First Report of Colletotrichum brevisporum Causing Anthracnose of Dalbergia odorifera in China.

Dalbergia odorifera T. Chen (family Fabaceae) is one of four prized species of mahogany plant in China. In June 2017, an investigation of the condition of anthracnose was carried out on apporximately 333 hectares of D. odorifera plantations in Haikou City, Hainan Province (110.19°E, 20.03°N). Approximately 40% of D. odorifera plants had disease symptoms. Lesions on leaves were brown to grayish-white containing black dots and dark-brown borders, occasionally surrounded by a yellowish-green halo. Leaf spots generally occurred along the edge of the leaf. Severely infected leaves became withered and died. Hyphal growth was recovered from symptomatic leaf tissue, surface-sterilized with a 75% ethanol solution for 30s, rinsed with sterile distilled water, plated on potato dextrose agar (PDA), and incubated at 26°C in the dark. The representative isolate JXHTC19 was recovered by transferring a hyphal tip to a fresh PDA plate to obtain a pure culture. Fungal colonies had white aerial mycelium initially, turning pale gray after 3 days. At 7 days, colonies had a cottony appearance ranging from white to dark gray with orange masses of conidia. The colony surface was slimy and aerial mycelium was sparse. Isolates displayed single-celled, cylindrical, hyaline conidia that were rounded at both ends, and were 9.7 - 16.4 µm long (avg. 13.5 µm) × 3.6 - 6.2 µm wide (vg. 4.8 µm) (n = 100). To further identify the fungus, genomic DNA was extracted from single conidial cultures of JXHTC19. The rDNA internal transcribed spacer (ITS) region, glutamine synthetase (GS) gene, partial sequence of glyceraldeyde-3-phosphate dehydrogenase-like (GAPDH) gene, actin (ACT) gene, and beta-tubulin (TUB2) gene were amplified using the primer pairs ITS4/ITS5, GS-F/GS-R, GDF1/GDR1, ACT-512F/ACT-783R, and TUB2-T1/Bt-2b (Weir et al 2012), respectively. The results showed that the ITS, GS, GAPDH, ACT and TUB2 genes of the target strain (JXHTC19) have 100%, 95%, 100%, 97% and 98% sequence homology with C. brevisporum, respectively. The sequences obtained were deposited in GenBank (MF993572, MN737615, MN737614, MG515612, and MG515615[LJ1]). All five sequences were analyzed together with representative sequences from type or ex-type specimens of the Colletotrichum genus (Yang et al. 2011, Weir et al. 2012) and a phylogenetic tree was generated via the neighbor-joining method using MEGA6. The tree placed the isolate in the same group as C. brevisporum. Thus, both morphological and molecular characteristics identified the pathogen as C. brevisporum. To verify Koch's postulates, two-year-old leaves of healthy potted D. odorifera plants (n = 6) were inoculated with a spore suspensions of JXHTC19 that contained 105 conidia/ml. Plants were sprayed with water to serve as mock-inoculated controls [LJ2](Garibaldi et al, 2020). Six plants per treatment were used in each test. The test was repeated once.Plants were incubated in moist chambers at 26°C and monitored daily for symptom development. After five days, eleven of twelve isolates [LJ3]caused lesions on all inoculated plants, whereas no symptoms developed on the mock-inoculated controls. Koch's postulates were fulfilled by reisolating the same fungus and verifying its colony and morphological characters as C. brevisporum. To our knowledge, this is the first report of this species causing anthracnose of D. odorifera in China. Corresponding measures must be adopted to manage this disease such as reducing the planting density of D. odorifera and increasing the species diversity of undergrowth vegetation. These results could help develop better monitoring and management practices for this disease.

[Characteristics of Light Absorption, Sources, and Seasonal Variation of Atmospheric Brown Carbon in Northern Nanjing].

Brown carbon (BrC) refers to a group of organic compounds in fine atmospheric particles (PM2.5) that are able to absorb light in the ultraviolet and visible range. They have a significant impact on the visibility of air and on the earth's climate. In this study, we used a black carbon analyzer (Model AE33) to conduct field measurements in northern suburban Nanjing from March 2021 to February 2022. We measured the light absorption coefficients of BrC in PM2.5 and quantified the contributions of primary (BrCpri) and secondary brown carbon (BrCsec) in BrC by using the minimum correlation method (MRS), combined with the backward trajectories,potential source contribution function (PSCF) analysis, and diurnal patterns to analyze the seasonal characteristics of BrC. The results showed that the annual average light absorption of BrC was(7.76±7.17)Mm-1 (at 370 nm), and its contribution to the total aerosol light absorption was (22.0±8.8)%. BrC light absorption at different wavelengths all showed a U-shape seasonal variation of high in spring and winter and low in summer and fall. MRS analysis showed that the annual average contributions of BrCpri and BrCsec were (62.9±21.4)% and (37.1±21.4)% (at 370 nm), respectively; however, the contribution of BrCsec increased with the increase in wavelength, and it became dominant in longer wavelengths such as 660 nm. Backward trajectory and PSCF analysis showed that BrC was heavily influenced by air masses from the sea in spring, summer, and fall but was influenced greatly by local and regional continental emissions in winter. Traffic emissions in spring, summer, and fall were more intense to contribute to BrCpri than that in winter, whereas coal and biomass combustion had a greater impact on BrCpri in winter. Detailed analysis revealed that gas-phase photochemistry and aqueous chemistry had different influences on BrCsec formation in different seasons. It was mainly from gas-phase photochemistry in summer but was dominated by aqueous process in winter; both processes, however, were important pathways to BrCsec in spring and fall.

Ursodeoxycholic acid alleviates experimental liver fibrosis involving inhibition of autophagy.

AIMS: Ursodeoxycholic acid (UDCA) has been widely used in the treatment of primary biliary cholangitis (PBC) with chronic liver fibrosis, but its detailed mechanism remains unclear. This study was aimed to determine whether autophagic signaling is involved in the therapeutic effect of UDCA on liver fibrosis. METHODS: By using hepatic stellate cell (HSC) line LX2 and CCl4-induced fibrotic rat model, autophagy signaling was investigated by western blotting and mRFP-EGFP-LC3 tandem fluorescent tagged plasmid (ptfLC3) transfection technique. Anti-fibrotic profile was determined by western blotting, qRT-PCR, MTT assay, trypan blue, hydroxyproline assay and Masson staining. KEY FINDINGS: TGFß1 treatment decreased P62 accumulation and increased both autophagosomes and autolysosomes in LX2 cells, thereby elevated autophagic flux. Hydroxychloroquine (HCQ), antagonist of autophagy, was found to dramatically inhibit COL1A2 mRNA expression and cell proliferation in a dose-dependent manner. This coincides with the effect of UDCA intervention on collagen aggradation and cell viability. Meanwhile, UDCA inhibited TGFß1-induced autophagy flux. And rapamycin, agonist of autophagy, was found to impair the anti-fibrotic effect of UDCA. Moreover, study in vivo showed that UDCA alone or in combination with HCQ restored the CCl4-induced liver fibrosis in rodent models with autophagy inhibited profile. SIGNIFICANCE: Taken together, our study revealed that UDCA displays anti-fibrotic role by protecting HSC against production of collagen and inhibiting cellular viability involving autophagy inhibition and provide a new insight into the pharmacological basis of UDCA treatment for hepatic fibrosis.

Autophagy inhibition contributes to the synergistic interaction between EGCG and doxorubicin to kill the hepatoma Hep3B cells.

(-)-Epigallocatechin-3-O-gallate(EGCG), the highest catechins from green tea, has promisingly been found to sensitize the efficacy of several chemotherapy agents like doxorubicin (DOX) in hepatocellular carcinoma (HCC) treatment. However, the detailed mechanisms by which EGCG augments the chemotherapeutic efficacy remain unclear. Herein, this study was designed to determine the synergistic impacts of EGCG and DOX on hepatoma cells and particularly to reveal whether the autophagic flux is involved in this combination strategy for the HCC. Electron microscopy and fluorescent microscopy confirmed that DOX significantly increased autophagic vesicles in hepatoma Hep3B cells. Western blot and trypan blue assay showed that the increasing autophagy flux by DOX impaired about 45% of DOX-induced cell death in these cells. Conversely, both qRT-PCR and western blotting showed that EGCG played dose-dependently inhibitory role in autophagy signaling, and that markedly promoted cellular growth inhibition. Amazingly, the combined treatment caused a synergistic effect with 40 to 60% increment on cell death and about 45% augmentation on apoptosis versus monotherapy pattern. The DOX-induced autophagy was abolished by this combination therapy. Rapamycin, an autophagic agonist, substantially impaired the anticancer effect of either DOX or combination with EGCG treatment. On the other hand, using small interference RNA targeting chloroquine autophagy-related gene Atg5 and beclin1 to inhibit autophagy signal, hepatoma cell death was dramatically enhanced. Furthermore, in the established subcutaneous Hep3B cells xenograft tumor model, about 25% reduction in tumor growth as well as 50% increment of apoptotic cells were found in combination therapy compared with DOX alone. In addition, immunohistochemistry analysis indicated that the suppressed tendency of autophagic hallmark microtubule-associated protein light chain 3 (LC3) expressions was consistent with thus combined usage in vitro. Taken together, the current study suggested that EGCG emerges as a chemotherapeutic augmenter and synergistically enhances DOX anticancer effects involving autophagy inhibition in HCC.

Expression of the Pokemon proto-oncogene in nasopharyngeal carcinoma cell lines and tissues.

To study the differentiated expression of the proto-oncogene Pokemon in nasopharyngeal carcinoma (NPC) cell lines and tissues, mRNA and protein expression levels of CNE1, CNE2, CNE3 and C666-1 were detected separately by reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and Western-blotting. The immortalized nasopharyngeal epithelial cell line NP69 was used as a control. The Pokemon protein expression level in biopsy specimens from chronic rhinitis patients and undifferentiated non keratinizing NPC patients was determined by Western-blotting and arranged from high to low: C666-1>CNE1>CNE2> CNE3>NP69. The Pokemon mRNA expression level was also arranged from high to low: CNE1>CNE2>NP69>C666-1>CNE3. Pokemon expression of NP69 and C666-1 obviously varied from mRNA to protein. The Pokemon protein level of NPC biopsy specimens was obviously higher than in chronic rhinitis. The data suggest that high Pokemon protein expression is closely associated with undifferentiated non-keratinizing NPC and may provide useful information for NPC molecular target therapy.