Sequence-independent cloning methods for long DNA fragments applied to synthetic biology.

Simplified methods to assemble DNA fragments by independent cloning sequence have helped in the progress of synthetic biology, allowing some biotechnological processes to become economically viable by genetic improvement of microorganisms. We compared three methods of assembling six DNA fragments: PCR fusion-based, isothermal NEBuilder and circular polymerase extension cloning (CPEC). Double and triple fusion occurs directly with the PCR products using PCR fusion-based and NEBuilder methods. For multiple fragments the results showed higher efficiency by the CPEC method which allowed assembly of six fragments previously purified by agarose gel extraction, after a sequence of 20 annealing/extension cycles without any primer.

Antimicrobial and antineoplasic activity of Pleurotus ostreatus.

The objectives of this work were to evaluate the antimicrobial and antineoplasic activity of Pleurotus ostreatus DSM 1833. To study the antimicrobial activity, the following extracts were prepared: water infusion of the fresh fruiting bodies (B1), dehydrated fruiting bodies (B2), fresh mycelium (M1), and dehydrated mycelium (M2). Polysaccharides from the fresh mycelium were also obtained by water infusion followed by ethanol treatment (EP). The extracts were tested against Candida albicans, Escherichia coli, and Bacillus subtilis. To investigate the antineoplasic effect of P. ostreatus, the culture broth in natura, the extract from the culture broth (ECB), and the extract from the fruiting bodies were tested in female Swiss albino mice inoculated with the Ehrlich ascitic tumor (EAT). B1, B2, and M1 showed more than 50.0% inhibition against C. albicans. M2 presented a high degree of inhibition against E. coli (87.5%) and B. subtilis (57.5%), while EP was not effective. All the tested substances inhibited the development of EAT at levels near 70% when injected intraperitoneally in mice. The highest tumor inhibition (76%) was achieved for the treatment with ECB. The intragastric treatment did not promote any reduction in tumor cell development, independent of the test substance.

Risk factors for methadone outside treatment programs: implications for HIV treatment among injection drug users.

BACKGROUND: Diversion of methadone outside treatment programs occurs, yet reasons for use of 'street methadone' are characterized poorly. Self-medication for withdrawal symptoms is one plausible hypothesis. Among HIV-infected drug users, some antiretroviral medications can reduce potency of methadone, yet any association between such effects and the use of supplemental methadone sources remains undetermined. OBJECTIVE: To estimate the frequency and risk factors for use of street methadone. METHODS: Injection drug users (IDUs) recruited through extensive community outreach in 1988-89 and 1994 were followed semi-annually with questionnaires about health history, use of licit and illicit drugs including methadone and HIV-related assays. Analyses were performed using generalized estimating equation logistic regression. RESULTS: Of 2811 IDUs enrolled and eligible for analysis, 493 people reported use of street methadone over 12 316 person-years of follow-up (4.0/100 person-years). In multivariate analyses, street methadone use was more common among women, whites, those 40-59 years old, those who reported withdrawal symptoms, past methadone program attendance (6-12 months before visit), recent heroin injection with or without cocaine (but not cocaine alone), smoking or sniffing heroin and reported trading sex. Street methadone was not associated with HIV infection or treatment. CONCLUSION: The results suggest that older IDUs still using heroin may be using street methadone to treat signs of withdrawal. The absence of a higher rate of street methadone use in HIV seropositive IDUs reveals that antiretroviral/methadone interactions are not a primary determinant of use outside of treatment settings.

Screening for microorganisms that produce only endo-inulinase.

Sixteen fungal strains reported in the literature as endo-inulinase producers and three bacterial strains, isolated from the dahlia rhizosphere, were analysed for endo-inulinase production. From four isolated strains (one fungus and three bacteria) the results were evaluated in terms of substrate consumption, cell growth and production of endo-inulinases. All three bacterial strains were sole endo-inulinase producers and, among these, strain Paenibacillus sp. CDB 003 was the most suitable for endo-inulinase production, as this enzyme produced inulobiose as the principal substrate as well as inulo-oligosaccharides with polymerisation degrees of 3-5.