CRISPR/Cas9-mediated knockout of NtMYC2a gene involved in resistance to bacterial wilt in tobacco.

MYC2 is a class of bHLH family transcription factors and a major regulatory factor in the JA signaling pathway, and its molecular function in tobacco has not been reported. In this study, CRISPR/Cas9-mediated MYC2 gene NtMYC2a knockout mutants at tobacco was obtained and its agronomic traits, disease resistance, and chemical composition were identified. Comparing with the WT, the leaf width of the KO-NtMYC2a was narrowed, the nornicotine content and mecamylamine content increased significantly and the resistance to Ralstonia solanacearum significantly decreased. The transcriptome sequencing results showed that DEGs related to immunity, signal transduction and growth and development were enriched between KO-NtMYC2a and WT. NtJAR1 and NtCOI1 in KO-NtMYC2a were down-regulated to regulating the JA signaling pathway, result in a significant decrease in tobacco's resistance to R. solanacearum. Our research provides theoretical support for the functional research of MYC2 and the study of the mechanism of tobacco bacterial wilt resistance.

Effects of Different Storage Times on the Stability of 12 Traditional Chinese Medicine Decoction Pieces.

As storage time increases, the quality of traditional Chinese medicines (TCMs) may change, and stability is an essential aspect of ensuring the safety and efficacy of TCMs. In this study, the effects of different storage times on the stability of 12 decoction pieces were evaluated. High-performance liquid chromatography was used to determine the contents of the active components in the 12 decoction pieces. The chemical composition data were analyzed using fingerprinting and clustering heatmap (CH). Results showed that during storage, significant variations (relative standard deviation > 10%) were observed in the levels of paeoniflorin in Paeoniae Radix Alba and Paeoniae Radix Rubra, hesperidin in Citri Reticulatae Pericarpium and Citri Reticulatae Pericarpium Viride, bufothionine in Siccus Bufo and chlorogenic acid in White Chrysanthemi Flos and Lonice Raejaponicae Caulis. However, calycosin-7-glucoside and calycosin in Astragali Radix Praeparata Cum Melle and chlorogenic acid in Lonicerae Japonicae Flos, Yellow Chrysanthemi Flos and Mori Folium were all <10%, which is consistent with the CH. Decoction pieces can be stored for up to six months, but it is recommended that volatile oil-containing and animal-based decoction pieces should not be stored for more than one month. This study provides new perspectives for the stability and quality control studies of TCM.

Overexpression of NtLHT1 affects the development of leaf morphology and abiotic tolerance in tobacco.

LYSINE HISTIDINE TRANSPORTER1 (LHT1) is a crucial broad-specificity and high-affinity amino acid transporter affecting the uptake of nitrogen and probably the tolerance to abiotic stress in plants. However, little is known about the phenotypic functions of LHT1 in plant growth and development and abiotic stress tolerance. In this study, we identified the NtLHT1 gene from the tobacco variety Honghuadajinyuan (HD) and determined its important roles in leaf morphological development and plant resistance to abiotic stress. Comprehensive functional analyses using knockout and overexpression transgenic lines (ntlht1 and OE) revealed overexpression of NtLHT1 accelerated leave senescence and increased plant height, leaf number and plant tolerance under cold, salt and drought stresses. In addition, NtLHT1 overexpression significantly decreased the leaf elongation of HD, causing the leaves to change from a long-elliptical shape to an elliptical shape. However silencing NtLHT1 decreased the seed germination rate under NaCl and PEG stresses. Moreover, NtLHT1 significantly affected the contents of various amino acids, such as the neutral, acidic, non-polar and aromatic amino acids, ethylene precursor (ACC), GA3 and IAA in tobacco. These results suggested that the amino acid and ethylene precursor ACC transport activities of NtLHT1 provide fine regulatory function for plant growth and development and plant tolerance to abiotic stress.

Knockout of CAFFEOYL-COA 3-O-METHYLTRANSFERASE 6/6L enhances the S/G ratio of lignin monomers and disease resistance in Nicotiana tabacum.

Background: Nicotiana tabacum is an important economic crop, which is widely planted in the world. Lignin is very important for maintaining the physiological and stress-resistant functions of tobacco. However, higher lignin content will produce lignin gas, which is not conducive to the formation of tobacco quality. To date, how to precisely fine-tune lignin content or composition remains unclear. Results: Here, we annotated and screened 14 CCoAOMTs in Nicotiana tabacum and obtained homozygous double mutants of CCoAOMT6 and CCoAOMT6L through CRSIPR/Cas9 technology. The phenotype showed that the double mutants have better growth than the wild type whereas the S/G ratio increased and the total sugar decreased. Resistance against the pathogen test and the extract inhibition test showed that the transgenic tobacco has stronger resistance to tobacco bacterial wilt and brown spot disease, which are infected by Ralstonia solanacearum and Alternaria alternata, respectively. The combined analysis of metabolome and transcriptome in the leaves and roots suggested that the changes of phenylpropane and terpene metabolism are mainly responsible for these phenotypes. Furthermore, the molecular docking indicated that the upregulated metabolites, such as soyasaponin Bb, improve the disease resistance due to highly stable binding with tyrosyl-tRNA synthetase targets in Ralstonia solanacearum and Alternaria alternata. Conclusions: CAFFEOYL-COA 3-O-METHYLTRANSFERASE 6/6L can regulate the S/G ratio of lignin monomers and may affect tobacco bacterial wilt and brown spot disease resistance by disturbing phenylpropane and terpene metabolism in leaves and roots of Nicotiana tabacum, such as soyasaponin Bb.

Gallic acid ameliorates dextran sulfate sodium-induced ulcerative colitis in mice via inhibiting NLRP3 inflammasome.

Background: Ulcerative colitis (UC) is a chronic recurrent inflammatory bowel disease (IBD). The conventional drugs for UC may induce severe side effects. Herbal medicine is considered as a complementary and alternative choice for UC. Purpose: This study aims to estimate the effect of natural polyphenol gallic acid (GA) on the NLRP3 inflammasome with dextran sulfate sodium (DSS)-induced colitis in mice. Study design: The body weights and symptoms of BALB/c mice were recorded. Histological evaluation, ELISA, q-PCR, immunohistochemistry, and western blotting were carried out to observe the morphology, cytokine contents, mRNA expressions, and protein expressions, respectively. Lipopolysaccharide (LPS)-induced RAW264.7 macrophage was used to probe GA's effect on relative protein expression. Results: GA attenuated weight loss (p < 0.05), relieved symptoms, and ameliorated colonic morphological injury (p < 0.05) in mice with colitis induced by DSS. GA also lowered the contents of TNF-α, IL-1ß, IL-18, IL-33, and IFN-γ in the serum and colon of mice, which were elevated by DSS, downregulated protein, and mRNA expressions of the NLRP3 pathway in the colon tissue. Furthermore, GA downregulated the expressions of NLRP3 (p < 0.05), iNOS (p < 0.01), COX2 (p < 0.01), and P-p65 (p < 0.05), and suppressed NO release (p < 0.001) in LPS-induced RAW264.7 cells. Conclusion: GA ameliorated DSS-induced UC in mice via inhibiting the NLRP3 inflammasome. These findings furnish evidence for the anti-inflammatory effect of herbal medicines containing GA on UC.

GC-MS combined with multivariate analysis for the determination of the geographical origin of Elsholtzia rugulosa Hemsl. in Yunnan province.

Elsholtzia rugulosa Hemsl., a Chinese herbal medicine, may have the potential to treat COVID-19. The geographical origin has a significant influence on the quality and application of E. rugulosa. In this paper, gas chromatography-mass spectrometry (GC-MS) combined with principal component analysis (PCA) and hierarchical cluster analysis (HCA) and other multivariate statistical analyses were performed for the identification of E. rugulosa. origins. The results showed that the volatile components of E. rugulosa. from different origins were significantly different. PCA and HCA can clearly distinguish the E. rugulosa of Lijiang and Fumin, and Dali and Yongsheng can be distinguished but with a certain overlap. The correlation of different components of was investigated by Pearson correlation. The results showed that E. rugulosa. characteristic component Elsholtzia ketone is regulated by terpenoid metabolism. The discriminant functions of different origins are constructed by Fisher stepwise discrimination, and its initial verification accuracy and leave-one-out cross-validation accuracy were 100% and 87.5%, respectively.

The Agronomic Traits, Alkaloids Analysis, FT-IR and 2DCOS-IR Spectroscopy Identification of the Low-Nicotine-Content Nontransgenic Tobacco Edited by CRISPR-Cas9.

In this study, the agricultural traits, alkaloids content and Fourier transform infrared spectroscopy (FT-IR) and two-dimensional correlation infrared spectroscopy (2DCOS-IR) analysis of the tobacco after Berberine Bridge Enzyme-Like Proteins (BBLs) knockout were investigated. The knockout of BBLs has limited effect on tobacco agricultural traits. After the BBLs knockout, nicotine and most alkaloids are significantly reduced, but the content of myosmine and its derivatives increases dramatically. In order to identify the gene editing of tobacco, principal component analysis (PCA) was performed on the FT-IR and 2DCOS-IR spectroscopy data. The results showed that FT-IR can distinguish between tobacco roots and leaves but cannot classify the gene mutation tobacco from the wild one. 2DCOS-IR can enhance the characteristics of the samples due to the increased apparent resolution of the spectra. Using the autopeaks in the synchronous map for PCA analysis, we successfully identified the mutants with an accuracy of over 90%.

Beware of the Potential Risks for Polygoni Multiflori Caulis-Induced Liver Injury.

Background: Reynoutria multiflora (Thunb.) Moldenke (PM) is a widely-used medicinal plant in China, whose root and stem are included in the Chinese Pharmacopoeia as Polygoni Multiflori Radix (RPM), Polygoni Multiflori Radix Preparata (PMP), and Polygoni Multiflori Caulis (PMC). The hepatotoxicity of RPM and PMP is concerned by the public, while the risk of PMC is ignored. Purpose: Here, we investigate the potential risks for PMC-induced liver injury from clinical, chemical, and animal features. Study design: First, we analyzed the 12-month usage of RPM, PMP, and PMC in Longhua Hospital. Second, we determined the contents of gallic acid, cis-2,3,5,4'-tetrahydroxy-stilbene-2-O-ß-D-glucoside (cis-SG), trans-2,3,5,4'-tetrahydroxy-stilbene-2-O-ß-D-glucoside (trans-SG), emodin-8-O-ß-D-glucoside (EG), physcion-8-O-ß-D-glucoside (PG), emodin, and physcion in the water extracts from 15 batches of RPM, PMP, and PMC. Third, we probed the hepatotoxic effect of RPM, PMP, and PMC in mice and explored the mechanism of cis-SG and trans-SG causing the liver injury at the dosages based on our results from the first and second parts. Results: PMC had nearly five times the amount of usage in both outpatient prescriptions and inpatient orders than RPM and PMP. Overall, 68% dosage of PMC was 30 g. The contents of cis-SG, trans-SG, and emodin in PMC water extracts were significantly lower than those in RPM and PMP water extracts. PMC induced milder idiosyncratic liver injury for its lower content of cis-SG and trans-SG than its root counterparts. Conclusion: The potential risks for PMC-induced liver injury should be fully aware of.

Fingerprinting and Determination of Hepatotoxic Constituents in Polygoni Multiflori Radix Praeparata of Different Producing Places by HPLC.

Polygoni Multiflori Radix Praeparata (PMRP) is used as Chinese herbal medicine with long history. However, reports about PMRP hepatotoxicity have increased recently, and producing area might be one reason. This article aims to figure out the relationship between producing area and hepatotoxic ingredients in PMRP. HPLC fingerprint for PMRP was established and the contents of gallic acid, trans-stilbene glycoside (TSG), emodin-8-O-ß-D-glucoside (EG), emodin and physcion were determined. Clustering heatmap was implemented by TCMNPAS software，and principal component analysis was implemented by SPSS and SIMCA-P software. Hepatotoxic constituents' contents of PMRP from separate producing area were different. PMRP from Guangxi had the highest content of gallic acid, TSG, EG, emodin and physcion, followed by Hubei, Guangdong, Guizhou, Yunnan. PMRP from Henan had the lowest contents of hepatotoxic components. Hepatotoxic components' contents of PMRP in southern were higher than central China. This study carried out a preliminary qualitative and quantitative investigation on the PMRP from different producing places, which provided a basis for safe medication of PMRP.

Influence of cigarette smoking on oral microbiota in patients with recurrent aphthous stomatitis.

Recurrent aphthous stomatitis (RAS) is a common recurrent ulcerative disease of the oral mucosa which is closely related to oral microbial composition. However, the specific effect and the mechanism of smoking in RAS are unclear. In this study, 16S rRNA sequencing technology was used to compare the differences in saliva microbial community between 28 non-smoking healthy controls (NSctrl), 31 non-smoking RAS patients (NSras), and 19 smoking RAS patients (Sras). The results showed that the bacterial community diversity in patients with RAS (NSras and Sras) was lower than that of NSctrl. The microbial community in smoking-associated RAS is less diverse and distinct from that of non-smokers. The RAS groups have higher abundance of Veillonella, Rothia, and Sneathia and lower abundance of Bacteroidales, Bacteroides, Wolinella, Moryella, Pyramidobacter, and Christensenellaceae at the genera level. A significantly different abundance of Anaerovorax, Candidatus Endomicrobium, Lactococcus, Sneathia, Veillonella, and Cloacibacterium was observed between the Sras and the NSras group. Notably, there was a significant difference in many species from the genus Prevotella and Treponema between the NSras and the Sras group. Further, the relative abundance of several taxa is correlated with smoking age or frequency, including Megasphaera, Haemophilus, Leptotrichia, and Rothia at the genera level, and Prevotella melaninogenica, Prevotella salivae, Megasphaera micronuciformis, Haemophilus parainfluenzae, Alloprevotella tannerae, Actinomyces naeslundii, Lautropia mirabilis, and Capnocytophaga sputigena at the species level. Among patients with RAS, smoking aggravated the pathways of respiration and human pathogens. Our results suggest that smoking is closely related to changes in the oral microbiota, which may contribute an opposite effect to the pathogenesis of RAS. This study provides new insight and theoretical basis for the cause and pathogenesis of RAS and better prevention and treatment.

Effects of Qing Chang Suppository Powder and its Ingredients on IL-17 Signal Pathway in HT-29 Cells and DSS-induced Mice.

BACKGROUND: Qingchang Suppository, a formula used for more than 30 years in Longhua Hospital, has shown satisfactory clinical effects on Ulcerative Colitis (UC). However, its therapeutic mechanism has not been fully elucidated. PURPOSE: The study aims to investigate the effects of Qingchang Suppository powder (QCSP) and its ingredients by regulating the IL-17A signaling pathway which plays an important role in the development of UC. METHODS: HPLC was used to analyze the main ingredients (Gallic acid, Indigo, Indirubin) in QCSP. HT-29 cells were induced by rhIL-17A and TNF-α, and IL-17A related protein expressions were determined by western blot. BALB/C mice were induced by 4% Dextran Sodium sulfate (DSS). The effects of QCSP and its ingredients were evaluated by measuring weight loss, disease activity index (DAI), colon length, histological analysis. Western blot was used for analysis of IL-17A and MAPK related proteins p-ERK, p-JNK, p-P38. Quantitative reverse transcription polymerase chain reaction (q-PCR) was used to detect the expression of IL-17A, HSP90 and ACT1 in colon tissue. Cytokines such as IL-17A, IL-1ß, IFN-γ and TNF-α were determinated by enzyme-linked immunosorbent assay (ELISA). RESULTS: QCSP had good therapeutic effect on DSS-induced colitis in mice. QCSP significantly relieved weight loss, restored colon length, repaired colon lesions, reduced histological scores and DAI, decreased TNF-α, IL-1ß, IL-17 and IFN-γ contents, significantly suppressed the gene expressions of IL-17A, ACT1 and HSP90, and up-regulated the expressions of tight junction proteins like ZO-1 and Occludin. IL-17A pathway related proteins such as IL-17A, IL-17RA, HSP90, MAPKs, P-iκbα and iNOS were significantly increased in vitro and in vivo. CONCLUSIONS: This paper reveals that QCSP inhibited the IL-17A signaling pathway in HT-29 cells and DSS induced mice, presenting a new mechanism of QCS on treating UC.

Bufothionine induces autophagy in H22 hepatoma-bearing mice by inhibiting JAK2/STAT3 pathway, a possible anti-cancer mechanism of cinobufacini.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinobufacini is extracted from the skins and parotid venom glands of the toad for treating symptoms like swelling and pain in ancient times. Nowadays, cinobifucini injection has also achieved satisfactory therapeutic effects on hepatocellular carcinoma (HCC) in China. AIM OF THE STUDY: Our previous work found that bufothionine, an alkaloid abundant in cinobufacini injection, induced mitochondria-mediated apoptosis. In this work, the underlying effects of bufothionine on autophagy in HCC and its possible dependent pathway were investigated. METHODS: CCK-8 and Hoechst staining assays were performed to verify effects of drugs on proliferation and apoptosis of SMMC7721 cell. H22-tumor-bearing mice model was established by inoculating ascites fluid. HE staining was used to observe pathological changes in liver and tumor tissues. ELISA and Western blot experiments were conducted to investigate IL-6/JAK2/STAT3 signaling pathway. The effects of drugs on expressions of autophagic relative proteins were investigated by Western blot in vitro and in vivo. RESULTS: In vitro, CCK-8 and Hoechst staining assays showed that bufothionine inhibited SMMC7721 cell proliferation and promoted apoptosis at 100 µM. In vivo, bufothionine relieved symptoms of H22-tumor-bearing mice and exerted anti-inflammation activity. ELISA and Western blot demonstrated that bufothionine significantly reduced serum IL-6 concentration, suppressed p-Stat3tyr705, p-Stat3ser727 and Jak2 expressions in tumor tissues and upregulated Atg5, Atg7 and LC3Ⅱ expressions in SMMC7721 cell and H22 tumor. CONCLUSION: This is the first report showing that bufothionine might induce autophagy in HCC by inhibiting JAK2/STAT3 pathway, presenting a possible anti-cancer mechanism of bufothionine in cinobufacini injection.

Three new diphenyl ether derivatives from the fermentation products of an endophytic fungus Phomopsis fukushii.

Three new diphenyl ethers (1-3), together with four known isopentylated diphenyl ethers derivatives (4-7), were isolated from the fermentation products of an endophytic fungus Phomopsis fukushii. Their structures were elucidated by spectroscopic methods, including extensive 1D and 2D NMR techniques. Compounds 1-3 were evaluated for their anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) activity. The results revealed that compounds 1 and 2 showed strong inhibitions with inhibition zone diameters (IZD) of 20.2 ± 2.5 mm and 17.9 ± 2.2 mm, respectively. Compound 3 also showed good inhibition with IZD 15.2 ± 1.8 mm. The IZD data of compound 1 is close to that of positive control with IZD 21.9 ± 2.1 mm.

[A new isoflavone derivative from Rosa Damascena and its antibacterial activity].

A new isoflavone derivative was isolated from Rosa damascena by using various chromatographic techniques including silica gel, Sephadex LH-20, and preparative RP-HPLC separation. Its structure was identified as 4'-hydroxy-7-(3-hydroxypropanoyl)-6-methoxy-isoflavone using combined examinations of their UV, IR, MS, and NMR spectroscopic data. Biological activity test showed that this compound showed prominent antibacterial activity with MIC90 value of (46±4) mg·L⁻¹ for methicillin resistant Staphylococcus aureus(MRSA) strain. This value is close to that of levofloxacin [with MIC90 value (53±5) mg·L⁻¹].

Isopentylated diphenyl ether derivatives from the fermentation products of an endophytic fungus Phomopsis fukushii.

Three new isopentylated diphenyl ethers, (1-3), together with two known isopentylated diphenyl ethers derivatives (4 and 5) were isolated from the fermentation products of an endophytic fungus Phomopsis fukushii. Their structures were elucidated by spectroscopic methods, including extensive 1D- and 2D NMR techniques. Compounds 1-3 were evaluated for their anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) activity. The results showed that compounds 1-3 showed strong activity with diameter of inhibition zone (IZD) of 21.8 ± 2.4 mm, 16.8 ± 2.2 mm, and 15.6 ± 2.0 mm, respectively.

[A new naphthalene derivative from Aloe vera and its antibacterial activity].

A new naphthalene derivative has been isolated from Aloe vera by using various chromatographic techniques, including silica gel, sephadex, MCI-gel resin, and RP-HPLC. The new compound was determined as 3-hydroxy-1-(1,7-dihydroxy-3,6-dimethoxynaphthalen-2-yl)propan-1-one (1). In the biological activity assay, compound 1 disglayed prominent antibacterial activity with a MIC90 value of (48±4) mg•L⁻¹ for methicillin resistant Staphylococcus aureus (MRSA) strain which was stronger than that of the positive control levofloxacin with a MIC90 value (58±5) mg•L⁻¹.