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Funding: This work was supported by grants from the Emergency Medicine Foundation Career Development Grant; AHA Mentored Clinical & Population Research Award (14CRP18860027); NIH/NINDS-R01-NS085419 (C.C., J.M.L.); NIH/NINDS-R37-NS107230, NIH/NINDS U24-NS107230 (J.M.L.); NIH/NINDS-K23-NS099487 (L.H.); NIH/NIA-K99-AG062723 (L.I.); Barnes-Jewish Hospital Foundation (J.M.L.); Biogen (C.C., J.M.L.); Bright Focus Foundation, US Department of Defense, Helsinki University Central Hospital; Finnish Medical Foundation; Finland government subsidiary funds; Spanish Ministry of Science and Innovation; Instituto de Salud Carlos III (grants ‘Registro BASICMAR’ Funding for Research in Health (PI051737), ‘GWALA project’ from Fondos de Investigación Sanitaria ISC III (PI10/02064, PI12/01238 and PI15/00451), JR18/00004); Fondos FEDER/EDRF Red de Investigación Cardiovascular (RD12/0042/0020); Fundació la Marató TV3; Genestroke Consortium (76/C/2011); Recercaixa’13 (JJ086116). Tomás Sobrino (CPII17/00027), Francisco Campos (CPII19/00020) and Israel Fernandez are supported by Miguel Servet II Program from Instituto de Salud Carlos III and Fondos FEDER. I.F. is also supported by Maestro project (PI18/01338) and Pre-test project (PMP15/00022) from Instituto de Salud Carlos III and Fondos Feder, Agaur; and Epigenesis project from Marató TV3 Foundation. J.C., J.M., A.D., J.M.-F., J.A. and I.F. are supported by Invictus plus Network (RD16/0019) from Instituto de Salud Carlos III and Fondos Feder. Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP-2013/07559-3) (I.L.-C.), Sigrid Juselius Foundation. The MEGASTROKE project received funding from sources specified at http://www.megastroke.org/acknowledgments.html. B.S., B.A. and F.S. are supported by NIH awards NS097000, NS101718, NS075035, NS079153 and NS106950. During the first hours after stroke onset, neurological deficits can be highly unstable: some patients rapidly improve, while others deteriorate. This early neurological instability has a major impact on long-term outcome. Here, we aimed to determine the genetic architecture of early neurological instability measured by the difference between the National Institutes of Health Stroke Scale (NIHSS) within 6h of stroke onset and NIHSS at 24h. A total of 5876 individuals from seven countries (Spain, Finland, Poland, USA, Costa Rica, Mexico and Korea) were studied using a multi-ancestry meta-analyses. We found that 8.7% of NIHSS at 24h of variance was explained by common genetic variations, and also that early neurological instability has a different genetic architecture from that of stroke risk. Eight loci (1p21.1, 1q42.2, 2p25.1, 2q31.2, 2q33.3, 5q33.2, 7p21.2 and 13q31.1) were genome-wide significant and explained 1.8% of the variability suggesting that additional variants influence early change in neurological deficits. We used functional genomics and bioinformatic annotation to identify the genes driving the association from each locus. Expression quantitative trait loci mapping and summary data-based Mendelian randomization indicate that ADAM23 (log Bayes factor = 5.41) was driving the association for 2q33.3. Gene-based analyses suggested that GRIA1 (log Bayes factor = 5.19), which is predominantly expressed in the brain, is the gene driving the association for the 5q33.2 locus. These analyses also nominated GNPAT (log Bayes factor = 7.64) ABCB5 (log Bayes factor = 5.97) for the 1p21.1 and 7p21.1 loci. Human brain single-nuclei RNA-sequencing indicates that the gene expression of ADAM23 and GRIA1 is enriched in neurons. ADAM23, a presynaptic protein and GRIA1, a protein subunit of the AMPA receptor, are part of a synaptic protein complex that modulates neuronal excitability. These data provide the first genetic evidence in humans that excitotoxicity may contribute to early neurological instability after acute ischaemic stroke. Postprint

Producción Científica, Stroke is one of the most common causes of death and disability. Reperfusion therapies are the only treatment available during the acute phase of stroke. Due to recent clinical trials, these therapies may increase their frequency of use by extending the time-window administration, which may lead to an increase in complications such as hemorrhagic transformation, with parenchymal hematoma (PH) being the more severe subtype, associated with higher mortality and disability rates. Our aim was to find genetic risk factors associated with PH, as that could provide molecular targets/pathways for their prevention/treatment and study its genetic correlations to find traits sharing genetic background. We performed a GWAS and meta-analysis, following standard quality controls and association analysis (fastGWAS), adjusting age, NIHSS, and principal components. FUMA was used to annotate, prioritize, visualize, and interpret the meta-analysis results. The total number of patients in the meta-analysis was 2034 (216 cases and 1818 controls). We found rs79770152 having a genome-wide significant association (beta 0.09, p-value 3.90 × 10−8) located in the RP11-362K2.2:RP11-767I20.1 gene and a suggestive variant (rs13297983: beta 0.07, p-value 6.10 × 10−8) located in PCSK5 associated with PH occurrence. The genetic correlation showed a shared genetic background of PH with Alzheimer’s disease and white matter hyperintensities. In addition, genes containing the ten most significant associations have been related to aggregated amyloid-β, tau protein, white matter microstructure, inflammation, and matrix metalloproteinases., Instituto de Salud Carlos III y Fondo Europeo de Desarrollo Regional (FEDER) - ( Projects PI 11/0176 and PI18/01338), Instituto de Salud Carlos III - (Project CM18/00198), Agencia de Gestión de Ayudas Universitarias y de Investigación (AGAUR) y Fondo Social Europeo - (Grant 2020FI_B1 00157), Instituto de Salud Carlos III y Fondo Europeo de Desarrollo Regional (FEDER) - (Project CD20/00043), Instituto de Salud Carlos III - (Projects FI19/00309, CP12/03298, CPII17/00027, CPII19/00020)

A recent study showed that antlers have evolved a high rate of growth due to the expression of proto-oncogenes and that they have also evolved to express several tumour suppressor genes to control the risk of cancer. This may explain why deer antler velvet (DAV) extract shows anti-tumour activity. The fast growth of antler innervation through the velvet in close association to blood vessels provides a unique environment to study the fast but non-cancerous proliferation of heterogeneous cell populations. We set out to study the anti-cancer effect of DAV in glioblastoma (GB) cell lines in comparison with temozolomide, a chemotherapeutic drug used to treat high-grade brain tumours. Here we report, for the first time, that DAV extract from the tip, but not from mid-parts of the antler, exhibits an anti-tumour effect in GB cell lines (T98G and A172) while being non-toxic in non-cancerous cell lines (HEK293 and HACAT). In T98G cells, DAV treatment showed reduced proliferation (37.5%) and colony-formation capacity (84%), inhibited migration (39%), induced changes in cell cycle progression, and promoted apoptosis. The anticancer activity of DAV extract as demonstrated by these results may provide a new therapeutic strategy for GB treatment., The authors are thankful to the Fundación del Hospital Nacional de Parapléjicos (Toledo, Spain) and the Spanish Ministry of Economy (Ministerio de Economía, Industria y Competitividad, Madrid, Spain) for financial support. L. C. is thankful to FNHP for the research fellowship (398764, 2018/6003). T. L. C., M. P., J. P., and A. G. are co-funded by the Estate program of Research, Innovation, and Development oriented towards Challenges of Society, by the European Union (reference number RTC-2016-5327-2) and by Counselling of Education, Culture, and Sports of Castilla-La Mancha [project no. SBPLY/19/180501/000115)].

During the first hours after stroke onset neurological deficits can be highly unstable: some patients rapidly improve, while others deteriorate. This early neurological instability has a major impact on long-term outcome. Here, we aimed to determine the genetic architecture of early neurological instability measured by the difference between NIH stroke scale (NIHSS) within six hours of stroke onset and NIHSS at 24h (ΔNIHSS). A total of 5,876 individuals from seven countries (Spain, Finland, Poland, United States, Costa Rica, Mexico and Korea) were studied using a multi-ancestry meta-analyses. We found that 8.7% of ΔNIHSS variance was explained by common genetic variations, and also that early neurological instability has a different genetic architecture than that of stroke risk. Seven loci (2p25.1, 2q31.2, 2q33.3, 4q34.3, 5q33.2, 6q26 and 7p21.1) were genome-wide significant and explained 2.1% of the variability suggesting that additional variants influence early change in neurological deficits. We used functional genomics and bioinformatic annotation to identify the genes driving the association from each loci. eQTL mapping and SMR indicate that ADAM23 (log Bayes Factor (LBF)=6.34) was driving the association for 2q33.3. Gene based analyses suggested that GRIA1 (LBF=5.26), which is predominantly expressed in brain, is the gene driving the association for the 5q33.2 locus. These analyses also nominated PARK2 (LBF=5.30) and ABCB5 (LBF=5.70) for the 6q26 and 7p21.1 loci. Human brain single nuclei RNA-seq indicates that the gene expression of ADAM23 and GRIA1 is enriched in neurons. ADAM23, a pre-synaptic protein, and GRIA1, a protein subunit of the AMPA receptor, are part of a synaptic protein complex that modulates neuronal excitability. These data provides the first evidence in humans that excitotoxicity may contribute to early neurological instability after acute ischemic stroke.RESEARCH INTO CONTEXTEvidence before this studyNo previous genome-wide association studies have investigated the genetic architecture of early outcomes after ischemic stroke.Added Value of this studyThis is the first study that investigated genetic influences on early outcomes after ischemic stroke using a genome-wide approach, revealing seven genome-wide significant loci. A unique aspect of this genetic study is the inclusion of all of the major ethnicities by recruiting from participants throughout the world. Most genetic studies to date have been limited to populations of European ancestry.Implications of all available evidenceThe findings provide the first evidence that genes implicating excitotoxicity contribute to human acute ischemic stroke, and demonstrates proof of principle that GWAS of acute ischemic stroke patients can reveal mechanisms involved in ischemic brain injury.

Saffron (Crocus sativus L.), as well as having notable culinary value, has been traditionally used as a medicinal plant due to its bioactivity. Furthermore, its antioxidant properties have been evaluated, and it may have cardiovascular benefits. Multiple sclerosis (MS) is a chronic disease of the central nervous system that causes a diminution in the daily physical exercise of patients. This sedentary lifestyle may contribute to an increased incidence of dyslipidaemia in this population, and could explain the increased cardiovascular morbidity. The aim of this study was to assess the effect of the intake of a saffron infusion over 96 days on the lipid profile of MS patients. Considering the extraction kinetics of bioactive metabolites, the best way to prepare the infusion was by using ground saffron (PDO &ldquo, Azafr&aacute, n de La Mancha&rdquo, ), passed through a 0.5 mm sieve and infused with water at the boiling point (95&ndash, 100 &deg, C). A total of 35 MS patients took a midafternoon infusion for 96 days, 16 of them completed the treatment. A significant decrease in total blood cholesterol levels from 199.5 to 179.5 mg/dL was observed. Additionally, a significant diminution of triglyceride levels, from 124.0 to 101.0 mg/dL, was observed. In conclusion, the intake of an infusion of 50 mg of &ldquo, for 96 days had a lipid-lowering effect in patients with MS.

Dysregulation of apoptosis may contribute to the etiology and/or progression of several prevalent diseases, including stroke and neurodegenerative pathologies. So, detection of the apoptotic processes in patients would be useful in daily clinical practice, However, thein vivoanalysis of apoptosis that occurs in tissue has limitations. We therefore propose to use circulating apoptotic bodies as biomarkers for measuring apoptotic death in patients. Since there is no scientific literature establishing the most appropriate method for measuring apoptotic bodies from human blood samples, we here describe a reproducible centrifugation-based method combined with electron microscopy, dynamic light scattering and flow cytometry studies to isolate, characterize and quantify plasma apoptotic bodies of patients with ischemic stroke, multiple sclerosis and Parkinson’s disease. The analysis revealed that our isolation protocol achieves notable recovery rates of highly-purified intact apoptotic bodies. This easy and rapid procedure would help physicians to implement the use of plasma apoptotic bodies analysis as a non-invasive tool to monitor apoptotic death in neurological patients for prognostic purposes and for following disease activity and assessing drug effectiveness.

Autophagy and exosome secretion play important roles in a variety of physiological and disease states, including the development of age‐related macular degeneration. Previous studies have demonstrated that these cellular mechanisms share common pathways of activation. Low oxidative damage in ARPE‐19 cells, alters both autophagy and exosome biogenesis. Moreover, oxidative stress modifies the protein and genetic cargo of exosomes, possibly affecting the fate of surrounding cells. In order to understand the connection between these two mechanisms and their impact on angiogenesis, stressed ARPE‐19 cells were treated with a siRNA‐targeting Atg7, a key protein for the formation of autophagosomes. Subsequently, we observed the formation of multivesicular bodies and the release of exosomes. Released exosomes contained VEGFR2 as part of their cargo. This receptor for VEGF—which is critical for the development of new blood vessels—was higher in exosome populations released from stressed ARPE‐19. While stressed exosomes enhanced tube formation, exosomes became ineffective after silencing VEGFR2 in ARPE‐19 cells and were, consequently, unable to influence angiogenesis. Moreover, vessel sprouting in the presence of stressed exosomes seems to follow a VEGF‐independent pathway. We propose that abnormal vessel growth correlates with VEGFR2‐expressing exosomes release from stressed ARPE‐19 cells, and is directly linked to autophagy. Sin financiación 4.658 JCR (2018) Q1, 31/136 Medicine, Research & Experimental; Q2, 60/193 Cell Biology 1.439 SJR (2018) Q1, 41/177 Molecular Medicine; Q2, 95/299 Cell Biology No data IDR 2018 UEV

Protein p56 encoded by the Bacillus subtilis phage φ29 inhibits host uracil-DNA glycosylase (UDG) activity. In previous studies, we suggested that this inhibition is likely a defense mechanism developed by phage φ29 to prevent the action of UDG if uracilation occurs in DNA either from deamination of cytosine or the incorporation of dUMP during viral DNA replication. In this work, we analyzed the ability of φ29 DNA polymerase to insert dUMP into DNA. Primer extension analysis showed that viral DNA polymerase incorporates dU opposite dA with a catalytic efficiency only 2-fold lower than that for dT. Using the φ29 DNA amplification system, we found that φ29 DNA polymerase is also able to carry out the extension of the dA:dUMP pair and replicate past uracil. Additionally, UDG and apurinic-apyrimidinic endonuclease treatment of viral DNA isolated from φ29-infected cells revealed that uracil residues arise in φ29 DNA during replication, probably as a result of misincorporation of dUMP by the φ29 DNA polymerase. On the other hand, the action of UDG on uracil-containing φ29 DNA impaired in vitro viral DNA replication, which was prevented by the presence of protein p56. Furthermore, transfection activity of uracil-containing φ29 DNA was significantly higher in cells that constitutively synthesized p56 than in cells lacking this protein. Thus, our data support a model in which protein p56 ensures an efficient viral DNA replication, preventing the deleterious effect caused by UDG when it eliminates uracil residues present in the φ29 genome., This work was supported by Spanish Ministry of Education and Science Grants BFU2005-00733/BMC and Consolider-Ingenio 24717 (to M.S.). The institutional help of Fundación Ramón Areces to the Centro de Biología Molecular “Severo Ochoa” is acknowledged. G.-S.H. had a Doctor contract from the Spanish National Research Council.

It becomes now apparent that prokaryotic DNA replication takes place at specific intracellular locations. Early studies indicated that chromosomal DNA replication, as well as plasmid and viral DNA replication, occurs in close association with the bacterial membrane. Moreover, over the last several years, it has been shown that some replication proteins and specific DNA sequences are localized to particular subcellular regions in bacteria, supporting the existence of replication compartments. Although the mechanisms underlying compartmentalization of prokaryotic DNA replication are largely unknown, the docking of replication factors to large organizing structures may be important for the assembly of active replication complexes. In this article, we review the current state of this subject in two bacterial species, Escherichia coli and Bacillus subtilis, focusing our attention in both chromosomal and extrachromosomal DNA replication. A comparison with eukaryotic systems is also presented, This work was supported by Grants 2R01 GM27242-24 (National Institutes of Health) and BMC2002-03818 (Ministerio de Ciencia y Tecnología) to M.S., as well as by grant 08.2/0025/2003 1 (Comunidad Autónoma de Madrid) to A.B. The institutional help of Fundación Ramón Areces to the Centro de Biología Molecular “Severo Ochoa” is acknowledged. A.B. was supported by the Ministerio de Ciencia y Tecnología (Programa Ramón y Cajal). G.S.-H. was a predoctoral-fellow from Ministerio de Ciencia y Tecnología.

Background Since atherogenesis is related to oxidative stress, our objective was to study the association of oxidative stress markers with the vascular recurrence in non-cardioembolic stroke. Methods Atherosclerotic and oxidative stress markers were evaluated on admission, in 477 patients suffering from a first non-cardioembolic stroke. Patients were followed at 6 and 12 months after inclusion, recording cardiovascular events. As markers of endothelial oxidative stress we used oxidized LDL, Cu/Zn superoxide dismutase and 8-OH deoxiguanosine. 136 patients were being treated with statins at the moment of serum samples acquisition. Results Patients who suffered vascular recurrence or vascular-origin death had higher levels of 8-OHDG (40.06±24.70vs33.11±15.18;p=0.003). We also found associations between vascular recurrence or vascular origin death and Cu/ZnSOD (OR,1.02; 95%CI,1.00-1.03;p=0.0001) and 8-OHDG (OR,1.12;95%CI,1.08-1.16;p Conclusions Specific oxidative stress markers were found to be markers of atherosclerosis plaque types and vascular recurrence in non-statins treated patients at admission.