Multiplexed DNA detection with a composite molecular beacon based on guanine-quenching.

We developed a multiplexed DNA detection method with a composite molecular beacon (MB) probe based on guanine-quenching by synchronous fluorescence analysis. It is demonstrated by two types of tumor-suppressor genes namely exon segments of p16 (T1) and p53 (T2) genes. The composite MB probe includes two loops and two stems, and two fluorophores of 6-carboxyfluorescein group (FAM) and tetramethyl-6-carboxyrhodamine (TAMRA) are connected to the two ends of molecular beacon. Every stem portion of MB include four continuous nucleotides with guanine (G) base as quencher, every loop portion is a probe sequence that is complementary to a corresponding target sequence. In the absence of target DNA, the composite MBs are in the stem-closed form, the fluorescence of FAM and TAMRA are quenched by G bases. At this time, the fluorescence signals of FAM and TAMRA are all very low. In the presence of target DNA, the MBs hybridize with the target DNA and form double-strands, FAM and TAMRA are separated from G bases, and the fluorescence of FAM and TAMRA recovers simultaneously. Thus, the simultaneous detection of two targets of DNA can be realized by measuring fluorescence signals of FAM and TAMRA, respectively. Under the optimum conditions, the fluorescence intensities of FAM and TAMRA all exhibit good linear dependence on their target DNA concentration in the range from 5 × 10(-11) to 5.5 × 10(-9) M. The detection limit of T1 is 4 × 10(-11) M (3σ), and that of T2 is 3 × 10(-11) M. This composite MB can be applied to detect the real sample, and can be applied to detect two aleatoric sequences of DNA. Compared with previously reported methods of detecting multiplexed target DNA with MBs, the proposed method has some advantages including easy synthesis of composite MB probes, low detection cost and shorter analytical time.

Network pharmacology to explore the molecular mechanisms of Prunella vulgaris for treating thyroid cancer.

BACKGROUND: Thyroid cancer (TC) is the most common endocrine malignancy that has rapidly increased in global incidence. Prunella vulgaris (PV) has manifested therapeutic effects in patients with TC. We aimed to investigate its molecular mechanisms against TC and provide potential drug targets by using network pharmacology and molecular docking. METHODS: The ingredients of PV were retrieved from Traditional Chinese Medicine Systematic Pharmacology Database. TC-related gene sets were established using the GeneCard and OMIM databases. The establishment of the TC-PV target gene interaction network was accomplished using the STRING database. Cytoscape constructed networks for visualization. Protein-protein interaction, gene ontology and the biological pathway Kyoto encyclopedia of genes and genomes enrichment analyses were performed to discover the potential mechanism. Molecular docking technology was used to analyze the effective compounds from PV for treating TC. RESULTS: 11 active compounds and 192 target genes were screened from PV. 177 potential targets were obtained by intersecting PV and TC gene sets. Network pharmacological analysis showed that the PV active ingredients including Vulgaxanthin-I, quercetin, Morin, Stigmasterol, poriferasterol monoglucoside, Spinasterol, kaempferol, delphinidin, stigmast-7-enol, beta-sitosterol and luteolin showed better correlation with TC target genes such as JUN, AKT1, mitogen-activated protein kinase 1, IL-6 and RELA. The gene ontology and Kyoto encyclopedia of genes and genomes indicated that PV can act by regulating the host defense and response to oxidative stress immune response and several signaling pathways are closely associated with TC, such as the TNF and IL-17. Protein-protein interaction network identified 8 hub genes. The molecular docking was conducted on the most significant gene MYC. Eleven active compounds of PV can enter the active pocket of MYC, namely poriferasterol monoglucoside, stigmasterol, beta-sitosterol, vulgaxanthin-I, spinasterol, stigmast-7-enol, luteolin, delphinidin, morin, quercetin and kaempferol. Further analysis showed that oriferasterol monoglucoside, followed by tigmasterol, were the potential therapeutic compound identified in PV for the treatment of TC. CONCLUSION: The network pharmacological strategy integrates molecular docking to unravel the molecular mechanism of PV. MYC is a promising drug target to reduce oxidative stress damage and potential anti-tumor effect. Oriferasterol monoglucoside and kaempferol were 2 bioactive compounds of PV to treat TC. This provides a basis to understand the mechanism of the anti-TC activity of PV.

Case report: Successful treatment with contezolid in a patient with tuberculous meningitis who was intolerant to linezolid.

Tuberculous meningitis (TBM) is the most common form of central nervous system tuberculosis (TB) and the most severe form of extrapulmonary TB. It often presents with non-specific symptoms initially and has a high mortality and disability rate. With good central nervous system penetration, linezolid is recommended for treating drug-resistant, severe, or refractory tuberculous meningitis in China. Despite the benefits of linezolid on TBM treatment, the adverse effects of long-term therapy, such as myelosuppression, peripheral neuritis, and optic neuritis, are notable and can be severe and even life-threatening, leading to discontinuation and compromising treatment expectations. Contezolid is a novel oxazolidinone antibacterial agent approved by the National Medical Products Administration of China in 2021, which has a more favorable safety profile than linezolid in terms of myelosuppression and monoamine oxidase inhibition. Here we first report a case of TBM in a patient who was intolerant to antituberculosis treatment with linezolid and achieved good efficacy and safety results after the compassionate use of contezolid. Given the widespread use of linezolid in TB treatment and the potential risks for long-term use, multi-center prospective controlled clinical trials in TB and TBM patients are needed to investigate the appropriate use of contezolid further.

[Hydrophilic interaction liquid chromatography for removal of pesticide residues in ginseng extracts].

Ginseng extracts are rich in a variety of ginseng monomer saponins, which have pharmacological functions of retarding aging, enhancing immunity, stimulating blood circulation, and lowering blood pressure. Ginseng is widely used in health products and dietary supplements in the domestic and foreign market. However, the amount of pesticide residues is an important index for measuring the quality of ginseng and ginseng extracts. Therefore, studies focused on methods for the removal of pesticide residues in ginseng extract are of great significance. Hydrophilic interaction liquid chromatography (HILIC) is used to improve the retention and separation selectivity of strongly polar substances, and it is widely employed in drug analysis, metabolomics, proteomics, etc. In this study, a method for the removal of pesticide residues was developed based on the difference in the retention behavior of pesticide residues and ginsenosides on the HILIC column. Using commercially available ginsenoside extracts, the retention behaviors of pesticide residues and ginsenosides on reverse chromatography and hydrophilic chromatographic columns were evaluated by high performance liquid chromatography. The results proved that on the reversed-phase liquid chromatography (RPLC) stationary phase, in addition to the strong retentions of quintozene and pentachloroaniline, which could be clearly separated from the saponins, the retentions of the other five pesticide residues including carbendazim, azoxystrobin, procymidone, iprodione and propiconazole were similar to total ginsenosides. The seven ginsenosides showed strong retention due to the formation of hydrogen bonds between the hydroxyl groups on the sugar chain and the carboxyl groups on the HILIC stationary phase. However, the pesticide residues were not well retained because of their poor hydrophilicity and small molecular weights. For this reason, the pesticide residues and ginsenosides could be completely separated on the HILIC column. Thus, enrichment of the seven ginsenosides and removal of the 14 pesticide residues was realized in one step on the HILIC column. In addition, the effects of loading amount, loading volume, and washing volume on the removal of pesticide residues in ginsenosides were investigated using the Click XIon SPE column. Then, taking the ginsenoside recoveries and pesticide residue removal rates into account, we confirmed the following: the ratio of the maximum sample loading mass to the filler mass was 1∶10; the optimal elution volume was twice the column volume; and the optimal loading volume was twice the column volume. The ginseng extracts were solvated with a 95% ethanol solution and loaded onto an HILIC column. The sample was subjected to pesticide residue removal, and ginsenoside purification and enrichment under the optimum removal conditions. Gradient elution was carried out using ethanol and water as the mobile phases. The total ginsenoside content in the final extracts was increased to 69.61%. The recovery of the total ginsenosides was 94.4%. The pesticide residues in the samples were quantitatively detected by gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) in the multiple reaction monitoring (MRM) mode. The 14 pesticide residues in the original ginsenoside extracts were effectively removed. The amounts of five residues were reduced to below 0.05 mg/kg, while the other nine residues were completely eliminated. This study demonstrates the application of HILIC to pesticide residue removal in traditional Chinese medicine extracts and reveals a new technique for the purification of natural products. The proposed method shows a high removal rate of pesticide residues and a high recovery of total ginsenosides. It is safe, efficient, and environment-friendly, and can aid the development of high-quality ginsenoside extracts.

Purification of tertiary and quaternary alkaloids from Rhizoma Corydalis using reversed-phase/weak cation-exchange mixed-mode class separation combined with preparative C18 and silica based strong cation-exchange chromatography.

A new optimization strategy for purification of alkaloids from Rhizoma Corydalis using preparative liquid chromatography was developed, featuring a selective separation of different types of alkaloids into different parts by a reversed-phase/weak cation-exchange mixed-mode column (named C18WCX) at first. The total alkaloids of Rhizoma Corydalis were divided into four fractions with fraction III and IV corresponding to the tertiary type medium bases and the quaternary type strong bases, respectively. For fraction III, a conventional C18 column was used to isolate tertiary alkaloids using acetonitrile and 0.1% phosphoric acid (adjusted with triethylamine to pH 6.0) as mobile phases. High selectivity and symmetrical peak shapes of tertiary alkaloids were obtained, resulting in six main tertiary alkaloids isolated in a single run. As strong bases, quaternary alkaloids often suffer from serious peak tailing problem on conventional C18 columns. Therefore, a silica-based strong cation-exchange (SCX) column was used for purification of fraction IV. On the SCX column, good peak shapes in high sample loading were achieved. Five main quaternary alkaloids were isolated and identified from the fraction in one-step. The procedures presented effective for the preparative isolation and purification of alkaloids from Rhizoma Corydalis.

[An analytical method for alkaloids of Coptis chinensis based on mixed-mode surface electrostatic exclusion/reversed-phase chromatography].

A mixed-mode chromatographic method based on surface electrostatic exclusion and reversed-phase chromatography was established for the determination of alkaloids present in Coptis chinensis. The effects of two mobile phase additives, formic acid and acetic acid, on retention, peak shape and selectivity of the alkaloids in Coptis chinensis were investigated using the self-made C18HCE column. Acetic acid (0.1%, v/v) used as the additive was found to be optimum for effective separation of the main alkaloids present in Coptis chinensis. The main chromatographic peaks of Coptis chinensis were recognized by the established method and references, which were coptisine, epiberberine, columbamine, jatrorrhizine, berberine and palmatine, respectively. With reference to the content determination method of Coptis chinensis in the 2015 edition of pharmacopoeia, the linear relationship of berberine in the range of 0.5-100 mg/L was good, the correlation coefficient was 0.9996, and the average recovery was 93.74%. The contents of alkaloids in Coptis chinensis in different batches of Hubei and Chongqing were determined. The method is simple, reliable and accurate, and can be used as reference for separation and analysis of other basic compounds.

Highly sensitive fluorescence quantitative detection of specific DNA sequences with molecular beacons and nucleic acid dye SYBR Green I.

A highly sensitive fluorescence method of quantitative detection for specific DNA sequence is developed based on molecular beacon (MB) and nucleic acid dye SYBR Green I by synchronous fluorescence analysis. It is demonstrated by an oligonucleotide sequence of wild-type HBV (target DNA) as a model system. In this strategy, the fluorophore of MB is designed to be 6-carboxyfluorescein group (FAM), and the maximum excitation wavelength and maximum emission wavelength are both very close to that of SYBR Green I. In the presence of targets DNA, the MBs hybridize with the targets DNA and form double-strand DNA (dsDNA), the fluorophore FAM is separated from the quencher BHQ-1, thus the fluorophore emit fluorescence. At the same time, SYBR Green I binds to dsDNA, the fluorescence intensity of SYBR Green I is significantly enhanced. When targets DNA are detected by synchronous fluorescence analysis, the fluorescence peaks of FAM and SYBR Green I overlap completely, so the fluorescence signal of system will be significantly enhanced. Thus, highly sensitive fluorescence quantitative detection for DNA can be realized. Under the optimum conditions, the total fluorescence intensity of FAM and SYBR Green I exhibits good linear dependence on concentration of targets DNA in the range from 2×10(-11) to 2.5×10(-9)M. The detection limit of target DNA is estimated to be 9×10(-12)M (3σ). Compared with previously reported methods of detection DNA with MB, the proposed method can significantly enhance the detection sensitivity.

FeCl3/NaNO2: an efficient photocatalyst for the degradation of aquatic steroid estrogens under natural light irradiation.

Assistance and acceleration of the environment's self-remediation of pollutants represent an important and long-standing goal for environmental chemistry communities. Here, a degradation route using a combination of a nitrite and a ferric salt as the photocatalyst is presented for catalytically removing 17beta-estradiol (E2), estriol (E3), and 17alpha-ethynylestradiol (EE2) in water under mimicked natural environmental conditions, i.e., in the phytotron. After a 1 day reaction, 86.6% of the estrogen E2 was degraded. Extending the incubation time to 30 days, more than 99.9% E2 was removed and a very small quantity of malonic acid observed as the residual organic compound, and estrogenic activity was determined. The results showed that the estrogenic activities of the intermediate products are negligible and that there is no secondary risk associated with increased the estrogenic activity. The degradation system demonstrated that FeCl3/NaN02 is an efficient photocatalyst which is active on natural light irradiation. This work highlights a promising development for in situ treatment of pollutants in natural-environment conditions.