Objective To investigate the effects of ischemic postconditioning on mitochondrial permeability transition and mitochondrial transmembrane potential(△Ψm)following hepatic ischemia-reperfusion(I/R)in rats.Methods Forty male SD rats weighing 220-260 g were randomly divided into 5 groups with 8 animals in each group:sham operation group(group S);atractyloside+sham operation group(group A+S);I/R group;ischemic postconditioning group(group IPO)and atractyloside+ischemic postconditioning group(group A+IPO).The animals were anesthetized with intramuscular injection of atropine 0.05 mg/kg.Hepatic I/R was produced by occlusion of hepatic blood flow for 60 min followed by 6 h reperfusion.In group A+S,atractyloside 5 mg/kg was injected intravenously before abdomen Was closed.In group IPO,the animals were subjected to 3 cycles of 1 min reperfusion interspersed with 1 min hepatic isehemia at the end of 60 min hepatic ischemia.In group A+IPO,atractyloside 5 mg/kg was injected intravenously before reperfusion. Venous blood samples were collected for determination of serum ALT and AST activities immediately before ischemia and at 6 h of reperfusion. The animals were then sacrificed.Their livers were removed for microscopic examination, detection of apoptosis and determination of cytochrome c (Cyt c) expression, △Ψm and mitochonerial permeability transition pore (MPTP)activity. Apoptosis index (AI) was calculated. Results There was no significant difference in serum ALT and AST activities, AI, Cyt c expression, △Ψm and MPTP activity between S and A + S groups (P＞0.05). Compared with group S, serum ALT and AST activities and AI were significantly increased, Cyt c expression was up-regulated, △Ψm was decreased and MPTP activity was increased in groups I/R, IPO and A+IPO(P＜0.05).Compared with group I/R, serum ALT and AST activities and AI were significantly decreased,Cyt c expression was down-regulated, △Ψm was increased and MPTP activity was decreased in group IPO(P＜0.05), while no significant change was found in group A+IPO(P＞0.05).Compared with group IPO,serum ALT and AST activities and AI were significantly increased, Cyt c expression was up-regulated, △Ψm was decreased and MPTP activity was increased in group A + IPO(P＜ 0.05).Microscopic examination showed that hepatic injury was reduced in group IPO compared with group I/R, while aggravated in group A+ IPO compared with group IPO. Conclusion Ischemic postconditioning can protect liver from I/R injury by attenuating the I/R-induced increase in MPTP opening and decrease in △Ψm in rats.

Objective To develop a murine model for infection by Cladosporium carrioni. Methods A total of 72 ICR mice were equally divided into 4 groups, group A (healthy mice inoculated by C. Carrioni suspension of 1 × 108 cfu conidia mL-1, group B (immune-suppressed mice inoculated by C. Carrioni sus pension of 1 × 108 cfu conidia mL-1), group C (immune-suppressed mice inoculated by C. Carrioni suspen-sion of 1 × 106 cfu conidia mL-1), group D (healthy mice inoculated by sodium chloride solution). C. Car-rioni suspension or sodium chloride solution was subcutaneously inoculated into foot pads of mice. On day 7, 30 and 60 after inoculation, 6 mice were killed in each group followed by the measurement of thickness of foot pads, pathology and mycology of skin samples taken from foot pads. Results In group A, B and C, there were swelling, blackening, ulceration and crusts at the inoculation site of all mice, with a morbidity of 100%. The thickness of foot pads in group A on day 30 was significantly higher than that on day 7 (2.40 ± 0.45 mm vs 2.85 ± 0.47 mm, P < 0.05), but lower than that on day 60 (1.64 ± 0.13 ram, P < 0.05). In group B, increased thickness of foot pads was observed on day 30 compared with that on day 7 and day 60 (2.19 ± 0.27 mm vs 1.80 ± 0.21 mm and 1.86 + 0.22 mm, respectively, both P < 0.05), which was the case with group C (1.98 ± 0.06 nun vs 1.51 ± 0.11 mm and 1.82 ± 0.09 mm, respectively, both P < 0.05). No significant changes occurred to the thickness of foot pads in group D from day 7 to day 60 (P > 0.05). Pathological changes in group A, B and C included necrosis, abscess and chronic granuloma formation; dark brown sclerotic bodies were observed on HE and PAS staining as well as on direct microscopy; cultures of tissue samples grew Cladosporium carrionii. The mice in group D remained uninfected. Conclusion Mouse model for chromoblastomycosis may be established by subcutaneous inoculation of Cladosporium carrionii suspension into foot pads of healthy or immuno-suppressed mice.

Objective To estimate the application value of colony PCR in the detection of pathogenic filamentous fungi. Methods Colony PCR was established and performed to amplify the internal transcribed spacer (ITS) region of 19 species (strains) of filamentous fungus followed by sequencing analysis. At the same time, DNA extracts from 8 of the 19 species of filamentous fungus were subjected to conventional PCR. Hha I and Hinf I endonucleases were used for restriction fragment length polymorphism (RFLP) analysis of the conventional and colony PCR products. Comparison analysis was carried out between the colony and conventional PCR. Results Of the 19 strains, 16(84.2%) yielded positive results by colony PCR; sequence analysis of the PCR products of ITS region revealed a 96% - 100% similarity with the reference sequence (NCBI database)of corresponding fungi. The amplification product length and RFLP profile of these products from the 8 species of filamentous fungus, except for those from Aspergillus nidulans, were consistent between the colony and conventional PCR. Conclusions Compared with conventional PCR, colony PCR-based detection of filamentous fungi is easy to operate, time and labor-saving, with high accuracy and reliability, and can be applied to the rapid identification of filamentous fungi.

Objective To investigate the growth inhibition of Aspergillns fumigatus by Candida albicans in vitro and to develop the selective medium for clinical isolation of Aspergillus fumigatus.Methods Aspergillus fumigatus and Candida albicans were single or co-cultured in sabouraud dextrose agar(SDA) medium and SDA broth in dark at 25 ℃,and fungal growth,pigmentation,as well as colony diameter weredocumented.Results ①The sensitivity of culture of Aspergillus fumigatus and Candida albicans on SDAplate was 100CFU/ml.②The growth of 106CFU/ml and 103CFU/ml Aspergills fumigatus was completely inhibited by 106CFU/ml Candida albicans.③Growth inhibition of Aspergillus fumigatus was correlated with the concentration of Candida albicans.④SDA containing 1 mg/L fluconazole inhibited growth of Candida albicans,and no Candida albicans was detected on SDA containing 5 mg/L and 25 mg/L fluconazole.Growth of Aspergillus fumigatus was partially inhibited on SDA containing 25 mg/L fluconazole.Conclusions Candida albicans can inhibit the growth of Aspergillus fumigatus in vitro.SDA containing 5 mg/L fluconazole can be used as the selective medium for the isolation of Aspergillus fumigatus.

Objective To investigate the effect of electron transfer system on the hyphal formation of Candida albicans. Methods Candida albicans was cultured in RPMI 1640 supplemented with 10% new-born calf serum in 5% CO2 at 37 ℃ with or without the presence of inhibitors or activators of electron transfer system. Growth curve, morphology and percent of filamentation were observed for Candida albicans. MTT assay was used to assess the viability of Candida albicans. Results The solvents (chloroform and dimethyl sulfoxide) had no significant effect on the growth of and filamentation in Candida albicans. After incubation with thenoyltrifluoroacetone (TTFA) or benzhydroxamic acid for 24 hours, yeast cells of Candida albicans predominated in the culture. The growth of Candida albicans was significantly inhibited in log phase by the incubation with classic respiratory chain inhibitors such as rotenone, antimycin A, oligomycin, sodium azide, TTFA and sodium malonate, compared with the controls (all P < 0.01). Benzhydroxamic acid, an inhibitor of alternative oxidative pathway, also significantly inhibited the growth of Candida albicans in log phase (t = 10.92, P < 0.01). After incubation with rotenone, antimycin A, oligomycin, sodium azide, TTFA, sodium malonate, benzhydroxamic acid and disodium gnanylate, the percentage of filamentation in Candida albicans at 12 hours was 87.49 ± 0.52, 48.75 ± 4.44, 50.33 ± 8.50, 99.00 ± 1.00, 1.60 ± 0.53, 94.01 ± 0.99, 0.00 ± 0.00 and 92.33 ± 2.08, respectively, and the growth of Candida albicans at 7 hours was inhibited by (1.34 ± 0.15)%, (70.61 ± 1.02)%, (50.63 ± 5.38)%, (17.80 ± 7.89)%, (45.17 ± 1.27)%, (10.75 ± 3.62)%, (72.46 ± 1.14)% and -(5.96 ± 4.07)%, respectively. Conclusions Hyphal formation of Candida albicans could be suppressed by inhibitors of classic respiratory chain or alternative oxidative pathway, and is mainly regulated by alternative oxidative pathway.

Objective To report a case of vaginal colonization due to Trichosporon inkin. Methods A 34-year-old female presented with increased vaginal discharge accompanied by abnormal odor for 2 months. Clinical laboratory examination was carried out. Cultures of vaginal discharge yielded yeast-like colony. Subsequently, the isolate underwent the following mycological examinations: purification, slide micro-culture, temperature test, urea enzyme test, biochemistry identification, antifungal susceptibility test, and gene sequencing. Results Gynecological examination revealed white homogeneous secretions attached to mucous membrane of the vagina. Nugent scores of vaginal discharge amounted to 5-6. Two rounds of culture of vaginal discharge resulted in stramineous, reductus and yeast-like colony. The isolate could grow in 42 ℃. Appressorium on the top of hypha and typical sarcinae formed in slide microculture of corn agar, and yeast malt agar was the optimal growth medium for it. Urea enzyme test was positive. API 20C AUX biochemical test and gene sequencing revealed that the isolate was consistent with Trichosporon inkin. The isolate was sensitive to amphotericin B and azoles such as clotrimazole and fluconazole, but resistant to flucytosine and caspofungin. Conclusions It is the first report of vaginal colonization due to T. Inkin in China. The accu-rate identification of T. Inkin relies on synthetic analysis of phenotype characteristics, biochemistry test and molecular sequencing.

Objective To develop a PCR-RFLP method to rapidly identify filamentous fungi causing deep infection. Methods Universal fungal primers were used to amplify the internal transcribed spacer (ITS) region of Aspergillus fumigatus, Aspergillus Bavus, Aspergillus terreus, Aspergillus niger, Aspergillus versicolor, Aspergillus nidulans, Scedosporium apiospermum and Fusarium moniliforme followed by restriction fragment length polymorphism (RFLP) analysis with restrictive endonucleases Hha I, Hae III, Hinf I, Taq I and Msp I. Then, 22 clinical and 2 environmental fungal isolates were identified with the developed PCR-RFLP method. Results The RFLP analysis of PCR products with restrictive endonucleases Hha I and Hinf I allowed discrimination of 8 filamentous fungi causing invasive infection, and it took only 1 day to carry out the whole procedure from DNA extraction to PCR and restriction digestion. The identification results of 22 clinical strains and 2 environmental isolates with this PCR-RFLP method were completely consistent with those with conventional morphological method. Conclusion PCR-RFLP analysis is an efficient method for rapid identification of cultured filamentous fungi.

Objective To compare broth microdilution and agar dilution methods for in vitro testing of activities of fluconazole,ketoconazole and itraconazole against clinical Malassezia isolates.Methods Broth microdilution and agar dilution methods were used to determine the minimal inhibitory concentration(MIC)of fluconazole,ketoconazole and itraconazole for 27 clinical strains(5 species)of Malassezia.Results The minimal inhibitory concentration(MIC)ranges of fluconazole,ketoconazole and itraconazole were 0.25-≥64 mg/L,≤0.03-0.5 mg/L and ≤0.03-0.125 mg/L respectively as shown by broth microdilution method,2-≥64 mg/L,≤0.03-0.5 mg/L and ≤0.03-0.25 mg/L respectively as revealed by agar dilution method.Both methods demonstrated that itraconazole possessed the strongest activity against Malassezia species,followed by ketoconazole and fluconazole.The agreement rate in MICs between the two methods was 78.8％,85.2％ and 88.9％,respectively for fluconazole,ketoconazole and itraconazole,with the intraclass correlation coefficients (ICCs)being 0.88,0.80 and 0.76 respectively.Conclusions Fluconazole,ketoconazole and itraconazole are highly active against Malassezia species in vitro,and itraconazole is the most active.Broth microdilution and agar dilution method coincide well in,and are applicable for,the antifungal susceptibility testing of Malassezia species in vitro.

Objective To determine the extracellular enzymatic activity of common pathogens for onychomycosis, in the hope of finding virulence factors associated with the pathogenesis of onychomycosis. Methods Strains tested in this study included standard strains of common dermatophyte and non-dermatophyte fungi as well as clinical isolates of Trichophyton rubrum from patients with onychomycosis. All the tested strains were cultured in medium containing nail fragments at 25 ℃ for 10 to 21 days followed by the determination of the nail fragment-containing medium, a significant increase was observed in the activities of esterase, esterase lipase, N-acetyl-β-glucosaminidase and α-mannosidase in dermatophytes compared with non-dermatophytes (all P ＜ 0.05 ), as well as in the activity of N-acetyl-β-glucosaminidase in Trichophyton rubrum compared with the other tested species of fungi (all P ＜ 0.05). No significant difference was noted in the activity of extracellular enzymes, except for that of naphthol-AS-BI-phosphohydrolase, between the isolates of Trichophyton rubrum from patients with different ranges of scoring clinical index for onychomycosis (SCIO). Conclusions In specific conditions, the extracellular enzymatic activity of fungi isolated from patients with onychomycosis is associated with fungal species, and may have a certain influence on the manifestations of anychomycosis.

A 19-year-old man was admitted to the hospital for erythema and nodules on the face and postauricular region for 6 years. Microscopic examination of lesion scrapings revealed brown septate hyphae. A restricted, velvety and black colony grew on Sabouraud's dextrose agar. Slide culture on potato dextrose agar plate showed flask-shaped phialides at the apex of or around the hyphae with clear collarettes and flaring apex,mucilage-encapsuled, round to oval, semi-endogenous phialosporae accumulating at the apex of the phialides,giving a flower-like appearance. Anti-fungal susceptibility test showed that the fungus was sensitive to itraconazole, terbinafine and amphotericin B, but resistant to fluconazole. Sequence analysis of the ITS1-ITS4 region revealed a 98% consistency with the reference sequence of ITS1-ITS4 of Phialophora verrucosa. On the basis of above findings, the patient was diagnosed with cutaneous phaeohyphomycosis. Clinical improvement was seen after treatment with oral itraconazole (400 mg/d).