Colletotrichum horii and Colletotrichum fioriniae Causing Anthracnose on Star Anise (Illicium verum) in China.

Star anise (Illicium verum) is an important economic and medical plant widely cultivated in Guangxi province, China. Its fruit can be used as spice and medicine (Wang et al. 2011). In recent years, anthracnose led to a serious decline in the production of star anise in Guangxi. In 2021, a survey conducted in CenwangLaoshan Reserve of Guangxi (24°21'N; 106°27'E) showed that the 2500 ha planting area had disease incidence greater than 80%. The leaf symptoms initially appeared as small spots, then expanded to round spots, finally becoming withered with grayish-white centers, surrounded by dark brown margins. Sometimes, small black acervuli were observed in the later stage. To explore the pathogen, infected leaves were collected and cut into small pieces (about 5 mm2) from the edge of the lesion, disinfected with 75% ethanol for 10 s, 1% NaClO for 1 min, washed with sterilized water and incubated on potato dextrose agar (PDA) plates at 28 °C in the dark. Ten single-spore isolates were obtained from the cultures. After 7 days on PDA at 28 °C, the colonies of 7 isolates were white with abundant aerial hyphae, gray-black with white-gray margins, and the other 3 isolates were light gray on the upper surface, and pink or orange on the underside. Representative isolates BS3-4 and BS3-1 were selected from 3 isolates and 7 isolates, respectively. Conidia of BS3-4 and BS3-1 were both hyaline, cylindrical, aseptate, smooth, apex obtuse, base truncate, and no significant differences (P > 0.05) in size between BS3-1 (13.22 to 5.38 × 3.89 to 1.99 µm) (n = 50) and BS3-4 (12.04 to 4.34 × 3.48 to 1.64 µm) (n = 50). These morphological characteristics were consistent with the Colletotrichum ssp. (Damm et al. 2012). The species identification of BS3-4 and BS3-1 was performed based on DNA sequence analysis. Genomic DNA was extracted as a template. Partial sequences of the rDNA internal transcribed spacer (ITS), actin gene (ACT), ß-tubulin2 (TUB2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified and sequenced (Weir et al. 2012). The sequences were deposited in GenBank (ITS:OQ062642-43; ACT:OQ067614-15; GAPDH:OQ067616-17;TUB2：OQ067618-19). Based on the concatenated sequences of the 4 genes (ITS-ACT- GAPDH -TUB2) of BS3-4 and BS3-1 as well as sequences of other Colletotrichum spp. obtained from GenBank, the Maximum likelihood (ML) tree which produced with IQ-TREE (Minh et al. 2020) revealed that the isolate BS3-1 was Colletotrichum horii, and BS3-4 was Colletotrichum fioriniae. Pathogenicity was confirmed on healthy leaves of 1-year-old star anise seedlings (cultivar Dahong), and the leaves were wounded by sterilized toothpicks, and were inoculated with 10 µl of conidial suspensions of BS3-1 and BS3-4 (106 conidia/ml). Control seedlings were inoculated with sterilized distilled water. Five leaves per plant and 3 plants per treatment were selected. All inoculated seedlings were maintained in the greenhouse (12/12h light/dark, 25 ± 2℃, 90% relative humidity). Wound sites inoculated with BS3-1 and BS3-4 both turned greenish-brown after 2 days and then turned light brown with water-soaked spots. Black (BS3-1) or orange (BS3-4) dots of acervuli developed after 6 days. The lesion diameter of BS3-1 (14.4 mm) was larger than that of BS3-4 (8.1 mm). No symptoms were observed on controls. BS3-1 and BS3-4 were re-isolated from inoculated leaves, fulfilling Koch's postulates. Anthracnose of star anise caused by C.horii has been reported in China (Liao et al. 2017). However, to our knowledge, this is the first report of C.fioriniae infecting star anise in China. Accurate pathogen identification in this study could provide a reference for the control of anthracnose on star anise.

lncRNA differentiation antagonizing nonprotein coding RNA overexpression accelerates progression and indicates poor prognosis in pancreatic ductal adenocarcinoma.

BACKGROUND: lncRNA differentiation antagonizing nonprotein coding RNA (lncRNA DANCR) has been suggested to play an oncogenic role in multiple cancers. However, to the best of our knowledge, the clinical significance and role of DANCR in pancreatic ductal adenocarcinoma (PDAC) has not been illuminated till now. The present study aims to identify the functional role of DANCR in PDAC. METHODS: The expression of DANCR was detected in PDAC cells and tissues. The correlation of DANCR expression and PDAC clinicopahological features was analysed. Kaplan-Meier method was used to depict the overall survival (OS) rate and shorter progression-free survival (PFS) of PDAC patients, and Log-rank test was performed to analyse the difference. Univariate and multivariate COX regression model were utilized to analyse the risk factors for prognosis. Transwell assay and Matrigel assay were conducted to detect the effect of DANCR on the migration and invasion of PDAC cells, respectively. Colony formation assay and Cell Counting Kit-8 (CCK-8) assay were performed to evaluate the function of DANCR on proliferation. The mechanisms of DANCR exerting its function were also explored. RESULTS: DANCR was revealed to promote PDAC progression, with relatively higher expression levels in PDAC cell lines and tissues. Correlation analysis of the clinicopathological features and DANCR expression found that high DANCR expression was statistically correlated with vascular invasion (P=0.013), advanced T stage (P=0.005), lymph node metastasis (P<0.001) and advanced TNM stage (P<0.001). Notably, survival analysis discovered that high DANCR expression predicted lower OS rate and shorter PFS period. In addition, high DANCR expression was identified as an independent risk factor for poor OS (HR=1.199, 95% CI=1.113-1.290, P<0.001) and PFS (HR=1.199, 95% CI=1.114-1.290, P<0.001) of PDAC. Moreover, in vitro assays detected that the migration and invasion of Panc1 cells with DANCR deficiency were significantly suppressed in the Transwell assay and the Matrigel assay. However, the motility of BxPC3 cells with DANCR overexpression was obviously increased. In addition, the loss of DANCR suppressed the proliferation of Panc1 cells in the CCK-8 assay and the colony formation assay, while ectopic expression of DANCR in BxPC3 cells promoted the proliferation. Besides, microRNA-33a-5p/AXL signaling pathway may be involved in mediating the function of DANCR. CONCLUSION: Overexpression of lncRNA DANCR in PDAC is associated with cancer progression and predicts poor OS and PFS. DANCR could promote the proliferation and metastasis of PDAC cells. DANCR may serve as a potential prognostic marker and therapeutic target in PDAC.

[Protection of liver against ischemia/reperfusion injury by Kupffer cell mediated emulsified isoflurane preconditioning: experiment with rats].

OBJECTIVE: To investigate the protective effect of pretreatment with Kupffer cell mediated emulsified isoflurane on ischemia/reperfusion injury in liver. METHODS: Thirty-two SD rats underwent partial obstruction of liver blood flow for 30 m and were randomly divided into 4 equal groups during the operation: lipid emulsion control group (Group C, pretreated with intravenous injection of lipid emulsion), inhibition of Kupffer cell and preconditioning with lipid emulsion control group (Group KC, intraperitoneally injected with gadolinium chloride, inhibitor of Kupffer cells, and then pretreated with intravenous injection of lipid emulsion), preconditioning with emulsified isoflurane group (Group IP, intravenously injected with emulsified isoflurane), and Inhibition of Kupffer cells and preconditioning with isoflurane group (Group IK, intraperitoneally injected with gadolinium chloride and then intravenously injected with emulsified isoflurane), Then liver perfusion was recovered for 2 h and the rats were killed. Blood samples were collected from the vena cava to detect the serum alanine transaminase (ALT) and aspartate transaminase (AST). Specimens of left liver tissue were collected to undergo light microscopy. The contents of malonyldialdehyde (MDA) and superoxide dismutase (SOD) in the liver homogenate were examined with relevant kits. RESULTS: Compared with Group C, the serum ALT and AST of Group IP were significantly lower (both P < 0.05). The MDA level of Group IP was significantly lower than that of Group C, and the SOD level of Group IP was significantly higher than that of Group C (both P < 0.05). However, there were no significant differences in the serum levels of ALT and AST and levels of MDA and SOD in lever homogenate among Group KC, IK, and C. The pathological changes in liver were remarkably milder in Group IP then in Group C, however, there were no significant differences in the pathological changes among Groups KC, IK, and C. CONCLUSION: Preconditioning with emulsified isoflurane protects the liver from ischemia/reperfusion injury and this effect me be mediated by Kupffer cells.

[Expression of IL-34 in chronic periapical lesions and its clinical significance].

PURPOSE: To observe the expression of IL-34 mRNA in chronic periapical lesions and healthy periodontal ligaments and discuss the role of IL-34 in the etiology of chronic apical periodontitis. METHODS: A total of 25 periapical tissues from chronic apical periodontitis and 22 normal periodontal ligament tissue from extracted healthy teeth for orthodontic reason were selected. The expression of IL-34mRNA was detected by real-time PCR; the expression of IL-34 protein was detected by immunohistochemical analysis. Statistical analysis was performed using SPSS 13.0 software package. RESULTS: The level of IL-34 mRNA expression in periapical lesions (3.53±3.07) was significantly higher than that of the normal control (1.07±0.76); IL-34 was positively expressed in lymphocytes, plasma cells and macrophages. Image analysis software indicated that the level of IL-34 protein was significantly higher in periapical lesions than that in normal control (P<0.01). CONCLUSIONS: IL-34 may be closely related to inflammation of chronic apical periodontitis.

[Effect of cone angle on fracture resistance force of root and post-core system].

OBJECTIVE: To study the effect of cone angle on fracture resistance force of root and post-core system. METHODS: Forty-two simulated tooth roots made of polymethacrylate (PMMA) were divided into six groups according to the root canal cone angles of 0 degrees, 3.93 degrees, 5.71 degrees, 7.48 degrees, 11.31 degrees and 14.71 degrees. Cast post and cores were manufactured and cemented with zinc polycarboxylate cement (PC). Casting the post and core to restore every simulated tooth root. Each specimen was embedded in acrylic resin and then fixed in a special jig on the universal load-testing machine. A compressive load was applied at a 90-degree angle to the long axis of the core until fracture, at a crosshead speed of 1 mm/min. The maximum of load was recorded. RESULTS: The mean values of load in root and post-core system were 0.1225 kN, 0.1498 kN, 0.1600 kN, 0.1893 kN, 0.1078 kN and 0.0945 kN. There were significant differences in the fracture resistance among groups. CONCLUSION: The fracture resistance force of cast post and core increased with the enlargement of post cone angles under certain conditions.

[Expression of Wnt5a in chronic apical periodontitis and its clinical significance].

PURPOSE: To detect the expression of Wnt5a in lesions of chronic apical periodontitis and determine the relationship between expression of Wnt5a and inflammation degree. METHODS: Ten patients with chronic apical periodontitis and 5 healthy controls were enrolled in this study. According to the inflammatory cell infiltration, the specimens were divided into 2 groups: severe inflammation group and mild inflammation group. The expression of Wnt5a was measured by real-time PCR (RT-PCR) and immunohistochemical analysis in the lesions of chronic apical periodontitis. The amount of Wnt5 expression was assayed and compared in different inflammation levels. Statistical analysis was performed using SPSS 13.0 software package. RESULTS: Wnt5a was detected in both groups. Expression of Wnt5a mRNA in patients were significantly higher than the controls (P<0.05). According to inflammation level, the positive expression rate of Wnt5a in severe inflammation group was significantly higher than the controls (P<0.01), and Wnt5a positive expression in mild inflammation group was also significantly higher than the controls (P<0.05). The expression of Wnt5a was significantly different between severe inflammation group and mild inflammation group (P<0.05). CONCLUSIONS: The expression of Wnt5a increases as the severity of tissue inflammation increases, which indicates that Wnt5a plays an important role in the development of chronic apical periodontitis.

GLAST expression on bullfrog Müller cells is regulated by dark/light.

Using immunofluorescence double-labeling, Western blotting and confocal laser scanning microscopy, we investigated if and how the expression of glial high-affinity glutamate/aspartate transporter (GLAST) in bullfrog Müller cells may be regulated by dark/light. Compared with light-adapted retinas, the expression of GLAST in Müller cells was overall up-regulated in retinas dark-adapted for 30 min but declined in retinas dark-adapted for longer (>30 min) periods. The declined expression level of GLAST during prolonged dark adaptation was raised by immersion with 1 mM glutamate. These results suggest that glutamate uptake mediated by GLAST could be regulated dynamically and efficiently in accord with dark/light-induced changes in glutamate release of retinal neurons.

Glycine receptors and transporters on bullfrog retinal Müller cells.

Glycine is a principal inhibitory neurotransmitter in the vertebrate retina. We have characterized glycine-induced currents from Müller cells in the bullfrog retinal slice preparation using whole-cell recordings. The glycine-induced current was partially suppressed by strychnine, and the remaining strychnine-resistant component was sacrosine-sensititve, suggesting that these two components may be mediated by glycine receptors and glycine transporters, respectively. Furthermore, the two components were maximal at the inner nuclear layer (INL) and declined asymmetrically as the application site was moved away from the INL to either the external or internal limiting membranes. It is suggested that Müller cells may be involved in glycinergic transmission by communicating with retinal neurons through these receptors and transporters.

[Effects of snow cover on water soluble and organic solvent soluble components during foliar litter decomposition in an alpine forest].

Seasonal snow cover may change the characteristics of freezing, leaching and freeze-thaw cycles in the scenario of climate change, and then play important roles in the dynamics of water soluble and organic solvent soluble components during foliar litter decomposition in the alpine forest. Therefore, a field litterbag experiment was conducted in an alpine forest in western Sichuan, China. The foliar litterbags of typical tree species (birch, cypress, larch and fir) and shrub species (willow and azalea) were placed on the forest floor under different snow cover thickness (deep snow, medium snow, thin snow and no snow). The litterbags were sampled at snow formation stage, snow cover stage and snow melting stage in winter. The results showed that the content of water soluble components from six foliar litters decreased at snow formation stage and snow melting stage, but increased at snow cover stage as litter decomposition proceeded in the winter. Besides the content of organic solvent soluble components from azalea foliar litter increased at snow cover stage, the content of organic solvent soluble components from the other five foliar litters kept a continue decreasing tendency in the winter. Compared with the content of organic solvent soluble components, the content of water soluble components was affected more strongly by snow cover thickness, especially at snow formation stage and snow cover stage. Compared with the thicker snow covers, the thin snow cover promoted the decrease of water soluble component contents from willow and azalea foliar litter and restrain the decrease of water soluble component content from cypress foliar litter. Few changes in the content of water soluble components from birch, fir and larch foliar litter were observed under the different thicknesses of snow cover. The results suggested that the effects of snow cover on the contents of water soluble and organic solvent soluble components during litter decomposition would be controlled by litter quality.

[Effects of snow patches on the release of N and P during foliar litter decomposition in an alpine forest of western Sichuan China].

A field experiment using litterbags was conducted in an alpine forest of western Sichuan in order to understand the effects of snow patches on the dynamics of N and P during decomposition of six representative species foliar litter in different periods of winter. Net N immobilization during foliar litter decomposition was observed in the whole snow cover season regardless of species. In contrast, P mainly released from foliar litter in the snow cover season, with a rapid rate of P release in the snow melt stage. Thick and moderate snow patches showed higher P release rates, but lower N release rates of foliar litter. The rate of N release was negatively related to daily mean temperature regardless of species, but the rate of P release was positively related to daily mean temperature with the exception of fir needle-litter. The decrease of snow cover in the scenario of global warming could inhibit P release but promote N release from foliar litter decomposition in winter in the alpine forest.

[Effect of Ca(OH)2 on the cytotoxicity of lipopolysaccharide extracted from Porphyromonas endodontalis in vitro].

PURPOSE: To detect the degradation of Ca(OH)2 on lipopolysaccharide (LPS) extracted from Porphyromonas endodontalis (P.e) in vitro and estimate the influence of P.e LPS pretreated with Ca(OH)2 on the proliferation of MC3T3-E1 cells. METHODS: The effect of Ca(OH)2 on MC3T3-E1 cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay. Then P.e LPS was treated with Ca(OH)2 for 30 mins or 60 mins at 37 degrees centigrade in vitro and the activity of P.e LPS was evaluated by Chromogenic End-point Tachypleus Amebocyte Lysate (CE TAL) test. Finally, MC3T3-E1 cells were exposed to P.e LPS pretreated with 15% Ca(OH)2 for 1, 3 and 5 d, and the cell proliferation was measured using the MTT assay comparing with the P.e LPS control group. SPSS 13.0 software package was used for statistical analysis. RESULTS: Compared with the negative control, exposing cells to 5%, 10% and 15% Ca(OH)2 had greatly promoted MC3T3-E1 cell proliferation. P.e LPS treated with 10% and 15% Ca(OH)2 both presented the best results by CE TAL and significant difference compared with P.e LPS control group. When 10 µg/mL P.e LPS was pretreated with 15% Ca(OH)2, no inhibition of MC3T3-E1 cell proliferation was noted. CONCLUSIONS: Ca(OH)2 detoxifies P.e LPS in vitro, mitigates the impact of P.e LPS on MC3T3-E1 cell proliferation. Supported by Science and Technology Projects of Liaoning Province (2011225020).

Comparative study between original and traditional method in establishing a chronic sinus node damage model in rabbit.

Sick Sinus Syndrome is a common and refractory arrhythmia, needing further study in which setting up a credible sinus node damage model is important. To explore the feasibility and superiority of an original formaldehyde pinpoint pressing permeation (FPPP) method for building a chronic sinus node damage (CSND) model, 5 rabbits were chosen from 35 as a sham-operation group, and the remaining were randomly divided into two groups: the formaldehyde wet compressing (FWC) group, in which models were established by applying a cotton bud dipped in 20% formaldehyde onto the sinus node (SN) area, and the FPPP group, in which models were established by injecting formaldehyde into the SN area through a self-made pinpointing and injecting electrode. We found that in both groups, the HR at 2 h, 24 h, 1 wk, and 2 wk after modeling decreased compared with premodeling; sinoatrial conduction time, sinus node recovery time, and corrected sinus node recovery time were prolonged compared with premodeling. The indexes mentioned shortened by 2 wk after modeling compared with 2 h in the FWC group, whereas they were stable after modeling in the FPPP group. The modeling achievement ratio in the FPPP group was higher and the death rate was lower. Under light microscope, paraffin sections of the SN tissue and cells showed severe injury in both groups. The results indicate that the CSND models in rabbits can be successfully established by the FPPP method, with higher achievement ratio, lower death rate, better stabilization effect, and less damaging comparing with the traditional method.