Clinical Utility of Liquid Biopsy-Based Actionable Mutations Detected via ddPCR.

Cancer is one of the leading causes of death worldwide and remains a major public health challenge. The introduction of more sensitive and powerful technologies has permitted the appearance of new tumor‐specific molecular aberrations with a significant cancer management improvement. Therefore, molecular pathology profiling has become fundamental not only to guide tumor diagnosis and prognosis but also to assist with therapeutic decisions in daily practice. Although tumor biopsies continue to be mandatory in cancer diagnosis and classification, several studies have demonstrated that liquid biopsies could be used as a potential tool for the detection of cancer‐specific biomarkers. One of the main advantages is that circulating free DNA (cfDNA) provides information about intra‐tumoral heterogeneity, reflecting dynamic changes in tumor burden. This minimally invasive tool has become an accurate and reliable instrument for monitoring cancer genetics. However, implementing liquid biopsies across the clinical practice is still ongoing. The main challenge is to detect genomic alterations at low allele fractions. Droplet digital PCR (ddPCR) is a powerful approach that can overcome this issue due to its high sensitivity and specificity. Here we explore the real‐world clinical utility of the liquid biopsy ddPCR assays in the most diagnosed cancer subtypes.post-print994 K

Utilización del contenido de vesículas extracelulares circulantes en sangre periférica para el manejo clínico de pacientes con gliomas

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina. Departamento de Bioquímica. Fecha de lectura: 24 de febrero, 2017Esta tesis tiene embargado el acceso al texto completo hasta el 24-08-2018Los gliomas son los tumores primarios más comunes en el Sistema Nervioso Central. El Glioblastoma (GBM) es el tumor primario de origen glial más frecuente y agresivo, con una supervivencia media que rara vez supera los 15 meses. El fracaso terapéutico se debe en parte a la existencia de una población celular denominada células iniciadoras de tumor (CIT) a las que se les atribuye la responsabilidad del inicio del tumor, de la invasividad y la resistencia a los tratamientos actuales. Por lo que las CIT se han propuesto como el mejor modelo pre-clínico para el estudio de los gliomas. En este trabajo nos planteamos el estudio de la evolución in vitro de cultivos primarios aislados de muestras quirúrgicas de pacientes diagnosticados con GBM, así como analizamos su patrón de diseminación en ratones inmunodeprimidos. Nuestros resultados muestran que las CIT son susceptibles de evolucionar a lo largo de los pases en cultivo, presentando eventos de inestabilidad cromosómica, un aumento en la expresión de marcadores específicos de CIT y de troncalidad, además de fluctuaciones importantes en su perfil de viabilidad y en el patrón de respuesta a fármacos. El hecho de que las CIT secreten vesículas extracelulares (VEs) al espacio extracelular y que estas VEs transporten material genético de las células productoras, ha hecho que se postulen como uno de los mejores tipos de biopsia líquida disponibles en la actualidad. Sin embargo, en la mayoría de los gliomas de bajo grado la barrera hematoencefálica (BHE) mantiene su estructura, por lo que su liberación al torrente sanguíneo puede estar limitada. Para ello creamos un modelo animal xenotrasplantado con CIT que presentaba la BHE intacta, de esta manera hemos creado una metodología indirecta que nos permite separar las VEs procedentes del tumor de las del propio ratón. En dicho modelo demostramos por primera vez que los tres tipos de VEs: cuerpos apoptóticos, vesículas de shedding, y exosomas, son capaces de atravesar la BHE y aparecer en el torrente sanguíneo llevando en su interior secuencias de ADNg secretadas por el tumor. Finalmente, demostramos la presencia de IDH1G395A en las VEs aisladas de sangre periférica procedente de una cohorte de pacientes diagnosticados con gliomas de bajo y alto grado, lo que supone un método minimamente invasivo para la detección de este marcador esencial para el diagnóstico, pronóstico y seguimiento de los pacientes.Gliomas are the most common primary tumors in the central nervous system. Glioblastoma (GBM) is the most frequent and aggressive glial primary tumor, with an average survival of 15 months. The therapeutic failure is in part due to the existence of a cell population call cancer initiating cells (CIT), which are considered to be responsible for tumor initiation, invasiveness and resistance to current therapy. For this reason, CIT have been proposed as the best pre-clinical model for glioma studies. In this work we investigated the in vitro evolution of primary cell cultures isolated from GBM patients’s surgical samples, as well as their dissemination pattern in immunocompromised mice. Our results demonstrated that CIT evolve along the passages in culture, showing chromosomic instability events, increased stemness marker expression, as well as significant variation in viability rates and response to drug treatment. CIT secrete extracellular vesicles (EVs), which carry genetic cargo of originating cells and have been proposed as one of the best type of liquid biopsy available nowadays. However, most low-grade gliomas maintain intact blood-brain barrier (BBB), which might limit EV release into the bloodstream. We used orthotopic xenotransplant mouse model of GBM-CIT featuring an intact BBB. We demonstrated for the first time, that all EV types — apoptotic bodies, shedding microvesicles and exosomes — cross the BBB and can be detected in peripheral blood carrying gDNA secreted by tumor cells. Finally, we demonstrated the presence of IDH1G395A mutation inside EVs isolated from from a cohort of low and high grade glioma patients’ peripheral blood, which provides a minimally invasive method to detect this marker essential for diagnosis, prognosis and patient follow-up

Oncolytic Virotherapy in Glioma Tumors.

Glioma tumors are one of the most devastating cancer types. Glioblastoma is the most advanced stage with the worst prognosis. Current therapies are still unable to provide an e ective cure. Recent advances in oncolytic immunotherapy have generated great expectations in the cancer therapy field. The use of oncolytic viruses (OVs) in cancer treatment is one such immune-related therapeutic alternative. OVs have a double oncolytic action by both directly destroying the cancer cells and stimulating a tumor specific immune response to return the ability of tumors to escape the control of the immune system. OVs are one promising alternative to conventional therapies in glioma tumor treatment. Several clinical trials have proven the feasibility of using some viruses to specifically infect tumors, eluding undesired toxic e ects in the patient. Here, we revisited the literature to describe the main OVs proposed up to the present moment as therapeutic alternatives in order to destroy glioma cells in vitro and trigger tumor destruction in vivo. Oncolytic viruses were divided with respect to the genome in DNA and RNA viruses. Here, we highlight the results obtained in various clinical trials, which are exploring the use of these agents as an alternative where other approaches provide limited hope.post-print832 K

Proteomics and metabolomics approach in adult and pediatric glioma diagnostics.

The diagnosis of glioma is mainly based on imaging methods that do not distinguish between stage and subtype prior to histopathological analysis. Patients with gliomas are generally diagnosed in the symptomatic stage of the disease. Additionally, healing scar tissue may be mistakenly identified based on magnetic resonance imaging (MRI) as a false positive tumor recurrence in postoperative patients. Current knowledge of molecular alterations underlying gliomagenesis and identification of tumoral biomarkers allow for their use as discriminators of the state of the organism. Moreover, a multiomics approach provides the greatest spectrum and the ability to track physiological changes and can serve as a minimally invasive method for diagnosing asymptomatic gliomas, preceding surgery and allowing for the initiation of prophylactic treatment. It is important to create a vast biomarker library for adults and pediatric patients due to their metabolic differences. This review focuses on the most promising proteomic, metabolomic and lipidomic glioma biomarkers, their pathways, the interactions, and correlations that can be considered characteristic of tumor grade or specific subtype.post-print2427 K

Unravelling glioblastoma heterogeneity by means of single-cell RNA sequencing.

Glioblastoma (GBM) is the most invasive and deadliest brain cancer in adults. Its inherent heterogeneity has been designated as the main cause of treatment failure. Thus, a deeper understanding of the diversity that shapes GBM pathobiology is of utmost importance. Single-cell RNA sequencing (scRNA-seq) technologies have begun to uncover the hidden composition of complex tumor ecosystems. Herein, a semi-systematic search of reference literature databases provided all existing publications using scRNA-seq for the investigation of human GBM. We compared and discussed findings from these works to build a more robust and unified knowledge base. All aspects ranging from inter-patient heterogeneity to intra-tumoral organization, cancer stem cell diversity, clonal mosaicism, and the tumor microenvironment (TME) are comprehensively covered in this report. Tumor composition not only differs across patients but also involves a great extent of heterogeneity within itself. Spatial and cellular heterogeneity can reveal tumor evolution dynamics. In addition, the discovery of distinct cell phenotypes might lead to the development of targeted treatment approaches. In conclusion, scRNA-seq expands our knowledge of GBM heterogeneity and helps to unravel putative therapeutic targets.post-print4967 K

Metabolic therapy and bioenergetic analysis: The missing piece of the puzzle.

Background Aberrant metabolism is recognized as a hallmark of cancer, a pillar necessary for cellular proliferation. Regarding bioenergetics (ATP generation), most cancers display a preference not only toward aerobic glycolysis (“Warburg effect”) and glutaminolysis (mitochondrial substrate level-phosphorylation) but also toward other metabolites such as lactate, pyruvate, and fat-derived sources. These secondary metabolites can assist in proliferation but cannot fully cover ATP demands. Scope of review The concept of a static metabolic profile is challenged by instances of heterogeneity and flexibility to meet fuel/anaplerotic demands. Although metabolic therapies are a promising tool to improve therapeutic outcomes, either via pharmacological targets or press-pulse interventions, metabolic plasticity is rarely considered. Lack of bioenergetic analysis in vitro and patient-derived models is hindering translational potential. Here, we review the bioenergetics of cancer and propose a simple analysis of major metabolic pathways, encompassing both affordable and advanced techniques. A comprehensive compendium of Seahorse XF bioenergetic measurements is presented for the first time. Major conclusions Standardization of principal readouts might help researchers to collect a complete metabolic picture of cancer using the most appropriate methods depending on the sample of interest.post-print3250 K

Ongoing and emerging questions in water erosion studies

Soil erosion is a threat to food security, especially in regions where the area of arable land is shrinking dramatically because of soil degradation. Research on soil erosion expanded progressively throughout the 20th centu\ry, although a number of unresolved problems persist despite this issue being crucial for the environment and the welfare of society. Some basic unresolved issues, including the absence of a universally accepted definition of soil erosion and disagreement about how to measure it have contributed to a degree of scientific stagnation. Accurate prediction of the response of soils to disturbance is hampered by the dependence of the erosion process on the spatial scale involved, the time lag between the disturbance and the erosion response and the short periods for which data are typically available. We argue that devoting increased attention to the following environmental, demographic, political and societal issues will reinvigorate progress in the field. (i) The relationships between on-site and off-site consequences of soil erosion need to be elucidated if the economic and environmental costs are to be adequately assessed. (ii) Effective measures for soil conservation need to focus on spatial patterns of plant cover that reduce sediment connectivity, and most importantly on the relationships between hillslopes and sediment transfer in eroded channels. (iii) The scientific community must be able to identify early warning signs of critical transitions, if irreversible soil degradation is to be prevented. (iv) Consensus needs to be reached concerning the contribution of soil erosion to the carbon cycle. (v) The consequences of climate change on erosion and sediment transport should be investigated in depth. (vi) The general society needs to perceive soil erosion as a critical matter requiring an urgent response

La evolución de las marcas de lujo en el desarrollo de estrategias de comunicación digital

Las marcas de lujo se encuentran en un punto en el que tienen que redefinir sus estrategias de marketing y de negocio para poder acercarse a sus públicos objetivos, un target con un elevado poder adquisitivo y con deseo de comprar productos exclusivos, es por ello que el marketing digital se transforma en una herramienta imprescindible para la comunicación de las marcas de moda de lujo. La presente investigación pretende conocer cuáles están siendo las estrategias actuales en marketing digital que están aplicando las marcas del sector de la moda de lujo. Queremos identificar los elementos más relevantes en su comunicación en un entorno virtual. Para conseguir nuestros objetivos hemos usado técnicas cualitativas, mediante análisis de contenidos y entrevistas en profundidad. Gracias a ello podemos destacar que el principio de “storytelling” es una de las estrategias clave en el marketing digital, puesto que las marcas buscan interactuar con el público y mostrar su historia

Normalizing the gene dosage of Dyrk1A in a mouse model of Down syndrome rescues several Alzheimer's disease phenotypes

The intellectual disability that characterizes Down syndrome (DS) is primarily caused by prenatal changes in central nervous system growth and differentiation. However, in later life stages, the cognitive abilities of DS individuals progressively decline due to accelerated aging and the development of Alzheimer's disease (AD) neuropathology. The AD neuropathology in DS has been related to the overexpression of several genes encoded by Hsa21 including DYRK1A (dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A), which encodes a protein kinase that performs crucial functions in the regulation of multiple signaling pathways that contribute to normal brain development and adult brain physiology. Studies performed in vitro and in vivo in animal models overexpressing this gene have demonstrated that the DYRK1A gene also plays a crucial role in several neurodegenerative processes found in DS. The Ts65Dn (TS) mouse bears a partial triplication of several Hsa21 orthologous genes, including Dyrk1A, and replicates many DS-like abnormalities, including age-dependent cognitive decline, cholinergic neuron degeneration, increased levels of APP and A?, and tau hyperphosphorylation. To use a more direct approach to evaluate the role of the gene dosage of Dyrk1A on the neurodegenerative profile of this model, TS mice were crossed with Dyrk1A KO mice to obtain mice with a triplication of a segment of Mmu16 that includes this gene, mice that are trisomic for the same genes but only carry two copies of Dyrk1A, euploid mice with a normal Dyrk1A dosage, and CO animals with a single copy of Dyrk1A. Normalizing the gene dosage of Dyrk1A in the TS mouse rescued the density of senescent cells in the cingulate cortex, hippocampus and septum, prevented cholinergic neuron degeneration, and reduced App expression in the hippocampus, A? load in the cortex and hippocampus, the expression of phosphorylated tau at the Ser202 residue in the hippocampus and cerebellum and the levels of total tau in the cortex, hippocampus and cerebellum. Thus, the present study provides further support for the role of the Dyrk1A gene in several AD-like phenotypes found in TS mice and indicates that this gene could be a therapeutic target to treat AD in DS.This work was supported by the Jerome Lejeune Foundation, Fundación Tatiana Pérez de Guzmán el Bueno and the Spanish Ministry of Economy and Competitiveness (PSI-2016-76194-R, AEI/FEDER, EU). The authors wish to express their gratitude to Mariona Arbonés for providing Dyrk1A +/− KO mice and to Eva García Iglesias for technical assistance

New Insights into the Medieval Hispano-Muslim Panel Painting: The Alfarje Found in a Balearic Casal (Spain)

Hispano-Muslim culture flourished during the Middle Ages in the Iberian Peninsula and Balearic Islands. During the restoration of a Balearic nobiliary building (casal), several panels with polychrome decoration on the back side were found. They were part of an old Muslim wooden ceiling (alfarje). A multi-technique strategy including optical microscopy, infrared and μRaman spectroscopies, field emission scanning electron microscopy-X-ray microanalysis (FESEM-EDX), focused ion beam (FIB-FESEM-EDX), atomic force microscopy nanoindentation (AFM-NI), and gas chromatography-mass spectrometry (GC-MS) has been applied in the analysis of these panel paintings and has provided morphological and compositional data that have led to the identification of the materials and artistic technique as well as the alteration mechanisms due to the natural aging and the adverse conditions of conservation. As a novelty, this study has confirmed the use of indigo as a blue pigment, an unusual material in Hispano-Muslim panel painting. Apart from the notable change in the visual appearance observed in the paintings, the study has also confirmed a change in the mechanical resistance in the paint layers. These changes have been induced by the combination of the chemical and microbiological alteration mechanisms identified