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Analysis of the spatio-temporal distribution of calcium sparks showed a preferential increase in sparks near the sarcolemma in atrial myocytes from patients with atrial fibrillation (AF), linked to higher ryanodine receptor (RyR2) phosphorylation at s2808 and lower calsequestrin-2 levels. Mathematical modeling, incorporating modulation of RyR2 gating, showed that only the observed combinations of RyR2 phosphorylation and calsequestrin-2 levels can account for the spatio-temporal distribution of sparks in patients with and without AF. Furthermore, we demonstrate that preferential calcium release near the sarcolemma is key to a higher incidence and amplitude of afterdepolarizations in atrial myocytes from patients with AF.

Aims: Atrial fibrillation (AF) has been associated with altered expression of the transcription factor Pitx2c and a high incidence of calcium release-induced afterdepolarizations. However, the relationship between Pitx2c expression and defective calcium homeostasis remains unclear and we here aimed to determine how Pitx2c expression affects calcium release from the sarcoplasmic reticulum (SR) and its impact on electrical activity in isolated atrial myocytes. Methods: To address this issue, we applied confocal calcium imaging and patch-clamp techniques to atrial myocytes isolated from a mouse model with conditional atrial-specific deletion of Pitx2c. Results: Our findings demonstrate that heterozygous deletion of Pitx2c doubles the calcium spark frequency, increases the frequency of sparks/site 1.5-fold, the calcium spark decay constant from 36 to 42 ms and the wave frequency from none to 3.2 min. Additionally, the cell capacitance increased by 30% and both the SR calcium load and the transient inward current (I) frequency were doubled. Furthermore, the fraction of cells with spontaneous action potentials increased from none to 44%. These effects of Pitx2c deficiency were comparable in right and left atrial myocytes, and homozygous deletion of Pitx2c did not induce any further effects on sparks, SR calcium load, I frequency or spontaneous action potentials. Conclusion: Our findings demonstrate that heterozygous Pitx2c deletion induces defects in calcium homeostasis and electrical activity that mimic derangements observed in right atrial myocytes from patients with AF and suggest that Pitx2c deficiency confers cellular electrophysiological hallmarks of AF to isolated atrial myocytes.

Cholesterol is a crucial component of membrane bilayers by regulating their structural and functional properties. Cholesterol traffics to different cellular compartments including mitochondria, whose cholesterol content is low compared to other cell membranes. Despite the limited availability of cholesterol in the inner mitochondrial membrane (IMM), the metabolism of cholesterol in the IMM plays important physiological roles, acting as the precursor for the synthesis of steroid hormones and neurosteroids in steroidogenic tissues and specific neurons, respectively, or the synthesis of bile acids through an alternative pathway in the liver. Accumulation of cholesterol in mitochondria above physiological levels has a negative impact on mitochondrial function through several mechanisms, including the limitation of crucial antioxidant defenses, such as the glutathione redox cycle, increased generation of reactive oxygen species and consequent oxidative modification of cardiolipin, and defective assembly of respiratory supercomplexes. These adverse consequences of increased mitochondrial cholesterol trafficking trigger the onset of oxidative stress and cell death, and, ultimately, contribute to the development of diverse diseases, including metabolic liver diseases (i.e. fatty liver disease and liver cancer), as well as lysosomal disorders (i.e. Niemann-Pick type C disease) and neurodegenerative diseases (i.e. Alzheimer's disease). In this review, we summarize the metabolism and regulation of mitochondrial cholesterol and its potential impact on liver and neurodegenerative diseases.

As the COVID-19 pandemic has progressed, long-COVID has emerged as a major problem that poses a significant challenge for attending physicians and health care policy makers. Therefore, we read with much interest the recently published unicentre study in the Journal of Infection by Righi et al.,1 carried out on 465 adult COVID-19 patients (235 [50.5%] hospital-admitted) followed-up during nine months, concluding that those with advanced age, intensive care unit (ICU) admission, and multiple symptoms at onset were more likely to have long-term COVID-19 symptoms, with negative impact on physical and mental wellbeing. Other studies have found that female gender, age, longer hospital stay, pre-existing hypertension, cardiovascular disease, diabetes, chronic obstructive pulmonary disease, smoking, obesity, and chronic alcoholism increase the likelihood of long-COVID.2,3 It is known that SARS-CoV-2 RNAemia is a predictor of COVID-19 severity and in-hospital complications.4,5 However, to the best of our knowledge, only two studies have assessed, up to one or three months after the acute COVID-19 onset, whether SARS-CoV-2 RNAemia may have an impact on long-COVID,6,7 both finding that RNAemia at presentation might predict the persistence of symptoms. However, these studies did not provide information regarding long-COVID symptoms nor the association with SARS-CoV-2 RNAemia beyond three months, and could not differentiate between “true” long-COVID and the convalescence phase of the SARS-CoV-2 infection.

Alcoholic (ASH) and nonalcoholic. (NASH).steatohepatitis are advanced.stages.of.fatty.liver.disease.Methionine adenosyltransferase 1A (MAT1A) plays a key role in hepatic methionine metabolism and germline Mat1a deletion in mice promotes NASH. Acid sphingomyelinase (ASMase) triggers hepatocellular apoptosis and liver fibrosis and has been shown to downregulate MAT1A expression in the context of fulminant liver failure. Given the role of ASMase in steatohepatitis development, we investigated the status of ASMase in Mat1a-/- mice and the regulation of ASMase by SAM/SAH. Consistent with its role in NASH, Mat1a-/- mice fed a choline-deficient (CD) diet exhibited macrosteatosis, inflammation, fibrosis and liver injury as well as reduced total and mitochondrial GSH levels. Our data uncovered an increased basal expression and activity of ASMase but not neutral SMase in Mat1a-/- mice, which further increased upon CD feeding. Interestingly, adenovirus-mediated shRNA expression targeting ASMase reduced ASMase activity and protected Mat1a-/- mice against CD diet-induced NASH. Similar results were observed in CD fed Mat1a-/- mice by pharmacological inhibition of ASMase with amitriptyline. Moreover, Mat1a/ASMase double knockout mice were resistant to CD-induced NASH. ASMase knockdown protected wild type mice against NASH induced by feeding a diet deficient in methionine and choline. Furthermore, Mat1a-/- mice developed acute-on-chronic ASH and this outcome was ameliorated by amitriptyline treatment. In vitro data in primary mouse hepatocytes revealed that decreased SAM/SAH ratio increased ASMase mRNA level and activity. MAT1A and ASMase mRNA levels exhibited an inverse correlation in liver samples from patients with ASH and NASH. Thus, disruption of methionine metabolism sensitizes to steatohepatitis by ASMase activation via decreased SAM/SAH. These findings imply that MAT1A deletion and ASMase activation engage in a self-sustained loop of relevance for steatohepatitis.

Obesity and its associated metabolic comorbidities are a rising global health and social issue, with novel therapeutic approaches urgently needed. Adipose tissue plays a key role in the regulation of energy balance and adipose tissue-derived mesenchymal stem cells (AT-MSCs) have gained great interest in cell therapy. Carnitine palmitoyltransferase 1A (CPT1A) is the gatekeeper enzyme for mitochondrial fatty acid oxidation. Here, we aimed to generate adipocytes expressing a constitutively active CPT1A form (CPT1AM) that can improve the obese phenotype in mice after their implantation. AT-MSCs were differentiated into mature adipocytes, subjected to lentivirus-mediated expression of CPT1AM or the GFP control, and subcutaneously implanted into mice fed a high-fat diet (HFD). CPT1AM-implanted mice showed lower body weight, hepatic steatosis and serum insulin and cholesterol levels alongside improved glucose tolerance. HFD-induced increases in adipose tissue hypertrophy, fibrosis, inflammation, endoplasmic reticulum stress and apoptosis were reduced in CPT1AM-implanted mice. In addition, the expression of mitochondrial respiratory chain complexes was enhanced in the adipose tissue of CPT1AM-implanted mice. Our results demonstrate that implantation of CPT1AM-expressing AT-MSC-derived adipocytes into HFD-fed mice improves the obese metabolic phenotype, supporting the future clinical use of this ex vivo gene therapy approach.

Lafora disease (LD; OMIM#254780) is a rare form of progressive myoclonus epilepsy (prevalence <1:1,000,000) characterized by the accumulation of insoluble deposits of aberrant glycogen (polyglucosans), named Lafora bodies, in the brain but also in peripheral tissues. LD is the most severe form of the group of progressive myoclonus epilepsies, since patients present a rapid deterioration and dementia with amplification of seizures, leading to death after a decade from the onset of the first symptoms. We have recently described that reactive glia-derived neuroinflammation should be considered a novel hallmark of LD since we observed a florid upregulation of differentially expressed genes in both LD mouse lines, which were mainly related to mediators of inflammatory response. In this work, we define an upregulation of the expression of mediators of the TNF and IL6/JAK2 signaling pathways in LD. In addition, we describe the activation of the non-canonical form of the inflammasome. Furthermore, we describe the infiltration of peripheral immune cells in the brain parenchyma, which could aggravate glia-derived neuroinflammation. Finally, we describe CXCL10 and S100b as blood biomarkers of the disease, which will allow the study of the progression of the disease using serum blood samples. We consider that the identification of these initial inflammatory changes in LD will be very important to implement possible anti-inflammatory therapeutic strategies to prevent the development of the disease.

Laforin and Malin are two proteins that are encoded by the genes EPM2A and EPM2B, respectively. Laforin is a glucan phosphatase and Malin is an E3-ubiquitin ligase, and these two proteins function as a complex. Mutations occurring at the level of one of the two genes lead to the accumulation of an aberrant form of glycogen meant to cluster in polyglucosans that go under the name of Lafora bodies. Individuals affected by the appearance of these polyglucosans, especially at the cerebral level, experience progressive neurodegeneration and several episodes of epilepsy leading to the manifestation of a fatal form of a rare disease called Lafora disease (LD), for which, to date, no treatment is available. Despite the different dysfunctions described for this disease, many molecular aspects still demand elucidation. An effective way to unknot some of the nodes that prevent the achievement of better knowledge of LD is to focus on the substrates that are ubiquitinated by the E3-ubiquitin ligase Malin. Some substrates have already been provided by previous studies based on protein-protein interaction techniques and have been associated with some alterations that mark the disease. In this work, we have used an unbiased alternative approach based on the activity of Malin as an E3-ubiquitin ligase. We report the discovery of novel bonafide substrates of Malin and have characterized one of them more deeply, namely PIP3-dependent Rac exchanger 1 (P-Rex1). The analysis conducted upon this substrate sets the genesis of the delineation of a molecular pathway that leads to altered glucose uptake, which could be one of the origin of the accumulation of the polyglucosans present in the disease.

[EN]: Quality indicators are crucial for standardizing and guaranteeing the quality of health care practices. The Spanish Academy of Dermatology and Venereology (AEDV) launched the CUDERMA Project to define quality indicators for the certification of specialized units in dermatology; the first 2 areas selected were psoriasis and dermato-oncology. The aim of this study was to achieve consensus on what should be evaluated by these indicators using a structured process comprising a literature review and selection of an initial list of indicators to be evaluated in a Delphi consensus study following review by a multidisciplinary group of experts. The selected indicators were evaluated by a panel of 28 dermatologists and classified as either «essential» or «of excellence». The panel agreed on 84 indicators, which will be standardized and used to develop the certification standard for dermato-oncology units., [ES]: Los indicadores de calidad son una herramienta clave como garantía de calidad y homogenización de la asistencia sanitaria. En este contexto, la Academia Española de Dermatología y Venereología ha diseñado el proyecto CUDERMA (Certificación de unidades de dermatología), una iniciativa que busca definir indicadores de calidad para certificar unidades de dermatología en distintos ámbitos, entre los que se seleccionaron psoriasis y dermato-oncología de forma inicial. Este estudio tuvo por objetivo consensuar los aspectos a evaluar por los indicadores, siguiendo un proceso estructurado para la revisión bibliográfica y elaboración de un set preliminar de indicadores, revisado por un grupo multidisciplinar de expertos, para su evaluación mediante un Consenso Delphi. Un panel de 28 dermatólogos evaluó los indicadores y los clasificó como «básicos» o «de excelencia», generando un conjunto de 84 indicadores consensuados que serán estandarizados para diseñar la norma con la que certificar las unidades de dermato-oncología.

Impaired function of organic cation transporter 1 (OCT1) in hepatocellular carcinoma (HCC) has been associated with unsatisfactory response to sorafenib. However, some patients lacking OCT1 at the plasma membrane (PM) of HCC cells still respond to sorafenib, suggesting that another transporter may contribute to take up this drug. The aim of this study was to investigate whether OCT3 could contribute to the uptake of sorafenib and other tyrosine kinase inhibitors (TKIs) and whether OCT3 determination can predict HCC response to sorafenib. Cells overexpressing OCT3 were used to determine the ability of this carrier to transport sorafenib. Immunostaining of OCT3 was performed in HCC samples obtained in the TRANSFER study. Considering the intensity of staining and the number of OCT3-positive cells, tumors were classified as having absent, weak, moderate, or strong OCT3 expression and were also categorized according to the presence or absence of PM staining. Functional in vitro studies revealed that OCT3 is also able to mediate sorafenib uptake. Other TKIs, such as regorafenib, lenvatinib, and cabozantinib can also interact with this transporter. In silico studies suggested that the expression of OCT3 is better preserved in HCC than that of OCT1. In HCC samples, OCT3 was expressed at the PM of cancer cells, and its presence, detected in 26% of tumors, was associated with better outcomes in patients treated with sorafenib. In conclusion, analysis by immunohistochemistry of OCT3 in the PM of tumor cells may help to predict the response of HCC patients to sorafenib and potentially to other TKIs.

Metabolic plasticity is the ability of a biological system to adapt its metabolic phenotype to different environmental stressors. We used a whole-body and tissue-specific phenotypic, functional, proteomic, metabolomic and transcriptomic approach to systematically assess metabolic plasticity in diet-induced obese mice after a combined nutritional and exercise intervention. Although most obesity and overnutrition-related pathological features were successfully reverted, we observed a high degree of metabolic dysfunction in visceral white adipose tissue, characterized by abnormal mitochondrial morphology and functionality. Despite two sequential therapeutic interventions and an apparent global healthy phenotype, obesity triggered a cascade of events in visceral adipose tissue progressing from mitochondrial metabolic and proteostatic alterations to widespread cellular stress, which compromises its biosynthetic and recycling capacity. In humans, weight loss after bariatric surgery showed a transcriptional signature in visceral adipose tissue similar to our mouse model of obesity reversion. Overall, our data indicate that obesity prompts a lasting metabolic fingerprint that leads to a progressive breakdown of metabolic plasticity in visceral adipose tissue.

Type 4 sphingosine-1-phosphate receptor, which mediates the activation of the Nod-like receptor (NLR) family pyrin domain containing 3 inflammasome, emerges as a new therapeutic target for nonalcoholic steatohepatitis. We developed a selective functional antagonist for type 4 sphingosine-1-phosphate receptor (SLB736), which protected mice against nonalcoholic steatohepatitis and fibrosis

Background: Antibodies against the P3 sequence (Gly1127-Cys1140) of LRP1 (anti-P3 Abs) specifically block cholesteryl ester (CE) accumulation in vascular cells. LRP1 is a key regulator of insulin receptor (InsR) trafficking in different cell types. The link between CE accumulation and the insulin response are largely unknown. Here, the effects of P3 peptide immunization on the alterations induced by a high-fat diet (HFD) in cardiac insulin response were evaluated. Methods: Irrelevant (IrP)- or P3 peptide-immunized rabbits were randomized into groups fed either HFD or normal chow. Cardiac lipid content was characterized by thin-layer chromatography, confocal microscopy, and electron microscopy. LRP1, InsR and glucose transporter type 4 (GLUT4) levels were determined in membranes and total lysates from rabbit heart. The interaction between InsR and LRP1 was analyzed by immunoprecipitation and confocal microscopy. Insulin signaling activity and glucose uptake were evaluated in HL-1 cells exposed to rabbit serum from the different groups. Findings: HFD reduces cardiac InsR and GLUT4 membrane levels and the interactions between LRP1/InsR. Targeting the P3 sequence on LRP1 through anti-P3 Abs specifically reduces CE accumulation in the heart independently of changes in the circulating lipid profile. This restores InsR and GLUT4 levels in cardiac membranes as well as the LRP1/InsR interactions of HFD-fed rabbits. In addition, anti-P3 Abs restores the insulin signaling cascade and glucose uptake in HL-1 cells exposed to hypercholesterolemic rabbit serum. Interpretation: LRP1-immunotargeting can block CE accumulation within the heart with specificity, selectivity, and efficacy, thereby improving the cardiac insulin response; this has important therapeutic implications for a wide range of cardiac diseases.

Drug-induced liver injury is a major cause of acute liver failure (ALF) and one of the most challenging liver disorders with respect to its prediction, diagnosis, and management. Acetaminophen (APAP) is one of the most widely used pain relievers worldwide. Although relatively safe, APAP is a dose-dependent hepatotoxin and APAP overdose is a major cause of ALF, far exceeding other causes of ALF in industrialized countries. APAP hepatotoxicity in humans can be modelled in rodents after administration of an acute or cumulative overdose. However, despite intensive efforts, the mechanisms involved in APAP hepatotoxicity are not fully understood, which has hampered the availability of effective therapy for APAP hepatotoxicity.1,2 N-acetylcysteine (NAC) is the standard treatment for APAP hepatotoxicity and other causes of ALF

Rank signaling enhances stemness in mouse and human mammary epithelial cells (MECs) and mediates mammary tumor initiation. Mammary tumors initiated by oncogenes or carcinogen exposure display high levels of Rank and Rank pathway inhibitors have emerged as a new strategy for breast cancer prevention and treatment. Here, we show that ectopic Rank expression in the mammary epithelia unexpectedly delays tumor onset and reduces tumor incidence in the oncogene-driven Neu and PyMT models. Mechanistically, we have found that ectopic expression of Rank or exposure to Rankl induces senescence, even in the absence of other oncogenic mutations. Rank leads to DNA damage and senescence through p16/p19. Moreover, RANK-induced senescence is essential for Rank-driven stemness, and although initially translates into delayed tumor growth, eventually promotes tumor progression and metastasis. We uncover a dual role for Rank in the mammary epithelia: Rank induces senescence and stemness, delaying tumor initiation but increasing tumor aggressiveness.

Nuclear receptors (NRs) are a superfamily of ligand-activated transcription factors that act as biological sensors and use a combination of mechanisms to modulate positively and negatively gene expression in a spatial and temporal manner. The highly orchestrated biological actions of several NRs influence the proliferation, differentiation, and apoptosis of many different cell types. Synthetic ligands for several NRs have been the focus of extensive drug discovery efforts for cancer intervention. This review summarizes the roles in tumour growth and metastasis of several relevant NR family members, namely androgen receptor (AR), estrogen receptor (ER), glucocorticoid receptor (GR), thyroid hormone receptor (TR), retinoic acid receptors (RARs), retinoid X receptors (RXRs), peroxisome proliferator-activated receptors (PPARs), and liver X receptors (LXRs). These studies are key to develop improved therapeutic agents based on novel modes of action with reduced side effects and overcoming resistance.

Von Willebrand Factor (vWF), a 300-kDa plasma protein key to homeostasis, is cleaved at a single site by multi-domain metallopeptidase ADAMTS-13. vWF is the only known substrate of this peptidase, which circulates in a latent form and becomes allosterically activated by substrate binding. Herein, we characterised the complex formed by a competent peptidase construct (AD13-MDTCS) comprising metallopeptidase (M), disintegrin-like (D), thrombospondin (T), cysteine-rich (C), and spacer (S) domains, with a 73-residue functionally relevant vWF-peptide, using nine complementary techniques. Pull-down assays, gel electrophoresis, and surface plasmon resonance revealed tight binding with sub-micromolar affinity. Cross-linking mass spectrometry with four reagents showed that, within the peptidase, domain D approaches M, C, and S. S is positioned close to M and C, and the peptide contacts all domains. Hydrogen/deuterium exchange mass spectrometry revealed strong and weak protection for C/D and M/S, respectively. Structural analysis by multi-angle laser light scattering and small-angle X-ray scattering in solution revealed that the enzyme adopted highly flexible unbound, latent structures and peptide-bound, active structures that differed from the AD13-MDTCS crystal structure. Moreover, the peptide behaved like a self-avoiding random chain. We integrated the results with computational approaches, derived an ensemble of structures that collectively satisfied all experimental restraints, and discussed the functional implications. The interaction conforms to a ‘fuzzy complex’ that follows a ‘dynamic zipper’ mechanism involving numerous reversible, weak but additive interactions that result in strong binding and cleavage. Our findings contribute to illuminating the biochemistry of the vWF:ADAMTS-13 axis.

Recent achievements in the field of immunotherapy, such as the development of engineered T cells used in adoptive cell therapy, are introducing more efficient strategies to combat cancer. Nevertheless, there are still many limitations. For example, these T cells are challenging to manufacture, manipulate, and control. Specifically, there are limitations in producing the large amounts of therapeutic T cells needed for these therapies in a short period of time and in an economically viable manner. In this study, three-dimensional (3D) poly(ethylene) glycol (PEG) hydrogels covalently combined with low molecular weight heparin are engineered to resemble the lymph nodes, where T cells reproduce. In these hydrogels, PEG provides the needed structural and mechanical properties, whereas heparin is used as an anchor for the cytokine CCL21, which is present in the lymph nodes, and can affect cell migration and proliferation. The 3D structure of the hydrogel in combination with its loading capacity result in an increased primary human CD4+ T cell proliferation compared to the state-of-the-art expansion systems consisting of artificial antigen presenting cells. Thus, we present a new tool for adoptive cell therapy to help achieving the large numbers of cells required for therapy of selected phenotypes targeted against cancer cells, by mimicking the lymph nodes.

[Objective]: Metainflammation is a chronic low-grade inflammatory state induced by obesity and associated comorbidities, including peripheral insulin resistance. Brown adipose tissue (BAT), a therapeutic target against obesity, is an insulin target tissue sensitive to inflammation. Therefore, it is necessary to find strategies to protect BAT against the effects of inflammation in energy balance. In this study, we explored the impact of moderate sirtuin 1 (SIRT1) overexpression on insulin sensitivity and ß-adrenergic responses in BAT and brown adipocytes (BA) under pro-inflammatory conditions., [Methods]: The effect of inflammation on BAT functionality was studied in obese db/db mice and lean wild-type (WT) mice or mice with moderate overexpression of SIRT1 (SIRT1Tg+) injected with a low dose of bacterial lipopolysaccharide (LPS) to mimic endotoxemia. We also conducted studies on differentiated BA (BA-WT and BA-SIRT1Tg+) exposed to a macrophage-derived pro-inflammatory conditioned medium (CM) to evaluate the protection of SIRT1 overexpression in insulin signaling and glucose uptake, mitochondrial respiration, fatty acid oxidation (FAO), and norepinephrine (NE)-mediated-modulation of uncoupling protein-1 (UCP-1) expression., [Results]: BAT from the db/db mice was susceptible to metabolic inflammation manifested by the activation of pro-inflammatory signaling cascades, increased pro-inflammatory gene expression, tissue-specific insulin resistance, and reduced UCP-1 expression. Impairment of insulin and noradrenergic responses were also found in the lean WT mice upon LPS injection. In contrast, BAT from the mice with moderate overexpression of SIRT1 (SIRT1Tg+) was protected against LPS-induced activation of pro-inflammatory signaling, insulin resistance, and defective thermogenic-related responses upon cold exposure. Importantly, the decline in triiodothyronine (T3) levels in the circulation and intra-BAT after exposure of the WT mice to LPS and cold was markedly attenuated in the SIRT1Tg+ mice. In vitro BA experiments in the two genotypes revealed that upon differentiation with a T3-enriched medium and subsequent exposure to a macrophage-derived pro-inflammatory CM, only BA-SIRT1Tg+ fully recovered insulin and noradrenergic responses., [Conclusions]: This study has ascertained the benefit of the moderate overexpression of SIRT1 to confer protection against defective insulin and ß-adrenergic responses caused by BAT inflammation. Our results have potential therapeutic value in combinatorial therapies for BAT-specific thyromimetics and SIRT1 activators to combat metainflammation in this tissue.

Zika virus (ZikV) is a flavivirus that infects neural tissues, causing congenital microcephaly. ZikV has evolved multiple mechanisms to restrict proliferation and enhance cell death, although the underlying cellular events involved remain unclear. Here we show that the ZikV-NS5 protein interacts with host proteins at the base of the primary cilia in neural progenitor cells, causing an atypical non-genetic ciliopathy and premature neuron delamination. Furthermore, in human microcephalic fetal brain tissue, ZikV-NS5 persists at the base of the motile cilia in ependymal cells, which also exhibit a severe ciliopathy. Although the enzymatic activity of ZikV-NS5 appears to be dispensable, the amino acids Y25, K28, and K29 that are involved in NS5 oligomerization are essential for localization and interaction with components of the cilium base, promoting ciliopathy and premature neurogenesis. These findings lay the foundation for therapies that target ZikV-NS5 multimerization and prevent the developmental malformations associated with congenital Zika syndrome.

Cardiomyocytes are subjected to the intense mechanical stress and metabolic demands of the beating heart. It is unclear whether these cells, which are long-lived and rarely renew, manage to preserve homeostasis on their own. While analyzing macrophages lodged within the healthy myocardium, we discovered that they actively took up material, including mitochondria, derived from cardiomyocytes. Cardiomyocytes ejected dysfunctional mitochondria and other cargo in dedicated membranous particles reminiscent of neural exophers, through a process driven by the cardiomyocyte's autophagy machinery that was enhanced during cardiac stress. Depletion of cardiac macrophages or deficiency in the phagocytic receptor Mertk resulted in defective elimination of mitochondria from the myocardial tissue, activation of the inflammasome, impaired autophagy, accumulation of anomalous mitochondria in cardiomyocytes, metabolic alterations, and ventricular dysfunction. Thus, we identify an immune-parenchymal pair in the murine heart that enables transfer of unfit material to preserve metabolic stability and organ function. Video Abstract: [Figure presented] A system of macrophages in the heart supports cardiomyocyte health by phagocytosing exopher particles ejected from cardiomyocytes that contain defective mitochondria, among other cellular contents.

Background and Aims: GAS6 signaling, through the TAM receptor tyrosine kinases AXL and MERTK, participates in chronic liver pathologies. Here, we addressed GAS6/TAM involvement in Non-Alcoholic SteatoHepatitis (NASH) development. Methods: GAS6/TAM signaling was analyzed in cultured primary hepatocytes, hepatic stellate cells (HSC) and Kupffer cells (KCs). Axl, Mertk and wild-type C57BL/6 mice were fed with Chow, High Fat Choline-Deficient Methionine-Restricted (HFD) or methionine-choline-deficient (MCD) diet. HSC activation, liver inflammation and cytokine/chemokine production were measured by qPCR, mRNA Array analysis, western blotting and ELISA. GAS6, soluble AXL (sAXL) and MERTK (sMERTK) levels were analyzed in control individuals, steatotic and NASH patients. Results: In primary mouse cultures, GAS6 or MERTK activation protected primary hepatocytes against lipid toxicity via AKT/STAT-3 signaling, while bemcentinib (small molecule AXL inhibitor BGB324) blocked AXL-induced fibrogenesis in primary HSCs and cytokine production in LPS-treated KCs. Accordingly; bemcentinib diminished liver inflammation and fibrosis in MCD- and HFD-fed mice. Upregulation of AXL and ADAM10/ADAM17 metalloproteinases increased sAXL in HFD-fed mice. Transcriptome profiling revealed major reduction in fibrotic- and inflammatory-related genes in HFD-fed mice after bemcentinib administration. HFD-fed Mertk mice exhibited enhanced NASH, while Axl mice were partially protected. In human serum, sAXL levels augmented even at initial stages, whereas GAS6 and sMERTK increased only in cirrhotic NASH patients. In agreement, sAXL increased in HFD-fed mice before fibrosis establishment, while bemcentinib prevented liver fibrosis/inflammation in early NASH. Conclusion: AXL signaling, increased in NASH patients, promotes fibrosis in HSCs and inflammation in KCs, while GAS6 protects cultured hepatocytes against lipotoxicity via MERTK. Bemcentinib, by blocking AXL signaling and increasing GAS6 levels, redu

Changes in the oxidation state of protein Cys residues are involved in cell signalling and play a key role in a variety of pathophysiological states. We had previously developed GELSILOX, an in-gel method that enables the large-scale, parallel analysis of dynamic alterations to the redox state of Cys sites and protein abundance changes. Here we present FASILOX, a further development of the GELSILOX approach featuring: i) significantly increased peptide recovery, ii) enhanced sensitivity for the detection of Cys oxidative alterations, and iii) streamlined workflow that results in shortened assay duration. In mitochondria isolated from the adipose tissue of obese, diabetic patients, FASILOX revealed a sexually dimorphic trait of Cys oxidation involving mainly mitochondrial oxidative phosphorylation complexes. These results provide the first evidence for a decreased efficiency in the antioxidant response of men as compared to women.

ErbB4 is a transmembrane receptor tyrosine kinase that binds to neuregulins to activate signaling. Proteolytic cleavage of ErbB4 results in release of soluble fragments of ErbB4 into the interstitial fluid. Disruption of the neuregulin-ErbB4 pathway has been suggested to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). This study assesses whether soluble proteolytic fragments of the ErbB4 ectodomain (ecto-ErbB4) can be detected in cerebrospinal fluid (CSF) and plasma, and if the levels are altered in ALS. Immunoprecipitation combined with mass spectrometry or western blotting analyses confirmed the presence of ecto-ErbB4 in human CSF. Several anti-ErbB4-reactive bands, including a 55 kDa fragment, were detected in CSF. The bands were generated in the presence of neuregulin-1 (Nrg1) and were absent in plasma from ErbB4 knockout mice. Ecto-ErbB4 levels were decreased in CSF from ALS patients (n = 20) and ALS with concomitant frontotemporal dementia patients (n = 10), compared to age-matched controls (n = 13). A similar decrease was found for the short ecto-ErbB4 fragments in plasma of the same subjects. Likewise, the 55-kDa ecto-ErbB4 fragments were decreased in the plasma of the two transgenic mouse models of ALS (SOD1G93A and TDP-43A315T). Intracellular ErbB4 fragments were decreased in the frontal cortex from SOD1G93A mice, indicating a reduction in Nrg-dependent induction of ErbB4 proteolytic processing, and suggesting impaired signaling. Accordingly, overexpression of Nrg1 induced by an adeno-associated viral vector increased the levels of the ecto-ErbB4 fragment in the SOD1G93A mice. We conclude that the determination of circulating ecto-ErbB4 fragments could be a tool to evaluate the impairment of the ErbB4 pathway and may be a useful biomarker in ALS.

In order to navigate through the surrounding environment many mammals, including humans, primarily rely on vision. The eye, composed of the choroid, sclera, retinal pigmented epithelium, cornea, lens, iris and retina, is the structure that receives the light and converts it into electrical impulses. The retina contains six major types of neurons involving in receiving and modifying visual information and passing it onto higher visual processing centres in the brain. Visual information is relayed to the brain via the axons of retinal ganglion cells (RGCs), a projection known as the optic pathway. The proper formation of this pathway during development is essential for normal vision in the adult individual. Along this pathway there are several points where visual axons face ‘choices’ in their direction of growth. Understanding how these choices are made has advanced significantly our knowledge of axon guidance mechanisms. Thus, the development of the visual pathway has served as an extremely useful model to reveal general principles of axon pathfinding throughout the nervous system. However, due to its particularities, some cellular and molecular mechanisms are specific for the visual circuit. Here we review both general and specific mechanisms involved in the guidance of mammalian RGC axons when they are traveling from the retina to the brain to establish precise and stereotyped connections that will sustain vision.

Numerous degenerative diseases affecting visual function, including glaucoma and retinitis pigmentosa, are produced by the loss of different types of retinal cells. Cell replacement therapy has emerged as a promising strategy for treating these and other retinal diseases. The retinal margin or ciliary body (CB) of mammals has been proposed as a potential source of cells to be used in degenerative conditions affecting the retina because it has been reported it might hold neurogenic potential beyond embryonic development. However, many aspects of the origin and biology of the CB are unknown and more recent experiments have challenged the capacity of CB cells to generate different types of retinal neurons. Here we review the most recent findings about the development of the marginal zone of the retina in different vertebrates and some of the mechanisms underlying the proliferative and neurogenic capacity of this fascinating region of the vertebrates eye. In addition, we performed experiments to isolate CB cells from the mouse retina, generated neurospheres and observed that they can be expanded with a proliferative ratio similar to neural stem cells. When induced to differentiate, cells derived from the CB neurospheres start to express early neural markers but, unlike embryonic stem cells, they are not able to fully differentiate in vitro or generate retinal organoids. Together with previous reports on the neurogenic capacity of CB cells, also reviewed here, our results contribute to the current knowledge about the potentiality of this peripheral region of the eye as a therapeutic source of functional retinal neurons in degenerative diseases.

Vitamin D (VitD) may modulate anti-HIV-1 responses modifying the risk to acquire the HIV-1-infection. We performed a nested case-control exploratory study involving 413 individuals; HIV-1-exposed seropositives (cases) and seronegatives (HESN) (controls) from three cohorts: sexually-exposed from Colombia and Italy and parenterally-exposed from Spain. The association and interactions of 139 variants in 9 VitD pathway genes, and in 14 antiviral genes with resistance/susceptibility (R/S) to HIV-1 infection was evaluated. Associations between variants and mRNA levels were also analyzed in the Colombian samples. Variants and haplotypes in genes of VitD and antiviral pathways were associated with R/S, but specific associations were not reproduced in all cohorts. Allelic heterogeneity could explain such inconsistency since the associations found in all cohorts were consistently in the same genes: VDR and RXRA of the VitD pathway genes and in TLR2 and RNASE4. Remarkably, the multi-locus genotypes (interacting variants) observed in genes of VitD and antiviral pathways were present in most HESNs of all cohorts. Finally, HESNs carrying resistance-associated variants had higher levels of VitD in plasma, of VDR mRNA in blood cells, and of ELAFIN and defensins mRNA in the oral mucosa. In conclusion, despite allelic heterogeneity, most likely due to differences in the genetic history of the populations, the associations were locus dependent suggesting that genes of the VitD pathway might act in concert with antiviral genes modulating the resistance phenotype of the HESNs. Although these associations were significant after permutation test, only haplotype results remained statistically significant after Bonferroni test, requiring further replications in larger cohorts and functional analyzes to validate these conclusions.

[Background] Studies evaluating leptin levels in patients with first-episode psychoses (FEP) have been inconclusive, and apparently, the high levels of leptin reported in patients with schizophrenia may be associated with weight gain. The aim of this study was to evaluate leptin levels at the early stages of the disease and the relationship between leptin and lifestyle habits, stress-related variables and metabolic parameters., [Methods] In total, 14 at-risk mental state (ARMS) patients, 39 FEP patients, 32 psychotic patients in the critical period (CP) and 21 healthy controls (HCs) were assessed. Anthropometric and biochemical parameters, as well as dietary intake, physical activity, stress-related variables and symptomatology, were collected., [Results] Leptin levels were higher in the ARMS, FEP and CP patients than in the HCs. After controlling for age, sex, BMI, physical exercise, tobacco use and dietary intake, the highest differences in leptin levels were observed between the ARMS patients and HCs (p = 0.025). In the whole sample, leptin levels were positively correlated with BMI (p < 0.001), waist circumference (p < 0.001), insulin levels (p = 0.020), levels of the inflammatory marker IL-6 (p = 0.007) and energy intake (p = 0.043) and negatively correlated with HDL cholesterol (p = 0.018). Interestingly, energy intake and food craving scores were positively correlated with levels of leptin only in females (p = 0.022 and p = 0.036, respectively)., [Discussion] The present study detected increased leptin levels in the early stages of psychosis and significant correlations between leptin levels and anthropometric, lipid, hormone, and cytokine parameters. We found higher leptin levels in women, and we identified dietary intake habits associated with leptin exclusively in females that advocate considering sex in future studies.

Implantable electrodes act with direct electrical contact although recent work has shown that electrostimulation is also possible through non-contact wireless settings, through the generation of dipoles at the borders of the material by bipolar electrochemistry. The experimental observations with neural cell cultures demonstrate a clear difference between insulator and conducting materials, but also between conducting and mixed conducting intercalation materials used as substrates of neural growth. Known bipolar electrochemistry effects may explain voltage profiles induced on conducting materials. Finite element studies shown here with the same configuration that the experimental processes described, evidence voltage profiles in qualitative agreement with known bipolar effects, although with a clear difference between intercalation materials and metals. Calculations also show a clear mapping of charge gradients at the material surface influencing growing neurons cells. While insulating materials only distort the electric field space distribution, the dipole generated at the borders of an implanted conducting material, inverted with respect to the insulating case, extends along the material interface, being relevant that is much smoother in intercalation materials. Mapping of the gradients as the distance is increased from the conducting material is also discussed. These observations may explain the differences in neural cell growth observed for various substrate materials.

[EN] Different studies have analyzed the potential of the off-the-shelf Microsoft Kinect, in its different versions, to estimate spatiotemporal gait parameters as a portable markerless low-cost alternative to laboratory grade systems. However, variability in populations, measures, and methodologies prevents accurate comparison of the results. The objective of this study was to determine and compare the reliability of the existing Kinect-based methods to estimate spatiotemporal gait parameters in healthy and post-stroke adults. Forty-five healthy individuals and thirty-eight stroke survivors participated in this study. Participants walked five meters at a comfortable speed and their spatiotemporal gait parameters were estimated from the data retrieved by a Kinect v2, using the most common methods in the literature, and by visual inspection of the videotaped performance. Errors between both estimations were computed. For both healthy and post-stroke participants, highest accuracy was obtained when using the speed of the ankles to estimate gait speed (3.6¿5.5 cm/s), stride length (2.5¿5.5 cm), and stride time (about 45 ms), and when using the distance between the sacrum and the ankles and toes to estimate double support time (about 65 ms) and swing time (60¿90 ms). Although the accuracy of these methods is limited, these measures could occasionally complement traditional tools.

Huntington’s disease (HD) is a dominantly inherited neurodegenerative disease caused by expansion of a polyglutamine tract in the huntingtin protein. HD symptoms include severe motor, cognitive, and psychiatric impairments that result from dysfunction and later degeneration of medium-sized spiny neurons (MSNs) in the striatum. A key early pathogenic mechanism is dysregulated synaptic transmission due to enhanced surface expression of juvenile NMDA-type glutamate receptors containing GluN3A subunits, which trigger the aberrant pruning of synapses formed by cortical afferents onto MSNs. Here, we tested the therapeutic potential of silencing GluN3A expression in YAC128 mice, a well-established HD model. Recombinant adeno-associated viruses encoding a short-hairpin RNA against GluN3A (rAAV-shGluN3A) were generated, and the ability of different serotypes to transduce MSNs was compared. A single injection of rAAV9-shGluN3A into the striatum of 1-month-old mice drove potent (>90%) and long-lasting reductions of GluN3A expression in MSNs, prevented dendritic spine loss and improved motor performance in YAC128 mice. Later delivery, when spine pathology is already apparent, was also effective. Our data provide proof-of-concept for GluN3A silencing as a beneficial strategy to prevent or reverse corticostriatal disconnectivity and motor impairment in HD and support the use of RNAi-based or small-molecule approaches for harnessing this therapeutic potential.

[Background]: Intrauterine growth restriction (IUGR) is associated with cardiovascular remodeling persisting into adulthood. Mitochondrial bioenergetics, essential for embryonic development and cardiovascular function, are regulated by nuclear effectors as sirtuins. A rabbit model of IUGR and cardiovascular remodeling was generated, in which heart mitochondrial alterations were observed by microscopic and transcriptomic analysis. We aimed to evaluate if such alterations are translated at a functional mitochondrial level to establish the etiopathology and potential therapeutic targets for this obstetric complication. [Methods]: Hearts and placentas from 16 IUGR-offspring and 14 controls were included to characterize mitochondrial function. [Results]: Enzymatic activities of complexes II, IV and II + III in IUGR-hearts (−11.96 ± 3.16%; −15.58 ± 5.32%; −14.73 ± 4.37%; p < 0.05) and II and II + III in IUGR-placentas (−17.22 ± 3.46%; p < 0.005 and −29.64 ± 4.43%; p < 0.001) significantly decreased. This was accompanied by a not significant reduction in CI-stimulated oxygen consumption and significantly decreased complex II SDHB subunit expression in placenta (−44.12 ± 5.88%; p < 0.001). Levels of mitochondrial content, Coenzyme Q and cellular ATP were conserved. Lipid peroxidation significantly decreased in IUGR-hearts (−39.02 ± 4.35%; p < 0.001), but not significantly increased in IUGR-placentas. Sirtuin3 protein expression significantly increased in IUGR-hearts (84.21 ± 31.58%; p < 0.05) despite conserved anti-oxidant SOD2 protein expression and activity in both tissues. [Conclusions]: IUGR is associated with cardiac and placental mitochondrial CII dysfunction. Up-regulated expression of Sirtuin3 may explain attenuation of cardiac oxidative damage and preserved ATP levels under CII deficiency.

[EN] Ageing is the main risk factor for cardiovascular disease (CVD). The increased prevalence of CVD is partly due to the global increase in life expectancy. In this context, it is essential to identify the mechanisms by which ageing induces CVD, with the ultimate aim of reducing its incidence. Both atherosclerosis and heart failure significantly contribute to age-associated CVD morbidity and mortality. Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder caused by the synthesis of progerin, which is noted for accelerated ageing and CVD. This mutant form of prelamin A induces generalised atherosclerosis, vascular calcification, and cardiac electrophysiological abnormalities, leading to premature ageing and death, mainly due to myocardial infarction and stroke. This review discusses the main vascular structural and functional abnormalities during physiological and premature ageing, as well as the mechanisms involved in the exacerbated CVD and accelerated ageing induced by the accumulation of progerin and prelamin A. Both proteins are expressed in non-HGPS individuals, and physiological ageing shares many features of progeria. Research into HGPS could therefore shed light on novel mechanisms involved in the physiological ageing of the cardiovascular system., [ES] El envejecimiento es el principal factor de riesgo de enfermedad cardiovascular (ECV) y su prevalencia está aumentando progresivamente debido en gran parte al incremento de la esperanza de vida a nivel mundial. En este contexto, es fundamental establecer cuáles son los mecanismos por los que el envejecimiento promueve el desarrollo de ECV, con el objetivo de reducir su incidencia. La aterosclerosis y la insuficiencia cardiaca contribuyen de manera significativa a la morbimortalidad por ECV asociada a la edad. El síndrome de progeria de Hutchinson-Gilford (HGPS) se caracteriza por un envejecimiento prematuro que cursa también con ECV acelerada. Se trata de un trastorno genético raro causado por la expresión de progerina, una forma mutada de la prelamina A. La progerina induce aterosclerosis masiva y alteraciones electrofisiológicas en el corazón, promueve el envejecimiento y finalmente la muerte prematura a una edad media de 14,6 anos, ˜ principalmente por infarto de miocardio o ictus cerebral. En esta revisión se discuten las principales alteraciones estructurales y funcionales que afectan al sistema vascular durante el envejecimiento fisiológico y prematuro, así como los mecanismos que subyacen a la aterosclerosis y al envejecimiento exagerados inducidos por la prelamina A y la progerina. Dado que ambas proteínas se expresan en individuos sin HGPS y muchas de las características del envejecimiento normal se presentan en la progeria, la investigación en el ámbito del HGPS podría contribuir a la identificación de nuevos mecanismos implicados en el envejecimiento cardiovascular fisiológico.

Neural electrode implants are made mostly of noble materials. We have synthesized a nanostructured material combining the good electrochemical properties of iridium oxide (IrOx) and carbon-nanotubes (CNT) and the properties of poly(3,4-ethylenedioxythiophene) (PEDOT). IrOx-CNT-PEDOT charge storage capacity was lower than that of IrOx and IrOx-CNT, but higher than that of other PEDOT-containing hybrids and Pt. Cyclic voltammetry, SEM, XPS and micro-Raman spectroscopy suggest that PEDOT encapsulates IrOx and CNT. In our search for a cell culture platform that could optimize modelling the in vivo environment, we determined cell viability, neuron and astrocyte functionality and the response of astrocytes to an inflammatory insult by using primary cultures of neurons, of astrocytes and co-cultures of both. The materials tested (based on IrOx, CNT and PEDOT, as well as Pt as a reference) allowed adhesion and proliferation of astrocytes and full compatibility for neurons grown in co-cultures. Functionality assays show that uptake of glutamate in neuron-astrocyte co-culture was significantly higher than the sum of the uptake in astrocytes and neurons. In co-cultures on IrOx, IrOx-CNT and IrOx-CNT-PEDOT, glutamate was released by a depolarizing stimulus and induced a significant increase in intracellular calcium, supporting the expression of functional NMDA/glutamate receptors. LPS-induced inflammatory response in astrocytes showed a decreased response in NOS2 and COX2 mRNA expression for IrOx-CNT-PEDOT. Results indicate that neuron-astrocyte co-cultures are a reliable model for assessing the biocompatibility and safety of nanostructured materials, evidencing also that hybrid IrOx-CNT-PEDOT nanocomposite materials may offer larger resistance to inflammatory insults.

Autosomal dominant hypercholesterolemia, being referred to as familial hypercholesterolemia (FH), is mainly due to defective LDL receptor (LDLR) function, but is also associated with variants in genes encoding APOB (LDLR ligand) and PCSK9, the catabolic regulator of LDLR. The signal-transducing adaptor family member 1 (STAP1) gene has been recently linked to FH. We describe the case of a 56-year-old male patient found to have hypercholesterolemia at age 34, but who did not continue follow-up nor received treatment with lipid-lowering drugs. At age 55 he suffered a myocardial infarction. A systematic NGS analysis did not show point mutations in the LDLR, APOB, LDLRAP1, or PCSK9 genes, nor large rearrangements of the LDLR gene, but revealed the heterozygous missense variant rs199787258 of STAP1 (c.526C > T; p.Pro176Ser). This variant was also found in heterozygosis in the two siblings of the index case, who also had hypercholesterolemia, but did not cosegregate in his progeny. A bioinformatics analysis and available structural information predicts p.Pro176Ser as the most damaging of all STAP1 missense variants associated with familial hypercholesterolemia. Our findings confirm and extend the linkage between STAP1 variants and FH, and point to an important role of this adaptor protein within a signaling pathway that affects cholesterol homeostasis.

Niemann Pick type C (NPC) disease is a progressive lysosomal storage disorder caused by mutations in genes encoding NPC1/NPC2 proteins, characterized by neurological defects, hepatosplenomegaly and premature death. While the primary biochemical feature of NPC disease is the intracellular accumulation of cholesterol and gangliosides, predominantly in endolysosomes, mitochondrial cholesterol accumulation has also been reported. As accumulation of cholesterol in mitochondria is known to impair the transport of GSH into mitochondria, resulting in mitochondrial GSH (mGSH) depletion, we investigated the impact of mGSH recovery in NPC disease. We show that GSH ethyl ester (GSH-EE), but not N-acetylcysteine (NAC), restored the mGSH pool in liver and brain of Npc1-/- mice and in fibroblasts from NPC patients, while both GSH-EE and NAC increased total GSH levels. GSH-EE but not NAC increased the median survival and maximal life span of Npc1-/- mice. Moreover, intraperitoneal therapy with GSH-EE protected against oxidative stress and oxidant-induced cell death, restored calbindin levels in cerebellar Purkinje cells and reversed locomotor impairment in Npc1-/- mice. High-resolution respirometry analyses revealed that GSH-EE improved oxidative phosphorylation, coupled respiration and maximal electron transfer in cerebellum of Npc1-/- mice. Lipidomic analyses showed that GSH-EE treatment had not effect in the profile of most sphingolipids in liver and brain, except for some particular species in brain of Npc1-/- mice. These findings indicate that the specific replenishment of mGSH may be a potential promising therapy for NPC disease, worth exploring alone or in combination with other options.

[EN] MicroRNAs (miRNAs) are a class of small noncoding RNA (20-25 nucleotides) involved in gene regulation. In recent years, miRNAs have emerged as a key epigenetic mechanism in the development and physiology ofthe cardiovascular system. Thesemolecular species regulate basic functions invirtually all celltypes, and are therefore directly associated with the pathophysiology of a large number of cardiovascular diseases. Since their relatively recent discovery in extracellular fluids, miRNAs have been studied as potential biomarkers of disease.A wide array of studies have proposed miRNAs as circulating biomarkers of different cardiovascular pathologies (eg, myocardial infarction, coronary heart disease, and heart failure, among others), which may have superior physicochemical and biochemical properties than the conventional protein indicators currently used in clinical practice. In the present review, we provide a brief introduction to the field of miRNAs, paying special attention to their potential clinical application. This includes their possible role as new diagnostic or prognostic biomarkers in cardiovascular disease, [ES] Los microARN (miARN) son una clase de ARN no codificante de pequen˜o taman˜o (20-25 nucleo´tidos) que participan en la regulacio´n ge´nica. En los u´ ltimos an˜os, los miARN han emergido como un mecanismo epigene´tico clave en el desarrollo y en la funcionalidad del sistema cardiovascular. Estas especies moleculares regulan funciones ba´ sicas en pra´ cticamente todos los tipos celulares y por ello esta´n directamente asociadas con la fisiopatologı´a de un gran nu´ mero de enfermedades cardiovasculares. Desde su relativamente reciente descubrimiento en fluidos extracelulares, se ha estudiado los miARN como potenciales biomarcadores de enfermedad. Son numerosos los estudios que proponen a los miARN como biomarcadores circulantes de diferentes patologı´as cardiovasculares (infarto de miocardio, enfermedad coronaria o insuficiencia cardiaca, entre otras), incluso con propiedades fisicoquı´micas y bioquı´micas superiores a los indicadores proteicos convencionales utilizados actualmente en la pra´ ctica clı´nica. En el presente trabajo se ofrece una breve introduccio´n al campo de los miARN, con especial atencio´n a su potencial aplicacio´n clı´nica. Esta incluye su posible papel como nuevos biomarcadores de diagno´ stico o de prono´ stico en la enfermedad cardiovascular.

Recent evidence suggests that obstructive sleep apnea (OSA) may increase the risk of Alzheimer´s disease (AD), with the latter promoting alterations in brain tissue stiffness, a feature of ageing. Here, we assessed the effects of age and intermittent hypoxia (IH) on brain tissue stiffness in a mouse model of OSA. Two-month-old and 18-month-old mice (N=10 each) were subjected to IH (20% O2 40 s – 6% O2 20 s) for 8 weeks (6 h/day). Corresponding control groups for each age were kept under normoxic conditions in room air (RA). After sacrifice, the brain was excised and 200-micron coronal slices were cut with a vibratome. Local stiffness of the cortex and hippocampus were assessed in brain slices placed in an Atomic Force Microscope. For both brain regions, the Young's modulus (E) in each animal was computed as the average values from 9 force-indentation curves. Cortex E mean (±SE) values were 442±122 Pa (RA) and 455±120 (IH) for young mice and 433±44 (RA) and 405±101 (IH) for old mice. Hippocampal E values were 376±62 (RA) and 474±94 (IH) for young mice and 486±93 (RA) and 521±210 (IH) for old mice. For both cortex and hippocampus, 2-way ANOVA indicated no statistically significant effects of age or challenge (IH vs. RA) on E values. Thus, neither chronic IH mimicking OSA nor ageing up to late middle age appear to modify local brain tissue stiffness in otherwise healthy mice.

Adverse cardiac remodeling after myocardial infarction (MI) causes impaired ventricular function and heart failure. Histopathological characterization is commonly used to detect the location, size and shape of MI sites. However, the information about chemical composition, physical structure and molecular mobility of peri- and infarct zones post-MI is rather limited. The main objective of this work was to explore the spatiotemporal biochemical and biophysical alterations of key cardiac components post-MI. The FTIR spectra of healthy and remote myocardial tissue shows amides A, I, II and III associated with proteins in freeze-died tissue as major absorptions bands. In infarcted myocardium, the spectrum of these main absorptions was deeply altered. FITR evidenced an increase of the amide A band and the distinct feature of the collagen specific absorption band at 1338 cm−1 in the infarct area at 21 days post-MI. At 21 days post-MI, it also appears an important shift of amide I from 1646 cm−1 to 1637 cm−1 that suggests the predominance of the triple helical conformation in the proteins. The new spectra bands also indicate an increase in proteoglycans, residues of carbohydrates in proteins and polysaccharides in ischemic areas. Thermal analysis indicates a deep increase of unfreezable water/freezable water in peri- and infarcted tissues. In infarcted tissue is evidenced the impairment of myofibrillar proteins thermal profile and the emergence of a new structure. In conclusion, our results indicate a profound evolution of protein secondary structures in association with collagen deposition and reorganization of water involved in the scar maturation of periand infarct zones post-MI.

Macrophages exert potent effector functions against invading microorganisms but constitute, paradoxically, a preferential niche for many bacterial strains to replicate. Using a model of infection by Salmonella Typhimurium, we have identified a molecular mechanism regulated by the nuclear receptor LXR that limits infection of host macrophages through transcriptional activation of the multifunctional enzyme CD38. LXR agonists reduced the intracellular levels of NAD+ in a CD38-dependent manner, counteracting pathogen-induced changes in macrophage morphology and the distribution of the F-actin cytoskeleton and reducing the capability of non-opsonized Salmonella to infect macrophages. Remarkably, pharmacological treatment with an LXR agonist ameliorated clinical signs associated with Salmonella infection in vivo, and these effects were dependent on CD38 expression in bone-marrow-derived cells. Altogether, this work reveals an unappreciated role for CD38 in bacterial-host cell interaction that can be pharmacologically exploited by activation of the LXR pathway.

Electrostimulation of the neural system in functional or repair therapies requires new materials that protect the living system from electric field (EF) effects at the interface. Intercalation materials offer an alternative to radical formation during stimulation. Furthermore, nanostructuring of the electroactive material used as electrode offers an enlargement of the charge capacity which in turn involves changes in the EF effect. In this work, electric field stimulation of cortical neuron cultures has been applied in an in vitro model of lesion, namely, a physical scratch in the cell culture creates a cell-free area reminiscent of a lesion where new neurites grow. Regeneration of the “wound” zone upon EF stimulation is observed for various types of electrode materials, and compared to the spontaneous process and to platinum electrodes. Significantly, electric field effects are highly dependent on the electrode material used, even for the same charge delivered and similar impedance values. Electrode coatings with large charge storage capacity yield significantly better results than that of bare Pt electrodes. Neurite outgrowth at the scratched “wound” zone is lowest, below spontaneous regeneration, when using Pt electrodes. On the other hand, electroactive materials, such as bilayers of PEDOT and polypyrrole with lysine counterions or iridium oxide-pristine graphene hybrids, promote further regeneration. Beyond impedance considerations, the optimal material is the nanostructured one with the largest charge capacity, even at low charge deliveries. It is remarkable that IrOx-graphene hybrids reach regenerations above spontaneous case in very short stimulation times, for equal charge deliveries and potential protocols. The implications from the results suggest that EF application using these new coatings, may have an immediate use in safer electrostimulation procedures, and open routes for much needed neural repair.

The retina of lower vertebrates grows continuously by integrating new neurons generated from progenitors in the ciliary margin zone (CMZ). Whether the mammalian CMZ provides the neural retina with retinal cells is controversial. Live imaging of embryonic retina expressing eGFP in the CMZ shows that cells migrate laterally from the CMZ to the neural retina where differentiated retinal ganglion cells (RGCs) reside. Because Cyclin D2, a cell-cycle regulator, is enriched in ventral CMZ, we analyzed Cyclin D2−/− mice to test whether the CMZ is a source of retinal cells. Neurogenesis is diminished in Cyclin D2 mutants, leading to a reduction of RGCs in the ventral retina. In line with these findings, in the albino retina, the decreased production of ipsilateral RGCs is correlated with fewer Cyclin D2+ cells. Together, these results implicate the mammalian CMZ as a neurogenic site that produces RGCs and whose proper generation depends on Cyclin D2 activity.

Background Prognostic biomarkers are needed to improve the management of the heart failure (HF) epidemic, being the brain natriuretic peptides the most valuable. Here we evaluate 3 biomarkers, high sensitivity troponin T (hs-TnT), galectin-3 (Gal-3) and C-terminal propeptide of type I procollagen (CICP), compare them with a recently described new candidate (sAXL), and analyze their relationship with BNP. Methods HF patients with reduced ejection fraction (n = 192) were included in this prospective observational study, with measurements of candidate biomarkers, functional, clinical and echocardiographic variables. A Cox regression model was used to determine predictors for clinical events, i.e. all-cause mortality and heart transplantation. Results Hs-TnT circulating values were correlated to clinical characteristics indicative of more advanced HF. When analyzing the event-free survival at a mean follow-up of 3.6 years, patients in the higher quartile of either BNP, hs-TnT, CICP and sAXL had increased risk of suffering a clinical event, but not Gal-3. Combination of high sAXL and BNP values had greater predictive value (HR 6.8) than high BNP alone (HR 4.9). In a multivariate Cox regression analysis, BNP, sAXL and NYHA class were independent risk factors for clinical events. Conclusions In this HF cohort, hs-TnT is a good HF marker and has a very significant prognostic value. The prognostic value of CICP and sAXL was of less significance. However, hs-TnT did not add predictive value to BNP, while sAXL did. This suggests that elevated troponin has a common origin with BNP, while sAXL could represent an independent pathological mechanism.

© 2014 Elsevier Ltd. All rights reserved. Hybrid materials based on iridium oxide and graphene oxide, and graphite oxide as reference, are reported as biocompatible electrodes for the neural system. An electrodeposition process based on dynamic potential sweeps, allows an optimal adherence of coatings to the platinum substrate. The resulting electrochemical properties evidence an order of magnitude increase in charge capacity with respect to iridium oxide, and a quasi reversible cyclability for the electrode that expands to more than a thousand cycles for the graphene oxide case. Such stability upon cycling is also an order of magnitude larger than that of IrOx. Neuronal cell cultures show full biocompatibility even in absence of growth factors, and sustain near 100% survival rates and optimal development of neurites. Together with previously reported hybrid IrOx-carbon nanotubes, IrOx-graphene oxide hybrids open the field of materials to be used in the neural system.

The need to enhance charge capacity in neural stimulation-electrodes is promoting the formation of new materials and coatings. Among all the possible types of graphene, pristine graphene prepared by graphite electrochemical exfoliation, is used in this work to form a new nanostructured IrOx-graphene hybrid (IrOx-eG). Graphene is stabilized in suspension by IrOx nanoparticles without surfactants. Anodic electrodeposition results in coatings with much smaller roughness than IrOx-graphene oxide. Exfoliated pristine graphene (eG), does not electrodeposit in absence of iridium, but IrOx-nanoparticle adhesion on graphene flakes drives the process. IrOx-eG has a significantly different electronic state than graphene oxide, and different coordination for carbon. Electron diffraction shows the reflection features expected for graphene. IrOx 1-2 nm cluster/nanoparticles are oxohydroxo-species and adhere to 10 nm graphene platelets. eG induces charge storage capacity values five times larger than in pure IrOx, and if calculated per carbon atom, this enhancement is one order magnitude larger than the induced by graphene oxide. IrOx-eG coatings show optimal in vitro neural cell viability and function as cell culture substrates. The fully straightforward electrochemical exfoliation and electrodeposition constitutes a step towards the application of graphene in biomedical systems, expanding the knowledge of pristine graphene vs. graphene oxide, in bioelectrodes. © 2015 Elsevier B.V.

The tubulin alpha 4a (TUBA4A) gene has been recently associated with amyotrophic lateral sclerosis. Interestingly, some of the mutation carriers were also diagnosed with frontotemporal degeneration (FTD) or mild cognitive impairment. With the aim to investigate the role of TUBA4A in FTD, we screened TUBA4A in a series of 814 FTD patients from Spain. Our data did not disclose any nonsense or missense variant in the cohort, thus suggesting that TUBA4A mutations are not associated with FTD., The tubulin alpha 4a (TUBA4A) gene has been recently associated with amyotrophic lateral sclerosis. Interestingly, some of the mutation carriers were also diagnosed with frontotemporal degeneration (FTD) or mild cognitive impairment. With the aim to investigate the role of TUBA4A in FTD, we screened TUBA4A in a series of 814 FTD patients from Spain. Our data did not disclose any nonsense or missense variant in the cohort, thus suggesting that TUBA4A mutations are not associated with FTD.

© 2015 European Association for the Study of the Liver. Background & Aims Liver fibrosis, an important health concern associated to chronic liver injury that provides a permissive environment for cancer development, is characterized by accumulation of extracellular matrix components mainly derived from activated hepatic stellate cells (HSCs). Axl, a receptor tyrosine kinase and its ligand Gas6, are involved in cell differentiation, immune response and carcinogenesis. Methods HSCs were obtained from WT and Axl-/- mice, treated with recombinant Gas6 protein (rGas6), Axl siRNAs or the Axl inhibitor BGB324, and analyzed by western blot and real-time PCR. Experimental fibrosis was studied in CCl4-treated WT and Axl-/- mice, and in combination with Axl inhibitor. Gas6 and Axl serum levels were measured in alcoholic liver disease (ALD) and hepatitis C virus (HCV) patients. Results In primary mouse HSCs, Gas6 and Axl levels paralleled HSC activation. rGas6 phosphorylated Axl and AKT prior to HSC phenotypic changes, while Axl siRNA silencing reduced HSC activation. Moreover, BGB324 blocked Axl/AKT phosphorylation and diminished HSC activation. In addition, Axl-/- mice displayed decreased HSC activation in vitro and liver fibrogenesis after chronic damage by CCl4 administration. Similarly, BGB324 reduced collagen deposition and CCl4-induced liver fibrosis in mice. Importantly, Gas6 and Axl serum levels increased in ALD and HCV patients, inversely correlating with liver functionality. Conclusions The Gas6/Axl axis is required for full HSC activation. Gas6 and Axl serum levels increase in parallel to chronic liver disease progression. Axl targeting may be a therapeutic strategy for liver fibrosis management.

Much effort is currently devoted to implementing new materials in electrodes that will be used in the central nervous system, either for functional electrostimulation or for tests on nerve regeneration. Their main aim is to improve the charge capacity of the electrodes, while preventing damaging secondary reactions, such as peroxide formation, occurring while applying the electric field. Thus, hybrids may represent a new generation of materials. Two novel hybrid materials are synthesized using three known biocompatible materials tested in the neural system: polypyrrole (PPy), poly(3,4- ethylenedioxythiophene) (PEDOT) and iridium oxide (IrO2). In particular, PPy-IrO2 and PEDOT-IrO2 hybrid nanocomposite materials are prepared by chemical polymerization in hydrothermal conditions, using IrO2 as oxidizing agent. The reaction yields a significant ordered new hybrid where the conducting polymer is formed around the IrO 2 nanoparticles, encapsulating them. Scanning electron microscopy and backscattering techniques show the extent of the encapsulation. Both X-ray photoelectron and Fourier transform infrared spectroscopies identify the components of the phases, as well as the absence of impurities. Electrochemical properties of the final phases in powder and pellet form are evaluated by cyclic voltammetry. Biocompatibility is tested with MTT toxicity tests using primary cultures of cortical neurons grown in vitro for 6 and 9 days. © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Postmenopausal women may be more vulnerable to cognitive loss and Alzheimer's disease (AD) than premenopausal women because of their deficiency in estrogens, in addition to their usually older age. Aerobic physical exercise has been proposed as a therapeutic approach for maintaining health and well-being in postmenopausal women, and for improving brain health and plasticity in populations at high risk for AD. To study the neuroprotective mechanisms of physical exercise in a postmenopausal animal model, we submitted previously ovariectomized, six-month old non-transgenic and 3xTg-AD mice to three months of voluntary exercise in a running wheel. At nine months of age, we observed lower grip strength and some exacerbation of the behavioral and psychological symptoms of dementia (BPSD)-like involving active exploratory activities. A similar major cognitive impairment was observed of ovariectomized 3xTg-AD mice in comparison with sham-operated 3xTg-AD mice. A reduction of bodily fitness and lack of retention of memory were observed in the ovariectomized non-transgenic mice. Physical exercise protected against all deleterious behaviors and normalized learning and memory. It also protected against body frailty, as expected. Analyses of hippocampal key markers of antioxidant and neuroplasticity signaling pathways, showed that ovariectomy impairs the activation of CREB through physical exercise. Furthermore, molecular and behavioral correlates suggested a central role of BDNF in the neuroprotection mediated by physical exercise therapy against apathy and memory loss induced by ovariectomy and the AD-genotype. © 2014 Elsevier Ltd.

© 2014 Acta Materialia Inc. Nanostructured iridium oxide-carbon nanotube hybrids (IrOx-CNT) deposited as thin films by dynamic electrochemical methods are suggested as novel materials for neural electrodes. Single-walled carbon nanotubes (SWCNT) serve as scaffolds for growing the oxide, yielding a tridimensional structure with improved physical, chemical and electrical properties, in addition to high biocompatibility. In biological environments, SWCNT encapsulation by IrOx makes more resistant electrodes and prevents the nanotube release to the media, preventing cellular toxicity. Chemical, electrochemical, structural and surface characterization of the hybrids has been accomplished. The high performance of the material in electrochemical measurements and the significant increase in cathodal charge storage capacity obtained for the hybrid in comparison with bare IrOx represent a significant advance in electric field application in biosystems, while its cyclability is also an order of magnitude greater than pure IrOx. Moreover, experiments using in vitro neuronal cultures suggest high biocompatibility for IrOx-CNT coatings and full functionality of neurons, validating this material for use in neural electrodes.