Establishment and preliminary evaluation of a fluorescent recombinase-aided amplification/CRISPR-Cas12a system for rapid detection of Plasmodium falciparum / 中国血吸虫病防治杂志

Objective To establish a fluorescent assay for rapid detection of Plasmodium falciparum based on recombinaseaided amplification (RAA) and CRISPR-Cas12a system,and to preliminarily evaluate the diagnostic efficiency of this system.. Methods The 18S ribosomal RNA (rRNA) gene of P. falciparum was selected as the target sequence, and three pairs of RAA primers and CRISPR-derived RNA (crRNA) were designed and synthesized. The optimal combination of RAA primers and crRNA was screened and the reaction conditions of the system were optimized to create a fluorescent RAA/CRISPR-Cas12a system. The plasmid containing 18S rRNA gene of the P. falciparum strain 3D7 was generated, and diluted into concentrations of 1 000, 100, 10, 1 copy/μL for the fluorescent RAA/CRISPR-Cas12a assay, and its sensitivity was evaluated. The genomic DNA from P. vivax, P. malariae, P. ovum, hepatitis B virus, human immunodeficiency virus and Treponema pallidum was employed as templates for the fluorescent RAA/CRISPR-Cas12a assay, and its specificity was evaluated. Fifty malaria clinical samples were subjected to the fluorescent RAA/CRISPR-Cas12a assay and nested PCR assay, and the consistency between two assays was compared. In addition, P. falciparum strain 3D7 was cultured in vitro. Then, the culture was diluted into blood samples with parasite densities of 1 000, 500, 200, 50, 10 parasites/μL with healthy volunteers’ O-positive red blood cells for the RAA/CRISPR-Cas12a assay, and the detection efficiency was tested. Results The Pf-F3/Pf-R3/crRNA2 combination, 2.5 μL as the addition amount of B buffer, 40 min as the RAA reaction time, 37 °C as the reaction temperature of the CRISPR-Cas12a system were employed to establish the fluorescent RAA/CRISPR-Cas12a system. Such a system was effective to detect the plasmid containing 18S rRNA gene of the P. falciparum strain 3D7 at a concentration of 1 copy/μL, and presented fluorescent signals for detection of P. falciparum, but failed to detect P. ovum, P. malariae, P. vivax, T. pallidum, hepatitis B virus or human immunodeficiency virus. The fluorescent RAA/CRISPR-Cas12a system and nested PCR assay showed completely consistent results for detection of 50 malaria clinical samples (kappa = 1.0, P < 0.001). Following 6-day in vitro culture of the P. falciparum strain 3D7, 10 mL cultures were generated and the fluorescent RAA/CRISPR-Cas12a system showed the minimal detection limit of 50 parasites/μL. Conclusion The fluorescent RAA/CRISPR-Cas12a system is rapid, sensitive and specific for detection of P. falciparum, which shows promising value for rapid detection and risk monitoring of P. falciparum.

Zhuanggu Guanjie herbal formula mitigates osteoarthritis via the NF-κB transduction mechanism.

The Zhuanggu Guanjie herbal formula has been a famous Chinese prescription for treating bone diseases since time immemorial. The anti-osteoarthritis (OA) properties of this botanical prescription are well documented in the Chinese Pharmacopoeia. However, the detailed mechanisms behind the phenomenon have not been elucidated. Hence, we aimed to investigate the anti-OA efficacy of the Zhuanggu Guanjie herbal formula and its underlying mechanism. The anti-OA properties of Zhuanggu Guanjie capsule (ZGC) were determined by the cytokine contents and inflammatory-related proteins, which were measured by RT-PCR, flow cytometry, Western blot, and laser confocal assay in ATDC5 cells. The levels of interleukin-6, tumor necrosis factor-α, inducible nitric oxide synthase, cyclooxygenase-2, and prostaglandin synthesis E2 have been markedly reduced after being treated with ZGC for 48 h in a dose-dependent manner. Furthermore, ZGC prevented the translocation of NF-κB from the cytosol to the nucleus. On the other hand, we used the mono-iodoacetate (MIA)-induced OA model to confirm the in vivo efficacies of this herbal formula. Oral administration of ZGC attenuated MIA-induced OA damage through changes in histopathological and knee joint volumes. The serum matrix metalloproteinase-13 contents in the ZGC treatment group declined as compared to those in the MIA model group. Through our in vitro and in vivo studies, we confirmed the anti-OA efficacy of ZGC and uncovered its detailed mechanism, and this treatment shed light on OA pathophysiology.

SPME-GC-MS combined with chemometrics to assess the impact of fermentation time on the components, flavor, and function of Laoxianghuang.

Laoxianghuang, fermented from Citrus medica L. var. Sarcodactylis Swingle of the Rutaceae family, is a medicinal food. The volatiles of Laoxianghuang fermented in different years were obtained by solid-phase microextraction combined with gas chromatography-mass spectrometry (SPME-GC-MS). Meanwhile, the evolution of its component-flavor function during the fermentation process was explored in depth by combining chemometrics and performance analyses. To extract the volatile compounds from Laoxianghuang, the fiber coating, extraction time, and desorption temperature were optimized in terms of the number and area of peaks. A polydimethylsiloxane/divinylbenzene (PDMS/DVB) with a thickness of 65 µm fiber, extraction time of 30 min, and desorption temperature of 200 °C were shown to be the optimal conditions. There were 42, 44, 52, 53, 53, and 52 volatiles identified in the 3rd, 5th, 8th, 10th, 15th, and 20th years of fermentation of Laoxianghuang, respectively. The relative contents were 97.87%, 98.50%, 98.77%, 98.85%, 99.08%, and 98.36%, respectively. Terpenes (mainly limonene, γ-terpinene and cymene) displayed the highest relative content and were positively correlated with the year of fermentation, followed by alcohols (mainly α-terpineol, ß-terpinenol, and γ-terpineol), ketones (mainly cyclohexanone, D(+)-carvone and ß-ionone), aldehydes (2-furaldehyde, 5-methylfurfural, and 1-nonanal), phenols (thymol, chlorothymol, and eugenol), esters (bornyl formate, citronellyl acetate, and neryl acetate), and ethers (n-octyl ether and anethole). Principal component analysis (PCA) and hierarchical cluster analysis (HCA) showed a closer relationship between the composition of Laoxianghuang with similar fermentation years of the same gradient (3rd-5th, 8th-10th, and 15th-20th). Partial least squares discriminant analysis (PLS-DA) VIP scores and PCA-biplot showed that α-terpineol, γ-terpinene, cymene, and limonene were the differential candidate biomarkers. Flavor analysis revealed that Laoxianghuang exhibited wood odor from the 3rd to the 10th year of fermentation, while herb odor appeared in the 15th and the 20th year. This study analyzed the changing pattern of the flavor and function of Laoxianghuang through the evolution of the composition, which provided a theoretical basis for further research on subsequent fermentation.

Inhibition of tau aggregation and associated cytotoxicity on neuron-like cells by calycosin.

In this study, the in vitro assembly of tau and anti-amyloidogenic properties of one naturally occurring phytoestrogen, calycosin, was investigated by spectroscopic techniques including ThT and ANS fluorescence, CD, Congo red absorbance as well as TEM analysis. Afterwards the cytotoxicity of different amyloid species against SH-SY5Y cells was evaluated by MTT assay. Fluorescence spectroscopic studies revealed that calycosin exerts its anti-amyloidogenic effects through increasing the lag time and reducing the apparent growth rate constant (kapp), the amount of fibrillation, and the exposure of hydrophobic regions. Congo red absorbance and CD studies indicated that calycosin prevented the formation of tau aggregate species and ß-sheets structures, respectively. TEM analysis also determined the capacity of calycosin to inhibit tau fibrillogenesis through formation of large amorphous aggregates. Furthermore, cellular assays disclosed that calycosin mitigated the cell mortality, LDH release, ROS level, and expression of Bax, Bcl-2, and Caspase-3 in both mRNA and protein levels induced by tau amyloid fibrils. In conclusion, this data may suggest that calycosin can prevent tau amyloid fibrillation and the associated cytotoxicity, mainly due to its effects on formation of lower content of oligomeric and fibrillar aggregates with lower solvent-exposed hydrophobic patches compared to those produced in the absence of calycosin.

Analysis on HPLC Fingerprints And Index Content Determination of Lao-Xiang-Huang of Chaozhou / 世界科学技术-中医药现代化

Lao-Xiang-Huang (LXH) of Chaozhou is the processed product of Fructus Citri sarcodactylis.LXH from different producing areas were used as research objects in the study for the establishment of HPLC fingerprints of LXH.Contents of hesperidin and 5,7-dimethoxycoumarin were also determined to provide scientific basis for the establishment of quality standard of LXH.Samples were separated by an Agilent Eclipse XDB C18 column (4.6 mm × 250 mm,5 μm) using acetonitrile-0.1％ phosphoric acid with water gradient system as a mobile phase.The flow rate was 1.0 mL· min-1.The injection volume was 20 μL.The detection wavelength was at 283 nm.The results showed that HPLC fingerprint of LXH was established with good separation and repeatability.The similarity evaluation on 27 batches samples of LXH showed that there was a certain similarity on the HPLC fingerprints of LXH.However,there was a certain difference as a whole.Contents of hesperidin and 5,7-dimethoxycoumarin in LXH were simultaneously determined.It was concluded that the established HPLC fingerprint of LXH and content determination of hesperidin and 5,7-dimethoxycoumarin method were accurate,sensitive and repeatable.It provided scientific evidence for the quality control standard of LXH of Chaozhou.

Effect and Underlying Mechanism of Sulfur-fumigation and Water-soaking on Total Ash of Dioscoreae Rhi-zoma / 中国药师

Objective:To study the effect and underlying mechanism of sulfur-fumigation and water-soaking on total ash of Di-oscoreae Rhizoma, find the key factor( s) affecting the total ash of Dioscoreae Rhizoma, and explore the rationality of ash limits of Di-oscoreae Rhizoma described in Chinese Pharmacopoeia. Methods:Dioscoreae Rhizoma was respectively dealt with sulfur-fumigation and water-soaking. The changes in total ash content of Dioscoreae Rhizoma was detected by the ash determination methods for total ash and SO2 described in the pharmacopoeia, and then the ash content change of inorganic salts was used to study the mechanism. Results:Sulfur-fumigation could slightly reduce the total ash content of Dioscoreae Rhizoma, while significantly reduce the ash content of calcium oxalate and calcium sulfate with the reduction degree of 7. 20% and 9. 90%, respectively. Calcium phosphate and calcium chloride were slightly affected by sulfur-fumigation, and the results indicated that the effect of sulfur-fumigation on ash content was mainly real-ized by increasing the decomposition rate of calcium oxalate and calcium sulfate. Water-soaking could decline the ash content of Di-oscoreae Rhizoma, and the phenomenon was common in the rhizome medicinal materials. The influence of water-soaking on total ash was more significant than that of sulfur fumigation. Conclusion:Sulfur-fumigation can reduce the total ash content of Dioscoreae Rhizo-ma by increasing the decomposition rate of calcium oxalate and calcium sulfate, however, the effect is mild and the process isn't the key influencing factor in the total ash content of Dioscoreae Rhizoma. During the preparation of Dioscoreae Rhizoma medicinal slices, water-soaking can cause the great loss of water-soluble mineral salts, such as Cl-, C2 O4 2-, NO3 - and SO4 2-, which leads to the reduction of total ash content, therefore, water-soaking is the key influencing factor in the total ash content of Dioscoreae Rhizoma.

Application of Sulfur in Traditional Chinese Medicine / 世界科学技术-中医药现代化

Sulfur , a major component of gunpowder , has been widely used in the engineering and military in-dustries since ancient times . In fact , the application of sulfur in traditional Chinese medicine ( TCM ) has a long history , which indicated the uniqueness of TCM theory and practice . Besides , sulfur has played an impor-tant role for the development of TCM in the history . In order to scientifically analyze the role of sulfur in TCM , this paper focused on the application and evolution of sulfur in the development process of TCM , which aimed to provide a reference for the study of the value and role of sulfur in TCM .