Objective To determine the genetic subtypes and susceptible population of Cryptoccocus neoformans in China. Methods We analyzed 129 strains of Cryptococcus neoformans isolated during the period from 1980 through 2006 in 18 Prov-inces and municipalities of central and eastern China using epidemiological method,PCR fingerprint,and multilocus sequence typing (MLST).Results About 71.5% (91/129)of the clinical strains were isolated from the patients without any recognizable immunodeficiency.Only 8.5% (11/129)of the strains were from AIDS patients.The remaining 20.9% (27/129)were from patients with underlying diseases other than HIV infection.Furthermore,PCR fingerprinting and MLST showed that serotype A strains exhibited a unique VNⅠsubtype,which was distinguishable from the reference VNⅠmolecular types.For convenience we named this unique genotype as VNⅠc.Conclusions People without any recognizable immunodeficiency are the main susceptible population of the 129 clinical strains of Cryptococcus neoformans ,which contrasts with the reports from other countries.The different genetic subtypes of Cryptococcus neoformans between China and other countries may contribute to such a difference.

Objective To review the clinical features and diagnosis of syphilitic aortic aneurysm .Methods A case of syphilitic aortic aneurysm was reported and the relevant literatures were reviewed .Results The patient showed progressive hoarse and heart murmurs and was diagnosed by computed tomographic angiography of aorta and syphilis serology test .In the era of antibiotics ,syphilitic aortic aneurysm is a very rare and dangerous case with covert onset ,various manifestations and high mortality .Conclusions The prevalence of late cardiovascular syphilis is increasing with the increase of early-onset syphilis and HIV epidemic .Syphilis serologic test should be available for the young patients with aortic aneurysm even though the risk factors of coronary heart disease are not present .Medical and surgical treatments for syphilis should be provided for the patients who are clinically suspected of syphilitic aortic aneurysm .

Objective To explore the mechanism of Cryptococcus neoformans mutant in CNLAC1and investigate the difference of CNLAC1between wild type(Mel+ )and the mutant (Mel- ).Methods The ab-normal PCR DNA product of Cryptococcus neoformans was cloned into sequencing vector PG EM-T for se-quencing.An expression vector was c onstructed for efficient expressio n in E.coli DH 5? .The gene was sequenced.Results The cloned DNA sequence was found to b e different between wild type(Mel+)and the mutant (Mel-)(between 1715bp and 2617bp).The differences were not only in len gth,but also in base sequence.Con-clusion The mechanism of the mutant in CNLAC1might be a change of sequence(between 1715bp and 2617bp),and not a DNA deletion.This finding might provide us a target gene for the prognosis and treatment of cryptococcosis.

The quorum sensing commonly exists in procaryote kingdom,regulating various biologic functions.Recently similar phenomenon was also found in fungi world,which affecting both biofilm formation and dimorphism.In this article,we focus on the recent progress on quorum sensing of pathogenic fungi and discuss the possibility of taking quorum sensing molecule as a potential target for antifungl therapy.

Objective To clarify the quorum sensing and identify the quorum sensing molecular of Cryptococcus neoformans. Methods The growth of Cryptococcus neoformans TUP1 △ strain at different inoculum size was observed. The culture filtrate of high cell density TUP1△ strain was collected and plated on medium with low density TUP1△ cell, and the growth of low cell density TUP1△ was recorded. Finally, the active molecule in culture filtrate was purified and identified.Results The growth of Cryptococcus neoformans TUP1△ strain was density dependent and the colony could be normally formed only above a specific density. The filtrate from a high cell density TUP1△culture could promote the growth of low cell density TUP1△, and the active molecule in this culture filtrate was identified to be an oligopeptide composed of 11 amino acids pepride, named quorum sensing peptide 1(QSP1). QSP1 was artificially synthesized, which could also promote the colony forming of TUP1△ strain inoculated at low cell density. Conclusion Quorum sensing exists in Cryptococcus neoformans, and the quorum sensing molecular is QSP1.

Objectives：To study the molecular mechanism of Cryptococcus neoformans mutation in the production of melanin.Methods：By comparing wild types(Mel+) and mutants(Mel-),we detected C.neoformans mutant laccase (CNLAC1) gene by PCR.We also cloned gene of the CNLAC1 containing about 450bp in length.E.coli DH5α was the host strain for the plasmid containing the DNA clone.Results：No deletion was found in wild type.However,CNLAC1 deletion (about 450 bp) was present (between 1 715 bp and 2 617 bp).CNLAC1 containing about 450 bp in length was cloned successfully.Conclusions：The present study was helpful in the biochemical and molecular study of CNLAC1 mutation.It might also provide us a target gene for the prognosis and gene therapy of Cryptocosis.

Objective:To study the role of RapIDYeast Plus (RYP) system in identifying common clinical yeasts.Methods: The target strains were cultured and passaged twice on Sabouraud dextrose agar,and were fed to RYP system after 24-48 h incubation at 30℃.Results: One hundred and thirty-nine of the 150 target strains were identified to the level of species correctly,and 8 undetermined strains were confirmed by additional tests.It was found that 3 strains had been incorrectly identified by RYP system.The accuracy of RYP system was calculated as 92% without additional tests and 98% with additional tests.Conclusion: RYP system is suitable for routine tests in clinical microbiological laboratory;it can accurately identify more than 40 kinds of yeasts and yeast-like bacteria in clinical practice.

To investigate the effect of catalpol on high-fat diet(HFD)-induced nonalcoholic fatty liver disease(NAFLD)and its underlying molecular mechanisms. Sixty C57BL/6J male mice were randomly divided into six groups:control group;HFD group;HFD+catalpol(100 mg/kg)group;HFD+catalpol(200 mg/kg)group;HFD+catalpol(400 mg/kg)group;and HFD+atorvastatin calcium(ATC)(30 mg/kg)group.The control group was fed a normal diet containing 4.4 kJ/g fat,whereas the other five groups were fed a high-fat diet containing 19.8 kJ/g fat.Mice in the catalpol or ATC treatment groups were administered by gavage for different doses of catalpol or ATC,whereas other mice were treated with saline.Body weight was measured once a week.Experiments were terminated after 18 weeks,and blood and liver samples were collected after an overnight fast(12 hours)for analysis.The body weight and liver weight were measured and the levels of serum total cholesterol(TC),triglyceride(TG),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),alanine aminotransferase(ALT),and aspartate transaminase(AST)as well as inflammatory factors tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,and IL-6 were determined by commercially available kits.Liver sections were stained with Oil Red O and HE to investigate the lipid accumulation and histopathological changes.The protein expressions of nuclear factor kappa-B(NF-κB)p65,inhibitor of nuclear factor kappa-B α(IκBα),B-cell lymphoma-2(Bcl-2),Bcl-2 associated x protein(Bax),and Caspase-3 were determined by Western blot. Compared to the model group,the body weight gains(all =0.001),liver index(=0.008,=0.001,=0.001),ALT(=0.004,=0.001,=0.001),and AST(=0.008,=0.001,=0.001)were significantly decreased in catalpol treatment groups,and the serum levels of TC(=0.005,=0.001),TG (all =0.001),and LDL-C(all =0.001)were also significantly decreased in middle and high dose groups,and the serum level of HDL-C was significantly increased in high group(=0.009).Moreover,compared to the model group,the degree of liver injury and lipid accumulation were obviously decreased in the catalpol treatment groups according to the pathology.Similarly,the release of inflammatory factors was significantly inhibited by the treatment with catalpol.The results of Western blot showed that the protein levels of NF-κB p65(=0.014,=0.001,=0.001)and Caspase-3(all =0.001)in the livers of HFD-fed mice were significantly reduced by catalpol treatment.In addition,the protein level of IκBα(=0.028,=0.001,=0.001)and the ratio of Bcl-2/Bax in high dose group(=0.003)was increased by treatment with catalpol. Catalpol can effectively improve the body weight gains,liver index,dyslipidemia,and lipid accumulation in HFD-fed mice and inhibit the release of inflammatory factors and hepatocyte apoptosis,thereby preventing the development of NAFLD induced by HFD.
Animals ; Diet, High-Fat ; Iridoid Glucosides ; Male ; Mice ; Mice, Inbred C57BL ; Non-alcoholic Fatty Liver Disease

Objective To analyze the differential expression of microRNA-146a (miR-146a) in monocyte-macrophage cell line (THP-1 cells) after induction by Cryptococcus neoformans (C.neoformans,reference strain WM148) or Cryptococcus gattii (C.gattii,reference strain R265),and investigate the mechanism of miR-146a in regulating the inflammatory response of cryptococcal meningitis.Methods The cultured THP-1 cells were divided into two groups to be induced by C.neoformans or C.gattii,respectively.THP-1 cells were induced with inactivated WN148 (or R265) strains at multiplicity of infection (MOI) of 5 in all experiments.The supematant and the cell pellet were collected separately after incubation.The expression of miR-146a was measured by real-time quantitative PCR (qRT-PCR) technique.The levels of TNF-u and IL-6 release were assayed by ELISA.Results The expression of miR-146a increased significantly in the C.neoformans induction group compared to 0 h.It reached peak at 3 h (P＜0.01),and then declined gradually.The level of TNF-α increased in supematant and reached peak at 12 h.The expression of IL-6 did not change significantly at each time point.The expression of miR-146a and TNF-α increased gradually and reached peak at 12 h in the C.gattii induction group (P ＜0.01),but the change did not reach statistical significance at 3 h,6 h time points.The expression of IL-6 gradually increased,and reached peak at 12 h time point.Conclusions Following stimulation with C.neoformans or C.gattii,the expression ofmiR-146a in THP-1 cells showed different patterns over time.The expression levels of TNF-α and IL-6 showed different patterns.These findings suggest that there may be different regulatory mechanisms in the THP-1 cells-associated inflammatory response after stimulation by inactivated C.neoformans and C.gattii strains.

Objective To explore the effects of triclosan (TCS) on the immune function of mice. Methods Forty male and female Kunming mice (25±2 g) were selected. The animals were divided into 5 groups according to body weight ratio, including a blank control (saline solution) group, dimethyl sulfoxide (DMSO) group, and three triclosan solution groups (59.375 mg/kg, 118.75 mg/kg, and 237.5 mg/kg, respectively). There were 8 mice in each group, half male and half female. Animals were treated with TCS by intragastric administration once a day for two weeks. Upon the completion of the treatment, animals were sacrificed, the spleen, thymus and other tissues were collected, and the ratios of their weight to body weigh were calculated. The peripheral blood was taken by eye-ball removal method, and the half hemolysis value was determined. Results Compared with the positive control group, the spleen index of male mice in the medium dose group and high dose group increased significantly (P < 0.05), and the spleen index of female mice in the high dose group showed significant difference (P < 0.05). Compared with the positive control group, the thymus index of male high dose group was significantly different (P < 0.05). The thymus indexes of female high, medium, and low dose groups all were significantly different compared to the control group (P < 0.05). HC50 results showed that the HC50 of both male and female mice decreased (P < 0.05). Conclusion High concentration of triclosan can inhibit the immune function of Kunming mice.