Porcine reproductive and respiratory syndrome virus triggers Golgi apparatus fragmentation-mediated autophagy to facilitate viral self-replication.

Macroautophagy/autophagy is a cellular degradation and recycling process that maintains the homeostasis of organisms. A growing number of studies have reported that autophagy participates in infection by a variety of viruses. Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe financial losses to the global swine industry. Although much research has shown that PRRSV triggers autophagy for its own benefits, the exact molecular mechanisms involved in PRRSV-triggered autophagy remain to be fully elucidated. In the current study, we demonstrated that PRRSV infection significantly induced Golgi apparatus (GA) fragmentation, which promoted autophagy to facilitate viral self-replication. Mechanistically, PRRSV nonstructural protein 2 was identified to interact with and degrade the Golgi reassembly and stacking protein 65 dependent on its papain-like cysteine protease 2 activity, resulting in GA fragmentation. Upon GA fragmentation, GA-resident Ras-like protein in brain 2 was disassociated from Golgi matrix protein 130 and subsequently bound to unc-51 like autophagy activating kinase 1 (ULK1), which enhanced phosphorylation of ULK1 and promoted autophagy. Taken together, all these results expand the knowledge of PRRSV-triggered autophagy as well as PRRSV pathogenesis to support novel potential avenues for prevention and control of the virus. More importantly, these results provide the detailed mechanism of GA fragmentation-mediated autophagy, deepening the understanding of autophagic processes.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) infection results in a serious swine disease affecting pig farming worldwide. Despite that numerous studies have shown that PRRSV triggers autophagy for its self-replication, how PRRSV induces autophagy is incompletely understood. Here, we identify that PRRSV Nsp2 degrades GRASP65 to induce GA fragmentation, which dissociates RAB2 from GM130 and activates RAB2-ULK1-mediated autophagy to enhance viral replication. This work expands our understanding of PRRSV-induced autophagy and PRRSV replication, which is beneficial for anti-viral drug development.

Ultrasound-guided microwave ablation as a palliative treatment for mycosis fungoides eyelid involvement: A case report.

BACKGROUND: Mycosis fungoides (MF) is a form of lymphoma derived from heterogeneous T cells, and eyelid involvement is extremely rare. The common methods to treat eyelid involvement are radiotherapy and chemotherapy, but their efficacies are limited. Herein, we report a case of advanced-stage MF eyelid involvement, propose ultrasound (US)-guided microwave ablation (MWA) therapy and present a literature review. CASE SUMMARY: A male patient was admitted to our hospital in June 2018 and diagnosed with MF via radiological and histopathological examinations. The patient's condition was not well controlled by various conventional chemotherapies. US-guided MWA was performed to relieve the patient's symptoms and improve his quality of life, showing satisfactory efficacy. CONCLUSION: Eyelid involvement is one of the most troublesome clinical problems for advanced-stage MF patients. This is the first report on the use of US-guided MWA as a palliative therapy for MF eyelid involvement; the treatment successfully relieved the patient's clinical symptoms and reduced his anxiety behaviours. Our study sheds new light on methods for improving the clinical management of eyelid involvement in MF.

A novel biphenyl compound IMB-S7 ameliorates hepatic fibrosis in BDL rats by suppressing Sp1-mediated integrin αv expression.

Chronic tissue injury with fibrosis results in the disruption of tissue architecture, organ dysfunction, and eventual organ failure. Therefore, the development of effective antifibrotic drugs is urgently required. IMB-S7 is novel biphenyl compound derived from bifendate (biphenyldicarboxylate) that is used for the treatment of chronic hepatitis in China. In the current study we investigated the potential of IMB-S7 as an antihepatic fibrosis agent. In bile duct ligation (BDL) rat model, oral administration of IMB-S7 (400 mg· kg-1· d-1, for 14 days) significantly ameliorated BDL-induced liver necrosis, bile duct proliferation, and collagen accumulation. We then showed that IMB-S7 treatment markedly suppressed the TGF-ß/Smad pathway in human hepatic stellate cell line LX2 and mouse primary HSCs, as well as in liver samples of BDL rats, thus inhibiting the transcription of most fibrogenesis-associated genes, including TGF-ß1, COL1A1, and ACTA2. Furthermore, IMB-S7 treatment significantly suppressed the expression of integrin αv at the mRNA and protein levels in TGF-ß-treated LX2 cells and liver samples of BDL rats. Using integrin αv overexpression and silencing, we demonstrated that integrin αv activity correlated positively with the activation of TGF-ß/Smad pathway. Based on dual luciferase assay and DNA affinity precipitation assay, we revealed that IMB-S7 inactivated integrin αv through competitively inhibiting the binding of Sp1, a transcription factor, to the integrin αv (ITGAV) promoter (-173/-163 bp). These results suggest that IMB-S7 inhibits HSCs activation and liver fibrosis through Sp1-integrin αv signaling, and IMB-S7 may be a promising candidate to combat hepatic fibrosis in the future.

A multivariable model of BRAFV600E and ultrasonographic features for predicting the risk of central lymph node metastasis in cN0 papillary thyroid microcarcinoma.

Background: Prophylactic central lymph node dissection (CLND) in papillary thyroid microcarcinoma (PTMC) patients without clinical evidence of central lymph node metastasis (CLNM) remains controversial. The purpose of our study is to identify preoperative predictive factors for finding CLNM in Chinese PTMC patients, which may allow tailored CLND. Methods: We retrospectively reviewed 182 consecutive Chinese PMTC patients with negative central lymph nodes who underwent total thyroidectomy plus central neck dissection from October 2015 to December 2017. Chi-squared and multivariate analysis were performed to evaluate the association of CLNM with ultrasonographic and clinicopathologic characteristics. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the utility of markers in predicting CLNM. Results: The CLNM was found in 39.0% (71 of 182) of cN0 PTMC patients. In multivariate analysis, tumor size>7 mm (OR: 3.636, 95% CI: 1.671-7.914), marked hypoechogenicity (OR: 2.686, 95% CI: 1.080-6.678), multifocality (OR: 4.184, 95% CI: 1.707-10.258) and BRAFV600E mutation (OR: 5.339, 95% CI: 2.529-11.272) were independent predictors of CLNM. In ROC analysis integrating these predictors, the sensitivity was 63.4% and specificity was 80.2%, and the area under the ROC (AUC) was 0.755. Conclusion: In conclusion, we found tumor size>7 mm, marked hypoechogenicity, multifocality, and BRAFV600E mutation were risk factors for CLNM. In term of these preoperative risk factors for CLNM, prophylactic CLND should be cautiously performed in cN0 PTMC patients.

Generation of Indoles with Agrochemical Significance through Biotransformation by Chaetomium globosum.

Six new (1-6) and two known (7 and 8) indole alkaloids were produced by the marine fish-derived fungus Chaetomium globosum 1C51 through biotransformation. The structures of these alkaloids were elucidated by a combination of MS, NMR, and X-ray crystallography analyses. Chaetoindolone A (1) was shown to inhibit the growth of the rice-pathogenic bacteria Xanthomonas oryzae pv. oryzae (xoo) both in vitro and in vivo. Chaetogline A (7) was found to be fungicidal against Sclerotinia sclerotiorum, a pathogen causing rape sclerotinia rot. Collectively, this work provides access to new indole alkaloids with potential agrochemical significance.

Design, synthesis, and antifungal activity of carboxamide derivatives possessing 1,2,3-triazole as potential succinate dehydrogenase inhibitors.

Succinate dehydrogenase (SDH) is demonstrably one of the most important molecular targets in development of new fungicide. In our continuous efforts to discover novel SDH inhibitors, forty-two carboxamide derivatives containing 1,2,3-triazole ring were designed and synthesized, which were precisely characterized by 1H NMR, ESI-MS, elemental analysis and X-ray single-crystal diffraction. The compounds were screened for antifungal activities against phytopathogenic fungi by mycelia growth inhibition assay in vitro. Compound A3-3 exhibited significant antifungal activity against Sclerotinia sclerotiorum, Botrytis cinerea, Rhizoctonia cerealis and Gaeumannomyces graminsis with EC50 values of 1.08, 8.75, 1.67 and 5.30 µg/mL, respectively, comparable to those of commercial SDHI boscalid. In vivo testing demonstrated that A3-3 was effective for suppressing rape sclerotinia rot, cucumber grey mould and wheat powdery mildew caused by S. sclerotiorum， B. cinerea and Blumeria graminis at a dosage of 200 µg/mL. Inhibition activities against SDH test proved the designed analogues were effective in the enzyme level. The molecular docking simulation revealed that A3-3 interacted with ARG43，TYR58 and TRP173 of the SDH through hydrogen bond and pi-pi interaction, which could explain the probable mechanism of action between the inhibitor and target protein.

New Metabolites from Endophytic Fungus Chaetomium globosum CDW7.

Five metabolites including two new ones, prochaetoviridin A (1) and chaetoindolin A (2), were isolated from the endophytic fungus Chaetomium globosum CDW7. Compounds 1 and 2 were characterized as an isocoumarin and an indole alkaloid derivative, respectively, with their structures elucidated by comprehensive spectroscopic analyses including high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), NMR, and circular dichroism (CD) comparison. Compounds 3⁻5 were identified as chaetoviridin A, chaetoglobosin R, and chaetoglobosin T, respectively. Chaetoviridin A (3) exhibited antifungal activity against Sclerotinia sclerotiorum with an EC50 value of 1.97 µg/mL. In vivo test showed that 3 displayed a protective efficacy of 64.3% against rape Sclerotinia rot at the dosage of 200 µg/mL, comparable to that of carbendazim (69.2%).

D-chiro-inositol effectively attenuates cholestasis in bile duct ligated rats by improving bile acid secretion and attenuating oxidative stress.

Cholestatic liver diseases are important causes of liver cirrhosis and liver transplantation, but few drugs are available for treatment. D-chiro-inositol (DCI), an isomer of inositol found in many Leguminosae plants and in animal viscera, is used clinically for the treatment of polycystic ovary syndrome (PCOS) and diabetes mellitus. In this study, we investigated whether DCI exerted an anti-cholestatic effect and its underlying mechanisms. A cholestatic rat model was established via bile duct ligation (BDL). After the surgery, the rats were given DCI (150 mg·kg-1·d-1) in drinking water for 2 weeks. Oral administration of DCI significantly decreased the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and attenuated bile duct proliferation, parenchymal necrosis and fibrosis in BDL rats. Furthermore, DCI treatment significantly increased the serum and bile levels of total bile acid (TBA), and decreased TBA levels in the liver. Moreover, DCI treatment significantly increased expression of the genes encoding bile acid transporters BSEP (Abcb11) and MRP2 (Abcc2) in liver tissues. DCI treatment also markedly decreased hepatic CD68 and NF-kappaB (NF-κB) levels, significantly decreased the serum and hepatic MDA levels, markedly increased superoxide dismutase activity in both serum and liver tissues. Using whole-genome oligonucleotide microarray, we revealed that DCI treatment altered the expression profiles of oxidation reduction-related genes in liver tissues. Collectively, DCI effectively attenuates BDL-induced hepatic bile acid accumulation and decreases the severity of injury and fibrosis by improving bile acid secretion, repressing inflammation and decreasing oxidative stress. The results suggest that DCI might be beneficial for patients with cholestatic disorders.

Chaetomium globosum CDW7, a potential biological control strain and its antifungal metabolites.

Screening for endophytic fungi with antifungal activity is an effective strategy for the discovery of novel biopesticides. Our previous work indicated that Chaetomium globosum CDW7, an endophyte from Ginkgo biloba, exhibited strong inhibitory activity against plant pathogenic fungi in vitro. In this study, we evaluated the CDW7 strain for its antifungal activities against nine phytopathogenic fungi and its biocontrol potential against rape sclerotinia rot caused by Sclerotinia sclerotiorum. The fermentation broth of CDW7 could successfully inhibit disease development in S. sclerotiorum-infected rape in vivo with 57.8% protective efficiency, which is comparable to that of carbendazim (59.8%) at 250 µg mL-1. The fermentation broth also expressed significant activity stability when exposed to 60°C and UV illumination, or when stored at 4°C. Furthermore, we found that 10% fermentation broth can promote the germination and growth of rape seedlings. Followed by the bioassay-guided approach, seven known metabolites were isolated and identified by spectroscopic analyses. Among them, chaetoglobosin A and D exhibited inhibitory activity against S. sclerotiorum with IC50 values of 0.35 and 0.62 µg mL-1, respectively, compared with carbendazim (0.17 µg mL-1). Therefore, our study demonstrated that CDW7 is a promising biocontrol fungus against S. sclerotiorum in agriculture.

The diagnostic performances of conventional strain elastography (SE), acoustic radiation force impulse (ARFI) imaging and point shear-wave speed (pSWS) measurement for non-calcified thyroid nodules.

BACKGROUND: Non-calcified thyroid nodules are relatively difficult to diagnose only relying on features of at conventional US images. OBJECTIVE: To investigate the diagnostic performances of conventional strain elastography (SE), acoustic radiation force impulse (ARFI) SE and point shear-wave speed (pSWS) measurement for non-calcified thyroid nodules. METHODS: A total of 201 non-calcified thyroid nodules in 195 patients were studied. They were examined with conventional ultrasound (US), conventional SE, ARFI SE and pSWS measurement. Their diagnostic performances and multivariable models were assessed with receiver operating characteristic (ROC) curve and logistic regression analyses respectively. RESULTS: There were 156 benign and 45 malignant non-calcified nodules proven by histopathology or cystology. The mean diameters of the nodules were 21.2±10.8 mm. Areas under receiver operating characteristic curve (AUCs) of elastography features (ranged, 0.488-0.745) were all greater than that of US (ranged, 0.111-0.332). At multivariate analysis, there were three predictors of malignancy for non-calcified nodules, including pSWS of nodule (odds ratio [OR], 34.960; 95% CI, 11.582-105.529), marked hypoechogenicity (OR, 16.223; 95% CI, 1.761-149.454) and ARFI SE grade (OR, 10.900; 95% CI, 3.567-33.310). US+SE+pSWS owned the largest AUC (0.936; 95% CI, 0.887-0.985; P < 0.05), followed by US+pSWS (0.889; 95% CI, 0.823-0.955), and the poorest was US (0.727; 95% CI, 0.635-0.819). CONCLUSIONS: ARFI SE and pSWS measurement had better diagnostic performances than conventional SE and US. When US combined with SE and pSWS measurement, it could achieve an excellent diagnostic performance and might contribute a better decision-making of FNA for non-calcified thyroid nodules.

Calcification of thyroid nodules increases shear-wave speed (SWS) measurement: using multiple calcification-specific SWS cutoff values outperforms a single uniform cutoff value in diagnosing malignant thyroid nodules.

Conventional ultrasound cannot satisfactorily distinguish malignant and benign thyroid nodules. Shear-wave elastography (SWE) can evaluate tissue stiffness and complement conventional ultrasound in diagnosing malignant nodules. However, calcification of nodules may affect the results of SWE. The purposes of this study are to compare the differences of shear-wave speed (SWS) measurement among different calcification groups and compare the diagnostic performance between using a single uniform SWS cutoff value and multiple individual calcification-specific cutoff values using technique of point SWS measurement. We retrospectively identified 517 thyroid nodules (346 benign and 171 malignant nodules) examined by conventional ultrasound and point SWS measurement. There were 177 non-calcified, 159 micro-calcified and 181 macro-calcified nodules. The diagnostic performance was evaluated by receiver operating characteristic (ROC) curve and area under the curve (AUC) was computed. The mean SWS in malignant nodules more than doubled that of benign nodules (4.81±2.03 m/s vs. 2.29±0.99 m/s, p<0.001). The mean SWS of nodules progressively increased from the non-calcification (2.60±1.49 m/s), to micro-calcification (3.27±1.85 m/s) and to macro-calcification (3.68±2.26 m/s) groups (p<0.001), which was true in both the benign and malignant nodules. If we used individual SWS cutoff values for non- (SWS >2.42 m/s), micro- (SWS >2.88 m/s) and macro-calcification (SWS >3.59 m/s) nodules in the whole group, the AUC was 0.859 (95% confidence interval [CI], 0.826-0.888), which was significantly better than the AUC of 0.816 (95% CI, 0.780-0.848) if a single uniform cutoff value (SWS >2.72 m/s) was applied to all the nodules regardless of calcification status (p=0.011). The cutoff values of SWS for different calcified nodules warrant future prospective validation.

Protective effect of bicyclol against bile duct ligation-induced hepatic fibrosis in rats.

AIM: To evaluate the protective effect of bicyclol against bile duct ligation (BDL)-induced hepatic fibrosis in rats. METHODS: Sprague-Dawley male rats underwent BDL and sham-operated animals were used as healthy controls. The BDL rats were divided into two groups which received sterilized PBS or bicyclol (100 mg/kg per day) orally for two consecutive weeks. Serum, urine and bile were collected for biochemical determinations. Liver tissues were collected for histological analysis and a whole genome oligonucleotide microarray assay. Reverse transcription-polymerase chain reaction and Western blotting were used to verify the expression of liver fibrosis-related genes. RESULTS: Treatment with bicyclol significantly reduced liver fibrosis and bile duct proliferation after BDL. The levels of alanine aminotransferase (127.7 ± 72.3 vs 230.4 ± 69.6, P < 0.05) and aspartate aminotransferase (696.8 ± 232.6 vs 1032.6 ± 165.8, P < 0.05) were also decreased by treatment with bicyclol in comparison to PBS. The expression changes of 45 fibrogenic genes and several fibrogenesis-related pathways were reversed by bicyclol in the microarray assay. Bicyclol significantly reduced liver mRNA and/or protein expression levels of collagen 1a1, matrix metalloproteinase 2, tumor necrosis factor, tissue inhibitors of metalloproteinases 2, transforming growth factor-ß1 and α-smooth muscle actin. CONCLUSION: Bicyclol significantly attenuates BDL-induced liver fibrosis by reversing fibrogenic gene expression. These findings suggest that bicyclol might be an effective anti-fibrotic drug for the treatment of cholestatic liver disease.

[Establishment and application of a high-throughput drug screening model based on COL1A1 promoter for anti-liver fibrosis].

For screening the potential drugs as anti-liver fibrosis candidates, we established a high- throughput drug screening cell model based on COL1A1 promoter. The activity of COL1A1 promoter and luciferase reporter gene can be elevated by TGF-ß1, and inhibited by candidate drugs. We constructed a recombined plasmid with COL1A1 promoter and luciferase reporter gene pGL4.17, the activity of COL1A1 promoter was reflected by fluorescence intensity. COL1A1 promoter activity was detected by Dual-Luciferase Reporter Assay System, it came that the relative luciferase activity of COL1A1 promoter was 15.98 times higher than that of control group induced by TGF-ß1, showing the recombined plasmid could be used in cell model. The recombined plasmid was transfected into human hepatic stellate cells LX2, detected the effect of potential drugs, and obtained a stable expression system through stable transfection and monoclonal cell culture. A sample which could reduce COL1A1 promoter activity signally by our cell model, decreased collagen I mRNA and protein expression detected by real-time RT-PCR and Western blotting. It indicates this novel cell model can be used in high-throughput drug screening of potential anti-liver fibrosis drugs.

The anti-fibrotic effects of epigallocatechin-3-gallate in bile duct-ligated cholestatic rats and human hepatic stellate LX-2 cells are mediated by the PI3K/Akt/Smad pathway.

AIM: (-)-Epigallocatechin-3-gallate (EGCG) is one of the most abundant polyphenols in green tea with strong antioxidant activity and various therapeutic effects. In this study, we investigated the anti-fibrotic effects of EGCG and underlying mechanisms in bile duct-ligated (BDL) rats and a liver fibrosis model in vitro. METHODS: BDL rats were treated with EGCG (25 mg·kg(-1)·d(-1), po) for 14 d, and then the serum, bile and liver samples were collected. Liver fibrosis was assessed by serum, urine and bile biochemistry analyses and morphological studies of liver tissues. TGF-ß1-stimulated human hepatic stellate LX-2 cells were used as a liver fibrosis model in vitro. The expression of liver fibrogenic genes and signaling proteins in the PI3K/Akt/Smad pathway was examined using Western blotting and/or real-time PCR. RESULTS: In BDL rats, EGCG treatment significantly ameliorates liver necrosis, inflammation and fibrosis, and suppressed expression of the genes associated with liver inflammation and fibrogenesis, including TNF-α, IL-1ß, TGF-ß1, MMP-9, α-SMA, and COL1A1. In LX-2 cells, application of EGCG (10, 25 µmol/L) dose-dependently suppressed TGF-ß1-stimulated expression of COL1A1, MMP-2, MMP-9, TGF-ß1, TIMP1, and α-SMA. Furthermore, EGCG significantly suppressed the phosphorylation of Smad2/3 and Akt in the livers of BDL rats and in TGF-ß1-stimulated LX-2 cells. Application of LY294002, a specific inhibitor of PI3K, produced similar effects as EGCG did in TGF-ß1-stimulated LX-2 cells, but co-application of EGCG and LY294002 did not produce additive effects. CONCLUSION: EGCG exerts anti-fibrotic effects in BDL rats and TGF-ß1-stimulated LX-2 cells in vitro via inhibiting the PI3K/Akt/Smad pathway.

Synthesis and cytotoxic evaluation of alkoxylated chalcones.

A series of chalcones a1-20 bearing a 4-OMe groups on the A-ring were initially synthesized and their anticancer activities towards HepG2 cells evaluated. Subsequently, a series of chalcones b1-42 bearing methoxy groups at the 2' and 6'-positions of the B-ring were synthesized and their anticancer activities towards five human cancer cell lines (HepG2, HeLa, MCF-7, A549 and SW1990) and two non-tumoral human cell lines evaluated. The results showed that six compounds (b6, b8, b11, b16, b18, b22, b23 and b29) displayed promising activities, with compounds b22 and b29 in particular showing higher levels of activity than etoposide against all five cancer cell lines. Compound b29 showed a promising SI value compared with both HMLE and L02 (2.1-6.5 fold in HMLE and > 33 > 103.1 fold in L02, respectively).

[Potential targets for anti-liver fibrosis].

Liver fibrosis is a pathological process of the excessive accumulation of extracellular matrix, especially collagen al (I) in liver. Ultimately, hepatic fibrosis leads to cirrhosis or hepatic failure. Liver fibrosis and early cirrhosis can be reversed, thus control of the development of liver fibrosis is very important for preventive treatment of cirrhosis and hepatic failure. This is a review of potential targets for anti-hepatic fibrosis based on plenty of publications, including TGF-ß1 and integrin α(v) and so on, aimed at providing novel therapeutic targets in liver fibrosis.

Calcineurin B-like protein CBL10 directly interacts with AKT1 and modulates K+ homeostasis in Arabidopsis.

Potassium transporters and channels play crucial roles in K+ uptake and translocation in plant cells. These roles are essential for plant growth and development. AKT1 is an important K+ channel in Arabidopsis roots that is involved in K+ uptake. It is known that AKT1 is activated by a protein kinase CIPK23 interacting with two calcineurin B-like proteins CBL1/CBL9. The present study showed that another calcineurin B-like protein (CBL10) may also regulate AKT1 activity. The CBL10-over-expressing lines showed a phenotype as sensitive as that of the akt1 mutant under low-K+ conditions. In addition, the K+ content of both CBL10-over-expressing lines and akt1 mutant plants were significantly reduced compared with wild-type plants. Moreover, CBL10 directly interacted with AKT1, as verified in yeast two-hybrid, BiFC and co-immunoprecipitation experiments. The results of electrophysiological analysis in both Xenopus oocytes and Arabidopsis root cell protoplasts demonstrated that CBL10 impairs AKT1-mediated inward K+ currents. Furthermore, the results from the yeast two-hybrid competition assay indicated that CBL10 may compete with CIPK23 for binding to AKT1 and negatively modulate AKT1 activity. The present study revealed a CBL-interacting protein kinase-independent regulatory mechanism of calcineurin B-like proteins in which CBL10 directly regulates AKT1 activity and affects ion homeostasis in plant cells.