Machine Learning Models and Pathway Genome Data Base for Trypanosoma cruzi Drug Discovery

BackgroundChagas disease is a neglected tropical disease (NTD) caused by the eukaryotic parasite Trypanosoma cruzi. The current clinical and preclinical pipeline for T. cruzi is extremely sparse and lacks drug target diversity.

Methodology/Principal Findings

In the present study we developed a computational approach that utilized data from several public whole-cell, phenotypic high throughput screens that have been completed for T. cruzi by the Broad Institute, including a single screen of over 300,000 molecules in the search for chemical probes as part of the NIH Molecular Libraries program. We have also compiled and curated relevant biological and chemical compound screening data including (i) compounds and biological activity data from the literature, (ii) high throughput screening datasets, and (iii) predicted metabolites of T. cruzi metabolic pathways. This information was used to help us identify compounds and their potential targets. We have constructed a Pathway Genome Data Base for T. cruzi. In addition, we have developed Bayesian machine learning models that were used to virtually screen libraries of compounds. Ninety-seven compounds were selected for in vitro testing, and 11 of these were found to have EC₅₀ < 10μM. We progressed five compounds to an in vivo mouse efficacy model of Chagas disease and validated that the machine learning model could identify in vitro active compounds not in the training set, as well as known positive controls. The antimalarial pyronaridine possessed 85.2% efficacy in the acute Chagas mouse model. We have also proposed potential targets (for future verification) for this compound based on structural similarity to known compounds with targets in T. cruzi.

Conclusions/ Significance

We have demonstrated how combining chemoinformatics and bioinformatics for T. cruzi drug discovery can bring interesting in vivo active molecules to light that may have been overlooked. The approach we have taken is broadly applicable to other NTDs.     

Production and characterization of human granulocyte–macrophage colony-stimulating factor (hGM-CSF) expressed in the oleaginous yeast Yarrowia lipolytica

Since its isolation, the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) has been proposed as a new class of therapeutic biological products in the treatment of various diseases. However, the toxicity of this cytokine towards its expression host constitutes a major obstacle to bioprocess development for large-scale production. In this work, the optimized gene encoding hGM-CSF was expressed in the yeast Yarrowia lipolytica in one and two copies under the control of the fatty acid-inducible POX2 promoter. Protein secretion was directed by the targeting sequence of the extracellular lipase (LIP2): preXALip2. After 48?h of induction, Western blot analysis revealed the presence of a nonglycosylated form of 14.5?kDa and a trail of hGM-CSF hyperglycosylated varying from 23?kDa to more than 60?kDa. The two-copy transformants produced hGM-CSF level which was sevenfold higher compared to the single-copy ones. Deglycosylation with PNGase F showed two forms: a mature form of 14.5?kDa and an unprocessed form of 18?kDa. The addition of two alanines to the signal sequence resulted in correct hGM-CSF processing. The production level was estimated at 250?mg/l after preliminary optimization studies of the cultivation and induction phases. The purified hGM-CSF was identified by N-terminal sequencing and LC-MS/MS analysis; its biological activity was confirmed by stimulating the proliferation of TF1 cell line. This study demonstrated that Y. lipolytica is a promising host for the efficient production of active toxic proteins like hGM-CSF.     

Parasites rather than phoronts: Teratorhabditis synpapillata nematodes reduce lifespan of their Rhynchophorus ferrugineus host in a life stage‐dependent manner

Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) red palm weevils are often reported in association with different organisms including nematodes. The significance of this interaction and whether nematodes can influence their life‐history traits is unclear. We collected Rhynchophorus ferrugineus red palm weevils at different developmental stages and locations in Tunisia, observed and dissected them in search for nematodes and other interacting organisms, established laboratory colonies and identified the nematodes associated with them, and conducted nematode–insect interaction assays to determine the capacity of the nematodes to influence their life‐history traits. We observed Beauveria bassiana fungi in larvae, nymph, and adults; Centrouropoda and Uroobovella acari associated with the adults, and Teratorhabditis synpapillata nematodes associated with larvae and adults. Nematode–insect interaction bioassays revealed that T. synpapillata nematodes reduce the lifespan of the insect larvae in a population‐dependent manner, but do not influence the lifespan of adults. Our study uncovers an important factor that may determine population dynamics of this important palm pests and provides evidence to conclude that these organisms establish a parasitic relationship, rather than a phoretic relationship.     

Phytosterols accumulation in the seeds of Linum usitatissimum L.

A comparative study was performed to determine the free sterols content and composition during the development of three varieties of linseed (H52, O116 and P129). Seed samples were collected at regular intervals from 7 to 60 days after flowering (DAF). Ten compounds were identified: cholesterol, campesterol, brassicasterol, stigmasterol, β-sitosterol, Δ5-avenasterol, cycloartenol; 24-methylene cycloartanol, obtusifoliol, citrostadienol. The maximum level of 4-desmethylsterols (1515 mg/100 g oil) was reached at 7 DAF in P129 variety. H52 had the highest level of 4-4 dimethylsterols (355 mg/100 g oil) at 28 DAF. The greatest amount of 4-monomethylsterols (35 mg/100 g oil) was detected in H52 at 14 DAF. During linseed development, β sitosterol (830 mg/100 g oil) was the major 4-desmethylsterols, followed by campesterol (564 mg/100 g oil) and stigmasterol (265 mg/100 g oil). Some of these compounds followed nearly the same accumulation pattern during linseed maturation.     

Proteome analysis of virus–host cell interaction: rabies virus replication in Vero cells in two different media

The use of Vero cells for rabies vaccine production was recommended from the WHO in 2005. A controlled production process is necessary to reduce the risk of contaminants in the product. One step towards this is to turn away from animal-derived components (e.g. serum, trypsin, bovine serum albumin) and face a production process in animal component-free medium. In this study, a proteomic approach was applied, using 2-D differential gel electrophoresis and mass spectrometry to compare rabies virus propagation in Vero cells under different cultivation conditions in microcarrier culture. Protein alterations were investigated for uninfected and infected Vero cells over a time span from 1 to 8 days post-infection in two different types of media (serum-free versus serum-containing media). For mock-infected cells, proteins involved in stress response, redox status, protease activity or glycolysis, and protein components in the endoplasmic reticulum were found to be differentially expressed comparing both cultivation media at all sampling points. For virus-infected cells, additionally changes in protein expression involved in general cell regulation and in calcium homeostasis were identified under both cultivation conditions. The fact that neither of these additional proteins was identified for cells during mock infection, but similar protein expression changes were found for both systems during virus propagation, indicates for a specific response of the Vero cell proteome on rabies virus infection.     

Cytoprotective Effects of the Red Marine Alga <Emphasis Type="Italic">Chondrus canaliculatus</Emphasis> Against Maneb-Induced Hematotoxicity and Bone Oxidative Damages in Adult Rats

The current study aimed at evaluating the ability of a mineral and antioxidant-rich extract from Chondrus canaliculatus to improve maneb (MB)-induced toxicity in adult rat. The animals were divided into four groups: group 1 used as a control group, group 2 received MB, group 3 received MB + C. canaliculatus extract, and group 4 received only the algal extract. MB, a Mn-containing ethylene-bis-dithiocarbamate fungicide, induced oxidative stress damages, mineral perturbations in the plasma, urine, and bone, and genotoxicity in rats. Hematological analysis revealed in the MB-treated group a disruption in the number of red blood cells, platelets, and white blood cells associated with a striking genotoxicity. Interestingly, a significant increase in malondialdehyde and advanced oxidation protein product levels in erythrocytes and bones were found. On the other hand, an impairment of the antioxidant status in both tissues was occurred. Along, our results revealed that MB injection caused a striking drop and disruption in bone’s mineral rates, especially calcium and phosphorus. These biochemical results were in accordance with the histological and molecular changes. However, co-treatment with C. canaliculatus extract showed, for the first time, that this alga was effective against MB-induced hematotoxicity, genotoxicity, and oxidative stress in the blood and bone and maintained osteomineral metabolism and bone histo-architecture. Such observations might be explained by the strong in vitro antioxidant and antibacterial activities exhibited by the alga, as well as by its high levels in several minerals: calcium, phosphorus, sodium, potassium, magnesium, iron, and zinc.     

Development of an animal‐component free medium for vero cells culture

This work describes the development of an animal‐component free medium (IPT‐AFM) that allows an optimal growth of Vero cells, an adherent cell line used for the production of viral vaccines. Statistical experimental design was applied to identify crucial nutrients that affect cell growth. Using Medium 199 or MEM as a basal medium, a serum‐free medium (SFM) referred as IPT‐SFM that only enclosed transferrin as a component of animal origin was developed at first. Then, the composition of IPT‐SFM was further improved to obtain an animal‐component free medium named IPT‐AFM. IPT‐AFM contains M199 as a basal medium, plant hydrolysates, epidermal growth factor, ethanolamine, ferric citrate, and vitamin C. Among various plant hydrolysates, specific combinations of soy (Hypep 1510) and wheat gluten (Hypeps 4601 and 4605) hydrolysates, were identified to promote cell growth; whereas individual Hypeps had a minor positive effect on cell growth. Nevertheless, the removal of serum did influence cell attachment. Coating tissue‐culture flasks with teleostean, a product extracted from cold water fish skin, had not only enhanced cell attachment but also improved cell growth performance in static cultures. Different non‐animal proteases were also assessed as an alternative to trypsin. TrypLE Select, a recombinant trypsin, gave the best cell growth performances. Kinetics of cell growth in IPT‐AFM were investigated in T‐flasks, cell growth was comparable with that obtained in MEM+10% fetal calf serum (FCS). A mean cell division number equal to 2.26 ± 0.18 and a specific growth rate μ 0.019 ± 0.003 h^?1 were achieved in IPT‐AFM. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Evolution of tocopherols in relation of unsaturated fatty acids during maturation of seeds of rapeseed (Brassica napus L.)

The oil content increases during the maturation of seeds (rise of 30%), but decreases at the end of seed maturation. Differences between SDS-PAGE total protein profiles were shown. Polyunsaturated fatty acids contents increase during middle-maturation. Contents of alpha and gamma tocopherols increase with time. This increase is explained by the fact that tocopherols participate actively in the protection of membranes whose phospholipids consist of polyunsaturated fatty acids (PUFAs).