Inhibition mechanism of cordycepin and ergosterol from Cordyceps militaris Link. against xanthine oxidase and cyclooxygenase-2.

Cordyceps militaris Link. (C. militaris) is an entomopathogenic fungus that parasitizes the pupa or cocoon of lepidopteran insect larvae, with various bioactive compounds. Cordycepin and ergosterol are the two active components in C. militaris. This study aimed to evaluate the inhibitory activity of cordycepin and ergosterol against xanthine oxidase (XO) and cyclooxygenase-2 (COX-2), as well as investigate the inhibition mechanism. Cordycepin could better inhibit XO (IC50 = 0.014 mg/mL) and COX-2 (IC50 = 0.055 mg/mL) than ergosterol. Additionally, surface hydrophobicity and circular dichroism (CD) spectra results confirmed the conformational changes in enzymes induced by cordycepin and ergosterol. Finally, cordycepin and ergosterol significantly decreased uric acid (UA) and inflammatory factors to normal level in mice with gouty nephropathy (GN). This study could provide theoretical evidence for utilization of C. militaris in hyperuricemia-management functional foods.

[Clinical efficacy and safety of vancomycin compared with linezolid for the treatment of neonatal gram-positive bacterial sepsis].

OBJECTIVE: To evaluate the clinical efficacy and safety of vancomycin and linezolid for the treatment of gram-positive neonatal bacterial sepsis. METHOD: The data of neonates diagnosed as gram-positive bacterial sepsis in neonatology department of Suzhou Municipal Hospital from June 2009 to December 2015 were retrospectively collected. These neonates were divided into vancomycin group and linezolid group. Propensity score matching (PSM) on baseline variables was used to balance the two groups by identifying a comparable group of neonates who received vancomycin and linezolid therapy. Clinical and microbiologic success rates were compared by chi-square test, and changes of laboratory parameters before and after treatment at the end of treatment were then directly compared by rank-sum test between the matched groups. In vancomycin group, correlation between trough concentration of vancomycin and clinical efficacy were evaluated. RESULT: Totally 108 and 209 cases were respectively selected in vancomycin and linezolid groups; 108 cases with well-matched baseline characteristics were included in matched linezolid group. The clinical success rates of vancomycin vs. linezolid therapy were 86.1%(93/108) and 88.9%(96/108)(P=0.681), and the microbiologic success rates were 91.7%(99/108) and 93.5%(101/108)(P=0.795). The average trough concentration of vancomycin was (12±8) mg/L. The rate of reaching the high trough concentration standard was only 33.3%(36/108). In 50 (46.3%) cases the dose was adjusted according to the initial concentration data. Compared to less than 10 mg/L, the clinical efficacy of trough concentration in 10-20 mg/L was much higher(93.9%(46/49) vs. 78.6%(33/42), P=0.031). Total bilirubin and platelet count had significant difference between the two-matched groups (34.1(14.9, 91.0)µmol/L vs. 53.0(27.0, 121.6)µmol/L, P=0.034; 301.0(198.8, 416.0)×10(9)/L vs. 195.5(94.0, 283.2)×10(9)/L, P=0.000). The incidence of linezolid related thrombocytopenia was 13.4%(28 cases). CONCLUSION: The clinical effect of vancomycin and linezolid on gram-positive bacterial sepsis in neonates is comparable. The rate of trough concentration of vancomycin reaching the high trough concentration standard is low, and the clinical efficacy is related to trough concentration. Linezolid have an effect on bilirubin and platelet count, the risk of thrombocytopenia should be monitored closely during linezolid treatment.

MTBDRplus results correlate with treatment outcome in previously treated tuberculosis patients.

BACKGROUND: Although MTBDRplus is validated for the detection of multidrug-resistant tuberculosis (MDR-TB), its role in the assessment of treatment outcome is less clear. We evaluated the association of MTBDRplus results with treatment outcome in new and previously treated patients in an endemic setting in China and determined factors associated with poor treatment outcomes. METHODS: We prospectively enrolled 298 smear-positive pulmonary TB patients who received the World Health Organization recommended initial treatment regimen or retreatment regimen. MTBDRplus was compared with conventional drug susceptibility testing and DNA sequencing for the detection of MDR-TB. Treatment responses were monitored using sputum smear, culture and chest radiography. RESULTS: MTBDRplus successfully identified all MDR-TB and had good concordance with sequencing. MDR-TB rates were low among new patients (4/187, 2.1%), but high in previously treated patients (12/28, 42.9%); 65.2% (15/23) of previously treated cases and 17.1% (27/158) of new cases were unsuccessfully treated (P < 0.001). Seven of eight (87.5%) previously treated MDR-TB patients failed the retreatment regimen. In addition to drug resistance, sputum smear positivity at week 8 and cavitation are associated with treatment failure. CONCLUSION: Not only did MTBDRplus correctly identify all MDR-TB cases, MTBDRplus results are also associated with treatment outcomes in previously treated patients. The retreatment regimen should no longer be used; treatment should be guided by molecular testing.

Aspergillus vertebral osteomyelitis and ureteral obstruction after liver transplantation.

Aspergillus osteomyelitis has been reported as a result of dissemination in solid organ transplant recipients. Vertebral osteomyelitis is one of the most common forms of Aspergillus osteomyelitis. An Aspergillus fungal ball is a rare cause of ureteral obstruction. We describe an unusual case of simultaneous vertebral osteomyelitis and ureteral obstruction caused by A. flavus in a hepatic transplant recipient, who was successfully treated with sequential intravenous and oral itraconazole solution.

Truncated Rv2820c enhances mycobacterial virulence ex vivo and in vivo.

Three hypervirulent strains of Mycobacterium tuberculosis isolated from patients suffering from tuberculous meningitis were shown to grow more rapidly inside human macrophages in our previous study. In the current investigation, genomic polymorphisms in these hypervirulent strains were examined using microarray-based comparative genomic hybridization. Among the five genomic polymorphisms identified, two are in-frame deletion (Rv0071/4 and Rv0613c/6c), two are frameshift deletion (Rv1758' and Rv2820c'), and one is gene replacement (Mb3159). The five genomic polymorphisms were transformed into Mycobacterium smegmatis strain mc(2)155 and the survivability of recombinants inside the human monocytic cell line THP-1 was measured. Interestingly, only the recombinant possessing the Rv2820c' survived significantly better than the vector control after 6 h of ex vivo infection (P < 0.001, one-way ANOVA). The Rv2820c' was later transformed into Mycobacterium marinum strain M and the recombinant was used to infect zebrafish. The in vivo infection also showed that the zebrafish infected with the recombinant possessing the Rv2820c' died significantly faster than the vector control (P = 0.006, log-rank test). The 3' truncation in the Rv2820c' was caused by the Beijing/W-defining deletion RD207 and is commonly found in the Beijing/W strains. The current study demonstrated that the truncated Rv2820c of Beijing/W strains could enhance mycobacterial virulence ex vivo and in vivo. This enhancement, however, was not observed for the intact Rv2820c of the non-Beijing/W strains. The presence of the 3' truncated portion of Rv2820c may interfere with overall protein folding and render the Rv2820c of the non-Beijing/W strains non-functional.

Differential fadE28 expression associated with phenotypic virulence of Mycobacterium tuberculosis.

Ability to persist in human macrophages is central to the virulence of Mycobacterium tuberculosis and is not invariable among various strains. Differential gene expression that is associated with phenotypic virulence may provide additional information of virulent genes involved in the pathogenesis of M. tuberculosis, which is not fully elucidated. Three hypervirulent strains of M. tuberculosis isolated from patients suffering with tuberculous meningitis were shown to grow more rapidly inside human macrophages in a previous study. In the current investigation, expression of 7 mycobacterial genes (fadE28, mce1A, mymA, acr, sigA, sugC, and Rv3723) of these strains during ex vivo macrophage challenge and in vitro acid shock was quantified by real-time PCR. Using rrs gene as a normalisation gene, fadE28 gene exhibited differential gene expression that is associated with phenotypic virulence, whereas the other 6 genes showed indistinguishable expression patterns. Up-regulation of fadE28 gene in the hypervirulent strains may account for virulence by increasing the efficiency of beta-oxidation, which is important for the persistence in macrophages as M. tuberculosis uses fatty acids preferably inside phagosome of macrophages. The fadE28 gene, together with its adjacent genes may also be critical in the process of lipid modification that could facilitate parasitism in human macrophages.

Molecular characterization of clinical isolates of Mycobacterium tuberculosis and their association with phenotypic virulence in human macrophages.

Among 125 clinical isolates of Mycobacterium tuberculosis collected in Hong Kong and Shanghai, China, between 2002 and 2004, IS6110 typing revealed that 71 strains (57%) belonged to the Beijing family. The intracellular growth of the strains in human peripheral blood monocyte-derived macrophages was measured ex vivo on days 0, 3, 6, and 10. Among all tested strains, three hypervirulent strains showed significant increases in intracellular growth after 10 days of incubation. With an initial bacterial load of 10(4) CFU, most of the clinical isolates and H37Ra (an avirulent strain) exhibited no intracellular survival on day 10, while the three hypervirulent strains together with H37Rv (a virulent strain) showed on average a two- to fourfold rise in CFU count. These three hypervirulent strains belonging to a non-Beijing family were isolated from patients suffering from tuberculosis meningitis. Cytokines secreted by gamma interferon-activated macrophages were measured daily after challenge with selected strains of M. tuberculosis. The levels of tumor necrosis factor alpha were elevated after 24 h of infection among all strains, but the levels were significantly lower among the three hypervirulent strains, whereas interleukin 10 (IL-10) and IL-12 were not detected. Results were concordant with the differential expression of the corresponding cytokine genes in activated macrophages, as monitored by real-time PCR. Our findings highlighted that these three hypervirulent strains may possess an innate mechanism for escaping host immunity, which accounts for their characteristic virulence in patients presenting with a more severe form of disease.

Antibody response to four secretory proteins from Mycobacterium tuberculosis and their complex antigen in TB patients.

OBJECTIVE: To evaluate antibody responses to a panel of four protein antigens secreted from Mycobacterium tuberculosis, CFP-21, ESAT-6, MPT-63 and MPT-64, and their complex antigen. DESIGN: Seventy-eight samples of serum and cerebral spinal fluid were tested by ELISA. RESULTS: Serum IgM antibody positive rates of protein antigens CFP-21, ESAT-6, MPT-63 and MPT-64 and their multi-antigen complex were respectively 34.6%, 60.3%, 52.6%, 78.2% and 96.2%; IgG antibody positive rates were respectively 46.2%, 64.1%, 93.6%, 57.7% and 84.6%. The specificity of the antibodies to the four proteins secreted from M. tuberculosis and its mixture antigen was 100%; sensitivity to the multi-antigen complex was higher than for the four single antigens. Among the IgM positive rates, the differences between the multi-antigen complex and CFP-21, ESAT-6, MPT-63 and MPT-64 were significant (P < 0.01); among the IgG positive rates, the differences between the multi-antigen complex and CFP-21, ESAT-6 and MPT-64 were significant (P < 0.01). Although the IgG positive rate for MPT-63 was higher than for the multi-antigen complex, the difference was not significantly different (P > 0.05). CONCLUSION: These findings indicate that the multi-antigen complex composed of CFP-21, ESAT-6, MPT-63 and MPT-64 may be useful in the diagnosis of TB.

Three-dimensional model and molecular mechanism of Mycobacterium tuberculosis catalase-peroxidase (KatG) and isoniazid-resistant KatG mutants.

Mycobacterium tuberculosis KatG enzyme functions both as catalase for removing hydrogen peroxide (H(2)O(2)) and as peroxidase for oxidating isoniazid (INH) to active form of anti-tuberculosis drug. Although mutations in M. tuberculosis KatG confer INH resistance in tuberculous patients, structural bases for INH-resistant mutations in the KatG gene remains poorly understood. Here, three M. tuberculosis KatG mutants bearing Arg418--> Gln, Ser315 --> Thr, or Trp321 --> Gly replacement were assessed for changes in catalase-peroxidase activities and possible structure bases relevant to such changes. These three M. tuberculosis KatG mutants exhibited a marked impairment or loss of catalase-peroxidase activities. The possible structural bases for the mutant-induced loss of enzyme activities were then analyzed using a three-dimensional model of M. tuberculosis KatG protein constructed on the basis of the crystal structure of the catalase-peroxidase from Burkholderia pseudomallei. The model suggests that three M. tuberculosis KatG mutants bearing Arg418 --> Gln, Ser315 -->Thr, or Trp321--> Gly replacement affect enzyme activities by different mechanisms, although each of them impacts consequently on a heme-associated structure, the putative oxidative site. Moreover, in addition to the widely accepted substrate-binding site, M. tuberculosis KatG may bear another H(2)O(2) binding site. This H(2)O(2) binding site appears to interact with the catalytic site by a possible electron-transfer chain, a Met255-Tyr229-Trp107 triad conserved in many catalase-peroxidases. The Ser315 --> Thr mutant may have direct effect on the catalytic site by interfering with electron transfer in addition to the previously proposed mechanism of steric constraint.

[Investigation of anti-Toxoplasma gondii antibodies in immunodeficient patients].

OBJECTIVE: To investigate the presence of anti-Taxoplasma gondii antibodies in immunodeficient patients. METHODS: T. gondii-specific immunoglobulin G (IgG) antibodies in serum samples from 371 immunodeficient patients were detected by enzyme-linked immunosorbent assay (ELISA). The patients were with solid malignancies (including untreated digestive system malignancies and solid malignancies received chemotherapy), chronic liver diseases, patients received immunosuppressant therapy (dermatomyositis, psoriasis, pemphigus, post-renal transplantation, systemic lupus erythematosus and other miscellanies), lymphoma, leukemia and diabetes. 100 normal serum samples served as controls. RESULTS: The positive rate of patients with solid malignancies received chemotherapy, solid malignancies received chemotherapy, chronic liver diseases, systemic lupus erythematosus and leukemia was 19.0%, 33.3%, 16.5%, 45.4% and 20.0%, respectively, being significantly higher than that of the control group(P < 0.05). CONCLUSION: The immunosuppressed patients are highly predisposing to secondary T. gondii infection.

Chinese herbal formula XQ-9302: pilot study of its clinical and in vitro activity against human immunodeficiency virus.

OBJECTIVES: To evaluate the effectiveness of XQ-9302--a purified, precise mixture of 20 Chinese herbs--against infection with human immunodeficiency virus in vitro and in the clinic. DESIGN: In vitro cell culture assay, heavy metal content analysis, and pilot non-randomised clinical trial. SETTING: Drug rehabilitation centre and municipal surveillance centre, Shanghai, China. PATIENTS: Forty-eight patients who had various clinical histories, such as drug abuse, cancer, and infection with human immunodeficiency virus, participated in the clinical study. INTERVENTION: During the clinical trial, multiple 15-day courses of XQ-9302 10.8 g/d were given to participants. MAIN OUTCOME MEASURES: CD4 count, P24 antigen level, level of antibody against human immunodeficiency virus, number of copies per millilitre of human immunodeficiency virus in the plasma (viral load), and any side effects. RESULTS: XQ-9302 protected cultured MT4 cells from infection with human immunodeficiency virus in vitro. Clinical tests showed that the herbal formula relieved the symptoms of acquired immunodeficiency syndrome and enhanced CD4 counts in patients infected by the human immunodeficiency virus. There were no observable side effects, even after taking the drug for several months. In three patients who had acquired immunodeficiency syndrome, treatment with XQ-9302 reduced the magnitude of the viral load by more than 1 log. CONCLUSION: XQ-9302 not only improves the immune function of patients infected with the human immunodeficiency virus, but also interrupts viral replication and slows the progression of the disease without detectable side effects. In addition, the heavy metal content of XQ-9302 is well within safety levels set by the Government of China.

[Extracellular ATP: effects, sources and fate].

The change of extracellular adenosine triphosphate (ATP) concentration affects many important physiological functions significantly. As a transmitter, ATP acts on neuro-effector junction directly and/or modulates the release of other neurotransmitters. By acting on the various P2-purinoceptors or ionic channels on the membrane, ATP also has some striking effects on cell activities. This review focused on the latest advances on the research of extracellular ATP in the respects of discovering and confirming ATP as a transmitter, the source, subtypes of P2-purinoceptors, the receptor-mediated reactions, and the degradation of extracellular ATP.

[Experimental study and case control study of nosocomial infection caused by Pseudomonas aeruginosa].

144 strains of Pseudomonas aeruginosa (Ps.a) were serotyped and phage-typed. Antibiotic sensitivity test and case control study of nosocomial infection caused by Ps.a were also done for these strains. Three epidemic strains were collected from the ICU in a neurosurgical ward. The etiology of a cross infection among tracheotomy patients was two epidemic strains isolated from the hands of a nurse and an attendant. Hospitalization days longer than 56 days, tracheotomy and indwelling catheterization were 3 risk factors for Ps.a infection shown by logistic analysis. The sensitivity rate of Ps.a to antibiotics was highest with ceftazidine, followed by amikacin and piperacillin. The most common resistant antibiogram was Gentamycin and tobramycin Resistance to gentamicin increased obviously.