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This study was intended to establish the predictive target of Shikonin (SK) against ovarian cancer using network pharmacology and to clarify the potential mechanism of SK in promoting apoptosis in ovarian cancer. Cell Counting Kit-8 assay, plate clone assays, LDH assay, flow cytometric analysis of Annexin V-fluorescein isothiocyanate/propidium iodide staining, and western blotting were used to assess the effect of SK on apoptosis of ovarian cancer cell lines (SKOV3 and A2780). Pharmacodynamic targets were used to predict the targets of SK and ovarian cancer. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analyses were used to analyze the biological functions and signal pathways of these targets. SK promoted apoptosis in ovarian epithelioid adenocarcinoma cells. SK-ovarian cancer pharmacodynamic target analysis screened 17 related genes. GO and KEGG analyses showed that SK affected the estrogen signaling pathway. SK inhibited the expression of GPER in SKOV3 and A2780 cells and downregulated the expression of EGFR, p-EGFR, PI3K, and p-AKT in a concentration-dependent manner. The apoptosis-promoting effect of SK was enhanced by GPER-specific agonist G1 and inhibited by the specific inhibitor G15. The expression of EGFR, p-EGFR, PI3K, and p-AKT was decreased by G1 and reversed by G15. SK also inhibited tumor growth in the SKOV3 xenograft model, and it acted synergistically with G1. However, the effect can be attenuated by G15 in vivo. In summary, SK may affect the apoptosis of ovarian cancer cells through GPER/EGFR/PI3K/AKT, and GPER may be a key target of SK in ovarian cancer cell apoptosis.

To determine the phase of NUDT15 sequence variants for more comprehensive star (*) allele diplotyping, we developed a novel long-read single-molecule real-time HiFi amplicon sequencing method. A 10.5 kb NUDT15 amplicon assay was validated using reference material positive controls and additional samples for specimen type and blinded accuracy assessment. Triplicate NUDT15 HiFi sequencing of two reference material samples had nonreference genotype concordances of99.9%, indicating that the assay is robust. Notably, short-read genome sequencing of a subset of samples was unable to determine the phase of star (*) allele-defining NUDT15 variants, resulting in ambiguous diplotype results. In contrast, long-read HiFi sequencing phased all variants across the NUDT15 amplicons, including a *2/*9 diplotype that previously was characterized as *1/*2 in the 1000 Genomes Project v3 data set. Assay throughput was also tested using 8.5 kb amplicons from 100 Ashkenazi Jewish individuals, which identified a novel NUDT15 *1 suballele (c.-121GA) and a rare likely deleterious coding variant (p.Pro129Arg). Both novel alleles were Sanger confirmed and assigned as *1.007 and *20, respectively, by the PharmVar Consortium. Taken together, NUDT15 HiFi amplicon sequencing is an innovative method for phased full-gene characterization and novel allele discovery, which could improve NUDT15 pharmacogenomic testing and subsequent phenotype prediction.

Background. Soft tissue sarcomas (STSs) are rare tumors and occur at any site in the body. Our goal was to identify a putative molecular mechanism for N6-methyladenosine (m6A) lncRNA alteration and to develop predictive biomarkers for sarcoma. Methods. The lncRNA levels were obtained from TCGA datasets. Pearson correlation analysis was used to select all the lncRNAs that are connected to m6A. An m6A-related lncRNA model was built using LASSO Cox regression. To assess the prognostic efficiency of the model and potential lncRNAs, we performed univariate survival analysis and receiver operating characteristic (ROC) analysis. We also performed enrichment analysis to evaluate the roles of the potential genes. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to confirm m6A-related lncRNA expression in tissues. Results. Following Pearson correlation analysis on TCGA datasets, we identified 78 m6A-related lncRNAs. Next, we used LASSO Cox regression analysis and identified 13 m6A-related lncRNAs as prognostic lncRNAs. After calculating risk scores, sarcoma patients were divided into high- and low-risk groups depending on the median of risk scores. We also found that these lncRNAs were immune associated via enrichment analysis. Conclusions. Here, we found that SNHG1, FIRRE, and YEATS2-AS1 could serve as biomarkers to predict overall survival of sarcoma patients, which provides a new insight into treatment of STS.

Background. The extract of freshwater clams has been used to protect the body against liver diseases in traditional folk medicine. This study aims at investigating the effects of freshwater clam extract on activated hepatic stellate cells (aHSCs), which are critical contributors to liver fibrosis. Methods. The aHSCs used in this study were derived from hepatic stellate cells that were isolated and purified from the livers of male Wistar rats and then transformed into the activated phenotype by culturing on uncoated plastic dishes. Freshwater clam extract (CE) was collected after the outflow from the live freshwater clams in a water bath at 100°C for 60 min. The effects of CE on aHSCs were analyzed by MTT assay, flow cytometry, Oil Red O (ORO) staining, western blot, and real-time RT-PCR. Results. The results indicated that CE suppressed the proliferation of aHSCs through G0/G1 cell cycle arrest by downregulating cyclin D1 and upregulating p27. The expression levels of a-SMA, collagen I, TGF-β, and TNF-α were inhibited in the CE-treated aHSCs. In addition, the CE treatment increased the lipid contents in aHSCs by promoting PPARγ expression. Furthermore, CE modulated the expression of ECM-related genes, i.e., by upregulating MMP-9 and downregulating TIMP-II. Conclusions. These data revealed that CE could induce the deactivation of aHSCs. We therefore suggest that CE has potential as an adjuvant therapeutic agent against hepatic fibrosis.

Firework-related eye injury is a horrible medical problem and creates huge health and social burdens. Herein, we explored the changing trends and demographic and clinical features of firework-related eye injury, in an effort to inform strategies to prevent this injury. We reviewed the data of 468 hospitalized patients who underwent surgery for firework-related eye injury at the Zhongshan Ophthalmic Center between January 2013 and December 2017. During this period, the trend in firework-related eye injuries was stable (mean, 93 ± 14 cases per year), and fireworks were the major cause of explosive eye injury. The average age of the patients was 24 ± 18 years and 87% of the patients were male, with boys under 10 years of age comprising the largest group (27% of patients). There were an average of 24 ± 7 cases per year from urban areas and 70 ± 8 cases from rural areas (P<0.05). Furthermore, 21 ± 5% of cases occurred during Spring Festival. After treatment, the best corrected visual acuity was increased compared to that before treatment, and the intraocular pressure tended to become normal by the final visit (P<0.05). The top three diagnoses were cataract (39%), retinal detachment (18%), and choroidal detachment (14%). Additionally, the most common surgery was cataract extraction (25%), followed by pars plana vitrectomy (19%) and pars plana vitrectomy plus pars plana lensectomy (10%). Over the five-year study period, day surgery hospitalization increased from 1% to 32%. This was associated with a corresponding decrease in the length of hospitalization, without adverse events, demonstrating that day surgery is feasible in firework-related eye injury cases. The present study results suggest that greater attention should be paid to firework-related eye injury, and a variety of measures should be taken to prevent this kind of ocular tragedy.

To improve the effectiveness and robustness of the existing semisupervised fuzzy clustering for segmenting image corrupted by noise, a kernel space semisupervised fuzzy C-means clustering segmentation algorithm combining utilizing neighborhood spatial gray information with fuzzy membership information is proposed in this paper. The mean intensity information of neighborhood window is embedded into the objective function of the existing semisupervised fuzzy C-means clustering, and the Lagrange multiplier method is used to obtain its iterative expression corresponding to the iterative solution of the optimization problem. Meanwhile, the local Gaussian kernel function is used to map the pixel samples from the Euclidean space to the high-dimensional feature space so that the cluster adaptability to different types of image segmentation is enhanced. Experiment results performed on different types of noisy images indicate that the proposed segmentation algorithm can achieve better segmentation performance than the existing typical robust fuzzy clustering algorithms and significantly enhance the antinoise performance.

The current study quantified the risk of releasing African swine fever virus (ASFV) into Taiwan from pork products illegally carried by international travellers from 157 countries or territories through six international airports and three international seaports. The association between various factors and the number of pork products detected by the border control authorities was also examined. The risk was estimated with a stochastic process after modelling the number of undetected illegal pork products, probability of pork product detection at international airports and seaports and probability of ASFV contamination of pork products from various countries. The overall annual probability of ASFV release to Taiwan was estimated to be 1 [95% confidence interval (CI): 1-1] under no enhanced mitigation measures. All the median airport-level risks were higher than .921, and four of them reached 1. The total annual risk was .570 (95% CI: .109-.937) for international seaports. The country or territory level risk was estimated to be 1 for Vietnam, China, Hong Kong, the Philippines and South Korea, .999 (95% CI: .628-1) for Macao and .967 (95% CI: .359-1) for Indonesia. After the total number of travellers was factored in, the number of detected illegal pork products was the highest in January and February, and travellers from Vietnam [risk ratio to Japan (RR): 80.45; 95% CI: 58.68-110.3], the Philippines (RR: 37.67; 95% CI: 26.9-52.74) and Cambodia (RR: 28.39; 95% CI: 12.69-63.51) were most likely to bring pork products to Taiwan. Our study indicated a high risk of ASFV introduction through international travellers and also identified the factors associated with the risk. This information can be used as empirical evidence for cost-effective risk mitigation practices.

Refractory dermatomyositis (DM) is defined as cases that do not show improvement after initial treatment with two different immunosuppressives combined with corticosteroids with or without intravenous immunoglobulins. In recent years, few studies have reported a positive response to the use of Janus kinase inhibitors (JAK-inhibitors) for the treatment of refractory DM. A systematic literature review was performed for articles studying the use of JAK-inhibitors for the treatment of refractory DM. We identified 38 females and 15 males treated with JAK-inhibitors without serious side effects. Tofacitinib was the most frequently used JAK-inhibitor followed by ruxolitinib. Significant improvement in CDASI score, muscle strength, body weight, and skin lesions were reported in most of the studies. The duration of follow-up ranged from 1 to 15 months without relapse. Therefore, the use of JAK-inhibitors looks promising in the treatment of refractory DM and further high volume research may be required to validate the current concept. As only case reports and series were identified without direct comparison for review, there is a potential risk of bias. Despite these limitations, we believe that the result of this analysis allows a better understanding of treatment options for refractory DM and will help generate a hypothesis that can be further tested.

Flood discharge containing excessive suspended sediments (SS) can result in total dissolved gas (TDG) supersaturation downstream of dams. It has been confirmed that high TDG levels and excessive SS cause serious threats to fish survival. To explore the effects of TDG supersaturation on the hatchability of Chinese sucker (Myxocyprinus asiaticus Bleeker) eggs, eggs were exposed to 100%, 125%, 130% and 135% TDG supersaturated water. The results showed that the hatching rates were 78.25%, 75.35%, 71.30% and 67.50% at the 100%, 125%, 130% and 135% TDG respectively. The hatching rate decreased with increasing TDG levels. Furthermore, acute lethality experiments were carried out to investigate the interaction of TDG supersaturation and SS on Chinese sucker. Half‐year‐old juveniles were exposed to TDG supersaturated water with SS concentrations of 0, 200, 600 and 1,000 mg/L, and the TDG levels were 125%, 130%, 135% and 140% respectively. The fish exhibited abnormal behaviours and signs of gas bubble disease. Mortality increased with increasing TDG levels and SS concentrations. The Chinese suckers only survived for 2.5 hr at 140% TDG and 1,000 mg/L SS. Moreover, an increase in SS in TDG supersaturated water could cause a decrease in the median lethal time (LT₅₀). When the TDG level reached 140%, the LT₅₀ declines significantly at 600 and 1,000 mg/L SS. The interaction of TDG supersaturation and SS was significant at LT₅₀ (F = 22.81, df = 9, p

Rhynchanthus beesianus is a medicinal, ornamental, and edible plant, and its essential oil has been used as an aromatic stomachic in China. In this study, the chemical constituents, antibacterial, and anti-inflammatory properties of flower essential oil (F-EO), leaf essential oil (L-EO), and stem essential oil (S-EO) of R. beesianus were investigated for the first time. According to the GC-FID/MS assay, the F-EO was mainly composed of bornyl formate (21.7%), 1,8-cineole (21.6%), borneol (9.7%), methyleugenol (7.7%), β-myrcene (5.4%), limonene (4.7%), camphene (4.5%), linalool (3.4%), and α-pinene (3.1%). The predominant components of L-EO were bornyl formate (33.9%), borneol (13.2%), 1,8-cineole (12.1%), methyleugenol (8.0%), camphene (7.8%), bornyl acetate (6.2%), and α-pinene (4.3%). The main components of S-EO were borneol (22.5%), 1,8-cineole (21.3%), methyleugenol (14.6%), bornyl formate (11.6%), and bornyl acetate (3.9%). For the bioactivities, the F-EO, L-EO, and S-EO exhibited significant antibacterial property against Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, Proteus vulgaris, Pseudomonas aeruginosa, and Escherichia coli with the inhibition zones (7.28–9.69 mm), MIC (3.13–12.50 mg/mL), and MBC (6.25–12.50 mg/mL). Besides, the F-EO, L-EO, and S-EO significantly inhibited the production of proinflammatory mediator nitric oxide (NO) (93.15–94.72%) and cytokines interleukin-6 (IL-6) (23.99–77.81%) and tumor necrosis factor-α (TNF-α) (17.69–24.93%) in LPS-stimulated RAW264.7 cells at the dose of 128 μg/mL in the absence of cytotoxicity. Hence, the essential oils of R. beesianus flower, leaf, and stem could be used as natural antibacterial and anti-inflammatory agents with a high application potential in the pharmaceutical and cosmetic fields.

Background. Baveno VI criteria, based on liver stiffness (LS) measured by transient elastography and platelet counts (PLT), have been proposed to avoid unnecessary endoscopy screening for high-risk varices (HRVs). However, the cut-off value of LS measured by 2D-SWE and PLT to predict HRVs in compensated hepatitis B-related cirrhotic patients remains unknown. Aims. To prospectively analyze the cut-off of the combination of LS measured by 2D-SWE and PLT in predicting HRVs and the influence of antiviral therapies in its efficacy. Methods. Serum parameters, LS, and endoscopy results were obtained from 160 compensated hepatitis B-related cirrhotic patients. The accuracy of the combined algorithm was assessed in the whole cohort and subgroups with or without consecutive antiviral therapies in the past 6 months. Results. In the whole cohort, the optimal cut-off value of LS for HRVs was 14.5 kPa. Patients with a LS value < 14.5 kPa with a PLT value > 110 × 10 9 / L can be excluded from HRVs ( NPV = 0.99 , endoscopy saved rates = 0.68 ). Conversely, a LS value of ≥14.5 kPa and a PLT value of ≤ 110 × 10 9 / L indicated HRVs, with accurate rates of 82.35%, and 10.63% of patients can avoid additional endoscopy screening. Moreover, antiviral therapy had no significant effect on the accuracy and rates saved from further endoscopy screening, when comparing patients with or without antiviral therapies (all p values > 0.05). Conclusions. The combination of LS (14.5 kPa) measured by 2D-SWE and PLT ( 110 × 10 9 / L ) can predict HRVs accurately in compensated hepatitis B-related cirrhotic patients without significant interference of antiviral therapy histories.

The effect of ultrasound on the extraction of bioactive aglycone isoflavones for the valorization of black soybean okara was investigated. A high extraction efficiency for total isoflavones was achieved with the total isoflavones extracted using water being 77.8% of that using 80% methanol as the solvent. The fraction of bioactive aglycones (daidzein and genistein) in total isoflavones using ultrasound (40 kHz/300 W) for 10 min was 81.7%, and further enhanced to 86.7% for 30 min. The profile of isoflavones was significantly influenced by temperature with the amounts of 351.00 μg/g‐okara of aglycones and 91.43 μg/g‐okara of glycosides using ultrasound at 60°C for 3 hr, and the fractions of aglycones in isoflavones were 96.4, 89.6, and 79.3% for 35, 50, and 60°C, respectively. An extraction model was proposed to successfully correlate the extraction rate coefficients and the limit concentrations of the four isoflavones in water. PRACTICAL APPLICATIONS: The bioactive aglycone isoflavones are beneficial for human health. The okara produced from food industries contains high‐quality isoflavones, which have to be recovered for the potential use in functional foods. The process developed in this study allows the recovery of high‐content aglycone isoflavones from black soybean okara without further biotransformation of glycoside isoflavones, and provides the effective valorization of okara and process sustainability in food applications.

The cytochrome P450-2D6 (CYP2D6) enzyme metabolizes ∼25% of common medications, yet homologous pseudogenes and copy number variants (CNVs) make interrogating the polymorphic CYP2D6 gene with short-read sequencing challenging. Therefore, we developed a novel long-read, full gene CYP2D6 single molecule real-time (SMRT) sequencing method using the Pacific Biosciences platform. Long-range PCR and CYP2D6 SMRT sequencing of 10 previously genotyped controls identified expected star (*) alleles, but also enabled suballele resolution, diplotype refinement, and discovery of novel alleles. Coupled with an optimized variant-calling pipeline, CYP2D6 SMRT sequencing was highly reproducible as triplicate intra- and inter-run nonreference genotype results were completely concordant. Importantly, targeted SMRT sequencing of upstream and downstream CYP2D6 gene copies characterized the duplicated allele in 15 control samples with CYP2D6 CNVs. The utility of CYP2D6 SMRT sequencing was further underscored by identifying the diplotypes of 14 samples with discordant or unclear CYP2D6 configurations from previous targeted genotyping, which again included suballele resolution, duplicated allele characterization, and discovery of a novel allele and tandem arrangement. Taken together, long-read CYP2D6 SMRT sequencing is an innovative, reproducible, and validated method for full-gene characterization, duplication allele-specific analysis, and novel allele discovery, which will likely improve CYP2D6 metabolizer phenotype prediction for both research and clinical testing applications.

Purpose. To report the causative organisms, management strategies, and visual outcomes of culture-proven endogenous fungal endophthalmitis in a case series from southern China. Methods. We reviewed the microbiological and medical records of patients with culture-positive endogenous fungal endophthalmitis visiting the Zhongshan Ophthalmic Center, Guangzhou, China, between January 1, 2006, and March 31, 2016. Results. The inclusion criteria were met in 32 eyes of 29 patients. Molds were a common causative organism in 15 patients (51.7%), while yeasts appeared in 14 patients (48.3%). Initial visual acuity (VA) at the level of finger counting or better was significantly related to a good visual outcome (P=0.002). Molds as a causative agent were significantly associated with worse visual outcome than yeasts (P=0.020). Conclusion. Molds were a common cause of culture-proven fungal endophthalmitis. Endogenous fungal endophthalmitis is generally associated with poor VA outcomes, especially if caused by molds and if the patient’s initial VA is too low to permit finger counting.

Ling-Zhi-8 (LZ-8), a low-molecular weight protein from Ganoderma lucidum, exerts immunomodulatory effects in vitro and in vivo. The aim of this study was to investigate the effects of exposure of LZ-8 to denaturing conditions on its ability to suppress inflammatory responses in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Ultraviolet, fluorescence and circular dichroism spectroscopy analyses showed that LZ-8 was not denatured by exposure to pH extremes, but was irreversibly denatured by temperatures above 55C. LZ-8 lowered the production of nitric oxide and interleukin-6, but had no effect on the level of tumor necrosis factor-α. Heat and acid/alkali-treated preparations of LZ-8 had comparable capacity to suppress proinflammatory mediator production. Since native and denatured LZ-8 were both anti-inflammatory, it appears that this particular biological property of LZ-8 is not dependent on the native structure of the protein, but, possibly on a relatively small peptide domain of LZ-8. Practical Applications Recent evidence suggests that LZ-8, a small protein originally isolated from G. lucidum, has immunomodulatory and anti-carcinogenic properties. In this study, LZ-8 exerted an anti-inflammatory effect on LPS-stimulated macrophages, but this effect was not dependent on the native protein structure, suggesting that the entire protein might not be responsible for the bioactive properties of LZ-8. Since the biological functions of LZ-8 were resistant to heating and exposure to acid or alkali, LZ-8 could have applications in food-processing industries and the production of pharmaceuticals. Furthermore, peptides derived from LZ-8 may be potential targets for development of novel anti-carcinogenic and immunomodulatory agents.

Summary The relationship between QT duration and its dispersion in patients with primary hyperaldosteronism is not clearly known. We studied 26 patients (nine males and 17 females) with primary hyperaldosteronism. The serum potassium levels were low (2.32 ± 0.52 mmol/l), did not correlate with serum renin or aldosterone levels, or aldosterone/renin ratio (ARR). The maximum QT intervals (QTmax) were prolonged (502 ± 62 ms), correlated well with ARRs (p = 0.005) and aldosterone levels (p = 0.019), but not to renin (p = 0.517) or potassium levels (p = 0.196). The QT dispersions (QTd) were small (60 ± 28.8 ms) and did not correlate with potassium, renin or aldosterone levels. QTmax but not QTd correlate with aldosterone levels in patients with primary aldosteronism. The maintenance of repolarisation homogeneity with relatively unchanged QT dispersion may contribute to our understanding of the clinical observation that ventricular tachydysrhythmia is rare among patients with primary aldosteronism.