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Hemophilia A is an X-linked hereditary disorder that results from deficient coagulation factor VIII (FVIII) activity, leading to spontaneous bleeding episodes, particularly in joints and muscles. FVIII deficiency has been associated with altered bone remodeling, dysregulated macrophage polarization, and inflammatory processes that are associated with the neoformation of abnormal blood vessels. Treatment based on FVIII replacement can lead to the development of inhibitors that render FVIII concentrate infusion ineffective. In this context, hemophilia has entered a new therapeutic era with the development of new drugs, such as emicizumab, that seek to restore the hemostatic balance by bypassing pathologically acquired antibodies. We discuss the potential extrahemostatic functions of FVIII that may be crucial for defining future therapies in hemophilia.

Resumen Las distonias son trastornos del movimiento caracterizados por contracciones musculares sostenidas que producen movimientos repetitivos de torsion o posturas anormales. Pueden clasificarse segun la etiologia como primarias (formas idiopaticas y geneticas) o secundarias. La presentacion asociada con episodios generalizados, intensos y con exacerbacion de contracturas musculares intensas que suelen ser refractarias a la farmacoterapia tradicional se conoce como status distonico o tormenta distonica. Se presenta el caso de un paciente de 33 anos, con antecedente de sordera congenita, trastorno por consumo de sustancias psicoactivas y tratamiento psicofarmacologico con antipsicoticos, que presento un cuadro distonico grave que evoluciono a un status distonico.

García-Silva et al. show for the first time that extracellular vesicles isolated from the exudative seroma obtained from the lymphatic drainage implanted in melanoma patients after lymphadenectomy can be interrogated for melanoma markers and BRAF mutations. Profiling the BRAFV600E mutation in this biofluid is a novel approach to predict disease relapse., Liquid biopsies from cancer patients have the potential to improve diagnosis and prognosis. The assessment of surrogate markers of tumor progression in circulating extracellular vesicles could be a powerful non-invasive approach in this setting. We have characterized extracellular vesicles purified from the lymphatic drainage also known as exudative seroma (ES) of stage III melanoma patients obtained after lymphadenectomy. Proteomic analysis showed that seroma-derived exosomes are enriched in proteins resembling melanoma progression. In addition, we found that the BRAFV600E mutation can be detected in ES-derived extracellular vesicles and its detection correlated with patients at risk of relapse., Graphical Abstract

The development of noninvasive biomarkers that reflect the state of immunosuppression (IS) remains an unmet need in liver transplantation (LT). Torque Teno virus (TTV) is a highly prevalent, nonpathogenic DNA virus whose plasma levels may be associated with the immune status of the host. The aim of this study was to assess the role of TTV as a biomarker of IS in LT recipients. TTV DNA in plasma was quantified by real-time polymerase chain reaction at different time points during the first year after transplant in a prospectively followed cohort of 63 de novo LT recipients, and any correlation between TTV DNA and biopsy-proven acute cellular rejection (ACR) and opportunistic infections was then evaluated. In addition, TTV DNA was studied in 10 longterm LT recipients in monotherapy with tacrolimus, 10 tolerant recipients, and 10 healthy controls. TTV was detected in the plasma of all patients. Among the 63 LT recipients, 20 episodes of ACR were diagnosed, and there were 28 opportunistic infections, 26 of them being cytomegalovirus (CMV) infections. TTV viremia was significantly lower during ACR (4.41 versus 5.95 log10 copies/mL; P = 0.002) and significantly higher during CMV infections (5.79 versus 6.59 log10 copies/mL; P = 0.009). The area under the receiver operating characteristic curve of TTV viral load for the diagnosis of moderate ACR was 0.869, with a sensitivity and negative predictive value of 100%, respectively, for a cutoff point of 4.75 log10 copies/mL. There were no statistically significant differences in TTV DNA in either longterm or tolerant patients and healthy controls. In conclusion, plasma TTV DNA levels are associated with immune-related events after LT and could constitute a potential biomarker of the state of IS during the first months after transplant.

The thermal quality of the habitat is key for the regulation of body temperature in terrestrial ectotherms and, therefore, permits them to carry out their fundamental biological activities. In thermally heterogeneous environments, ectotherms might follow different behavioral or physiological strategies to maintain their body temperature within biologically adequate boundaries, for which they depend on microhabitat selection. These aspects are, thus, relevant in the context of habitat degradation and land-use change. In this study, we characterized the thermal ecology of three lizard species (genus Xantusia) that differ in microhabitat use along the Baja California peninsula, Mexico. We made three predictions: (1) the three species will follow different thermoregulatory strategies according to habitat thermal quality; (2) the thermal requirements and tolerances of these species will match the environmental or microenvironmental thermal conditions; and (3) due to their habitat and range restriction, the species studied will be highly vulnerable to climate change. Our results indicate the existence of thermoregulatory mechanisms in Xantusia to face thermal heterogeneity, including behavioral thermoregulation by choosing different microhabitats, shifts in activity periods, and adaptation to particular high thermal quality microhabitats. Furthermore, despite their association to specific microhabitats and specialized physiology, the studied species will not be adversely affected by climate change, as the increased microenvironmental temperatures will lead to a higher habitat thermal quality and lower costs of thermoregulation. However, we do not discard other indirect adverse effects of climate change not considered in this study.

Background One hundred fifty million contagions, more than 3 million deaths and little more than 1 year of COVID-19 have changed our lives and our health management systems forever. Ageing is known to be one of the significant determinants for COVID-19 severity. Two main reasons underlie this: immunosenescence and age correlation with main COVID-19 comorbidities such as hypertension or dyslipidaemia. This study has two aims. The first is to obtain cut-off points for laboratory parameters that can help us in clinical decision-making. The second one is to analyse the effect of pandemic lockdown on epidemiological, clinical, and laboratory parameters concerning the severity of the COVID-19. For these purposes, 257 of SARSCoV2 inpatients during pandemic confinement were included in this study. Moreover, 584 case records from a previously analysed series, were compared with the present study data. Results Concerning the characteristics of lockdown series, mild cases accounted for 14.4, 54.1% were moderate and 31.5%, severe. There were 32.5% of home contagions, 26.3% community transmissions, 22.5% nursing home contagions, and 8.8% corresponding to frontline worker contagions regarding epidemiological features. Age > 60 and male sex are hereby confirmed as severity determinants. Equally, higher severity was significantly associated with higher IL6, CRP, ferritin, LDH, and leukocyte counts, and a lower percentage of lymphocyte, CD4 and CD8 count. Comparing this cohort with a previous 584-cases series, mild cases were less than those analysed in the first moment of the pandemic and dyslipidaemia became more frequent than before. IL-6, CRP and LDH values above 69 pg/mL, 97 mg/L and 328 U/L respectively, as well as a CD4 T-cell count below 535 cells/μL, were the best cut-offs predicting severity since these parameters offered reliable areas under the curve. Conclusion Age and sex together with selected laboratory parameters on admission can help us predict COVID-19 severity and, therefore, make clinical and resource management decisions. Demographic features associated with lockdown might affect the homogeneity of the data and the robustness of the results.

In mammals, the KRAS locus encodes two protein isoforms, KRAS4A and KRAS4B, which differ only in their C terminus via alternative splicing of distinct fourth exons. Previous studies have shown that whereas KRAS expression is essential for mouse development, the KRAS4A isoform is expendable. Here, we have generated a mouse strain that carries a terminator codon in exon 4B that leads to the expression of an unstable KRAS4B154 truncated polypeptide, hence resulting in a bona fide Kras4B-null allele. In contrast, this terminator codon leaves expression of the KRAS4A isoform unaffected. Mice selectively lacking KRAS4B expression developed to term but died perinatally because of hypertrabeculation of the ventricular wall, a defect reminiscent of that observed in embryos lacking the Kras locus. Mouse embryonic fibroblasts (MEFs) obtained from Kras4B-/- embryos proliferated less than did wild-type MEFs, because of limited expression of KRAS4A, a defect that can be compensated for by ectopic expression of this isoform. Introduction of the same terminator codon into a Kras FSFG12V allele allowed expression of an endogenous KRAS4AG12V oncogenic isoform in the absence of KRAS4B. Exposure of Kras +/FSF4AG12V4B- mice to Adeno-FLPo particles induced lung tumors with complete penetrance, albeit with increased latencies as compared with control Kras +/FSFG12V animals. Moreover, a significant percentage of these mice developed proximal metastasis, a feature seldom observed in mice expressing both mutant isoforms. These results illustrate that expression of the KRAS4AG12V mutant isoform is sufficient to induce lung tumors, thus suggesting that selective targeting of the KRAS4BG12V oncoprotein may not have significant therapeutic consequences. We thank Marta San Roman, Raquel Villar, and Nuria Cabrera for excellent technical assistance; Mayte Lamparero and Isabel Blanco (Animal Facility) for mouse work; the Histopathology Unit for processing of mouse tissues; Lola Martinez (Flow Cytometry Unit) for her help with flow cytometry analyses; Diego Megias and Manuel Perez (Confocal Microscopy Unit) for assistance with confocal microscopy; and the Mouse Genome Editing Unit for support with the generation of the mouse strains described here. We also thank Ignacio Perez de Castro (Instituto de Salud Carlos III, Madrid, Spain) for sharing the EGFP-KRAS4B plasmid and Orlando Dominguez (Genomics Unit) and Pedro P. Lopez-Casas (Clinical Research Program) for their advice on exome sequencing. This work was supported by grants from the European Research Council (ERC-2015-AdG/695566, THERACAN), the Spanish Ministry of Science, Innovation and Universities (RTC-2017-6576-1), and the Autonomous Community of Madrid (B2017/BMD-3884 iLUNG-CM); a grant from the CRIS Cancer Foundation (to M.B.); and a grant from the Spanish Ministry of Science, Innovation and Universities (RTI2018-094664-B-I00, to M.B. and M.M.). M.B. is a recipient of an Endowed Chair from the AXA Research Fund. M.S. was supported by predoctoral contract "Severo Ochoa" (BES-2016-079096) from the SpanishMinistry of Science, Innovation and Universities. G.P. was a recipient of a "Young Ph.D." grant from the Government of the Community of Madrid. F.F.-G. was supported by a formacion de profesorado universitario (FPU) fellowship from the Spanish Ministry of Science, Innovation and Universities. Sí

International audience; Macrophages are specialized cells that control tissue homeostasis. They include non-resident and tissue-resident macrophage populations which are characterized by the expression of particular cell surface markers and the secretion of molecules with a wide range of biological functions. The differentiation and polarization of macrophages relies on specific growth factors and their receptors. Macrophage-colony stimulating factor (CSF-1) and interleukine-34 (IL-34), also known as "twin" cytokines, are part of this regluatory landscape. CSF-1 and IL-34 share a common receptor, the macrophage-colony stimulating factor receptor (CSF-1R), which is activated in a similar way by both factors and turns on identical signaling pathways. However, there is some discrete differential activation leading to specific activities. In this review, we disscuss recent progress in understanding of the role of the twin cytokines in macrophage differentiation, from their interaction with CSF-1R and the activation of signaling pathways, to their implication in macrophage polarization of non-resident and tissue-resident macrophages. A special focus on IL-34, its involvement in pathophsyiological contexts, and its potential as a theranostic target for macrophage therapy will be proposed.

Objectives: to verify which organizational, methodological, and resource-related characteristics of Continuing Health Education (CHE) help to best predict the professionals´ satisfaction. Method: a cross-sectional study with multivariate logistic regressions to predict a high mean satisfaction with different dimensions of educational actions used: Overall satisfaction, Utility, Methodology, Organization and resources, and Teaching Capacity. 25,281 satisfaction questionnaires have been analysed completed by health professionals attending 1,228 training activities in Andalusia (Spain), during the period from March 2012 to April 2015. Results: the characteristics that best predict a high overall satisfaction are the following: clinical session type as opposed to the workshop (Odds Ratio [OR]=2.07, p

Objetivo: realizar un estudio descriptivo sobre Cáncer Colorrectal en Cova da Beira Portugal y el valor pronóstico de BCL2 en asociación con la localización del tumor, Estadificación TNM, y tipo histológico. Materiales y Métodos: se realizó un estudio en 29 pacientes que tuvieron cirugía curativa para la escisión de Cáncer Colon Rectal (CCR) en Centro Hospitalario Cova da Beira con el objetivo de verificar si la presencia de la oncoproteína BCL2 en células neoplásicas es un factor predictivo del pronóstico, verificando también si es predictivo de la estadificación TNM, localización y diferenciación celular en el Cáncer Colon Rectal. Resultados: los hallazgos de este estudio coinciden con lo reportado en la literatura, se encontró que la incidencia del CCR es mayor en hombres que en mujeres, el riesgo de la enfermedad aumenta con la edad y la localización más frecuente de lesión neoplásica es en la porción izquierda del colon, con un total de 26 casos (89,65%). Además, se encontró asociación estadística de la expresión de BCL2 con el pronóstico y con la diferenciación histológica. Conclusión: a pesar de las limitaciones de este estudio, los datos hallados guardan relación con lo reportado en la literatura y establecen una asociación estadística de la expresión de BCL2 con el pronóstico y con la diferenciación histológica.

Ischemic-type biliary lesions (ITBLs) arise most frequently after donation after circulatory death (DCD) liver transplantation and result in high morbidity and graft loss. Many DCD grafts are discarded out of fear for this complication. In theory, microvascular thrombi deposited during donor warm ischemia might be implicated in ITBL pathogenesis. Herein, we aim to evaluate the effects of the administration of either heparin or the fibrinolytic drug tissue plasminogen activator (TPA) as means to improve DCD liver graft quality and potentially avoid ITBL. Donor pigs were subjected to 1 hour of cardiac arrest (CA) and divided among 3 groups: no pre-arrest heparinization nor TPA during postmortem regional perfusion; no pre-arrest heparinization but TPA given during regional perfusion; and pre-arrest heparinization but no TPA during regional perfusion. In liver tissue sampled 1 hour after CA, fibrin deposition was not detected, even when heparin was not given prior to arrest. Although it was not useful to prevent microvascular clot formation, pre-arrest heparin did offer cytoprotective effects during CA and beyond, reflected in improved flows during regional perfusion and better biochemical, functional, and histological parameters during posttransplantation follow-up. In conclusion, this study demonstrates the lack of impact of TPA use in porcine DCD liver transplantation and adds to the controversy over whether the use of TPA in human DCD liver transplantation really offers any protective effect. On the other hand, when it is administered prior to CA, heparin does offer anti-inflammatory and other cytoprotective effects that help improve DCD liver graft quality. Liver Transplantation 24 665-676 2018 AASLD.

Highlights • 3D cell cultures present similar drugs resistance features than in vivo tumours. • Unlike 2D cell cultures, 3D culture approaches mimic better the tumour microenvironment. • Collagen scaffolds result in a better osteosarcoma 3D proliferation than soft hydrogels. • Osteosarcoma 3D models constitute an appealing approach for bone mineralisation studies. • Combination of microfluidic/bioprinting technology with bone 3D cultures as ideal tools to study bone sarcomas., Bone sarcomas are rare tumour entities that arise from the mesenchyme most of which are highly heterogeneous at the cellular, genetic and epigenetic levels. The three main types are osteosarcoma, Ewing sarcoma, and chondrosarcoma. These oncological entities are characterised by high morbidity and mortality and an absence of significant therapeutic improvement in the last four decades. In the field of oncology, in vitro cultures of cancer cells have been extensively used for drug screening unfortunately with limited success. Indeed, despite the massive knowledge acquired from conventional 2D culture methods, scientific community has been challenged by the loss of efficacy of drugs when moved to clinical trials. The recent explosion of new 3D culture methods is paving the way to more relevant in vitro models mimicking the in vivo tumour environment (e.g. bone structure) with biological responses close to the in vivo context. The present review gives a brief overview of the latest advances of the 3D culture methods used for studying primary bone sarcomas.

Background The aim of this study is to determine values of insulinaemia, homeostasis model assessment (HOMA) index and quantitative insulin sensitivity check index (QUICKI) among a population of prepubertal Caucasian children, to analyse factors associated with insulin resistance (IR), and to study its association with cardiovascular risk factors. Materials and Methods Population-based study conducted on a randomly selected sample of prepubescent Caucasian subjects aged 2.00 to 9.99 years old. Anthropometric measurements, blood pressure, and fasting blood samples were obtained, including fasting glucose, triglycerides, High Density Lipoprotein (HDL)-cholesterol, and insulin. In addition, QUICKI and HOMA indices were calculated. Generalised additive models for location, scale and shape (GAMLSS) was used to calculate centiles curves and multivariate logistic regression analysis to assess factors associated with IR. Results A total of 654 subjects were included. Mean values obtained for insulinaemia, HOMA index, and QUICKI were 3.74 μIU/mL, 0.73, and 0.44, respectively, in the overall population and 3.32 μIU/mL, 0.64 and 0.46, respectively, in normal weight subjects. The main factor associated with IR was abdominal obesity (odds ratio [OR] 3.38 [95% CI 1.44-7.94] in the subgroup aged 2.00-5.99 years and OR 9.14 [3.42-24.41] for those aged 6.00-9.99 years). An increased risk of hyperglycaemia (P = 0.043), hypertriglyceridaemia (P

Purpose To evaluate the development of an extracorporeal membrane oxygenation (ECMO) program for the treatment of acute respiratory distress syndrome (ARDS) in adults. Methods a) Descriptive study of 15 cases treated since the program approval from 2010 to 2016. b) Case–control study matching the 15 ECMO cases with the 52 severe ARDS treated between 2005 and 2011 in which alternative rescue treatments (prone ventilation, tracheal gas insufflation (TGI) and/or the administration of inhaled nitric oxide (iNO)) were used. Results ECMO experience : Mortality 47% (7/15). Four patients died due to complications directly related to ECMO therapy. ICU stay 46.6 ± 45 days (range 4–138). Hospital stay 72.4 ± 98 days (range 4–320). Case–control : The mortality in the control group was 77% (44/52). The ECMO group practically doubled the mean days of ICU and hospital stay ( p Economic analysis : The hospital cost per patient in the ECMO group doubled compared to that of the control group (USD 77,099 vs USD 37,660). However, the cost per survivor was reduced by 4% (USD 144,560 vs USD 150,640, respectively). Conclusions Our results endorse the use of ECMO as a rescue therapy in adults with ARDS, although there are some risks associated with a learning curve as well as an important increase in the days of patient stay. The justification for the maintenance of an ECMO program in adults should be based on future studies of efficacy and cost effectiveness.

Mutations in genes encoding subunits of the SWI/SNF chromatin remodeling complex are frequently found in different human cancers. While the tumor suppressor function of this complex is widely established in solid tumors, its role in hematologic malignancies is largely unknown. Recurrent point mutations in BCL7A gene, encoding a subunit of the SWI/SNF complex, have been reported in diffuse large B-cell lymphoma (DLBCL), but their functional impact remains to be elucidated. Here we show that BCL7A often undergoes biallelic inactivation, including a previously unnoticed mutational hotspot in the splice donor site of intron one. The splice site mutations render a truncated BCL7A protein, lacking a portion of the amino-terminal domain. Moreover, restoration of wild-type BCL7A expression elicits a tumor suppressor-like phenotype in vitro and in vivo. In contrast, splice site mutations block the tumor suppressor function of BCL7A by preventing its binding to the SWI/SNF complex. We also show that BCL7A restoration induces transcriptomic changes in genes involved in B-cell activation. In addition, we report that SWI/SNF complex subunits harbor mutations in more than half of patients with germinal center B-cell (GCB)-DLBCL. Overall, this work demonstrates the tumor suppressor function of BCL7A in DLBCL, and highlights that the SWI/SNF complex plays a relevant role in DLBCL pathogenesis.

A primary limitation in hepatic surgery is leaving a remnant liver of adequate size and function. Experimental models have been designed to study processes of liver injury and regeneration in this context, yet a formula to accurately calculate liver mass in an animal model is lacking. This study aims to create a novel and simple formula to estimate the mass of the native liver in a species of pigs commonly used in experimental liver surgery protocols. Using data from 200 male weanling Landrace-Large White hybrid pigs, multiple linear regression analysis is used to generate the formula. Clinical features used as variables for the predictive model are body mass and length. The final formula for pig liver mass is as follows: Liver mass (g) = 26.34232 * Body mass (kg) – 1.270629 * Length (cm) + 163.0076; R2 = 0.7307. This formula for porcine liver mass is simple to use and may be helpful in studies using animals of similar characteristics to evaluate restoration of liver mass following major hepatectomy.

Objective: To determine blood lead levels (BLL) in a healthy paediatric population and to analyse related sociodemographic, dietary and haematological factors. Methods: A cross-sectional study was made of 1427 healthy subjects aged 1–16 years from the city of Almería (south-eastern Spain). BLL, iron parameters and erythropoietin were determined, and sociodemographic and dietary data obtained. The study paramateters was analyses in BLL toxic and BLL no toxic group by multiple logistic regression. Results: The mean BLL was 1.98 ± 1.1 µg/dL (95% CI:1.91–2.04). For 5.7% of the population, mean BLL was 2–5 µg/dL, for 2.1% it was &gt, 5 µg/dL and for 0.15% it was &gt, 10 µg/dL. Multivariate analysis showed that immigrant origin (OR:11.9, p &lt, 0.0001), low level of parental education (OR:4.6, 0.02) and low dietary iron bioavailability (OR: 3.2, 0.02) were all risk factors for toxic BLL. Subjects with toxic and non-toxic BLL presented similar iron and erythropoiesis-related parameters, except erythrocyte protoporphyrin, which was significantly higher in the BLL &gt, 5 µg/dL group. Conclusions: BLL and the prevalence of toxic BLL in healthy subjects aged 1–16 years living in south-eastern Spain are low and similar to those found in other developed countries. The factors associated with toxic BLL are immigrant origin, low level of parental education and dietary iron deficiency. The toxicity of BLL was not related to changes in the analytical parameters studied.

Epithelial malignancies are effectively treated by antiangiogenics; however, acquired resistance is a major problem in cancer therapeutics. Epithelial tumors commonly have mutations in the MAPK/Pi3K-AKT pathways, which leads to high-rate aerobic glycolysis. Here, we show how multikinase inhibitor antiangiogenics (TKIs) induce hypoxia correction in spontaneous breast and lung tumor models. When this happens, the tumors downregulate glycolysis and switch to long-term reliance on mitochondrial respiration. A transcriptomic, metabolomic, and phosphoproteomic study revealed that this metabolic switch is mediated by downregulation of HIF1α and AKT and upregulation of AMPK, allowing uptake and degradation of fatty acids and ketone bodies. The switch renders mitochondrial respiration necessary for tumor survival. Agents like phenformin or ME344 induce synergistic tumor control when combined with TKIs, leading to metabolic synthetic lethality. Our study uncovers mechanistic insights in the process of tumor resistance to TKIs and may have clinical applicability. M.Q.-F. is a recipient of the following grants: FIS PI10/0288 and FIS PI13/00430 from the Ministry of Health (Spain), 2010-BECA-RETORNO from theAECC Scientific Foundation, and donations from the Rosae Foundation andAvon Espan ̃a S.A.U. I.Z. is a recipient of La Caixa-CNIO PhD 2011 fellowship.M.Q.-F. received research funds from Boehringer-Ingelheim and Novartis. Sí

Post-transcriptional regulatory processes may change transcript levels and affect cell-to-cell variability or noise. We study small-RNA downregulation to elucidate its effects on noise in the iron homeostasis network of Escherichia coli. In this network, the small-RNA RyhB undergoes stoichiometric degradation with the transcripts of target genes in response to iron stress. Using single-molecule fluorescence in situ hybridization, we measured transcript numbers of the RyhB-regulated genes sodB and fumA in individual cells as a function of iron deprivation. We observed a monotonic increase of noise with iron stress but no evidence of theoretically predicted, enhanced stoichiometric fluctuations in transcript numbers, nor of bistable behavior in transcript distributions. Direct detection of RyhB in individual cells shows that its noise is much smaller than that of these two targets, when RyhB production is significant. A generalized two-state model of bursty transcription that neglects RyhB fluctuations describes quantitatively the dependence of noise and transcript distributions on iron deprivation, enabling extraction of in vivo RyhB-mediated transcript degradation rates. The transcripts' threshold-linear behavior indicates that the effective in vivo interaction strength between RyhB and its two target transcripts is comparable. Strikingly, the bacterial cell response exhibits Furdependent, switch-like activation instead of a graded response to iron deprivation. Israel Science Foundation [514415 to J.S.]; Feinberg Foundation Visiting Faculty Program ( to J.M.-G.); MICINN (Spain) [FIS2012-32349 to J.M.-G.]; Intramural Research Program of the National Institutes of Health (to D.L.C.); National Cancer Institute (to D.L.C.); Center for Cancer Research (to D.L.C.); Siegfried and Irma Ullman Professorial Chair ( to J. S.). Funding for open access charge: Israel Science Foundation.

Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates various aspects of DNA replication. We previously showed that the chromatin around replisomes is rich in SUMO and depleted in Ub, whereas an opposite pattern is observed in mature chromatin. How this SUMO-rich/Ub-low environment is maintained at sites of DNA replication is not known. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Chemical inhibition or genetic deletion of USP7 leads to the accumulation of Ub on SUMOylated proteins, which are displaced to chromatin away from replisomes. Our findings provide a model to explain the differential accumulation of SUMO and Ub at replication forks, and identify an essential role of USP7 in DNA replication that should be taken into account for the use of USP7 inhibitors as anticancer agents.

Despite significant efforts to improve pancreatic ductal adenocarcinoma (PDAC) clinical outcomes, overall survival remains dismal. The poor response to current therapies is partly due to the existence of pancreatic cancer stem cells (PaCSCs), which are efficient drivers of PDAC tumorigenesis, metastasis and relapse. To find new therapeutic agents that could efficiently kill PaCSCs, we screened a chemical library of 680 compounds for candidate small molecules with anti-CSC activity, and identified two compounds of a specific chemical series with potent activity in vitro and in vivo against patient-derived xenograft (PDX) cultures. The anti-CSC mechanism of action of this specific chemical series was found to rely on induction of lysosomal membrane permeabilization (LMP), which is likely associated with the increased lysosomal mass observed in PaCSCs. Using the well characterized LMP-inducer siramesine as a tool molecule, we show elimination of the PaCSC population in mice implanted with tumors from two PDX models. Collectively, our approach identified lysosomal disruption as a promising anti-CSC therapeutic strategy for PDAC., This study was financially supported by a Ramón y Cajal Merit Award (RYC-2012-12104) from the Ministerio de Economía y Competitividad, Spain (B.S.Jr.); funding fromThe Fero Foundation (B.S.Jr.); a Coordinated grant from the Fundación Asociación Española Contra el Cáncer (AECC) GC16173694BARB (B.S.,Jr.); Fondo de Investigaciones Sanitarias (FIS) grant PI15/01507 and PI18/00757 (B.S.,Jr.), (cofinanced through Fondo Europeo de Desarrollo Regional (FEDER) “Una manera de hacer Europa”). ETP activities were supported by funds of The Spanish National Cancer Research Centre (CNIO).Work in the laboratory of M.S. was funded by the CNIO, the IRB and “laCaixa” Foundation, and by grants from the Spanish Ministry of Economy co-funded by the European Regional Development Fund (ERDF) (SAF2017-82613-R), the European Research Council (ERC-2014-AdG/669622) and, Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement of Catalonia (Grup de Recerca consolidat 2017 SGR 282).

For ectotherms, thermal physiology plays a fundamental role in the establishment and success of invasive species in novel areas and, ultimately, in their ecological interactions with native species. Invasive species are assumed to have a greater ability to exploit the thermal environment, higher acclimation capacities, a wider thermal tolerance range, and better relative performance under a range of thermal conditions. Here we compare the thermal ecophysiology of two species that occur in sympatry in a tropical dry forest of the Pacific coast of Mexico, the microendemic species Benedetti's Leaf-toed Gecko (Phyllodactylus benedettii) and the invasive Common House Gecko (Hemidactylus frenatus). We characterized their patterns of thermoregulation, thermoregulatory efficiency, thermal tolerances, and thermal sensitivity of locomotor performance. In addition, we included morphological variables and an index of body condition to evaluate their effects on the thermal sensitivity of locomotor performance in these species. Although the two species had similar selected temperatures and thermal tolerances, they contrasted in their thermoregulatory strategies and thermal sensitivity of locomotor performance. Hemidactylus frenatus had a higher performance than the native species, P. benedettii, which would represent an ecological advantage for the former species. Nevertheless, we suggest that given the spatial and temporal limitations in habitat use of the two species, the probability of agonistic interactions between them is reduced. We recommend exploring additional biotic attributes, such as competition, behavior and niche overlap in order assess the role of alternative factors favoring the success of invasive species.

La hiperplasia idiopatica difusa de celulas endocrinas es considerada una lesion premaligna por la Organizacion Mundial de la Salud desde 1999. Ya han sido publicados numerosos casos a lo largo de los anos sobre este tema, describiendo sus caracteristicas. El primer caso, una mujer de mediana edad, diagnosticada de asma, con un patron nodular bilateral en las pruebas de imagen y de curso clinico favorable, es buen ejemplo-resumen de la forma mas frecuente de presentacion. El segundo caso describe por primera vez un posible acumulo familiar con dos pacientes en la misma familia. Confiando en aumentar la casuistica sobre esta patologia, y en anadir alguna novedad, presentamos los siguientes casos clinicos.

MASTL, a Ser/Thr kinase that inhibits PP2A-B55 complexes during mitosis, is mutated in autosomal dominant thrombocytopenia. However, the connections between the cell cycle machinery and this human disease remain unexplored. We report here that, whereas Mastl ablation in megakaryocytes prevented proper maturation of these cells, mice carrying the thrombocytopenia-associated mutation developed thrombocytopenia as a consequence of aberrant activation and survival of platelets. Activation of mutant platelets was characterized by hyper-stabilized pseudopods mimicking the effect of PP2A inhibition and actin polymerization defects. These aberrations were accompanied by abnormal hyper-phosphorylation of multiple components of the actin cytoskeleton and were rescued both in vitro and in vivo by inhibiting upstream kinases such as PKA, PKC, or AMPK. These data reveal an unexpected role of Mastl in actin cytoskeleton dynamics in postmitotic cells, and suggest that the thrombocytopenia-associated mutation in MASTL is a pathogenic dominant mutation that mimics decreased PP2A activity resulting in altered phosphorylation of cytoskeletal regulatory pathways. We thank Peter Storz (Mayo Clinic; Jacksonville, FL) for sharin g reagents and Sheila Rueda for her support with the management of the mouse colony. B.H. and R.S.-M. were supported by the Juan de la Cierva Programme from the Spanish M inistry of Economy and Competitiveness (MINECO). M.T. was supported by Foundation La Caixa. A.E.B. was supported by the Programa de Empleo Juvenil, Comunidad de M adrid. M.A.-F. received a young investigator g rant from MINECO (SAF2014-60442- JIN; co-financed by FEDER funds). P.G.dF. was supported by Fundació la Marató de TV3 (project 080121 and project 20153031). J.M. was supported by the Ramon y Cajal programme (MINECO; RYC-2012-10651). M.M. lab. is supported by grants from the MINE CO (SAF2015- 69920-R), Programa iLUNG from the Comunidad de Madrid (B2017/BM D-3884), and Worldwide Cancer Research (15-0278). CNIO is a Severo Ochoa Cen ter of Excellence (MINECO awards SEV-2015-0510) Sí

Background Community-acquired pneumonia (CAP) is a lung infection that can be acquired during day-to-day activities in the community (not while receiving care in a hospital). Community-acquired pneumonia poses a significant public health burden in terms of mortality, morbidity, and costs. Shorter antibiotic courses for CAP may limit treatment costs and adverse effects, but the optimal duration of antibiotic treatment is uncertain. Objectives To evaluate the efficacy and safety of short-course versus longer-course treatment with the same antibiotic at the same daily dosage for CAP in non-hospitalised adolescents and adults (outpatients). We planned to investigate non-inferiority of short-course versus longer-term course treatment for efficacy outcomes, and superiority of short-course treatment for safety outcomes. Search methods We searched CENTRAL, which contains the Cochrane Acute Respiratory Infections Group Specialised Register, MEDLINE, Embase, five other databases, and three trials registers on 28 September 2017 together with conference proceedings, reference checking, and contact with experts and pharmaceutical companies. Selection criteria Randomised controlled trials (RCTs) comparing short- and long-courses of the same antibiotic for CAP in adolescent and adult outpatients. Data collection and analysis We planned to use standard Cochrane methods. Main results Our searches identified 5260 records. We did not identify any RCTs that compared short- and longer-courses of the same antibiotic for the treatment of adolescents and adult outpatients with CAP.We excluded two RCTs that compared short courses (five compared to seven days) of the same antibiotic at the same daily dose because they evaluated antibiotics (gemifloxacin and telithromycin) not commonly used in practice for the treatment of CAP. In particular, gemifloxacin is no longer approved for the treatment of mild-to-moderate CAP due to its questionable risk-benefit balance, and reported adverse effects. Moreover, the safety profile of telithromycin is also cause for concern.We found one ongoing study that we will assess for inclusion in future updates of the review. Authors' conclusions We found no eligible RCTs that studied a short-course of antibiotic compared to a longer-course (with the same antibiotic at the same daily dosage) for CAP in adolescent and adult outpatients. The effects of antibiotic therapy duration for CAP in adolescent and adult outpatients remains unclear.

OBJECTIVES: Respiratory infections (RIs) are an important cause of morbidity and excessive antibiotic prescriptions in children attending day care centers (DCCs). We aimed to assess the effectiveness of an educational and hand hygiene program in DCCs and homes in reducing RI incidence and antibiotic prescriptions in children. METHODS: A cluster, randomized, controlled, and open study of 911 children aged 0 to 3 years attending 24 DCCs in Almería (Spain) with an 8-month follow-up. Two intervention groups of DCC families performed educational and hand hygiene measures, 1 with soap and water (SWG; n = 274), another with hand sanitizer (HSG; n = 339), and the control group (CG; n = 298) followed usual hand-washing procedures. RI episode rates were compared through multilevel Poisson regression models. The percentage of days missed were compared with Poisson exact tests. RESULTS: There were 5211 RI episodes registered. Children in the HSG had less risk of RI episodes (incidence rate ratio [IRR]: 0.77; 95% confidence interval [CI]: 0.68–0.88) and antibiotic prescriptions (IRR: 0.69; 95% CI: 0.57–0.84) compared with the those in the CG. Children in the SWG had a higher risk of RI episodes (IRR: 1.21; 95% CI: 1.06–1.39) and antibiotic prescriptions (IRR: 1.31; 95% CI: 1.08–1.56) than those in the HSG. Pupils missed 5186 DCC days because of RIs, and the percentage of days absent was significantly lower in the HSG compared with the CG (P < .001) and the SWG (P < .001). CONCLUSIONS: Hand hygiene programs that include hand sanitizer and educational measures for DCC staff, children, and parents, reduce absent days, RIs, and antibiotic prescriptions for these infections in children at DCCs.

SUMMARYSenescence is a cellular phenotype characterized by an irreversible cell cycle arrest and the secretion of inflammatory proteins, denominated senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, until now this role has been mainly attributed to soluble factors. Here, we report that extracellular vesicles also alter the environment by transmitting the senescent phenotype to other cells via exosomes (extracellular vesicles of endocytic origin). A combination of functional assays, Cre-/oxP reporter systems, proteomic analysis and RNAi screens confirm that exosomes form part of the senescent secretome and mediate paracrine senescence via the activation of a non-canonical interferon (IFN) pathway. Altogether, we speculate that exosomes could be drivers of tissue degeneration both locally and systemically during aging and age- related disease.

Ibuprofen (IBU-H), a widely used anti-inflammatory, also shows a marked antimicrobial effect against several bacterial species, including those involved in cystic fibrosis such as Pseudomona aeruginosa, methicillin resistant Staphylococcus aureus and Burkholderia cepacia complex. Additionally, our results show significant synergy between water soluble Na-ibuprofen (IBU-Na) and ionic strength. Salt concentrations above 0.5 M modify the zeta potential promoting the action of Na-IBU; thus, with 1 M sodium chloride, IBU-Na is ten times more efficient than in the absence of ionic strength, and the minimum effective contact time is reduced from hours to minutes. In short time periods, where neither IBU-Na nor controls with 1 M NaCl show activity, the combination of both leads to a reduction in the bacterial load. We also analyzed whether the changes caused by salt on the bacterial membrane also promoted the activity of other microbicide compounds used in cystic fibrosis like gentamicin, tobramycin and phosphomycin. The results show that the presence of ionic strength only enhanced the bactericidal activity of the amphipathic molecule of IBU-Na. In this respect, the effect of saline concentration was also reflected in the surface properties of IBU-Na, where, in addition to the clear differences observed between 145 mM and 1 M, singular behaviors were also found, different in each condition. The combination of anti-inflammatory activity and this improved bactericidal effect of Na-IBU in hypertonic solution provides a new alternative for the treatment of respiratory infections of fibrotic patients based on known and widely used compounds.

Microfluidic technologies have become a standard tool in cell biological studies, offering unprecedented control of the chemical and physical environment of cells grown in microdevices, the possibility of multiplexing assays, as well as the capacity to monitor the behavior of single cells in real time while dynamically manipulating their growth medium. However, the properties of the materials employed for the fabrication of microchips that are compatible with live-cell imaging has limited the use of these techniques for a broad range of experiments. In particular, the strong absorption of a large panel of small molecules by these materials prevents the accurate delivery of compounds of interest. Here we describe a novel microsystem dedicated to live-cell imaging that (1) uses alternative materials devoid of absorptive properties, and (2) allows for dynamic in-chip control of sample temperature. Based on a proof-of-concept design that we have routinely used with non-adherent fission yeast cells, this chapter details all the steps for the fabrication and utilization of these microdevices.

El presente estudio responde a la necesidad de obtener datos a escala global sobre los profesionales del español como lengua extranjera (ELE) o segunda (EL2), ante la escasez de información de carácter empírico sobre la profesión. Para ello, se dan a conocer las respuestas de 1675 informantes de 84 países obtenidas mediante un cuestionario en línea estructurado en tres bloques temáticos: a) perfil laboral y docente, b) perfil formativo, y c) perfil investigador. A partir del análisis cuantitativo y cualitativo de las respuestas, ofrecemos perspectivas actuales sobre los profesionales del español en el mundo y sobre los diversos ámbitos en los que se desenvuelven. El cotejo de los resultados permite dilucidar algunos de los retos y desafíos inmediatos a los que se enfrentan los profesionales del idioma. El estudio concluye con unas líneas de actuación concretas para cada perfil analizado. Esta es la primera investigación empírica que ofrece datos sobre la profesión desde tres puntos de vista distintos pero complementarios. This study fills the gap in the availability of empirical data on L2 Spanish language professionals around the world. We surveyed 1,675 individuals from 84 countries through an online questionnaire divided into three thematic sections: a) professional profile, b) educational profile, and c) research profile. The analysis of this unique set of quantitative and qualitative data enables us to offer current and novel perspectives on L2 Spanish language professionals worldwide and on the different environments in which they work, as well as to analyse the main challenges and opportunities that L2 Spanish language professionals face. Based on these data, the study concludes with concrete recommended lines of action for each of the three areas. This is the first empirical study to offer data on the profession from three different, but complementary, angles. La publicación de este artículo se enmarca en la beca otorgada a Javier Muñoz-Basols como investigador visitante de la Universidad de Salamanca en el Programa “CEI 15-11 USAL en red” asociado al Centro Internacional del Español. La información aquí presentada pertenece también a los proyectos I+D+i “Identidades y culturas digitales en la educación lingüística” (EDU2014-57677-C2-1-R) y “Género, humor e identidad: desarrollo, consolidación y aplicabilidad de mecanismos lingüísticos en español” (FFI2015-64540-C2-1-P), del programa estatal de investigación, desarrollo e innovación orientado a los retos de la sociedad, del Ministerio de Economía y Competitividad de España. Asimismo, la elaboración del estudio y su publicación han sido posibles gracias al proyecto I+D+i concedido a Alberto Rodríguez-Lifante, “Competencia existencial en el profesorado de español como lengua extranjera en Grecia” (ACIE16-8).

The brain microenvironment imposes a particularly intense selective pressure on metastasis-initiating cells, but successful metastases bypass this control through mechanisms that are poorly understood. Reactive astrocytes are key components of this microenvironment that confine brain metastasis without infiltrating the lesion. Here, we describe that brain metastatic cells induce and maintain the co-option of a pro-metastatic program driven by signal transducer and activator of transcription 3 (STAT3) in a subpopulation of reactive astrocytes surrounding metastatic lesions. These reactive astrocytes benefit metastatic cells by their modulatory effect on the innate and acquired immune system. In patients, active STAT3 in reactive astrocytes correlates with reduced survival from diagnosis of intracranial metastases. Blocking STAT3 signaling in reactive astrocytes reduces experimental brain metastasis from different primary tumor sources, even at advanced stages of colonization. We also show that a safe and orally bioavailable treatment that inhibits STAT3 exhibits significant antitumor effects in patients with advanced systemic disease that included brain metastasis. Responses to this therapy were notable in the central nervous system, where several complete responses were achieved. Given that brain metastasis causes substantial morbidity and mortality, our results identify a novel treatment for increasing survival in patients with secondary brain tumors. Reactive astrocytes expressing STAT3 support the metastatic growth of tumor cells colonizing the brain and can be pharmacologically targeted to improve the clinical management of patients with secondary brain tumors.

Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms Activation of the PTEN-PI3K-mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation,. Here we show that mTORC1 regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. Through the use of integrative metabolomics in a mouse model and human biopsies of prostate cancer, we identified alterations in tumours impacting on the production of decarboxylated S-adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S-adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation was validated in murine and human cancer specimens. AMD1 was upregulated in prostate cancer specimens with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus exhibited a predominant decrease in AMD1 immunoreactivity that was associated to a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program.

The purpose of this study was to evaluate the most appropriate conditions to generate silver nanoparticles (AgNPs) loaded with a potent antimycotic drug like amphotericin B (AmB), characterize the physicochemical properties, and to evaluate the cytotoxic effect and biological activity of these new nanostructures as a potential nanocarrier for hydrophobic drugs. It was determined that the optimal molar ratio between Ag and AmB is 1/1 given the uniformity of size around 170 nm of the nanoparticles generated as well as their strongly negative ζ potential of -35 mV, a condition that favors repulsions between AgNPs and inhibiting their aggregation. In this condition, only 0.8 mg.mL-1 of Ag is needed to solubilize 5 mg.mL-1 of AmB, a concentration currently used in commercial formulations. It is important to emphasize that the loading capacity (w/w) of this nanostructure is much higher than that of micellar and liposomal formulations. These AgNP-AmB nanoparticles retain both the bactericidal effect of silver and the cytotoxic and antifungal effect of AmB. However, it was shown that these nanoparticles are spontaneously associated with plasma lipoproteins (LDL and HDL), inhibiting their cytotoxic effects on red blood cells and on at least two cell lines, Vero and H1299 and slightly reducing its bactericidal effect on P. aeruginosa. In contrast, the antifungal effect of the formulation is maintained and is even higher than that when the nanoparticle is not associated with lipoproteins, indicating that this association is of the reversible type. The characterization of these nanoparticles is discussed as a potential new model formulation able to improve the antifungal therapeutic efficiency of AmB. Fil: Leonhard, Victoria. Provincia de Córdoba. Ministerio de Ciencia y Técnica. Centro de Excelencia en Productos y Procesos de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina Fil: Alasino, Roxana Valeria. Provincia de Córdoba. Ministerio de Ciencia y Técnica. Centro de Excelencia en Productos y Procesos de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina Fil: Muñoz, Adrián Javier. Provincia de Córdoba. Ministerio de Ciencia y Técnica. Centro de Excelencia en Productos y Procesos de Córdoba; Argentina Fil: Beltramo, Dante Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Universidad Católica de Córdoba; Argentina. Provincia de Córdoba. Ministerio de Ciencia y Técnica. Centro de Excelencia en Productos y Procesos de Córdoba; Argentina

Cutaneous melanoma is a type of cancer with an inherent potential for lymph node colonization, which is generally preceded by neolymphangiogenesis1,2,3. However, sentinel lymph node removal does not necessarily extend the overall survival of patients with melanoma4,5. Moreover, lymphatic vessels collapse and become dysfunctional as melanomas progress6,7. Therefore, it is unclear whether (and how) lymphangiogenesis contributes to visceral metastasis. Soluble and vesicle-associated proteins secreted by tumours and/or their stroma have been proposed to condition pre-metastatic sites in patients with melanoma8,9,10,11,12,13,14. Still, the identities and prognostic value of lymphangiogenic mediators remain unclear2,14. Moreover, our understanding of lymphangiogenesis (in melanomas and other tumour types) is limited by the paucity of mouse models for live imaging of distal pre-metastatic niches15. Injectable lymphatic tracers have been developed7, but their limited diffusion precludes whole-body imaging at visceral sites16. Vascular endothelial growth factor receptor 3 (VEGFR3) is an attractive ‘lymphoreporter’17 because its expression is strongly downregulated in normal adult lymphatic endothelial cells, but is activated in pathological situations such as inflammation and cancer17,18. Here, we exploit this inducibility of VEGFR3 to engineer mouse melanoma models for whole-body imaging of metastasis generated by human cells, clinical biopsies or endogenously deregulated oncogenic pathways. This strategy revealed early induction of distal pre-metastatic niches uncoupled from lymphangiogenesis at primary lesions. Analyses of the melanoma secretome and validation in clinical specimens showed that the heparin-binding factor midkine is a systemic inducer of neo-lymphangiogenesis that defines patient prognosis. This role of midkine was linked to a paracrine activation of the mTOR pathway in lymphatic endothelial cells. These data support the use of VEGFR3 reporter mice as a ‘MetAlert’ discovery platform for drivers and inhibitors of metastasis., M.S.S. is funded by grants from the Spanish Ministry of Economy and Innovation (project SAF2014-56868-R), the Asociación Española Contra el Cáncer (AECC), the Worldwide Cancer Research, an Established Investigator Award from the Melanoma Research Alliance (MRA), and a L’Oréal Paris USA-MRA Team Science Award for Woman in Scientific Research. The CNIO Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. N.I. and J.M. are funded by SAF2013-45504-R (MINECO). J.M. is also supported by Ramon y Cajal Programme (MINECO) RYC-2012-10651. J.L.R.-P and P.O.-R are funded by grants FIS 2014/1737, 11/02568 and FIS 2014/01784, 11/1759, respectively, from the Spanish Ministry of Health. F.M. is funded by the AMIT Project/CDTI/CENIT Programme (MICINN), S.O. by SAF2013-44866-R (MINECO), and J.J.B.-C. by an NCI K22CA196750 grant and the TCI Young Scientist Cancer Research Award JJR Fund (P30 CA196521). J.D.M. is the recipient of a postdoctoral fellowship from the ARC Foundation and E.R.-F. from Fundación Científica de la Asociación Española Contra el Cáncer. D.C.-W. is the recipient of a predoctoral fellowship from Fundación La Caixa, and M.C.-A. and X.C. are recipients of the Immutrain Marie Skłodowska-Curie ITN Grant.

Summary Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells. Analysis of individual cells internalizing sEVs, using a Cre-reporter system, show a positive correlation between sEV uptake and senescence activation. We find an increase in the number of multivesicular bodies during senescence in vivo. sEV protein characterization by mass spectrometry (MS) followed by a functional siRNA screen identify interferon-induced transmembrane protein 3 (IFITM3) as being partially responsible for transmitting senescence to normal cells. We find that sEVs contribute to paracrine senescence., Graphical Abstract, Highlights • Small extracellular vesicles (sEVs) mediate paracrine senescence • Inhibition of sEV biogenesis prevents paracrine senescence • Multivesicular bodies and CD63 are increased during senescence in vivo • MS analysis identifies IFITM3 as partially responsible for sEV-paracrine senescence, Borghesan et al. show that the soluble fraction and small extracellular vesicles (sEVs) mediate paracrine senescence. RNA sequencing and loxP reporter systems confirm sEV-mediated paracrine senescence, while preventing sEV release averts senescence. Mass spectrometry and functional analysis show that the IFN protein, IFITM3, is partially responsible for this phenotype.

López-Contreras, Andrés J. et al., DNA replication is facilitated by multiple factors that concentrate in the vicinity of replication forks. Here, we developed an approach that combines the isolation of proteins on nascent DNA chains with mass spectrometry (iPOND-MS), allowing a comprehensive proteomic characterization of the human replisome and replisome-associated factors. In addition to known replisome components, we provide a broad list of proteins that reside in the vicinity of the replisome, some of which were not previously associated with replication. For instance, our data support a link between DNA replication and the Williams-Beuren syndrome and identify ZNF24 as a replication factor. In addition, we reveal that SUMOylation is widespread for factors that concentrate near replisomes, which contrasts with lower UQylation levels at these sites. This resource provides a panoramic view of the proteins that concentrate in the surroundings of the replisome, which should facilitate future investigations on DNA replication and genome maintenance., A.J.L. is the recipient of a postdoctoral fellowship from the Spanish Association Against Cancer (AECC). M.N. and S.R. are funded by Ph.D. fellowships from the La Caixa Foundation. Work in O.F.’s laboratory is supported by grants from the Spanish Ministry of Economy (SAF2011-23753 and CSD2007-00017), the Association for International Cancer Research (12-0229), the Howard Hughes Medical Institute, and the European Research Council (ERC-210520). Work in J.M.’s laboratory is supported by grants from the Spanish Ministry of Economy (BFU2010-21467 and CSD2007-00015).

Despite the maturity reached by targeted proteomic strategies, reliable and standardized protocols are urgently needed to enhance reproducibility among different laboratories and analytical platforms, facilitating a more widespread use in biomedical research. To achieve this goal, the use of dimensionless relative retention times (iRT), defined on the basis of peptide standard retention times (RT), has lately emerged as a powerful tool. The robustness, reproducibility and utility of this strategy were examined for the first time in a multicentric setting, involving 28 laboratories that included 24 of the Spanish network of proteomics laboratories (ProteoRed-ISCIII). According to the results obtained in this study, dimensionless retention time values (iRTs) demonstrated to be a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups both intra- and inter-laboratories. iRT values also showed very low variability over long time periods. Furthermore, parallel quantitative analyses showed a high reproducibility despite the variety of experimental strategies used, either MRM (multiple reaction monitoring) or pseudoMRM, and the diversity of analytical platforms employed. [Biological significance]: From the very beginning of proteomics as an analytical science there has been a growing interest in developing standardized methods and experimental procedures in order to ensure the highest quality and reproducibility of the results. In this regard, the recent (2012) introduction of the dimensionless retention time concept has been a significant advance. In our multicentric (28 laboratories) study we explore the usefulness of this concept in the context of a targeted proteomics experiment, demonstrating that dimensionless retention time values is a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups., All laboratories from Spain are members of ProteoRed (Plataforma de Recursos Biomoleculares y Bioinformáticos) and are supported bygrant PT13/0001 funded by Instituto de Salud Carlos III (ISCIII) andFEDER

Cyanobacteria forming one-dimensional filaments are paradigmaticmodel organisms of the transition between unicellular andmulticellular living forms. Under nitrogen-limiting conditions, infilaments of the genus Anabaena, some cells differentiate into heterocysts,which lose the possibility to divide but are able to fixenvironmental nitrogen for the colony. These heterocysts form aquasiregular pattern in the filament, representing a prototype ofpatterning and morphogenesis in prokaryotes. Recent years haveseen advances in the identification of the molecular mechanism regulatingthis pattern. We use these data to build a theory on heterocystpattern formation, for which both genetic regulation and theeffects of cell division and filament growth are key components. Thetheory is based on the interplay of three generic mechanisms: localautoactivation, early long-range inhibition, and late long-range inhibition.These mechanisms can be identified with the dynamics ofhetR, patS, and hetN expression. Our theory reproduces quantitativelythe experimental dynamics of pattern formation and maintenancefor wild type and mutants. We find that hetN alone is notenough to play the role as the late inhibitory mechanism: a secondmechanism, hypothetically the products of nitrogen fixation suppliedby heterocysts, must also play a role in late long-range inhibition.The preponderance of even intervals between heterocysts arisesnaturally as a result of the interplay between the timescales of geneticregulation and cell division.We also find that a purely stochasticinitiation of the pattern, without a two-stage process, is enoughto reproduce experimental observations. Funding from the Spanish Ministry of Economy and Competitiveness through Grant PHYSDEV (FIS2012-32349) and the Ramón y Cajal Program (to S.A.).

Two types of stem cells are currently defined in small intestinal crypts: cycling crypt base columnar (CBC) cells and quiescent ‘+4’ cells. Here, we combine transcriptomics with proteomics to define a definitive molecular signature for Lgr5 + CBC cells. Transcriptional profiling of FACS‐sorted Lgr5 + stem cells and their daughters using two microarray platforms revealed an mRNA stem cell signature of 384 unique genes. Quantitative mass spectrometry on the same cell populations identified 278 proteins enriched in intestinal stem cells. The mRNA and protein data sets showed a high level of correlation and a combined signature of 510 stem cell‐enriched genes was defined. Spatial expression patterns were further characterized by mRNA in‐situ hybridization, revealing that approximately half of the genes were expressed in a gradient with highest levels at the crypt bottom, while the other half was expressed uniquely in Lgr5 + stem cells. Lineage tracing using a newly established knock‐in mouse for one of the signature genes, Smoc2 , confirmed its stem cell specificity. Using this resource, we find—and confirm by independent approaches—that the proposed quiescent/‘+4’ stem cell markers Bmi1 , Tert , Hopx and Lrig1 are robustly expressed in CBC cells.

Introduction Differences between the opinions of patients and physicians on the impact of asthma are common. We hypothesised that patient–physician discordance may negatively affect asthma outcome. Methods A total of 2902 patients (61% women, mean age 47 years) with moderate–severe asthma and 231 physicians participated in a prospective study. At the baseline visit, data on demographics, clinical variables, degree of asthma control according to the Asthma Control Test (ACT), basic spirometry and the Hospital Anxiety and Depression Scale (HADS) were collected and an ad hoc questionnaire was completed that allowed the degree of concordance between doctors and patients to be assessed. A scheduled telephone call after 3 months was used to elicit the ACT score and the future risk of asthma. At the final visit at 6 months, the following data were recorded: ACT score, spirometry, HADS score and an ad hoc questionnaire to assess the agreement between the doctor and the patient. Changes in study variables according to patient–physician concordance or discordance were analysed. Results The rate of patient–physician discordance was 27.2%, with overestimation of disease impact by the physician in 12.3% and underestimation in 14.9%. Patient–physician opinion discordance, particularly in the case of physicians underestimating the impact of asthma, showed worse results with statistically significant differences in ACT score, a higher percentage of patients with poor asthma control and lower HADS scores. The need for hospital and emergency department admissions was also higher. Conclusion Patient–physician opinion discordance may be contributing to lower symptomatic control and increased future risk, with a higher impact when physicians underestimate the impact of asthma on their patients.

Contextual cues associated with previous drug exposure can trigger drug craving and seeking, and form a substantial obstacle in substance use recovery. Using in vivo imaging in mice, we found that cocaine administration induced a rapid increase in the formation and accumulation of new dendritic spines, and that measures of new persistent spine gain correlated with cocaine conditioned place preference. Our data suggest that new persistent spine formation in the frontal cortex may be involved in stimulant-related learning driving appetitive behavior.

In an active, self-ubiquitinated state, the Ring1B ligase monoubiquitinates histone H2A playing a critical role in Polycomb-mediated gene silencing. Following ubiquitination by external ligases, Ring1B is targeted for proteosomal degradation. Using biochemical data and computational modeling, we show that the Ring1B ligase can exhibit abrupt switches, overshoot transitions and self-perpetuating oscillations between its distinct ubiquitination and activity states. These different Ring1B states display canonical or multiply branched, atypical polyubiquitin chains and involve association with the Polycomb-group protein Bmi1. Bistable switches and oscillations may lead to all-or-none histone H2A monoubiquitination rates and result in discrete periods of gene (in)activity. Switches, overshoots and oscillations in Ring1B catalytic activity and proteosomal degradation are controlled by the abundances of Bmi1 and Ring1B, and the activities and abundances of external ligases and deubiquitinases, such as E6-AP and USP7., Author Summary The generation of polyubiquitin chains on target proteins as a degradation signal was a landmark discovery rewarded by the 2004 Nobel Prize in Chemistry. However, emerging evidence suggests that protein ubiquitination is more versatile. Different types of ubiquitin chains serve numerous non-proteolytic functions, among them regulation of the biological activities of target proteins. Here we demonstrate a flexible role of ubiquitination in the dynamic control of Ring1B, a ubiquitin ligase that monoubiquitinates histone H2A, which in turn silences gene expression. Remarkably, Ring1B increases its own activity by self-ubiquitination. A binding partner of Ring1B, Bmi1, facilitates Ring1B self-ubiquitination and protects both proteins from rapid degradation. We use computational modeling to show that the Ring1B/Bmi1 system can act as analog-digital converter, generating abrupt switches, multistable dynamics, oscillations and excitable overshoots. For instance, an increase in Bmi1 abundance brings about an abrupt “On” switch of Ring1B monoubiquitinating activity and downregulation of H2A-controlled genes, while a decrease in Bmi1 leads to an “Off” switch. These digital responses can display hysteresis, creating the biological memory. Distinct types of Ring1B activity responses (oscillatory, bistable and excitable) facilitate signal discrimination and allow the Ring1B/Bmi1/H2A system to distinctly affect gene silencing and potentially trigger different cell fates.