Boosting life sciences research in Brazil: building a case for a local Drosophila stock center.

Drosophila melanogaster is undoubtedly one of the most useful model organisms in biology. Initially used in solidifying the principles of heredity, and establishing the basic concepts of population genetics and of the synthetic theory of evolution, it can currently offer scientists much more: the possibility of investigating a plethora of cellular and biological mechanisms, from development and function of the immune system to animal neurogenesis, tumorigenesis and beyond. Extensive resources are available for the community of Drosophila researchers worldwide, including an ever-growing number of mutant, transgenic and genomically-edited lines currently carried by stock centers in North America, Europe and Asia. Here, we provide evidence for the importance of stock centers in sustaining the substantial increase in the output of Drosophila research worldwide in recent decades. We also discuss the challenges that Brazilian Drosophila scientists face to keep their research projects internationally competitive, and argue that difficulties in importing fly lines from international stock centers have significantly stalled the progression of all Drosophila research areas in the country. Establishing a local stock center might be the first step towards building a strong local Drosophila community that will likely contribute to all areas of life sciences research.

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry.

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, Drosophila research lacks popularity in developing countries, possibly due to the misinformed idea that establishing a lab and performing relevant experiments with such tiny insects is difficult and requires expensive, specialized apparatuses. Here, we describe how to build an affordable flylab to quantitatively analyze a myriad of behavioral parameters in D. melanogaster, by 3D-printing many of the necessary pieces of equipment. We provide protocols to build in-house vial racks, courtship arenas, apparatuses for locomotor assays, etc., to be used for general fly maintenance and to perform behavioral experiments using adult flies and larvae. We also provide protocols on how to use more sophisticated systems, such as a high resolution oxygraph, to measure mitochondrial oxygen consumption in larval samples, and show its association with behavioral changes in the larvae upon the xenotopic expression of the mitochondrial alternative oxidase (AOX). AOX increases larval activity and mitochondrial leak respiration, and accelerates development at low temperatures, which is consistent with a thermogenic role for the enzyme. We hope these protocols will inspire researchers, especially from developing countries, to use Drosophila to easily combine behavior and mitochondrial metabolism data, which may lead to information on genes and/or environmental conditions that may also regulate human physiology and disease states.

Effects of intermittent dietary supplementation with conjugated linoleic acid and fish oil (EPA/DHA) on body metabolism and mitochondrial energetics in mice.

Understanding the mitochondrial processes that contribute to body energy metabolism may provide an attractive therapeutic target for obesity and co-morbidities. Here we investigated whether intermittent dietary supplementation with conjugated linoleic (CLA, 18:2n-6), docosahexaenoic (22:6n-3, DHA) and eicosapentaenoic (20:5n-3, EPA) acids, either alone or in combination, changes body metabolism associated with mitochondrial functions in the brain, liver, skeletal muscle and brown adipose tissue (BAT). Male C57Bl/6 mice were divided into groups: CLA (50% cis-9, trans-11; 50% trans-10, cis-12), EPA/DHA (64% EPA; 28% DHA), CLA plus EPA/DHA or control (linoleic acid). Each mouse received 3 g/kg b.w. of the stated oil by gavage on alternating days for 60 days. Dietary supplementation with CLA or EPA/DHA increased body VO2 consumption, VCO2 production and energy expenditure, being fish oil (FO) the most potent even in combination with CLA. Individually, both oils reduced mitochondrial density in BAT. CLA supplementation alone also a) elevated the expression of uncoupling proteins in soleus, liver and hippocampus and the uncoupling activity in the last two, ad this effect was associated with reduced hydrogen peroxide production in hippocampus; b) increased proteins related to mitochondrial fission in liver. EPA/DHA supplementation alone also a) induced mitochondrial biogenesis in liver, soleus and hippocampus associated with increased expression of PGC1-α; b) induced proteins related to mitochondrial fusion in the liver, and fission and fusion in the hippocampus. Therefore, this study shows changes on mitochondrial mechanisms induced by CLA and/or EPA/DHA that can be associated with elevated body energy expenditure.

Triglyceride depletion of brown adipose tissue enables analysis of mitochondrial respiratory function in permeabilized biopsies.

The research on mitochondrial functions in adipocytes has increasingly evidenced that mitochondria plays an important role in the onset and/or progression of obesity and related pathologies. Mitochondrial function in brown adipose tissue (BAT) has been classically assessed by measuring either the levels/activity of mitochondrial enzymes, or the respiration in isolated mitochondria. Isolation of mitochondria is not advantageous because it demands significant time and amount of tissue and, as tissue homogenates, disrupts biochemical and physical connections of mitochondria within the cell. Here, we described a new and efficient protocol to analyze the mitochondrial respiratory states in BAT biopsies that relies on intracellular triglyceride depletion followed by tissue permeabilization. In addition to minimizing tissue requirements to ∼17 mg wet weight, the proposed protocol enabled analysis of all mitochondrial respiratory states, including phosphorylation (OXPHOS), no-phosphorylation (LEAK), and uncoupled (ETS) states, as well as the use of substrates for complex I, complex II, and cytochrome c; together, these features demonstrated mitochondrial integrity and validated the preparation efficacy. Therefore, the protocol described here increases the possibilities of answering physiological questions related to small BAT regions of human and animal models, which shall help to unravel the mechanisms that regulate mitochondrial function in health and disease.