Establishment of a multicomponent quality control method and the transfer characteristics of five markers from Qidongning Formula to rat tissues by HPLC-QQQ-MS/MS.

Introduction: Traditional Chinese medicine compound preparations have become an increasingly utilized strategy for tumour treatment. Qidongning Formula (QDN) is a kind of antitumour compound preparation used in hospitals, and it can inhibit the growth of lung cancer cells. However, due to the complexity of botanical drugs, the quality evaluation of QDN is inconsistent, affecting clinical efficacy and posing potential safety risks for clinical application. Additionally, tissue distribution is an integral part of the drug development process. Methods: To study the distribution characteristics of markers in compound preparations and rat tissues, a novel HPLC-QQQ-MS/MS quantitative analytical method was established to determine five markers in QDN simultaneously, and the method was verified. Results and discussion: The analytical results showed that the contents of salidroside (51.6 ± 5.75 µg/g), calycosin-7-O-ß-D-glucoside (94.2 ± 15.4 µg/g), specnuezhenide (371 ± 72.5 µg/g), formononetin (23.8 ± 5.39 µg/g), and polyphyllin I (87.7 ± 10.6 µg/g) were stable in different batches of QDN. After intragastric administration (13.5 g/kg) in rats for 1 h, four markers in the QDN, except polyphyllin I, were distributed in most tissues. QDN was distributed chiefly in the stomach and small intestine, followed by the liver or kidney. The study also found that specnuezhenide had the highest concentration in both QDN and rat tissues (102 ± 22.1 µg/g in the stomach), while formononetin had the highest transfer rate (0.351%) from QDN to rat intestines. The above research lays a quality research foundation for the antitumour application of QDN and provides a scientific reference for the quality control of Chinese medicine compound preparations.

Investigation of the fall armyworm (Spodoptera frugiperda) gut microbiome and entomopathogenic fungus-induced pathobiome.

The gut microflora plays an important role in insect development and physiology. The gut bacterial microbiome of the fall armyworm (FAW), Spodoptera frugiperda, in both cornfield and laboratory-reared populations was investigated using a 16S metagenomic approach. The alpha- and beta-diversity of the cornfield FAW populations varied among sampling sites and were higher than those of the laboratory-reared FAW population, indicating that different diets and environments influence the gut bacterial composition. To better understand the interaction between the microbiome and entomopathogenic fungi (EPF), FAWs from organic and conventionally managed corn fields and from the laboratory-reared colony were inoculated with Beauveria bassiana NCHU-153 (Bb-NCHU-153). A longer median lethal time (LT50) was observed in the Bb-NCHU-153-infected cornfield FAW population than in the laboratory-reared FAWs. In terms of the microbiome, three Bb-NCHU-153-infected FAW groups showed different gut bacterial compositions compared to noninfected FAW. Further investigation of the cooccurrence network and linear discriminant analysis (LDA) of effect size (LEfSe) revealed that the enriched bacterial genera, such as Enterococcus, Serratia, Achromobacter, and Tsukamurella, in the gut might play the role of opportunistic pathogens after fungal infection; in contrast, some gut bacteria of Methylobacterium, Marinomonas, Paenochrobactrum, Pseudomonas, Acinetobacter, Delftia, Dietzia, Gordonia, Leucobacter, Paracoccus, and Stenotrophomonas might be probiotics against EPF infection. These results indicated that EPF infection can change the gut bacterial composition and lead to a pathobiome in the FAW and that some bacterial species might protect the FAW from EPF infection. These findings could be applied to the design of pathobiome-inducing biocontrol strategies.

Iristectorigenin C suppresses LPS-induced macrophages activation by regulating mPGES-1 expression and p38/JNK pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Nonsteroidal anti-inflammatory drugs (NSAIDs) have been used clinically to treat inflammatory diseases clinically. However, the adverse effects of NSAIDs cannot be ignored. Therefore, it is critical for us to find alternative anti-inflammatory drugs that can reduce adverse reactions to herbal medicine, such as Iris tectorum Maxim., which has therapeutic effects and can treat inflammatory diseases and liver-related diseases. AIM OF THE STUDY: This study aimed to isolate active compounds from I. tectorum and investigate their anti-inflammatory effects and action mechanisms. MATERIALS AND METHODS: Fourteen compounds were isolated from I. tectorum using silica gel column chromatography, Sephadex LH-20, ODS and high performance liquid chromatography, and their structures were identified by examining physicochemical properties, ultraviolet spectroscopy, infrared spectroscopy, mass spectrometry, and nuclear magnetic resonance spectroscopy. Classical inflammatory cell models were established using lipopolysaccharide (LPS)-stimulated RAW264.7 cells and rat primary peritoneal macrophages to examine the effect of these compounds. To examine the action mechanisms, the nitric oxide (NO) levels were measured by Griess reagent and the levels of inflammatory cytokines in the supernatant were measured by ELISA; The expressions of major proteins in prostaglandin E2 (PGE2) synthesis and the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways were examined by Western blotting, and the mRNA expression levels were measured by quantitative real-time polymerase chain reaction; and the nuclear translocation of p65 was examined by high content imaging. Molecular docking was used to predict the binding of active compound to target protein. RESULTS: Our findings revealed that Iristectorigenin C (IT24) significantly inhibited the levels of NO and PGE2 without affecting cyclooxygenase (COX)-1/COX-2 expression in LPS-induced RAW264.7 cells and rat peritoneal macrophages. Furthermore, IT24 was shown to decrease the expression of microsomal prostaglandin synthetase-1 (mPGES-1) in LPS-induced rat peritoneal macrophages. IT24 did not suppress the phosphorylation and nuclear translocation of proteins in the NF-κB pathway, but it inhibited the phosphorylation of p38/JNK in LPS-stimulated RAW264.7 cells. Additionally, molecular docking analysis indicated that IT24 may directly bind to the mPGES-1 protein. CONCLUSION: IT24 might inhibit mPGES-1 and the p38/JNK pathway to exert its anti-inflammatory effects and could be also developed as an inhibitor of mPGES-1 to prevent and treat mPGES-1-related diseases, such as inflammatory diseases, and holds promise for further research and drug development.

The Metabolic Pathways and Products of Ten Aconitum Alkaloids in Sanwujiao Pills from Eight Organs of Mice by UHPLC-Q-TOF-MS/MS.

BACKGROUND: Sanwujiao pill (SWJP) is a Chinese herbal preparation widely used in China. It is an essential medicine for treating rheumatism and blood stasis. However, its safety in clinical use has always been the focus of patients because it contains toxic herbs of Aconitum carmichaelii Debx. and A. vilmorinianum Kom. OBJECTIVE: To further reveal the pharmaceutical and toxic effect substances and the action mechanism of SWJPs, the metabolites and their pathways of ten Aconitum alkaloids (AAs) in the preparation at different time points after oral administration in eight organs of mice were investigated. METHOD: The biosamples were investigated by a four-step strategy of UPLC-Q-TOF-MS /MS technology. RESULTS: Aconitine (AC), mesaconitine (MA), and hypaconitine (HA) were not detected in any organs. The highest concentrations of the other seven AAs occurred at 0.5 h. Yunaconitine (YAC) was not detected in the brain; all seven AAs had the lowest concentration in the brain, and the metabolism was slow in the stomach. Twelve predicted metabolites were identified, the kidney and stomach were their primary distribution locations, and the most metabolites were found at 0.5h. The main metabolic pathways of the ten AAs were demethylation, deethylation, deoxygenation, hydroxylation, and deacetylation. CONCLUSION: This is the first report about the metabolism of ten AAs in SWJPs in mice. Significantly, the metabolic pathways and products of four hidden toxic AAs were analyzed in vivo for the first time. The results were of great significance for the safety and effectiveness of SWJPs in clinical application.

Synergistic antibacterial effects of 5-fluorouracil or thioridazine in combination with phytopolyphenols on cultured Porphyromonas gingivalis.

Background/purpose: 5-Fluorouracil (5FU) is a commonly used anticancer drug. However, the severe oral mucositis induced by 5FU in about 60-70% of patients was a major cause of discontinuous therapy. Since oral dysbiosis induced by 5FU was well correlated with severity of oral mucositis and Porphyromonas gingivalis (P.g.) was a keystone pathogen of dysbiosis. Thus, in this study, we aimed to explore the novel regimens of 5FU combined with phytopolyphenols (curcumin, green tea polyphenols) as well as ZnSO4 on antibacterial effects of cultured P.g. growth. In addition, similar regimens containing thioridazine (TRZ) were also tested for their antibacterial efficacy. Materials and methods: The synergistic (Combination Index (CI) < 1) antiproliferation and anti-protease efficacies (IC50) of novel regimens on cultured P.g. were evaluated by OD600 and colorimetric method respectively. Results: The results obtained indicated that both novel regimens of 5FU and TRZ exhibited potent synergistic antibacterial effects against growth and protease of P.g. Conclusion: These novel regimens of 5-FU and TRZ were potent antibacterial agents which merit for further preclinical and clinical trials in management of oral mucositis, cancers and infectious diseases.

First Report of Target Spot Caused by Rhizoctonia solani AG-5 on Tobacco in China.

Tobacco is one of the vital economic crops in China. Nevertheless, tobacco diseases cause substantial economic losses each year. Tobacco target spot is a fungal disease which commonly found on the leaves. While both sexual and asexual reproduction can occur, asexual reproduction is much more common in tobacco. In June 2022, target spot was found on tobacco leaf samples from Yibin, Sichuan Province and Meitan, Guizhou Province, China. The typical symptoms were light brown tissue with concentric ring marks, and the necrotic part of the disease spot was fragile and forming perforation after falling off. The diseased tissue in the sample was cut off and sterilized in 75% ethanol for 1 min, and rinsed three times in sterilized distilled water. Finally, the tissues were placed on potato glucose agar (PDA) medium with kanamycin (0.1 mg/mL). After incubation at 28 °C in darkness for 3 days,the culture of the isolate grew in the form of radial mycelium on PDA dishes, the mycelium was white initially, turned brown generally at the later stage, and finally thickened and separated with the growth of the culture. Nine pathogenic strains were isolated, including four isolates from Yibin and five from Meitan. They were all used for pathogen identification. Genomic DNA of each isolate was extracted using the CATB method, and PCR analysis was performed with primers specifically designed to detect individual fusion groups or fusion subgroups of solani: AG-1 IA, IB, and IC; AG-3 PT; AG-4 HG-I, HG-II and HG-III; AGs-5-6 and P-21-22. Among the 11 specific primer pairs, only AG-5-specific primer amplified the fungal DNA, indicating that the nine isolates tested all belonged to the R. solani AG-5 fusion group. BLASTn search was performed on the gene sequences obtained from these strains and they deposited in GenBank under accession no. OP647851-OP647859. These gene sequences were aligned with the voucher specimen R. solani AG-5, with more than 99% similarity . The nine isolates were then tested for mycelial anastomosis reactions using the R. solani AG-5 standard strain following the method described by Ogoshi (1987). A decrease in the diameter of the mycelia at the anastomosis site and death of adjacent cells were observed, indicating their anastomosis response. Therefore, these nine strains were identified as R. solani AG-5 based on morphological and genetic analysis. Subsequently, one pathogenic strain from Meitan and another one from Yibin were selected for pathogenicity verification. Mycelial PDA blocks (6 mm in diameter) of the two isolates were inoculated on healthy tobacco plants, while leaves containing only PDA blocks were used as controls. A total of 6 replicates were conducted. After inoculation, they were incubated at 85% relative humidity and 15 to 25 °C. Koch's hypothesis was confirmed by reisolating pathogens from diseased leaves 5 days after inoculation. Typical symptoms were observed on tobacco plants inoculated with the pathogen strains but not on control tobacco plants. To the best of our knowledge, tobacco target spot has been reported caused by R. solani AG-3, AG-6 and AG-2.1 groups in the field in China and in Argentina. Up until now, this is the first report of R. solani AG-5 causing tobacco target spot on tobacco in the field in China. It was also found to be highly virulent to chickpea in Turkey. Due to serious damages caused by this disease in the last five years in China, more attention should be paid in disease control measures to avoid economic losses. In addition, it also provides some theoretical help for the damage caused by this pathogen on other hosts and helps people to better understand Rhizoctonia solani AG-5.

Simultaneous determination of twenty-nine active compounds in fuzhengjiedu granules by HPLC-QQQ-MS/MS.

As an empirical medicine of traditional Chinese medicine, Fuzhengjiedu Granules have shown an effect against COVID-19 in clinical and inflammatory animal models. It is formulated with eight herbs, including Aconiti Lateralis Radix Praeparata, Zingiberis Rhizoma, Glycyrrhizae Radix Et Rhizoma, Lonicerae Japonicae Flos, Gleditsiae Spina, Fici Radix, Pogostemonis Herba, and Citri Reticulatae Pericarpium. This study established a high-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-QQQ-MS/MS) method by simultaneously determining 29 active compounds in the granules with significant content differences. Separation by gradient elution using acetonitrile and water (0.1% formic acid) as mobile phases was performed on a Waters Acquilty UPLC T3 column (2.1 mm × 100 mm, 1.7 µm). A triple quadrupole mass spectrometer, operating in positive and negative ionization modes, was used for multiple reaction monitoring to detect the 29 compounds. All calibration curves showed good linear regression (r2 > 0.998). RSDs of precision, reproducibility, and stability of active compounds were all lower than 5.0%. The recovery rates were 95.4-104.9%, with RSDs< 5.0%. This method was successfully used to analyze the samples, and the results showed that 26 representative active components from 8 herbs were detected in the granules. While aconitine, mesaconitine, and hypaconitine were not detected, indicating that the existing samples were safe. The granules had the maximum and minimum content of hesperidin (27.3 ± 0.375 mg/g) and benzoylaconine (38.2 ± 0.759 ng/g). To conclude, a fast, accurate, sensitive, and reliable HPLC-QQQ-MS/MS method was established, which can simultaneously detect 29 active compounds that have a considerable difference in the content of Fuzhengjiedu Granules. This study can be used to control the quality and safety of Fuzhengjiedu Granules and provide a basis and guarantee for further experimental research and clinical application.

Causal relationships between gut microbiota and programmed cell death protein 1/programmed cell death-ligand 1: A bidirectional Mendelian randomization study.

Background: Multiple clinical studies have indicated that the gut microbiota influences the effects of immune checkpoint blockade (ICB) therapy comprising PD-1/PD-L1 inhibitors, but the causal relationship is unclear. Because of numerous confounders, many microbes related to PD-1/PD-L1 have not been identified. This study aimed to determine the causal relationship between the microbiota and PD-1/PD-L1 and identify possible biomarkers for ICB therapy. Method: We used bidirectional two-sample Mendelian randomization with two different thresholds to explore the potential causal relationship between the microbiota and PD-1/PD-L1 and species-level microbiota GWAS to verify the result. Result: In the primary forward analysis, genus_Holdemanella showed a negative correlation with PD-1 [ßIVW = -0.25; 95% CI (-0.43 to -0.07); PFDR = 0.028] and genus_Prevotella9 showed a positive correlation with PD-1 [ßIVW = 0.2; 95% CI (0.1 to 0.4); PFDR = 0.027]; order_Rhodospirillales [ßIVW = 0.2; 95% CI (0.1 to 0.4); PFDR = 0.044], family_Rhodospirillaceae [ßIVW = 0.2; 95% CI (0 to 0.4); PFDR = 0.032], genus_Ruminococcaceae_UCG005 [ßIVW = 0.29; 95% CI (0.08 to 0.5); PFDR = 0.028], genus_Ruminococcus_gnavus_group [ßIVW = 0.22; 95% CI (0.05 to 0.4); PFDR = 0.029], and genus_Coprococcus_2 [ßIVW = 0.4; 95% CI (0.1 to 0.6); PFDR = 0.018] were positively correlated with PD-L1; and phylum_Firmicutes [ßIVW = -0.3; 95% CI (-0.4 to -0.1); PFDR = 0.031], family_ClostridialesvadinBB60group [ßIVW = -0.31; 95% CI (-0.5 to -0.11), PFDR = 0.008], family_Ruminococcaceae [ßIVW = -0.33; 95% CI (-0.58 to -0.07); PFDR = 0.049], and genus_Ruminococcaceae_UCG014 [ßIVW = -0.35; 95% CI (-0.57 to -0.13); PFDR = 0.006] were negatively correlated with PD-L1. The one significant species in further analysis was species_Parabacteroides_unclassified [ßIVW = 0.2; 95% CI (0-0.4); PFDR = 0.029]. Heterogeneity (P > 0.05) and pleiotropy (P > 0.05) analyses confirmed the robustness of the MR results.

Whole-genome DNA methylome analysis of different developmental stages of the entomopathogenic fungus Beauveria bassiana NCHU-157 by nanopore sequencing.

The entomopathogenic fungus (EPF), Beauveria bassiana, is an important and commonly used EPF for microbial control. However, the role of DNA methylation has not been thoroughly studied. Therefore, the whole genomic DNA methylome of one promising EPF isolate, B. bassiana NCHU-157 (Bb-NCHU-157), was investigated by Oxford Nanopore Technologies (ONT). First, the whole genome of Bb-NCHU-157 was sequenced by next-generation sequencing (NGS) and ONT. The genome of Bb-NCHU-157 contains 16 contigs with 34.19 Mb and 50% GC content, which are composed of 10,848 putative protein-coding genes. Two putative DNA methyltransferases (DNMTs) were found, including Dim-2 and C-5 cytosine-specific DNA methylases. Both DNMTs showed higher expression levels in the mycelium stage than in the conidia stage, indicating that development of DNA methylation in Bb-NCHU-157 might occur in the mycelium stage. The global methylation level of the mycelium stage (5 mC = 4.56%, CG = 3.33%, CHG = 0.74%, CHH = 0.49%) was higher than that of the conidial stage (5 mC = 2.99%, CG = 1.99%, CHG = 0.63%, CHH = 0.37%) in both the gene and transposable element (TE) regions. Furthermore, the TE regions showed higher methylation frequencies than the gene regions, especially for CHH site methylation, suggesting regulation of genomic stabilization during mycelium development. In the gene regions, high methylation frequencies were found around the transcription start site (TSS) and transcription end site (TES). Moreover, CG and CHG methylation mainly occur in the promoter and intergenic regions, while CHH methylation occurs in the TE region. Among the methylated regions, 371, 661, and 756 differentially DNA methylated regions (DMRs) were hypermethylated in the mycelium in CG, CHG, and CHH, while only 13 and 7 DMRs were hypomethylated in the mycelium in CHG, and CHH, respectively. Genes located in the DMR shared the GO terms, DNA binding (GO: 0003677), and sequence-specific DNA binding (GO: 0043565) for hypermethylation in the mycelium, suggesting that methylation might regulate gene expression from the initial process. Evaluation of the DNA methylome in Bb-NCHU-157 by ONT provided new insight into this field. These data will be further validated, and epigenetic regulation during the development of B. bassiana will be explored.

Onset of oral lichen planus following dental treatments: A nested case-control study.

INTRODUCTION: The exposure to amalgam restorations has been reported to bring about altered immunity followed by inflammation and infection. AIMS: This study aimed at identifying whether patients who received restorative or endodontic treatments, or tooth extraction, would have altered odds of developing oral lichen planus (OLP). MATERIAL AND METHODS: In this population-based nested case-control study, 421 cases of OLP and 1,684 controls were included after propensity score matching. Logistic regression was used to estimate the adjusted odds ratio (aOR) of OLP in individuals who had received amalgam and composite resin restorations, root canal therapy, and tooth extraction over a follow-up duration of five years. RESULTS: There were no significantly different odds of OLP for those who underwent either amalgam (aOR = 0.948, 95% CI = 0.853-1.053, p = 0.3170) or resin restorations (aOR = 1.007, 95% CI = 0.978-1.037, p = 0.6557) in both anterior and posterior teeth in an observational period of five  years after restorations. Root canal therapy was associated with significantly lower odds of OLP, with each additional root canal therapy attenuating the risk of OLP at an aOR of 0.771 (95% CI = 0.680-0.874, p = 0.0001) for both anterior (aOR = 0.786, 95% CI = 0.626-0.986, p = 0.0372) and posterior teeth (aOR = 0.762, 95% CI = 0.650-0.893, p = 0.0008). Likewise, each tooth extraction reduced the risk of OLP, with an aOR of 0.846 (95% CI = 0.772-0.927, p = 0.0003), especially for anterior teeth (aOR = 0.733, 95% CI = 0.595-0.904, p = 0.0037). CONCLUSIONS: We reported no significant association between dental restorations and consequent OLP, and significantly lower odds of OLP following both root canal therapy and tooth extraction.

Is OLP potentially malignant? A clue from ZNF582 methylation.

OBJECTIVE: Whether oral lichen planus (OLP) was potentially malignant remains controversial. Here, we examined associations of ZNF582 methylation (ZNF582m ) with OLP lesions, dysplastic features and squamous cell carcinoma (OSCC). MATERIALS AND METHODS: This is a case-control study. ZNF582m was evaluated in both lesion and adjacent normal sites of 42 dysplasia, 90 OSCC and 43 OLP patients, whereas ZNF582m was evaluated only in one mucosal site of 45 normal controls. High-risk habits affecting ZNF582m such as betel nut chewing and cigarette smoking were also compared in those groups. RESULTS: OLP lesions showed significantly lower ZNF582m than those of dysplasia and OSCC. At adjacent normal mucosa, ZNF582m increased from patients of OLP, dysplasia, to OSCC. In addition, ZNF582m at adjacent normal sites in OLP patients was comparable to normal mucosa in control group. Dysplasia/OSCC patients with high-risk habits exhibited significantly higher ZNF582m than those without high-risk habits. However, ZNF582m in OLP patients was not affected by those high-risk habits. CONCLUSIONS: OLP is unlikely to be potentially malignant based on ZNF582m levels. ZNF582m may also be a potential biomarker for distinguishing OLP from true dysplastic features and OSCC, and for monitoring the malignant transformation of OLP, potentially malignant disorders with dysplastic features and OSCC.

Novel regimens of phytopolyphenols with cisplatin or memantine and ZnSO4 for synergistic inhibition of growth and gingipains of the cultured Porphyromonas gingivalis.

Background/purpose: Porphyromonas gingivalis (P.g.) played a keystone pathogen not only in initiation and progression of periodontitis but also as a risk factor involved in systemic diseases (Alzheimer's disease, cancers, diabetes, osteoporosis etc.). Developments of effective and safe drugs to inhibit P.g. growth are urgent. In this study, we aimed at approaching novel regimens so called (PTM) by combination of repurposing drugs including phytopolyphenols (P) (curcumin, tea polyphenols), targeting drugs (T) such as cisplatin or memantine and metal ions(M) (ZnSO4). Materials and methods: The synergistic (combination Index (CI) < 1) antiproliferation and anti-protease efficacies (IC50) of novel regimens on cultured P.g. were evaluated by OD600 and colorimetric method respectively. Results: The results obtained revealed that these novel regimens (PTM) synergistically (combination index, CI < 1) exerted not only antiproliferative but also anti-gingipain protease effects of P.g. The concentrations for 50% inhibition (IC50) of novel regimens on P.g. growth and gingipains were greatly decreased as compared with those of cisplatin and memantine alone. Conclusion: Since these novel regimens exerted potent anti-bacterial effects on both planktonic and biofilm P.g., it is encouraged for further preclinical and clinical trials.

Transcriptome of Nosema ceranae and Upregulated Microsporidia Genes during Its Infection of Western Honey Bee (Apis mellifera).

Nosema ceranae is one of the fungal parasites of Apis mellifera. It causes physical and behavioral effects in honey bees. However, only a few studies have reported on gene expression profiling during A. mellifera infection. In this study, the transcriptome profile of mature spores at each time point of infection (5, 10, and 20 days post-infection, d.p.i.) were investigated. Based on the transcriptome and expression profile analysis, a total of 878, 952, and 981 differentially expressed genes (DEGs) (fold change ≥ 2 or ≤ -2) were identified in N. ceranae spores (NcSp) at 5 d.p.i., 10 d.p.i., and 20 d.p.i., respectively. Moreover, 70 upregulated genes and 340 downregulated genes among common DEGs (so-called common DEGs) and 166 stage-specific genes at each stage of infection were identified. The Gene Ontology (GO) analysis indicated that the DEGs and corresponding common DEGs are involved in the functions of cytosol (GO:0005829), cytoplasm (GO:0005737), and ATP binding (GO:0005524). Furthermore, the pathway analysis found that the DEGs and common DEGs are involved in metabolism, environmental information processing, and organismal systems. Four upregulated common DEGs with higher fold-change values, highly associated with spore proteins and transcription factors, were selected for validation. In addition, the stage-specific genes are highly involved in the mechanism of pre-mRNA splicing according to GO enrichment analysis; thus, three of them showed high expression at each d.p.i. and were also subjected to validation. The relative gene expression levels showed a similar tendency as the transcriptome predictions at different d.p.i., revealing that the gene expression of N. ceranae during infection may be related to the mechanism of gene transcription, protein synthesis, and structural proteins. Our data suggest that the gene expression profiling of N. ceranae at the transcriptomic level could be a reference for the monitoring of nosemosis at the genetic level.

Improving the Diagnostic Performance by Adding Methylation Marker to Conventional Visual Examination in Identifying Oral Cancer.

BACKGROUND: Visual oral examination (VOE) is a conventional oral cancer screening method. This study aimed to evaluate the value of methylation marker to assist VOE in identifying oral epithelial dysplasia and oral squamous cell carcinoma (OED/OSCC) from non-cancerous lesions in a real-world situation. METHODS: 201 patients with high-risk personal habits who self-perceived oral anomaly were VOE examined, ZNF582 methylation (ZNF582m) tested, and histologically diagnosed. RESULTS: Among them, 132 patients (65.7%) were histologically diagnosed OED/OSCC. Using VOE, 56.1% OED/OSCC patients had possible oral cancer, whereas 37.7% non-OED/OSCC patients had leukoplakia. ZNF582m-positive was detected in 90.2% OED/OSCC patients and 44.9% non-OED/OSCC patients. Various logistic regression models were postulated to evaluate the diagnostic performance of conventional VOE and new strategies using ZNF582m. ROC analysis and its corresponding C-index demonstrated that either triage or co-testing models of VOE and ZNF582m could improve diagnostic performance and discriminative abilities compared with the VOE only approach. CONCLUSIONS: In conclusion, methylation marker test shows equivalent performance to an experienced judgment by oral maxillofacial surgeons and plays a significantly supplementary role in increasing the efficacy in identifying oral malignant lesions. ZNF582m may be an especially important tool for family physicians or general dentists to properly diagnose suspicious oral lesions.

(9S,13R)-12-oxo-phytodienoic acid attenuates inflammation by inhibiting mPGES-1 and modulating macrophage polarization via NF-κB and Nrf2/HO-1 pathways.

Non-steroidal anti-inflammatory drugs (NSAIDs) relieve inflammation by suppressing prostaglandin E2/cyclooxygenase 2 (PGE2/COX-2) with cardiovascular and gastrointestinal bleeding risk. Theoretically, suppressing PGE2 through inhibiting the terminal synthase microsomal prostaglandin E2 synthase-1 (mPGES-1) instead of upstream COX-2 is ideal for inflammation. Here, (9S,13R)-12-oxo-phytodienoic acid (AA-24) extracted from Artemisia anomala was first screened as an anti-inflammatory candidate and decreased inducible nitric oxide synthase (iNOS), nitric oxide (NO), mPGES-1, and PGE2 without affecting COX-1/2, thromboxane A2 (TXA2) and prostaglandin I2 (PGI2). Besides, AA-24 suppressed the differentiation of M0 macrophages to M1 phenotype but enhanced it to M2 phenotype, blocked the activation of NF-κB pathway, and increased the activation of Nrf2 and heme oxygenase-1 (HO-1). Moreover, AA-24 selectively inhibited mPGES-1 and reduced inflamed paw edema in carrageenan-induced mice. In conclusion, AA-24 attenuates inflammation by inhibiting mPGES-1 and modulating macrophage polarization via the NF-κB and Nrf2/HO-1 pathways and could be a promising candidate for developing anti-inflammatory drugs.

The functional roles of microRNAs in the pathogenesis of oral submucous fibrosis.

Oral potentially malignant disorders (OPMD) are lesions that may precede the onset of cancers in the oral cavity, and oral submucosal fibrosis (OSF) is one of the OPMD that is usually found in the buccal mucosa. Considerable effort has been made to elucidate the pathogenesis of OSF, and emerging evidence has suggested that microRNAs may play significant roles in the development of OSF. Several studies demonstrated that aberrant expression of miRNAs is also observed in the fibrotic BMFs (fBMFs) derived from OSF tissues. For instance, it has been shown that miR-10b, miR-21, and miR-1246 are significantly elevated, and miR-29b, miR-200b, and miR-200c are reduced in fBMFs. This review systematically summarizes the current knowledge regarding the aberrant expression of microRNAs, molecular mechanisms underlying oral fibrogenesis by the dysregulated microRNAs, and how the interaction between microRNAs and long non-coding RNAs contributes to the progression of OSF. An overview of the modes of action by these microRNAs will provide a fundamental basis for clinical application.

Sinomenine increases the methylation level at specific GCG site in mPGES-1 promoter to facilitate its specific inhibitory effect on mPGES-1.

Prostaglandin E2 (PGE2) in cancer and inflammatory diseases is a key mediator of disease progression. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to inhibit the expression of PGE2 by depressing cyclooxygenase (COX) in inflammatory treatments. However, the inhibition to COXs may cause serious side effects. Thus, it is urgent to develop new anti-inflammatory drugs aiming new targets to inhibit PGE2 production. Microsomal prostaglandin E synthase 1 (mPGES-1) catalyzes the final step of PGE2 biosynthesis. Therefore, the selective inhibition of mPGES-1 has become a promising strategy in the treatments of cancer and inflammatory diseases. Our previous studies confirmed that sinomenine (SIN) is a specific mPGES-1 inhibitor. However, the exact mechanism by which SIN inhibits mPGES-1 remains unknown. This study aimed to explain the regulation effect of SIN to mPGES-1 gene expression by its DNA methylation induction effect. We found that the demethylating agent 5-azacytidine (5-AzaC) reversed the inhibitory effect of SIN to mPGES-1. Besides, SIN selectively increased the methylation level of the promoter region in the mPGES-1 gene while the pretreatment of 5-AzaC suppressed this effect. The results also shows that pretreatment with SIN increased the methylation level of specific GCG sites in the promoter region of mPGES-1. This specific methylation site may become a new biomarker for predicting and diagnosing RA and cancer with high expression of mPGES-1. Also, our research provides new ideas and solutions for clinical diagnosis and treatment of diseases related to mPGES-1 and for targeted methylation strategy in drug development.

Establishment of a High Content Image Platform to Measure NF-κB Nuclear Translocation in LPS-induced RAW264.7 Macrophages for Screening Anti-inflammatory Drug Candidates.

BACKGROUND: High Content Image (HCI), an automatic imaging and analysis system, provides a fast drug screening method by detecting the subcellular distribution of protein in intact cells. OBJECTIVE: This study established the first standardized HCI platform for lipopolysaccharide (LPS)-induced RAW264.7 macrophages to screen anti-inflammatory compounds by measuring nuclear factor-κB (NF-κB) nuclear translocation. METHODS: The influence of the cell passages, cell density, LPS induction time and concentration, antibody dilution, serum, dimethyl sulfoxide, and analysis parameters on NF-κB nuclear translocation and HCI data quality was optimized. The BAY-11-7085, the positive control for inhibiting NF-κB, and the Western blot assay were separately employed to verify the stability and reliability of the platform. Lastly, the effect of BHA on NO release, iNOS expression, IL-1ß, IL-6, and TNF-α mRNA in LPS-induced RAW264.7 cells was detected. RESULTS: The optimal conditions for measuring NF-κB translocation in LPS-induced RAW264.7 cells by HCI were established. Cells that do not exceed 22 passages were seeded at a density of 10 k cells/well and pretreated with compounds following 200 ng/mL LPS for 40 min. Parameters including the nuclear area of 65 µm2, cell area of 80 µm2, collar of 0.9 µm, and sensitivity of 25% were recommended for image segmentation algorithms in the analysis workstation. Benzoylhypaconine from aconite was screened for the first time as an anti-inflammatory candidate by the established HCI platform. The inhibitory effect of benzoylhypaconine on NF-κB translocation was verified by Western blot. Furthermore, benzoylhypaconine reduced the release of NO, inhibited the expression of iNOS, and decreased the mRNA levels of IL-1ß, IL-6, and TNF-α. CONCLUSION: The established HCI platform could be applied to screen anti-inflammatory compounds by measuring the NF-κB nuclear translocation in LPS-induced RAW264.7 cells.

Photothermal/NO combination therapy from plasmonic hybrid nanotherapeutics against breast cancer.

In this study, a plasmon-semiconductor nanotheranostic system comprising Au nanostars/graphene quantum dots (AuS/QD) hybrid nanoparticles loaded with BNN6 and surface modified with PEG-pyrene was developed for the photo-triggered hyperthermia effect and NO production as the dual modality treatment against orthotopic triple-negative breast cancer. The structure and morphology of the hybrid nanodevice was characterized and the NIR-II induced thermal response and NO production was determined. The hybrid nanodevice has shown enhanced plasmonic energy transfer from localized surface plasmonic resonance of Au nanostars to QD semiconductor that activates the BNN6 species loaded on QD surfaces, leading to the effective NO production and the gas therapy in addition to the photothermal response. The increased accumulation of the NIR-II-responsive hybrid nanotheranostic in tumor via the enhanced permeation and retention effects was confirmed by both in vivo fluorescence and photoacoustic imaging. The prominent therapeutic efficacy of the photothermal/NO combination therapy from the BNN6-loaded AuS@QD nanodevice with the NIR-II laser irradiation at 1064 nm against 4T1 breast cancer was observed both in vitro and in vivo. The NO therapy for the cancer treatment was evidenced with the increased cellular nitrosative and oxidative stress, nitration of tyrosine residues of mitochondrial proteins, vessel eradication and cell apoptosis. The efficacy of the photothermal treatment was corroborated directly by severe tissue thermal ablation and tumor growth inhibition. The NIR-II triggered thermal/NO combination therapy along with the photoacoustic imaging-guided therapeutic accumulation in tumor shows prominent effect to fully inhibit tumor growth and validates the promising strategy developed in this study.