[Differential proteomic screening of plasma exosomes before and after magnesium isoglycyrrhizinate treatment in chronic hepatitis B].

Objective: To screen the differential proteomic of plasma exosomes before and after magnesium isoglycyrrhizinate (MgIG) treatment in chronic hepatitis B patients. Methods: Plasma samples were collected from 36 cases with chronic hepatitis B before and after MgIG treatment (2 ml/case). Plasma exosomes were extracted by ultracentrifugation. Exosomal particles concentration and inner diameter were detected by Nanosight NS300 particle size analyzer. Three cases of plasma exosomes were randomly selected before and after MgIG treatment. Proteins were extracted after lysis and digested with trypsin. Label-free differential proteomics analysis was performed by liquid chromatography-tandem mass spectrometry to screen out differential proteins that changed more than 1.5 times. Enzyme linked immunosorbent assay (ELISA) was used to verify the quantitative differential protein expression (n = 30). Measurement data were compared by paired sample t-test. Results: The average particle concentration of the extracted exosomes was 2.2×10(9)/ml, and the average size was (107 ± 52) nm, which was consistent with the theoretical value of plasma exosome size, proving that the plasma exosomes were successfully extracted. Proteomics results showed that before and after MgIG treatment in chronic hepatitis B patients, a total of 153 differentially expressed proteins were screened, including 85 up-regulated and 68 down-regulated proteins. Enzyme-linked immunosorbent assay results showed that compared with the MgIG before and after treatment group of chronic hepatitis B patients, the differences in the concentrations of hepatocyte growth factor activator and hepatocyte growth factor like protein in plasma exosomes were statistically significant (P < 0.05). Hepatocyte growth factor activator concentration in the plasma exosomes before and after MgIG treatment group was (45.9 ± 9.4) µg/ml and (13.9 ± 2.0) µg/ml, respectively, and it was down-regulated by about 3 times. Hepatocyte growth factor-like protein concentration in the plasma exosomes before and after MgIG treatment group was (23.4 ± 4.9) µg/ml and (13.8 ± 2.2) µg/ml, respectively, and it was down-regulated by about 2 times. Enzyme-linked immunosorbent assay results had consistency with the proteomics results. Conclusion: This study successfully screened the differential proteomic of plasma exosomes before and after MgIG treatment in chronic hepatitis B, and provided experimental basis for studying the molecular mechanism of MgIG treatment for chronic hepatitis B.

First Report of Cylindrocarpon destructans var. destructans Causing Black Root Rot of Sanqi (Panax notoginseng) in China.

Sanqi (Panax notoginseng (Burk.) F. H. Chen) is planted on >10,000 ha in China and is a popular Chinese medicinal material (2). Black root rot is a recently identified but worsening problem on Sanqi since 2010 in Wenshan, China. Of the plant tubers examined from 185 ha, 8.5 to 27.4% were black with necrotic lesions. The base of leaves of infected plants had brown, sunken, necrotic lesions, and symptomatic plants had one to three chlorotic leaves. A fungus was isolated consistently from the basal leaves, bulb, and tubers of symptomatic plants. Six single-spore isolates were cultured on potato sucrose agar (PSA) at 25 ± 1°C in the dark. The mycelium of each culture was white initially on PSA, and then became rust-colored. The adaxial surfaces of the plates were black. Conidiophores were 13.6 to 167.3 × 1.4 to 21.8 µm (avg. 68.6 × 2.9 µm), single or with up to four levels of branching and two to three branches (or phialides) per level. The basal branches were often divergent, whereas the terminal branches were usually more appressed. Sporodochia were not present. Microconidia were 0-septate, 4.1 to 9.5 × 2.7 to 4.1 µm (avg. 8.2 × 2.9 µm). Conidia were 1- to 3-septate and occasionally 4-septate. One- to 3-septate conidia were clavate, with a truncate or slightly protruding conidial base, 9.2 to 40.8 × 3.5 to 6.8 µm (avg. 26.7 × 5.2 µm); whereas 4-septate conidia were 32.6 to 50.3 × 5.4 to 6.8 µm (avg. 40.9 × 6.5 µm). Chlamydospores were abundant, golden to brown, single or in chains or clumps, and up to 21.8 µm in diameter. PCR amplification was carried out for one isolate, RR926, using rDNA internal transcribed spacer (ITS) primer pairs ITS1F and ITS4 (4). Sequencing of the PCR product (GenBank Accession No. KC904953) revealed 99% similarity (99% coverage) with the ITS sequence of Cylindrocarpon destructans var. destructans (AM419065). Phylogenetic analysis (MEGA 4.1) using the neighbor-joining algorithm placed the isolate in a well-supported cluster (>90% bootstrap value based on 1,000 replicates) with AM419065. Therefore, the pathogen was identified as C. destructans (Zinssm.) Scholten var. destructans (teleomorph Ilyonectria radicicola (Gerlach & L. Nilsson) P. Chaverri & C. Salgado) based on morphological characteristics and rDNA-ITS sequence analysis (1,3). Pathogenicity tests of the six isolates were conducted on five 1-year-old and five 3-year-old plants/isolate. The roots of all plants were washed with sterilized water, and then surface-sterilized with 75% ethanol. Inoculum (1 ml of 106 conidia/ml) of each isolate was brushed onto the roots of each plant with a paintbrush. Inoculated plants were planted in pots in a mixture of sterilized quartz sand:vermiculite:pearlite (2:1:1, v/v). The pots were placed under black shadecloth. The roots of five 1-year-old and five 3-year-old plants were brushed similarly with sterilized water as control treatments. After 30 days, symptoms similar to those on the original diseased plants were observed on the roots of all plants inoculated with the six isolates. The roots of non-inoculated plants remained healthy. The experiment was repeated. The same pathogen was re-isolated from the inoculated plants, but no pathogen was isolated from roots of the control plants. C. destructans var. destructans is widely distributed in soils (1), but to our knowledge, this is the first report of this fungus causing black root rot of Sanqi in China. References: (1) P. Charerri et al. Stud. Mycol. 68:57, 2011. (2) C. Y. Hu. New Rural Technol. 2:59, 2013 (in Chinese). (3) K. A. Seifert and P. E. Axelrood. Can. J. Plant Pathol. 20:115, 1998. (4) K. A. Seifert et al. Phytopathology 93:1533, 2003.

Physiological and biochemical responses of in vitro Fusarium oxysporum f.sp. niveum to benzoic acid.

The allelopathic potential of an artificially applied allelochemical, benzoic acid, on in vitro Fusarium oxysporum f.sp. niveum (a soil-borne pathogen causing watermelon wilt) was evaluated. Benzoic acid strongly inhibited its growth, sporulation and conidia germination, whereas it stimulated virulence factors of this pathogen. The biomass was reduced by 83-96 % and the conidia germinating rate and conidia production rate were decreased by 100 % at a concentration of >200 mg/L. However, phytopathogenic enzyme activities and mycotoxin production were stimulated with an increase of 10.2-1250 % for enzyme activities and 610-2630 % for mycotoxin yield.