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Table S3: Treatment regimens of the Avatar patient derived xenograft models carrying the VEGFR2 WT and L840F genotypes.

Figure S2: Read alignments from WES-cfDNA, showing somatic mutations in APC and TP53 genes.

Detailed methods

INTRODUCTION: Alpha-1 antitrypsin deficiency (AATD) is a genetic condition resulting in lung and liver disease with a great clinical variability. MicroRNAs have been identified as disease modifiers; therefore miRNA deregulation could play an important role in disease heterogeneity. Members of miR-320 family are involved in regulating of multiple processes including inflammation, and have potential specific binding sites in the 3'UTR region of SERPINA1 gene. In this study we explore the involvement of miR-320c, a member of this family, in this disease. METHODS: Firstly in vitro studies were carried out to demonstrate regulation of SERPINA1 gene by miR-320. Furthermore, the expression of miR-320c was analyzed in the blood of 98 individuals with different AAT serum levels by using quantitative PCR and expression was correlated to clinical parameters of the patients. Finally, HL60 cells were used to analyze induction of miR-320c in inflammatory conditions. RESULTS: Overexpression of miR-320 members in human HepG2 cells led to inhibition of SERPINA1 expression. Analysis of miR-320c expression in patient's samples revealed significantly increased expression of miR-320c in individuals with pulmonary disease. Additionally, HL60 cells treated with the pro-inflammatory factor lipopolysaccharide (LPS) showed increase in miR-320c expression, suggesting that miR-320c responds to inflammation. CONCLUSION: Our findings demonstrate that miR-320c inhibits SERPINA1 expression in a hepatic cell line and its levels in blood are associated with lung disease in a cohort of patients with different AAT serum levels. These results suggest that miR-320c can play a role in AAT regulation and could be a biomarker of inflammatory processes in pulmonary diseases.

The SARS-CoV-2 variant of concern (VOC) Delta (B.617.2 lineage) displaced the predominant VOC Alpha (B.1.1.7 lineage) in the United Kingdom. In Madrid, recent start of the decline of predominant VOC Alpha suggested an equivalent phenomenon. However, 11 different variants, none overrepresented in frequency, occupied progressively over a period of 7 weeks the niche previously dominated by VOC Alpha. Only after these 7 weeks, VOC Delta started to emerge. Viral competition due to the entry of VOC Delta is not the major force driving the start of VOC Alpha decline in Madrid. IMPORTANCE Our data indicate that the dynamics of SARS-CoV-2 VOCs turnover in our setting differ from those proposed for other countries. A systematic genomic analysis, updated on a weekly basis, of representative randomly selected samples of SARS-CoV-2 circulating variants allowed us to define a lapse of 7 weeks between the start of VOC Alpha decline and the final emergence of VOC Delta. During this period, VOC Alpha showed a sustained decline, while 11 VOCs, variants of interest (VOIs), and other identified variants, none overrepresented, occupied the niche left by VOC Alpha. Only after these 7 weeks, emergence of VOC Delta occurred, indicating that viral competition involving VOC Delta was not the exclusive direct driving force behind the starting of VOC Alpha decline.

Background: Functional Fitness Training (FFT) is a new exercise modality prioritizing functional multi-joint movements executed at high intensity as a circuit. Objective: To examine the impacts of introducing rest intervals in a FFT workout compared to “rounds for time” (RFT) FFT. Materials and Methods: Participants were 25 resistance-trained adults who completed two FFT workouts 1 week apart. The study design was crossover such that in a given session half the participants completed the standard and the other half the adapted FFT (FFTadapted). The workouts consisted of the same exercises (circuit of four rounds of exercises), but one (FFTadapted) included preset rest intervals (three sets of 1 min after each completed round). Before and after the workouts, countermovement jump ability and blood lactate were measured. Heart rate (HR) and ratings of perceived exertion (RPE) were measured post-exercise. Results: For both the standard and adapted protocols, mean HR was 90% age-predicted maximum. Final RPE was also similar for both workouts (~15–15.5) and indicated a “hard” work intensity. Both FFTs took the same time to complete (~13 min). Furthermore, no significant differences were observed in jump ability between FFTs. In contrast, lactate (15.11 ± 3.64 vs. 13.48 ± 3.64 mmol·L−1, p &lt, 0.05), measured 3 min post-exercise, was significantly lower in FFTadapted. Conclusions: In FFTadapted, there was a significant reduction in RPE and blood lactate concentrations after exercise, while there were no significant differences in either HR or jumping ability, compared to a FFT workout in RFT methodology.

Reverse transcription-PCRs (RT-PCRs) targeting SARS-CoV-2 variant of concern (VOC) mutations have been developed to simplify their tracking. We evaluated an assay targeting E484K/N501Y to identify B.1.351/P1. Whole-genome sequencing (WGS) confirmed only 72 (59.02%) of 122 consecutive RT-PCR P.1/B.1.351 candidates. Prescreening RT-PCRs must target a wider set of mutations, updated from WGS data from emerging variants.

Evapotranspiration (ET) is a key process in hydrological systems and, consequently, in agroecosystems. It can be measured or derived with a large variety of models at scales ranging from leaf to catchment. MODIS16A2 is a satellite ET product with 500 meters / 8-day spatio-temporal resolution worldwide. It is based on the Penman-Monteith equation and considers the effect of vegetation dynamics, albedo and land cover. This technical paper compares the ET estimated from MODIS16A2 against the ET estimated at different scales from three reference methods: (1) the INIA-GRAS Water Balance on a country-scale, (2) the SWAT model of the Santa Lucia basin on the catchment scale, and (3) the Eddy Covariance Flux located in Colonia on a farmer scale. The analysis shows similarities between MODIS16A2 and the reference methods depending on seasonality, geographic location and scale of ET estimation. The assumptions about vegetation cover, vegetation dynamics, meteorological forcing and soil characteristics of the reference methods compared with MODIS16A2 ones could explain some deviations in the ET estimations. The results of this work contribute with a first approximation towards the quantification of the uncertainty of MODIS16A2 in Uruguay. Resumen: La evapotranspiración (ET) es un proceso clave en los sistemas hidrológicos y consecuentemente en los agroecosistemas. Puede ser medida o simulada con una gran variedad de modelos implementados a diferentes escalas que abarcan desde pequeñas escalas a nivel de la hoja de una planta, hasta escalas más grandes en una cuenca hidrográfica. El MODIS16A2 es un producto satelital que estima la ET. Tiene una resolución espaciotemporal de 500 metros y 8 días en todo el mundo. Se basa en la ecuación de Penman-Monteith y considera el efecto de la dinámica de la vegetación, el albedo y la cobertura del suelo. Esta nota técnica compara la estimación de la ET del MODIS16A2 con la ET estimada a diferentes escalas con tres métodos de referencia: (1) el Balance Hídrico INIA-GRAS a escala país; (2) el modelo SWAT de la cuenca Santa Lucía en la escala de cuenca, y (3) la Torre Eddy Covariance ubicada en Colonia a escala de chacra. La comparación muestra que la similitud del MODIS16A2 con estos métodos de referencia depende de la estacionalidad, la geolocalización de la estimación de ET, así como de la escala. Los supuestos sobre la cobertura vegetal, la dinámica de la vegetación, el forzamiento meteorológico y las características del suelo de los métodos de referencia en relación con los de MODIS16A2 podrían explicar algunas de las desviaciones en las estimaciones de ET. Los resultados de este trabajo contribuyen con una primera aproximación a la cuantificación de la incertidumbre de MODIS16A2 en Uruguay. Resumo: Evotranspiração (ET) é um processo chave nos sistemas hidrológicos. A mesma pode ser estimada utilizando medições indiretas ou simulada com uma grande variedade de modelos, que representam escala finas desde a folha de uma planta até escalas amplas em uma bacia hidrográfica. O MODIS16A2 é um produto de satélite que estima a ET. Apresenta uma resolução espaço-temporal de 500 metros e 8 dias no mundo inteiro. Está baseado na equação de Penman-Monteith e contempla o efeito da dinâmica da vegetação, o albedo e a cobertura do solo. Este documento técnico compara o modelo MODIS16A2 com três métodos de referência: (1) o balanço hídrico INIA-GRAS na escala país, (2) o modelo SWAT da bacia do Rio Santa Lucia na escala da bacia e (3) a Torre Eddy Covariance localizada em Colonia na escala de chacara. A comparação mostra que a similitude do modelo MODIS16A2 com os métodos de referência depende da estacionalidade e localização espacial da estimação de ET. O MODIS16A2 emprega diferentes suposições dos métodos de referência. Os pressupostos sobre cobertura vegetal, dinâmica da vegetação, variáveis meteorológicas e características do solo dos métodos de referência em relação aos do MODIS16A2, poderia explicar alguns dos desvios nas estimativas de ET. Os resultados deste trabalho contribuem com uma primeira aproximação na quantificação da incerteza do modelo MODIS16A2 no Uruguai.

ObjectivesThe large number of infected patients requiring mechanical ventilation has led to the postponement of scheduled neurosurgical procedures during the first wave of the COVID-19 pandemic. The aims of this study were to investigate the factors that influence the decision to postpone scheduled neurosurgical procedures and to evaluate the effect of the restriction in scheduled surgery adopted to deal with the first outbreak of the COVID-19 pandemic in Spain on the outcome of patients awaiting surgery.DesignThis was an observational retrospective study.SettingsA tertiary-level multicentre study of neurosurgery activity between 1 March and 30 June 2020.ParticipantsA total of 680 patients awaiting any scheduled neurosurgical procedure were enrolled. 470 patients (69.1%) were awaiting surgery because of spine degenerative disease, 86 patients (12.6%) due to functional disorders, 58 patients (8.5%) due to brain or spine tumours, 25 patients (3.7%) due to cerebrospinal fluid (CSF) disorders and 17 patients (2.5%) due to cerebrovascular disease.Primary and secondary outcome measuresThe primary outcome was mortality due to any reason and any deterioration of the specific neurosurgical condition. Second, we analysed the rate of confirmed SARS-CoV-2 infection.ResultsMore than one-quarter of patients experienced clinical or radiological deterioration. The rate of worsening was higher among patients with functional (39.5%) or CSF disorders (40%). Two patients died (0.4%) during the waiting period, both because of a concurrent disease. We performed a multivariate logistic regression analysis to determine independent covariates associated with maintaining the surgical indication. We found that community SARS-CoV-2 incidence (OR=1.011, pConclusionsPatients awaiting neurosurgery experienced significant collateral damage even when they were considered for scheduled procedures.

ObjectiveTo assess the effect of the first wave of the SARS-CoV-2 pandemic on the outcome of neurosurgical patients in Spain.SettingsThe initial flood of COVID-19 patients overwhelmed an unprepared healthcare system. Different measures were taken to deal with this overburden. The effect of these measures on neurosurgical patients, as well as the effect of COVID-19 itself, has not been thoroughly studied.ParticipantsThis was a multicentre, nationwide, observational retrospective study of patients who underwent any neurosurgical operation from March to July 2020.InterventionsAn exploratory factorial analysis was performed to select the most relevant variables of the sample.Primary and secondary outcome measuresUnivariate and multivariate analyses were performed to identify independent predictors of mortality and postoperative SARS-CoV-2 infection.ResultsSixteen hospitals registered 1677 operated patients. The overall mortality was 6.4%, and 2.9% (44 patients) suffered a perioperative SARS-CoV-2 infection. Of those infections, 24 were diagnosed postoperatively. Age (OR 1.05), perioperative SARS-CoV-2 infection (OR 4.7), community COVID-19 incidence (cases/105people/week) (OR 1.006), postoperative neurological worsening (OR 5.9), postoperative need for airway support (OR 5.38), ASA grade ≥3 (OR 2.5) and preoperative GCS 3–8 (OR 2.82) were independently associated with mortality. For SARS-CoV-2 postoperative infection, screening swab test 5people/week) (OR 1.011), preoperative cognitive impairment (OR 2.784), postoperative sepsis (OR 3.807) and an absence of postoperative complications (OR 0.188) were independently associated.ConclusionsPerioperative SARS-CoV-2 infection in neurosurgical patients was associated with an increase in mortality by almost fivefold. Community COVID-19 incidence (cases/105people/week) was a statistically independent predictor of mortality.Trial registration numberCEIM 20/217.

© 2020 The Authors., The distribution and role of tumor-infiltrating leucocytes in glioblastoma (GBM) remain largely unknown. Here, we investigated the cellular composition of 55 primary (adult) GBM samples by flow cytometry and correlated the tumor immune profile with patient features at diagnosis and outcome. GBM single-cell suspensions were stained at diagnosis (n = 44) and recurrence following radiotherapy and chemotherapy (n = 11) with a panel of 8-color monoclonal antibody combinations for the identification and enumeration of (GFAPCD45) tumor and normal astrocytic cells, infiltrating myeloid cells —i.e. microglial and blood-derived tumor-associated macrophages (TAM), M1-like, and M2-like TAM, neutrophils. and myeloid-derived suppressor cells (MDSC)— and tumor-infiltrating lymphocytes (TIL) —i.e. CD3T-cells and their TCD4, TCD8, TCD4CD8, and (CD25CD127) regulatory (T-regs) subsets, (CD19CD20) B-cells, and (CD16) NK-cells—. Overall, GBM samples consisted of a major population (mean ± 1SD) of tumor and normal astrocytic cells (73% ± 16%) together with a significant but variable fraction of immune cells (24% ± 18%). Within myeloid cells, TAM predominated (13% ± 12%) including both microglial cells (10% ± 11%) and blood-derived macrophages (3% ± 5%), in addition to a smaller proportion of neutrophils (5% ± 9%) and MDSC (4% ± 8%). Lymphocytes were less represented and mostly included TCD4 (0.5% ± 0.7%) and TCD8 cells (0.6% ± 0.7%), together with lower numbers of TCD4CD8 T-cells (0.2% ± 0.4%), T-regs (0.1% ± 0.2%), B-lymphocytes (0.1% ± 0.2%) and NK-cells (0.05% ± 0.05%). Overall, three distinct immune profiles were identified: cases with a minor fraction of leucocytes, tumors with a predominance of TAM and neutrophils, and cases with mixed infiltration by TAM, neutrophils, and T-lymphocytes. Untreated GBM patients with mixed myeloid and lymphoid immune infiltrates showed a significantly shorter patient overall survival versus the other two groups, in the absence of gains of the EGFR gene (p = 0.02). Here we show that immune cell infiltrates are systematically present in GBM, with highly variable levels and immune profiles. Patients with mixed myeloid and T-lymphoid infiltrates showed a worse outcome., Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain and fondos FEDER, Grant/Award Number: CB16/12/00400 and ISCIII PI16/0476; Consejería de Sanidad Junta de Castilla y León, Gerencia Regional de Salud, Spain, Grant/Award Number: GRS2049/A/19

Diagnosis and classification of gliomas mostly relies on histopathology and a few genetic markers. Here we interrogated microarray gene expression profiles (GEP) of 268 diffuse astrocytic gliomas—33 diffuse astrocytomas (DA), 52 anaplastic astrocytomas (AA) and 183 primary glioblastoma (GBM)—based on multivariate analysis, to identify discriminatory GEP that might support precise histopathological tumor stratification, particularly among inconclusive cases with II–III grade diagnosed, which have different prognosis and treatment strategies. Microarrays based GEP was analyzed on 155 diffuse astrocytic gliomas (discovery cohort) and validated in another 113 tumors (validation set) via sequential univariate analysis (pairwise comparison) for discriminatory gene selection, followed by nonnegative matrix factorization and canonical biplot for identification of discriminatory GEP among the distinct histological tumor subtypes. GEP data analysis identified a set of 27 genes capable of differentiating among distinct subtypes of gliomas that might support current histological classification. DA + AA showed similar molecular profiles with only a few discriminatory genes overexpressed (FSTL5 and SFRP2) and underexpressed (XIST, TOP2A and SHOX2) in DA vs AA and GBM. Compared to DA + AA, GBM displayed underexpression of ETNPPL, SH3GL2, GABRG2, SPX, DPP10, GABRB2 and CNTN3 and overexpression of CHI3L1, IGFBP3, COL1A1 and VEGFA, among other differentially expressed genes., MG-T, AO and MDT were supported by the following Grants: CB16/12/00400 (CIBER-ONC, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain and FONDOS FEDER), ISCIII PI16/0476 (Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain) and GRS2049/A/19 (Consejería de Sanidad Junta de Castilla y León, Gerencia Regional de Salud, Spain).

This article belongs to the Special Issue Brain Tumors., [Background]: The prognostic impact of the expression profile of genes recurrently amplified in glioblastoma multiforme (GBM) remains controversial., [Methods]: We investigated the RNA gene expression profile of epidermal growth factor receptor (EGFR), cyclin-dependent kinase 4 (CDK4), murine doble minute 4 (MDM4), and platelet derived growth factor receptor alpha (PDGFRA) in 83 primary GBM tumors vs. 42 normal brain tissue samples. Interphase FISH (iFISH) analysis for the four genes, together with analysis of intragenic deletions in EGFR and PDGFRA, were evaluated in parallel at the DNA level. As validation cohort, publicly available RNA gene expression data on 293 samples from 10 different GBM patient series were also studied., [Results]: At the RNA level, CDK4 was the most frequently overexpressed gene (90%) followed by EGFR (58%) and PDGFRA (58%). Chromosome 7 copy number alterations, i.e., trisomy (49%) and polysomy (44%), showed no clear association with EGFR gene expression levels. In turn, intragenic EGFR deletions were found in 39 patients (47%), including EGFRvIII (46%) in association with EGFRvIVa (4%), EGFRvII (2%) or other EGFR deletions (3%) and PDGFRA deletion of exons 8–9 was found in only two tumors (2%)., [Conclusions]: Overall, none of the gene expression profiles and/or intragenic EGFR deletions showed a significant impact on overall survival of GBM supporting the notion that other still unraveled features of the disease might play a more relevant prognostic role in GBM, This research was funded by Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Ministerio de Economía y Competitividad (RD12/0036/0048, AES PI16/00476-FONDOS FEDER and CB16/12/00400).

Purpose: Despite the wide use of antiangiogenic drugs in the clinical setting, predictive biomarkers of response to these drugs are still unknown. Experimental Design: We applied whole-exome sequencing of matched germline and basal plasma cell-free DNA samples (WES-cfDNA) on a RAS/BRAF/PIK3CA wild-type metastatic colorectal cancer patient with primary resistance to standard treatment regimens, including inhibitors to the VEGF:VEGFR2 pathway. We performed extensive functional experiments, including ectopic expression of VEGFR2 mutants in different cell lines, kinase and drug sensitivity assays, and cell- and patient-derived xenografts. Results: WES-cfDNA yielded a 77% concordance rate with tumor exome sequencing and enabled the identification of the KDR/VEGFR2 L840F clonal, somatic mutation as the cause of therapy refractoriness in our patient. In addition, we found that 1% to 3% of samples from cancer sequencing projects harbor KDR somatic mutations located in protein residues frequently mutated in other cancer-relevant kinases, such as EGFR, ABL1, and ALK. Our in vitro and in vivo functional assays confirmed that L840F causes strong resistance to antiangiogenic drugs, whereas the KDR hot-spot mutant R1032Q confers sensitivity to strong VEGFR2 inhibitors. Moreover, we showed that the D717V, G800D, G800R, L840F, G843D, S925F, R1022Q, R1032Q, and S1100F VEGFR2 mutants promote tumor growth in mice. Conclusions: Our study supports WES-cfDNA as a powerful platform for portraying the somatic mutation landscape of cancer and discovery of new resistance mechanisms to cancer therapies. Importantly, we discovered that VEGFR2 is somatically mutated across tumor types and that VEGFR2 mutants can be oncogenic and control sensitivity/resistance to antiangiogenic drugs. Clin Cancer Res; 24(15); 3550–9. ©2018 AACR.

Several classification systems have been proposed to address genomic heterogeneity of glioblastoma multiforme, but they either showed limited prognostic value and/or are difficult to implement in routine diagnostics. Here we propose a prognostic stratification model for these primary tumors based on tumor gene amplification profiles, that might be easily implemented in routine diagnostics, and potentially improve the patients management. Gene amplification profiles were prospectively evaluated in 80 primary glioblastoma multiforme tumors using single-nucleotide polymorphism arrays and the results obtained validated in publicly available data from 267/347 cases. Gene amplification was detected in 45% of patients, and chromosome 7p11.2 including the EGFR gene, was the most frequently amplified chromosomal region – either alone (18%) or in combination with amplification of DNA sequences in other chromosomal regions (10% of cases). Other frequently amplified DNA sequences included regions in chromosomes 12q(10%), 4q12(7%) and 1q32.1(4%). Based on their gene amplification profiles, glioblastomas were subdivided into: i) tumors with no gene amplification (55%); ii) tumors with chromosome 7p/EGFR gene amplification (with or without amplification of other chromosomal regions) (38%); and iii) glioblastoma multiforme with a single (11%) or multiple (6%) amplified DNA sequences in chromosomal regions other than chromosome 7p. From the prognostic point of view, these amplification profiles showed a significant impact on overall survival of glioblastoma multiforme patients (p>0.001). Based on these gene amplification profiles, a risk-stratification scoring system was built for prognostic stratification of glioblastoma which might be easily implemented in routine diagnostics, and potentially contribute to improved patient management., This work was supported by RETICC RD06/0020/0035, RD06/0020/0059 and RD12/0036/0048 grants from Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, (Madrid, Spain and FONDOS FEDER), AES PI16/000476 (Instituto de Salud Carlos III, Madrid, Spain and FONDOS FEDER), GRS909A14 (JCYL) and CB16/12/00400 grant (CIBERONC, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain and FONDOS FEDER).

A2 or A4 annular finger pulley tears are common injuries in rock climbers. This study reviews the measurement procedures used and tendon-to-bone distance data obtained on high-resolution ultrasound images when diagnosing isolated rupture of the A2 or A4 pulleys. Out of 3447 records extracted, only 7 remained after applying the exclusion criteria. In diagnosing a complete rupture, tendon-to-bone distance used varied widely from 1.9 to 5.1 mm for A2 and from 1.8 to 3.1 mm for A4. Our findings point to a lack of consensus diagnostic criteria for pulley injuries and identify technical details needing further research.

This work considers the problem of forecasting the normal solar irradiance with high spatial and temporal resolution (5 minutes). The forecasting is based on a dataset registered during one year from the high resolution radiometric network at a operational solar power plan at Almeria, Spain. In particular, we show a technique for forecasting the irradiance in the next few minutes from the irradiance values obtained on the previous hour. Our proposal employs a type of recurrent neural network known as LSTM, which can learn complex patterns and that has proven its usability for forecasting temporal series. The results show a reasonable improvement with respect to other prediction methods typically employed in the studies of temporal series., Es este trabajo se aborda el problema de la predicción de radiación global sobre superficie horizontal con alta resolución espacial y temporal (5 minutos) a partir de los datos registrados durante un año en la red radiométrica de alta resolución ubicada en la Plataforma Solar de Almería. En particular se muestra un método capaz de predecir el valor de radiación en los siguientes minutos a partir de los valores de los minutos anteriores. El método emplea el tipo de red neuronal recurrente conocido como LSTM, capaz de aprender patrones complejos y predecir el próximo elemento de una serie temporal. Los resultados muestran una mejora apreciable en con respecto a los métodos de predicción empleados habitualmente en el estudio de series temporales., Facultad de Informática

In recent years, important advances have been achieved in the understanding of the molecular biology of glioblastoma multiforme (GBM); thus, complex genetic alterations and genomic profiles, which recurrently involve multiple signaling pathways, have been defined, leading to the first molecular/genetic classification of the disease. In this regard, different genetic alterations and genetic pathways appear to distinguish primary (eg, EGFR amplification) versus secondary (eg, IDH1/2 or TP53 mutation) GBM. Such genetic alterations target distinct combinations of the growth factor receptor–ras signaling pathways, as well as the phosphatidylinositol 3-kinase/phosphatase and tensin homolog/AKT, retinoblastoma/cyclin-dependent kinase (CDK) N2A-p16 INK4A , and TP53/mouse double minute (MDM) 2/MDM4/CDKN2A-p14 ARF pathways, in cells that present features associated with key stages of normal neurogenesis and (normal) central nervous system cell types. This translates into well-defined genomic profiles that have been recently classified by The Cancer Genome Atlas Consortium into four subtypes: classic, mesenchymal, proneural, and neural GBM. Herein, we review the most relevant genetic alterations of primary versus secondary GBM, the specific signaling pathways involved, and the overall genomic profile of this genetically heterogeneous group of malignant tumors.

Summary Connexin43 (CX43), a protein that forms gap junction channels and hemichannels in astrocytes, is downregulated in high-grade gliomas. Its relevance for glioma therapy has been thoroughly explored; however, its positive effects on proliferation are counterbalanced by its effects on migration and invasion. Here, we show that a cell-penetrating peptide based on CX43 (TAT-Cx43266-283) inhibited c-Src and focal adhesion kinase (FAK) and upregulated phosphatase and tensin homolog in glioma stem cells (GSCs) derived from patients. Consequently, TAT-Cx43266-283 reduced GSC motility, as analyzed by time-lapse microscopy, and strongly reduced their invasive ability. Interestingly, we investigated the effects of TAT-Cx43266-283 on freshly removed surgical specimens as undissociated glioblastoma blocks, which revealed a dramatic reduction in the growth, migration, and survival of these cells. In conclusion, a region of CX43 (amino acids 266–283) exerts an important anti-tumor effect in patient-derived glioblastoma models that includes impairment of GSC migration and invasion., Highlights • TAT-Cx43266-283 exerts anti-tumor effects in patient-derived glioblastoma models • TAT-Cx43266-283 targets Src, PTEN, and FAK • TAT-Cx43266-283 inhibits glioma stem cell migration and invasion, In this article, Arantxa Tabernero and colleagues show that a short region of Connexin43 exerts an important anti-tumor effect in patient-derived glioblastoma models that includes the impairment of glioma stem cell migration and invasion. This peptide targets important proteins for glioblastoma, such as Src, FAK, and PTEN, highlighting its relevance for therapeutic development against this incurable disease.

Introduction The expression of connexin43 (Cx43), the main gap junction channel-forming protein, is down-regulated in glioma stem cells (GSC). Our previous studies showed that a cell-penetrating peptide containing the region of Cx43 that interacts with the oncoprotein c-Src (TAT-Cx43266–283), inhibits its oncogenic activity and up-regulates the tumour suppressor PTEN. Through this pathway, TAT-Cx43266–283 reduces glioma cell proliferation and reverses the GSC phenotype. Our next goal was to uncover the mechanism of action and the effects of TAT-Cx43266–283 on the migration and invasion of human GSCs. Material and methods Human primary GSCs (G9, G12, G13, G15 and G16) were obtained from glioblastoma patients’ biopsies from the Neurosurgery Service of the Hospital Universitario de Salamanca, Spain. The migration was analysed in these primary GSCs by tracking individual cell trajectories in cultures recorded by Time-Lapse Live-cell Imaging and the invasion with Matrigel-treated transwell inserts. The same tumour biopsies were used to study the effect of TAT-Cx43266–283 on fresh undissociated tumour blocks by time-lapse microscopy. The molecular interactions were studied in G166 GSCs by pull-down assays of biotinylated TAT-Cx43266–283 (TAT-Cx43266–283-B) and Western blot analysis. Results and discussions Our results confirmed that TAT-Cx43266–283 strongly reduces the migration and invasion of human primary GSCs by inhibiting c-Src activity and up-regulating PTEN that consequently, lowers the levels of phosphorylation of focal adhesion kinase tyrosines 397, 576 and 577. In addition, fresh undissociated tumour blocks revealed a dramatic reduction on the survival of these glioblastoma cells when exposed to TAT-Cx43266–283. By pull-down assays, we showed that TAT-Cx43266–283-B recruits c-Src together with its inhibitors PTEN and Csk, confirming the proposed mechanism for Cx43. Conclusion In conclusion, TAT-Cx43266–283 exerts an important antitumor effect by inhibiting c-Src and up-regulating PTEN with the subsequent reduction in FAK phosphorylation required to establish appropriate focal adhesions for migration, proliferation and survival. This inhibition is mediated by the recruitment of the c-Src intrinsic inhibitors, PTEN and Csk. All together, these results reinforce the relevance of this sequence of Cx43 that interacts with c-Src for the development of new therapies against glioblastoma.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Meningiomas are usually considered to be benign central nervous system tumors; however, they show heterogenous clinical, histolopathological and cytogenetic features associated with a variable outcome. In recent years important advances have been achieved in the identification of the genetic/molecular alterations of meningiomas and the signaling pathways involved. Thus, monosomy 22, which is often associated with mutations of the NF2 gene, has emerged as the most frequent alteration of meningiomas; in addition, several other genes (e.g. AKT1, KLF4, TRAF7, SMO) and chromosomes have been found to be recurrently altered often in association with more complex karyotypes and involvement of multiple signaling pathways. Here we review the current knowledge about the most relevant genes involved and the signaling pathways targeted by such alterations. In addition, we summarize those proposals that have been made so far for classification and prognostic stratification of meningiomas based on their genetic/genomic features., This work was partially supported by grants from the Fundação para a Ciência e Tecnologia (PIC/IC/83108/2007, FCT, Portugal), Fondo de Investigaciones Sanitarias (RD12/0036/0048, Instituto de Salud Carlos III (ISCIII/FEDER), Ministerio de Sanidad y Consumo, Madrid, Spain), and Consejeria Sanidad Junta de Castilla y León, Gerencia Regional de Salud: GRS689/A/11, and Proyecto Intramural-IBSAL IB14-05. Patrícia Domingues is partially supported by a grant (SFRH/BD/64799/2009) from FCT. Maria Dolores Tabernero is supported by IECSCYL (Soria, Spain).

[Background]: Tumor recurrence remains the major clinical complication of meningiomas, the majority of recurrences occurring among WHO grade I/benign tumors. In the present study, we propose a new scoring system for the prognostic stratification of meningioma patients based on analysis of a large series of meningiomas followed for a median of >5 years. [Methods]: Tumor cytogenetics were systematically investigated by interphase fluorescence in situ hybridization in 302 meningioma samples, and the proposed classification was further validated in an independent series of cases (n = 132) analyzed by high-density (500K) single-nucleotide polymorphism (SNP) arrays. [Results]: Overall, we found an adverse impact on patient relapse-free survival (RFS) for males, presence of brain edema, younger patients (50 mm, tumor localization at intraventricular and anterior cranial base areas, WHO grade II/III meningiomas, and complex karyotypes; the latter 5 variables showed an independent predictive value in multivariate analysis. Based on these parameters, a prognostic score was established for each individual case, and patients were stratified into 4 risk categories with significantly different (P, This work was partially supported by grants from the Fundação para a Ciência e Tecnologia (PIC/IC/83108/2007, FCT, Portugal); Fondo de Investigaciones Sanitarias (FIS/FEDER 06/0312 and RETICC RD06/0020/0035, RD06/0020/0059 and RD12/0036/0048, Instituto de Salud Carlos III (ISCIII/FEDER), Ministerio de Sanidad y Consumo, Madrid, Spain), Caja Burgos (Spain), Fundación MMA (exp 75312010 and 87692011, Madrid, Spain), and Consejeria Sanidad Junta de Castilla y León, Gerencia Regional de Salud: GRS689/A/11. Patrícia Domingues is supported by grant (SFRH/BD/64799/2009) from FCT. Maria Dolores Tabernero is supported by IECSCYL (Fundación Instituto de Estudios Ciencias de la Salud de Castilla y León, Soria, Spain).

[ES]La evacuación endoscópica se considera una alternativa a la craneotomía convencional en el tratamiento de los hematomas intracerebrales, sobre todo si se localizan en estructuras profundas. La valoración de la eficacia de la técnica y sus modificaciones en el animal experimental es fundamental como paso previo a la introducción en la clínica. Los objetivos del estudio fueron desarrollar en el cerdo un modelo de hematoma intracerebral, valorar la eficacia de la evacuación endoscópica y evaluar un nuevo modelo de vaina transparente como instrumento accesorio a la endoscopia. Se generaron hematomas intracerebrales en la sustancia blanca del lóbulo frontal derecho de 17 cerdos mediante la infusión de entre 7 y 20 cc de sangre autóloga. Los animales fueron sacrificados a las 4 horas, 24 horas y 5 días tras la formación del hematoma. Se determinaron los volúmenes residuales y los hallazgos anatomopatológicos en los cerebros fijados tanto en el grupo a los que no se les evacuó el hematoma (grupo control, 6 animales) como el grupo de evacuación endoscópica (grupo problema, 10 animales). La evacuación endoscópica en el grupo problema tuvo lugar a las 2 horas y 12 horas de haberse generado el hematoma. Como accesorio del endoscopio, se utilizó una vaina transparente con un componente externo, con final romo y cerrado, y un componente interno, a través del cual se introducía el endoscopio. Además, a 7 animales se les administró Azul de Evans tras la formación del hematoma (grupo problema de Evans). El volumen residual en el grupo problema fue significativamente menor que el del grupo control (56.25 cc comparado con 565 cc del grupo control, p, [EN] Endoscopic removal is considered an alternative to conventional craniotomy in the treatment of intracerebral hematomas , especially if located in deep structures . The assessment of the effectiveness of the technique and its modifications in the experimental animal is essential as a pre- step introduction into the clinic . The objectives of the study were to develop in the pig model of intracerebral hematoma, assess the efficacy of endoscopic evacuation and evaluate a new model of transparent sleeve as an adjunct to endoscopic instrument. Intracerebral hematomas were generated in the white matter of the right frontal lobe of 17 pigs by infusion of between 7 and 20 cc of autologous blood . The animals were sacrificed at 4 hours, 24 hours and 5 days after hematoma formation . Residual volumes and pathological findings in the brains fixed in both the group to which they were not evacuated the hematoma (control group , 6 animals ) and the endoscopic drainage group ( problem group , 10 animals ) were determined. Endoscopic evacuation in the problem group took place at 2 hours and 12 hours of the hematoma have been generated . As an accessory of the endoscope , a transparent shell with an external component , blunt , closed end , and an internal component is used , through which the endoscope is introduced . In addition , 7 animals were dosed after Evans Blue hematoma formation ( Evans problem group).

This is an open-access article distributed under the terms of the Creative Commons Attribution License., [Background]: Meningioma was the first solid tumor shown to contain a recurrent genetic alteration e.g. monosomy 22/del(22q), NF2 being the most relevant gene involved. Although monosomy 22/del(22q) is present in half of all meningiomas, and meningiomas frequently carry NF2 mutations, no study has been reported so far in which both alterations are simultaneously assessed and correlated with the features of the disease. [Methods]: Here, we analyzed the frequency of both copy number changes involving chromosome 22 and NF2 mutations in 20 sporadic meningiomas using high-density SNP-arrays, interphase-FISH and PCR techniques. [Results]: Our results show a significant frequency of NF2 mutations (6/20 patients, 30%), most of which (5/6) had not been previously reported in sporadic meningiomas. NF2 mutations involved five different exons and led to a truncated protein (p.Leu163CysfsX46, p.Phe62LeufsX61, p.Asp281MetfsX15, p.Phe285LeufsX11, p.Gln389ArgfsX37) and an in frame deletion of Phe119. Interestingly, all NF2 mutated cases were menopausal women with monosomy 22 but not del(22q). [Conclusions]: These results confirm and extend on previous observations about the high frequency and heterogeneity of NF2 mutations in sporadic meningiomas and indicate they could be restricted to a well-defined cytogenetic and clinical subgroup of menopausal women. Further studies in large series of patients are required to confirm our observations., This work has been partially supported by grants from Consejeria Sanidad Junta de Castilla y León, Gerencia Regional de Salud: GRS689/A/11. Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Madrid, Spain: RTICC RD06/0020/0035, RD06/0020/0059 and RD12/0036/0048. MD Tabernero is supported by IECSCYL (Fundación Instituto de Estudios Ciencias de la Salud de Castilla y León).

Meningiomas are primary tumors of the central nervous system composed of both neoplastic and other infiltrating cells. We determined the cellular composition of 51 meningioma samples by multiparameter flow cytometric (MFC) immunophenotyping and investigated the potential relationship between mRNA and protein expression levels of neoplastic cells. For immunophenotypic, morphologic, and cytogenetic characterization of individual cell populations, a large panel of markers was used together with phagocytic/endocytic functional assays and MFC sorting. Overall, our results revealed coexistence of CD45 − neoplastic cells and CD45 + immune infiltrating cells in all meningiomas. Infiltrating cells included tissue macrophages, with an HLA-DR + CD14 + CD45 + CD68 + CD16 −/+ CD33 −/+ phenotype and high phagocytic/endocytic activity, and a small proportion of cytotoxic lymphocytes (mostly T CD8 + and natural killer cells). Tumor cells expressed multiple cell adhesion proteins, tetraspanins, HLA-I/HLA-DR molecules, complement regulatory proteins, cell surface ectoenzymes, and growth factor receptors. Noteworthy, the relationship between mRNA and protein levels was variable, depending on the proteins evaluated and the level of infiltration by immune cells. In summary, our results indicate that MFC immunophenotyping provides a reliable tool for the characterization of the patterns of protein expression of different cell populations coexisting in meningioma samples, with a more accurate measure of gene expression profiles of tumor cells at the functional/protein level than conventional mRNA microarray, independently of the degree of infiltration of the tumor by immune cells.