ATP-P2X7 signaling mediates brain pathology while contributing to viral control in perinatal Zika virus infection.

Zika virus (ZIKV), the causative agent of Zika fever, is a flavivirus transmitted by mosquitoes of the Aedes genus. Zika virus infection has become an international concern due to its association with severe neurological complications such as fetal microcephaly. Viral infection can induce the release of ATP in the extracellular environment, activating receptors sensitized by extracellular nucleotides, such as the P2X7 receptor. This receptor is the primary purinergic receptor involved in neuroinflammation, neurodegeneration, and immunity. In this work, we investigated the role of ATP-P2X7 receptor signaling in Zika-related brain abnormalities. Wild-type mice (WT) and P2X7 receptor-deficient (P2X7-/-) C57BL/6 newborn mice were subcutaneously inoculated with 5 × 106plaque-forming units of ZIKV or mock solution. P2X7 receptor expression increased in the brain of Zika virus-infected mice compared to the mock group. Comparative analyses of the hippocampi from WT and P2X7-/-mice revealed that the P2X7 receptor increased hippocampal damage in CA1/CA2 and CA3 regions. Doublecortin expression decreased significantly in the brains of ZIKV-infected mice. WT ZIKV-infected mice showed impaired motor performance compared to P2X7-/- infected mice. WT ZIKV-infected animals showed increased expression of glial markers GFAP (astrocytes) and IBA-1 (microglia) compared to P2X7-/- infected mice. Although the P2X7 receptor contributes to neuronal loss and neuroinflammation, WT mice were more efficient in controlling the viral load in the brain than P2X7 receptor-deficient mice. This result was associated with higher induction of TNF-α, IFN-ß, and increased interferon-stimulated gene expression in WT mice than P2X7-/-ZIKV-infected. Finally, we found that the P2X7 receptor contributes to inhibiting the neuroprotective signaling pathway AKT/mTOR while stimulating the caspase-3 activation, possibly two distinct pathways contributing to neurodegeneration. These findings suggest that ATP-P2X7 receptor signaling contributes to the antiviral response in the brain of ZIKV-infected mice while increasing neuronal loss, neuroinflammation, and related brain abnormalities.

Toward the mechanism of jarastatin (rJast) inhibition of the integrin αVß3.

Disintegrins are a family of cysteine-rich small proteins that were first identified in snake venom. The high divergence of disintegrins gave rise to a plethora of functions, all related to the interaction with integrins. Disintegrins evolved to interact selectively with different integrins, eliciting many physiological outcomes and being promising candidates for the therapy of many pathologies. We used NMR to determine the structure and dynamics of the recombinant disintegrin jarastatin (rJast) and its interaction with the cancer-related integrin αVß3. rJast displayed the canonical fold of a medium-sized disintegrin and showed complex dynamic in multiple timescales. We used NMR experiments to map the interaction of rJast with αVß3, and molecular docking followed by molecular dynamics (MD) simulation to describe the first structural model of a disintegrin/integrin complex. We showed that not only the RGD loop participates in the interaction, but also the N-terminal domain. rJast plasticity was essential for the interaction with αVß3 and correlated with the main modes of motion depicted in the MD trajectories. In summary, our study provides novel structural insights that enhance our comprehension of the mechanisms underlying disintegrin functionality.

P2X7 receptor contributes to long-term neuroinflammation and cognitive impairment in sepsis-surviving mice.

Introduction: Sepsis is defined as a multifactorial debilitating condition with high risks of death. The intense inflammatory response causes deleterious effects on the brain, a condition called sepsis-associated encephalopathy. Neuroinflammation or pathogen recognition are able to stress cells, resulting in ATP (Adenosine Triphosphate) release and P2X7 receptor activation, which is abundantly expressed in the brain. The P2X7 receptor contributes to chronic neurodegenerative and neuroinflammatory diseases; however, its function in long-term neurological impairment caused by sepsis remains unclear. Therefore, we sought to evaluate the effects of P2X7 receptor activation in neuroinflammatory and behavioral changes in sepsis-surviving mice. Methods: Sepsis was induced in wild-type (WT), P2X7-/-, and BBG (Brilliant Blue G)-treated mice by cecal ligation and perforation (CLP). On the thirteenth day after the surgery, the cognitive function of mice was assessed using the novel recognition object and Water T-maze tests. Acetylcholinesterase (AChE) activity, microglial and astrocytic activation markers, and cytokine production were also evaluated. Results: Initially, we observed that both WT and P2X7-/- sepsis-surviving mice showed memory impairment 13 days after surgery, once they did not differentiate between novel and familiar objects. Both groups of animals presented increased AChE activity in the hippocampus and cerebral cortex. However, the absence of P2X7 prevented partly this increase in the cerebral cortex. Likewise, P2X7 absence decreased ionized calcium-binding protein 1 (Iba-1) and glial fibrillary acidic protein (GFAP) upregulation in the cerebral cortex of sepsis-surviving animals. There was an increase in GFAP protein levels in the cerebral cortex but not in the hippocampus of both WT and P2X7-/- sepsis-surviving animals. Pharmacological inhibition or genetic deletion of P2X7 receptor attenuated the production of Interleukin-1ß (IL-1ß), Tumor necrosis factor-α (TNF-α), and Interleukin-10 (IL-10). Conclusion: The modulation of the P2X7 receptor in sepsis-surviving animals may reduce neuroinflammation and prevent cognitive impairment due to sepsis-associated encephalopathy, being considered an important therapeutic target.

SARS-CoV-2 Spike protein alters microglial purinergic signaling.

Despite long-term sequelae of COVID-19 are emerging as a substantial public health concern, the mechanism underlying these processes still unclear. Evidence demonstrates that SARS-CoV-2 Spike protein can reach different brain regions, irrespective of viral brain replication resulting in activation of pattern recognition receptors (PRRs) and neuroinflammation. Considering that microglia dysfunction, which is regulated by a whole array of purinergic receptors, may be a central event in COVID-19 neuropathology, we investigated the impact of SARS-CoV-2 Spike protein on microglial purinergic signaling. Here, we demonstrate that cultured microglial cells (BV2 line) exposed to Spike protein induce ATP secretion and upregulation of P2Y6, P2Y12, NTPDase2 and NTPDase3 transcripts. Also, immunocytochemistry analysis shows that spike protein increases the expression of P2X7, P2Y1, P2Y6, and P2Y12 in BV2 cells. Additional, hippocampal tissue of Spike infused animals (6,5ug/site, i.c.v.) presents increased mRNA levels of P2X7, P2Y1, P2Y6, P2Y12, NTPDase1, and NTPDase2. Immunohistochemistry experiments confirmed high expression of the P2X7 receptor in microglial cells in CA3/DG hippocampal regions after spike infusion. These findings suggest that SARS-CoV-2 Spike protein modulates microglial purinergic signaling and opens new avenues for investigating the potential of purinergic receptors to mitigate COVID-19 consequences.

rJararacin, a recombinant disintegrin from Bothrops jararaca venom: Exploring its effects on hemostasis and thrombosis.

Integrins are a family of heterodimeric transmembrane receptors which link the extracellular matrix to the cell cytoskeleton. These receptors play a role in many cellular processes: adhesion, proliferation, migration, apoptosis, and platelet aggregation, thus modulating a wide range of scenarios in health and disease. Therefore, integrins have been the target of new antithrombotic drugs. Disintegrins from snake venoms are recognized by the ability to modulate the activity of integrins, such as integrin αIIbß3, a fundamental platelet glycoprotein, and αvß3 expressed on tumor cells. For this reason, disintegrins are unique and potential tools for examining integrin-matrix interaction and the development of novel antithrombotic agents. The present study aims to obtain the recombinant form of jararacin and evaluate the secondary structure and its effects on hemostasis and thrombosis. rJararacin was expressed in the Pichia pastoris (P. pastoris) expression system and purified the recombinant protein with a yield of 40 mg/L of culture. The molecular mass (7722 Da) and internal sequence were confirmed by mass spectrometry. Structure and folding analysis were obtained by Circular Dichroism and 1H Nuclear Magnetic Resonance spectra. Disintegrin structure reveals properly folded with the presence of ß-sheet structure. rJararacin significantly demonstrated inhibition of the adhesion of B16F10 cells and platelets to the fibronectin matrix under static conditions. rJararacin inhibited platelet aggregation induced by ADP (IC50 95 nM), collagen (IC50 57 nM), and thrombin (IC50 22 nM) in a dose-dependent manner. This disintegrin also inhibited 81% and 94% of the adhesion of platelets to fibrinogen and collagen under continuous flow, respectively. In addition, rjararacin efficaciously prevents platelet aggregation in vitro and ex vivo with rat platelets and thrombus occlusion at an effective dose (5 mg/kg). The data here provides evidence that rjararacin possesses the potential as an αIIbß3 antagonist, capable of preventing arterial thrombosis.

The recombinant disintegrin, jarastatin, inhibits platelet adhesion and endothelial cell migration.

Integrins are transmembrane heterodimeric glycoproteins, present in most cell types that act as mechanoreceptors, connecting extracellular matrix proteins to the cytoskeleton of the cell, mediating several physiological and pathological processes. The disintegrins are peptides capable of modulating the activity of integrins, such as αIIbß3, responsible for the platelet aggregation and αvß3, related to angiogenesis. The aim of this study was to produce the recombinant disintegrin jarastatin (rJast), to evaluate its secondary structure and biological activity. rJast was expressed in the yeast Komagataella phaffii (earlier Pichia pastoris) purified using molecular exclusion chromatography and the internal sequence and molecular mass were confirmed by mass spectrometry. The yield was approximately 40 mg/L of culture. rJast inhibited platelet aggregation induced by 2-4 µM ADP, 10 nM thrombin, and 1 µg/mL collagen (IC50 of 244.8 nM, 166.3 nM and 223.5 nM, respectively). It also blocked the adhesion of platelets to collagen under continuous flow in approximately 60% when used 1 µM. We also evaluated the effect of rJast on HMEC-1 cells. rJast significantly inhibited the adhesion of these cells to vitronectin, as well as cell migration (IC50 1.77 µM) without changing the viability. Conclusions: rJast was successfully expressed with activity in human platelets aggregation identical to the native molecule. Also, rJast inhibits adhesion and migration of endothelial cells. Thus, being relevant for the development of anti-thrombotic and anti-angiogenic drugs.

Characterization of Aspergillus nidulans Biofilm Formation and Structure and Their Inhibition by Pea Defensin Psd2.

Approximately four million people contract fungal infections every year in Brazil, primarily caused by Aspergillus spp. The ability of these fungi to form biofilms in tissues and medical devices complicates treatment and contributes to high rates of morbidity and mortality in immunocompromised patients. Psd2 is a pea defensin of 5.4 kDa that possesses good antifungal activity against planktonic cells of representative pathogenic fungi. Its function depends on interactions with membrane and cell wall lipid components such as glucosylceramide and ergosterol. In the present study, we characterized Aspergillus nidulans biofilm formation and determined the effect of Psd2 on A. nidulans biofilms. After 4 hours, A. nidulans conidia adhered to polystyrene surfaces and formed a robust extracellular matrix-producing biofilm at 24 h, increasing thickness until 48 h Psd2 inhibited A. nidulans biofilm formation in a dose-dependent manner. Most notably, at 10 µM Psd2 inhibited 50% of biofilm viability and biomass and 40% of extracellular matrix production. Psd2 significantly decreased the colonized surface area by the biofilm and changed its level of organization, causing a shortening of length and diameter of hyphae and inhibition of conidiophore formation. This activity against A. nidulans biofilm suggests a potential use of Psd2 as a prototype to design new antifungal agents to prevent biofilm formation by A. nidulans and related species.

P2X7 Receptor Triggers Lysosomal Leakage Through Calcium Mobilization in a Mechanism Dependent on Pannexin-1 Hemichannels.

The P2X7 receptor is a critical purinergic receptor in immune cells. Its activation was associated with cathepsin release into macrophage cytosol, suggesting its involvement in lysosomal membrane permeabilization (LMP) and leakage. Nevertheless, the mechanisms by which P2X7 receptor activation induces LMP and leakage are unclear. This study investigated cellular mechanisms associated with endosomal and lysosomal leakage triggered by P2X7 receptor activation. We found that ATP at 500 µM and 5 mM (but not 50 µM) induced LMP in non-stimulated peritoneal macrophages. This effect was not observed in P2X7-deficient or A740003-pretreated macrophages. We found that the P2X7 receptor and pannexin-1 channels mediate calcium influx that might be important for activating specific ion channels (TRPM2 and two-pore channels) on the membranes of late endosomes and lysosomes leading to LMP leakage and consequent cathepsin release. These findings suggest the critical role of the P2X7 receptor in inflammatory and infectious diseases via lysosomal dysfunction.

15N, 13C, and 1H resonance assignments of Jarastatin: a disintegrin of Bothrops jararaca.

Disintegrins are a group of cysteine-rich proteins found in a wide variety of snake venoms. These proteins selectively bind to integrins, which play a fundamental role in the regulation of many physiological and pathological processes. Here, we report the NMR chemical shift assignments for 1H, 15N, and 13C nuclei in the backbone and side chains of recombinant disintegrin Jarastatin (rJast), which was further validated by secondary structure prediction using the TALOS-N server. Taken together, these data are essential to perform NMR-based experiments, including structure determination, backbone dynamics, mapping ligand sites and enabling a deeper understanding of the effect of hydrophobic surface clusters, which are important elements to stabilize some 3D proteins structure/folding.

Pisum sativum Defensin 1 Eradicates Mouse Metastatic Lung Nodules from B16F10 Melanoma Cells.

Psd1 is a pea plant defensin which can be actively expressed in Pichia pastoris and shows broad antifungal activity. This activity is dependent on fungal membrane glucosylceramide (GlcCer), which is also important for its internalization, nuclear localization, and endoreduplication. Certain cancer cells present a lipid metabolism imbalance resulting in the overexpression of GlcCer in their membrane. In this work, in vitroassays using B16F10 cells showed that labeled fluorescein isothiocyanate FITC-Psd1 internalized into live cultured cells and targeted the nucleus, which underwent fragmentation, exhibiting approximately 60% of cells in the sub-G0/G1 stage. This phenomenon was dependent on GlcCer, and the participation of cyclin-F was suggested. In a murine lung metastatic melanoma model, intravenous injection of Psd1 together with B16F10 cells drastically reduced the number of nodules at concentrations above 0.5 mg/kg. Additionally, the administration of 1 mg/kg Psd1 decreased the number of lung inflammatory cells to near zero without weight loss, unlike animals that received melanoma cells only. It is worth noting that 1 mg/kg Psd1 alone did not provoke inflammation in lung tissue or weight or vital signal losses over 21 days, inferring no whole animal cytotoxicity. These results suggest that Psd1 could be a promising prototype for human lung anti-metastatic melanoma therapy.

P2X7 receptor activation increases expression of caveolin-1 and formation of macrophage lipid rafts, thereby boosting CD39 activity.

Macrophages are tissue-resident immune cells that are crucial for the initiation and maintenance of immune responses. Purinergic signaling modulates macrophage activity and impacts cellular plasticity. The ATP-activated purinergic receptor P2X7 (also known as P2RX7) has pro-inflammatory properties, which contribute to macrophage activation. P2X7 receptor signaling is, in turn, modulated by ectonucleotidases, such as CD39 (also known as ENTPD1), expressed in caveolae and lipid rafts. Here, we examined P2X7 receptor activity and determined impacts on ectonucleotidase localization and function in macrophages primed with lipopolysaccharide (LPS). First, we verified that ATP boosts CD39 activity and caveolin-1 protein expression in LPS-primed macrophages. Drugs that disrupt cholesterol-enriched domains - such as nystatin and methyl-ß-cyclodextrin - decreased CD39 enzymatic activity in all circumstances. We noted that CD39 colocalized with lipid raft markers (flotillin-2 and caveolin-1) in macrophages that had been primed with LPS followed by treatment with ATP. P2X7 receptor inhibition blocked these ATP-mediated increases in caveolin-1 expression and inhibited the colocalization with CD39. Further, we found that STAT3 activation is significantly attenuated caveolin-1-deficient macrophages treated with LPS or LPS+BzATP. Taken together, our data suggest that P2X7 receptor triggers the initiation of lipid raft-dependent mechanisms that upregulates CD39 activity and could contribute to limit macrophage responses restoring homeostasis.

Nuclear magnetic resonance solution structure of Pisum sativum defensin 2 provides evidence for the presence of hydrophobic surface-clusters.

Pisum sativum defensin 2 (Psd2) is a small (4.7 kDa) antifungal peptide whose structure is held together by four conserved disulfide bridges. Psd2 shares the cysteine-stabilized alpha-beta (CSαß) fold, which lacks a regular hydrophobic core. All hydrophobic residues are exposed to the surface, except for leucine 6. They are clustered in the surface formed by two loops, between ß1 and α-helix and ß2 and ß3 sheets. The observation of surface hydrophobic clusters reveals a remarkable evolution of the CSαß fold to expose and reorganize hydrophobic residues, which facilitates creating versatile binding sites.

Determining Maximal Muscle Strength in Mice: Validity and Reliability of an Adapted Swimming Incremental Overload Test.

Gouvêa, AL, Martinez, CG, and Kurtenbach, E. Determining maximal muscle strength in mice: validity and reliability of an adapted swimming incremental overload test. J Strength Cond Res 34(8): 2360-2368, 2020-At present, there are no reliable methods to determine maximal muscle strength in small rodents. Here, we established an adapted swimming incremental overload test (SIOT) as an instrument for this purpose. First, to validate the test, BALB/c mice received 20 mg·kg·d of dexamethasone (DEXA group) or water (control group). After 14 days, with a cumulative dose of 120 mg·kg of dexamethasone, the SIOT could detect a decrease of approximately 7% in muscle strength. In addition, this decrease was consistent with a significant reduction in body (above 13.5%) and muscle (approximately 15%) weight in DEXA atrophic animals. To establish the SIOT reliability, another group of animals was evaluated for 5 consecutive days. In this second protocol, the SIOT was executed with an initial load corresponding to 12% of the mouse body weight (BW) fixed to the tail. Increments between 1 and 5% of the BW were added during each attempt to obtain the highest load that was tolerated for a time interval of 5-7 seconds. On the last day, the SIOT reliability test was performed by 2 different raters to obtain the inter-rater reproducibility. The adapted SIOT was shown to be reliable when measured by the same rater (intraclass correlation coefficient [ICC] = 0.939) and by 2 different raters (ICC = 0.830). The Bland-Altman graphical representation did not demonstrate heteroscedastic errors. Therefore, the SIOT proved to be a sensitive and reliable method to measure muscle strength, and it can be applied to small animals in different models of muscle atrophy.

Expression of soluble, glycosylated and correctly folded dengue virus NS1 protein in Pichia pastoris.

The dengue virus (DENV) non structural protein 1 (NS1) is a 46-55 kDa protein that exists as homodimer inside cells and as hexamer in the extracellular milieu. Several lines of evidence have demonstrated that the biochemical and structural properties of recombinant NS1 (rNS1) vary depending on the protein expression system used. Aiming to improve current tools for studying NS1 multiple roles, a recombinant tag-free NS1 protein was expressed and purified from P. pastoris yeast cells. The best expression condition was achieved using GS115 strain and induction for 72 h with 0.7% methanol addition every 24 h. Total secreted rNS1 reached 2.18 mg in 1 L culture and the final yield of the purified protein was 1 mg per liter of culture (recovery yield of approximately 45.9%). Size-exclusion chromatography and treatment with EndoH and PNGase indicate that it exists as an N-glycosylated homodimer. Moreover, an ELISA assay with specific DENV anti-NS1 antibody that recognizes conformational epitopes revealed that rNS1 has most of its conformational epitopes preserved. The expression of rNS1 in its native conformation is an important tool for further studies of its role in DENV pathogenesis and replication.

Psd2 pea defensin shows a preference for mimetic membrane rafts enriched with glucosylceramide and ergosterol.

Psd2 is a pea defensin with 47 amino acid residues that inhibits the growth of fungal species by an uncharacterized mechanism. In this work, Psd2 interactions with model membranes mimicking the lipid compositions of different organisms were evaluated. Protein-lipid overlay assays indicated that Psd2 recognizes Fusarium solani glucosylceramide (GlcCerF.solani) and ergosterol (Erg) in addition to phosphatidylcholine (POPC) and some phosphatidylinositol species, such as PtdIns (3)P, (5)P and (3,5)P2, suggesting that these lipids may play important roles as Psd2 targets. Assays using lipid vesicles were also performed to study the behaviour and dynamics that occur after peptide-membrane interactions. Surface plasmon resonance analysis showed that Psd2 has a higher affinity for pure POPC and POPC-based vesicles containing GlcCer and Erg at a 70:30 proportion than for vesicles containing cholesterol (Chol). Partition experiments by fluorescence spectroscopy showed a decrease in Trp42 quantum yield of Psd2 in the presence of GlcCerF.solani and Erg, individually or in simultaneously enriched membranes. The partition coefficient (Kp) obtained indicated a Psd2 partition preference for this vesicles, confirmed by quenching assays using acrylamide and 5/16-doxyl-stearic acid. Furthermore, we showed that the presence of C8C9 double bonds and a methyl group at position C9 of the sphingoid base backbone of GlcCer was relevant to Psd2 activity against Aspergillus nidulans. These results are consistent with the selectivity of Psd2 against fungi and its lack of toxicity in human erythrocytes. Psd2 represents a promising natural compound for the treatment of fungal infections.

Immunization with plasmids encoding M2 acetylcholine muscarinic receptor epitopes impairs cardiac function in mice and induces autophagy in the myocardium.

Autoantibodies against the M2 subtype of muscarinic acetylcholine receptors with functional activities have been found in the sera of patients with dilated cardiomyopathy (DCM), and the second extracellular loop has been established as the predominant epitope. However, it has been shown that the third intracellular loop is recognized by Chagas disease patients with severe cardiac dysfunction. In this work, BALB/c mice were immunized with plasmids encoding these two epitopes, and a control group received the empty plasmid (pcDNA3 vector). Serum from these DNA-immunized animals had elevated and persistent titres of antibodies against respective antigens. Heart echocardiography indicated diminished left ventricular wall thickness and reduced ejection fraction for both epitope-immunized groups, and ergospirometry tests showed a significant decrease in the exercise time and oxygen consumption. Transfer of serum from these immunized mice into naïve recipients induced the same alterations in cardiac structure and function. Furthermore, electron microscopy analysis of donor-immunized animals revealed several ultrastructural alterations suggestive of autophagy and mitophagy, suggesting novel roles for these autoantibodies. Overall, greater functional and structural impairment was observed in the donor and recipient epitope groups, implicating the third intracellular loop epitope in the pathological effects for the first-time. Therefore, the corresponding peptides could be useful for autoimmune DCM diagnosis and targeted therapy.

Psd1 Effects on Candida albicans Planktonic Cells and Biofilms.

Candida albicans is an important human pathogen, causing opportunistic infections. The adhesion of planktonic cells to a substrate is the first step for biofilm development. The antimicrobial peptide (AMP) Psd1 is a defensin isolated from Pisum sativum seeds. We tested the effects of this AMP on C. albicans biofilms and planktonic cells, comparing its activity with amphotericin B and fluconazole. Three C. albicans variants were studied, one of them a mutant deficient in glucosylceramide synthase, conferring resistance to Psd1 antifungal action. Atomic force microscopy (AFM) was used to assess morphological and biomechanical changes on fungal cells. Surface alterations, with membrane disruption and leakage of cellular contents, were observed. Cytometry assays and confocal microscopy imaging showed that Psd1 causes cell death, in a time and concentration-dependent manner. These results demonstrate Psd1 pleiotropic action against a relevant fungal human pathogen, suggesting its use as natural antimycotic agent.

Acute Carnosine Administration Increases Respiratory Chain Complexes and Citric Acid Cycle Enzyme Activities in Cerebral Cortex of Young Rats.

Carnosine (ß-alanyl-L-histidine) is an imidazole dipeptide synthesized in excitable tissues of many animals, whose biochemical properties include carbonyl scavenger, anti-oxidant, bivalent metal ion chelator, proton buffer, and immunomodulating agent, although its precise physiological role(s) in skeletal muscle and brain tissues in vivo remain unclear. The aim of the present study was to investigate the in vivo effects of acute carnosine administration on various aspects of brain bioenergetics of young Wistar rats. The activity of mitochondrial enzymes in cerebral cortex was assessed using a spectrophotometer, and it was found that there was an increase in the activities of complexes I-III and II-III and succinate dehydrogenase in carnosine-treated rats, as compared to vehicle-treated animals. However, quantitative real-time RT-PCR (RT-qPCR) data on mRNA levels of mitochondrial biogenesis-related proteins (nuclear respiratory factor 1 (Nrf1), peroxisome proliferator-activated receptor-γ coactivator 1-α (Ppargc1α), and mitochondrial transcription factor A (Tfam)) were not altered significantly and therefore suggest that short-term carnosine administration does not affect mitochondrial biogenesis. It was in agreement with the finding that immunocontent of respiratory chain complexes was not altered in animals receiving carnosine. These observations indicate that acute carnosine administration increases the respiratory chain and citric acid cycle enzyme activities in cerebral cortex of young rats, substantiating, at least in part, a neuroprotector effect assigned to carnosine against oxidative-driven disorders.

P2×7 purinergic signaling in dilated cardiomyopathy induced by auto-immunity against muscarinic M2 receptors: autoantibody levels, heart functionality and cytokine expression.

Autoantibodies against the M2 receptors (M2AChR) have been associated with Dilated Cardiomyopathy (DCM). In the heart, P2×7 receptors influence electrical conduction, coronary circulation and response to ischemia. They can also trigger pro-inflammatory responses and the development of neurological, cardiac and renal disorders. Here, P2×7(-/-) mice displayed an increased heart rate and ST segment depression, but similar exercise performance when compared to wild type (WT) animals. After immunization with plasmid containing M2AChR cDNA sequence, WT mice produced anti-M2AChR antibodies, while P2×7(-/-) mice showed an attenuated production. Despite this, WT and P2×7(-/-) showed left ventricle cavity enlargement and decreased exercise tolerance. Transfer of serum from M2AChR WT immunized mice to näive recipients led to an alteration in heart shape. P2×7(-/-) mice displayed a significant increase in the frequency of spleen regulatory T cells population, which is mainly composed by the FoxP3(+)CD25(-) subset. M2AChR WT immunized mice showed an increase in IL-1ß, IFNγ and IL-17 levels in the heart, while P2×7(-/-) group produced lower amounts of IL-1ß and IL-17 and higher amounts of IFNγ. These results pointed to previously unnoticed roles of P2×7 in cardiovascular and immune systems, and underscored the participation of IL-17 and IFNγ in the progress of autoimmune DCM.

High Intensity Interval Training (HIIT) Induces Specific Changes in Respiration and Electron Leakage in the Mitochondria of Different Rat Skeletal Muscles.

High intensity interval training (HIIT) is characterized by vigorous exercise with short rest intervals. Hydrogen peroxide (H2O2) plays a key role in muscle adaptation. This study aimed to evaluate whether HIIT promotes similar H2O2 formation via O2 consumption (electron leakage) in three skeletal muscles with different twitch characteristics. Rats were assigned to two groups: sedentary (n=10) and HIIT (n=10, swimming training). We collected the tibialis anterior (TA-fast), gastrocnemius (GAST-fast/slow) and soleus (SOL-slow) muscles. The fibers were analyzed for mitochondrial respiration, H2O2 production and citrate synthase (CS) activity. A multi-substrate (glycerol phosphate (G3P), pyruvate, malate, glutamate and succinate) approach was used to analyze the mitochondria in permeabilized fibers. Compared to the control group, oxygen flow coupled to ATP synthesis, complex I and complex II was higher in the TA of the HIIT group by 1.5-, 3.0- and 2.7-fold, respectively. In contrast, oxygen consumed by mitochondrial glycerol phosphate dehydrogenase (mGPdH) was 30% lower. Surprisingly, the oxygen flow coupled to ATP synthesis was 42% lower after HIIT in the SOL. Moreover, oxygen flow coupled to ATP synthesis and complex II was higher by 1.4- and 2.7-fold in the GAST of the HIIT group. After HIIT, CS activity increased 1.3-fold in the TA, and H2O2 production was 1.3-fold higher in the TA at sites containing mGPdH. No significant differences in H2O2 production were detected in the SOL. Surprisingly, HIIT increased H2O2 production in the GAST via complex II, phosphorylation, oligomycin and antimycin by 1.6-, 1.8-, 2.2-, and 2.2-fold, respectively. Electron leakage was 3.3-fold higher in the TA with G3P and 1.8-fold higher in the GAST with multiple substrates. Unexpectedly, the HIIT protocol induced different respiration and electron leakage responses in different types of muscle.