Glycolysis regulator PFKP induces human melanoma cell proliferation and tumor growth

Purpose Cutaneous melanoma is an aggressive and deadly cancer resulting from malignant transformation of cells involved in skin pigmentation. Glycolysis is widely implicated in cancer progression, but its precise role in melanoma has not been extensively studied. Here, we investigated the role of the glycolysis regulator phosphofructokinase 1 platelet isoform (PFKP) in melanoma progression. Methods PFKP expression in human melanoma tissues was analyzed by immunohistochemistry. Knockdown of PFKP by siRNA and overexpression of PFKP were performed to evaluate its functions in vitro. CCK-8 assay was used to assess cell proliferation. Glycolytic activity was determined via measurement of extracellular acidification rate (ECAR), lactic acid level, and ATP content. A tumor xenograft model was used to test the function of PFKP in vivo. Results PFKP upregulation was observed in human melanoma tissues and correlated with poor patient survival. Knockdown of PFKP in human melanoma cells suppressed cell proliferation and reduced ECAR, ATP levels, and lactic acid levels, while overexpression of PFKP displayed the opposite effects. In vivo, knockdown of PFKP in melanoma cells markedly reduced tumorigenesis. Inhibitory effects on cell proliferation, glycolysis, and tumorigenesis due to PFKP knockdown were further augmented upon treatment with the glycolysis inhibitor 2-deoxy-D-glucose (2-DG). Conclusion Collectively, these results indicate that PFKP expression in melanoma cells increases proliferation and glycolytic activity in vitro and promotes tumorigenesis in vivo, suggesting that suppression of PKFP and inhibition of glycolysis may potently suppress melanoma progression (AU)

Rutin ameliorates inflammatory pain by inhibiting P2X7 receptor in mast cells

Rutin is a natural anti-inflammatory ingredient widely found in medicinal plants. Studies have shown that rutin inhibits mast cell degranulation and the release of inflammatory mediators. Mast cell P2X7 receptor mediates mast cell degranulation and serves as a therapeutic target for inflammatory pain. Herein, the aim of this study was to investigate whether the anti-inflammatory mechanism of rutin is related to the mast cell P2X7 receptor. Our results showed that rutin could inhibit [Ca2+]i elevation induced by 5 mM ATP or 30 μM BZATP in a concentration-dependent manner in mouse peritoneal mast cells. Rutin also suppressed the inward current mediated by P2X7 receptor. In vivo, rutin could significantly inhibit the mechanical hypersensitivity induced by 100 mM ATP that is associated with P2X7 receptor in mast cells. Moreover, molecular docking revealed the high affinity between rutin and the P2X7 receptor crystal structure. Collectively, this study demonstrated that rutin attenuated inflammatory pain by inhibiting the activity of P2X7 receptor in mast cells. (AU)

P2X and P2Y Receptor Antagonists Reduce Inflammation in ATPinduced Microglia

Background: P2 receptors have been implicated in the release of neurotransmitter and pro-inflammatory cytokines due to their response to neuroexcitatory substances in the microglia. The P2X4, P2X7 and P2Y12 receptors are involved in the development of pain behavior induced by peripheral nerve injury. However, it is not known if blocking P2X4, P2X7 and P2Y12 receptors is associated with the expression and the release of interleukin-1B (IL-1β), interleukin-6 (IL-6), or tumor necrosis factor-α (TNF-α) in cultured neonatal spinal cord microglia. Objective: For this reason, we examined the effects of P2X4, P2X7 and P2Y12 antagonists on the expression and the release of IL-1β, IL-6, and TNF-α in ATP-stimulated microglia. Methods: In this study, we observed the effect of A-740003, PSB-12062 and MRS 2395 (P2X4, P2X7 and P2Y12 receptors antagonist, respectively), on the expression and release of IL-1β, IL-6 and TNF-α by using real-time fluorescence quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Results: ATP induced the increased expression of IL-1β, IL-6 and TNF-α at the level of messenger RNA (mRNA). ATP-evoked increase in IL-1β, IL-6 and TNF-α mRNA expression was inhibited by the P2X4 receptor antagonist A-740003 or P2X7 receptor antagonist PSB-12062, respectively. Similarly, ATP-evoked release of IL-1β, IL-6 and TNF-α was inhibited by A-740003 and PSB-12062. Furthermore, ATP-evoked increased expression of Iba-1, IL-1β, IL-6 and TNF-α mRNA, and release of IL-1β, IL-6 and TNF-α were nearly all blocked after co-administration of A-740003 plus PSB-12062. Finally, ATP-evoked increased gene expression and release of IL-1β, IL-6 and TNF-α were also inhibited by MRS 2395 (P2Y12 antagonist). Conclusion: These observations suggest a new clue for therapeutic strategies to treat the neuro-inflammation. (AU)

Biofilms en instalaciones alimentarias militares. Comparación de técnicas rápidas de vigilancia y verificación / Biofilms in military food facilities. Comparison of rapid surveillance and verification techniques

Introducción:Los patógenos formadores de biofilms incrementan el riesgo de contaminación biológica de los alimentos en las industrias alimentarias, por lo que es necesario cuantificar y cualificar los peligros presentes en los mismos.Objetivo:Se pretende demostrar la presencia de biofilms bacterianos en las superficies de trabajo de instalaciones alimentarias militares (IAM) mediante distintas técnicas rápidas de control higiénico.Material y métodos:Se analizaron un total de 550 muestras entre las 10 IAM: Cada IAM contó con 5 puntos de muestreo (n = 50) y en cada uno de ellos se obtuvieron 11 muestras sobre una superficie de 100 cm2, para poner de manifiesto la presencia de biofilms y establecer la correlación entre las siguientes técnicas analíticas: determinación de adenosín trifosfato (ATP) mediante luminómetro 3M Clean-Trace™ NG3, valoración de residuos proteicos con hisopos Clean-Trace™ Surface Protein Plus, recuentos de microorganismos aerobios mesófilos (AC) mediante sistema TEMPO® y Petrifilm™, y visualización de biofilms mediante reacción enzimática con Biofinder®.Resultados:Se encontró correlación estadísticamente significativa (p < 0,05) entre las determinaciones de ATP y la presencia de biofilms y de residuos proteicos. También se halló linealidad con significación estadística (R2 0,422, p < 0,05) entre el ATP y el recuento de AC por TEMPO®. Se observó correlación y concordancia entre los recuentos de AC mediante el sistema TEMPO® y a través de Petrifilm™ (coeficiente de correlación intraclase 0,463, IC 95%: 0,179-0,673, p < 0,05). En ningún punto de muestreo (PM) se detectaron bacterias patógenas.Conclusión:Se detectaron biofilms en una tercera parte de los PM inspeccionados. Su monitorización combinando técnicas rápidas debe tenerse en consideración en los procedimientos de vigilancia del sistema de Análisis de Peligros y Puntos de Control Crítico (APPCC). (AU)

Introduction:Biofilm-forming pathogens increase the risk of biological food contamination in food industries, thus it is necessary to quantify and qualify the dangers present in them.Objective:To demonstrate the presence of bacterial biofilms on the work surfaces of military food facilities using different rapid hygienic control techniques.Materials and methods:An amount of 550 samples were analyzed among 10 military food facilities: Each one had 5 sampling points (n = 50) and in each of them 11 samples were obtainded on a surface of 100 cm2 to assess the presence of biofilms and to establish the correlation between different analytical techniques: determination of adenosin triphosphate (ATP) using 3M Clean-Trace ™ NG3 luminometer, protein residue titration with Clean-Trace ™ Surface Protein Plus swabs, mesophilic aerobic microorganism (AC) counts using TEMPO® and Petrifilm ™ system, and visualization of biofilms using enzymatic reaction with Biofinder®.Results:A statistically significant correlation (p <0.05) was found between ATP determinations and the presence of biofilms and protein residues. Linearity with statistical significance (R2 0.422, p <0.05) was also found between ATP and AC count by TEMPO®. Correlation and concordance between AC counts were observed using the TEMPO® system and through Petrifilm ™ (intraclass correlation coefficient 0.463, 95% CI: 0.179-0.673, p <0.05). At no sampling point (SP) were pathogenic bacteria detected.Conclusion:Combining quick techniques should be considered in surveillance procedures of Hazard Analysis and Critical Control Points (HACCP). (AU)

El ácido bempedoico como activador PPAR alfa: Nuevas perspectivas para el tratamiento de la esteatosis hepática en un modelo experimental de rata hembra / Bempedoic acid as a PPAR alfa activator: new perspectives for hepatic steatosis treatment in a female rat experimental model

INTRODUCCIÓN: La enfermedad del hígado graso no alcohólico cursa, en sus fases iniciales, con hipertrigliceridemia y acúmulo de lípidos en el hígado (esteatosis hepática). El ácido bempedoico es un inhibidor de la ATP:citrato liasa que promueve una inhibición dual de la síntesis de colesterol y ácidos grasos. Sin embargo, no se ha investigado su efecto en la prevención/tratamiento de la esteatosis hepática y la hipertrigliceridemia. El objetivo de nuestro trabajo ha sido elucidar si el ácido bempedoico, mediante un mecanismo diferente/alternativo a la inhibición de la ATP:citrato liasa, revierte estas alteraciones metabólicas. DISEÑO EXPERIMENTAL: El estudio se realizó con un modelo animal de rata Sprague-Dawley hembra alimentada, durante 3 meses, con una dieta rica en grasa saturada suplementada con fructosa al 10% (p/v) en el agua de bebida. Se administró, durante el último mes, ácido bempedoico (30mg/kg/día) a un grupo de animales. Se analizaron parámetros zoométricos, se realizaron valoraciones plasmáticas, de expresión génica y proteica en muestras de hígado y se determinó la actividad de unión PPAR-PPRE. RESULTADOS: Nuestro modelo de intervención dietética desarrolló esteatosis hepática e hipertrigliceridemia. A pesar de un aumento en la ingesta calórica total, no se observó un incremento de peso corporal de los animales. La administración de ácido bempedoico redujo significativamente la esteatosis hepática y promovió una marcada hipertrofia de los hepatocitos. Se observó un incremento del 66% en el peso del hígado de los animales tratados con el fármaco, que no se acompañó de modificaciones en los marcadores de inflamación, estrés oxidativo o estrés de retículo endoplasmático. El ácido bempedoico activó el receptor nuclear activado por proliferadores peroxisómicos (PPARα) y sus genes diana.

INTRODUCTION: In its initial stages, nonalcoholic fatty liver disease presents hypertriglyceridemia and accumulation of lipids in the liver (hepatic steatosis). Bempedoic acid is an ATP:citrate lyase inhibitor that promotes a dual inhibition of the synthesis of cholesterol and fatty acids. However, its effect in the prevention / treatment of hepatic steatosis and hypertriglyceridemia has not been investigated. The aim of our work has been to elucidate whether bempedoic acid, through a mechanism other than ATP:citrate lyase inhibition, reverses these metabolic alterations. EXPERIMENTAL DESIGN: The study was carried out in female Sprague-Dawley rats fed, for three months, with a high fat diet supplemented with fructose (10% w/v) in drinking water. During the last month, bempedoic acid (30mg/kg/day) was administered to a group of animals. Zoometric and plasmatic parameters were analyzed, gene and protein expression analysis were performed in liver samples and PPAR-PPRE binding activity was determined. RESULTS: Our interventional model developed hepatic steatosis and hypertriglyceridemia. Despite an increase in total caloric intake, there was no increase in body weight of the animals. The administration of bempedoic acid significantly reduced hepatic steatosis and promoted a marked hepatocyte hypertrophy. There was a 66% increase in the liver weight of the animals treated with the drug that was not accompanied by modifications in the markers of inflammation, oxidative stress, or endoplasmic reticulum stress. Bempedoic acid activated the peroxisome proliferator activated nuclear receptor (PPARα) and its target genes.

Predictive Value of Immune Cell Functional Assay for Non-Cytomegalovirus Infection in Lung Transplant Recipients: A Multicenter Prospective Observational Study / Valor predictivo del test de función de la inmunidad celular para infecciones distintas a CMV en receptores de un trasplante pulmonar: un estudio multicéntrico, prospectivo y observacional

Introduction: Immune cell functional assay (ImmuKnow®) is a non-invasive method that measures the state of cellular immunity in immunosuppressed patients. We studied the prognostic value of the assay for predicting non-cytomegalovirus (CMV) infections in lung transplant recipients. Methods: A multicenter prospective observational study of 92 patients followed up from 6 to 12 months after transplantation was performed. Immune cell functional assay was carried out at 6, 8, 10, and 12 months. Results: Twenty-three patients (25%) developed 29 non-CMV infections between 6 and 12 months post-transplant. At 6 months, the immune response was moderate (ATP 225–525ng/mL) in 14 (15.2%) patients and low (ATP<225ng/mL) in 78 (84.8%); no patients had a strong response (ATP≥525ng/mL). Only 1 of 14 (7.1%) patients with a moderate response developed non-CMV infection in the following 6 months compared with 22 of 78 (28.2%) patients with low response, indicating sensitivity of 95.7%, specificity of 18.8%, positive predictive value (PPV) of 28.2%, and negative predictive value (NPV) of 92.9% (AUC 0.64; p=0.043). Similar acute rejection rates were recorded in patients with mean ATP≥225 vs. <225ng/mL during the study period (7.1% vs. 9.1%, p=0.81). Conclusion: Although ImmuKnow® does not seem useful to predict non-CMV infection, it could identify patients with a very low risk and help us define a target for an optimal immunosuppression. (AU)

Introducción: El test de función de la inmunidad celular (ImmuKnow®) es un método que mide el estado de la inmunidad celular en pacientes inmunosuprimidos. Se estudió su valor pronóstico para predecir infecciones diferentes a citomegalovirus (CMV) en receptores de un trasplante pulmonar. Métodos: Se realizó un estudio observacional prospectivo multicéntrico de 92 pacientes seguidos desde los 6 a los 12 meses postrasplante. El test se realizó a los 6, 8, 10 y 12 meses. Resultados: Veintitrés pacientes (25%) desarrollaron 29 infecciones no debidas a CMV entre los 6 y los 12 meses posteriores al trasplante. A los 6 meses, la respuesta inmune fue moderada (ATP 225-525ng/ml) en 14 (15,2%) pacientes y baja (ATP<225ng/ml) en 78 (84,8%); ningún paciente tuvo una respuesta fuerte (ATP>525ng/ml). Solo uno de 14 (7,1%) pacientes con una respuesta moderada desarrolló una infección diferente a CMV en los 6 meses siguientes a la realización del test en comparación con 22 de 78 (28,2%) con respuesta baja, indicando una sensibilidad del 95,7%, una especificidad del 18,8%, un valor predictivo positivo del 28,2% y un valor predictivo negativo del 92,9% (AUC 0,64; p=0,043). Se registraron tasas de rechazo agudo similares en pacientes con ATP medio >225 frente a <225ng/ml durante el período de estudio (7,1 frente al 9,1%; p=0,81). Conclusión: Aunque el test ImmuKnow® no parece útil para predecir infecciones diferentes al CMV, podría identificar pacientes con riesgo muy bajo y ayudarnos a definir un objetivo de inmunosupresión óptima. (AU)

Remdesivir, la esperanza antiviral frente al SARS-CoV-2 / Remdesivir, the antiviral hope against SARS-CoV-2

El 31 de diciembre de 2019 se detectó en la ciudad de Wuhan (China) un brote neumonía causado por un nuevo coronavirus (SARS-CoV-2). Debido a la elevada capacidad de difusión e infección humana se ha convertido en una nueva pandemia zoónotica. La ausencia de una vacuna ha determinado la búsqueda de fármacos antivirales con capacidad para inhibir la replicación del nuevo virus. De entre ellos, remdesivir, un análogode la adenosina, es el que parece tener un futuro más prometedor. Este fármaco ha mostrado in vitro y en animales una elevada capacidad para bloquear la infección y replicación viralcon unas concentraciones alcanzables en el plasma humano.Aunque todos los estudios se han realizado con el SARS-CoV y el MERS-CoV, parece que por analogía virológica y funcional, remdesivir es de los pocos antivirales con demostrada eficacia. Sin embargo, se precisan estudios y ensayos clínicos en humanospara conocer el resultado de su aplicación en los mismos

On December 31, 2019 a pneumonia outbreak caused by a new coronavirus (SARS-CoV-2) was detected in the city of Wuhan (China). Due to the high capacity of diffusion and human infection it has become a new zoonotic pandemic. The absence of a vaccine has determined the search for antiviral drugs with the capacity to inhibit the replication of the new virus. Among them, remdesivir, an analogue of adenosine, is what seems to have a more promising future. This drug has shown in vitro and in animals a high capacity to block infection and viral replication with attainable concentrations in human plasma. Although all studies have been carried out with SARS-CoV and MERS-CoV, it seems that by virological and functional analogy, remdesivir is one of the few antiviral drugs with proven efficacy.However, studies and clinical trials in humans are required to know the result of their application in them

Interacciones neuroinmunológicas en el ictus / Neuroimmunological interactions in stroke

Introducción: El ictus es una de las principales causas de mortalidad en el mundo y debido al incremento en la expectativa de vida su incidencia va en aumento; sin embargo, el desarrollo de nuevos medicamentos con utilidad clínica ha sido prácticamente nulo, por lo que hasta la fecha el tratamiento de estos pacientes es muy limitado. Desarrollo: La evidencia básica y clínica en el área señala que tras un infarto cerebral se producen una serie de cambios neuroquímicos, entre los que se encuentran: la depleción energética, la producción de radicales libres, la acumulación de calcio, la desregulación de neurotransmisores, la excitotoxicidad, y de manera tardía, la activación del sistema inmune caracterizada como inflamación. Esta respuesta del sistema inmunológico ha mostrado ser un evento central en la progresión de la patología, en el que destaca la participación de las citocinas proinflamatorias como TNF, que aumentan el daño por excitotoxicidad y por acumulación de calcio, favorecen la formación de radicales libres y en general promueven la muerte celular. Por otro lado, algunas citocinas antiinflamatorias como IL-10 e IL-4 han mostrado tener efectos neuroprotectores e incluso favorecen la recuperación de sinapsis y la neurogénesis, haciendo de la modulación de la respuesta inmunológica un área con mucho potencial terapéutico. Conclusiones: El entendimiento de las relaciones entre el sistema inmunológico y el sistema nervioso no solo nos permite entender con mayor profundidad el fenómeno del ictus, sino que también nos ofrece un nuevo arsenal de estrategias diagnósticas, pronósticas y terapéuticas que podrían mejorar la calidad de vida de las personas aquejadas por esta terrible enfermedad

Introduction: Stroke is one of the leading causes of death in the world; its incidence is increasing due to increased life expectancy. However, treatment options for these patients are limited since no clinically effective drugs have been developed to date. Development: According to clinical evidence, a number of neurochemical changes take place after stroke, including energy depletion, increased free radical synthesis, calcium accumulation, neurotransmitter imbalance, excitotoxicity, and, at a later stage, immune system activation leading to inflammation. Immune response has been shown to be a major factor in disease progression. The release of proinflammatory cytokines such as TNF increase brain damage secondary to excitotoxicity and calcium accumulation, and promote free radical synthesis and cell death through various mechanisms. On the other hand, certain anti-inflammatory cytokines, such as IL-10 and IL-4, have been shown to have a neuroprotective effect and even promote neurogenesis and synapse remodeling, which makes immune modulation a promising treatment approach. Conclusions: Understanding the relationship between the immune system and the nervous system not only deepens our knowledge of stroke but also provides new diagnostic, prognostic, and therapeutic strategies that may increase the quality of life of stroke patients

Role of sodium-hydrogen exchanger isoform 1 in regulating hepatocyte apoptosis induced by hyperammonaemia / Papel de la isoforma 1 del intercambiador iónico de sodio/hidrógeno en la regulación de la apoptosis de hepatocitos inducida por hiperamoniaquemia

Background: The "secondary injury" theory of liver failure indicated that hyperammonaemia due to liver failure causes further deterioration of hepatocytes. Our previous studies have demonstrated that high blood ammonia levels may lead to hepatocyte apoptosis, as NH4Cl loading caused metabolic acidosis and an increase in sodium-hydrogen exchanger isoform 1 (NHE1). In this study, we established a hyperammonia hepatocyte model to determine the role of NHE1 in the regulation of hepatocyte apoptosis induced by NH4Cl. Materials and methods: In current studies, intracellular pH (pHi) and NHE1 activity were analyzed using the pHi-sensitive dye BCECF-AM. The results showed that intracellular pH dropped and NHE1 activity increased in hepatocytes under NH4Cl treatment. As expected, decreased pHi induced by NH4Cl was associated with increased apoptosis, low cell proliferation and ATP depletion, which was exacerbated by exposure to the NHE1 inhibitor cariporide. We also found that NH4Cl treatment stimulated PI3K and Akt phosphorylation and this effect was considerably reduced by NHE1 inhibition. Conclusion: This study highlighted the significant role of NHE1 in the regulation of cell apoptosis induced by hyperammonaemia

Antecedentes: La teoría de la «lesión secundaria» de la insuficiencia hepática mostró que la hiperamoniaquemia provocada por la insuficiencia hepática causa mayor deterioro de los hepatocitos. Nuestros anteriores estudios previos han demostrado que los niveles altos de amoníaco en sangre pueden conducir a la apoptosis de los hepatocitos. Como la carga de NH4Cl provocó acidosis metabólica y un aumento de la isoforma 1 del intercambiador de sodio/hidrógeno (NHE1). En este estudio, establecimos un modelo de hepatocitos de hiperamonia para establecer el papel de NHE1 en la regulación de la apoptosis de hepatocitos inducida por NH4Cl. Materiales y métodos: En los estudios actuales, el pH intracelular (pHi) y la actividad del NHE1 se analizaron con el colorante BCECF-AM, sensible al pHi. Los resultados mostraron que el pH intracelular disminuyó y la actividad del NHE1 aumentó en hepatocitos con tratamiento del NH4Cl. Como se esperaba, la disminución del pHi inducido por NH4Cl se relacionó con un aumento de la apoptosis, baja proliferación celular y reducción del ATP, que se exacerbó por la exposición a cariporide, inhibidor del NHE1. También encontramos que el tratamiento del NH4Cl estimuló la fosforilación de PI3K y Akt, y este efecto se redujo considerablemente por la inhibición del NHE1. Conclusión: Este trabajo ha destacado el importante papel del NHE1 en la regulación de la apoptosis celular inducida por hiperamoniaquemia

Protective effects of mitochondrion-targeted peptide SS-31 against hind limb ischemia-reperfusion injury

Hind limb ischemia-reperfusion injury is an important pathology in vascular surgery. Reactive oxygen species are thought to be involved in the pathogenesis of hind limb ischemia-reperfusion injury. SS-31, which belongs to a family of mitochondrion-targeted peptide antioxidants, was shown to reduce mitochondrial reactive oxygen species production. In this study, we investigated whether the treatment of SS-31 could protect hind limb from ischemia-reperfusion injury in a mouse model. The results showed that SS-31 treatment either before or after ischemia exhibited similar protective effects. Histopathologically, SS-31 treatment prevented the IR-induced histological deterioration compared with the corresponding vehicle control. SS-31 treatment diminished oxidative stress revealed by the reduced malondialdehyde level and increased activities and protein levels of Sod and catalase. Cellular ATP contents and mitochondrial membrane potential increased and the level of cytosolic cytC was decreased after SS-31 treatment in this IR model, demonstrating that mitochondria were protected. The IR-induced increase of levels of inflammatory factors, such as Tnf-α and Il-1β, was prevented by SS-31 treatment. In agreement with the reduced cytosolic cytC, cleaved-caspase 3 was kept at a very low level after SS-31 treatment. Overall, the effect of SS-31 treatment before ischemia is mildly more effective than that after ischemia. In conclusion, our results demonstrate that SS-31 confers a protective effect in the mouse model of hind limb ischemia-reperfusion injury preventatively and therapeutically

Reducing mitochondrial bound hexokinase II mediates transition from non-injurious into injurious ischemia/reperfusion of the intact heart

Ischemia/reperfusion (I/R) of the heart becomes injurious when duration of the ischemic insult exceeds a certain threshold (approximately ≥20 min). Mitochondrial bound hexokinase II (mtHKII) protects against I/R injury, with the amount ofmtHKII correlating with injury. Here, we examine whether mtHKII can induce the transition from non-injurious to injurious I/R, by detaching HKII from mitochondria during a non-injurious I/R interva l . Additionally, we examine possible underlying mechanisms (increased reactive oxygen species (ROS), increased oxygen consumption (MVO2) and decreased cardiac energetics) associated with this transition. Langendorff perfused rat hearts were treated for 20 min with saline, TAT-only or 200 nM TAT-HKII, a peptide that translocates HKII from mitochondria. Then, hearts were exposed to non-injurious 15-min ischemia, followed by 30-min reperfusion. I/R injury was determined by necrosis (LDH release) and cardiac mechanical recovery. ROS were measured by DHE fluorescence. Changes in cardiac respiratory activity (cardiac MVO2 and efficiency and mitochondrial oxygen tension (mitoPO2) using protoporphyrin IX) and cardiac energetics (ATP, PCr, ΔGATP) were determined following peptide treatment.When exposed to 15-min ischemia, control hearts had no necrosis and 85% recovery of function. Conversely, TAT-HKII treatment resulted in significant LDH release and reduced cardiac recovery (25%), indicating injurious I/R. This was associated with increased ROS during ischemia and reperfusion. TAT-HKII treatment reducedMVO2 and improved energetics (increased PCr) before ischemia, without affecting MVO2/RPP ratio or mitoPO2. In conclusion, a reduction in mtHKII turns non-injurious I/R into injurious I/R. Loss of mtHKII was associated with increased ROS during ischemia and reperfusion, but not with increased MVO2 or decreased cardiac energetics before damage occurs

Octodon degus, a new model to study the agonist and plexus-induced response in the urinary bladder

Urinary bladder function consists in the storage and controlled voiding of urine. Translational studies require animal models that match human characteristics, such as Octodon degus, a diurnal rodent. This study aims to characterize the contractility of the detrusor muscle and the morphology and code of the vesical plexus from O. degus. Body temperature was measured by an intra-abdominal sensor, the contractility of detrusor strips was evaluated by isometric tension recording, and the vesical plexus was studied by electrical field stimulation (EFS) and immunofluorescence. The animals showed a diurnal chronotype as judged from core temperature. The myogenic contractile response of the detrusor muscle to increasing doses of KCl reached its maximum (31.04 mN/mm2) at 60 mM. In the case of cumulative dose-response of bethanecol, the maximum response (37.42 mN/mm2) was reached at 3.2 × 10-4 M. The response to ATP was clearly smaller (3.8 mN/mm2). The pharmacological dissection of the EFS-induced contraction identified ACh and sensory fibers as the main contributors to this response. The neurons of the vesical plexus were located mainly in the trigone area, grouped in big and small ganglia. Out of them, 48.1 % of the neurons were nitrergic and 62.7 % cholinergic. Our results show functional and morphological similarities between the urinary bladder of O. degus and that of humans (AU)

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ATAD2 is overexpressed in gastric cancer and serves as an independent poor prognostic biomarker

Objective: ATPase family, AAA domain containing 2 (ATAD2) has been found overexpressed in various cancer types and correlated with malignant status and poor prognosis. However, little is known about the clinical significance of ATAD2 in gastric cancer patients. The aim of this study was to explore the clinical and prognostic significance of ATAD2 in gastric cancer. Methods: The mRNA and protein levels expression of ATAD2 were detected in clinical tissue samples by qRTPCR and immunohistochemistry, respectively. We examined the ATAD2 protein expression by immunohistochemistry. Furthermore, we analyzed the association between ATAD2 expression and clinicopathological features including prognosis in 166 gastric cancer samples. Results: In our results, ATAD2 mRNA and protein were highly expressed in gastric cancer samples. ATAD2 overexpression was correlated with advanced clinical stage, tumor depth, lymph node metastasis, and distant metastasis. According to the survival analysis, ATAD2 protein overexpression was a poor independent prognostic factor for gastric cancer patients. Conclusions: In summary, ATAD2 could serve as a prognostic biomarker for gastric cancer patients (AU)

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Activation of p38 in C2C12 myotubes following ATP depletion depends on extracellular glucose

Muscle cells adjust their glucose metabolism in response to myriad stimuli, and particular attention has been paid to glucose metabolism after contraction, ATP depletion, and insulin stimulation. Each of these requires translocation of GLUT4 to the cell membrane, and may require activation of glucose transporters by p38. In contrast, AICAR stimulates glucose transport without activation of p38, suggesting that p38 activation may be an indirect consequence of accelerated glucose transport or metabolism. This study was designed to investigate the contribution of AMPK and p38 to ATP homeostasis and glucose metabolism to test the hypothesis that p38 reflects glycolytic activity rather than controls glucose uptake. Treating mature myotubes with rotenone caused transient ATP depletion in 15 min with recovery by 120 min, associated with increased lactate production. Both ACC and p38 were rapidly phosphorylated, but ACC remained phosphorylated while p38 phosphorylation declined as ATP recovered. AMPK inhibition blocked ATP recovery, lactate production, and phosphorylation of p38 and ACC. Inhibition of p38 had little effect. AICAR induced ACC phosphorylation, but not lactate production or p38 phosphorylation. Finally, removing extracellular glucose potentiated rotenone-induced AMPK activation, but reduced lactate generation, ATP recovery and p38 activation. Thus, glucose metabolism is highly sensitive to ATP homeostasis via AMPK activity, but p38 activity is dispensable. Although p38 is strongly phosphorylated during ATP depletion, this appears to be an indirect consequence of accelerated glycolysis

Utilidad de la medición de adenosina 5'-trifosfato intracelular en células CD4+ en trasplante renal / The usefulness of intracellular adenosine-5'-triphosphate measurement in CD4+ cells in renal transplant

ImmuKnow® es un método diagnóstico in vitro que emplea sangre completa del paciente estimulada policlonalmente con fitohemaglutinina y mide la producción de adenosina 5'-trifosfato (ATP) por las células T CD4+. La prueba tiene como objetivo proporcionar una medida objetiva y global de la respuesta inmunitaria celular de cada individuo. El ensayo se diseñó con la idea de monitorizar la inmunosupresión administrada al paciente trasplantado de forma individualizada intentando ayudar a conseguir el equilibrio para evitar un exceso de inmunosupresión con los efectos adversos que conlleva (infecciones, cáncer, etc.) o un defecto de la inmunosupresión con el consiguiente riesgo de rechazo del aloinjerto. La mayoría de los trabajos que han evaluado su utilidad clínica muestra una gran diversidad en cuanto al modo de reclutamiento de los pacientes, el tratamiento inmunosupresor recibido, las variables clínicas analizadas y, sobre todo, el tiempo entre la realización de ImmuKnow® y el evento clínico evaluado. Los datos más consistentes muestran que este ensayo de función de las células T CD4+ resulta útil para predecir el riesgo de infección en trasplantados renales. Sin embargo, no está claro su empleo como marcador de riesgo de rechazo. Por último, dada la enorme variabilidad de la respuesta inmunitaria entre individuos y las publicaciones existentes, se deduce que un valor aislado de ImmuKnow® no tiene capacidad diagnóstica y solo un seguimiento seriado individualizado ayudaría más definitivamente a tomar decisiones clínicas y de cambios en el tratamiento inmunosupresor. Otros aspectos en relación con la aplicación de ImmuKnow® en la rutina clínica, como por ejemplo la periodicidad de la realización de la prueba, precisan estudios prospectivos aleatorizados para una más completa información (AU)

ImmuKnow® is an in vitro diagnosis method that uses patient samples of whole blood polyclonally stimulated with phytohaemagglutinin. It also measures adenosine-5'-triphosphate (ATP) production by CD4+ T cells. The test aims to offer an objective and overall measurement of each individual's cellular immune response. The assay was designed with the idea of individually monitoring the immunosuppression administered to transplant patients. At the same time, it aims to help achieve a balance as a way of avoiding immunosuppression excess and the associated adverse effects (infections, cancer, etc.) or an immunosuppression defect and the subsequent risk of allograft rejection. The majority of studies that have evaluated its clinical usefulness display great diversity in terms of patient recruitment, the immunosuppressant treatment received, the clinical variables analysed and, above all, the time between performing ImmuKnow® and the evaluated clinical event. The most consistent data show that this assay on CD4+ T cell functioning is useful for predicting the risk of infection in renal transplant patients. However, its use as a rejection risk indicator is unclear. Lastly, given the great variability of immune response amongst individuals and that of existing publications, it can be deduced that the isolated ImmuKnow® value does not have diagnostic capacity and only individual serial monitoring could provide definitive assistance in clinical decision making and immunosuppressant treatment changes. Other aspects of ImmuKnow® application in the clinical routine, such as assay cycles, require randomised prospective studies for more comprehensive information (AU)

Enfermedad de Wilson / Wilson´s disease

La enfermedad de Wilson es un trastorno del metabolismo del cobre que se hereda de forma autosómica recesiva. Está causada por mutaciones en el gen ATP7B que codifica para una ATPasa tipo P implicada en el transporte de cobre dentro del hepatocito, tanto al interior del aparato de Golgi para su incorporación a la apoceruloplasmina como en la excreción biliar del exceso de metal del organismo. El defecto en la función de esta proteína da lugar a la acumulación progresiva de cobre, primero en el hígado y posteriormente en el encéfalo y en otros tejidos, con manifestaciones clínicas principalmente hepáticas, neurológicas, psiquiátricas y oftalmológicas. Actualmente sigue representando un desafío diagnóstico, debido a que es una patología poco común, con manifestaciones clínicas inespecíficas y limitaciones en la exactitud de las diversas pruebas diagnósticas disponibles. El riesgo de que permanezca sin diagnosticar y progrese a muerte, junto con la existencia de un tratamiento eficaz, ponen de manifiesto la importancia de que se realice un diagnóstico correcto y temprano, siendo esencial para ello la aportación del laboratorio clínico (AU)

Wilson's disease is an autosomal recessive disorder of copper metabolism. It is caused by mutations in the ATP7B gene, which encodes a P-type ATPase that functions in the transport of copper inside the hepatocyte, both into the trans-Golgi compartment for incorporation into apo-caeruloplasmin, and into the bile, for excretion of the excess metal. Defective function of this protein leads to progressive copper accumulation, first in the liver but ultimately in the brain and other tissues, with mainly hepatic, neurological, psychiatric and ophthalmologic signs and symptoms. Nowadays, it still represents a diagnostic challenge due to it being an uncommon disease, with unspecific clinical manifestations, and limitations in the accuracy of the available diagnostic tests. The risk that it remains undiagnosed together with the availability of effective treatments stresses the importance of an early and correct diagnosis, with the clinic laboratory playing an essential the role (AU)

The role of iron in tumour cell proliferation

Iron has a pivotal role in homeostasis due to its participation in virtually all of the body's oxidation-reduction processes. However, iron can also be considered a double-edged weapon, as its excess may lead to an increased risk of developing cancer, presumably by the generation of reactive oxygen species, and its role as substrate to enzymes that participate in cell proliferation. Thus, iron might as well be considered a cofactor in tumour cell proliferation. In certain pathological conditions, such as haemochromatosis, hepatitis B and C virus infection, asbestosis and endometriosis, iron overload may increase the risk of cancer. By contrast, iron depletion could be considered a useful adjunct in antitumour therapy. This paper reviews the current scientific evidence behind iron's role as a protumoral agent, and the potential benefit of a state of iron depletion in patients with cancer (AU)

Relación hepatosomática y depósito de hierro en hígado durante la instauración de anemia ferropénica nutricional / Hepatosomatic index and iron liver stores during the instauration of nutritional ferropenic anaemia

El hígado es el principal órgano de almacén de hierro en el organismo y juega un papel crucial en la homeostasis de dicho mineral. Los niveles de hierro en se consideran un fiel reflejo del estatus de hierro en el organismo, hecho que nos indujo a determinar la relación hepatosomática y el contenido de hierro en hígado durante la instauración de la anemia ferropénica nutricional inducida experimentalmente en ratas en crecimiento. Los depósitos de hierro estaban profundamente deplecionados, el peso corporal y el peso hepático fue menor en animales anémicos. Como consecuencia, la relación hepatosomática se incrementó en animales ferrodeficientes. Durante la ferrodeficiencia, varios factores reguladores de la hepcidina se alteran, aumenta la demanda eritropoyética por disminución de los parámetros hematológicos, hay un menor aporte de oxígeno a los tejidos y se deplecionan los depósitos corporales, alterándose el metabolismo de hierro, hechos que conducen a una disminución de dicha hormona, lo cual se traducirá en una menor interacción con la ferroportina 1, evitando su internalización y degradación, de manera que aumenta el flujo de salida de hierro ferroso desde los hepatocitos y consecuentemente se reduce su depósito. Por otra parte, la ferrodeficiencia afectó el peso corporal, hecho que se puede atribuir a los menores niveles de hormonas tiroideas encontrados en esta patología. Puesto que hay una clara reducción de la hemoglobina y recuento de hematíes, el suministro de oxígeno a las células se limita considerablemente e incide negativamente en la síntesis de ATP e incremento de peso(AU)

Liver is main storage organ of iron in the organism and plays a crucial role in the homeostasis of this mineral. The levels of iron are considered a routine index of the iron status in the body, fact that encouraged us to asses the hepatosomatic index and the iron content in liver, during iron-deficiency anaemia in growing rats. In rats with iron-deficiency anaemia, iron deposits were deeply depleted, body weight and hepatic weight were lower, moreover the hepatic iron deposits were lower in anaemicrats. During the iron-deficiency, several regulatory factors of the hepcidin are impaired (the erythropoietic demand increase due to the decrease of the haematological parameters, there is a minor supply of oxygen to the tissues and the body stores are depleted, being the iron metabolism altered), facts that leads to a decrease of the above mentioned hormone, which will be translated in a minor interaction with the ferroportin1, avoiding its internalization and degradation, therefore increases the outflow of ferrous iron from the hepatocytes and consistently its storage diminishes in the above mentioned organ. On the other hand, the iron-deficiency impaired the body weight, fact that can be related with the lower levels of thyroid hormones found in this pathology. Moreover, since in iron deficiency situation the haemoglobin and red blood cells count diminish drastically, the supply of oxygen to the cells limits itself considerably, which affects in a negative way to the ATP synthesis andincrease of weight(AU)

¿Son todas las partículas de lipoproteínas de alta densidad iguales? / Are all high-density lipoprotein particles equal?

El análisis de las modificaciones de genes que influencian el metabolismo y la función de las lipoproteínas de alta densidad (HDL) en modelos murinos, demuestra que los cambios (aumentos o disminución) de colesterol unido a HDL no son un predictor adecuado de la susceptibilidad a la arteriosclerosis de éstos. Por tanto, los diferentes tipos de partículas de HDL generados por intervención sobre distintas potenciales dianas terapéuticas no son iguales en cuanto a su potencial antiaterogénico. El análisis del transporte reverso de colesterol específico de macrófagos, y de la capacidad de las HDL de proteger frente a la modificación oxidativa de lipoproteínas de baja densidad, aporta una mejor predicción del efecto de la modificación genética efectuada sobre la susceptibilidad a la arteriosclerosis (AU)

Analysis of the genetic modifications that influence high-density lipoprotein (HDL) metabolism and function in murine models shows that changes (whether increases or decreases) in HDL-cholesterol do not accurately predict susceptibility to arteriosclerosis in these models. Therefore, the distinct types of HDL particles generated by interventions on the various potential therapeutic targets differ in their antiatherogenic potential. Analysis of macrophage-specific reverse cholesterol transport and the ability of HDL to protect LDL against oxidative modification is better able to predict the effect of genetic modification on susceptibility to arteriosclerosis (AU)

Interacción entre receptores de glutamato y receptores de nucleótidos en neuronas granulares de cerebelo en cultivo / Cross-talk between glutamate and nucleotide receptors in cerebellar granule neurons in culture

ATP elicits Ca2+ transients in cultured cerebellar granule neuronsacting through specific ionotropic (P2X) and metabotropic (P2Y)purinergic receptors. In these neurons, application of L-Glutamate(L-Glu) immediately before ATP induced a prolonged reductionof ATP-mediated responses that remains at least 5 minutes afterL-Glu wash out. alpha-amino-3-hydro-5-methyl-4-isoxazolpropionicacid (AMPA), N-methyl-D-aspartate (NMDA) and 3,5-dihydroxyphenyl-glycine (DHPG), selective agonists of ionotropic non-NMDA,NMDA and Group I metabotropic glutamate receptors respectively,mimicked Glu-induced attenuating effects. The activity of calciumcalmodulindependent protein kinase II (CaMKII) seems to beinvolved, at least at long term, because inhibitors of CaMKII, 1-[N,Obis(5-isoquinolinesulfonyl)-N-methyl-L-(KN-62) and N-[2-[[[3-(4'-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4'-methoxybenzenesulfonamide(KN-93), abolished the inhibitory effect of L-Glu on ATP-mediatedresponses. However, it is likely that other protein kinases could beinvolved in the cross-talk process between both groups of receptorsat short term. Therefore, these results demonstrate that the activationof glutamate receptors is able to modulate nucleotide responses incerebellar granule neurons(AU)

Interacción entre receptores de glutamato y receptores denucleótidos en neuronas granulares de cerebelo en cultivoEl ATP induce un incremento de Ca2+ en neuronas granulares decerebelo en cultivo actuando a través de receptores purinérgicos específicosionótropicos (P2X) y metabotrópicos (P2Y). En estas neuronas,la aplicación de L-Glutamato (L-Glu) inmediatamente antes del ATPinduce una prolongada disminución de las respuestas mediadas porATP que se mantiene al menos durante cinco minutos tras el lavadodel L-Glu. Los agonistas selectivos de los receptores ionotrópicosde glutamato no-NMDA, NMDA y del Grupo I, el ácido alfa-amino-3-hidro-5-metil-4-isoxazolpropiónico (AMPA), el N-metil-D-aspartato(NMDA) y el 3,5-dihidroxifenil-glicina (DHPG), respectivamente, mimetizanlos efectos atenuantes inducidos por el glutamato. La actividadde la proteína calcio-calmodulina quinasa II (CaMKII) pareceestar implicada en este proceso, al menos a largo plazo, puesto quelos inhibidores de la CaMKII, 1-[N,O-bis(5-isoquinolinesulfonil)-Nmetil-L-tirosil]-4fenilpiperazine (KN-62) y N-[2-[[[3-(4'-clorofenil)-2-propenil]metilamino]metil]fenil]-N-(2-hidroxietil)-4'-metoxibenzenosulfonamida(KN-93), revierten el efecto inhibitorio del L-Glu sobrelas respuestas mediadas por ATP. Sin embargo, es probable que puedanestar implicadas otras proteín quinasas en los procesos de interacciónentre ambos grupos de receptores a corto plazo. Por lo tanto,estos resultados demuestran que la activación de los receptores deglutamato son capaces de modular las respuestas a nucleótidos enneuronas granulares de cerebelo(AU)