Identification and Fungicide Screening of Fungal Species Associated with Walnut Anthracnose in Shaanxi and Liaoning Provinces, China.

Walnut is cultivated around the world for its precious woody nut and edible oil. Recently, walnut infected by Colletotrichum spp. resulted in a great yield and quality loss. In August and September 2014, walnut fruits with anthracnose were sampled from two commercial orchards in Shaanxi and Liaoning provinces, and five representative isolates were used in this study. To identify the pathogen properly, four genes per region (internal transcribed spacer, glyceraldehyde-3-phosphate dehydrogenase, actin, and chitin synthase) were sequenced and used in phylogenetic studies. Based on multilocus phylogenetic analysis, five isolates clustered with Colletotrichum fioriniae, including its ex-type, with 100% bootstrap support. The results of multilocus phylogenetic analyses, morphology, and pathogenicity confirmed that C. fioriniae was one of the walnut anthracnose pathogens in China. All 13 fungicides tested inhibited mycelial growth and spore germination. Flusilazole, fluazinam, prochloraz, and pyraclostrobin showed the strongest suppressive effects on the mycelial growth than the others, the average EC50 values ranged from 0.09 to 0.40 µg/ml, and there was not any significant difference (P < 0.05). Pyraclostrobin, thiram, and azoxystrobin were the most effective fungicides on spore germination (P < 0.05), and the EC50 values ranged from 0.01 to 0.44 µg/ml. Pyraclostrobin, azoxystrobin, fluazinam, flusilazole, mancozeb, thiram, and prochloraz exhibited a good control effect on walnut anthracnose caused by C. fioriniae, and preventive activities were greater than curative activities. Pyraclostrobin at 250 a.i. µg/ml and fluazinam at 500 a.i. µg/ml provided the highest preventive and curative efficacy, and the values ranged from 81.3 to 82.2% and from 72.9 to 73.6%, respectively. As a consequence, mancozeb and thiram could be used at the preinfection stage, and pyraclostrobin, azoxystrobin, flusilazole, fluazinam, and prochloraz could be used at the early stage for effective prevention and control of walnut anthracnose caused by C. fioriniae. The results will provide more significant instructions for controlling the disease effectively in northern China.

First report of Colletotrichum fioriniae causing leaf spot on Schima superba in the world.

Schima superba Gardn. et Champ. is a subtropical evergreen tree species naturally distributed mainly in China, Japan, and Vietnam. It is primarily planted for its timber and urban landscaping in China (Ni, 1996). In September 2018, leaves necrotic spots were observed on S. superba in Jiangxi Forest Breeding Center (28°57'19.52" N, 115°39'21.32" E), Jiangxi Province, China. The disease incidence was about 30%. Initially, spots were circular to semicircular, grayish-brown in the center with dark brown margin, then expanded and eventually collapsed into sunken necrotic lesions. To identify the agent, diseased leaves were collected randomly. Pieces (5 × 5 mm) from the lesion borders were surfaced sterilized in 70% ethanol (30 s), 3% NaOCl (60 s), and rinsed 3 times in sterile water. These pieces were put on potato dextrose agar (PDA) and cultured at 25 °C. Pure cultures were obtained by monosporic isolation, and 3 isolates (MH-1, MH-2, MH-3) were used for morphological studies and phylogenetic analyses. On PDA, colonies were initially white, cottony, then became pinkish to deep-pink at the center and pink on the reverse. Conidia were fusiform with acute ends, smooth-walled, hyaline, 13.7-18.5 × 4.6-6.1 µm (16.4 ± 1.3× 5.3 ± 0.6 µm, n = 100). Conidiophores were colorless to pale brown, smooth, septate. Conidiogenous cells were colorless to pale brown, smooth, cylindrical to ampulliform. The morphological characteristics fit the descriptions of Colletotrichum acutatum J. H. Simmonds sensu lato (Damm et al., 2012). For accurate identification, genomic DNA of 3 isolates was extracted, and the internal transcribed spacer (ITS), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin 2 (TUB2), and chitin synthase (CHS-1) were amplified and sequenced using the corresponding primers (Weir et al., 2012). The sequences were deposited in GenBank (ITS: MZ325946, MZ325947, MW584318; ACT: MZ399375, MZ419566, MW661171; CHS-1: MZ399376, MZ419567, MW661172; MZ399377, GAPDH: MZ419568, MW661173; TUB2: MZ399378, MZ419569, MW661174). Five loci were concatenated, and the aligned sequences (1528bp) were 99.89% homologous to ex-type C. fioriniae (Marcelino & Gouli) R. G. Shivas & Y. P. Tan CBS128517. Phylogenetic analysis using the maximum likelihood showed that 3 isolates were clustered in C. fioriniae clade with 100% bootstrap support. Based on the multi-locus phylogeny and morphology, 3 isolates were identified as C. fioriniae. Pathogenicity tests were performed on 36 seedlings of S. superba (2-year-old). The leaves were wounded slightly and inoculated with a drop of spore suspension (106 conidia/mL). The sterile water was used as controls. All the tested leaves were covered with black plastic bags to keep them moist for 2 days. All seedlings were placed in the greenhouse (25 °C, 12 h light/dark) for 10 days, and all inoculated leaves had typical symptoms. The controls were asymptomatic. The same fungus was reisolated from the lesions, fulfilling Koch's postulates. Colletotrichum fioriniae was described as a new species from the C. acutatum s. l. (Shivas et al., 2009), and it was an important plant pathogen, such as Pyrus spp. (Pavlovic et al., 2019), Morus alba L. (Xue et al., 2019), and so on. This is the first report of the newly emerging disease of S. superba caused by C. fioriniae in the world, and its potential threat should be evaluated in the future. This study provided crucial information for epidemiologic studies and appropriate control strategies.

Curcumin Inhibits Viability of Clear Cell Renal Cell Carcinoma by Down-Regulating ADAMTS18 Gene Methylation though NF-κ B and AKT Signaling Pathway.

OBJECTIVE: To investigate the effect of curcumin on viability of clear cell renal cell carcinoma (ccRCC) and analyze its possible mechanism. METHODS: In cell lines of A498 and 786-O, the effects of curcumin (1.25, 2.5, 5 and 10 µ mol/L) on the viability of ccRCC were analyzed at 24, 48 and 72 h by MTT assay. The protein expression levels of ADAMTS18 gene, p65, phosphorylation p65 (pp65), AKT, phosphorylation AKT (pAKT) and matrix metallopeptidase 2 (MMP-2) before and after curcumin (10 µ mol/L) treatment were examined by Western blotting. Real-time PCR and methylation specific PCR (MSP) were applied to analyze the expression and methylation level of ADAMTS18 gene before and after curcumin treatment (10 µ mol/L). RESULTS: Curcumin significantly inhibited the viability of A498 and 786-O cell lines in a dose- and time-dependent manner (P<0.01). Up-regulation of ADAMTS18 gene expression with down-regulation of ADAMTS18 gene methylation was reflected after curcumin treatment, accompanied by down-regulation of nuclear factor κ B (NF-κ kB) related protein (p65 and pp65), AKT related protein (AKT and pAKT), and NF-κ B/AKT common related protein MMP-2. With ADAMTS18 gene overexpressed, the expression levels of p65, AKT and MMP2 were downregulated, of which were conversely up-regulated in silenced ADAMTS18 (sh-ADAMTS18). The expression of pp65, pAKT and MMP2 in sh-ADAMTS18 was down-regulated after being treated with PDTC (NF-κ B inhibitor) and LY294002 (AKT inhibitor). CONCLUSIONS: Curcumin could inhibit the viability of ccRCC by down-regulating ADAMTS18 gene methylation though NF-κ B and AKT signaling pathway.

Learning Curve of an Innovative "3-Port" Laparoscopic Radical Prostatectomy: A Single-Center Analysis from 2016 to 2019.

BACKGROUND: "Three-port" laparoscopic radical prostatectomy (LRP) has been applied as a substitution for the conventional 4- to 5-port LRP to treat prostate cancer (PCa) patients in our institution. OBJECTIVE: To evaluate the learning curve of an innovative "3-port" LRP for PCa patients. METHODS: 206 patients who received "3-port" LRP were retrospectively reviewed between January 2016 and December 2019 at our institution. According to the different years of operations performed, all of the patients were divided into group A (No. 1-50), group B (No. 51-107), group C (No. 108-160), and group D (No. 161-206). A learning curve was depicted by analyzing the parameters of operative time (OT), estimated blood loss (EBL), hospitalization, and drainage indwelling days. RESULTS: All groups were comparable with regard to the preoperative characteristics (p > 0.05). The sloping learning curve for the surgeon showed that OT and EBL were strongly correlated with an accumulated experience when compared between group A and the other groups (p < 0.05), denoting that the surgical skill of the "3-port" LRP can be fully mastered after around 50 cases. Although no significant correlation with additional experience was observed in the hospitalization and drainage indwelling days among groups, a tendency towards less hospitalization and drainage indwelling days was still reflected. CONCLUSIONS: Our 4-year analysis based on a single-center experience exhibits that the innovative "3-port" LRP appears to be favorable with decreasing tendency in OT and EBL with experience accumulation. In view of its advantage of perioperative parameters with an evidently improved learning curve, it should be recommended in the clinical practice!

Comparison of functional and oncological outcomes of innovative "three-port" and traditional "four-port" laparoscopic radical prostatectomy in patients with prostate cancer.

BACKGROUND: To compare the functional and oncological outcomes between innovative "three-port" and traditional "four-port" laparoscopic radical prostatectomy (LRP) in patients with prostate cancer (PCa). METHODS: We retrospectively collected the data of PCa patients treated at our institutions from June 2012 to May 2016. According to the inclusion criteria, a total of 234 patients were included in the study, including 112 in group A (four-port) and 122 in group B (three-port). The perioperatively surgical characteristics, functional and oncological outcomes were compared between groups. RESULTS: There were no statistical differences in the baseline parameters between these two groups. Compared with group A, the operative time (OT) and estimated blood loss (EBL) were significantly less in group B. On follow-up, the rate of positive surgical margin (PSM), prostate specific antigen (PSA) biochemical recurrence and continence after LRP did not show any statistically significant difference between the groups. An identical conclusion was also received in comparison of overall survival (OS) and biochemical recurrence-free survival (BRFS) between both groups. CONCLUSIONS: Innovative "three-port" LRP can significantly shorten the OT and reduce the EBL compared with the traditional "four-port" LRP. Meanwhile, it does not increase the rate of PSM and PSA biochemical recurrence. "Three-port" LRP could be popularized in the future in view of its superior surgical technique, considerably better functional outcomes and remarkable oncological control.

Aberrant methylation of the 16q23.1 tumor suppressor gene ADAMTS18 promotes tumorigenesis and progression of clear cell renal cell carcinoma.

BACKGROUND: The 16q23.1 tumor suppressor gene (TSG) of ADAMTS18 has been identified to be aberrant methylated in clear cell renal cell carcinoma (ccRCC), and there still exists an unclear situation between its methylation and the progression of ccRCC. OBJECTIVE: To analyze the biological function and mechanism of ADAMTS18 gene in the tumorigenesis and progression of ccRCC. METHODS: We examined ADAMTS18 gene methylation using methylation- specific polymerase chain reaction (MSP) in 92 ccRCC primary tumors from September 2017 to May 2018. Using reverse transcriptase PCR (RT-PCR) and immunohistochemical (IHC) assay, the relative expression level of ADAMTS18 was measured in the representative tumor samples with their adjacent normal tissues. Meanwhile, colony formation, cell viability, wound healing, transwell chamber, flow cytometry, and PI staining were performed to confirm the tumor-suppressive function and mechanism of ADAMTS18 gene. RESULTS: Aberrant methylation was further detected in 47 of the 92 (51.1%) primary tumors and in 8 of the 92 (8.7%) adjacent normal tissues (p < 0.05). Due to the phenomenon of aberrant methylation, ectopic low-level expression of ADAMTS18 gene could result in the promotion of tumorigenesis and progression in ccRCC. CONCLUSION: The aberrantly methylated ADAMTS18 gene may be involved in the tumorigenesis and progression of ccRCC.

Curcumin reverses the sunitinib resistance in clear cell renal cell carcinoma (ccRCC) through the induction of ferroptosis via the ADAMTS18 gene.

BACKGROUND: To explore the possible mechanism by which curcumin reverses the sunitinib resistance in clear cell renal cell carcinoma (ccRCC). METHODS: A sunitinib-resistant ccRCC cell model was established. The MTT assay was used to determine the half maximal inhibitory concentration (IC50) and drug resistance (DR) index. The effects of curcumin plus sunitinib or sunitinib alone on drug-resistant cell lines were verified by the cell counting kit-8 (CCK-8)assay, colony formation assay, and apoptosis assay. The concentration of iron ions in the cell lines was analyzed using an Abcam Iron Assay Kit. The expressions of ADAMTS18 gene and ferroptosis-related proteins (NCOA4, FTH1 and p53) after curcumin plus sunitinib treatment were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. After transfection of curcumin plus sunitinib/sunitinib alone-treated drug-resistant cell lines with si-ADAMTS18, cell proliferation activity was assessed by the CCK-8 assay, and the protein expression levels of ADAMTS18, NCOA1, FTH1 and p53 were analyzed by Western blotting. After treatment with ferroptosis-1 (Fer-1; a ferroptosis inhibitor), the cell proliferation activity of drug-resistant cell lines treated with curcumin plus sunitinib/sunitinib alone was reassessed using the CCK-8 assay. RESULTS: Curcumin plus sunitinib inhibited the proliferation of sunitinib-resistant ccRCC cells (P<0.05). Curcumin significantly decreased the concentration of iron ions and increased the expression of ADAMTS18 gene, while significantly inhibited ferroptosis-related protein expression (P<0.05). After silencing the ADAMTS18 gene, there was no significant difference in cell proliferation or ferroptosis-related protein expression between curcumin plus sunitinib and sunitinib-treated drug-resistant cell lines (P>0.05). Ferroptosis inhibitors reversed the inhibitory effect of curcumin on sunitinib-resistant ccRCC cell lines. CONCLUSIONS: Curcumin can reverse the sunitinib resistance in ccRCC, possibly by upregulating the expression of the ADAMTS18 gene to induce ferroptosis.

A novel "three-port" trocar placement technique for laparoscopic radical prostatectomy.

BACKGROUND: To introduce a novel "three-port" trocar placement technique for laparoscopic radical prostatectomy (LRP) in prostate cancer (PCa) patients. METHODS: We retrospectively reviewed 300 patients with PCa who received surgical treatment between November 2010 and June 2015 at our institution. They were divided into group A, three-port LRP; group B, conventional four-five-port LRP; group C, open RP (ORP); and group D, robotic-assisted RP (RARP). A learning curve was analyzed by dividing patients of group A into the early and late stages. RESULTS: All groups were comparable with regard to the preoperative characteristics except for the relatively smaller prostate volume in group A. The three-port LRP operations were performed successfully with only 8 cases of conversion to the conventional LRP. None of any severe complications or conversion to ORP occurred. In group A, the mean operative time (OT) duration was 113.8 min, the mean estimated blood loss (EBL) was 94.2 ml, the mean drainage days was 4.0 days, the mean hospitalization was 5.1 days, and 27.8% of the prostate specimen margins (PSM) were positive. The differences of OT, EBL, drainage days, hospitalization, and transfusion in group A were statistically significant among the majority of the other groups (p < 0.05). After undergoing the early stages of a learning curve analysis in three-port LRP, the EBL was obviously decreased. CONCLUSIONS: Three-port LRP is a novel technique that exhibits superior intraoperative advantages to the conventional LRP. Due to its less OT, EBL, drainage days, hospitalization, and transfusion with a shorter learning curve, it should be recommended and popularized in the clinical practice.