Integrating UPLC-MS/MS with in Silico and in Vitro Screening Accelerates the Discovery of Active Compounds in Stephania epigaea.

Traditional Chinese medicines (TCMs) are popular in clinic because of their safety and efficacy. They contain abundant natural active compounds, which are important sources of new drug discovery. However, how to efficiently identify active compounds from complex ingredients remains a challenge. In this study, a method combining UHPLC-MS/MS characterization and in silico screening was developed to discover compounds with dopamine D2 receptor (D2R) activity in Stephania epigaea (S. epigaea). By combining the compounds identified in S. epigaea by UHPLC-MS/MS with reported compounds, a virtual library of 80 compounds was constructed for in silico screening. Potentially active compounds were chosen based on screening scores and subsequently tested for in vitro activity on a transfected cell line CHO-K1-D2 model using label-free cellular phenotypic assay. Three D2R agonists and five D2R antagonists were identified. (-)-Asimilobine, N-nornuciferine and (-)-roemerine were reported for the first time as D2R agonists, with EC50 values of 0.35 ± 0.04 µM, 1.37 ± 0.10 µM and 0.82 ± 0.22 µM, respectively. Their target specificity was validated by desensitization and antagonism assay. (-)-Isocorypalmine, (-)-tetrahydropalmatine, (-)-discretine, (+)-corydaline and (-)-roemeroline showed strong antagonistic activity on D2R with IC50 values of 92 ± 9.9 nM, 1.73 ± 0.13 µM, 0.34 ± 0.02 µM, 2.09 ± 0.22 µM and 0.85 ± 0.08 µM, respectively. Their kinetic binding profiles were characterized using co-stimulation assay and they were both D2R competitive antagonists. We docked these ligands with human D2R crystal structure and analyzed the structure-activity relationship of aporphine-type D2R agonists and protoberberine-type D2R antagonists. These results would help to elucidate the mechanism of action of S. epigaea for its analgesic and sedative efficacy and benefit for D2R drug design. This study demonstrated the potential of integrating UHPLC-MS/MS with in silico and in vitro screening for accelerating the discovery of active compounds from TCMs.

Development and Characterization of Fluorescent Probes for the G Protein-Coupled Receptor 35.

The orphan G protein-coupled receptor 35 (GPR35) is a potential target for the treatment of pain, inflammation, and metabolic diseases. Although many GPR35 agonists have been discovered, research on functional GPR35 ligands, such as fluorescent probes, is still limited. Herein, we developed a series of GPR35 fluorescent probes by conjugating a BODIPY fluorophore to DQDA, a known GPR35 agonist. All probes exhibited excellent GPR35 agonistic activity and desired spectroscopic properties, as determined by the DMR assay, bioluminescence resonance energy transfer (BRET)-based saturation, and kinetic binding experiments. Notably, compound 15 showed the highest binding potency and the weakest nonspecific BRET binding signal (K d = 3.9 nM). A BRET-based competition binding assay with 15 was also established and used to determine the binding constants and kinetics of unlabeled GPR35 ligands.

Offline preparative three-dimensional HPLC for systematic and efficient purification of alkaloids from Gelsemium elegans Benth.

The natural compound library is the most productive source in drug discovery and alkaloids are the most potential drug leads in natural compounds. Presently, systematic purification of alkaloids remains a substantial challenge. In this study, we developed an offline preparative three-dimensional HPLC (3D-HPLC) method to resolve the problem of systematic purification of alkaloids. Ten Gelsemium standards were used in the construction of the method to evaluate several factors, including column selectivity, column loadability and separation orthogonality. The offline 3D-HPLC method achieved great orthogonal selectivity and resolution power using different stationary phases, mobile phases at different pH, and different mobile phase additives. Application of this 3D-HPLC method to Gelsemium elegans Benth. was evaluated, and 24 indole alkaloids were finally isolated, including four new alkaloids and one first-identified in this plant. Moreover, a total of 229 compounds were estimated to be obtained in this plant, almost twice the number of known Gelsemium alkaloids. Therefore, this 3D-HPLC method will be efficient for systematic purification of alkaloids from Gelsemium elegans Benth. and has the potential for alkaloid preparation from other plants.

[Biomimetic polymer material for glycopeptide high selective enrichment].

The study of protein glycosylation is important to deepening the current understanding of its biological functions as well as to elucidate novel biomarkers of glycosylation. However, glycopeptides must be enriched prior to analysis due to their naturally low abundance. In this study, tryptophan functionalized polymer materials (denoted as Poly-Trp) were synthesized to serve as a novel biomimetic polymers. The resulting materials were characterized by various methods including scanning electron microscopy, thermal analysis, and infrared spectrometry, validating the successful synthesis of Poly-Trp. The retention of glycopeptides on Poly-Trp was investigated revealing that the retention ability decreased as the acetonitrile content of the mobile phase decreased and its acidity increased. Poly-Trp exhibited higher enrichment selectivity for glycopeptides from bovine fetuin than the commercial material ZIC-HILIC, and aminated silica which could defect the interference of 100-fold amount of substance ratios of bovine serum albumin. Poly-Trp is expected to have further applications in the purification of biological samples for the study of glycosylation.

Interfacially Polymerized Particles with Heterostructured Nanopores for Glycopeptide Separation.

Porous polymer materials are extensively used for biomolecule separation. However, conventional homogeneous porous polymer materials cannot efficiently separate specific low-abundance biomolecules from complex samples. Here, particles fabricated by emulsion interfacial polymerization featuring heterostructured nanopores with tunable size are reported, which can be used to realize low-abundance glycopeptide (GP) separation from complex biofluids. The heterostructured surface inside the nanopores allows solvent-dependent local adsorption of biomolecules onto hydrophilic or hydrophobic regions. Low-abundance hydrophilic GPs in complex biofluids can be efficiently separated via the hydrophilic region of nanopores in low-polarity solvent after the hydrophobic region removes high-abundance hydrophobic proteins and non-glycopeptides in high-polarity solvent. It is expected that these particles with heterostructured nanopores can be used for separation of nucleic acids, saccharides, and proteins, and downstream clinical diagnosis.

Novel nanoporous covalent organic frameworks for the selective extraction of endogenous peptides.

Endogenous peptides are important biomarkers, but their low abundance and abundant interference in biosamples impede their analysis. In this study, a novel nanoporous covalent organic framework (COF) was prepared and successfully applied for selective extraction of endogenous peptides from human serum. This novel COF exhibited strong retention and high adsorption capacity toward peptides, as well as efficient exclusion of large proteins, ascribed to its strong hydrophobicity, uniform pore size (∼2.5 nm) and large surface area (826.5 m2 g-1). These features facilitated the extraction of endogenous peptides from complex biosamples, resulting in 27 identified peptides from tryptic digests of bovine serum albumin (BSA) mixed with 1000 mass folds of BSA protein. Moreover, the adsorption rate of the peptides was 3.6-fold faster than that of proteins on this novel COF. After application the novel COF to 5 µL human serum, 416 unique peptides were unambiguously identified. These results demonstrated the excellent properties of the novel COF in extraction of endogenous peptides. We envisage that COFs with adjustable organic building units and unique physicochemical properties will qualify their potential applications in peptidomics research.

[Therapeutic effect of submental flap in repairing of approaching circumferential defects after hypopharyngeal cancer ablation with laryngeal function unpreserved].

OBJECTIVE: To study the effectiveness of repairing nearly circumferential defect with the submental flaps after resection of laryngeal function unpreserved hypopharyngeal cancer. METHOD: All the cases were treated with the submental flaps after resection of hypopharyngeal cancer with laryngeal function unpreserved. RESULT: All 13 flaps were alive. Hypopharyngeal fistula occurred in 2 cases. All patients had normal swallowing function. The patients were followed up 6-42 months. Of 13 cases,3 had recurrence at neck Lymph node, but no local hypopharyngeal recurrence was found. Seven cases were followed up more than 3 years, and only 3 of them survived. CONCLUSION: Submental flap is an ideal tissue flap submental flap in repairing of approaching circumferential defects after hypopharyngeal cancer ablation with laryngeal function unpreserved for the repairment of after approaching circumferential defects after hypopharyngeal cancer ablation with laryngeal function unpreserved, For it is close to the defect region, safe, easy-to-obtain and easy-to-survive.

Kinetic resolution of primary allylic amines via palladium-catalyzed asymmetric allylic alkylation of malononitriles.

A range of primary allylic amines were resolved with selectivity factors of up to 491 through [Pd(allyl)Cl]2/(S)-BINAP-catalyzed and mesitylsulfonyl hydrazide-accelerated asymmetric allylic alkylation of malononitriles involving enantioselective C-N bond cleavage under aerobic conditions. Moreover, the reaction proved useful for the asymmetric synthesis of α-branched allyl-substituted malononitriles with high enantiopurity.

Palladium/copper-catalyzed oxidative arylation of terminal alkenes with aroyl hydrazides.

An unprecedented oxidative arylation reaction of terminal alkenes with simple aroyl hydrazides has been developed under aerobic conditions for the stereoselective synthesis of 1,2-disubstituted alkenes. A range of aroyl hydrazides underwent palladium/copper-catalyzed oxidative Mizoroki-Heck reaction with terminal alkenes open to air in a 1:1 mixture of dimethyl sulfoxide and acetonitrile to give structurally diverse 1,2-disubstituted alkenes in moderate to excellent yields with excellent regio- and E-selectivity. The reaction tolerated a wide variety of functional groups, such as alkoxy, hydroxy, amino, fluoro, chloro, bromo, cyano, nitro, ester, amide, imide, phosphine oxide, and sulfone groups, and, moreover, molecular oxygen and dimethyl sulfoxide were demonstrated to serve as terminal oxidants. This study provides a useful method for the stereoselective synthesis of 1,2-disubstituted alkenes through direct transformation of the vinylic CH bonds in terminal alkenes.

[Expression of SOX2 protein and its clinical significance in laryngeal carcinoma].

OBJECTIVE: To investigate the expression of SOX2 in laryngeal carcinoma and analyze the relation of SOX2 and clinical factors. METHOD: We measured the expression of SOX2 protein in 45 laryngeal carcinoma fresh samples and 45 paracarcinoma tissues (cutting margin > 0.5 cm) with flow cytometer (Epics-XL II), 20 normal laryngeal mucosa samples were also studied as controls. RESULT: The quantitative and qualitative expression of SOX2 protein in laryngeal carcinoma tissues was obviously higher than those in paracarcinoma and in normal laryngeal mucosa tissues respectively (P < 0 05). There was no significant difference between the expression of paracarcinoma and normal laryngeal mucosa tissues. In laryngeal carcinoma, the expression of SOX2 protein wasn't significantly related to patients' clinical classification, tumor size, smoking history, patients' age and sex but related to metastasis, pathological grade and clinical stage. CONCLUSION: The high expression of SOX2 may contribute to the carcinogenesis and development of laryngeal carcinoma. It is an important index of judging metastasis and staging and prognosis of laryngeal carcinoma to measure the expression of SOX2 protein.

Intracellular and current source density analysis of pretectal input to the optic tectum of the frog.

OBJECTIVE: Electrophysiological examination of the ipsilateral pretectotectal projection has proved that pretectal cells elicit strong suppressive responses to the ipsilateral tectum. However, the neural mechanisms underlying the contralateral pretectotectal prejection are still obscure. The present study aimed to examine the synaptic nature of pretectal nuclei and contralateral tectal cells, and to demonstrate the spatiotemporal pattern of neuronal activity in the 2 main brain structures. METHODS: Intracellular recording and current source density (CSD) analysis were used to test the complexity of neuronal mechanism of pretectotectal information transfer. RESULTS: The pretectal stimulation elicited only one type of response on the contralateral tectum, the inhibitory postsynaptic potential (IPSP). The majority of contra-induced IPSPs were assumed to be polysynaptically driven. In the CSD analysis, only one sink with short latency was observed in each profile. The ipsilateral projection produced a prominent monosynaptic sink in layer 8 of tectum. Recipient neurons were located in layers 6 and 7 of tectum. The result confirmed former findings from ipsilateral intracellular recordings. CONCLUSION: These results suggest the following neuronal circuit: afferents from the pretectal nuclei broadly inhibit both tectal neuron, and since no second sink occurs in tectal layers, the pretectotectal excitatory afferents probably do not extend over the whole tectum, but are within limited state. The results of intracellular recording and CSD analysis further provide evidence of how pretectal afferent activity flows within the tectal laminae.

Response properties and receptive field organization of collision-sensitive neurons in the optic tectum of bullfrog, Rana catesbeiana.

OBJECTIVE: Many studies have reported that animals will display collision avoidance behavior when the size of retinal image of an object reaches a threshold. The present study aimed to investigate the neural correlates underlying the frog collision avoidance behavior. METHODS: Different types of visual stimuli simulating the retinal image of an approaching or a recessing object were generated by a computer and presented to the right eye of frog. A multielectrode array was used to examine the activity of collision-sensitive neurons, and single electrode recordings were employed to quantify visual parameter(s) of the frog collision-sensitive neurons. RESULTS: The multielectrode array revealed that 40 neurons in the optic tectum showed selective responsiveness to objects approaching on a direct collision course. The response profiles of these collision-sensitive neurons were similar to those of lobula giant movement detector (LGMD) in the locust or to those of neurons in the pigeon. However, the receptive field (RF) size of the frog neurons [(18.5+/-3.8) degrees, n=33)] was smaller than those of collision-sensitive neurons of the locust and the pigeon. Multielectrode recordings also showed that the collision-sensitive neurons were activated only when the focus of expansion of a looming retinal image was located within the center of its RF. There was a linear relationship between the parameter l/v (l denotes half-size of the object, v denotes approaching velocity) and time-to-collision (time difference between the peak of the neuronal activity and the predictive collision) in 16 collision-sensitive neurons. Theoretical consideration showed that the peak firing rate always occurred at a fixed delay of (60.1 +/- 39.5) ms (n=16) after the object had reached a constant angular size of (14.8 +/- 3.4) degrees (n=16) on the retina. CONCLUSION: The results may help clarify the mechanisms underlying the collision avoidance behavior in bullfrog.

An intracellular study of pretectal influence on the optic tectum of the frog, Rana catesbeiana.

OBJECTIVE: A few investigations have been reported about pretectal suppressive influences on the optic tectum of frog, but characteristics of tectal activity to pretectal input are left unknown. We made intracellular recordings to demonstrate the unexpected complexity in synaptic mechanisms involved in the suppressive influences of pretecal stimulation on the tectal cells. METHODS: In the present study, we investigated the neuronal activity evoked by pretectal (Lpd/P) nuclei stimulation using intracellular recording technique. RESULTS: The pretectal stimulation mainly elicited two types of responses in the ipsilateral tectum: an excitatory postsynaptic potential (EPSP) followed by an inhibitory postsynaptic potential (IPSP) and a pure IPSP. The latter predominated in the tectal cells responding to pretectal stimulation. In a few cells, biphasic hyperpolarization appeared under stronger stimulus intensities. The spikes of tecto-pretectal projecting cells elicited by antidromical stimulation were recorded in the ipsilateral tectum, which revealed reciprocal connections between the tectum and particular pretectal nuclei. The synaptic natures underlying pretecto-tectal information transformation have also been demonstrated. EPSPs with short latencies were concluded to be monosynaptic. Most IPSPs were generated through polysynaptic paths, but monosynaptic IPSPs were also recorded in the tectum. Nearly 98% of impaled tectal cells (except for antidromically projecting cells) showed inhibitory responses to pretectal stimulation. CONCLUSION: The results provide strong evidence that pretectal cells broadly inhibit tectal neurons as that has suggested by behavioral and extracellular recording studies.