the implied mechanism remains unclear. Herein, extensive molecular dynamics simulations together with multiple analytical approaches are utilized to unravel the mechanism of PTH1R allosteric inhibition. The results suggest that the binding of NAM destabilizes the structure of the PTH1R–PTH–spep/qpep (the C terminus of Gs/Gq proteins) complexes. Moreover, the presence of NAM weakens the binding of PTH/peps (spep and qpep) and PTH1R. The intra- and inter-molecular couplings are also weakened in PTH1R upon NAM binding. Interestingly, compared with our previous study of the positive allosteric effects induced by extracellular Ca2+, the enhanced correlation between the PTH and G-protein binding sites is significantly reduced by the replacement of this negative allosteric regulator. Our findings might contribute to the development of new therapeutic agents for diseases caused by the abnormal activation of PTH1R.

Detection and identification of the in vivo metabolites of traditional Chinese medicine by untargeted profiling strategies are often confronted with severe interference from complex endogenous substances. Here we developed an integral approach, by combining untargeted data-dependent MS2 (dd-MS2) of Q-Orbitrap mass spectrometry and predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion scan (pMRM-IDA-EPI) of triple quadrupole-linear ion trap (QTRAP) mass spectrometry, aiming to detect and identify more extensive metabolites in bio-samples. Ecliptae Herba (EH) is a widely consumed medicinal herb with the effects of nourishing liver/kidney, but its metabolites in vivo have not been fully elucidated. Firstly, after UHPLC separation on an HSS T3 column, chemical fingerprinting of 70% ethanolic extract of EH was performed by untargeted dd-MS2 in negative ion mode. We could characterize 41 compounds from EH, and 24 were detectable in the plasma of rats (prototypes) after oral administration of EH extract (1 g/kg). Secondly, using echinocystic acid (triterpene), wedelolactone (coumarin), and apigenin (flavonoid) as the different parent templates, an MRM list containing 150 predicted ion-pairs was established to enhance MS2 scan by pMRM-IDA-EPI, which enabled the primary identification of up to 200 metabolites. The biotransformations mainly involve oxidation, hydrogenation, methylation, glucuronidation, sulfonation etc. Thirdly, the rat plasma samples obtained after oral administration of three pure compounds (echinocystic acid, wedelolactone and apigenin) were analyzed to verify the reliability of metabolites identification, and 11, 4, and 10 metabolites were found individually. This is the first comprehensive research on the metabolism of EH in vivo.

Processing of traditional Chinese medicine (TCM) can enhance the efficacy and/or reduce the toxicity. Currently available approaches regarding TCM processing generally focus on a few markers, rendering a one-sided strategy that fail to unveil the involved global chemical transformation. We herein present a strategy, by integrating enhanced multicomponent characterization, untargeted metabolomics, and mass spectrometry imaging (MSI), to visualize the chemical transformation and identify the markers associated with the wine steaming of Ligustri Lucidi Fructus (LLF), as a case. An ultra-high-performance liquid chromatography/quadrupole-Orbitrap mass spectrometry-based polarity-switching (between the negative and positive modes), precursor ions list-including data-dependent acquisition approach was developed, which enabled the simultaneous targeted/untargeted characterization of 158 components from LLF via one injection analysis. Holistic, continuous, and time-dependent chemical variation trajectory, among different processing time (0–12 h) for LLF, was depicted by principle component analysis. Pattern recognition chemometrics could unveil 20 markers, among which the peak area ratios of eight components to oleuropein aglycone, used as an internal standard, were diagnostic to identify the processed (both the commercial and in-house prepared) from the raw LLF. Four markers (10-hydroxyoleoside dimethylester, 8-demethyl-7-ketoliganin, elenolic acid, and salidroside) showed an increasing trend, while another four (neonuezhenide/isomer, verbascoside/isomer, luteoline, and nuzhenal A) decreased in LLF after processing. MSI visualized the spatial distribution in the fruit and indicated consistent variation trends for four major markers deduced by the untargeted metabolomics approach. This integral strategy, in contrast to the conventional approaches, gives more convincing data supporting the processing mechanism investigations of TCM from a macroscopic perspective.

Novel Tyrosinase Inhibitors of 4-functionalized Thiophene-2-carbaldehyde thiosemicarbazone (TCT) derivatives (1-8) had been synthesized and Spectrofluorimetry, 1H and 13C NMR titration and Molecular docking had been used to investigate their inhibitory activities and mechanisms on tyrosinase. The results showed that the inter-molecular interactions or hydrogen bond formation by increasing length of carbon chain or introducing benzene ring to the 4-functionalized ester group promoted or stabilized formation of complexes between modifier and tyrosinase, and enhanced the inhibitory activity of modifiers. The inhibitory activity of 4-benzoy methoxy-TCT was much stronger than that of any other synthesized tested modifiers, which was well explained by molecular docking and further verified by spectrofluorimetry and NMR titration by assuming that there existed an inter-molecular interaction besides formation of hydrogen bonds between the amino acid residues ASN260, GLU256, HIS85 of enzyme and the modifier.We concluded that 4-benzoy methoxy substitution of TCT was a good route obtaining novel tyrosinase inhibitors and deserved further studies.

The identification and quantization of traditional Chinese medicine (TCM) are a challenge for researchers and industry. Using untargeted analytical methods, the in vivo detection and identification of TCM compounds are difficult because of the significant interference of endogenous substances. Fortunately, the ongoing development of new analytical technologies, especially Q-Orbitrap-MS, offers some solutions. Our team developed a holistic MS method, combining untargeted data-dependent MS2 (dd-MS2 ) modes to extensively identify TCM prototypes in vivo. The method was successfully applied to the analysis of Ligustri Lucidi Fructus (LLF). LLF is a widely used TCM with a remarkable nourishing effect on the liver and kidney. In the study, we aimed to identify the prototypes in rat plasma after oral administration of LLF extract. Following separation on an HSS T3 column, LLF extract and rat plasma were performed in untargeted dd-MS2 mode. Forty-seven compounds were characterized in rats plasma as prototypes of LLF extract. Furthermore, seven major prototypes were chosen as pharmacokinetic markers to investigate LLF's pharmacokinetic properties. The results provides comprehensive determination of compounds in LLF both in vitro and in vivo, which is important for quality control, pharmacology studies and clinical use of LLF.

The Japanese ornamental carp (Cyprinus carpio var. Koi) is famous for multifarious colors and patterns, making it commonly culture and trade across the world. Although functional genes and inheritance of color traits have been commonly studied, seldom attentions were focused on the genetic regulation during the developmental process of pigmentation. To better understand the mechanism of skin color development, we observed the morphogenesis of pigment cells during the post-embryonic stages and analysed the temporal expression pattern of mRNAs/miRNAs profiles in four distinct developmental stages. 59 and 103 differentially expressed genes/miRNAs (DEGs/DEMs) associated with pigmentation and skin were identified, including pax7, mitf, tyr, tyrp1, etc., and the highest DEGs were detected at 11 days post hatching (dph). In addition, the functional characteristics of mRNAs/miRNAs associated with pteridine and carotenoid pathway were also examined. Furthermore, 65 miRNA-mRNA interaction pairs related to pigmentation, pteridines and carotenoids metabolism were detected between different stages. Interestingly, the largest pairs appeared in the transition from 11 dph to 48 dph, which had the similar trend with DEGs further manifesting the importance of 11 dph. This study produced a comprehensive programme of DEGs/DEMs during color development, which will provide resources to understand the regulation mechanism in color formation. The understanding of genetic basis in color formation might promote the production and breeding of the Koi carp.

Asthma is a chronic inflammatory disease that requires adherence to both preventative and therapeutic interventions in disease management. Children with asthma are likely to discontinue inhaled corticosteroids (ICS), especially when symptoms are under control. We aimed to investigate the impact of ICS adherence in children whose symptoms were under control. The study is cohort study; 35 children with controlled asthma that had undergone 3 years of follow-up were included. Serum eosinophil count, serum total IgE (tIgE), and lung function (FEV1, FEV1/FVC, PEF, FEF20–75%, and PC20) were evaluated at the beginning and end of the follow-up. At baseline, patients in both the adherent and nonadherent groups were similar. After 3 years, the nonadherent group who had discontinued ICS had a decrease in FEV1 (P

Background: To date, no predictive methods for the clinical response to allergy immunotherapy (AIT) are available. We sought to evaluate the pretreatment parameters used in diagnosing allergic asthma and/or rhinitis including allergen skin-prick test responses, serum specific and total IgE (sIgE and tIgE) levels and blood eosinophil counts, and to identify whether these can be used to predict clinical effectiveness in children treated with immunotherapy. Methods: This study involved 185 children who had undergone 3 years of standardized-quality house-dust mite subcutaneous immunotherapy. Clinical characteristics and laboratory parameters were analyzed. A multivariate unconditional logistic regression model and receiver operating characteristic curves were used. Predicted probabilities and predictive areas under the curve were calculated. Results: The clinical response to AIT was effective in 129/185 (69.7%) patients. Four variables were associated with clinical response by multivariate logistic analysis: tobacco smoke exposure [odds ratio (OR) 2.845 and 95% confidence interval (CI) 1.147-7.058; p = 0.024], a family history of atopy (OR 2.881 and 95% CI 1.240-6.692; p = 0.014), a serum tIgE level ≥965 kU/l (OR 5.917 and 95% CI 2.320-15.089; p = 0.000) and an sIgE/tIgE ratio ≥6% (OR 0.336 and 95% CI 0.124-0.911; p = 0.032). The sensitivity and specificity of the area under the curve of the serum tIgE were higher than those of serum sIgE and sIgE/tIgE ratio alone. Conclusion: Tobacco smoke exposure, atopic family history, serum tIgE and sIgE/tIgE ratio were in significant correlation with clinical response to AIT in children, which may be helpful for patient selection before immunotherapy. The serum tIgE is superior to both the serum sIgE/tIgE ratio and sIgE levels alone in predicting clinical effectiveness.