In Vitro and in Vivo Activity of mTOR Kinase and PI3K Inhibitors Against Leishmania donovani and Trypanosoma brucei.

Kinetoplastid parasites, including Leishmania and Trypanosoma spp., are life threatening pathogens with a worldwide distribution. Next-generation therapeutics for treatment are needed as current treatments have limitations, such as toxicity and drug resistance. In this study, we examined the activities of established mammalian target of rapamycin (mTOR)/phosphoinositide 3-kinase (PI3K) inhibitors against these tropical diseases. High-throughput screening of a library of 1742 bioactive compounds against intracellular L. donovani was performed, and seven mTOR/PI3K inhibitors were identified. Dose-dilution assays revealed that these inhibitors had half maximal effective concentration (EC50) values ranging from 0.14 to 13.44 µM for L. donovani amastigotes and from 0.00005 to 8.16 µM for T. brucei. The results of a visceral leishmaniasis mouse model indicated that treatment with Torin2, dactolisib, or NVP-BGT226 resulted in reductions of 35%, 53%, and 54%, respectively, in the numbers of liver parasites. In an acute T. brucei mouse model using NVP-BGT226 parasite numbers were reduced to under the limits of detection by five consecutive days of treatment. Multiple sequence and structural alignment results indicated high similarities between mTOR and kinetoplastid TORs; the inhibitors are predicted to bind in a similar manner. Taken together, these results indicated that the TOR pathways of parasites have potential for the discovery of novel targets and new potent inhibitors.

Autophagy-regulating small molecules and their therapeutic applications.

Autophagy or self-eating is a complicated cellular process that is involved in protein and organelle digestion occurring via a lysosome-dependent pathway. This process is of great importance in maintaining normal cellular homeostasis. However, disruption of autophagy is closely associated with various human diseases such as cancer, neurodegenerative disorders, heart disease and pathogen infection. Therefore, small molecules that modulate autophagy can be employed to dissect this complex process and ultimately could have high potential for the treatment of a variety of diseases. This critical review discusses general aspects of autophagy, autophagy-associated diseases and autophagy regulators for biological research and therapeutic applications (207 references).

Monitoring bacterial population dynamics using real-time PCR during the bioremediation of crude-oil-contaminated soil.

We evaluated the activity and abundance of the crude oil- degrading bacterium Nocardia sp. H17-1 during bioremediation of oil-contaminated soil, using real-time PCR. The total petroleum hydrocarbon (TPH) degradation rate constants (k) of the soils treated with and without H17-1 were 0.103 d-1 and 0.028 d-1, respectively. The degradation rate constant was 3.6 times higher in the soil with H17-1 than in the soil without H17-1. In order to detect and quantify the Nocardia sp. H17-1 in soil samples, we quantified the genes encoding 16S ribosomal RNA (16S rRNA), alkane monooxygenase (alkB4), and catechol 2,3-dioxygenase (23CAT) with real-time PCR using SYBR green. The amounts of H17-1 16S rRNA and alkB4 detected increased rapidly up to 1,000-folds for the first 10 days, and then continued to increase only slightly or leveled off. However, the abundance of the 23CAT gene detected in H17-1-treated soil, where H17-1 had neither the 23CAT gene for the degradation of aromatic hydrocarbons nor the catechol 2,3-dioxygenase activity, did not differ significantly from that of the untreated soil (alpha=0.05, p>0.22). These results indicated that H17-1 is a potential candidate for the bioaugmentation of alkane-contaminated soil. Overall, we evaluated the abundance and metabolic activity of the bioremediation strain H17-1 using real-time PCR, independent of cultivation.

Monitoring of microbial diversity and activity during bioremediation of crude oil-contaminated soil with different treatments.

The present study compared the microbial diversity and activity during the application of various bioremediation processes to crude oil-contaminated soil. Five different treatments, including natural attenuation (NA), biostimulation (BS), biosurfactant addition (BE), bioaugmentation (BA), and a combined treatment (CT) of biostimulation, biosurfactant addition, and bioaugmentation, were used to analyze the degradation rate and microbial communities. After 120 days, the level of remaining hydrocarbons after all the treatments was similar, however, the highest rate (k) of total petroleum hydrocarbon (TPH) degradatioN was observed with the CT treatment (P < 0.05). The total bacterial counts increased during the first 2 weeks with all the treatments, and then remained stable. The bacterial communities and alkane monooxygenase gene fragment, alkB, were compared by denaturing gradient gel electrophoresis (DGGE). The DGGE analyses of the BA and CT treatments, which included Nocardia sp. H17-1, revealed a simple dominant population structure, compared with the other treatments. The Shannon-Weaver diversity index (H') and Simpson dominance index (D), calculated from the DGGE profiles using 16S rDNA, showed considerable qualitative differences in the community structure before and after the bioremediation treatment as well as between treatment conditions.

Effects of crude oil, oil components, and bioremediation on plant growth.

The phytotoxic effects of crude oil and oil components on the growth of red beans (Phaseolus nipponesis OWH1) and corn (Zea mays) was investigated. In addition, the beneficial effects of bioremediation with the oil-degrading microorganism, Nocardia sp. H17-1, on corn and red bean growth in oil-contaminated soil was also determined. It was found that crude oil-contaminated soil (10,000mg/kg) was phytotoxic to corn and red beans. In contrast, obvious phytotoxicity was not observed in soils contaminated with 0-1000 mg/kg of aliphatic hydrocarbons such as decane (C10) and eicosane (C20). Phytotoxicity was observed in soils contaminated with 10-1000mg/kg of the poly aromatic hydrocarbons (PAHs) naphthalene, phenanthrene, and pyrene. It was observed that phytotoxicity increased with the number of aromatic rings, and that corn was more sensitive than red beans to PAH-contaminated soil. Bioremediation with Nocardia sp. H17-1 reduced phytotoxicity more in corn than in red bean, suggesting that this microbial species might degrade PAHs to some degree.