Susceptibility to obesity differs depending on the genetic background and housing temperatures. We have recently reported that CETP expressing female mice are leaner due to increased lipolysis, brown adipose tissue (BAT) activity, and body energy expenditure compared to nontransgenic (NTg) littermates under standard housing temperature (22 degrees C). The aim of this study is to evaluate how CETP expression affects body temperature, composition, and metabolism during cold exposure (4 degrees C) and thermoneutrality (30 degrees C). When submitted to cold, CETP mice maintained rectal temperature, body weight, and food intake similarly to NTg mice along acute or chronic exposure to 4 degrees C. The body oxygen consumption in response to an isoproterenol challenge was 21% higher at 22 degrees C, and 41% higher after 7 days of cold exposure in CETP than in NTg mice. In addition, BAT biopsies from CETP mice showed reduced lipid content and increased basal oxygen consumption rates. Under thermoneutrality (30 degrees C), when BAT activity is inhibited, CETP mice showed higher rectal and tail temperatures, increased food intake, and increased energy expenditure. Lean mass was elevated and fat mass reduced in CETP mice kept at 30 degrees C. In this thermoneutral condition, soleus muscle, but not gastrocnemius or liver of CETP mice, showed increased mitochondrial respiration rates. These data indicate that CETP expression confers a greater capacity of elevating body metabolic rates at both cold exposure, through BAT activity, and at thermoneutrality, through increased muscle metabolism. Thus, the CETP expression levels in females should be considered as a new influence in the contexts of obesity and metabolic disorders propensity.

Overall health relies on features of skeletal muscle that generally decline with age, partly due to mechanisms associated with mitochondrial redox imbalance and bioenergetic dysfunction. Previously, aged mice genetically devoid of the mitochondrial NAD(P)+ transhydrogenase (NNT, encoded by the nicotinamide nucleotide transhydrogenase gene), an enzyme involved in mitochondrial NADPH supply, were shown to exhibit deficits in locomotor behavior. Here, by using young, middle-aged, and older NNT-deficient (Nnt−/−) mice and age-matched controls (Nnt+/+), we aimed to investigate how muscle bioenergetic function and motor performance are affected by NNT expression and aging. Mice were subjected to the wire-hang test to assess locomotor performance, while mitochondrial bioenergetics was evaluated in fiber bundles from the soleus, vastus lateralis and plantaris muscles. An age-related decrease in the average wire-hang score was observed in middle-aged and older Nnt−/− mice compared to age-matched controls. Although respiratory rates in the soleus, vastus lateralis and plantaris muscles did not significantly differ between the genotypes in young mice, the rates of oxygen consumption did decrease in the soleus and vastus lateralis muscles of middle-aged and older Nnt−/− mice. Notably, the soleus, which exhibited the highest NNT expression level, was the muscle most affected by aging, and NNT loss. Additionally, histology of the soleus fibers revealed increased numbers of centralized nuclei in older Nnt−/− mice, indicating abnormal morphology. In summary, our findings suggest that NNT expression deficiency causes locomotor impairments and muscle dysfunction during aging in mice.

Heavy metal exposure leads to multiple system dysfunctions. The mechanisms are likely multifactorial and involve inflammation and oxidative stress. The aim of this study was to evaluate markers and risk factors for atherosclerosis in the LDL receptor knockout mouse model chronically exposed to inorganic mercury (Hg) in the drinking water. Results revealed that Hg exposed mice present increased plasma levels of cholesterol, without alterations in glucose. As a major source and target of oxidants, we evaluated mitochondrial function. We found that liver mitochondria from Hg treated mice show worse respiratory control, lower oxidative phosphorylation efficiency and increased H2O2 release. In addition, Hg induced mitochondrial membrane permeability transition. Erythrocytes from Hg treated mice showed a 50% reduction in their ability to take up oxygen, lower levels of reduced glutathione (GSH) and of antioxidant enzymes (SOD, catalase and GPx). The Hg treatment disturbed immune system cells counting and function. While lymphocytes were reduced, monocytes, eosinophils and neutrophils were increased. Peritoneal macrophages from Hg treated mice showed increased phagocytic activity. Hg exposed mice tissues present metal impregnation and parenchymal architecture alterations. In agreement, increased systemic markers of liver and kidney dysfunction were observed. Plasma, liver and kidney oxidative damage indicators (MDA and carbonyl) were increased while GSH and thiol groups were diminished by Hg exposure. Importantly, atherosclerotic lesion size in the aorta root of Hg exposed mice were larger than in controls. In conclusion, in vivo chronic exposure to Hg worsens the hypercholesterolemia, impairs mitochondrial bioenergetics and redox function, alters immune cells profile and function, causes several tissues oxidative damage and accelerates atherosclerosis development.

Mitochondria are proposed to be the main target of photobiomodulation (PBM) at the cell level, where photon absorption would lead to therapeutic results. However, there is still plenty to be explored and understood about the effects of photons on mitochondria. This study investigates photon interactions at 635 nm on mitochondrial bioenergetics measuring mitochondrial respiration through high-resolution respirometry (HRR) techniques. Mice (C57BL/6 females) liver mitochondria were isolated and immediately irradiated with a LASER beam emitting at 635 nm while measuring mitochondrial respiration. The selected protocol for respiration analysis allowed the evaluation of oxidative phosphorylation, non-oxidative phosphorylation and the maximal capacity of electron transport chain. The following experimental conditions were applied: temperature (37 and 28 °C), sample concentration (0.5 and 0.25 mg.mL-1) and LASER power intensities (200 to 800 mW). At 37 °C and 0.5 mg.mL-1, the effects were small, but indicated that higher powers might be more promising, and subsequent experiments followed with power intensities ranging from 600 to 800 mW. At 28 °C and 0.25 mg.mL-1 there was a photothermal influence of irradiation, interfering with the oxygen consumption measurements. These results entail the search for a way to correct such effect, aiming to bring the experimental conditions closer to those idealized for this study, that proposes to measure light effects in real time.

To investigate whether ovariectomy affects mitochondrial respiratory function, gene expression of the biogenesis markers and mitochondrial dynamics of the vastus lateralis muscle, female Wistar rats divided into ovariectomized (OVX) and intact (INT) groups were kept sedentary (SED) or submitted to resistance training (RT) performed for thirteen weeks on a vertical ladder in which animals climbed with a workload apparatus. RT sessions were performed with four climbs with 65, 85, 95, and 100 % of the rat's previous maximum workload. Mitochondrial Respiratory Function data were obtained by High-resolution respirometry. Gene expression of FIS1, MFN1 and PGC1-α was evaluated by real-time PCR. There was a decrease on oxidative phosphorylation capacity in OVX-SED compared to other groups. Trained groups presented increase on oxidative phosphorylation capacity when compared to sedentary groups. For respiratory control ratio (RCR), OVX-SED presented lower values when compared to INT-SED and to trained groups. Trained groups presented RCR values higher compared to INT-SED. Exercise increased the values of FIS1, MFN1 and PGC1-α expression compared to OVX-SED. Our results demonstrated that in the absence of ovarian hormones, there is a great decrease in oxidative phosphorylation and electron transfer system capacities of sedentary animals. RT was able to increase the expression of genes related to mitochondrial dynamics markers, reversing the condition determined by ovariectomy.

Molecular assays have been widely used for the detection and quantification of bovine babesiosis due to their high sensitivity and specificity. However, variations in the sensitivity of pathogen detection may occur depending on the selected target gene. Thus, this study aimed to compare the detection sensitivity (DS) of Babesia bovis and B. bigemina infection levels in artificially and naturally infected cattle using quantitative PCR (qPCR) and six target genes. For B. bovis, the merozoite surface antigen genes 2b and 2c (msa-2b and msa-2c), and the mitochondrial cytochrome b gene (cybmt) were used. For B. bigemina, the genes encoding the proteins associated with rhoptry 1c (rap-1c), rap-1a, and cybmt were used. Six bovines, free of babesiosis, were artificially infected with 1 × 10-8 red blood cells infected (iRBC) with B. bovis (n = 3) or 1 × 10-6B. bigemina iRBC (n = 3). The animals were evaluated daily until parasitemia was confirmed (≥ 2.0%). The quantity of iRBC present in each animal was determined by examining blood smears. Blood samples were then subjected to DNA extraction, serial dilution, and qPCR analysis to determine the DS of each target gene. In addition, 30 calves naturally infected by Babesia spp. were also evaluated using the same six target genes. Regarding the artificial infection, B. bovis cybmt showed 25-fold higher sensitivity than the msa-2b and msa-2c genes, while the B. bigemina cybmt exhibited 5-fold and 25-fold higher sensitivity than the rap-1a and rap-1c genes, respectively. The rap-1a gene was found to be 5 times more sensitive than the rap-1c gene, while the B. bovis msa-2b and msa-2c genes exhibited similar DS. The positive frequencies of naturally infected calves for the target cybmt, msa-2b, and msa-2c genes (B. bovis) were: 100%, 33.3% and 50%, while cybmt, rap-1a, and rap-1c genes (B. bigemina) were 90%, 83.3%, and 63.3%, respectively. This study may contribute to the selection of suitable genes for molecular monitoring of bovine babesiosis. Mitochondrial genes could be considered as an alternative to improve the sensitivity of B. bovis and B. bigemina detection using qPCR.

AIM:Atherosclerosis is responsible for high morbidity and mortality rates around the world. Local arterial oxidative stress is involved in all phases of atherosclerosis development. Mitochondria is a relevant source of the oxidants, particularly under certain risky conditions, such as hypercholesterolemia. The aim of this study was to test whether lowering the production of mitochondrial oxidants by induction of a mild uncoupling can reduce atherosclerosis in hypercholesterolemic LDL receptor knockout mice.METHODS:The mice were chronically treated with very low doses of DNP (2,4-dinitrophenol) and metabolic, inflammatory and redox state markers and atherosclerotic lesion sizes were determined.RESULTS:The DNP treatment did not change the classical atherosclerotic risk markers, such as plasma lipids, glucose homeostasis, and fat mass, as well as systemic inflammatory markers. However, the DNP treatment diminished the production of mitochondrial oxidants, systemic and tissue oxidative damage markers, peritoneal macrophages and aortic rings oxidants generation. Most importantly, development of spontaneous and diet-induced atherosclerosis (lipid and macrophage content) were significantly decreased in the DNP-treated mice. In vitro, DNP treated peritoneal macrophages showed decreased H2O2 production, increased anti-inflammatory cytokines gene expression and secretion, increased phagocytic activity, and decreased LDL-cholesterol uptake.CONCLUSIONS:These findings are a proof of concept that activation of mild mitochondrial uncoupling is sufficient to delay the development of atherosclerosis under the conditions of hypercholesterolemia and oxidative stress. These results promote future approaches targeting mitochondria for the prevention or treatment of atherosclerosis.

The interaction between supraphysiological cytosolic Ca2+ levels and mitochondrial redox imbalance mediates the mitochondrial permeability transition (MPT). The MPT is involved in cell death, diseases and aging. This study compared the liver mitochondrial Ca2+ retention capacity and oxygen consumption in the long-lived red-footed tortoise (Chelonoidis carbonaria) with those in the rat as a reference standard. Mitochondrial Ca2+ retention capacity, a quantitative measure of MPT sensitivity, was remarkably higher in tortoises than in rats. This difference was minimized in the presence of the MPT inhibitors ADP and cyclosporine A. However, the Ca2+ retention capacities of tortoise and rat liver mitochondria were similar when both MPT inhibitors were present simultaneously. NADH-linked phosphorylating respiration rates of tortoise liver mitochondria represented only 30% of the maximal electron transport system capacity, indicating a limitation imposed by the phosphorylation system. These results suggested underlying differences in putative MPT structural components [e.g. ATP synthase, adenine nucleotide translocase (ANT) and cyclophilin D] between tortoises and rats. Indeed, in tortoise mitochondria, titrations of inhibitors of the oxidative phosphorylation components revealed a higher limitation of ANT. Furthermore, cyclophilin D activity was approximately 70% lower in tortoises than in rats. Investigation of critical properties of mitochondrial redox control that affect MPT demonstrated that tortoise and rat liver mitochondria exhibited similar rates of H2O2 release and glutathione redox status. Overall, our findings suggest that constraints imposed by ANT and cyclophilin D, putative components or regulators of the MPT pore, are associated with the enhanced resistance to Ca2+-induced MPT in tortoises.

The mechanisms by which a high-fat diet (HFD) promotes non-alcoholic fatty liver disease (NAFLD) appear to involve liver mitochondrial dysfunction and redox imbalance. The functional loss of the enzyme NAD(P)+ transhydrogenase, a main source of mitochondrial NADPH, results in impaired mitochondrial peroxide removal, pyruvate dehydrogenase inhibition by phosphorylation, and progression of NAFLD in HFD-fed mice. The present study aimed to investigate whether pharmacological reactivation of pyruvate dehydrogenase by dichloroacetate attenuates the mitochondrial redox dysfunction and the development of NAFLD in NAD(P)+ transhydrogenase-null (Nnt-/-) mice fed an HFD (60% of total calories from fat). For this purpose, Nnt-/- mice and their congenic controls (Nnt+/+) were fed chow or an HFD for 20 weeks and received sodium dichloroacetate or NaCl in the final 12 weeks via drinking water. The results showed that HFD reduced the ability of isolated liver mitochondria from Nnt-/- mice to remove peroxide, which was prevented by the dichloroacetate treatment. HFD-fed mice of both Nnt genotypes exhibited increased body and liver mass, as well as a higher content of hepatic triglycerides, but dichloroacetate treatment attenuated these abnormalities only in Nnt-/- mice. Notably, dichloroacetate treatment decreased liver pyruvate dehydrogenase phosphorylation levels and prevented the aggravation of NAFLD in HFD-fed Nnt-/- mice. Conversely, dichloroacetate treatment elicited moderate hepatocyte ballooning in chow-fed mice, suggesting potentially toxic effects. We conclude that the protection against HFD-induced NAFLD by dichloroacetate is associated with its role in reactivating pyruvate dehydrogenase and reestablishing the pyruvate-supported liver mitochondrial capacity to handle peroxide in Nnt-/- mice.

The aim of this study was to determine the functional and biochemical changes at the neuromuscular junction (NMJ) induced by sepsis.Male Sprague–Dawley rats were divided into three groups as follows: control, denervation, and sepsis. The rats were subjected to cecal ligation and puncture (CLP) or tibias nerve transection. NMJ function and the area of end plates were assessed, and the protein level of acetylcholine receptors and axonal neuregulin-1 was evaluated on postoperative days 1, 7, and 14.In the control group, the amplitude of compound muscle action potential (CMAP) was 16.51 ± 2.53 mV. In the sepsis group, the amplitude of CMAP decreased, and duration was prolonged on postoperative days 7 and 14 (P < 0.01). Meanwhile, motor conduction velocity decreased significantly (P < 0.01). CMAP was lost in the denervation group. The twitch tension magnitude gradually declined (P < 0.05) in the sepsis group, although it could not be recorded after lesion. Sepsis and denervation upregulated the expression of γ-nicotinic acetylcholine receptor (nAChR) and α7-nAChR in muscle membrane, compared with those in normal NMJ (261.4 ± 26.5 μm2). The NMJ area decreased from 254.6 ± 23.8 μm2 (1 d after CLP) to 275.4 ± 22.6 μm2 (7 d after CLP) to 322.7 ± 34.4 μm2 (14 d after CLP). The postsynaptic NMJ had more discrete fragments (3.84 ± 0.6) compared with the control group (2.13 ± 0.4; P < 0.01). After denervation, NMJ underwent fragmentation and the number of discrete fragments increased (5.57 ± 1.2; P < 0.01). NMJ area increased from 254.6 ± 23.8 μm2 (1 d after CLP) to 275.4 ± 22.6 μm2 (7 d after CLP) to 322.7 ± 34.4 μm2 (14 d after CLP). Sepsis induced neuregulin-1 to decrease from 1 d up to 2 wk compared with the control group (P < 0.05).Chronic sepsis has a denervation-like effect on the NMJ, which was indicated by upregulation of heterogeneous nAChRs, the increased area of end plates, and demyelination of the motoneuron axon.

Significance: Millions of people are infected with trypanosomatids and new therapeutic approaches are needed. Trypanosomatids possess one mitochondrion per cell and its study has led to discoveries of general biological interest. These mitochondria, as in their animal counterparts, generate reactive oxygen species (ROS) and have evolved enzymatic and nonenzymatic defenses against them. Mitochondrial calcium ion (Ca2+) overload leads to generation of ROS and its study could lead to relevant information on the biology of trypanosomatids and to novel drug targets. Recent Advances: Mitochondrial Ca2+ is normally involved in maintaining the bioenergetics of trypanosomes, but when Ca2+ overload occurs, it is associated with cell death. Trypanosomes lack key players in the mechanism of cell death described in mammalian cells, although mitochondrial Ca2+ overload results in collapse of their membrane potential, production of ROS, and cytochrome c release. They are also very resistant to mitochondrial permeability transition, and cell death after mitochondrial Ca2+ overload depends on generation of ROS. Critical Issues: In this review, we consider the mechanisms of mitochondrial oxidant generation and removal and the involvement of Ca2+ in trypanosome cell death. Future Directions: More studies are required to determine the reactions involved in generation of ROS by the mitochondria of trypanosomatids, their enzymatic and nonenzymatic defenses against ROS, and the occurrence and composition of a mitochondrial permeability transition pore.

Species identification in dairy products has a notable importance in food traceability and adulteration control and consequently has a significant effect on the final economic value of foods. In the present study, we developed a method based on real-time quantitative PCR (qPCR) for detection and quantification of cow DNA in DNA samples from milk and dairy products from buffaloes, goats, and sheep. The qPCR reactions showed high specificity, and the amplifications only occurred to species-specific primers. The calibration curves allowed for the quantification of the amount of DNA of each species-specific primer, and the established detection limit was 0.016 ng for the four species. The detection limit of cow DNA in buffalo, goat and sheep DNA samples was 0.1% (0.01 ng). Although the present study aimed to detect and quantify cow DNA in buffalo, goat, and sheep dairy products, we believe that the qPCR assays can also be directed to differentiate and quantify goat × sheep, and/or buffalo × goat/sheep.

Statins are successful drugs used to treat hypercholesterolemia, a primary cause of atherosclerosis. In this work, we investigated how hypercholesterolemia and pravastatin treatment impact macrophage and mitochondria functions, the key cell involved in atherogenesis. By comparing bone marrow-derived macrophages (BMDM) of wild-type (WT) and LDL receptor knockout (LDLr-/-) mice, we observed hypercholesterolemia increased the number of contact sites at mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), enhanced mitochondrial hydrogen peroxide release, altered the gene expression of inflammatory markers, and increased oxidized LDL (ox-LDL) uptake and phagocytic activity. Three months of in vivo pravastatin treatment of LDLr-/- mice reversed the number of contact sites at the MAM, ox-LDL uptake, and phagocytosis in LDLr-/- BMDM. Additionally, pravastatin increased BMDM mitochondrial network branching. In peritoneal macrophages (PMs), hypercholesterolemia did not change MAM stability, but stimulated hydrogen peroxide production and modulated gene expression of pro- and anti-inflammatory markers. It also increased mitochondrial branching degree and had no effects on ox-LDL uptake and phagocytosis in PM. Pravastatin treatment increased superoxide anion production and changed inflammation-related gene expression in LDLr-/- PM. In addition, pravastatin increased markedly the expression of the mitochondrial dynamics-related genes Mfn2 and Fis1 in both macrophages. In summary, our results show that hypercholesterolemia and pravastatin treatment affect macrophage mitochondria network structure as well as their interaction with the endoplasmic reticulum (ER). These effects impact on macrophage conversion rates to foam cell and macrophage phagocytic capacity. These findings associate MAM stability changes with known mechanisms involved in atherosclerosis progression and resolution.

The occurrence of hepatic lipidosis is commonly reported in different reptilian species, especially in animals under captivity. Liver accumulation of fat is associated with disorders, better described in mammals as non-alcoholic fatty liver diseases (NAFLD), ranging from simple steatosis, to non-alcoholic steatohepatitis (NASH), and to more severe lesions of cirrhosis and hepatocellular carcinoma. Mitochondria play a central role in NAFLD pathogenesis, therefore in this study we characterized livers of ad libitum fed captive red-footed tortoise Chelonoidis carbonaria through histological and mitochondrial function evaluations of juvenile and adult individuals. Livers from adult tortoises exhibited higher levels of lipids, melanomacrophages centers and melanin than juveniles. The observed high score levels of histopathological alterations in adult tortoises, such as microvesicular steatosis, inflammation and fibrosis, indicated the progression to a NASH condition. Mitochondrial oxygen consumption at different respiratory states and with different substrates was 30 to 58% lower in adult when compared to juvenile tortoises. Despite citrate synthase activity was also lower in adults, cardiolipin content was similar to juveniles, indicating that mitochondrial mass was unaffected by age. Mitochondrial Ca2+ retention capacity was reduced by 70% in adult tortoises. Overall, we found that aggravation of NAFLD in ad libitum fed captive tortoises is associated with compromised mitochondrial function, indicating a critical role of the organelle in liver disease progression in reptiles.

Significance: Aging is a natural process that affects most living organisms, resulting in increased mortality. As the world population ages, the prevalence of age-associated diseases, and their associated health care costs, has increased sharply. A better understanding of the molecular mechanisms that lead to cellular dysfunction may provide important targets for interventions to prevent or treat these diseases. Recent Advances: Although the mitochondrial theory of aging had been proposed more than 40 years ago, recent new data have given stronger support for a central role for mitochondrial dysfunction in several pathways that are deregulated during normal aging and age-associated disease. Critical Issues: Several of the experimental evidence linking mitochondrial alterations to age-associated loss of function are correlative and mechanistic insights are still elusive. Here, we review how mitochondrial dysfunction may be involved in many of the known hallmarks of aging, and how these pathways interact in an intricate net of molecular relationships. Future Directions: As it has become clear that mitochondrial dysfunction plays causative roles in normal aging and age-associated diseases, it is necessary to better define the molecular interactions and the temporal and causal relationship between these changes and the relevant phenotypes seen during the aging process. Antioxid. Redox Signal. 36, 824-843.

Plasma cholesteryl ester transfer protein (CETP) activity diminishes HDL-cholesterol levels and thus may increase atherosclerosis risk. Experimental evidence suggests CETP may also exhibit anti-inflammatory properties, but local tissue-specific functions of CETP have not yet been clarified. Since oxidative stress and inflammation are major features of atherogenesis, we investigated whether CETP modulates macrophage oxidant production, inflammatory and metabolic profiles. Comparing macrophages from CETP-expressing transgenic mice and non-expressing littermates, we observed that CETP expression reduced mitochondrial superoxide anion production and H2O2 release, increased maximal mitochondrial respiration rates, and induced elongation of the mitochondrial network and expression of fusion-related genes (mitofusin-2 and OPA1). The expression of pro-inflammatory genes and phagocytic activity were diminished in CETP-expressing macrophages. In addition, CETP-expressing macrophages had less unesterified cholesterol under basal conditions and after exposure to oxidized LDL, as well as increased HDL-mediated cholesterol efflux. CETP knockdown in human THP1 cells increased unesterified cholesterol and abolished the effects on mitofusin-2 and TNFα. In summary, the expression of CETP in macrophages modulates mitochondrial structure and function to promote an intracellular antioxidant state and oxidative metabolism, attenuation of pro-inflammatory gene expression, reduced cholesterol accumulation, and phagocytosis. These localized functions of CETP may be relevant for the prevention of atherosclerosis and other inflammatory diseases.

Pyruvate is the final metabolite of glycolysis and can be converted into acetyl coenzyme A (acetyl-CoA) in mitochondria, where it is used as the substrate for the tricarboxylic acid cycle. Pyruvate availability in mitochondria depends on its active transport through the heterocomplex formed by the mitochondrial pyruvate carriers 1 and 2 (MPC1/MPC2). We report here studies on MPC1/MPC2 of Trypanosoma cruzi, the etiologic agent of Chagas disease. Endogenous tagging of T. cruziMPC1 (TcMPC1) and TcMPC2 with 3×c-Myc showed that both encoded proteins colocalize with MitoTracker to the mitochondria of epimastigotes. Individual knockout (KO) of TcMPC1 and TcMPC2 genes using CRISPR/Cas9 was confirmed by PCR and Southern blot analyses. Digitonin-permeabilized TcMPC1-KO and TcMPC2-KO epimastigotes showed reduced O2 consumption rates when pyruvate, but not succinate, was used as the mitochondrial substrate, while α-ketoglutarate increased their O2 consumption rates due to an increase in α-ketoglutarate dehydrogenase activity. Defective mitochondrial pyruvate import resulted in decreased Ca2+ uptake. The inhibitors UK5099 and malonate impaired pyruvate-driven oxygen consumption in permeabilized control cells. Inhibition of succinate dehydrogenase by malonate indicated that pyruvate needs to be converted into succinate to increase respiration. TcMPC1-KO and TcMPC2-KO epimastigotes showed little growth differences in standard or low-glucose culture medium. However, the ability of trypomastigotes to infect tissue culture cells and replicate as intracellular amastigotes was decreased in TcMPC-KOs. Overall, T. cruzi MPC1 and MPC2 are essential for cellular respiration in the presence of pyruvate, invasion of host cells, and replication of amastigotes.IMPORTANCETrypanosoma cruzi is the causative agent of Chagas disease. Pyruvate is the end product of glycolysis, and its transport into the mitochondrion is mediated by the mitochondrial pyruvate carrier (MPC) subunits. Using the CRISPR/Cas9 technique, we generated individual T. cruziMPC1 (TcMPC1) and TcMPC2 knockouts and demonstrated that they are essential for pyruvate-driven respiration. Interestingly, although glycolysis was reported as not an important source of energy for the infective stages, MPC was essential for normal host cell invasion and intracellular replication.

Mitochondrial calcium ion (Ca2+) uptake is important for buffering cytosolic Ca2+ levels, for regulating cell bioenergetics, and for cell death and autophagy. Ca2+ uptake is mediated by a mitochondrial Ca2+ uniporter (MCU) and the discovery of this channel in trypanosomes has been critical for the identification of the molecular nature of the channel in all eukaryotes. However, the trypanosome uniporter, which has been studied in detail in Trypanosoma cruzi, the agent of Chagas disease, and T. brucei, the agent of human and animal African trypanosomiasis, has lineage-specific adaptations which include the lack of some homologues to mammalian subunits, and the presence of unique subunits. Here, we review newly emerging insights into the role of mitochondrial Ca2+ homeostasis in trypanosomes, the composition of the uniporter, its functional characterization, and its role in general physiology.

The rising consumption of A2 milk and its derivatives in recent years has garnered attention from both consumers and producers, mainly due its possible health benefits, such as enhanced digestion and easier absorption. Thus, a novel real-time PCR using a combination of locked nucleic acid modified (LNA) conjugated probes was developed to genotype A1 and A2 alleles of β-casein gene (CSN2) and to detect and quantify the A1 presence in A2 samples. The limit of detection for each probe (A1 and A2) was evaluated using decreasing serial dilutions. Besides, the sensitivity of A1 allele detection in the A2 samples was also tested. The limits of detection of A1 and A2 alleles were 6 copies, while for A1 allele detection in A2 samples was 7.5 copies (1%). The LNA-probe based method was found to be rapid, robust, highly sensitive, cost effective, and can be employed as screening test to certificate the A2 dairy products.