Exploring the Roles of m6A-Modified circRNAs in Myasthenia Gravis Based on Multi-Omics Analysis.

Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibodies. The important roles of circRNAs modified by m6A methylation have been reported in the pathogenesis of other autoimmune diseases, but remain unclear in MG. To address this point, we collected peripheral blood mononuclear cells from six MG patients and six healthy controls and performed m6A­circRNA epitranscriptomic microarray and RNA sequencing. Differentially m6A-modified circRNAs and differentially expressed genes (DEGs) were analyzed. A network was constructed containing 17 circRNAs, 30 miRNAs, and 34 DEGs. The GSE85452 dataset was downloaded. DEGs that were differentially expressed in the GSE85452 dataset were selected as seed genes. Finally, four candidate m6A-modified circRNAs (hsa_circ_0084735, hsa_circ_0018652, hsa_circ_0025731, and hsa_circ_0030997) were identified through a random walk with restart. We found that they had different degree correlations with different immune cells. The results of MeRIP-qPCR showed that the m6A methylated levels of hsa_circ_0084735 and hsa_circ_0025731 were downregulated in MG patients, while the other two circRNAs were not significantly different between MG and control group. For the first time, we explored the pathogenesis of MG at the epigenetic transcriptome level. Our results will open new perspectives for MG research and identify potential biomarkers and therapeutic targets for MG.

Optimization of preparation method of hepatoprotective active components from Coreopsis tinctoria Nutt. and its action mechanism in vivo.

Capitula of Coreopsis tinctoria are widely used as a flower tea with great health benefits due to rich content of flavonoids and phenolic acids. The hepatoprotective effect of C. tinctoria and its bioactive basis have seldom been investigated until now. In the present study, capitula of C. tinctoria were extracted with a method optimized by response surface methodology (RSM) and BoxBehnken design (BBD) and further purified by macroporous resin HPD-300 to obtain a fraction (CE) enriched with flavonoids and phenolic acids. The contents of the four most abundant compounds, isookanin-7-O-ß-d-glucoside (1), quercetigetin-7-O-ß-d-glucoside (2), okanin (3), and marein (4), were determined by HPLC as 9.98, 5.21, 41.78 and 1.85 mg/g, respectively. Seventy-four compounds including fifity-five flavonoids, fifteen organic acids (twelve of them were phenolic compounds), and three coumarins were tentatively identified in CE by LC-HRMS/MS. In vivo hepatoprotective effect and potential mechanism of CE were studied with a high-fat diet-induced NASH mouse model. CE administration decreased the amount of weight gain, hepatic lipid, and sequentially improved dyslipidemia, inflammation, oxidative stress, and IR in HFD-fed mice. Molecular data revealed that CE inhibited hepatic inflammation by reducing NFκB/iNOS/COX-2/NLRP3/MAPK in the liver tissues and ameliorated oxidative stress by activating the Nrf2/HO-1 pathway. Modulation of inflammation and oxidative stress with CE may represent a promising target for the treatment of NAFLD and provide insight into the mechanism by which CE protects against obesity.

Pyrrolizidine alkaloids from Jacobaea vulgaris Gaertn and theoretical studies on intramolecular interactions.

Two undescribed pyrrolizidine alkaloids, 13-dehydrosenkirkine (1) and chloromethylretrorsine (2), along with three known analogues, onetine (3), retrorsine (4), and usaramine N-oxide (5), were isolated from Jacobaea vulgaris Gaertn. The structures of the undescribed compounds were elucidated by extensive spectrometric and spectroscopic techniques, including HRESIMS, NMR, calculated 13C-NMR DP4+ analysis and comparison with experimental and calculated ECD spectra. The undescribed compounds were evaluated for their antitumour activity against HT29, HeLa, and HepG2 cells. In addition, the intramolecular interactions and quadrupolar couplings were revealed by investigating the geometrical and electronic properties of three typical otonecine-type PAs in DFT theory.

Chlorine-containing guaianolide sesquiterpenoids from Achillea millefolium L. with inhibitory effects against LPS-induced NO release in BV-2 microglial cells.

Chlorine-containing guaianolide sesquiterpenoids are rare natural compounds that are mainly derived from plants of the Asteraceae family. Our previous investigations on the whole plant of Achillea millefolium L. resulted in the discovery of two chlorine-containing guaianolide sesquiterpenoids. In the present study, a LC‒MS tracking strategy based on the characteristic isotropic peak of atomic chlorine was employed to isolate nine undescribed chlorine-containing guaianolide sesquiterpenoids, Millefolactons B1-B9, from A. millefolium L.. The structures of these compounds were elucidated by spectroscopic data analysis, and the corresponding absolute configurations were determined by single-crystal X-ray crystallography and ECD data analysis. Millefolactons B2-B7 displayed inhibitory activities against LPS-induced NO release in BV-2 microglial cells.

Anti-Melanogenesis Effect of Polysaccharide from Saussurea involucrata on Forskolin-Induced Melanogenesis in B16F10 Melanoma Cells.

As one of the prominent medicinal plants listed in the Chinese pharmacopoeia (2020), Saussurea involucrata (Kar. et Kir.) Sch.-Bip was demonstrated to possess various therapeutic effects. In our recent research, we extracted the polysaccharides from S. involucrata (SIP) at optimal conditions and conducted further structure elucidation on the main fraction as well as the confirmation of its possible anti-inflammatory activity. Hence, in this work, we assessed the in vitro antioxidant activity and anti-melanogenesis effects of the crude SIP in forskolin-induced B16F10 melanoma cells. The results show that SIP possessed strong antioxidant activity and was effective in concentration-dependently decreasing melanin formation and inhibiting tyrosinase activity in forskolin-induced B16F10 cells. Based on these results, the inhibitory mechanism of melanogenesis was investigated by measuring Tyrosinase (TYR), Tyrosinase related protein-1 (TRP-1), Tyrosinase related protein-2 (TRP-2), Microphthalmia-associated transcription factor (MITF), cAMP-response element binding protein (CREB), mitogen-activated protein kinases (MAPK) signaling protein members, and ß-catenin degradation in forskolin-induced B16F10 cells. The anti-melanogenesis response of SIP might be attributed to the regulation of c-Jun N-terminal kinase (JNK) phosphorylation and ß-catenin degradation pathways. These results suggest that polysaccharides from S. involucrata possess a strong anti-melanogenic effect, and thus could be used as a high-value natural material for skin whitening in cosmeceutical industries.

Isolation, structure elucidation, and biological activity of polysaccharides from Saussurea involucrata.

The optimum extraction condition for the Saussurea involucrata polysaccharide (SIP) was determined to be a temperature of 80 °C, time 2 h, and a liquid-solid ratio of 30 mL/g with a yield of 11.37 %. An acidic homogenous polysaccharide, namely SIP-II was isolated from Saussurea involucrate through anion exchange and gel permeation column chromatography. The structure of the SIP-II was elucidated through the combination of HPLC, GC-MS, IC, peroxide oxidation, smith degradation, methylation, NMR analysis, it was mainly composed of arabinose, rhamnose, galactose, galacturonic acid, and glucose with the molar ratio of 19.85:20.30: 27.12:11.95:8.69 with a molecular weight of 237,570 Da. The glycosidic linkages of SIP-II mainly composed of →1)-α-L-Rhap-(2→, T-Araf, →1)-ß-D-GalpA-(4→, →1)-ß-D-Galp-(3,6→, →1)-ß-D-Galp-(6→, →1)-α-L-Rhap-(2,4→, T-Galp, and →1)-α-L-Araf-(5→. Meanwhile, the structures were characterized through extensive analysis of UV, FT-IR, SEM-EDX, CD, XRD, and TG. SIP-II possessed a remarkable anti-inflammatory activity by effectively inhibiting the expression of pro-inflammatory cytokines and inflammation-related mediators in LPS-stimulated RAW264.7 macrophages, and the anti-inflammatory response of SIP-II might be attributed to the regulation of the NF-κB, MAPK and JAK/STAT pathways. The results showed that polysaccharides from Saussurea involucrate could be a potential ingredient in the functional food and pharmaceutical industry.

Megastigmane sesquiterpenoids from whole plants of Viola kunawurensis.

Investigation on the chemical constituents of Viola kunawurensis resulted in the isolation of seven undescribed megastigmane sesquiterpenoids including four bicyclic megastigmane glucosides, kunawuronoside A-D, two megastigmane glucosides, kunawuronoside E-F, and a megastigmane, kunawurone A, together with ten known megastigmane sesquiterpenoids. Their structures were established by comprehensive 1D, 2D-NMR and HRESIMS analyses, and their absolute configurations were determined by comparing their calculated ECD data with the experimental ones. Evaluations of the anti-inflammatory activity revealed that kunawuronoside A-D and compounds 14-15 inhibited COX-2 expression with inhibition rates ranging from 36.7% to 58.5%, while the NO production induced by lipopolysaccharide (LPS) was suppressed by the kunawuronoside A-D in a dose-dependent manner in RAW264.7 macrophage cell line.

Phenolic glycosides from Nitraria sibirica leaves and their in vitro biological activities.

Seventeen phenolic glycosides were isolated from the Nitraria sibirica. Their structures were identified by the spectroscopic data and comparison with literatures as isovanillyl alcohol-7-O-ß-d-glucopyranoside (1), benzyl ß-primeveroside (2), benzyl-O-ß-d-glucopyranoside (3), 1-O-ß-d-glucopyranosyl-4-(8-hydroxyethyl)-2-methoxyphenyl (4), dehydrosyringin (5), trans-ferulic acid-4-O-ß-d-glucoside (6), cis-ferulic acid 4-O-ß-d-glucopyranoside (7), glucosyringic acid (8), 1-O-feruloyl-ß-d-glucoside (9), sachaloside VII (10), (3S, 5R, 6R, 7E, 9S)-megastigmane-7-ene-3-hydroxy-5,6-epoxy-9-O-ß-d-glucopyranoside(11), 3-hydroxy-4,5-dimethoxybenzyl alcohol (12), pinoresinol 4-O-ß-d-glucopyranoside (13), eucommin A (14), isoeucommin A (15), acanthoside (16), liriodendrin (17). All these compounds except compound 13 were isolated from the Nitraria genus for the first time. In bioactivity assays for all compounds, the compounds 8 and 15 were exhibited strong antioxidant activity (IC50 = 18.11 and 16.29 µM respectively), while compounds 3 and 11 were exhibited strong PTP1B enzymatic inhibition (IC50 = 6.97 and 11.76 µM, respectively). Furthermore, the compounds 10 and 17 were presented strong inhibitory capacities against Candida albicans (14.5 and 13.5 mm, respectively).

Quantitative Determination of Marker Compounds and Fingerprint Analysis of the Seeds of Vernonia anthelmintica.

In traditional Chinese medicine, the seeds of Vernonia anthelmintica (L.) Willd. have been widely used for treatment of cough, skin diseases, diarrhea, fever, schistosomiasis, amoebic dysentery, and gastrointestinal problems, especially in the treatment of vitiligo for thousands of years in China. In this study, an effective, reliable, and accurate high-performance liquid chromatography diode array detector (HPLC-DAD) method was developed for quantitative analysis of 3 marker bioactive compounds and chemical fingerprint of the seeds of V. anthelmintica. Data corresponding to common peak areas and HPLC chromatographic fingerprints were analyzed by exploratory hierarchical cluster analysis (HCA) and principal component analysis (PCA) to extract information of the most significant variables contributing to characterization and classification of the analyzed samples. Based on variety and origin, the high-performance thin layer chromatography (HPTLC) method validated the chemical fingerprint results used to screen the in vitro antioxidant activity of V. anthelmintica. The results show that the developed method has potential application values for the quality consistency evaluation and identification of similar instant V. anthelmintica samples. When considered collectively, this research results provide a scientific basis for the improvement of standardization and specification of V. anthelmintica medicinal materials and provide a pathway for the development and utilization of references for the identification of V. anthelmintica herbs.

Identification and quantification of Meiguihua oral solution using liquid chromatography combined with hybrid quadrupole-orbitrap and triple quadrupole mass spectrometers.

Meiguihua oral solution (MOS), a classical Chinese medicinal formula, was approved by the China Food and Drug Administration for production. However, the quality evaluation of MOS has not been reported. In this present study, qualitative and quantitative analysis of MOS were conducted by ultra high performance liquid chromatography coupled to quadrupole-orbitrap high resolution mass spectrometry (UHPLC-Q-orbitrap-HRMS) and high performance liquid chromatography-tri-quadrupole linear ion trap mass spectrometry (HPLC-tri-Q-LIT-MS). Totally 46 phenolic compounds (21 flavonoids and 25 tannins) were identified in the MOS, among them 14 polyphenols were not reported in raw plant materials of MOS. The simultaneous quantification of ten compounds including gallic acid, quercetin-3-O-sophoroside, ellagic acid, sophoraflavonoloside, hyperoside, isoquercitrin, avicularin, astragalin, quercitrin and juglanin, which were completed in 16 min in the negative electrospray ionization (ESI) mode under multiple reaction monitoring (MRM) method. Linearity was reached fine determination coefficient (r2 > 0.9995). Precisions, repeatability, stability (inter-day and intra-day), and recovery were validated and the relative standard deviations (RSD) were less than 2.9%, 4.7%, 3.6% and 1.79%, respectively. This result proved the high sensitivity and efficiency of the method. The quantitative and qualitative analysis of MOS would provide the substantial basis for further quality control and medicinal values.

Novel amides modified rupestonic acid derivatives as anti-influenza virus reagents.

In spired by the important role of amide groups of anti-influenza drugs oseltamivir, zanamivir and peramivir in bioactivity, a series of novel amides modified rupestonic acid derivatives were designed and synthesized. The absolute configuration of critical intermediate bearing chloride with newly formed stereocenter was confirmed by X-ray crystallographic analysis. And all new compounds were evaluated for their in vitro inhibitory activities against influenza A (H1N1 and H3N2) and influenza B viruses. The bioassay results showed that 5h with 4-fluorbenzylsulfonyl modified to 2 position of methyl rupestonate displayed the highest activity against influenza A (H1N1 and H3N2) viruses, even stronger than reference drugs oseltamivir and ribavirin (RVB), and might be recommended as a lead compound to further develop the new anti-influenza reagent.

Jatrophane Diterpenoids from Euphorbia glomerulans.

In a phytochemical investigation of the whole plant of Euphorbia glomerulans, 17 new (1-17) and five known jatrophane diterpenoids (18-22) were identified. The X-ray crystallographic data of compounds 1, 4, and 21 permitted the definition of the absolute configurations of these compounds. The cytotoxicity and multidrug resistance reversal activities of the 17 new compounds were evaluated on multidrug-resistant MCF-7/ADR cells overexpressing P-glycoprotein. Several compounds showed different chemoreversal activities and considerably decreased cytotoxicity. Compounds 11 (IC50 value of 5.0 ± 0.8 µM) and 12 (IC50 value of 5.2 ± 2.0 µM) possessed MDR reversal activities that were as good as that of verapamil (IC50 value of 4.7 ± 0.6 µM).

Multiomics analysis profile acute liver injury module clusters to compare the therapeutic efficacy of bifendate and muaddil sapra.

The pathogenesis of acute liver injury has been plagued by biologists and physicians. We know little about its therapeutic mechanism. Therefore, this study explored the mechanism of bifendate and muaddil sapra in the treatment of acute liver injury. Firstly, co-expression and cluster analysis of disease-related genes were carried out, and the Go function and KEGG pathway of modules and related genes were identified. Secondly, pivot analysis of modules can identify key regulators. On the other hand, based on the acute liver injury induced by CCl4, we use the combined analysis of proteomics and transcriptome to find therapeutic targets and related mechanisms of drugs. A total of 21 dysfunction modules were obtained, which were significantly involved in immune system, hepatitis and other related functions and pathways. Transcriptome analysis showed 117 targets for bifendate treatment, while 119 for muaddil sapra. Through exploring the mechanism, we found that the two drugs could modulate the module genes. Moreover, bifendate regulate the dysfunction module through ncRNA (SNORD43 and RNU11). Muaddil sapra can mediate dysfunction modules not only by regulating ncRNA (PRIM2 and PIP5K1B), but also by regulating TF (STAT1 and IRF8), thus having a wider therapeutic potential. On the other hand, proteome analysis showed that bifendate mainly regulated Rac2, Fermt3 and Plg, while muaddil sapra mainly regulated Sqle and Stat1. In addition, muaddil sapra regulates less metabolic related proteins to make them more effective. Overall, this study not only provides basic theory for further study of the complex pathogenesis of acute liver injury, but also provides valuable reference for clinical use of bifendate and muaddil sapra in the treatment of acute liver injury.

Evaluation of DNA barcode candidates for the discrimination of Artemisia L.

Because of the very similar morphologies and wide diversity of Artemisia L. varieties, they are difficult to identify, and there have been many arguments about the systematic classification Artemisia L., especially concerning the division of species. DNA barcode technology is used to rapidly identify species based on standard short DNA sequences. To evaluate seven candidate DNA barcodes (ITS, ITS2, psbA-trnH, rbcL, matK, rpoB, and rpoC1) regarding their ability to identify closely related species of the Artemisia genus in Xinjiang. The corresponding PCR amplification efficiency, detectable genetic divergence, identification efficiency and phylogenetic tree were assessed. We found that the internal transcribed spacer (ITS) region exhibited the highest interspecific divergence, which was significantly higher than the observed intraspecific variation and showed the highest identification efficiency, followed by ITS2, psbA-trnH and, finally, rpoB. matK, rbcL, and rpoC1 performed poorly in this evaluation. ITS, ITS2, and psbA-trnH were able to perfectly identify the tested species Artemisia annua, A. absinthium, A. rupestris, A. tonurnefortiana, A. austriaca, A. dracunculus, A. vulgaris, and A. macrocephala. Therefore, we propose the ITS, ITS2, and psbA-trnH regions as promising DNA barcodes for the closely related species of Artemisia L. in Xinjiang.

Manganese-mitigation of cadmium toxicity to seedling growth of Phytolacca acinosa Roxb. is controlled by the manganese/cadmium molar ratio under hydroponic conditions.

Manganese (Mn) can interact with cadmium (Cd) in environments and influence the toxic effect of Cd on plants. However, few studies have investigated the relationship between the Mn/Cd ratio and plant Cd-toxicity along Cd concentrations. In this paper, we studied the effects of external Mn/Cd molar ratios (0, 10, 30, 50 and 60) on Cd toxicity in the Mn hyperaccumulator and Cd tolerant plant, Phytolacca acinosa Roxb., at three Cd levels (50, 100 and 200 µM) under hydroponic conditions. Our result showed that seedling growth (y) under Cd stress was strongly positively related to the solution Mn/Cd molar ratio (SMCR). The relationship between the two variables under solution Cd concentrations was well explained by the linear regression model y=a+b1 (SMCR)+b2 (Solution-Cd). Increasing SMCR significantly reduced the Cd concentration and increased the Mn concentration in plant tissues. However, seedling growth was consistent with the shoot Mn/Cd molar ratio rather than with the Mn or Cd concentrations in plant tissues. At low levels of SMCR (e.g. 0 and 10), elevation of Mn distribution in shoot tissues might be a mechanism in P. acinosa seedlings to defend against Cd-toxicity. In comparison with low levels of SMCR, high levels of SMCR (e.g. 50 and 60) greatly alleviated lipid peroxidation and plant water-loss, and enhanced photosynthesis. However, the alleviated lipid peroxidation in the Mn-mitigation of Cd toxicity was likely to be the secondary effect resulting from the antagonism between Mn and Cd in the plant.

Phytochelatin synthase of Thlaspi caerulescens enhanced tolerance and accumulation of heavy metals when expressed in yeast and tobacco.

Phytochelatin synthase (PCS) is key enzyme for heavy metal detoxification and accumulation in plant. In this study, we isolated the PCS gene TcPCS1 from the hyperaccumulator Thlaspi caerulescens. Overexpression of TcPCS1 enhanced PC production in tobacco. Cd accumulation in the roots and shoots of TcPCS1 transgenic seedlings was increased compared to the wild type (WT), while Cd translocation from roots to shoots was not affected under Cd treatment. The root length of the TcPCS1 transgenic tobacco seedlings was significantly longer than that of the WT under Cd stress. These data indicate that TcPCS1 expression might increase Cd accumulation and tolerance in transgenic tobacco. In addition, the malondialdehyde content in TcPCS1 plants was below that of the wild type. However, the antioxidant enzyme activities of superoxide dismutase, peroxidase and catalase were found to be significantly higher than those of the WT when the transgenic plant was exposed to Cd stress. This suggests that the increase in PC production might enhance the Cd accumulation and thus increase the oxidative stress induced by the cadmium. The production of PCs could cause a transient decrease in the cytosolic glutathione (GSH) pool, and Cd and lower GSH concentration caused an increase in the oxidative response. We also determined TcPCS1 in Thlaspi caerulescens was regulated after exposure to various concentrations of CdCl(2) over different treatment times. Expression of TcPCS1 leading to increased Cd accumulation and enhanced metal tolerance, but the Cd contents were restrained by adding zinc in Saccharomyces cerevisiae transformants.

[Heavy metal absorption, transportation and accumulation mechanisms in hyperaccumulator Thlaspi caerulescens].

Thlaspi caerulescens, the famous model plant of heavy-metal hyperaccumulator, can uptake and accumulate large amount of heavy metals in its above-ground part of the plants. However, the very low biomass in Thlaspi caerulescens makes this plant unfit for direct application in phytoremediation. In recent years, there are many reports about the physiological and molecular characterization of Thlaspi caerulescens under heavy metals stresses, including absorption, transport and intracellular detoxification processes (e.g., chelation and compartmentation). Research teams have conducted many studies of chelators in plants, such as organ acid, amino acid, phytochelatins, metallothioneins and nicotianamine, and so on. Several transport protein families, such as Zinc Regulated Protein, Cation Diffusion Facilitator, Natural Resistance and Macrophage Protein and Heavy Metal ATPase, play important role in short/long distance transport in the plant. In this review, we summarize the current knowledge of the physiological and molecular mechanisms of heavy metals accumulation in Thlaspi caerulescens, with particular emphasis on the roles of transporters and chelatins in modulating plant heave-metal-stress responses.

[Progress in the plant GH3 gene family].

Phytohormone auxins play important roles in plant growth and development. The primary auxin-response genes can be classified into three major groups: Aux/IAAs, SAURs and GH3s. Significant progress has been made in understanding these gene families by approaches of the functional genomics, molecular genetics and molecular biology. In this review, we focused on the structures, functions and models of the expressional regulation of plant GH3 genes. The interactions in the signal transduction pathways between auxins and other signals mediated by the GH3 genes, the relationship between the GH3 genes and the stress adaptation responses of plants are emphasized.