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The family of two-pore domain potassium (K2P) channels is critically involved in central cellular functions such as ion homeostasis, cell development, and excitability. K2P channels are widely expressed in different human cell types and organs. It is therefore not surprising that aberrant expression and function of K2P channels are related to a spectrum of human diseases, including cancer, autoimmune, CNS, cardiovascular, and urinary tract disorders. Despite homologies in structure, expression, and stimulus, the functional diversity of K2P channels leads to heterogeneous influences on human diseases. The role of individual K2P channels in different disorders depends on expression patterns and modulation in cellular functions. However, an imbalance of potassium homeostasis and action potentials contributes to most disease pathologies. In this review, we provide an overview of current knowledge on the role of K2P channels in human diseases. We look at altered channel expression and function, the potential underlying molecular mechanisms, and prospective research directions in the field of K2P channels.

BACKGROUND/AIMS: Arginine vasopressin (AVP) neurons play an important role for sensing a change in the plasma osmolarity and thereby responding with regulated AVP secretion in order to maintain the body fluid homeostasis. The osmo-sensing processes in magnocellular neurosecretory cells (MNCs) including AVP and oxytocin (OXT) neurons of the hypothalamus were reported to be coupled to sustained osmotic shrinkage or swelling without exhibiting discernible cell volume regulation. Since increasing evidence has shown some important differences in properties between AVP and OXT neurons, osmotic volume responses are to be reexamined with distinguishing these cell types from each other. We previously reported that AVP neurons identified by transgenic expression of enhanced green fluorescence protein (eGFP) possess the ability of regulatory volume decrease (RVD) after hypoosmotic cell swelling. Thus, in the present study, we examined the ability of regulatory volume increase (RVI) after hyperosmotic cell shrinkage in AVP neurons. METHODS: Here, we used eGFP-identified AVP neurons acutely dissociated from AVP-eGFP transgenic rats. We performed single-cell size measurements, cytosolic RT-PCR analysis, AVP secretion measurements, and patch-clamp studies. RESULTS: The AVP neurons were found to respond to a hyperosmotic challenge with physiological cell shrinkage caused by massive secretion of AVP, called a secretory volume decrease (SVD), superimposed onto physical osmotic cell shrinkage, and also to exhibit the ability of RVI coping with osmotic and secretory cell shrinkage. Furthermore, our pharmacological and molecular examinations indicated that AVP secretion and its associated SVD event are triggered by activation of T-type Ca2+ channels, and the RVI event is attained by parallel operation of Na+/H+ exchanger and Cl-/HCO3- anion exchanger. CONCLUSION: Thus, it is concluded that AVP neurons respond to hyperosmotic stimulation with the regulatory volume increase and the secretory volume increase by activating ion transporters and Ca2+ channels, respectively.

Despite advances in diagnostics and therapy of non-small cell lung cancer (NSCLC), the problem of prognosis and prevention of tumor progression is still highly important. Even if NSCLC is diagnosed in the early stages, almost a quarter of patients develop relapse; most of them die from recurrent disease. A large number of different markers have been proposed to predict the risk of NSCLC progression; however, none of them are used in clinical practice. It is obvious that this situation is related to the economic and methodological complexity of the proposed markers and/or their insufficient efficiency due to a lack of effective study models and tumor heterogeneity. Another reason may be that potential markers are developed for NSCLC progression in general, which is represented by at least four pathogenetically-distinct processes: synchronous lymph node metastasis, local, regional, and distant recurrence. In this review, we summarize data from published literature on clinicopathological, genetic, and molecular factors associated with different types of NSCLC progression and emphasize challenges and approaches to developing prognostic factors. In conclusion, we highlight the importance of further studies to reveal molecular mechanisms of NSCLC progression and the need for differential analysis of markers of local, regional, and distant recurrences for disease prognosis.

The regulation of cell volume is an essential cellular process in nearly every living organism. The importance of volume regulation in immune cells cannot be understated, as it ensures proper cellular function and effective immune response. These cells utilize ion channels and transporters to maintain volume homeostasis through rapid ion transport across the cell membrane. Immune cells express mechanisms controlling regulatory volume decrease (RVD), regulatory volume increase (RVI), proliferative RVD, and apoptotic volume decrease (AVD). In this review, we summarize recent studies examining the importance of several ion channels, particularly potassium and chloride channels in regulating ion transport during osmotic stress, and in immune cell function, activation, proliferation, and death. We also review the key mechanisms functioning in immune cell proliferation and apoptosis. They serve a crucial role in maintaining adequate ionic concentrations, mediating immune cell activation, and generating proliferative pathways. These regulatory mechanisms play key roles in the function and survival of immune cells, as impaired volume regulation contributes to the pathophysiology of various disorders. A complete understanding of immune cell volume regulatory mechanisms may be a starting point for the development of therapeutic agents targeting these ion channels to treat inflammatory diseases.

BACKGROUND/AIMS: Signaling and metabolic perturbations contribute to dysregulated skeletal muscle protein homeostasis and secondary sarcopenia in response to a number of cellular stressors including ethanol exposure. Using an innovative multiomics-based curating of unbiased data, we identified molecular and metabolic therapeutic targets and experimentally validated restoration of protein homeostasis in an ethanol-fed mouse model of liver disease. METHODS: Studies were performed in ethanol-treated differentiated C2C12 myotubes and physiological relevance established in an ethanol-fed mouse model of alcohol-related liver disease (mALD) or pair-fed control C57BL/6 mice. Transcriptome and proteome from ethanol treated-myotubes and gastrocnemius muscle from mALD and pair-fed mice were analyzed to identify target pathways and molecules. Readouts including signaling responses and autophagy markers by immunoblots, mitochondrial oxidative function and free radical generation, and metabolic studies by gas chromatography-mass spectrometry and sarcopenic phenotype by imaging. RESULTS: Multiomics analyses showed that ethanol impaired skeletal muscle mTORC1 signaling, mitochondrial oxidative pathways, including intermediary metabolite regulatory genes, interleukin-6, and amino acid degradation pathways are β-hydroxymethyl-butyrate targets. Ethanol decreased mTORC1 signaling, increased autophagy flux, impaired mitochondrial oxidative function with decreased tricarboxylic acid cycle intermediary metabolites, ATP synthesis, protein synthesis and myotube diameter that were reversed by HMB. Consistently, skeletal muscle from mALD had decreased mTORC1 signaling, reduced fractional and total muscle protein synthesis rates, increased autophagy markers, lower intermediary metabolite concentrations, and lower muscle mass and fiber diameter that were reversed by β-hydroxymethyl-butyrate treatment. CONCLUSION: An innovative multiomics approach followed by experimental validation showed that β-hydroxymethyl-butyrate restores muscle protein homeostasis in liver disease.

In order to cope with external stressors such as changes in humidity and temperature or irritating substances, the epidermis as the outermost skin layer forms a continuously renewing and ideally intact protective barrier. Under certain circumstances, this barrier can be impaired and epidermal cells have to counteract cell swelling or shrinkage induced by osmotic stress via regulatory volume decrease (RVD) or increase (RVI). Here, we will review the current knowledge regarding the molecular machinery underlying RVD and RVI in the epidermis. Furthermore, we will discuss the current understanding how cell volume changes and its regulators are associated with epidermal renewal and barrier formation.

BACKGROUND/AIMS: Microglia are the dynamic motile phagocytes of the brain considered the first line of defense against threats or disturbances to the Central Nervous System (CNS). Microglia help orchestrate the immunological response by interacting with others immune cells. Mast cells (MCs) are effector cells of the innate immune system distributed in all organs and vascularized tissues, brain included. Several molecular mechanisms for potential interactions between MCs and microglia have been determined. However, the effect of MCs on regulated exocytosis and phagocytic clearance in microglia has not been explored. METHODS: Cocktails of MCs mediators (MCM) obtained at 37°C and 53°C were used to induce microglia activation. Changes in intracellular calcium [Ca2+]i and ATP release were studied by calcium and quinacrine fluorescence imaging. Fluorescent latex beads were used to assay phagocytosis in microglia after MCM treatment and compared to that measured in the presence of histamine, ATP and lipopolysaccharide (LPS). Iba-1 expression and area were quantified by immunofluorescence and histamine levels evaluated by ELISA techniques. RESULTS: Local application onto microglia of the MC mediator cocktail elicited Ca2+ transients and exocytotic release associated with quinacrine dye de-staining. Ca2+ signals were mimicked by histamine and blocked by the H1 receptor (H1R) antagonist, cetirizine. Hydrolysis of ATP by apyrase also affected Ca2+ transients to a lesser extent. Iba-1 fluorescence, cell area and phagocytosis were enhanced by histamine through H1R. However, ATP prevented iba-1 expression and microglial phagocytosis. MCM showed combined effects of histamine and ATP, increasing the number of internalized microbeads per cell and area without raising iba1 expression. CONCLUSION: Our results highlight the relevance of MC-derived histamine and ATP in the modulation of secretory and phagocytic activities that would explain the heterogeneity of microglial responses in different pathological contexts.

Background/Aims: The mineral-dust-induced gene mdig is a lung-cancer-associated oncogene. The focus of this study is to evaluate the expression status of mdig in lung cancer and to assess its influence in predicting the patient’s overall survival. Methods: Using high-density tissue microarrays and clinical samples of synchronous multiple primary lung cancer (SMPLC), we investigated the expression of mdig through immunohistochemistry and utilized the open-access lung cancer patient databases containing genomic and transcriptomic data from the UCSC Xena and TCGA web platforms to determine the prognostic values of mdig expression status among different subtypes of lung cancer. Results: mdig is upregulated in smokers and in lung squamous cell carcinoma. High mdig expression predicted poor overall survival in lung squamous cell carcinoma and female smokers. Among tumor tissues from SMPLC patients, we not only unraveled the highest positive rate of mdig expression, but also revealed a unique cytoplasmic, rather than nuclear localization of mdig protein. Furthermore, by inspecting some pathological but not cancerous lung tissues, we believe that mdig is required for the transformation of non-cancerous lung cells to the fully-fledged cancer cells. Conclusion: These data suggested that mdig is involved in various stages of lung carcinogenesis, possibly through the epigenetic regulation on some critical cancer-associated genes, and increased mdig expression is an important prognostic factor for some types of lung cancer.

High density of intracellular macromolecules creates a special condition known as macromolecular crowding (MC). One well-established consequence of MC is that only a slight change in the concentration of macromolecules (e.g., proteins) results in a shift of chemical equilibria towards the formation of macromolecular complexes and oligomers. This suggests a physiological mechanism of converting cell density changes into cellular responses. In this review, we start by providing a general overview of MC; then we examine the available experimental evidence that MC may act as a direct signaling factor in several types of cellular activities: mechano- and osmosensing, cell volume recovery in anisosmotic solutions, and apoptotic shrinkage. The latter phenomenon is analyzed in particular detail, as persistent shrinkage is known both to cause apoptosis and to occur during apoptosis resulting from other stimuli. We point to specific apoptotic reactions that involve formation of macromolecular complexes and, therefore, may provide a link between shrinkage and downstream responses.

BACKGROUND/AIMS: Exposure to particulate air pollution is associated with increased cardiovascular morbidity and mortality. These effects are particularly aggravated in patients with pre-existing kidney diseases. Cerium oxide nanoparticles (CNPs), used as diesel fuel additives, are emitted in vehicle exhaust and affect humans when inhaled. However, thrombotic and cardiac injury resulting from pulmonary exposure to CNPs in experimental acute kidney injury (AKI) is not fully understood. The objective of the present study was to evaluate the thrombotic and cardiac injury effects of CNPs in a rat model of AKI. METHODS: AKI was induced in rats by a single intraperitoneal injection of cisplatin (CDDP, 6 mg/kg). Six days after injection, rats were intratracheally (i.t.) instilled with either CNPs (1 mg/kg) or saline (control), and various cardiovascular variables and markers of inflammation, oxidative stress and DNA injury were assessed by enzyme linked immunosorbent assay, colorimetric assay, single-cell gel electrophoresis assay and immunohistochemistry, the following day. RESULTS: Compared with individual CDDP or CNPs treatments, the combined CDDP + CNPs treatment elevated significantly the coagulation function, relative heart weight, and troponin I, lactate dehydrogenase, interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and total nitric oxide levels in the plasma. In heart homogenates, the combination of CDDP and CNPs induced a significant increase in IL-6, TNFα, catalase, and glutathione. Furthermore, significantly more DNA damage was observed in this group than in the CDDP or CNPs groups. Immunohistochemical analysis of the heart revealed that expression of nuclear factor erythroid-derived 2-like 2 (Nrf2) and glutathione peroxidase by cardiac myocytes and endothelial cells was increased in the CDDP + CNPs group more than in either CDDP or CNPs group. CONCLUSION: I.t. administration of CNPs in rats with AKI exacerbated systemic inflammation, oxidative stress, and coagulation events. It also aggravated cardiac inflammation, DNA damage, and Nrf2 expression.

BACKGROUND/AIMS: Tea, produced from the evergreen Camellia sinensis, has reported therapeutic properties against multiple pathologies, including hypertension. Although some studies validate the health benefits of tea, few have investigated the molecular mechanisms of action. The KCNQ5 voltage-gated potassium channel contributes to vascular smooth muscle tone and neuronal M-current regulation.METHODS: We applied electrophysiology, myography, mass spectrometry and in silico docking to determine effects and their underlying molecular mechanisms of tea and its components on KCNQ channels and arterial tone.RESULTS: A 1% green tea extract (GTE) hyperpolarized cells by augmenting KCNQ5 activity >20-fold at resting potential; similar effects of black tea were inhibited by milk. In contrast, GTE had lesser effects on KCNQ2/Q3 and inhibited KCNQ1/E1. Tea polyphenols epicatechin gallate (ECG) and epigallocatechin-3-gallate (EGCG), but not epicatechin or epigallocatechin, isoform-selectively hyperpolarized KCNQ5 activation voltage dependence. In silico docking and mutagenesis revealed that activation by ECG requires KCNQ5-R212, at the voltage sensor foot. Strikingly, ECG and EGCG but not epicatechin KCNQ-dependently relaxed rat mesenteric arteries.CONCLUSION: KCNQ5 activation contributes to vasodilation by tea; ECG and EGCG are candidates for future anti-hypertensive drug development.

The regulation of cellular volume in response to osmotic change has largely been studied at the whole cell level. Such regulation occurs by the inhibition or activation of ionic and organic solute transport pathways at the cell surface and is coincident with remodelling of the plasma membrane. However, it is only in rare instances that osmotic insults are experienced by cells and tissues. By contrast, the relatively minute luminal volumes of membrane-bound organelles are constantly subject to shifts in their solute concentrations as exemplified in the endocytic pathway where these evolve alongside with maturation. In this review, we summarize recent evidence that suggests trafficking events are in fact orchestrated by the solute fluxes of organelles that briefly impose osmotic gradients. We first describe how hydrostatic pressure and the resultant tension on endomembranes can be readily dissipated by controlled solute efflux since water is obliged to exit. In such cases, the relief of tension on the limiting membrane of the organelle can promote its remodelling by coat proteins, ESCRT machinery, and motors. Second, and reciprocally, we propose that osmotic gradients between organellar lumens and the cytosol may persist or be created. Such gradients impose osmotic pressure and tension on the endomembrane that prevent its remodelling. The control of endomembrane tension is dysregulated in lysosomal storage disorders and can be usurped by pathogens in endolysosomes. Since trafficking and signaling pathways conceivably sense and respond to endomembrane tension, we anticipate that understanding how cells control organellar volumes and the movement of endocytic fluid in particular will be an exciting new area of research.

Many factors including growth factors (GF), scaffold materials, and chemical and physical cues determine the cell behaviors. For many years, growth factors have been considered as the pivotal cell behavior regulators, whereas recent studies emphasize also the key role of physical factors such as mechanical forces, cell shape, surface topographies, and extracellular matrix (ECM) in regulating the cell proliferation, apoptosis, differentiation, etc. through mechanotransduction pathways. In this process, the cell morphology and mechanical properties of the cell's micro/ nano-environments and ECM can be conveyed to the nucleus by regulating transcriptional factors such as Yes-associated protein and transcriptional coactivator with PDZ-binding motif (TAZ). Generally, YAP/TAZ activity is considered as the key factor for the growth of whole organs, however, recent studies have also repeatedly addressed the role of YAP/TAZ in mechanotransduction. In this review, the biological functions of the YAP/TAZ pathway and its contribution to the mechanotransduction and cell behavior regulation in response to the mechanical cues have been summarized. Also, the role of key mechanical checkpoints in the cell including focal adhesions, cytoskeletal tension, Rho small GTPases, and nuclear membrane protein elements involved in the transfer of environmental mechanical cues from the cell surface to the nucleus and their effect in regulating the YAP/TAZ activity are discussed.

BACKGROUND/AIMS: Histamine is an important chemical transmitter involved in inflammatory processes, including asthma and other chronic inflammatory diseases. Its inflammatory effects involve mainly the histamine H4 receptor (H4R), whose role in several studies has already been demonstrated. Our group have explored the effects of 1-[(2,3-dihydro-1-benzofuran-2-yl)methyl]piperazines as antagonists of H4R, and herein the compounds LINS01005 and LINS01007 were studied with more details, considering the different affinity profile on H4R and the anti-inflammatory potential of both compounds. METHODS: We carried out a more focused evaluation of the modulatory effects of LINS01005 and LINS01007 in a murine asthma model. The compounds were given i.p. (1-7 mg/kg) to ovalbumin sensitized BALB/c male mice (12 weeks old) 30 min before the antigen challenging, and after 24 h the cell analysis from the bronchoalveolar lavage fluid (BALF) was performed. The lung tissue was used for evaluation by western blot (COX-2, 5-LO, NF-κB and STAT3 expressions) and histological analysis. RESULTS: Treatment with the more potent H4R antagonist LINS01007 significantly decreased the total cell count and eosinophils in BALF at lower doses when compared to LINS01005. The expression of COX-2, 5-LO, NF-κB and STAT3 in lung tissue was significantly reduced after treatment with LINS01007. Morphophysiological changes such as mucus and collagen production and airway wall thickening were significantly reduced after treatment with LINS01007. CONCLUSION: These results show important down regulatory effect of novel H4R antagonist (LINS01007) on allergic lung inflammation.

The release of insulin in response to increased levels of glucose in the blood strongly depends on CaWe used INS-1 cells as a beta cell model in which we analysed the occurrence of TRPM3 isoformes by immunoprecipitation and western blotting and by cloning of RT-PCR amplified cDNA fragments. We applied pharmacological as well as CRISPR/Cas9-based strategies to analyse the interplay of TRPM3 and voltage-gated CaTRPM3 channels triggered the activity of voltage-gated CaThe present study suggests an important role for TRPM3 channels in the control of glucose-dependent insulin release.

BACKGROUND/AIMS: Compelling evidence indicates that CK2α, which is one of the two catalytic isoforms of protein kinase CK2, is required for cell viability and plays an important role in cell proliferation and differentiation. While much is known on CK2 in the context of disease states, particularly cancer, its critical role in non-cancerous cell growth has not been extensively investigated. METHODS: In the present study, we have employed a cell line derived from rat heart with inducible down-regulation of CK2α and CK2α-knockout mouse tissue to identify CK2-mediated molecular mechanisms regulating cell growth. For this, we have performed Incucyte® live-cell analysis and applied flow cytometry, western blot, immunoprecipitation, immunohistochemistry, RT-qPCR and luciferase-based methods. RESULTS: Here, we show that lack of CK2α results in significantly delayed cell cycle progression through G1, inhibition of cyclin E-CDK2 complex, decreased phosphorylation of Rb protein at S795, and inactivation of E2F transcription factor. These events are accompanied by nuclear accumulation and up-regulation of the cyclin-dependent kinase inhibitor p27KIP1 in cells and CK2α-knockout mouse tissues. We found that increased levels of p27KIP1 are mainly attributable to post-translational modifications, namely phosphorylation at S10 and T197 amino acid residues catalyzed by Dyrk1B and AMPK, respectively, as silencing of FoxO3A transcription factor, which activates CDKN1B the gene coding for p27KIP1, does not result in markedly decreased expression levels of the corresponding protein. Interestingly, simultaneous silencing of CK2α and p27KIP1 significantly impairs cell cycle progression without increasing cell death. CONCLUSION: Taken together, our study sheds light on the molecular mechanisms controlling cell cycle progression through G1 phase when myoblasts proliferation potential is impaired by CK2α depletion. Our results suggest that elevated levels of p27KIP1,which follows CK2α depletion, contribute to delay the G1-to-S phase transition. Effects seen when p27KIP1 is down-regulated are independent of CK2α and reflect the protective role exerted by p27KIP1 under unfavorable cell growth conditions.

BACKGROUND/AIMS: Excessive consumption of dietary fat and sugar is associated with an elevated risk of nonalcoholic fatty liver disease (NAFLD). Hepatocytes exposed to saturated fat or sugar exert effects on nearby hepatic stellate cells (HSCs); however, the mechanisms by which this occurs are poorly understood. We sought to determine whether paracrine effects of hepatocytes exposed to palmitate and fructose produced profibrotic transcriptional responses in HSCs. METHODS: We performed expression profiling of mRNA and lncRNA from HSCs treated with conditioned media (CM) from human hepatocytes treated with palmitate (P), fructose (F), or both (PF). RESULTS: In HSCs exposed to CM from palmitate-treated hepatocytes, we identified 374 mRNAs and 607 lncRNAs showing significant differential expression (log2 foldchange ≥ |1|; FDR ≤0.05) compared to control cells. In HSCs exposed to CM from PF-treated hepatocytes, the number of differentially expressed genes was much higher (1198 mRNAs and 3348 lncRNAs); however, CM from fructose-treated hepatocytes elicited no significant changes in gene expression. Pathway analysis of differentially expressed genes showed enrichment for hepatic fibrosis and hepatic stellate cell activation in P- (FDR =1.30E-04) and PF-(FDR =9.24E-06) groups. We observed 71 lncRNA/nearby mRNA pairs showing differential expression under PF conditions. There were 90 mRNAs and 264 lncRNAs strongly correlated between the PF group and differentially expressed transcripts from a comparison of activated and quiescent HSCs, suggesting that some of the transcriptomic changes occurring in response to PF overlap with HSC activation. CONCLUSION: The results reported here have implications for dietary modifications in the prevention and treatment of NAFLD.

Made available in DSpace on 2021-06-25T10:13:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Background/Aims: Considering the importance of inflammation on obesity-related disorders pathogenesis, including cardiac dysfunction, the interest in natural anti-inflammatory therapeutic strategies has emerged. The lycopene is a carotenoid presents in tomato and red fruits that displays anti-inflammatory properties. In this sense, we will evaluate the anti-inflammatory effect of tomato-oleoresin supplementation on obesity- related cardiac dysfunction by modulating myocardial calcium kinetic. Methods: Male Wistar rats were initially randomized into 2 experimental groups: (Control, n= 20) or high sugar- fat diet (HSF, n=20) for 20 weeks. At week 20th, once detected the cardiac dysfunction (cardiac remodeling, systolic and diastolic dysfunction) by echocardiography in HSF group, animals were randomly divided to begin the treatment with tomato-oleoresin, performing 4 groups: Control (n= 10); Control + tomato tomato-oleoresin supplementation (Control + Ly, n= 10); HSF (n= 10) or HSF + tomato tomato-oleoresin supplementation (HSF + Ly, n= 10). Tomato oleoresin was mixed with maize oil equivalent to 10mg lycopene/kg body weight (BW) per day and given orally, by gavage, every morning for a 10-week period. It was analyzed cardiac inflammatory parameters by the enzyme-linked immunosorbent assay (ELISA) and in vivo (echocardiography) and in vitro (studying isolated papillary muscles from the left ventricle) cardiac function. The groups were compared by Two-Way analysis of variance (ANOVA). Results: The HSF diet induced cardiac dysfunction (FS(%) C: 60.4±1.3; C+Ly: 60.9±1.3; HSF: 51.7±1.3; HSF+Ly: 59.4±1.4) and inflammation (TNF-a: C: 1.88±0.41; C+Ly: 1.93±1.01; HSF: 4.58±1.99; HSF+Ly: 2.03±0.55; IL-6: C: 0.58±0.16; C+Ly: 0.40±0.16; HSF: 2.00±0.45; HSF+Ly: 0.53±0.26; MCP-1: C: 0.31±0.08; C+Ly: 0.43±0.22; HSF: 1.54±0.32; HSF+Ly: 0.50±0.16). Tomato-oleoresin supplementation improved cardiac remodeling and dysfunction, cardiac inflammation and myocardial calcium kinetic. Conclusion: the anti-inflammatory effect of tomato-oleoresin supplementation treated the obesity-induced cardiac dysfunction by modulating myocardial calcium handling. Medical School Sao Paulo State University (UNESP) Institute of Biosciences Sao Paulo State University (UNESP) Medical School Sao Paulo State University (UNESP) Institute of Biosciences Sao Paulo State University (UNESP) FAPESP: 2020/12578-0

Background/aims The disruption of redox equilibrium by oxidative stress, which is characterized by an overproduction of reactive oxygen species (ROS), is considered to be associated with fibroblast death in severe lung diseases. Indole-6-carboxaldehyde (I6CA) is a natural indole derivative isolated from Sargassum thunbergii, which a type of brown algae. However, the antioxidative effects of I6CA, and their mechanisms, have not been identified. This study was conducted to investigate the potential protective effects of I6CA against oxidative stress in V79-4 Chinese hamster lung fibroblasts. Methods Cell viability and mechanisms related to antioxidant activity of I6CA (ROS production, cell cycle, DNA damage, mitochondrial membrane potential (MMP) and apoptosis) were studied. Western blot analysis was carried out to understand the involvement of various genes at protein level. Results Our results demonstrated that I6CA inhibited hydrogen peroxide (H2O2)-induced cytotoxicity by blocking abnormal ROS accumulation. H2O2 treatment of V79-4 fibroblasts caused cell cycle arrest at the G2/M phase, which was accompanied by increased expression of the cyclin-dependent kinase (Cdk) inhibitor p21WAF1/CIP1 and decreased expression of cyclin B1 and cyclin A. However, these effects were attenuated by treatment with I6CA. I6CA also effectively protected V79-4 cells against H2O2-induced apoptosis by increasing the Bcl-2/Bax ratio and suppressing the loss of MMP and the cytosolic release of cytochrome c. In addition, the activation of nuclear factor-erythroid-2-related factor 2 (Nrf2) was markedly promoted by I6CA, which was associated with enhanced expression and activity of heme oxygenase-1 (HO-1). However, inhibiting the activity of HO-1 by zinc protoporphyrin IX, a potent inhibitor of HO-1, eliminated the ROS scavenging and anti-apoptotic effects of I6CA, indicating that I6CA was able to protect V79-4 lung fibroblasts from H2O2-induced oxidative stress by activating the Nrf2 signaling pathway. Conclusion We suggest that I6CA may be useful as a candidate therapeutic agent for the treatment of oxidative stress-related lung diseases.

Background/aims Down syndrome associated disorders are caused by a complex genetic context where trisomy 21 is a central component in relation to other changes involving epigenetic regulators and signaling molecules. This unique genetic context is responsible for the predisposition of people with Down syndrome to acute leukemia. Although, the research in this field has discovered some important pathogenic keys, the exact mechanism of this predisposition is not known. Methods In this study we applied functional enrichment analysis to evaluate the interactions between genes localized on chromosome 21, genes already identify as having a key role in acute leukemia of Down syndrome, miRNAs and signaling pathways implicated in cancer and cell development and found that miR-155 has a high impact in genes present on chromosome 21. Forward, we performed next generation sequencing on DNA samples from a cohort of patients diagnosed with acute leukemia of Down syndrome and in vitro functional assay using a CMK-86 cell line, transfected with either mimic or inhibitor of the microRNA-155-5p. Results Our results show that the epigenetic alteration of the TNF superfamily receptors in Down syndrome, which can be correlated to microRNA-155-5p aberrant activity, may play an important role in cell signaling and thus be linked to acute myeloid leukemia. Conclusion Some genes, already shown to be mutated in AML-DS, are potential targets for miR-155. Our results show that the epigenetic alteration of the TNF superfamily receptors in Down syndrome may play an important role in cell signaling and thus be linked to acute myeloid leukemia.

Background/aims Although adhesion formation is a frequent adverse event following intraperitoneal surgery, efficient prophylactic interventions have not yet been established. We recently reported that blockade of interleukin (IL)-6 prevented postoperative adhesion after cecum cauterization. Intriguingly, this intervention dampened tumor necrosis factor (TNF) induction in the injured serosa. Herein, we addressed whether TNF might be a key target and, if so, how TNF blockade rescued adhesion formation. Methods Mice were administered an anti-TNF biologic (etanercept) on days -2 and -1 before and upon cecal cauterization. The adhesion scores were evaluated at day 7 postoperatively. Histological alterations were examined by immunochemistry/immunofluorescence studies. We incubated human neutrophils and mesothelial cell line cells with recombinant TNF in the presence of etanercept and measured transcript levels of cytokines and chemokines by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Results Etanercept rescued mice from adhesion formation, accompanied by a robust reduction of neutrophilia in the injured serosa. Immunofluorescence revealed a substantial formation of neutrophil extracellular traps (NETs) with the potential to induce tissue damage and profibrotic responses. In contrast, the etanercept-treated mice lacked NET formation. In addition, etanercept inhibited TNF-induced IL-6, TNF, and neutrophil-recruiting chemokines in neutrophils and mesothelial cells, a major cellular source of myofibroblasts in the adhesion band. Conclusion Prophylactic administration of etanercept might be a potential strategy for preventing postoperative adhesion formation.

Background/aims We recently described a novel regulatory role for histone deacetylase 2 (HDAC2) in protecting endothelial cells from oxidized low-density lipoprotein (OxLDL)-induced injury. In this study, we examined the effects of endothelial-specific HDAC2 overexpression on endothelial-dependent vasorelaxation and atherogenesis in vivo. Methods Endothelial-specific HDAC2-overexpressing transgenic mice (HDAC2-Tg) were generated under control of the Tie2 promoter. An atherosclerosis model was produced by injecting HDAC2-Tg and wild-type (WT) mice with adeno-associated virus encoding a PCSK9 gain-of-function mutant under control of a liver-specific promoter and feeding them a high-fat diet for 12 weeks. Aortic stiffness in vivo was determined by measuring pulse wave velocity. Wire myography was used to measure endothelium dependent (acetylcholine) and independent (sodium nitroprusside) relaxation in isolated mice aortas. Atherosclerotic plaque burden in aortas was determined by Oil Red O staining and protein expression was determined by western blotting. Results At baseline, HDAC2-Tg mice had normal mean arterial blood pressure (MAP) and body weight, but pulse wave velocity (PWV), an inverse measure of vascular health and stiffness, was decreased, suggesting that their vessels were more compliant. Moreover, basal nitric oxide production was enhanced in the vessels of HDAC2-Tg mice as compared to that in WT controls, although no significant differences in acetylcholine (endothelial component)- or sodium nitroprusside (non-endothelial component)-mediated relaxation were observed. However, after exposure to OxLDL, aortas from HDAC2-Tg mice exhibited greater acetylcholine-induced relaxation than did those from WT mice. Thus, endothelial-specific vasodilator production was enhanced despite oxidative injury. Atherosclerosis induction in WT mice led to a significant increase in PWV, but in HDAC2-Tg mice, PWV and MAP remained unchanged. Further, aortic rings from HDAC2-Tg exhibited better endothelial-dependent vascular relaxation than did those from WT mice, but not when treated with nitric oxide synthase inhibitor L-NAME. Finally, plaque burden, determined by Oil red O staining, was significantly increased in WT, but not HDAC2-Tg mice, subjected to the atherogenic model. Deletion of endothelial HDAC2 led to impaired endothelial cell-dependent vascular relaxation and increased PWV, compared with those in littermate controls. Conclusion HDAC2 protects against endothelial dysfunction and atherogenesis induced by oxidized lipids. Hence, overexpression or activation of HDAC2 represents a novel therapy for endothelial dysfunction and atherosclerosis. HDAC2-Tg mice provide an opportunity to determine the role of endothelial HDAC2 in vascular endothelial homeostasis.

Background/aims Trace amines (TA) are small organic compounds that have neuromodulator activity due to their interaction with some neuron-related receptors, such as trace amine associated receptors (TAARs), α2-adrenergic receptor (α2-AR) and s-adrenergic receptor (s-AR). However, there is little information on whether TA and dopamine (DOP) can interact with other adrenergic receptors (ARs) such as the mammalian α1-AR and the bacterial counterpart QseC, which is involved in quorum sensing of some Gram-negative pathogens. The aim of this study was to investigate the interaction of TA and DOP with α1-AR and QseC. Methods We performed an in silico study using 3D structure from SWISS MODEL and analyzed the protein interaction via molecular docking using PyMol, PoseView and PyRX 8.0. For the in vitro study, we investigated the QseC kinase activity by measuring the remaining ATP in a reaction containing QseC-enriched membrane incubated together with purified QseB and EPI, TA, DOP, or PTL respectively. We also measured the intracellular Ca++ levels, which represents the α1-AR activation, in LNCAP (pancreatic cell line) cells treated with EPI, TA, DOP and PTL respectively using a fluorescence-based assay. The LNCAP cell proliferation was measured using an MTT-based assay. Results Our in silico analysis revealed that TAs and DOP have high binding affinity to the human α1-AR and the bacterial adrenergic receptor (QseC), comparable to epinephrine (EPI). Both are membrane-bound kinases. Experimental studies with pancreatic cell line (LNCAP) showed that the TAs and DOP act as α1-AR antagonist by counteracting the effect of EPI. In the presence of EPI, TA and DOP trigger an increase of the intracellular Ca++ levels in the LNCAP cells leading to an inhibition of cell proliferation. Although in silico data suggest an interaction of TA and DOP with QseC, they do not inhibit the kinase activity of QseC, a histidine kinase receptor involved in quorum sensing which is also sensitive to EPI. Conclusion Our study showed that the TAs and DOP act as α1-AR antagonist but no effect was observed for QseC.

Background/aims The role of VDAC1, the most abundant mitochondrial outer membrane protein, in cell death depends on cell types and stimuli. Both silencing and upregulation of VDAC1 in various type of cancer cell lines can stimulate apoptosis. In contrast, in mouse embryonic stem (MES) cells and mouse embryonic fibroblasts (MEFs), the roles of VDAC1 knockout (VDAC1-/-) in apoptotic cell death are contradictory. The contribution and underlying mechanism of VDAC1-/- in oxidative stress-induced cell death in cardiac cells has not been established. We hypothesized that VDAC1 is an essential regulator of oxidative stress-induced cell death in H9c2 cells. Methods We knocked out VDAC1 in this rat cardiomyoblast cell line with CRISPR-Cas9 genome editing technique to produce VDAC1-/- H9c2 cells, and determined if VDAC1 is critical in promoting cell death via oxidative stress induced by tert-butylhydroperoxide (tBHP), an organic peroxide, or rotenone (ROT), an inhibitor of mitochondrial complex I by measuring cell viability with MTT assay, cell death with TUNEL stain and LDH release. The mitochondrial and glycolytic stress were examined by measuring O2 consumption rate (OCR) and extracellular acidification rate (ECAR) with a Seahorse XFp analyzer. Results We found that under control conditions, VDAC1-/- did not affect H9c2 cell proliferation or mitochondrial respiration. However, compared to the wildtype (WT) cells, exposure to either tBHP or ROT enhanced the production of ROS, ECAR, and the proton (H+) production rate (PPR) from glycolysis, as well as promoted apoptotic cell death in VDAC1-/- H9c2 cells. VDAC1-/- H9c2 cells also exhibited markedly reduced mitochondria-bound hexokinase II (HKII) and Bax. Restoration of VDAC1 in VDAC1-/- H9c2 cells reinstated mitochondria-bound HKII and concomitantly decreased tBHP and ROT-induced ROS production and cell death. Interestingly, mitochondrial respiration remained the same after tBHP treatment in VDAC1-/- and WT H9c2 cells. Conclusion Our results suggest that VDAC1-/- in H9c2 cells enhances oxidative stress-mediated cell apoptosis that is directly linked to the reduction of mitochondria-bound HKII and concomitantly associated with enhanced ROS production, ECAR, and PPR.

Background/aims Kv1.3 channel is the only voltage-dependent potassium channel in plasma membrane of human lymphocytes. Bearing in mind a rather steep voltage-dependence of Kv1.3 activation and inactivation, its modulation by B and T cells activation and by co-culture with stromal bone-marrow cells was addressed. Methods Patch-clamp technique in the whole cell mode was applied to human resting and activated human B and T cells, in monoculture and co-culture with stromal OP9 cells. Results Polyclonal activation of B and T cells in monoculture caused Kv1.3 current in B cells to activate at more negative and in T cells at more positive potentials, whereas the inactivation of Kv1.3 current in resting T cells occurred at more negative voltages. Co-culture with OP9 cells abolished the shift of voltage dependence upon the polyclonal activation but fixed the substantial difference between B and T cells, resting or activated, with both activation and inactivation negatively shifted by 15 mV for T lymphocytes. However, activated B cells displayed an incomplete inactivation, which was augmented by the co-culture. Neither activation nor co-culture caused substantial changes in the Kv1.3 current density. Conclusion The combination of activation and inactivation processes yields the fraction of steady-state Kv1.3 current (window current), which was higher in activated B cells, partly due to an incomplete inactivation. A relatively smaller window current in resting B cells and resting T cells in co-culture correlated with a more depolarized resting membrane potential. Rather than insignificant changes in the Kv1.3 channels functional expression, the modulation of their voltage dependence by activation and co-culture with bone-marrow stromal cells was essential for the control of membrane potential.

Background/aims Light-induced control of the cell membrane potential has enabled important advances in the study of biological processes involving the nervous system and muscle activity. The use of these light-induced modifications is expected in various medical applications, including the control of physiological responses and the recovery of lost functions by regulating nerve activity. In particular, charge-separating linkage molecules (Charge-Separation (CS) molecules) can depolarize cells by photoexcitation without genetic processing. However, the molecular mechanisms underlying cell membrane depolarization are unknown and have hindered its application. Here, we show that CS molecules localized in the cell membrane of PC12 cells using a high-density lipoprotein (HDL)-based drug carrier can excite the cells through a novel membrane current regulation mechanism by light irradiation. Methods Membrane potential, channel activity, and membrane capacitance were measured by patch clamp method in rat adrenal gland pheochromocytoma (PC12) cells and KV-overexpressing PC12 cells. CS molecules localized in the cell membrane of PC12 cells using HDL-based drug carrier. The localization of CS molecule was measured by a confocal microscopy. The mRNA expression was tested by RT-PCR. Results Current clamp measurements revealed that the photo-activated CS molecule causes a sharp depolarization of about 15 mV. Furthermore, it was shown by voltage clamp measurement that this mechanism inactivates the voltage-dependent potassium current and simultaneously generates photo-activated CS molecule induced (PACS) current owing to the loss of the cell membrane capacitance. This activity continues the depolarization of the target cell, but is reversible via a regenerative mechanism such as endocytosis and exocytosis because the cell membrane is intact. Conclusion Thus, the mechanism of photo-induced depolarization concludes that photo-activated TC1 causes depolarization by generating PACS current in parallel with the suppression of the K+ current. Moreover, the depolarization slowly restores by internalization of TC1 from the membrane and insertion of new lipids into the cell membrane, resulting in the restoration of KV to normal activity and eliminating PACS currents, without cell damage. These results suggest the possibility of medical application that can safely control membrane excitation.

Background/aims GnIH receptors (GnIHRs) belong to the family of G-protein coupled receptors (GPCRs) and play a key role in the regulation of reproduction from fishes to mammals, either by inhibiting or stimulating the expression of gonadotropins. The aim of this study was to characterize GnIH receptor (GnIHR2) from Indian Major Carp, Labeo catla and its docking and simulation with GnIH antagonist RF313. Methods The full length sequence of GnIHR2 was obtained with RACE PCR. The docking analysis of RF313 with GnIHR2 receptor was performed with AutoDock v. 4.2.6 and molecular dynamics (MD) simulation with GROMACS 5.0. Results In the present study, we cloned full-length cDNA (1733 bp) of GnIHR2 from the brain of L. catla. The phylogenetic analysis showed clustering of catla GnIHR2 with goldfish and zebrafish in the GPR147 group. L. catla GnIHR2 receptor comprised seven transmembrane domains and the 3D-structure was predicted by I-TASSER tool. The docking analysis revealed high binding affinity (-11.6 kcal/mol) of GnIH antagonist, RF313 towards GnIHR2 receptor. The primary bonds involved were alkyl and hydrogen bonds while the amino acids participated were proline 43, 210, 339, cysteine 214, leucine 211, serine 213 and phenylalanine 338. The MD simulation analysis of docked complex for 100 nano-seconds (ns) in the lipid membrane environment showed the stability of the complex with time. Conclusion Our study showed that GnIH antagonist, RF313 interact tightly with the GnIH receptor, GnIHR2 of L. catla. To the best of our knowledge, this is the first report on computational modelling and MD simulation of GnIH receptor in fishes. This will help in functional characterization studies of GnIH/GnIHR system in vertebrates.

The pandemic of the severe acute respiratory syndrome coronavirus (SARS-CoV)-2 at the end of 2019 marked the third outbreak of a highly pathogenic coronavirus affecting the human population in the past twenty years. Cross-species zoonotic transmission of SARS-CoV-2 has caused severe pathogenicity and led to more than 655,000 fatalities worldwide until July 28, 2020. Outbursts of this virus underlined the importance of controlling infectious pathogens across international frontiers. Unfortunately, there is currently no clinically approved antiviral drug or vaccine against SARS-CoV-2, although several broad-spectrum antiviral drugs targeting multiple RNA viruses have shown a positive response and improved recovery in patients. In this review, we compile our current knowledge of the emergence, transmission, and pathogenesis of SARS-CoV-2 and explore several features of SARS-CoV-2. We emphasize the current therapeutic approaches used to treat infected patients. We also highlight the results of in vitro and in vivo data from several studies, which have broadened our knowledge of potential drug candidates for the successful treatment of patients infected with and discuss possible virus and host-based treatment options against SARS-CoV-2. CA - Khairnar

Background/Aims: High-molecular-weight advanced glycation end-products (HMW-AGEs) are abundantly present in our Western diet. There is growing evidence reporting that HMW-AGEs contribute to the development of cardiovascular dysfunction in vivo, next to the well-known low-molecular-weight AGEs. The goal of our study is to assess the ultrastructure and function of cardiomyocytes after chronic exposure to HMW-AGEs. A better understanding of underlying mechanisms is essential to create new opportunities for further research on the specific role of HMW-AGEs in the development and progression of cardiovascular diseases. Methods: Adult male rats were randomly assigned to daily intraperitoneal injection for six weeks with either HMW-AGEs (20 mg/kg/day) or a control solution. Hemodynamic measurements were performed at sacrifice. Single cardiomyocytes from the left ventricle were obtained by enzymatic dissociation through retrograde perfusion of the aorta. Unloaded cell shortening, time to peak and time to 50% relaxation were measured during field stimulation and normalized to diastolic length. L-type Ca 2+ current density (I CaL) and steady-state inactiva-tion of I CaL were measured during whole-cell ruptured patch clamp. Myofilament functional properties were measured in membrane-permeabilized cardiomyocytes. Ultrastructural examination of cardiac tissue was performed using electron microscopy. Results: Rats injected with HMW-AGEs displayed in vivo cardiac dysfunction, characterized by significant changes in Background/Aims: High-molecular-weight advanced glycation end-products (HMW-AGEs) are abundantly present in our Western diet. There is growing evidence reporting that HMWAGEs contribute to the development of cardiovascular dysfunction in vivo, next to the wellknown low-molecular-weight AGEs. The goal of our study is to assess the ultrastructure and function of cardiomyocytes after chronic exposure to HMW-AGEs. A better understanding of underlying mechanisms is essential to create new opportunities for further research on the specific role of HMW-AGEs in the development and progression of cardiovascular diseases. Methods: Adult male rats were randomly assigned to daily intraperitoneal injection for six weeks with either HMW-AGEs (20 mg/kg/day) or a control solution. Hemodynamic measurements were performed at sacrifice. Single cardiomyocytes from the left ventricle were obtained by enzymatic dissociation through retrograde perfusion of the aorta. Unloaded cell shortening, time to peak and time to 50% relaxation were measured during field stimulation and normalized to diastolic length. L-type Ca2+ current density (ICaL) and steady-state inactivation of ICaL were measured during whole-cell ruptured patch clamp. Myofilament functional properties were measured in membrane-permeabilized cardiomyocytes. Ultrastructural examination of cardiac tissue was performed using electron microscopy. Results: Rats injected with HMW-AGEs displayed in vivo cardiac dysfunction, characterized by significant changes in eft ventricular peak rate pressure rise and decline accompanied with an increased heart mass. Single cardiomyocytes isolated from the left ventricle revealed concentric hypertrophy, indicated by the increase in cellular width. Unloaded fractional cell shortening was significantly reduced in cells derived from the HMW-AGEs group and was associated with slower kinetics. Peak L-type Ca2+ current density was significantly decreased in the HMW-AGEs group. L-type Ca2+ channel availability was significantly shifted towards more negative potentials after HMW-AGEs injection. The impact of HMW-AGEs on myofilament function was measured in membrane-permeabilized cardiomyocytes showing a reduction in passive force, maximal Ca2+ activated force and rate of force development. Ultrastructural examination of cardiac tissue demonstrated adverse structural remodeling in HMW-AGEs group characterized by a disruption of the cyto-architecture, a decreased mitochondrial density and altered mitochondrial function. Conclusion: Our data indicate that HMW-AGEs induce structural and functional cellular remodeling via a different working mechanism as the well-known LMW-AGEs. Results of our research open the door for new strategies targeting HMW-AGEs to improve cardiac outcome. Acknowledgements We thank Marc Jans for assisting with the EM. The authors also thank Petra Bex, Rosette Beenaerts and Kanigula Mubagwa for their skillful technical assistance. The graphical abstract was created using images from Servier Medical Art Commons Attribution 3.0 Unported License (http://smart.servier.com). Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License. Funding This work was supported by a Bijzonder onderzoeksfonds (BOF) grant from Hasselt University (15NI06-BOF). Statement of Ethics The animal protocol was approved by the Local Ethical Committee (Ethical Commission for Animal Experimentation, UHasselt, Diepenbeek, Belgium). All animal procedures were performed conforming to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Only trained researchers, certified with a Laboratory Animal Science course according to the Federation of European Laboratory Animal Science Associations, performed animal handling and procedures.

Background/aims The study of the effects of simulated microgravity on primary cultures of human satellite cells represents a reliable model for identifying the biomolecular processes involved in mechanic load-related muscle mass loss. Therefore, this study aims to investigate the role of myostatin and Bone Morphogenetic Protein-2 in human satellite cells response to simulated microgravity condition. Methods In order to identify the main molecules involved in the phenomena of degeneration/regeneration of muscle tissue related to the alteration of mechanic load, we performed a morphological and immunohistochemical study on 27 muscle biopsies taken from control, osteoporotic and osteoarthritic patients, underwent hip arthroplasty. For each patient, we set up primary satellite cell cultures subjected to normogravity and simulated microgravity (110h) regimens. Cellular functionality has been studied through a morphological evaluation performed by optical microscopy, and an ultrastructural evaluation carried out by transmission electron microscopy. Furthermore, we evaluated the expression of Bone Morphogenetic Protein-2 and myostatin through immunocytochemical reactions. Results Our results showed that in the very early phases of simulated microgravity condition the satellite cells are more active than those subjected to the normogravity regime, as demonstrated by both the increase in the number of myotubes and the significant increase in the expression of Bone Morphogenetic Protein-2 in all experimental groups. However, with prolongated exposure to simulated microgravity regime (>72h), satellite cells and new formed myotubes underwent to cell death. It is important to note that, in early phases, simulated microgravity can stimulate the formation of new myotubes from satellite cells derived by osteoporotic patients. Furthermore, we observed that simulated microgravity can induce changes in myostatin expression levels by group-dependent variations. Conclusion The results obtained allowed us to hypothesize a possible molecular mechanism of response to simulated microgravity, confirming the importance of Bone Morphogenetic Protein-2 and myostatin in the physio-pathogenesis of muscle tissue. In addition, these data can lay the foundation for new therapeutic approached in the prevention/cure of osteoporosis and sarcopenia.

Background/aims The beneficial effect of aerobic exercise training (ET) on cardiac remodeling caused by supravalvar aortic stenosis (AS) has been demonstrated in experimental studies; however, the mechanisms responsible for improving cardiac function are not entirely understood. We evaluated whether ET-generated cardioprotection in pressure-overloaded rats is dependent on cardiomyocyte proliferation, increased angiotensin-(1-7) (Ang-1-7) levels, and its receptor in the myocardium. Methods Eighteen weeks after ascending AS surgery, Wistar rats were randomly assigned to four groups: sedentary control (C-Sed), exercised control (C-Ex), sedentary aortic stenosis (AS-Sed) and exercised aortic stenosis (AS-Ex) groups. The moderate treadmill exercise protocol was performed for ten weeks. The functional capacity was assessed by treadmill exercise testing. Cardiac structure and function were evaluated by echocardiogram. Cardiomyocyte proliferation was evaluated by flow cytometry. Expression of cell cycle regulatory genes as CCND2, AURKB, CDK1, and MEIS1 was verified by RT-qPCR. Cardiac and plasma angiotensin I (Ang I), angiotensin II (Ang II), and Ang-(1-7) levels were analyzed by high-performance liquid chromatography (HPLC). The angiotensin-converting enzyme (ACE) activity was assessed by the fluorometric method and protein expression of AT1 and Mas receptors by Western blot. Results The AS-Ex group showed reduced left ventricular wall relative thickness and improved ejection fraction; also, it showed decreased gene expression of myocyte cell cycle regulators, ACE, Ang I, Ang II and Ang II/Ang-(1-7) ratio levels compared to AS-Sed group. However, ET did not induce alterations in Ang-(1-7) and cardiac Mas receptor expression and myocyte proliferation. Conclusion Aerobic exercise training improves systolic function regardless of myocyte proliferation and Ang-(1-7)/Mas receptor levels. However, the ET negatively modulates the vasoconstrictor/hypertrophic axis (ACE/Ang II) and decreases the expression of negative regulatory genes of the cell cycle in cardiomyocytes of rats with supravalvular aortic stenosis.

Background/aims Suicidal erythrocyte death (eryptosis) is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface following a Ca2+ entry in the cell. Eryptosis is stimulated by increased cytosolic Ca2+ ([Ca2+]i), oxidative stress, energy depletion, or high osmotic shock. Eryptosis signaling includes p38 mitogen-activated protein kinase (MAPK), caspases, casein kinase 1 (CK1), janus kinase 3 (JAK3), and protein kinase C (PKC). Dog and human erythrocytes have different characteristics, for example, dog erythrocytes lack Na+/K+- ATPase activity. Whether eryptosis occurs in dog erythrocytes in an analogous way as that in humans remains unclear. Eryptosis in dogs has not been investigated. This study aimed to explore which stimulator and signaling molecules are involved in eryptosis in healthy dog erythrocytes. Methods Erythrocytes were isolated from 10 dogs, and eryptosis was stimulated by oxidative stress with tert-butyl hydroperoxide (tBOOH), high osmotic shock with excessive sucrose condition, energy depletion with minus glucose condition, and high [Ca2+]i with ionomycin. Phosphatidylserine exposure was estimated using annexin V binding. Erythrocyte volume and [Ca2+]i were measured by forward scatter and Fluo3-fluorescence, respectively. In addition, the role of certain mediators was assessed using the following inhibitors to determine the detailed mechanisms of eryptosis in dog erythrocytes: p38MAPK, caspase family, CK1, JAK3, and PKC inhibitors. Results All eryptosis-inducing factors resulted in phosphatidylserine exposures, except for ionomycin. In addition, the erythrocyte volume increased with ionomycin and tBOOH but decreased with excessive sucrose and minus glucose condition. All treatments increased [Ca2+]i. Furthermore, WH1-P154 and chelerythrine significantly blunted the increase of annexin V binding erythrocytes following the tBOOH treatment. Conclusion Eryptosis in dogs is triggered by oxidative stress, hyperosmotic shock, and energy depletion. It is suggested that JAK3 and PKC play an important role in eryptosis following an oxidative stress in dog erythrocytes.

Background/aims The induction of necroptosis, a form of caspase-independent cell death, represents one of the most promising anticancer therapeutic modalities, as necroptosis serves as an alternative way to eliminate apoptosis-resistant tumor cells. Here, we investigated whether protein-bound polysaccharides (PBPs) derived from the fungus Coriolus versicolor (CV) induce the necroptotic death pathway in breast cancer and melanoma cells. Methods MCF-7 and SKMel-188 cells were exposed to PBPs either alone or in combination with necrostatin-1 (Nec-1), GSK'872 or necrosulfonamide (NSA), pharmacological inhibitors of the kinases receptor-interacting protein 1 kinase (RIPK1), receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL), respectively, which are involved in necroptotic processes. The effects of cellular treatment with these inhibitors were quantified by measuring cell viability and reactive oxygen species (ROS) generation via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and 2',7'-dichlorofluorescein diacetate (DCF-DA) assays, respectively. The morphological changes induced in these cells were detected using holotomographic (HT) microscopy. Activation of the TNF-α/TNFR1 pathway in the PBP-stimulated cells was evaluated using TNF-α-neutralizing antibody, qRT-PCR and immunofluorescence-based assays. Results PBPs showed effective antitumor activity against MCF-7 and SKMel-188 cells. Cotreatment of the cells with Nec-1, GSK'872 or NSA abrogated PBP-induced cell death, and the cells were protected against membrane rupture. Moreover, breast cancer cell death caused by PBPs was mediated by induced activation of the TNF-α/TNFR1 pathway. Interestingly, the melanoma cells did not express TNF-α or TNFR1 after PBP stimulation; instead, PBPs triggered intracellular ROS generation, which was partially diminished by the inhibitors Nec-1, GSK'872 and NSA. Conclusion These results suggest that PBPs from the fungus CV induce RIPK1/RIPK3/MLKL-mediated necroptosis in breast cancer and melanoma cells, providing novel insights into the molecular effects of PBPs on cancer cells.

Background/aims The tumour microenvironment is rich in multiple cells that influence cancer development. Among them, macrophages are the most abundant immune cells, which secrete factors involved in carcinogenesis. Since protein-bound polysaccharides (PBP) from the Coriolus versicolor fungus are believed to inhibit the growth of cancers, in the present study, we investigated whether these PBP influence crosstalk between triple-negative 4T1 breast cancer cells and RAW 264.7 macrophages. Methods 4T1 cells were cultured in conditioned media (CM) collected after: stimulation of the macrophages with PBP (CM-PBP) or incubation of non-treated macrophages (CM-NT). A co-cultured model of both cell lines was also employed to investigate the crosstalk between the cells. Cell viability was measured using the MTT assay. The levels of cytokines and chemokines were determined by ELISA methods. Commercial assay kits were used to assess the activity of both arginase 1 and inducible nitric oxide synthase (iNOS) and the level of cell migration. Results The results revealed that CM-NT promotes proliferation and migration of 4T1 cells, and increases the secretion of pro-angiogenic factors (VEGF, MCP-1) by cancer cells. In contrast, CM-PBP inhibits 4T1 cell growth and migration, decreases the secretion of pro-angiogenic factors (VEGF, MCP-1) and upregulates the production of pro-inflammatory mediators (IL-6, TNF-α) with certain anti-tumoral properties Moreover, PBP-treated CM significantly decreases the level of M2 macrophage markers (arginase 1 activity, IL-10 and TGF-β concentrations), but upregulates iNOS activity and IL-6 and TNF-α production, which are M1 cell markers. Conclusion The results suggest that PBP suppress the favourable tumour microenvironment by inhibiting the crosstalk between 4T1 cells and macrophages through the regulation of production of angiogenic and inflammatory mediators, and modulating the M1/M2 macrophage subtype.

Neutrophils were traditionally considered as short-lived cells with abundant secretory and protein synthetic activity. Recent studies, however, indicate neutrophils are in reality a heterogeneous population of cells. Neutrophils differentiate from pluripotent stem cells in the bone marrow, and can further mature in the blood stream and can have different phenotypes in health and disease conditions. Neutrophils undergo primary functions such as phagocytosis, production of reactive oxygen species (ROS), release of lipid mediators and inflammatory proteins (mainly cytokines), and apoptosis. Neutrophils stimulate other neutrophils and trigger a cascade of immune and inflammatory responses. The underpinning intracellular metabolisms that support these neutrophil functions are herein reported. It has been known for many decades that neutrophils utilize glucose as a primary fuel and produce lactate as an end product of glycolysis. Neutrophils metabolize glucose through glycolysis and the pentose- phosphate pathway (PPP). Mitochondrial glucose oxidation is very low. The PPP provides the reduced nicotinamide adenine dinucleotide phosphate (NADPH) for the NADPH-oxidase (NOX) complex activity to produce superoxide from oxygen. These cells also utilize glutamine and fatty acids to produce the required adenosine triphosphate (ATP) and precursors for the synthesis of molecules that trigger functional outcomes. Neutrophils obtained from rat intraperitoneal cavity and incubate for 1 hour at 37°C metabolize glutamine at higher rate than that of glucose. Glutamine delays neutrophil apoptosis and maintains optimal NOX activity for superoxide production. Under limited glucose provision, neutrophils move to fatty acid oxidation (FAO) to obtain the required energy for the cell function. FAO is mainly associated with neutrophil differentiation and maturation. Hypoxia, hormonal dysfunction, and physical exercise markedly change neutrophil metabolism. It is now become clear that neutrophil metabolism underlies the heterogeneity of neutrophil phenotypes and should be intense focus of investigation.

Background/aims The subcellular localization of ERK1 and ERK2 (ERKs) in cells, which is important for proper signaling, may be regulated through protein-protein interactions. However, the proteins involved and the way they are regulated to affect localization is not entirely understood. Methods In order to identify the interacting proteins upon varying conditions, we used co-immunoprecipitation of ERK, active ERK and its binding CRS mutant. In addition, we examined the effect of intracellular calcium on the binding using calcium chelators and ionophores, analyzing the binding using silver stain, mass spectrometry and immunoblotting. The effect of calcium on ERK localization was examined using immunofluorescent staining and Western blotting. Results We found that inactive ERK2 interacts with a large number of proteins through its CRS/CD domain, whereas the phospho-ERK2 interacts with only few substrates. Varying calcium concentrations significantly modified the repertoire of ERK2-interacting proteins, of which many were identified. The effect of calcium on ERKs' interactions influenced also the localization of ERKs, as calcium chelators enhanced nuclear translocation, while elevated calcium levels prevented it. This effect of calcium was also apparent upon the physiological lysophosphatidic acid stimulation, where ERKs translocation was delayed compared to that induced by EGF in a calcium-dependent manner. In vitro translocation assay revealed that high calcium concentrations affect ERKs' translocation by preventing the shuttling machinery through the nuclear envelope, probably due to higher binding to nuclear pore proteins such as NUP153. These results are consistent with a model in which ERKs in quiescent cells are bound to several cytoplasmic proteins. Conclusion Upon stimulation, ERKs are phosphorylated and released from their cytoplasmic anchors to allow shuttling into the nucleus. This translocation is delayed when calcium levels are increased, and this modifies the localization of ERKs and therefore also their spatiotemporal regulation. Thus, calcium regulates ERKs localization, which is important for the compartmentalization of ERKs with their proper substrates, and thereby their signaling specificity.

Background/aims Sodium is a key player in the fundamental cell functions. Fluorescent probes are indispensable tools for monitoring intracellular sodium levels in single living cells. Since the fluorescence of sodium-sensitive dyes in cells is significantly different from that in an aqueous solution, the fluorescence signal is calibrated in situ indirectly using ionophores for equalizing external and intracellular ion concentration. Attempts to compare data obtained using fluorescent probes and by direct flame emission analysis are sparse and results are inaccurate. Methods We determined the intracellular sodium concentration in U937 cells by flow cytometry using the Na+-sensitive probe Asante Natrium Green-2 (ANG), and by standard flame emission photometry combined with the cellular water determination by cell density in Percoll gradient. The intracellular Na+ concentrations was modified using known ionophores or, alternatively, by blocking the sodium pump with ouabain or by causing cell apoptosis with staurosporine. Results It is revealed that both methods are comparable when intracellular sodium concentration was modified by ouabain-mediated blockage of the sodium pump or staurosporine-induced apoptosis. The ANG fluorescence of cells treated with ionophores is approximately two times lower than that in cells with the same Na+ concentration but not treated with ionophores. Although the mechanism is still unknown, this effect should be taken into account when a quantitative assessment of the concentration of intracellular sodium is required. Conclusion The sodium sensitive dye ANG-2 is a sensitive and useful probe for determination changes in Na+ content and concentration both in single cells and subcellular microparticles. The ANG fluorescence determined in the studied cells in the absence of ionophores, cannot be used as a measure of the real intracellular concentration of Na+ if calibration was carried out in the presence of ionophores.

Background/aims Src kinase family members, including c-Src, are involved in numerous signaling pathways and have been observed inside different cellular compartments. Notably, c-Src modulates carbohydrate and fatty acid metabolism and is involved in the metabolic rewiring of cancer cells. This kinase is found within mitochondria where it targets different proteins to impact on the organelle functions and overall metabolism. Surprisingly, no global metabolic characterization of Src has been performed although c-Src knock-out mice have been available for 30 years. Considering that c-Src is sensitive to various metabolites, c-Src might represent a crucial player in metabolic adjustments induced by nutrient stress. The aim of this work was to characterize the impact of c-Src on mitochondrial activity and overall metabolism using multi-omic characterization. Methods Src+/+ and Src-/- mice were fed ad libitum or fasted during 24h and were then analyzed using multi-omics. Results We observed that deletion of c-Src is linked to lower phosphorylation of Y412-NDUFA8, inhibition of oxygen consumption and accumulation of metabolites involved in glycolysis, TCA cycle and amino acid metabolism in mice fed ad libitum. Finally, metabolomics and (phosphotyrosine) proteomics are differently impacted by Src according to nutrient availability. Conclusion The findings presented here highlight that c-Src reduces mitochondrial metabolism and impacts the metabolic adjustment induced by nutrient stress.

Background/aims This study aimed to establish a precise and well-defined working model, assessing pharmaceutical effects on vascular smooth muscle cell monolayer in-vitro. It describes various analysis techniques to determine the most suitable to measure the biomechanical impact of vasoactive agents by using CellDrum technology. Methods The so-called CellDrum technology was applied to analyse the biomechanical properties of confluent human aorta muscle cells (haSMC) in monolayer. The cell generated tensions deviations in the range of a few N/m² are evaluated by the CellDrum technology. This study focuses on the dilative and contractive effects of L-type Ca2+ channel agonists and antagonists, respectively. We analyzed the effects of Bay K8644, nifedipine and verapamil. Three different measurement modes were developed and applied to determine the most appropriate analysis technique for the study purpose. These three operation modes are called, particular time mode" (PTM), "long term mode" (LTM) and "real-time mode" (RTM). Results It was possible to quantify the biomechanical response of haSMCs to the addition of vasoactive agents using CellDrum technology. Due to the supplementation of 100nM Bay K8644, the tension increased approximately 10.6% from initial tension maximum, whereas, the treatment with nifedipine and verapamil caused a significant decrease in cellular tension: 10nM nifedipine decreased the biomechanical stress around 6,5% and 50nM verapamil by 2,8%, compared to the initial tension maximum. Additionally, all tested measurement modes provide similar results while focusing on different analysis parameters. Conclusion The CellDrum technology allows highly sensitive biomechanical stress measurements of cultured haSMC monolayers. The mechanical stress responses evoked by the application of vasoactive calcium channel modulators were quantified functionally (N/m²). All tested operation modes resulted in equal findings, whereas each mode features operation-related data analysis.

Background/aims Galectin 3 (GAL-3) is a beta galactoside binding lectin that has different roles in normal and pathophysiological conditions. GAL-3 has been associated with heart failure and was linked to increased risk of death in a number of studies. GAL-3 was found to be up regulated in animal models of heart failure as well as myocardial infarction (MI). The objective of his study is to test if high GAL-3 after myocardial infarction has a protective role on the heart through its anti-apoptotic and anti-necrotic functions. Methods Male C57B6/J mice and GAL-3 knockout (KO) mice were used for permanent ligation of the left anterior descending artery of the heart to create infarction in the anterior myocardium. Heart and plasma samples were collected 24 hours after the induction of MI and were used for immunohistochemistry, Tunnel procedure, electron microscopy and enzyme linked immunosorbent assay (ELISA). Results Our results show that the significant increase in GAL-3 levels in the left ventricle at 24-hour following MI is associated with significant lower levels of pro-apoptotic proteins; cytochrome c, Bax, annexin V, cleaved caspase-3 and a higher levels of anti-apoptotic protein Bcl2 in GAL-3 wild MI group than GAL-3 KO group. We also have identified the anti-apoptotic activity of GAL-3 is mediated through a significant increase in Akt-1, NF kappa-B and beta- catenin proteins. In addition, we have identified the antiapoptotic activity is mediated through a significant lower levels of cathepsin-D protein. Conclusion We conclude that the increased levels of GAL-3 at 24-hour following MI regulate antiapoptotic mechanisms in the myocardium that will shape the future course of the disease. We also identified that the anti-apoptotic mechanisms are likely mediated through interaction of GAL-3 with Akt-1, NF kappa-B, beta- catenin and cathepsin D proteins.

Background/aims Heart failure with preserved ejection fraction (HFpEF) is recognised as an important cause of cardiovascular mortality and morbidity, accounting for approximately 50% of heart failure cases. Metabolic-related complications, such as obesity, have been associated with the pathophysiology of this complex syndrome. The anatomic proximity between cardiac visceral adipose tissue (CVAT) and the myocardium has been drawing attention due to its potential pathogenic role in cardiac diseases. Thus, we aimed to characterise the phenotypic and proteomic differences between CVAT from ZSF1 lean (control) and ZSF1 obese (HFpEF) rats as well as to evaluate the myocardial impact of conditioned media derived from CVAT of these 2 groups. Methods CVAT of 20-weeks-old lean and obese ZSF1 rats was collected for: 1) 24h DMEM incubation to obtain conditioned media, 2) separation of proteins to mass spectrometry identification, 3) adipokines' expression, 4) adipocytes cross-sectional area assessment. Organotypic cultures were prepared from 7 days-old Wistar Han cardiac explants and incubated for 24h with the conditioned media. After incubation, cross-section area of cardiomyocytes and fibrosis were evaluated. Cardiomyocytes were isolated from Wistar Han and incubated with conditioned media for viability studies. Results CVAT from lean rats presented a higher expression of uncoupling protein-1 (UCP-1) protein, associated with a multilocular appearance and an increased expression of brown adipose tissue markers. Contrarily, CVAT from obese rats revealed a white adipose tissue-like phenotype accompanied by hypertrophy of adipocytes. The analysis of the CVAT proteome reinforced the phenotypic differences between lean and obese CVAT, showing enrichment of proteins involved in triglyceride metabolic processes in obese CVAT. In contrast, mitochondrial proteins were prominent in lean CVAT, further suggesting a brown adipose tissue-like phenotype. The twenty-four hours-long incubation of myocardial organo-cultures with conditioned media obtained from CVAT obese (CM-obese) rats significantly reduced cell viability, induced cardiomyocytes hypertrophy and fibrosis, in stark contrast with the incubation with the conditioned media from lean rats CVAT (CM-lean). Furthermore, the deleterious effect imposed by CM-obese was associated with a pro-inflammatory profile, characterised by an increased expression of several pro-inflammatory adipokines. Conclusion Obesity promotes alterations in CVAT proteome signature, structure, composition and secretome, translating into dramatic myocardial consequences.

Background/aims Pericytes (PCs) are multipotent vascular precursors that play a critical physiological role in the development and maintenance of blood vessel integrity. In this study, we aim to characterize PCs isolated from human abdominal adipose tissue and develop an integration-free induced pluripotent stem cells (iPSCs) using episomal vectors. Methods The ultrastructure of adipose tissue-derived PCs was determined using scanning and transmission electron microscopy. The expression of mesenchymal stem cells (MSCs) and pericyte markers were examined using flow cytometry and PCR analysis. PCs were induced to adipogenic, osteogenic and myogenic lineages, and their angiogenic potential was determined using tube formation assay. We further established pericyte reprogramming protocol using episomal vectors. Results Our data showed that human adipose tissue-derived PCs uniformly expressed MSCs, CD105 and CD73, and PCs markers, desmin, and alpha smooth muscle actin (α-SMA), while lacked the expression of HLA-DR and the hematopoietic markers CD34, CD11b and CD45. Ultrastructure analysis showed typical internal structure for the PCs with a characteristic prominent eccentric nuclei and cytoplasmic invaginations forming a caveolar system. Functional analysis showed efficient differentiation into adipocytes, osteocytes, and myocyte-like cells. Adipose tissue-derived PCs showed angiogenic potential using tube-forming assay. To determine further application of these cells for personalized therapy, we reprogrammed PCs into induced pluripotent stem cells (iPSCs) using episomal vectors. Reprogrammed cells gradually lost their fusiform shape, acquired the epithelial cell morphology and formed colonies. Furthermore, reprogrammed cells successfully expressed the pluripotency markers OCT4, Nanog, SSEA-4, and β-catenin, an early reprogramming marker. Conclusion The accessibility and abundance of human fat supports the application of adipose derived PCs as a novel and promising source of cell therapy and regenerative medicine.

BACKGROUND/AIMS: Cell migration and extracellular matrix remodeling underlie normal mammalian development and growth as well as pathologic tumor invasion. Skeletal muscle is no exception, where satellite cell migration replenishes nuclear content in damaged tissue and extracellular matrix reforms during regeneration. A key set of enzymes that regulate these processes are matrix metalloproteinases (MMP)s. The collagenase MMP-13 is transiently upregulated during muscle regeneration, but its contribution to damage resolution is unknown. The purpose of this work was to examine the importance of MMP-13 in muscle regeneration and growth in vivo and to delineate a satellite cell specific role for this collagenase. METHODS: Mice with total and satellite cell specific Mmp13 deletion were utilized to determine the importance of MMP-13 for postnatal growth, regeneration after acute injury, and in chronic injury from a genetic cross with dystrophic (mdx) mice. We also evaluated insulin-like growth factor 1 (IGF-1) mediated hypertrophy in the presence and absence of MMP-13. We employed live-cell imaging and 3D migration measurements on primary myoblasts obtained from these animals. Outcome measures included muscle morphology and function. RESULTS: Under basal conditions, Mmp13(−/−) mice did not exhibit histological or functional deficits in muscle. However, following acute injury, regeneration was impaired at 11 and 14 days post injury. Muscle hypertrophy caused by increased IGF-1 was blunted with minimal satellite cell incorporation in the absence of MMP-13. Mmp13(−/−) primary myoblasts displayed reduced migratory capacity in 2D and 3D, while maintaining normal proliferation and differentiation. Satellite cell specific deletion of MMP-13 recapitulated the effects of global MMP-13 ablation on muscle regeneration, growth and myoblast movement. CONCLUSION: These results show that satellite cells provide an essential autocrine source of MMP-13, which not only regulates their migration, but also supports postnatal growth and resolution of acute damage.

Background/aims Adverse effects of cigarette smoke on health are widely known. Heating rather than combusting tobacco is one of strategies to reduce the formation of toxicants. The sensitive nature of mitochondrial dynamics makes the mitochondria an early indicator of cellular stress. For this reason, we studied the morphology and dynamics of the mitochondrial network in human bronchial epithelial cells (BEAS-2B) exposed to total particulate matter (TPM) generated from 3R4F reference cigarette smoke and from aerosol from a new candidate modified risk tobacco product, the Tobacco Heating System (THS 2.2). Methods Cells were subjected to short (1 week) and chronic (12 weeks) exposure to a low (7.5 µg/mL) concentration of 3R4F TPM and low (7.5 µg/mL), medium (37.5 µg/mL), and high (150 µg/mL) concentrations of TPM from THS 2.2. Confocal microscopy was applied to assess cellular and mitochondrial morphology. Cytosolic Ca2+ levels, mitochondrial membrane potential and mitochondrial mass were measured with appropriate fluorescent probes on laser scanning cytometer. The levels of proteins regulating mitochondrial dynamics and biogenesis were determined by Western blot. Results In BEAS-2B cells exposed for one week to the low concentration of 3R4F TPM and the high concentration of THS 2.2 TPM we observed clear changes in cell morphology, mitochondrial network fragmentation, altered levels of mitochondrial fusion and fission proteins and decreased biogenesis markers. Also cellular proliferation was slowed down. Upon chronic exposure (12 weeks) many parameters were affected in the opposite way comparing to short exposure. We observed strong increase of NRF2 protein level, reorganization of mitochondrial network and activation of the mitochondrial biogenesis process. Conclusion Comparison of the effects of TPMs from 3R4F and from THS 2.2 revealed, that similar extent of alterations in mitochondrial dynamics and biogenesis is observed at 7.5 µg/mL of 3R4F TPM and 150 µg/mL of THS 2.2 TPM. 7 days exposure to the investigated components of cigarette smoke evoke mitochondrial stress, while upon chronic, 12 weeks exposure the hallmarks of cellular adaptation to the stressor were visible. The results also suggest that mitochondrial stress signaling is involved in the process of cellular adaptation under conditions of chronic stress caused by 3R4F and high concentration of THS 2.2.

BACKGROUND/AIMS Still in 1999 the first hints were published for the pharmacoresistant Cav2.3 calcium channel to be involved in the generation of epileptic seizures, as transcripts of alpha1E (Cav2.3) and alpha1G (Cav3.1) are changed in the brain of genetic absence epilepsy rats from Strasbourg (GAERS). Consecutively, the seizure susceptibility of mice lacking Cav2.3 was analyzed in great detail by using 4-aminopyridine, pentylene-tetrazol, N-methyl-D-aspartate and kainic acid to induce experimentally convulsive seizures. Further, γ-hydroxybutyrolactone was used for the induction of non-convulsive absence seizures. For all substances tested, Cav2.3-competent mice differed from their knockout counterparts in the sense that for convulsive seizures the deletion of the pharmacoresistant channel was beneficial for the outcome during experimentally induced seizures [1]. The antiepileptic drug lamotrigine reduces seizure activity in Cav2.3-competent but increases it in Cav2.3-deficient mice. In vivo, Cav2.3 must be under tight control by endogenous trace metal cations (Zn2+ and Cu2+). The dyshomeostasis of either of them, especially of Cu2+, may alter the regulation of Cav2.3 severely and its activity for Ca2+ conductance, and thus may change hippocampal and neocortical signaling to hypo- or hyperexcitation. METHODS To investigate by telemetric EEG recordings the mechanism of generating hyperexcitation by kainate, mice were tested for their sensitivity of changes in neuronal (intracerebroventricular) concentrations of the trace metal cation Zn2+. As the blood-brain barrier limits the distribution of bioavailable Zn2+ or Cu2+ into the brain, we administered micromolar Zn2+ ions intracerebroventricularly in the presence of 1 mM histidine as carrier and compared the effects on behavior and EEG activity in both genotypes. RESULTS Kainate seizures are more severe in Cav2.3-competent mice than in KO mice and histidine lessens seizure severity in competent but not in Cav2.3-deficient mice. Surprisingly, Zn2+ plus histidine resembles the kainate only control with more seizure severity in Cav2.3-competent than in deficient mice. CONCLUSION Cav2.3 represents one important Zn2+-sensitive target, which is useful for modulating convulsive seizures.

BACKGROUND/AIMS Store-operated Ca2+ entry (SOCE) through plasma membrane Ca2+ channel Orai1 is essential for many cellular processes. SOCE, activated by ER Ca2+ store-depletion, relies on the gating function of STIM1 Orai1-activating region SOAR of the ER-anchored Ca2+-sensing protein STIM1. Electrophysiologically, SOCE is characterized as Ca2+ release-activated Ca2+ current (ICRAC). A major regulatory mechanism that prevents deleterious Ca2+ overload is the slow Ca2+-dependent inactivation (SCDI) of ICRAC. Several studies have suggested a role of Ca2+/calmodulin (Ca2+/CaM) in triggering SCDI. However, a direct contribution of STIM1 in regulating Ca2+/CaM-mediated SCDI of ICRAC is as yet unclear. METHODS The Ca2+/CaM binding to STIM1 was tested by pulling down recombinant GFP-tagged human STIM1 C-terminal fragments on CaM sepharose beads. STIM1 was knocked out by CRISPR/Cas9 technique in HEK293 cells stably overexpressing human Orai1. Store-operated Ca2+ influx was measured using Fluorometric Imaging Plate Reader and whole-cell patch clamp in cells transfected with STIM1 CaM binding mutants. The involvement of Ca2+/CaM in SCDI was investigated by including recombinant human CaM in patch pipette in electrophysiology. RESULTS Here we identified residues Leu374/Val375 (H1) and Leu390/Phe391 (H2) within SOAR that serve as hydrophobic anchor sites for Ca2+/CaM binding. The bifunctional H2 site is critical for both Orai1 activation and Ca2+/CaM binding. Single residue mutations of Phe391 to less hydrophobic residues significantly diminished SOCE and ICRAC, independent of Ca2+/CaM. Hence, the role of H2 residues in Ca2+/CaM-mediated SCDI cannot be precisely evaluated. In contrast, the H1 site controls exclusively Ca2+/CaM binding and subsequently SCDI, but not Orai1 activation. V375A but not V375W substitution eliminated SCDI of ICRAC caused by Ca2+/CaM, proving a direct role of STIM1 in coordinating SCDI. CONCLUSION Taken together, we propose a mechanistic model, wherein binding of Ca2+/CaM to STIM1 hydrophobic anchor residues, H1 and H2, triggers SCDI by disrupting the functional interaction between STIM1 and Orai1. Our findings reveal how STIM1, Orai1, and Ca2+/CaM are functionally coordinated to control ICRAC.

Background/aims Mitochondrial ATP synthase, in addition to being involved in ATP synthesis, is involved in permeability transition pore (PTP) formation, which precedes apoptosis in mammalian cells and programmed cell death in yeast. Mutations in genes encoding ATP synthase subunits cause neuromuscular disorders and have been identified in cancer samples. PTP is also involved in pathology. We previously found that in Saccharomyces cerevisiae, two mutations in ATP synthase subunit a (atp6-P163S and atp6-K90E, equivalent to those detected in prostate and thyroid cancer samples, respectively) in the OM45-GFP background affected ROS and calcium homeostasis and delayed yeast PTP (yPTP) induction upon calcium treatment by modulating the dynamics of ATP synthase dimer/oligomer formation. The Om45 protein is a component of the porin complex, which is equivalent to mammalian VDAC. We aimed to investigate yPTP function in atp6-P163S and atp6-K90E mutants lacking the e and g dimerization subunits of ATP synthase. Methods Triple mutants with the atp6-P163S or atp6-K90E mutation, the OM45-GFP gene and deletion of the TIM11 gene encoding subunit e were constructed by crossing and tetrad dissection. In spores capable of growing, the original atp6 mutations reverted to wild type, and two compensatory mutations, namely, atp6-C33S-T215C, were selected. The effects of these mutations on cellular physiology, mitochondrial morphology, bioenergetics and permeability transition (PT) were analyzed by fluorescence and electron microscopy, mitochondrial respiration, ATP synthase activity, calcium retention capacity and swelling assays. Results The atp6-C33S-T215C mutations in the OM45-GFP background led to delayed growth at elevated temperature on both fermentative and respiratory media and increased sensitivity to high calcium ions concentration or hydrogen peroxide in the medium. The ATP synthase activity was reduced by approximately 50% and mitochondrial network was hyperfused in these cells grown at elevated temperature. The atp6-C33S-T215C stabilized ATP synthase dimers and restored the yPTP properties in Tim11∆ cells. In OM45-GFP cells, in which Tim11 is present, these mutations increased the fraction of swollen mitochondria by up to 85% vs 60% in the wild type, although the time required for calcium release doubled. Conclusion ATP synthase subunit e is essential in the S. cerevisiae atp6-P163S and atp6-K90E mutants. In addition to subunits e and g, subunit a is critical for yPTP induction and conduction. The increased yPTP conduction decrease the S. cerevisiae cell fitness.

Background/aims We performed co-culture experiments between human RPE cells (ARPE-19) and human umbilical vascular endothelial cells (HUVEC) in order to evaluate how anti-VEGF drugs could affect NO release, mitochondrial function, the oxidative status, proliferation and migration of RPE cells through modulation of their cross talk with vascular endothelial cells. Methods The co-culture HUVEC/RPE, was exposed to Ranibizumab/Aflibercept in the absence/presence of the NO synthase (NOS) inhibitor, the phosphatidylinositol 3'-kinase (PI3K), the extracellular-signal-regulated kinases 1/2 (ERK1/2) and the p38 mitogen-activated protein kinase (p38 MAPK) blockers. Specific kits were used for cell viability, mitochondrial membrane potential, NO, ROS and GSH production. Western blot was performed for apoptosis markers, NOS isoforms, and others kinases detection. Cell migration was analyzed by scratch assay, whereas cell proliferation and cell cycle through xCELLigence and flow cytometry. Results In RPE cells co-cultured with HUVEC in physiological conditions, Aflibercept/Ranibizumab increased NO release in a dose and time-dependent way. Opposite results were obtained in peroxidative conditions. Both anti-VEGF agents were able to prevent the fall of cell viability and mitochondrial membrane potential, an effect which was reduced by various inhibitors, and increased cell migration. Aflibercept/Ranibizumab counteracted the changes of apoptosis markers, NOS expression/activation, PI3K and ERK1/2 activation caused by peroxidation. These results were confirmed by cell cycle analysis. Conclusion This study has shown new mechanisms at the basis of protective effects elicited by Aflibercept/Ranibizumab in RPE cells. HUVEC stimulated with Aflibercept/Ranibizumab, could release some paracrine factors that can modulate the RPE cells response in both physiologic and peroxidative conditions.