High-Throughput Sequencing of microRNAs in Peripheral Blood Mononuclear Cells: Identification of Potential Weight Loss Biomarkers

INTRODUCTION: MicroRNAs (miRNAs) are being increasingly studied in relation to energy metabolism and body composition homeostasis. Indeed, the quantitative analysis of miRNAs expression in different adiposity conditions may contribute to understand the intimate mechanisms participating in body weight control and to find new biomarkers with diagnostic or prognostic value in obesity management. OBJECTIVE: The aim of this study was the search for miRNAs in blood cells whose expression could be used as prognostic biomarkers of weight loss. METHODS: Ten Caucasian obese women were selected among the participants in a weight-loss trial that consisted in following an energy-restricted treatment. Weight loss was considered unsuccessful when <5% of initial body weight (non-responders) and successful when >5% (responders). At baseline, total miRNA isolated from peripheral blood mononuclear cells (PBMC) was sequenced with SOLiD v4. The miRNA sequencing data were validated by RT-PCR. RESULTS: Differential baseline expression of several miRNAs was found between responders and non-responders. Two miRNAs were up-regulated in the non-responder group (mir-935 and mir-4772) and three others were down-regulated (mir-223, mir-224 and mir-376b). Both mir-935 and mir-4772 showed relevant associations with the magnitude of weight loss, although the expression of other transcripts (mir-874, mir-199b, mir-766, mir-589 and mir-148b) also correlated with weight loss. CONCLUSIONS: This research addresses the use of high-throughput sequencing technologies in the search for miRNA expression biomarkers in obesity, by determining the miRNA transcriptome of PBMC. Basal expression of different miRNAs, particularly mir-935 and mir-4772, could be prognostic biomarkers and may forecast the response to a hypocaloric diet.

Lymphangioleiomyomatosis Biomarkers Linked to Lung Metastatic Potential and Cell Stemness

Lymphangioleiomyomatosis (LAM) is a rare lung-metastasizing neoplasm caused by the proliferation of smooth muscle-like cells that commonly carry loss-of-function mutations in either the tuberous sclerosis complex 1 or 2 (TSC1 or TSC2) genes. While allosteric inhibition of the mechanistic target of rapamycin (mTOR) has shown substantial clinical benefit, complementary therapies are required to improve response and/or to treat specific patients. However, there is a lack of LAM biomarkers that could potentially be used to monitor the disease and to develop other targeted therapies. We hypothesized that the mediators of cancer metastasis to lung, particularly in breast cancer, also play a relevant role in LAM. Analyses across independent breast cancer datasets revealed associations between low TSC1/2 expression, altered mTOR complex 1 (mTORC1) pathway signaling, and metastasis to lung. Subsequently, immunohistochemical analyses of 23 LAM lesions revealed positivity in all cases for the lung metastasis mediators fascin 1 (FSCN1) and inhibitor of DNA binding 1 (ID1). Moreover, assessment of breast cancer stem or luminal progenitor cell biomarkers showed positivity in most LAM tissue for the aldehyde dehydrogenase 1 (ALDH1), integrin-beta 3 (ITGB3/CD61), and/or the sex-determining region Y-box 9 (SOX9) proteins. The immunohistochemical analyses also provided evidence of heterogeneity between and within LAM cases. The analysis of Tsc2-deficient cells revealed relative over-expression of FSCN1 and ID1; however, Tsc2-deficient cells did not show higher sensitivity to ID1-based cancer inhibitors. Collectively, the results of this study reveal novel LAM biomarkers linked to breast cancer metastasis to lung and to cell stemness, which in turn might guide the assessment of additional or complementary therapeutic opportunities for LAM.

Biochemical biomarkers in samples processed by different methods in Environmental Specimen Banks

In the last decades the creation of new Environmental Specimen Banks (ESB) is increasing due to the necessity of knowing the effects of pollutants in both the environment and human populations. ESBs analyze and store samples in order to understand the effects of chemicals, emerging substances and the environmental changes in biota. For a correct analysis of the effect induced by these variables, there is a need to add biological endpoints, such as biomarkers, to the endpoints based on chemical approaches which have being used until now. It is essential to adapt ESB´s sampling strategies in order to enable scientists to apply new biological methods. The present study was performed to obtain biochemical endpoints from samples stored in the BBEBB (Biscay Bay Environmental Biospecimen Bank) of the Marine Station of Plentzia (PIE - UPV/EHU). The main objective of the present work was to study the variability caused in biochemical biomarkers by different processing methods in mussels (Mytilus galloprovincialis) from two localities (Plentzia and Arriluze) with different pollution history. It can be concluded that the selected biomarkers (glutathione S-transferase and acetylcholinesterase) can be accurately measured in samples stored for years in the ESBs. The results also allowed the discrimination of both sampling sites. However, in a further step, the threshold levels and baseline values should be characterized for a correct interpretation of the results in relation to the assessment of the ecosystem health status.

Stability of the factorial structure of metabolic syndrome from childhood to adolescence : a 6-year follow-up study

Background: Metabolic syndrome (MS) is a clustering of cardiometabolic risk factors that is considered a predictor of cardiovascular disease, type 2 diabetes and mortality. There is no consistent evidence on whether the MS construct works in the same way in different populations and at different stages in life. Methods: We used confirmatory factor analysis to examine if a single-factor-model including waist circumference, triglycerides/HDL-c, insulin and mean arterial pressure underlies metabolic syndrome from the childhood to adolescence in a 6-years follow-up study in 174 Swedish and 460 Estonian children aged 9 years at baseline. Indeed, we analyze the tracking of a previously validated MS index over this 6-years period. Results: The estimates of goodness-of-fit for the single-factor-model underlying MS were acceptable both in children and adolescents. The construct stability of a new model including the differences from baseline to the end of the follow-up in the components of the proposed model displayed good fit indexes for the change, supporting the hypothesis of a single factor underlying MS component trends. Conclusions: A single-factor-model underlying MS is stable across the puberty in both Estonian and Swedish young people. The MS index tracks acceptably from childhood to adolescence.

Early Functional Deficit and Microglial Disturbances in a Mouse Model of Amyotrophic Lateral Sclerosis

11 p.

DNA Damage and Transcriptional Changes in the Gills of Mytilus galloprovincialis Exposed to Nanomolar Doses of Combined Metal Salts (Cd, Cu, Hg)

[ENG]Aiming at an integrated and mechanistic view of the early biological effects of selected metals in the marine sentinel organism Mytilus galloprovincialis, we exposed mussels for 48 hours to 50, 100 and 200 nM solutions of equimolar Cd, Cu and Hg salts and measured cytological and molecular biomarkers in parallel. Focusing on the mussel gills, first target of toxic water contaminants and actively proliferating tissue, we detected significant dose-related increases of cells with micronuclei and other nuclear abnormalities in the treated mussels, with differences in the bioconcentration of the three metals determined in the mussel flesh by atomic absorption spectrometry. Gene expression profiles, determined in the same individual gills in parallel, revealed some transcriptional changes at the 50 nM dose, and substantial increases of differentially expressed genes at the 100 and 200 nM doses, with roughly similar amounts of up- and down-regulated genes. The functional annotation of gill transcripts with consistent expression trends and significantly altered at least in one dose point disclosed the complexity of the induced cell response. The most evident transcriptional changes concerned protein synthesis and turnover, ion homeostasis, cell cycle regulation and apoptosis, and intracellular trafficking (transcript sequences denoting heat shock proteins, metal binding thioneins, sequestosome 1 and proteasome subunits, and GADD45 exemplify up-regulated genes while transcript sequences denoting actin, tubulins and the apoptosis inhibitor 1 exemplify down-regulated genes). Overall, nanomolar doses of co-occurring free metal ions have induced significant structural and functional changes in the mussel gills: the intensity of response to the stimulus measured in laboratory supports the additional validation of molecular markers of metal exposure to be used in Mussel Watch programs

Assessment of phytostabilization of Pb/Zn mine soils with organic amendments and a native metallicolous ecotype of Festuca Rubra l.: implications for amendment selection and recovery of soil health

La contaminación del suelo es una de las principales amenazas para los ecosistemas y la salud humana. Actualmente, desde un punto de vista tanto económico como ambiental, la fitoestabilización es la mejor tecnología para remediar suelos contaminados con elevadas concentraciones de metales como son los suelos mineros. La fitoestabilización asistida consiste en el empleo de plantas y enmiendas orgánicas y/o inorgánicas con el fin de reducir la movilidad y la biodisponibilidad de los contaminantes y recuperar la salud de suelo. En este trabajo se han realizado ensayos en microcosmos y en campo centrándonos en la salud del suelo minero contaminado con Pb y Zn durante un proceso de fitoestabilización empleando enmiendas orgánicas (purines vacunos, gallinaza, estiércol de oveja y lodos de papelera mezclados con gallinaza) y/o la especie metalífera Festuca rubra con el objetivo de (i) estudiar las interacciones suelo-enmienda responsables de los cambios inducidos por el proceso de quimioestabilización en las propiedades físicoquímicas y biológicas del suelo, (ii) evaluar la efectividad del proceso de fitoestabilización sobre suelos vegetados y de la revegetación sobre suelos desnudos (iii) valorar la idoneidad de distintos indicadores químicos y biológicos (parámetros microbianos y de la vegetación) para monitorizar la efectividad de la fitoestabilización asistida en términos de reducción de la biodisponibilidad de metales en el suelo, mejora de la vegetación y de la recuperación de la salud del suelo. La aplicación de enmiendas al suelo minero supone una entrada de materia orgánica y nutrientes que conduce a una disminución de la biodisponibilidad de metales, facilitando la colonización de las plantas y el crecimiento de la vegetación nativa, además de estimular la actividad microbiana del suelo. El pH del suelo es un factor crítico que condiciona la movilidad de los metales y la toxicidad del suelo. Las poblaciones microbianas de las enmiendas no modificaron la diversidad funcional de las comunidades microbianas nativas de la mina. Los purines vacunos y los lodos de papelera mezclados con gallinaza son los tratamientos más efectivos en el proceso de fitoestabilización asistida bajo condiciones de campo. La gallinaza fue el tratamiento que más estimuló el crecimiento de la vegetación nativa y la colonización en los suelos desnudos. El bioensayo de elongación radical de lechuga es un test sensible, sencillo y barato para evaluar la biodisponibilidad de metal y la ecotoxicidad del suelo. Los tocoferoles son biomarcadores de exposición a metales con potencial para su implementación en bioensayos de toxicidad. Este trabajo permite concluir que la población metalífera de F. rubra, combinada con enmiendas orgánicas, es una excelente candidata para los proyectos de fitoestabilización asistida. Además, la monitorización simultánea de los parámetros fisicoquímicos y microbiológicos del suelo y de su ecotoxicidad permite una evaluación adecuada de la salud del suelo, así como la selección de enmiendas apropiadas para el desarrollo de un proceso fitoestabilizador.

On the Selection of Non-Invasive Methods Based on Speech Analysis Oriented to Automatic Alzheimer Disease Diagnosis

The work presented here is part of a larger study to identify novel technologies and biomarkers for early Alzheimer disease (AD) detection and it focuses on evaluating the suitability of a new approach for early AD diagnosis by non-invasive methods. The purpose is to examine in a pilot study the potential of applying intelligent algorithms to speech features obtained from suspected patients in order to contribute to the improvement of diagnosis of AD and its degree of severity. In this sense, Artificial Neural Networks (ANN) have been used for the automatic classification of the two classes (AD and control subjects). Two human issues have been analyzed for feature selection: Spontaneous Speech and Emotional Response. Not only linear features but also non-linear ones, such as Fractal Dimension, have been explored. The approach is non invasive, low cost and without any side effects. Obtained experimental results were very satisfactory and promising for early diagnosis and classification of AD patients.

Análisis histológico e inmunohistoquímico de los subtipos de enfermedad liquenoide oral

176 p. : il.

Análisis bioquímico e histoquímico de la variabilidad producida por cambios de temperatura en muestras del banco de especimenes.

[es]En las últimas décadas está proliferando la creación de nuevos Bancos de Especímenes Ambientales. Es fundamental en ellos disponer de métodos de congelación que garanticen la óptima conservación de las muestras, de forma que sean fiables los resultados obt enidos en las diversas investigaciones realizadas con ellas, para ser utilizadas por futuros investigadores. Este trabajo se ha realizado para conseguir dicho objetivo en relación a el BBEBB (Biscay Bay Environmental Bioespecimen Bank) de la Estación Marin a de Plentzia (PIE - U P V/EHU). Como muestra se han utilizado mejillones ( Mytilus galloprovincialis ) y mediante análisis bioquímico e histoquímico se ha estudiado la variabilidad producida por diferentes métodos de congelación en una serie de biomarcadores pr eviamente seleccionados. La conclusión ha sido que los biomarcadores varían dependiendo del método de congelación. Pero, en general, de forma poco significativa. Por lo tanto cualquiera de los procedimientos de congelación puede ser utilizado en los difere ntes biobancos con garantías. No obstante y para una mayor fiabilidad es conveniente reincidir en este trabajo utilizando otras variables como animales de localidades y/o épocas diferentes

Autophagic Marker MAP1LC3B Expression Levels Are Associated with Carotid Atherosclerosis Symptomatology

Objectives: The mechanism by which atheroma plaque becomes unstable is not completely understood to date but analysis of differentially expressed genes in stable versus unstable plaques may provide clues. This will be crucial toward disclosing the mechanistic basis of plaque instability, and may help to identify prognostic biomarkers for ischaemic events. The objective of our study was to identify differences in expression levels of 59 selected genes between symptomatic patients (unstable plaques) and asymptomatic patients (stable plaques). Methods: 80 carotid plaques obtained by carotid endarterectomy and classified as symptomatic (>70% stenosis) or asymptomatic (>80% stenosis) were used in this study. The expression levels of 59 genes were quantified by qPCR on RNA extracted from the carotid plaques obtained by endarterectomy and analyzed by means of various bioinformatic tools. Results: Several genes associated with autophagy pathways displayed differential expression levels between asymptomatic and symptomatic (i.e. MAP1LC3B, RAB24, EVA1A). In particular, mRNA levels of MAP1LC3B, an autophagic marker, showed a 5-fold decrease in symptomatic samples, which was confirmed in protein blots. Immune system-related factors and endoplasmic reticulum-associated markers (i.e. ERP27, ITPR1, ERO1LB, TIMP1, IL12B) emerged as differently expressed genes between asymptomatic and symptomatic patients. Conclusions: Carotid atherosclerotic plaques in which MAP1LC3B is underexpressed would not be able to benefit from MAP1LC3B-associated autophagy. This may lead to accumulation of dead cells at lesion site with subsequent plaque destabilization leading to cerebrovascular events. Identified biomarkers and network interactions may represent novel targets for development of treatments against plaque destabilization and thus for the prevention of cerebrovascular events.

Mugilid Fish Are Sentinels of Exposure to Endocrine Disrupting Compounds in Coastal and Estuarine Environments

Effects on fish reproduction can result from a variety of toxicity mechanisms first operating at the molecular level. Notably, the presence in the environment of some compounds termed endocrine disrupting chemicals (EDCs) can cause adverse effects on reproduction by interfering with the endocrine system. In some cases, exposure to EDCs leads to the animal feminization and male fish may develop oocytes in testis (intersex condition). Mugilid fish are well suited sentinel organisms to study the effects of reproductive EDCs in the monitoring of estuarine/marine environments. Up-regulation of aromatases and vitellogenins in males and juveniles and the presence of intersex individuals have been described in a wide array of mullet species worldwide. There is a need to develop new molecular markers to identify early feminization responses and intersex condition in fish populations, studying mechanisms that regulate gonad differentiation under exposure to xenoestrogens. Interestingly, an electrophoresis of gonad RNA, shows a strong expression of 5S rRNA in oocytes, indicating the potential of 5S rRNA and its regulating proteins to become useful molecular makers of oocyte presence in testis. Therefore, the use of these oocyte markers to sex and identify intersex mullets could constitute powerful molecular biomarkers to assess xenoestrogenicity in field conditions.

RARRES3 suppresses breast cancer lung metastasis by regulating adhesion and differentiation

In estrogen receptor-negative breast cancer patients, metastatic relapse usually occurs in the lung and is responsible for the fatal outcome of the disease. Thus, a better understanding of the biology of metastasis is needed. In particular, biomarkers to identify patients that are at risk of lung metastasis could open the avenue for new therapeutic opportunities. Here we characterize the biological activity of RARRES3, a new metastasis suppressor gene whose reduced expression in the primary breast tumors identifies a subgroup of patients more likely to develop lung metastasis. We show that RARRES3 downregulation engages metastasis-initiating capabilities by facilitating adhesion of the tumor cells to the lung parenchyma. In addition, impaired tumor cell differentiation due to the loss of RARRES3 phospholipase A1/A2 activity also contributes to lung metastasis. Our results establish RARRES3 downregulation as a potential biomarker to identify patients at high risk of lung metastasis who might benefit from a differentiation treatment in the adjuvant programme.

Subtypes of Patients Experiencing Exacerbations of COPD and Associations with Outcomes

Chronic obstructive pulmonary disease (COPD) is a complex and heterogeneous condition characterized by occasional exacerbations. Identifying clinical subtypes among patients experiencing COPD exacerbations (ECOPD) could help better understand the pathophysiologic mechanisms involved in exacerbations, establish different strategies of treatment, and improve the process of care and patient prognosis. The objective of this study was to identify subtypes of ECOPD patients attending emergency departments using clinical variables and to validate the results using several outcomes. We evaluated data collected as part of the IRYSS-COPD prospective cohort study conducted in 16 hospitals in Spain. Variables collected from ECOPD patients attending one of the emergency departments included arterial blood gases, presence of comorbidities, previous COPD treatment, baseline severity of COPD, and previous hospitalizations for ECOPD. Patient subtypes were identified by combining results from multiple correspondence analysis and cluster analysis. Results were validated using key outcomes of ECOPD evolution. Four ECOPD subtypes were identified based on the severity of the current exacerbation and general health status (largely a function of comorbidities): subtype A (n = 934), neither high comorbidity nor severe exacerbation; subtype B (n = 682), moderate comorbidities; subtype C (n = 562), severe comorbidities related to mortality; and subtype D (n = 309), very severe process of exacerbation, significantly related to mortality and admission to an intensive care unit. Subtype D experienced the highest rate of mortality, admission to an intensive care unit and need for noninvasive mechanical ventilation, followed by subtype C. Subtypes A and B were primarily related to other serious complications. Hospitalization rate was more than 50% for all the subtypes, although significantly higher for subtypes C and D than for subtypes A and B. These results could help identify characteristics to categorize ECOPD patients for more appropriate care, and help test interventions and treatments in subgroups with poor evolution and outcomes.

Current Approaches for Predicting a Lack of Response to Anti-EGFR Therapy in KRAS Wild-Type Patients

Targeting epidermal growth factor receptor (EGFR) has been one of the most effective colorectal cancer strategies. Anti-EGFR antibodies function by binding to the extracellular domain of EGFR, preventing its activation, and ultimately providing clinical benefit. KRAS mutations in codons 12 and 13 are recognized prognostic and predictive biomarkers that should be analyzed at the clinic prior to the administration of anti-EGFR therapy. However, still an important fraction of KRAS wild-type patients do not respond to the treatment. The identification of additional genetic determinants of primary or secondary resistance to EGFR targeted therapy for further improving the selection of patients is urgent. Herein, we review the latest published literature highlighting the most important genes that may predict resistance to anti-EGFR monoclonal antibodies in colorectal cancer patients. According to the available findings, the evaluation of BRAF, NRAS, PIK3CA, and PTEN status could be the right strategy to select patients who are likely to respond to anti-EGFR therapies. In the future, the combination of those biomarkers will help establish consensus that can be introduced into clinical practice.