African trypanosomes are protozoan parasites transmitted by a tsetse fly vector to a mammalian host. The life cycle includes highly proliferative forms and quiescent forms, the latter being adapted to host transmission. The signaling pathways controlling the developmental switch between the two forms remain unknown. Trypanosoma brucei contains two target of rapamycin (TOR) kinases, TbTOR1 and TbTOR2, and two TOR complexes, TbTORC1 and TbTORC2. Surprisingly, two additional TOR kinases are encoded in the T. brucei genome. We report that TbTOR4 associates with an Armadillo domain-containing protein (TbArmtor), a major vault protein, and LST8 to form a unique TOR complex, TbTORC4. Depletion of TbTOR4 caused irreversible differentiation of the parasite into the quiescent form. AMP and hydrolysable analogs of cAMP inhibited TbTOR4 expression and induced the stumpy quiescent form. Our results reveal unexpected complexity in TOR signaling and show that TbTORC4 negatively regulates differentiation of the proliferative form into the quiescent form.parasitology | cell biology | kinetoplastida

In the interest of identification of new kinase-targeting chemotypes for target and pathway analysis and drug discovery in Trypanosomal brucei, a high-throughput screen of 42,444 focused inhibitors from the GlaxoSmithKline screening collection was performed against parasite cell cultures and counter-screened against human hepatocarcinoma (HepG2) cells. In this way, we have identified 797 sub-micromolar inhibitors of T. brucei growth that are at least 100-fold selective over HepG2 cells. Importantly, 242 of these hit compounds acted rapidly in inhibiting cellular growth, 137 showed rapid cidality. A variety of in silico and in vitro physicochemical and drug metabolism properties were assessed, and human kinase selectivity data were obtained, and, based on these data, we prioritized three compounds for pharmacokinetic assessment and demonstrated parasitological cure of a murine bloodstream infection of T. brucei rhodesiense with one of these compounds (NEU-1053). This work represents a successful implementation of a unique industrial-academic collaboration model aimed at identification of high quality inhibitors that will provide the parasitology community with chemical matter that can be utilized to develop kinase-targeting tool compounds. Furthermore these results are expected to provide rich starting points for discovery of kinase-targeting tool compounds for T. brucei, and new HAT therapeutics discovery programs.

The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis, a disease potentially fatal if not treated. Current available treatments have major limitations, and new and safer drugs are urgently needed. In recent years, advances in high-throughput screening technologies have enabled the screening of millions of compounds to identify new antileishmanial agents. However, most of the compounds identified in vitro did not translate their activities when tested in in vivo models, highlighting the need to develop more predictive in vitro assays. In the present work, we describe the development of a robust replicative, high-content, in vitro intracellular L. donovani assay. Horse serum was included in the assay media to replace standard fetal bovine serum, to completely eliminate the extracellular parasites derived from the infection process. A novel phenotypic in vitro infection model has been developed, complemented with the identification of the proliferation of intracellular amastigotes measured by EdU incorporation. In vitro and in vivo results for miltefosine, amphotericin B, and the selected compound 1 have been included to validate the assay.T he leishmaniases are a complex of diseases, with visceral and cutaneous manifestations caused by protozoan parasites of the genus Leishmania. Visceral leishmaniasis (VL) has been the main focus for drug research and development over the past 2 decades, due to the large disease burden in East Africa and South Asia (1) and potential patient death if not treated. For VL, there has been progress in treatment over the past decade, with clinical evidence for efficacy of and registration for use of oral miltefosine, paromomycin, and the liposomal formulation of amphotericin B (AmBisome, Gilead, USA) in South Asia (2), as well as combinations of these standard drugs (3). The need for new drugs to treat VL remains, as (i) miltefosine is the only approved oral treatment but requires 28 days of treatment and potential teratogenicity limits its use (4), (ii) paromomycin requires 21 days of treatment and intramuscular administration (http://www.dndi.org/diseases-projects/diseases /vl/current-treatment/current-treatment-vl.html), and (iii) liposomal amphotericin B formulations, which have successful cure rates with a single dose (5), require intravenous (i.v.) infusion, have a high cost if not donated, and have a requirement for cold storage, limiting use in countries where the disease is endemic (6). As part of the drive to find new treatments, there has been a refocus on the assays and models used to identify and develop new molecules as antileishmanial drugs. For in vitro screens and assays, this has ranged from the need to develop methods that (i) are adaptable to and enable high-throughput screens against the replicative intracellular-macrophage amastigote stage of Leishmania donovani, one of the causative species of VL (7); and (ii) include high-throughput technologies that enable the collection of more information compared to the traditionally use...

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The objective of this work was to evaluate the ability of the white rot basidiomycete Coriolopsis rigida to detoxify the water soluble fraction from "alpeorujo" (WSFA), a solid by-product produced by the olive oil extraction industry and characterized by a high concentration of phenols which limits its use as fertilizer and/or amendment. C. rigida reduced the phenol content in the liquid media supplemented with WSFA at 10 and 20% (v/v) after 15d of incubation. The analysis of WSFA toxicity after fungal treatment showed that C. rigida was responsible for a significant increase in the survival rate of Azospirillum brasiliense, a N(2) fixing soil rhizobacterium which promotes plant growth. Supplementation of culture medium with CuSO(4) (300 microM) resulted in strong laccase induction thus facilitating higher phenol reduction and detoxification of WSFA. In vitro reactions using a crude extracellular preparation from laccase-active C. rigida showed phenol removal as well as detoxification of the WSFA at 20%. These results suggest that C. rigida reduces the phenol content of the WSFA through the effect of laccase on free phenolic compounds consequently decreasing the toxic effect on A. brasiliense, which suggests that the enzyme plays an important role in the process. These findings have implications in the management and revalorization of olive-mill residues treated with laccase-producing fungi and their potential impact on integrative agricultural systems including organic residues and the co-inoculation with microorganisms which can facilitate the growth of plants of agricultural interest.

Compound NVP-BEZ235 (1) is a potent inhibitor of human phospoinositide-3-kinases and mammalian target of rapamycin (mTOR) that also showed high inhibitory potency against Trypanosoma brucei cultures. With an eye toward using 1 as a starting point for anti-trypanosomal drug discovery, we report efforts to reduce host cell toxicity, to improve the physicochemical properties, and to improve the selectivity profile over human kinases. In this work, we have developed structure–activity relationships for analogues of 1 and have prepared analogues of 1 with improved solubility properties and good predicted central nervous system exposure. In this way, we have identified 4e, 9, 16e, and 16g as the most promising leads to date. We also report cell phenotype and phospholipidomic studies that suggest that these compounds exert their anti-trypanosomal effects, at least in part, by inhibition of lipid kinases.

Two laccase isoenzymes were purified and characterized from the basidiomycete Coriolopsis rigida during transformation of the water-soluble fraction of "alpeorujo" (WSFA), a solid residue derived from the olive oil production containing high levels of toxic compounds. Zymogram assays of laccases secreted by the fungus growing on WSFA and WSFA supplemented with glucose showed two bands with isoelectric points of 3.3 and 3.4. The kinetic studies of the two purified isoenzymes showed similar affinity on 2,6-dimethoxyphenol and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid), used as phenolic and non-phenolic model substrate, respectively. The molecular mass of both proteins was 66 kDa with 9% N-linked carbohydrate. Physico-chemical properties of the purified laccases from media containing WSFA were similar to those obtained from medium with glucose as the main carbon source. In-vitro studies performed with the purified laccases revealed a 42% phenol reduction of WSFA, as well as changes in the molecular mass distribution. These findings indicate that these laccases are involved in the process of transformation, via polymerization by the oxidation of phenolic compounds present in WSFA. A single laccase gene, containing an open reading frame of 1,488 bp, was obtained in PCR amplifications performed with cDNA extracted from mycelia grown on WSFA. The product of the gene shares 90% identity (95% similarity) with a laccase from Trametes trogii and 89% identity (95% similarity) with a laccase from Coriolopsis gallica. This is the first report on purification and molecular characterization of laccases directly involved in the transformation of olive oil residues.

Vovlas, N., Rapoport, H. F., Jiménez Díaz, R. M., and Castillo, P. 2005. Differences in feeding sites induced by root-knot nematodes, Meloidogyne spp., in chickpea. Phytopathology 95:368-375.Root-knot nematodes (Meloidogyne spp.) are sedentary, obligate endoparasites in plants, where they induce specialized feeding sites. The feeding sites act as strong metabolic sinks to which photosynthates are mobilized. The histopathological modifications in the nematode-induced feeding sites of artificially inoculated chickpea cv. UC 27 were qualitatively and quantitatively compared using five isolates of M. artiellia and one isolate each of M. arenaria, M. incognita, and M. javanica. All Meloidogyne isolates infected chickpea plants, but root gall thickening was significantly less for M. artiellia isolates than for the other Meloidogyne species. Nevertheless, neither the number of giant cells in the feeding site (averaging four to six) nor the area of individual giant cells was influenced by nematode species or isolate. However, the number of nuclei per giant cell was significantly smaller, and the maximum diameters of nuclei and nucleoli were significantly greater, in giant cells induced by M. artiellia isolates than in those induced by M. arenaria, M. incognita, or M. javanica. In a second experiment, M. artielliainduced giant cells in faba bean and rapeseed also contained a small number of large nuclei.Additional keywords: Brassica napus var. oleifera, Cicer arietinum, food legumes, histopathology, Vicia faba.Sedentary endoparasitic root-knot nematodes of the genus Meloidogyne are among nature's most successful parasites. These nematodes infect thousands of different herbaceous and woody monocotyledonous and dicotyledonous plants and cause serious losses to numerous agricultural crops worldwide (13,28). Parasitism by root-knot nematodes is characterized by the establishment of permanent feeding sites comprised of giant cells in the cortex, endodermis, pericycle, and vascular parenchyma of the host root tissues. These feeding sites are sinks for the plant photosynthates, and they thus impair plant growth and development. In addition, deformation and blockage of vascular tissues at the feeding sites limit translocation of water and nutrients, further suppressing plant growth and crop yield (20). Management of root-knot nematodes has primarily depended on nematicides and resistant cultivars. Use of nematicides is questioned because of their short-term efficacy as well as their potentially negative impact on the environment and human health. Therefore, there is a need for new root-knot nematode management strategies, which are likely to require better understanding of interactions between the plant and the nematode.Infection by root-knot nematodes begins with penetration of the root tissues by the second-stage juvenile (J2) at the zone of root elongation. The nematode then migrates in the apoplast, ultimately reaching the vascular cylinder where giant cells are induced.Thereafter, the J2 undergo three molts to devel...