RESUMEN
Electromagnetic signals from everyday wireless technologies are an ever-present environmental stressor, affecting biological systems. In this article, we substantiate this statement based on the weight of evidence from papers collated within the ORSAA database (ODEB), focusing on the biological and health effects of electromagnetic fields and radiation. More specifically, the experiments investigating exposures from real-world devices and the epidemiology studies examining the effects of living near mobile phone base stations were extracted from ODEB and the number of papers showing effects was compared with the number showing no effects. The results showed that two-thirds of the experimental and epidemiological papers found significant biological effects. The breadth of biological and health categories where effects have been found was subsequently explored, revealing hundreds of papers showing fundamental biological processes that are impacted, such as protein damage, biochemical changes and oxidative stress. This understanding is targeted toward health professionals and policy makers who have not been exposed to this issue during training. To inform this readership, some of the major biological effect categories and plausible mechanisms of action from the reviewed literature are described. Also presented are a set of best practice guidelines for treating patients affected by electromagnetic exposures and for using technology safely in health care settings. In conclusion, there is an extensive evidence base revealing that significant stress to human biological systems is being imposed by exposure to everyday wireless communication devices and supporting infrastructure. This evidence is compelling enough to warrant an update in medical education and practice.
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Teléfono Celular , Tecnología Inalámbrica , Humanos , Campos Electromagnéticos , Estrés Oxidativo , Atención a la SaludRESUMEN
Genetic variation in the FAM13A (Family with Sequence Similarity 13 Member A) locus has been associated with several glycemic and metabolic traits in genome-wide association studies (GWAS). Here, we demonstrate that in humans, FAM13A alleles are associated with increased FAM13A expression in subcutaneous adipose tissue (SAT) and an insulin resistance-related phenotype (e.g. higher waist-to-hip ratio and fasting insulin levels, but lower body fat). In human adipocyte models, knockdown of FAM13A in preadipocytes accelerates adipocyte differentiation. In mice, Fam13a knockout (KO) have a lower visceral to subcutaneous fat (VAT/SAT) ratio after high-fat diet challenge, in comparison to their wild-type counterparts. Subcutaneous adipocytes in KO mice show a size distribution shift toward an increased number of smaller adipocytes, along with an improved adipogenic potential. Our results indicate that GWAS-associated variants within the FAM13A locus alter adipose FAM13A expression, which in turn, regulates adipocyte differentiation and contribute to changes in body fat distribution.
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Adipocitos/metabolismo , Distribución de la Grasa Corporal , Proteínas Activadoras de GTPasa/genética , Adipogénesis/genética , Animales , Diferenciación Celular/genética , Proteínas Activadoras de GTPasa/metabolismo , Técnicas de Silenciamiento del Gen , Sitios Genéticos , Estudio de Asociación del Genoma Completo , Células HEK293 , Humanos , Resistencia a la Insulina/genética , Grasa Intraabdominal/metabolismo , Masculino , Metabolómica , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Polimorfismo de Nucleótido Simple/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Grasa Subcutánea/metabolismoRESUMEN
BACKGROUND: Molecular and cellular changes are intrinsic to aging and age-related diseases. Prior cross-sectional studies have investigated the combined effects of age and genetics on gene expression and alternative splicing; however, there has been no long-term, longitudinal characterization of these molecular changes, especially in older age. RESULTS: We perform RNA sequencing in whole blood from the same individuals at ages 70 and 80 to quantify how gene expression, alternative splicing, and their genetic regulation are altered during this 10-year period of advanced aging at a population and individual level. We observe that individuals are more similar to their own expression profiles later in life than profiles of other individuals their own age. We identify 1291 and 294 genes differentially expressed and alternatively spliced with age, as well as 529 genes with outlying individual trajectories. Further, we observe a strong correlation of genetic effects on expression and splicing between the two ages, with a small subset of tested genes showing a reduction in genetic associations with expression and splicing in older age. CONCLUSIONS: These findings demonstrate that, although the transcriptome and its genetic regulation is mostly stable late in life, a small subset of genes is dynamic and is characterized by a reduction in genetic regulation, most likely due to increasing environmental variance with age.
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Envejecimiento/genética , Empalme Alternativo , Regulación de la Expresión Génica , Anciano , Anciano de 80 o más Años , Envejecimiento/metabolismo , Femenino , Humanos , MasculinoRESUMEN
BACKGROUND: The prevalence of obesity and its comorbidities, including type 2 diabetes mellitus (T2DM), is dramatically increasing throughout the world; however, the underlying aetiology is incompletely understood. Genome-wide association studies (GWAS) have identified hundreds of genec susceptibility loci for obesity and T2DM, although the causal genes and mechanisms are largely unknown. SPRY2 is a candidate gene identified in GWAS of body fat percentage and T2DM, and has recently been linked to insulin production in pancreatic ß-cells. In the present study, we aimed to further understand SPRY2 via functional characterisation in HepG2 cells, an in vitro model of human hepatocytes widely used to investigate T2DM and insulin resistance. METHODS: CRISPR-Cas9 genome editing was used to target SPRY2 in HepG2 cells, and the functional consequences of SPRY2 knockout (KO) and overexpression subsequently assessed using glucose uptake and lipid droplet assays, measurement of protein kinase phosphorylation and RNA sequencing. RESULTS: The major functional consequence of SPRY2 KO was a significant increase in glucose uptake, along with elevated lipid droplet accumulation. These changes were attenuated, but not reversed, in cells overexpressing SPRY2. Phosphorylation of protein kinases across key signalling pathways (including Akt and mitogen activated protein kinases) was not altered after SPRY2 KO. Transcriptome profiling in SPRY2 KO and mock (control) cells revealed a number of differentially expressed genes related to cholesterol biosynthesis, cell cycle regulation and cellular signalling pathways. Phospholipase A2 group IIA (PLA2G2A) mRNA level was subsequently validated as significantly upregulated following SPRY2 KO, highlighting this as a potential mediator downstream of SPRY2. CONCLUSION: These findings suggest a role for SPRY2 in glucose and lipid metabolism in hepatocytes and contribute to clarifying the function of this gene in the context of metabolic diseases.
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Sistemas CRISPR-Cas , Glucosa/metabolismo , Hepatocitos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Gotas Lipídicas/metabolismo , Lipogénesis , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Perfilación de la Expresión Génica , Células Hep G2 , Hepatocitos/citología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Fosforilación , Transducción de SeñalRESUMEN
Insulin resistance (IR) predisposes to type 2 diabetes and cardiovascular disease but its causes are incompletely understood. Metabolic challenges like the oral glucose tolerance test (OGTT) can reveal pathogenic mechanisms. We aimed to discover associations of IR with metabolite trajectories during OGTT. In 470 non-diabetic men (age 70.6 ± 0.6 years), plasma samples obtained at 0, 30 and 120 minutes during an OGTT were analyzed by untargeted liquid chromatography-mass spectrometry metabolomics. IR was assessed with the hyperinsulinemic-euglycemic clamp method. We applied age-adjusted linear regression to identify metabolites whose concentration change was related to IR. Nine trajectories, including monounsaturated fatty acids, lysophosphatidylethanolamines and a bile acid, were significantly associated with IR, with the strongest associations observed for medium-chain acylcarnitines C10 and C12, and no associations with L-carnitine or C2-, C8-, C14- or C16-carnitine. Concentrations of C10- and C12-carnitine decreased during OGTT with a blunted decline in participants with worse insulin resistance. Associations persisted after adjustment for obesity, fasting insulin and fasting glucose. In mouse 3T3-L1 adipocytes exposed to different acylcarnitines, we observed blunted insulin-stimulated glucose uptake after treatment with C10- or C12-carnitine. In conclusion, our results identify medium-chain acylcarnitines as possible contributors to IR.
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Carnitina/análogos & derivados , Glucosa/metabolismo , Resistencia a la Insulina , Células 3T3-L1 , Anciano , Animales , Carnitina/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Prueba de Tolerancia a la Glucosa , Humanos , Lipólisis , Masculino , Metabolómica , RatonesRESUMEN
AIM: To assess practical implications of genotype-based recall (GBR) studies, an increasingly popular approach for in-depth characterization of genotype-phenotype relationships. METHODS: We genotyped 2500 participants from the Swedish EpiHealth cohort and considered loss-of-function and missense variants in genes with relation to cardiometabolic traits as the basis for our GBR study. Therefore, we focused on carriers and non-carriers of the PPARG Pro12Ala (rs1801282) variant, as it is a relatively common variant with a minor allele frequency (MAF) of 0.14. It has also been shown to affect ligand binding and transcription, and carriage of the minor allele (Ala12) is associated with a reduced risk of type 2 diabetes. We re-invited 39 Pro12Pro, 34 Pro12Ala, and 30 Ala12Ala carriers and performed detailed anthropometric and serological assessments. RESULTS: The participation rates in the GBR study were 31%, 44%, and 40%, and accordingly we included 12, 15, and 13 individuals with Pro12Pro, Pro12Ala, and Ala12Ala variants, respectively. There were no differences in anthropometric or metabolic variables among the different genotype groups. CONCLUSIONS: Our report highlights that from a practical perspective, GBR can be used to study genotype-phenotype relationships. This approach can prove to be a valuable tool for follow-up findings from large-scale genetic discovery studies by undertaking detailed phenotyping procedures that might not be feasible in large studies. However, our study also illustrates the need for a larger pool of genotyped or sequenced individuals to allow for selection of rare variants with larger effects that can be examined in a GBR study of the present size.
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PPAR gamma/genética , Anciano , Femenino , Estudio de Asociación del Genoma Completo , Genotipo , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Proyectos PilotoRESUMEN
Social and emotional impairment, school dysfunction, and neurobehavioral impairment are highly prevalent in survivors of childhood craniopharyngioma and negatively affect quality of life. As surgical resection of craniopharyngioma typically impairs hypothalamic/pituitary function, it has been postulated that perhaps post-operative deficiency of the hormone oxytocin may be the etiology of social/emotional impairment. Research on the benefits of oxytocin treatment as a hormone facilitating social interaction is well established. However, no research has yet been conducted on patients with known pituitary/hypothalamic dysfunction due to structural lesions or surgery. This case report investigates the effects of oxytocin therapy on a youngster with pituitary/hypothalamic dysfunction after craniopharyngioma removal. In this individual, treatment with low dose intranasal oxytocin resulted in increased desire for socialization and improvement in affection towards family. In light of these findings, the authors believe that further research into the potential benefits of intranasal oxytocin therapy for patients with panhypopituitarism is necessary to determine whether a broader population may also benefit from intranasal oxytocin therapy.
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Craneofaringioma/tratamiento farmacológico , Relaciones Interpersonales , Oxitocina/uso terapéutico , Padres , Neoplasias Hipofisarias/tratamiento farmacológico , Calidad de Vida , Conducta Social , Niño , Craneofaringioma/cirugía , Femenino , Humanos , Neoplasias Hipofisarias/cirugía , Periodo Posoperatorio , Pronóstico , SobrevivientesRESUMEN
Phosphorylation of protein tyrosine residues is critical to cellular processes, and is regulated by kinases and phosphatases (PTPs). PTPs contain a redox-sensitive active site Cys residue, which is readily oxidized. Myeloperoxidase, released from activated leukocytes, catalyzes thiocyanate ion (SCN(-)) oxidation by H2O2 to form hypothiocyanous acid (HOSCN), an oxidant that targets Cys residues. Dysregulated phosphorylation and elevated MPO levels have been associated with chronic inflammatory diseases where HOSCN can be generated. Previous studies have shown that HOSCN inhibits isolated PTP1B and induces cellular dysfunction in cultured macrophage-like cells. The present study extends this previous work and shows that physiologically-relevant concentrations of HOSCN alter the activity and structure of other members of the wider PTP family (including leukocyte antigen-related PTP, PTP-LAR; T-cell PTP, TC-PTP; CD45 and Src homology phosphatase-1, Shp-1) by targeting Cys residues. Isolated PTP activity, and activity in lysates of human monocyte-derived macrophages (HMDM) was inhibited by 0-100 µM HOSCN with this being accompanied by reversible oxidation of Cys residues, formation of sulfenic acids or sulfenyl-thiocyanates (detected by Western blotting, and LC-MS as dimedone adducts), and structural changes. LC-MS/MS peptide mass-mapping has provided data on the modified Cys residues in PTP-LAR. This study indicates that inflammation-induced oxidants, and particularly myeloperoxidase-derived species, can modulate the activity of multiple members of the PTP superfamily via oxidation of Cys residues to sulfenic acids. This alteration of the balance of PTP/kinase activity may perturb protein phosphorylation and disrupt cell signaling with subsequent induction of apoptosis at sites of inflammation.
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Cisteína/metabolismo , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Tiocianatos/farmacología , Secuencia de Aminoácidos , Células Cultivadas , Humanos , Datos de Secuencia Molecular , Oxidación-ReducciónRESUMEN
OBJECTIVES: Cystic fibrosis liver disease (CFLD), resulting from progressive hepatobiliary fibrosis, causes significant morbidity and mortality in up to 20% of children with cystic fibrosis (CF). Both pathogenesis and early detection of CFLD are elusive. Current diagnostic procedures to detect early CFLD and stage fibrosis severity are inadequate. Recent studies highlight a role for microRNAs (miRNAs) in the pathogenesis of many diseases and have suggested that serum miRNAs could be used as diagnostic biomarkers. METHODS: We profiled circulating serum miRNA levels in patients with CFLD (nâ=â52), patients with CF without liver disease (CFnoLD, nâ=â30), and non-CF pediatric controls (nâ=â20). Extracted RNA was subjected to polymerase chain reaction (PCR) array of 84 miRNAs detectable in human serum. Seven candidate miRNAs identified were validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), normalizing data to geNorm-determined stable reference genes, miR-19b and miR-93. RESULTS: miR-122 was significantly elevated in patients with CFLD versus patients with CFnoLD and controls (Pâ<â0.0001). miR-25 (Pâ=â0.0011) and miR-21 (Pâ=â0.0133) were elevated in patients with CFnoLD versus patients with CFLD and controls. CFLD was discriminated by both miR-122 (area under the curve [AUC] 0.71, Pâ=â0.002) and miR-25 (AUC 0.65, Pâ=â0.026). Logistic regression combining 3 miRNAs (-122, -25, -21) was greatly predictive of detecting CFLD (AUC 0.78, Pâ<â0.0001). A combination of 6 miRNAs (-122, -21, -25, -210, -148a, -19a) distinguished F0 from F3-F4 fibrosis (AUC 0.73, Pâ=â0.04), and miR-210 combined with miR-22 distinguished F0 fibrosis from any fibrosis, that is, F1-F4 (AUC 0.72, Pâ=â0.02). CONCLUSIONS: These data provide the first evidence of changes to circulating miRNA levels in CF, suggesting that serum-based miRNA analysis may complement and extend current CFLD screening strategies with potential to predict early hepatic fibrosis.
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Fibrosis Quística/complicaciones , Cirrosis Hepática/sangre , Cirrosis Hepática/diagnóstico , MicroARNs/sangre , Adolescente , Área Bajo la Curva , Biomarcadores/sangre , Niño , Preescolar , Femenino , Humanos , Cirrosis Hepática/etiología , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Valor Predictivo de las Pruebas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Índice de Severidad de la EnfermedadRESUMEN
PURPOSE/BACKGROUND: Researchers have identified sex-differences in lower extremity muscle activation during functional activities that involve landing and cutting maneuvers. However, less research has been conducted to determine if muscle activation differences occur during rehabilitation exercises. The purpose of this investigation was to determine if sex-differences exist for activation amplitudes of the trunk and hip muscles during four single leg squat (SLS) exercises. METHODS: Eighteen males and 16 females participated. Surface electromyography (EMG) was used to determine muscle activity of the abdominal obliques (AO), lumbar extensors (LE), gluteus maximus (GMX), and gluteus medius (GM) during four SLS exercises. Data were expressed as a percentage of a maximum voluntary isometric contraction (% MVIC). A 2 X 4 mixed-model analysis of variance with repeated measures was used to determine the interaction between sex and exercise on each muscle's activity. RESULTS: No interaction effect existed between sex and exercise. A main effect for sex existed for the GM and LE. On average, females generated 39% greater GM (27.6 ± 10.4 % MVIC versus 19.8 ± 10.5 % MVIC) and 40% greater LE (8.0 ± 2.8 % MVIC versus 5.7 ± 2.8 % MVIC) activity than males. All subjects, regardless of sex, demonstrated similar GMX and AO activity. Overall EMG values ranged from 11.0 % MVIC to 14.7 % MVIC for the GMX and 5.7 % MVIC to 8.8 % MVIC for the AO. CONCLUSIONS: None of the subjects generated sufficient EMG activity for strength gains. Females generated a moderate level of GM activity appropriate for neuromuscular re-education/endurance. Males generated a low level of GM activity that may not necessarily be sufficient to improve GM function. Subjects exhibited low levels of EMG activity for the other muscles. These findings suggest that clinicians modify and/or prescribe different exercises than those studied herein for the purpose of improving GM, GMX, AO, and LE function. LEVEL OF EVIDENCE: 3b.
RESUMEN
Oxidative damage catalysed by the heme enzyme myeloperoxidase (MPO) has been linked with multiple inflammatory pathologies. The major oxidant species generated by MPO are hypochlorous (HOCl) and hypothiocyanous acids (HOSCN). The damage induced by HOCl is well characterized and has been linked to multiple diseases, however the role of HOSCN is less well understood. It is known that HOSCN can cause selective damage, as this oxidant selectively targets thiol (e.g. Cys) residues and selenium-containing species. The aim of the current study was to assess whether HOCl and HOSCN can disrupt the [4Fe-4S] cluster of aconitase causing iron release and loss of activity. It is shown that HOSCN induces rapid and efficient release of iron from aconitase, with 80% removed at an oxidant concentration of 3 micromoles/mg protein; this is markedly more efficient than HOCl. In contrast the extent of loss of enzymatic activity was comparable between the two oxidants at the same concentration. Blocking the [4Fe-4S] cluster inhibited HOSCN-mediated inactivation, but did not have dramatic effects on HOCl-mediated damage, consistent with HOSCN, but not HOCl, interacting with the cluster. This data is supported by peptide mass mapping studies that indicate that HOSCN oxidises Cys385 of the [4Fe-4S] cluster. In contrast HOCl damaged multiple sites. Exposure of human coronary artery endothelial cells (HCAEC) to 0-50 micromolar HOCl or 0-150 micromolar HOSCN resulted in an increase in intracellular iron, loss of aconitase activity and a loss of mitochondrial aconitase protein. In contrast cytosolic aconitase was not affected. These data indicate that aconitase - and particularly the mitochondrial form - is a target for MPO-mediated damage with HOSCN showing a selectivity for the [4Fe-4S] cluster and inducing greater iron release. This damage, and the release of iron, may exacerbate oxidative stress in cells at sites of inflammation where active MPO is present.
RESUMEN
Zinc is an abundant cellular transition metal ion, which binds avidly to protein cysteine (Cys) and histidine (His) residues to form zinc-Cys/His clusters; these play a key role in the function of many proteins (e.g., DNA binding and repair enzymes, transcription factors, nitric oxide synthase). Leukocyte-derived myeloperoxidase generates powerful oxidants including hypochlorous (HOCl), hypobromous (HOBr), and hypothiocyanous (HOSCN) acids from H(2)O(2) and (pseudo)halide ions. Excessive or misplaced formation of these species is associated with cellular dysfunction, apoptosis and necrosis, and multiple inflammatory diseases. HOCl and HOBr react rapidly with sulfur-containing compounds, and HOSCN reacts specifically with thiols. Consequently, we hypothesized that zinc-Cys/His clusters would be targets for these oxidants, and the activity of such enzymes would be perturbed. This hypothesis has been tested using yeast alcohol dehydrogenase (YADH), which contains a well-characterized Zn(1)Cys(2)His(1) cluster. Incubation of YADH with pathologically relevant concentrations of HOSCN, HOCl, and HOBr resulted in rapid oxidation of the protein (rate constants, determined by competition kinetics, for reaction of HOCl and HOSCN with YADH being (3.3±0.9)×10(8) and (2.9±0.4)×10(4) M(-1) s(-1) per YADH monomer, respectively), loss of enzyme activity, Zn(2+) release, changes in protein structure (particularly formation of disulfide cross-links), and oxidation of Cys residues. The loss of enzyme activity correlated with Zn(2+) release, loss of thiols, and changes in protein structure. We conclude that exposure of zinc-Cys/His clusters to inflammatory oxidants can result in impaired protein activity, thiol oxidation, and Zn(2+) release. These reactions may contribute to inflammation-induced tissue damage.
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Complejos de Coordinación/química , Cisteína/química , Histidina/química , Oxidantes/química , Peroxidasa/química , Alcohol Deshidrogenasa/antagonistas & inhibidores , Alcohol Deshidrogenasa/química , Sitios de Unión , Bromatos/química , Dominio Catalítico , Ácido Hipocloroso/química , Cinética , Oxidación-Reducción , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/química , Tiocianatos/químicaRESUMEN
The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) plays a critical role in Ca(2+) homeostasis via sequestration of this ion in the sarco/endoplasmic reticulum. The activity of this pump is inhibited by oxidants and impaired in aging tissues and cardiovascular disease. We have shown previously that the myeloperoxidase (MPO)-derived oxidants HOCl and HOSCN target thiols and mediate cellular dysfunction. As SERCA contains Cys residues critical to ATPase activity, we hypothesized that HOCl and HOSCN might inhibit SERCA activity, via thiol oxidation, and increase cytosolic Ca(2+) levels in human coronary artery endothelial cells (HCAEC). Exposure of sarcoplasmic reticulum vesicles to preformed or enzymatically generated HOCl and HOSCN resulted in a concentration-dependent decrease in ATPase activity; this was also inhibited by the SERCA inhibitor thapsigargin. Decomposed HOSCN and incomplete MPO enzyme systems did not decrease activity. Loss of ATPase activity occurred concurrent with oxidation of SERCA Cys residues and protein modification. Exposure of HCAEC, with or without external Ca(2+), to HOSCN or HOCl resulted in a time- and concentration-dependent increase in intracellular Ca(2+) under conditions that did not result in immediate loss of cell viability. Thapsigargin, but not inhibitors of plasma membrane or mitochondrial Ca(2+) pumps/channels, completely attenuated the increase in intracellular Ca(2+) consistent with a critical role for SERCA in maintaining endothelial cell Ca(2+) homeostasis. Angiotensin II pretreatment potentiated the effect of HOSCN at low concentrations. MPO-mediated modulation of intracellular Ca(2+) levels may exacerbate endothelial dysfunction, a key early event in atherosclerosis, and be more marked in smokers because of their higher SCN(-) levels.
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Calcio/metabolismo , Vasos Coronarios/patología , Retículo Endoplásmico/enzimología , Células Endoteliales/enzimología , Ácido Hipocloroso/metabolismo , Oxidantes/metabolismo , Peroxidasa/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Tiocianatos/metabolismo , Señalización del Calcio , Células Cultivadas , Cloraminas/metabolismo , Células Endoteliales/metabolismo , Humanos , Ácido Hipocloroso/farmacología , Oxidantes/farmacología , Oxidación-Reducción , Retículo Sarcoplasmático/enzimología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/química , Compuestos de Sulfhidrilo/metabolismo , Tiocianatos/farmacologíaRESUMEN
Traumatic brain injury (TBI) elicits a sequence of complex biochemical changes including oxidative stress, oedema, inflammation and excitotoxicity. These factors contribute to the high morbidity and mortality following TBI, although their underlying molecular mechanisms remain poorly understood. Transient receptor potential melastatin 2 (TRPM2) is a non-selective cation channel, highly expressed in the brain and immune cells. Recent studies have implicated TRPM2 channels in processes involving oxidative stress, inflammation and cell death. However, no studies have investigated the role of TRPM2 in TBI pathophysiology. In the present study, we have characterised TRPM2 mRNA and protein expression following experimental TBI. Adult male Sprague Dawley rats were injured using the impact-acceleration model of diffuse TBI with survival times between 5 and 5 days. Real-time RT-PCR (including reference gene validation studies) and semi-quantitative immunohistochemistry were used to quantify TRPM2 mRNA and protein levels, respectively, following TBI. Significant increases in TRPM2 mRNA and protein expression were observed in the cerebral cortex and hippocampus of injured animals, suggesting that TRPM2 may contribute to TBI injury processes such as oxidative stress, inflammation and neuronal death. Further characterisation of how TRPM2 may contribute to TBI pathophysiology is warranted.
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Lesiones Encefálicas/genética , Lesiones Encefálicas/fisiopatología , Canales Catiónicos TRPM/biosíntesis , Canales Catiónicos TRPM/genética , Regulación hacia Arriba , Animales , Lesiones Encefálicas/metabolismo , Muerte Celular/genética , Corteza Cerebral/lesiones , Corteza Cerebral/patología , Corteza Cerebral/fisiología , Modelos Animales de Enfermedad , Encefalitis/genética , Encefalitis/metabolismo , Encefalitis/fisiopatología , Regulación de la Expresión Génica/fisiología , Hipocampo/lesiones , Hipocampo/patología , Hipocampo/fisiología , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/fisiología , Masculino , Neuronas/patología , Estrés Oxidativo/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Canales Catiónicos TRPM/fisiología , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: The mechanisms of brain injury following intracerebral haemorrhage (ICH) are incompletely understood. Gene expression studies using quantitative real-time RT-PCR following ICH have increased our understanding of these mechanisms, however the inconsistent results observed may be related to inappropriate reference gene selection. Reference genes should be stably expressed across different experimental conditions, however, transcript levels of common reference genes have been shown to vary considerably. Reference gene panels have therefore been proposed to overcome this potential confounder. RESULTS: The present study evaluated the stability of seven candidate reference genes in the striatum and overlying cortex of collagenase-induced ICH in rodents at survival times of 5 and 24 hours. Transcript levels of the candidate reference genes were quantified and ranked in order of stability using geNorm. When our gene of interest, transient receptor potential melastatin 2 (TRPM2), was normalised against each reference gene individually, TRPM2 mRNA levels were highly variable. When normalised to the four most stable reference genes selected for accurate normalisation of data, we found no significant difference between ICH and vehicle rats. CONCLUSION: The panel of reference genes identified in the present study will enable more accurate normalisation of gene expression data in the acute phase of experimental ICH.
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Hemorragia Cerebral/genética , Perfilación de la Expresión Génica/normas , Animales , Hemorragia Cerebral/inducido químicamente , Hemorragia Cerebral/metabolismo , Colagenasas , ARN Mensajero/metabolismo , Ratas , Estándares de Referencia , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismoRESUMEN
While brain free magnesium levels have been shown to decline in a number of acute and chronic brain pathologies, the mechanisms of such decline and the potential for magnesium administration as a therapeutic intervention are still unclear. In acute brain injury, magnesium therapy has failed in recent clinical trials of trauma, presumably because of an intact blood brain barrier at the time of administration reducing central penetration. Under such conditions, magnesium's peripheral effects on cardiovascular parameters may dominate over the central, and potentially neuroprotective, effects of the compound. In contrast, magnesium has been demonstrated to be beneficial in lacunar strokes, albeit that recent animal studies indicate that this effect is without any significant reduction of lesion size. Postnatal magnesium has also been shown to improve neurological outcome in term neonates with perinatal asphyxia, although this may be limited to cases of mild to moderate brain injury; no effect is observed following severe brain injury. Prenatal magnesium has been reported to be beneficial for outcome in very preterm infants, although this may only be at low doses. Combination therapies are also showing promise in experimental studies, with combined magnesium and mild hypothermia as well as magnesium and polyethylene glycol proving effective in ischemic stroke and in spinal cord injury, respectively. With respect to chronic brain injury, recent results indicate that magnesium deficient mice are susceptible to developing Parkinson's disease, which is consistent with earlier findings that magnesium deficiency over a number of generations is associated with the development of Parkinson's disease. The latter was associated with the appearance of variants of the TRPM channels. Our recent studies have shown that Parkinson's disease is associated with reduced TRPM2 and TRPM7 channel mRNA expression. Taken together, a more complete picture is emerging of the role of magnesium in brain injury, its therapeutic potential as well the mechanisms associated with its decline.
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Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/patología , Magnesio/uso terapéutico , Animales , Lesión Encefálica Crónica/tratamiento farmacológico , Lesión Encefálica Crónica/patología , Humanos , Metaanálisis como Asunto , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/patología , Fármacos Neuroprotectores/uso terapéutico , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/patologíaRESUMEN
Traumatic brain injury (TBI) confers a major burden to Western society and effective treatments are urgently required to improve the long-term deficits that inflict TBI survivors. Depletion of intracellular Mg(2+) is a well-known phenomenon occurring after TBI and is associated with poor neurological outcome. However, despite success in pre-clinical experimental studies, therapies utilizing Mg(2+) have not always proven to be clinically effective. Recent evidence implicates members of the transient receptor potential melastatin (TRPM) channel family in processes leading to neuronal cell death following ischemic injury, however, the exact mechanism by which this occurs is not completely understood. Specifically, TRPM7 and TRPM6 are two channels that have been identified as potentially playing a role in regulating Mg(2+) homeostasis, although whether this role in magnesium regulation and neuronal injury is significant is controversial. The purpose of this review is to explore the relationship between TRPM family members and Mg(2+) homeostasis, including their potential involvement in secondary injury processes leading to cell death following TBI.
Asunto(s)
Lesiones Encefálicas/fisiopatología , Magnesio/fisiología , Canales Catiónicos TRPM/fisiología , Lesiones Encefálicas/mortalidad , Lesiones Encefálicas/patología , Lesiones Encefálicas/terapia , Calcio/fisiología , Muerte Celular , Homeostasis , Humanos , Receptores de N-Metil-D-Aspartato/fisiología , SobrevivientesRESUMEN
Careful validation of reference genes used for the normalization of real-time RT-PCR data is required to obtain accurate results regarding gene expression. We evaluated the stability of seven commonly used reference genes in the cerebral cortex and hippocampus of rats 3 days following traumatic brain injury (TBI). HPRT, SDHA, and GUSB were found to be the most stable reference genes in the cerebral cortex, whereas B2MG, TBP, and GAPDH were the most stable in the hippocampus. The use of three reference genes was determined to be the optimal number for accurate normalization of data. To illustrate this point, when our gene of interest, substance P (SP), was normalized against the three most stable reference genes in both brain areas, we found no significant difference between injured and uninjured rats at the 3-day time point. However, when our SP data were normalized to each reference gene individually, SP mRNA level was highly variable depending on the reference gene chosen. The results of the present study highlight the importance of validating reference genes to be used for real-time RT-PCR analysis. The use of the most stable reference genes presented here will allow more accurate normalization of gene expression data in TBI.