Lopinavir and Nelfinavir Induce the Accumulation of Crystalloid Lipid Inclusions within the Reservosomes of Trypanosoma cruzi and Inhibit Both Aspartyl-Type Peptidase and Cruzipain Activities Detected in These Crucial Organelles

Several research groups have explored the repositioning of human immunodeficiency virus aspartyl peptidase inhibitors (HIV-PIs) on opportunistic infections caused by bacteria, fungi and protozoa. In Trypanosoma cruzi, HIV-PIs have a high impact on parasite viability, and one of the main alterations promoted by this treatment is the imbalance in the parasite’s lipid metabolism. However, the reasons behind this phenomenon are unknown. In the present work, we observed by transmission electron microscopy (TEM) that the treatment of T. cruzi epimastigotes with the HIV-PIs lopinavir and nelfinavir induced a huge accumulation of crystalloid-shaped lipids within the reservosomes, most of them deforming these key organelles. As previously reported, those structures are characteristic of lipid inclusions formed mostly of cholesterol and cholesterol-esters. The fractionation of nontreated epimastigotes generated two distinct fractions enriched in reservosomes: one mostly composed of lipid inclusion-containing reservosomes (Fraction B1) and one where lipid inclusions were much less abundant (Fraction B2). Interestingly, the extract of Fraction B2 presented enzymatic activity related to aspartyl-type peptidases 3.5 times higher than that found in the extract obtained from Fraction B1. The cleavage of cathepsin D substrate by this class of peptidases was strongly impaired by pepstatin A, a prototypical aspartyl PI, and the HIV-PIs lopinavir and nelfinavir. In addition, both HIV-PIs also inhibited (to a lesser extent) the cruzipain activity present in reservosomes. Finally, our work provides new evidence concerning the presence and supposed participation of aspartyl peptidases in T. cruzi, even as it adds new information about the mechanisms behind the alterations promoted by lopinavir and nelfinavir in the protozoan.

Immobilization of Daurian Rosehip Antioxidants by Protein-Lipid Inclusion

Introduction. Antioxidant system is one of the main cellular mechanisms of adaptation. It is able to neutralize the destructive effect of free radicals, both external and internal. Functional products, especially of meat origin, can provide necessary antioxidant properties. Some types of plant raw materials possess a high content of polyphenol complexes with antioxidant properties. Therefore, introduction of phytonutrients into the composition of meat products can help to expand the range of foods with an antioxidant effect. Daurian rosehip is rich in biologically active substances and can increase their safety during processing. The research objective was to study the possibility of immobilization of Daurian rosehip antioxidants by protein-lipid inclusion. Study objects and methods. The research featured fruits of Daurian rosehip, its aqueous alcoholic extract, and protein-lipid complex. Results and discussion. The article presents experimental data on the chemical composition of Daurian rosehip. The optimal method with the best antioxidant results was a microwave phytoextraction by an aqueous alcoholic solution at a power of 800 W for 6–7 min. The antioxidants were immobilized by including the protein-lipid complex into the gel. The experiment demonstrated excellent functional and technological characteristics of the resulting system. Conclusion. The research revealed the optimal modes of microwave extraction of biologically active substances of Daurian rosehip. The rosehip extract was included into the protein-lipid complex and immobilized antioxidants, thus increasing their efficiency.

Development of a Fluorescent Assay to Search New Drugs Using Stable tdTomato-Leishmania, and the Selection of Galangin as a Candidate With Anti-Leishmanial Activity

Antimonials continue to be considered the first-line treatment for leishmaniases, but its use entails a wide range of side effects and serious reactions. The search of new drugs requires the development of methods more sensitive and faster than the conventional ones. We developed and validated a fluorescence assay based in the expression of tdTomato protein by Leishmania, and we applied this method to evaluate the activity in vitro of flavonoids and reference drugs. The pIR1SAT/tdTomato was constructed and integrated into the genome of Leishmania (Leishmania) amazonensis. Parasites were selected with nourseothricin (NTC). The relation of L. amaz/tc3 fluorescence and the number of parasites was determined; then the growth in vitro and infectivity in BALB/c mice was characterized. To validate the fluorescence assay, the efficacy of miltefosine and meglumine antimoniate was compared with the conventional methods. After that, the method was used to assess in vitro the activity of flavonoids; and the mechanism of action of the most active compound was evaluated by transmission electron microscopy and ELISA. A linear correlation was observed between the emission of fluorescence of L. amaz/tc3 and the number of parasites (r2 = 0.98), and the fluorescence was stable in the absence of NTC. No differences were observed in terms of infectivity between L. amaz/tc3 and wild strain. The efficacy of miltefosine and meglumine antimoniate determined by the fluorescence assay and the microscopic test showed no differences, however, in vivo the fluorescence assay was more sensitive than limiting dilution assay. Screening assay revealed that the flavonoid galangin (GAL) was the most active compound with IC50 values of 53.09 µM and 20.59 µM in promastigotes and intracellular amastigotes, respectively. Furthermore, GAL induced mitochondrial swelling, lipid inclusion bodies and vacuolization in promastigotes; and up-modulated the production of IL-12 p70 in infected macrophages. The fluorescence assay is a useful tool to assess the anti-leishmanial activity of new compounds. However, the assay has some limitations in the macrophage-amastigote model that might be related with an interfere of flavanol aglycones with the fluorescence readout of tdTomato. Finally, GAL is a promising candidate for the development of new treatment against the leishmaniasis.

Effect of dietary lipid inclusion from byproduct-based pellets on dry matter intake, ruminal fermentation, and nutrient digestion in finishing beef heifers

This study was designed to evaluate dry matter intake (DMI), ruminal fermentation, ruminal digesta outflow, omasal flow of N constituents, and ruminal and total-tract digestibility in response to increasing dietary lipid content derived from high-lipid byproduct-based pellets for finishing beef cattle. Five ruminally cannulated heifers were used in a 5 × 5 Latin square design. Dietary treatments were iso-nitrogenous with increasing ether extract (EE) concentrations resulting in dietary concentrations of 3.5, 4.2, 4.7, 5.1 and 5.9% of dry matter. Periods were 28 d in duration with the first 19 d used for dietary adaptation and the last 9 d for sample and data collection. Dry matter intake, ruminal pH, omasal flow of N constituents, and SCFA concentrations were not affected by increasing dietary EE content (P ≥ 0.10). Increasing dietary EE content increased outflow of EE from the rumen (P < 0.001). Apparent ruminal and total-tract digestibility of EE increased linearly (P ≤ 0.03) with increasing dietary EE levels. Results indicate that increasing dietary EE content from 3.5 up to 5.9% of DM by including high-lipid byproduct-based pellets does not alter site of nutrient digestibility with no negative effects on nutrient intake, ruminal fermentation, or apparent ruminal and total-tract digestibility.

Control of Adipose Cell Browning and Its Therapeutic Potential

Adipose tissue is the largest endocrine organ in humans and has an important influence on many physiological processes throughout life. An increasing number of studies have described the different phenotypic characteristics of fat cells in adults. Perhaps one of the most important properties of fat cells is their ability to adapt to different environmental and nutritional conditions. Hypothalamic neural circuits receive peripheral signals from temperature, physical activity or nutrients and stimulate the metabolism of white fat cells. During this process, changes in lipid inclusion occur, and the number of mitochondria increases, giving these cells functional properties similar to those of brown fat cells. Recently, beige fat cells have been studied for their potential role in the regulation of obesity and insulin resistance. In this context, it is important to understand the embryonic origin of beige adipocytes, the response of adipocyte to environmental changes or modifications within the body and their ability to transdifferentiate to elucidate the roles of these cells for their potential use in therapeutic strategies for obesity and metabolic diseases. In this review, we discuss the origins of the different fat cells and the possible therapeutic properties of beige fat cells.

Utilisation of a mix of powdered oils as fat supplement in nursery- and growing-pig diets

Two experiments were conducted to challenge nursery and growing pigs to increased levels of dietary fat (5–10% as fed), using a crystallised powdered oil mix (CPOM), produced by a modified freeze-drying process. Growth performance of nursery pigs was determined and a digestibility trial was also conducted with growing pigs (Experiment 2). The CPOM was compared, at similar levels of lipid inclusion (10% total lipids), with other fat sources commonly used in swine diet, namely soybean oil (SBO) and hydrogenated palm oil. For the growth assay (Experiment 1), the CPOM was prepared and added at different levels (0%, 2.6% and 3.8%) commonly used in commercial diets (Phase 1–4 diets). Seventy-five weaning pigs (28 days of age) were housed in 15 pens (5 pigs per pen) and randomly assigned into the three dietary treatments until 70 days of age. Growth performance and feed utilisation were compared weekly among experimental groups. The use of CPOM improved average daily gain (~60%), and increased average daily feed intake by 40% and 50% at the 2.6% and 3.8% CPOM levels respectively. In the digestibility trial (Experiment 2), pigs fed with 5% incorporation of CPOM (10% of total lipid content) had digestibility of energy and ether extract similar to those fed the SBO-supplemented diet, although the SBO diet had a significantly greater polyunsaturated fatty acid concentration. The powdered crystallisation process of the CPOM fat allowed an equivalent digestibility of this fat source with more saturated fatty acids, and the physical-property effect of this processed oil source on apparent total-tract digestibility should be further studied.

Multiscale Model of Mycobacterium tuberculosis Infection Maps Metabolite and Gene Perturbations to Granuloma Sterilization Predictions

Granulomas are a hallmark of tuberculosis. Inside granulomas, the pathogenMycobacterium tuberculosismay enter a metabolically inactive state that is less susceptible to antibiotics. UnderstandingM. tuberculosismetabolism within granulomas could contribute to reducing the lengthy treatment required for tuberculosis and provide additional targets for new drugs. Two key adaptations ofM. tuberculosisare a nonreplicating phenotype and accumulation of lipid inclusions in response to hypoxic conditions. To explore how these adaptations influence granuloma-scale outcomesin vivo, we present a multiscalein silicomodel of granuloma formation in tuberculosis. The model comprises host immunity,M. tuberculosismetabolism,M. tuberculosisgrowth adaptation to hypoxia, and nutrient diffusion. We calibrated our model toin vivodata from nonhuman primates and rabbits and apply the model to predictM. tuberculosispopulation dynamics and heterogeneity within granulomas. We found that bacterial populations are highly dynamic throughout infection in response to changing oxygen levels and host immunity pressures. Our results indicate that a nonreplicating phenotype, but not lipid inclusion formation, is important for long-termM. tuberculosissurvival in granulomas. We used virtualM. tuberculosisknockouts to predict the impact of both metabolic enzyme inhibitors and metabolic pathways exploited to overcome inhibition. Results indicate that knockouts whose growth rates are below ∼66% of the wild-type growth rate in a culture medium featuring lipid as the only carbon source are unable to sustain infections in granulomas. By mapping metabolite- and gene-scale perturbations to granuloma-scale outcomes and predicting mechanisms of sterilization, our method provides a powerful tool for hypothesis testing and guiding experimental searches for novel antituberculosis interventions.

Ultrastructural Changes and Death ofLeishmania infantumPromastigotes Induced byMorinda citrifoliaLinn. Fruit (Noni) Juice Treatment

The search for new treatments against leishmaniasis has increased due to high frequency of drug resistance registered in endemics areas, side effects, and complications caused by coinfection with HIV.Morinda citrifoliaLinn., commonly known as Noni, has a rich chemical composition and various therapeutic effects have been described in the literature. Studies have shown the leishmanicidal activity ofM. citrifolia; however, its action on the parasite has not yet been elucidated. In this work, we analyzed leishmanicidal activity and ultrastructural changes inLeishmania infantumpromastigotes caused byM. citrifoliafruit juice treatment.M. citrifoliafruit extract showed a yield of 6.31% and high performance liquid chromatography identified phenolic and aromatic compounds as the major constituents. IC50values were 260.5 µg/mL for promastigotes and 201.3 µg/mL for intracellular amastigotes ofL. infantumtreated withM. citrifolia. Cytotoxicity assay with J774.G8 macrophages showed thatM. citrifoliafruit juice was not toxic up to 2 mg/mL. Transmission electron microscopy showed cytoplasmic vacuolization, lipid inclusion, increased exocytosis activity, and autophagosome-like vesicles inL. infantumpromastigotes treated withM. citrifoliafruit juice.M. citrifoliafruit juice was active againstL. infantumin thein vitromodel used here causing ultrastructural changes and has a future potential for treatment against leishmaniasis.