Introduction: Extracellular vesicles (EVs) participate in cell-to-cell paracrine signaling and can be biomarkers of the pathophysiological processes underlying disease. In intracerebral hemorrhage, the study of the number and molecular content of circulating EVs may help elucidate the biological mechanisms involved in damage and repair, contributing valuable information to the identification of new therapeutic targets. Discussion: The protein content of circulating EVs at different time points following an ICH may reflect evolutionary changes in the pathophysiology of the disease. Volume 16 - 2022 | https://doi.org/10.3389/fncel.2022.1058546 Introduction: Extracellular vesicles (EVs) participate in cell-to-cell paracrine signaling and can be biomarkers of the pathophysiological processes underlying disease the study of the number and molecular content of circulating EVs may help elucidate the biological mechanisms involved in damage and repair contributing valuable information to the identification of new therapeutic targets Methods: The objective of this study was to describe the number and protein content of blood-derived EVs following an intracerebral hemorrhage (ICH) an experimental ICH was induced in the striatum of Sprague-Dawley rats and EVs were isolated and characterized from blood at baseline The protein content in the EVs was analyzed by mass spectrometric data-dependent acquisition; protein quantification was obtained by sequential window acquisition of all theoretical mass spectra data and compared at pre-defined time points Results: Although no differences were found in the number of EVs the proteomic study revealed that proteins related to the response to cellular damage such as deubiquitination regulation of MAP kinase activity (UCHL1) and signal transduction (NDGR3) were up-expressed at 24 h compared to baseline; and that at 28 days the protein expression profile was characterized by a higher content of the proteins involved in healing and repair processes such as cytoskeleton organization and response to growth factors (COR1B) and the regulation of autophagy (PI42B) Discussion: The protein content of circulating EVs at different time points following an ICH may reflect evolutionary changes in the pathophysiology of the disease in this study we sought to describe the number and protein content of EVs obtained from serum at different time points following the induction of an ICH in an experimental model in rats The presence of hemorrhage was confirmed by T2-weighted spin echo magnetic resonance imaging (MRI) at 48 h post-ICH using a 7-Tesla horizontal bore magnet (Bruker Pharmascan Blood samples were obtained from the tail vein at baseline Blood tubes were centrifuged at 3,000 g for 15 min at 4°C and the serum was stored at −80°C until its use for the analysis of the EVs Animals were subjected to an ICH and blood-derived EVs were isolated at baseline Images were acquired in a resolution of 7 Tesla (Bruker Pharmascan 7 Tesla USA) with a rapid acquisition with relaxation enhancement (RARE) sequence in axial orientations and the following parameters: number of echo images = 2 [echo time (TE): 29.54 ms and 88.61 ms] acquisition matrix = 256 × 256 corresponding to an in-plane resolution of 137 × 137 μm2 slice thickness = 1.00 mm without a gap and number of slices = 16 All data were acquired using a Hewlett-Packard console running Paravision 6.0.1 software (Bruker Medical Gmbh Lesion volumes were visualized with Image J v1.52 (National Institutes of Health EVs were isolated from serum samples using the ExoQuick ULTRA EV isolation kit (System Biosciences, Cat #EQULTRA-20A-1, USA) following manufacturer instructions as previously described (Coughlan et al., 2020) Isolated EVs were stored at −80°C until analysis EVs were characterized by: (1) Western blot; (2) transmission electron microscopy (TEM); and (3) nanoparticle tracking analysis (NTA) Western blot was performed using specific antibodies for the detection of surface antigens in EVs: anti-CD9 (1:750 Cat #PA5-89332 USA) as a purity control followed by an HRP-secondary antibody (1:10,000 UK) in a 4%–10% sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) for electrophoresis with 20 μg of protein per lane Band quantification was performed using a chemiluminescence method according to the manufacturer (ECL Pierce chemiluminescence; Thermo Fisher Scientific USA) in an Uvitec-Cambridge image acquisition system Samples were observed under the TEM (80 kV; S-3000N Japan) to verify the size of the isolated EVs ranging from 30 to 200 nm the EVs were fixed in 2.5% glutaraldehyde 0.1 M sodium cacodylate solution for 1 h at 4°C and postfixed with 2% osmium tetroxide for 1 h at 4°C The pellet was dehydrated with a graded acetone series and embedded in resin obtaining 60-nm thick sections for observation UK) was performed to determine the size and number of EVs The sample of isolated EVs was diluted in 500 μl of sterile Phosphate Buffered Saline (PBS) for a working concentration of 1 × 107–109 particles/ml Three videos of 60 s each were recorded at a shutter speed of 30.00 ms and camera level of 13 A detection threshold of three was employed for the analyses An ExoELISA-ULTRA assay kit (System Biosciences USA) was used for EVs quantification based on the presence of the anti-CD63 marker A Western blot to confirm the specific proteins found in the proteomic study was performed using the same samples of EVs Specific antibodies for the detection of UCHL1 (anti-UCHL1; 1:500 USA) and β-Actin as loading control (anti- β-Actin; 1:5,000 The methodology for the Western blot analysis has been previously explained for extracellular vesicle characterization Results were expressed as mean ± standard deviation (SD) The Kruskal–Wallis test followed by the Mann–Whitney U test were used to compare the number of EVs in each of the different study time points; p-values of <0.05 were considered statistically significant at a 95% confidence interval Analyses were conducted using SPSS 23 (IBM and the figures were obtained using GraphPad Prism 8 (GraphPad software The results from the proteomic studies were then analyzed using SWATH principal component analysis (PCA) and cluster analysis Austria) with “base,” “stats,” “gplots,” “Hmisc,” “dplyr,” and “car” packages The PCA was applied considering the correlation matrix due to its pairwise two-sided p-values being 0 for the entire matrix all of them therefore being statistically significant which could account for the successful application of the PCA The total variability of the data was 87.70% and is explained with the first two principal components a Student’s t-test for means comparison between samples and Euclidean distance suitable for quantitative variables and complete linkage as cluster criteria were applied For differentially expressed protein selection p-values were adjusted using a Benjamini-Hochberg correction selecting proteins with a FC of >2 or <0.5 and an adjusted p-value of <0.05 Twenty-nine rats were used over the course of the study Five were not included because they did not develop an ICH Four other rats died before completion of the observation period and were therefore excluded from the study Animals excluded from the study were replaced by new subjects until the predefined sample size of 20 was reached Blood-derived EVs showed proteins, morphology, and size (<200 nm) specific to traditional EVs by Western blot (Figure 2A, raw data available in Supplementary Figure S1.1), TEM (Figure 2B) and NTA (Figure 2C) Extracellular vesicles (EVs) characterization (A) Presence of specific EVs proteins (CD9 Alix) and albumin as a purity control by Western blot analysis following isolation (B) Transmission electron microscopy (TEM) images illustrating the characteristic size and form of the EVs (C) NTA illustrating the size and concentration of the EVs The number of blood-derived EVs at the pre-defined study time points is illustrated in Figure 3 No significant differences were found in the number of EVs at baseline (5.95 × 106 ± 1.02 × 106 EVs/ml) 24 h (4.42 × 106 ± 6.76 × 105 EVs/ml) and 28 days (6.09 × 106 ± 9.12 × 106 EVs/ml; p > 0.05) A preliminary analysis showed that our sample shared 26 proteins with the top 100 Vesiclepedia proteins (Figure 4; Pathan et al., 2019) Common proteins present in the EVs study sample compared to the top 100 proteins in Vesiclepedia No proteins were found down-expressed compared to baseline at 24 h or 28 days (A,C) The volcano plots represent proteins present in the EVs compared by time point Biological processes in which the differential proteins as described by UniProt appear to be involved are shown on the right (B,D) Immunoblot assays of the selected proteins from the proteomic study Protein detection cropped image and graphs from band quantification are shown it is highly likely that only the specific EVs from cells affected in the pathological process but not the total circulating EVs may change We did not analyze the differential number of circulating EVs based on their cellular origin (i.e. which may explain why we did not find significant changes in the total number of circulating EVs following ICH in our study this suggests that the number of EVs in and of itself is not relevant as a biomarker for ICH the differential protein cargo of these EVs in the acute and post-acute phases of an ICH compared to baseline supports the hypothesis that the protein content of circulating EVs reflects the pathophysiological mechanisms triggered as a response to the cerebral hemorrhage and thus could be used as biomarkers to better understand these processes we confirmed that our study results shared 26 proteins with the top 100 Vesiclepedia proteins supporting the assertion that the results of the proteomic analysis in fact reflected the EVs protein content Considering the objective to analyze the differential protein cargo in the EVs as a source of information about the pathophysiological mechanisms underlying damage and repair following an ICH the comparison was done between the acute and post-acute phases with baseline Proteins in the acute phase may represent mechanisms of damage while those at later stages could be involved in mechanisms of repair Given the biological characteristics of EVs as carriers of molecules this discrepancy with our results can be explained because EVs can protect their content from degradation and may also contain a higher concentration of specific proteins expressed as a result of the disease The proteomic analysis of EVs content therefore is very likely to increase the probability of finding specific biomarkers compared to the analysis in body fluids especially in animal models in which the total amount of these biomarkers can be low Although to our knowledge NDGR3 has not been previously described as involved in hemorrhagic stroke the high levels of this protein in EVs suggest that it could be related to the initial cellular damage produced by the ICH we have not found any previous works linking the biological functions of these proteins with an ICH or brain damage and further research is needed to determine their specific role in the post-acute phase of an ICH and their potential relation to restorative processes and outcomes this study reveals the differential protein content of circulating EVs in the acute and post-acute phases following an ICH compared to baseline with a higher expression of proteins related to the response to cellular damage in the early period and of proteins related to healing and removal of debris and repair processes in later stages These findings support the role of circulating EVs as biomarkers of the pathophysiological processes underlying ICH Further research on the proteins described is warranted to define the molecular functions in which they are involved and their role in determining evolution and outcomes following a cerebral hemorrhage The original contributions presented in the study are publicly available. This data can be found here: https://www.ebi.ac.uk/pride/archive/projects/PXD037246 PROEX 159/17) pursuant to Spanish and European Union regulations [(86/609/CEE 2010/63/EU) and Spanish Royal Decree (RD 1201/2005 and RD53/2013)] Investigation and writing—original draft: FL-G All authors contributed to the article and approved the submitted version This work was supported by the Spanish Ministry of Health-Carlos III Health Institute (ISCIII) and the European Regional Development Fund (FEDER Funding) under grant PI17/01922 and PI20/00243 the Invictus Plus network under grant RD16/0019/0005 Miguel Servet under grant CPII20/00002 to MG-F; CP20/00024 to LO-O Sara Borrell under grant CD19/00033 to MP-M Universidad Autónoma de Madrid under grant CA1/RSUE/2021-00753 to DP and the Spanish Ministry of Health-Carlos III Health Institute (ISCIII) under grant FI18/00026 to FL-G We appreciate the support of Morote Traducciones SL for its assistance in the editing of this manuscript JF-V is a shareholder of Biomedica Molecular Medicine SL The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fncel.2022.1058546/full#supplementary-material Proteomics: technologies and their applications Serum GFAP and UCH-L1 for prediction of absence of intracranial injuries on head CT (ALERT-TBI): a multicentre observational study Epidemiology of stroke in Europe and trends for the 21st century Chantada-Vázquez Exosome isolation by ultracentrifugation and precipitation and 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hemorrhage Received: 30 September 2022; Accepted: 30 December 2022; Published: 19 January 2023 distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: María Alonso de Leciñana, bWFsZWNpbmFuYWNhc2VzQHNhbHVkLm1hZHJpZC5vcmc=; María Gutiérrez-Fernández, bWd1dGllcnJlemZlcm5hbmRlekBzYWx1ZC5tYWRyaWQub3Jn † These authors share first authorship ‡ These authors have contributed equally to this work Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish Egan Bernal takes second place ahead of Jefferson Cepeda Sebastian Berwick (Caja Rural-Seguros RGA) Davide Bais and Andrea Pietrobon (both Team Polti Kometa) Alex Molenaar (Team Illes Balears Arabay Cycling) After enduring heavy rain for much of the stage the Tour de France champion signalled his intent as he doffed his rain gear on the Alto de San Pedro de Líncora When he went with 5km to go only Jefferson Cepeda (Caja Rural-Seguros RGA) and a resurgent Egan Bernal (Ineos Grenadiers) could follow Vingegaard left the two challengers behind before the summit with 3.4km to go and soloed on to the stage victory and race lead after the opening time trial was neutralised due to high wind Bernal out-paced Cepeda for second on the stage in another sign he is on his way to being back to his best The pair came through 24 seconds behind Vingegaard leaving Bernal in second overall at 28 seconds after the time bonuses and Cepeda third at 30 seconds I'm very happy with the day and with the team We did all the work today and the team did amazing and I want to thank all my teammates," Vingegaard said at the finish So I'm happy I could pay a bit back and yeah in the end a good day for us - a cold day but we're happy so I'm very happy to take the win today and looking forward to the next coming days as well." Vingegaard took the overall lead with a 28-second advantage over Bernal, and 30 seconds over Cepeda. Q36.5 rider Xabier Azparren took the points classification lead After the opening time trial stage was partially neutralised because of rough weather with only placings but not time gaps counting all 118 riders that completed the stage 1 rolled out of the start village of Taboada shortly after 1 p.m with a heavy rain storm just as they were getting underway an unwelcome reminder of the tough conditions of the day before On a day with nearly 3,000 metres of vertical climbing and where the dauntingly steep and narrow category 2 ascent of San Pedro de Licora was always likely to decide the stage winner there were few takers for an early break.  The first intermediate sprint of the day was snapped up by Ruben Guerreiro (Movistar) and for nearly an hour only a quickly extinguished move of eight briefly interrupted the unspoken unofficial truce in the peloton On a winding descent off an unclassified climb with around 110 kilometres to go 12 riders managed to establish a bridgehead: Jorge Arcas (Movistar) Sebastian Berwick (Caja Rural-Seguros RGS) Davie Bais and Andrea Pietrobon (Polti-Kometa) Alex Molenaar (Illes Balears-Arabay Cycling) Frederico Figueiredo (Sabgal) and Embret Svestad-Bardseng (Arkea-B&B Hotels).  Such a large group had few problems establishing a gap of just over 2:00 before Movistar then began establishing a watching brief at the front of the peloton and the umpteenth rain shower teemed down The holding pattern continued until well into the second half of the race the 12 continued to hold their advantage well that none of the five WorldTour teams present had riders in the break When Movistar were joined by Visma-Lease A Bike at the front of the peloton it began to look increasingly complicated for the move A lone attack by Xabier Azparren just before the first of the two ascents of the Alto de San Pedro de Licora left the front group divided and as the sun began to shine for the first time in the stage Visma-Lease A Bike began to shred the peloton and sweeping up stragglers from the break Molenaar then joined Azparren close to the summit and the two swept through the finish line with 1:05 advantage on the remnants of the Visma-led peloton with Carlos Rodriguez (Ineos Grenadiers) one notable name already five minutes down Visma continued to force the pace behind on the rain-soaked backroads Results powered by FirstCycling This website is using a security service to protect itself from online attacks. 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Please include what you were doing when this page came up and the Cloudflare Ray ID found at the bottom of this page. highlighted by the public as the best tapa of the competition won first place in a tapas competition in Galicia 'Patacón de Abadía' is the name of their gastronomic proposal a delicious interpretation of the recipe for stuffed plantains with shredded beef The Cuban woman works at "La Cervecería de Chantada" in the province of Lugo This bar was one of the participants in the Chantapas contest The event was organized by the association of entrepreneurs and the Open Commercial Center (CCA) of Chantada which celebrated the return of the competition after several years of absence The public is in charge of voting for their favorite tapa and was delighted with Yailé's proposal who has been living in Galicia for twelve years I love this town," she commented in an interview with El Progreso She confessed that she did not think she would win "Many people came to congratulate me on the dish and there were even people of Latin origin who were moved because the dish reminded them of their homeland," he commented It is popular in other Caribbean countries such as Venezuela it is prepared in a concave shape in order to include the filling of ropa vieja "Ropa vieja is shredded and stewed beef flank I made the sauce with the Abadía beer that we sell here," explained Yailé The final step to complete the dish is to fry ripe plantains creating a perfect base for the tasty meat "We entered a tapas dish into the competition just to participate because there were very diverse options in all the bars and of a high standard we did not expect to win," commented Antonio García Olivero we made 77 and they were gone in an hour and a half," Olivero recalled The second place in the competition was awarded to "Juicy from land and sea" from A Viñoteca and the third place was won by "Focaccia San Xoán" from Café Cantón Hundreds of people attended the eight participating venues stamping their passports to vote for their favorite bites The Chantapas has not only been a celebration of local gastronomy but also a demonstration of how cuisine can be a bridge between cultures bringing a piece of Cuba to the hearts and palates of Galicians PhD in Sciences from the University of Alicante and Bachelor in Sociocultural Studies PhD in Sciences from the University of Alicante and Bachelor of Sociocultural Studies Metrics details DNA-dependent protein kinase (DNA-PK) is a critical player in the DNA damage response (DDR) and instrumental in the non-homologous end-joining pathway (NHEJ) used to detect and repair DNA double-strand breaks (DSBs) We demonstrate that the potent and highly selective DNA-PK inhibitor is an efficient sensitizer of radiation- and doxorubicin-induced DNA damage with combinations in xenograft and patient-derived xenograft (PDX) models inducing sustained regressions in combination with the PARP inhibitor olaparib resulting in cell growth inhibition and apoptosis AZD7648 enhanced olaparib efficacy across a range of doses and schedules in xenograft and PDX models enabling sustained tumour regression and providing a clear rationale for its clinical investigation Through its differentiated mechanism of action as an NHEJ inhibitor AZD7648 complements the current armamentarium of DDR-targeted agents and has potential in combination with these agents to achieve deeper responses to current therapies respectively (trial identifiers: VX-984; NCT02644278 particularly if those effects are significantly enhanced by tumour-specific DDR deficiencies such as in ATM signalling tumour regression at tolerated doses in vivo was observed and regulatory approval has now been obtained to progress these combinations in the clinic (trial identifier: NCT03907969) AZD7648 is a potent radiosensitizer in vitro a Optimization of screening hit 1 into the potent and selective DNA-PK inhibitor b Western blot analysis of A549 cells treated with 8 Gy IR ± 1 h pre-treatment with increasing concentrations of AZD7648 c High-content imaging analysis of indicated DNA damage markers in A459 cells Cells were treated with 2 Gy IR ± AZD7648 pre-treatment (1 h) and immunofluorescently stained at indicated time points micronuclei per cell or relative nuclear fragmentation from two independent experiments (n = 2 Black dotted lines indicate AZD7648 cellular IC50 concentration (91 nM) Representative images are shown for 2 Gy IR ± 1.25 μM AZD7648 at 72 h (scale bars D represents DMSO vehicle-treated controls d Cell cycle analysis was performed based on DAPI staining intensity of nuclei detected by imaging analysis Graphs represent cell cycle distribution (percentage population) from a representative experiment (n = 2) N/A (non-assigned) represents cell populations where signal intensities exceeded the threshold to accurately determine the cell cycle phase Dotted lines indicate AZD7648 cellular IC50 concentration (91 nM) e Colony formation assays performed with A549 or NCI-H1299 cells treated with an ionizing radiation dose response ± AZD7648 Graphs represent mean surviving fraction normalized to the single-agent activity of AZD7648 Data were fitted to the linear quadratic model (mean ± SD (n = 2); unpaired t-test where P ≤ 0.05 is significant) Mean dose enhancement factor values (DEF37) are shown Together these data demonstrate in vitro that AZD7648 leads to the persistence of DNA damage following IR resulting in G2/M DNA damage checkpoint activation genome instability and reduced cellular survival t-test with unequal variances was used and for tumour regression d Pharmacodynamic modulation of DNA-PK biomarkers and pharmacokinetics (PK) of AZD7648 after dosing of AZD7648 and IR in A549 xenografts pDNA-PKcs Ser2056 and γH2AX were measured by immunohistochemistry (image scale bars Several comparisons between the IR and IR + AZD7648 treatments were found to be statistically significant using a one-way ANOVA For pDNA-PKcs Ser2056: 0.5 h (p < 0.0001) For γH2AX: 0.5 h (p = 0.05) and 7 h (p = 0.02) AZD7648 and doxorubicin have synergistic combination activity in breast and ovarian cancer cell lines a Western blot analysis of OAW42 cells treated with doxorubicin (100 nM) ± AZD7648 (3 μM) b Concentration-dependent response to AZD7648 ± doxorubicin was measured by a Live/Dead assay Graphs represent percentage viable cells ± SD following 5 days’ treatment relative to DMSO vehicle-treated controls from a representative experiment (n = 3) c High-content imaging analysis of indicated DNA damage markers in OAW42 cells Cells were treated with increasing AZD7648 concentrations ± doxorubicin (3 nM) and immunofluorescently stained at indicated time points Graphs represent mean ± 2 SEM γH2AX signal intensity or micronuclei per cell from three independent experiments Representative images are shown for AZD7648 (1 μM) ± doxorubicin (3 nM) at 48 h (scale bars d Synergy scores for the AZD7648 and doxorubicin combination in a panel of four ovarian and seven breast cancer cell lines Cells were treated for 5–7 days and viability was measured by the Live/Dead assay A synergy score of >5 is indicative of synergistic activity e Activity heatmaps from representative experiments for MDA-MB-468 Experimental activity heatmap represents growth inhibitory (0–100) and cytotoxic activity (100–200) following treatment Loewe additivity model fit heatmap represents expected activity values for an additive combination Concentrations where combination activity occurred in excess of the expected activity are boxed in pink Synergy scores from the representative experiment are indicated in brackets c Pharmacokinetics of AZD7648 and pharmacodynamic modulation of DNA-PK biomarkers pDNA-PKcs Ser2056 and γH2AX after dosing of AZD7648 and liposomal doxorubicin in BT474 xenografts Mice were dosed once with liposomal doxorubicin and with AZD7648 for 3 days Samples were then collected 8 h after a last morning dose of AZD7648 on the fourth day Significance assessed with two-sided t-tests performed on log-transformed data assuming unequal variance d Pharmacokinetics of AZD7648 and pharmacodynamic modulation of DNA-PK biomarkers after dosing of AZD7648 and olaparib in FaDu ATM KO xenografts Mice were taken from the efficacy study described above (c) between days 11 and 18 and samples were collected 2 h after the first dose of AZD7648 (1 h after the dose of olaparib) AZD7648 in combination with olaparib affects genome stability and induces apoptosis in ATM KO cells a Frequency of micronuclei formation in FaDu WT and ATM KO cells following 72 h treatment Data are shown as mean number of micronuclei per cell (mean ± SEM; n = 2; unpaired t-test b Chromosomal aberrations in FaDu WT and ATM KO cells treated with olaparib (1 μM) or the combination for 48 h were detected by metaphase spread analysis chromosome breaks and chromosome fusions were quantified separately Data are shown as mean aberrations per metaphase spread (mean ± SD; n = 3; paired t-test c Caspase 3/7 activity of FaDu ATM KO and WT cells following 72 h treatment Graph represents mean fluorescence levels normalized for total cell confluency and relative to the DMSO vehicle-treated control (mean ± SD; n = 3; two-way ANOVA The inhibition of NHEJ is an important additional strategy to realize the full potential of DDR-targeted therapies and their combinations clinically we report an inhibitor of DNA-PK kinase activity with a considerably improved selectivity profile to previously reported inhibitors We demonstrate that AZD7648 is both an efficacious combination partner with the standard-of-care therapies IR and doxorubicin as well as report robust anti-tumour activity of DNA-PK inhibition in combination with the PARP inhibitor the anti-tumour activity of AZD7648 in combination with IR or doxorubicin is associated with a decrease in DNA repair and an increase in DNA damage demonstrated by elevated levels of γH2AX foci and an accumulation of the G2/M cell cycle population compared with the respective single-agent controls This potentiation activity is reflected in our observations whereby AZD7648 in combination with IR or liposomal doxorubicin resulted in significant tumour growth inhibition and sustained regression in vivo the profound tumour regressions achieved in FaDu ATM KO xenografts show a clear combination benefit for DNA-PK and PARP inhibition in the ATM-null genetic background the combination benefit observed in olaparib-sensitive HR-proficient as well as in BRCA1 and BRCA2-deficient PDX models suggest that there are likely other genetic vulnerabilities that sensitize to the combination We are hopeful that future preclinical and clinical studies will elucidate what these might be The magnitude of the AZD7648 and olaparib combination treatment response in the FaDu ATM KO xenografts in vivo (where tumour volumes started to regress after 10 days of treatment) was more striking than the combination in vitro suggesting that the length of treatment and the accumulation of DNA damage is an important factor in achieving significant anti-tumour effects The continuous dosing of the combination treatment of up to 70 days was well tolerated in mice we demonstrated that AZD7648 administered intermittently alongside continuous olaparib dosing can also achieve tumour regression or tumour growth inhibition (stable disease) that significantly improves upon olaparib dosed on its own What appears to discriminate regressions from stable disease using these schedules is the total amount of drug delivered in a 21-day period although future preclinical studies may help elucidate this further The ability to dose in an intermittent or continuous manner allows various schedules of AZD7648 in combination with olaparib to achieve anti-tumour efficacy which provides options for adapting AZD7648 treatment in the clinic depending on the tolerability achieved these observations underscore the need for the clinical evaluation of DNA-PK inhibitors such as AZD7648 for cancer therapy a potent and selective inhibitor of DNA-PK that has broad potential for development as an anticancer agent acting as a sensitizer to a range of DNA DSB-inducing agents cytotoxic chemotherapy and the PARP inhibitor Through its differentiated mechanism-of-action as an inhibitor of NHEJ AZD7648 has potential to be combined with other DDR-targeted agents to treat a broader range of tumour types and achieve deeper and more sustained responses to current therapies Detailed synthesis protocols and characterization of compounds can be found in the Supplementary Materials and Methods section of the Supplementary Information All cell lines tested negative for Mycoplasma and were authenticated by short tandem repeat analysis. A full list of cell lines, their origins, cell growth media and assay media used in this study can be found in Supplementary Table 4 The FaDu cell line with all three copies of ATM knocked out was generated using transcription activator-like effector nucleases in-house (Discovery Sciences The A549 cell line with ATM knocked out was generated by CRISPR-Cas9 in-house (Oncology R&D Primary potency of AZD7648 on DNA-PK activity was evaluated in A549 cells following IR by measuring DNA-PKcs autophosphorylation on Ser2056 The selectivity of AZD7648 was evaluated in other cell assays for its pharmacological activity on ATM (pATM Ser1981) in HT29 and PI3K isoforms (pAKT Thr308) in BT474 (PI3Kα) IC50 was defined as the concentration required to give a 50% reduction in the phosphorylation of the downstream target for each respective kinase High-content imaging assays were performed in A549 and OAW42 cells to evaluate the cellular response to combination treatment of AZD7648 with IR and doxorubicin 53BP1 and pATM Ser1981 and imaged on the CV7000 high-content imaging platform (Yokogawa) Analysis was performed on a Columbus™ image data storage and analysis system (Perkin Elmer) A score of >0 is indicative of an additive combination effect and ≥5 is indicative of synergistic combination activity Immunocompromised SCID (C.B-17/IcrHan®Hsd-Prkdcscid) or Hsd:Athymic Nude-Foxn1nu female mice (Envigo) were used for tumour implantation AZD7648 was formulated in 0.5% hydroxypropyl methylcellulose/0.1% Tween80 (HPMC/T) and orally dosed (4–100 mg kg−1) the time between the morning and evening doses was 8 h Targeted irradiation of 2 Gy was delivered over 2 min daily over the first 5 days of treatment Liposomal doxorubicin (Doxoves) was diluted in physiological saline and intravenously dosed at 2.5 or 5 mg kg−1 once per week Olaparib was formulated in 10% DMSO/30% Kleptose and orally dosed at 100 mg kg−1 once daily All three combinations were dosed 1 h after the morning dose of AZD7648 or its vehicle HPMC/T Tumour growth inhibition from start of treatment was assessed by comparison of the mean change in tumour volume for the control and treated groups using the Mousetrap application and represented as TGI Statistical significance was evaluated using a one-tailed t-test All in vivo studies complied with all relevant ethical regulations for animal testing and research followed AstraZeneca’s global bioethics policy and received ethical approval from the AstraZeneca ethical committee HBCx-17 PDX study was carried out at XenTech France in accordance with French regulatory legislation concerning the protection of laboratory animals CTG-0828 and CTG-0149 PDX studies were carried out at Champions Oncology USA in accordance to the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Champions Oncology and the USA regulatory legislation CDX and OV2022 PDX studies were conducted in the UK in accordance with UK Home Office legislation the Animal Scientific Procedures Act 1986 and the Home Office project licences 70/8839 Further information on research design is available in the Nature Research Reporting Summary linked to this article The DNA-damage response in human biology and disease Targeting the DNA damage response in cancer The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway DNA-PK: a dynamic enzyme in a versatile DSB repair pathway Non-homologous DNA end joining and alternative pathways to double-strand break repair The DNA-dependent protein kinase: a multifunctional protein kinase with roles in DNA double strand break repair and mitosis ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation Autophosphorylation of DNA-dependent protein kinase regulates DNA end processing and may also alter double-strand break repair pathway choice Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks Beyond DNA repair: DNA-PK function in cancer and Chk1 in countering replication stress during S phase DNA-PKcs is required to maintain stability of Chk1 and Claspin for optimal replication stress response ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress Requirement for the kinase activity of human DNA-dependent protein kinase catalytic subunit in DNA strand break rejoining Absence of p350 subunit of DNA-activated protein kinase from a radiosensitive human cell line Differential and common DNA repair pathways for topoisomerase I- and II-targeted drugs in a genetic DT40 repair cell screen panel Targeting DNA-dependent protein kinase for cancer therapy a novel PI3K/mTOR dual inhibitor eliciting transient target modulation to impede tumor growth a dual inhibitor of mTOR kinase and DNA-PK blocks DNA damage repair pathways and selectively inhibits ATM-deficient cell growth in vitro Abstract 3716: potent radiation enhancement with VX-984 a selective DNA-PKcs inhibitor for the treatment of NSCLC Abstract 4198: highly potent and selective DNA-PK inhibitor M3814 with sustainable anti-tumor activity in combination with radiotherapy a novel investigational DNA-PK inhibitor: enhancing the effect of fractionated radiotherapy leading to complete regression of tumors in mice Essential role for DNA-PKcs in DNA double-strand break repair and apoptosis in ATM-deficient lymphocytes A functional cancer genomics screen identifies a druggable synthetic lethal interaction between MSH3 and PRKDC Therapeutic targeting of a robust non-oncogene addiction to PRKDC in ATM-defective tumors Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers Candidate biomarkers of PARP inhibitor sensitivity in ovarian cancer beyond the BRCA genes Modulation of DNA repair by pharmacological inhibitors of the PIKK protein kinase family Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks Mitotic progression following DNA damage enables pattern recognition within micronuclei Cell cycle dependence of DNA-dependent protein kinase phosphorylation in response to DNA double strand breaks ATM phosphorylates histone H2AX in response to DNA double-strand breaks Analysis of drug combinations: current methodological landscape Laying a trap to kill cancer cells: PARP inhibitors and their mechanisms of action Deficiency in the repair of DNA damage by homologous recombination and sensitivity to poly(ADP-ribose) polymerase inhibition The PARP inhibitor olaparib induces significant killing of ATM-deficient lymphoid tumor cells in vitro and in vivo ATM orchestrates the DNA-damage response to counter toxic non-homologous end-joining at broken replication forks Nonhomologous end joining drives poly(ADP-ribose) polymerase (PARP) inhibitor lethality in homologous recombination-deficient cells PARP inhibitors: extending benefit beyond BRCA-mutant cancers Chemosensitization of cancer cells by KU-0060648 Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase-1 A novel DNA-dependent protein kinase inhibitor potentiates the cytotoxicity of topoisomerase II poisons used in the treatment of leukemia Preclinical evaluation of a potent novel DNA-dependent protein kinase inhibitor NU7441 PARP inhibitors: synthetic lethality in the clinic Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks Synthetic lethality between mutation in Atm and DNA-PK(cs) during murine embryogenesis Enhanced cytotoxicity of PARP inhibition in mantle cell lymphoma harbouring mutations in both ATM and p53 Differential induction of chromosomal aberrations by topoisomerase inhibitors in cultured Chinese hamster cells Role of DNA-dependent protein kinase catalytic subunit in cancer development and treatment Download references We thank the AstraZeneca Laboratory Animal Sciences and Oncology in vivo teams for their expert technical assistance We thank all past and present members of the DNA-PK project team We thank XenTech SAS and Champions Oncology for their assistance with PDX studies We also thank Mudskipper for editorial support Simon Barry and Viia Valge-Archer for critical reading of the manuscript Medical writing assistance was provided by Martin Goulding from Mudskipper Business Ltd These authors contributed equally: Jacqueline H Mercedes Vazquez-Chantada & Stephanie Ling conducted most of the experiments and analysis of the data contributed to the design and/or synthetic route towards the inhibitor were responsible for conception and oversight of the project All authors discussed the results and reviewed the manuscript are or were employees of AstraZeneca at the time of conducting these studies Peer review information Nature Communications thanks Anette Duensing and the other reviewers for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-019-12836-9 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Functional & Integrative Genomics (2025) Journal of Pharmacokinetics and Pharmacodynamics (2025) Naunyn-Schmiedeberg's Archives of Pharmacology (2025) Sign up for the Nature Briefing newsletter — what matters in science The magic which envelops every part of the Ribeira Sacra is most clearly represented in the variety of festivals celebrated in its different municipalities Carnival and traditional trades and exalting wine and cuisine are pilgrimages: festivals which began as religious and commercial celebrations and which are now excellent excuses to come together and enjoy food Some of these celebrations have been declared Galician Festivals of Tourist Interest such as the Feria do Viño de Amandi (Amandi Wine Fair) in Sober or the Folión de Carros(Cart Festival) in Chantada: a celebration dating back to the Middle Ages which represents the agricultural trades and work carried out in the area The Festa das Fachas de Castelo (Castelo Torch Burning Festival) in Taboada has also been awarded this title seeing locals and visitors build a large torch made of straw before its flames light up the night we have compiled a list of the main festivals celebrated in the different municipalities of the Ribeira Sacra Fiesta de la carne ao caldeiro (Stewed Beef Festival) Fiestas de la Asunción (Assumption of Mary Festival) Magosto de San Martiño (San Martiño Chestnut Festival) Fiesta de la Ternera Gallega (Galician Beef Festival) Fiesta de la cereza y del aceite (Cherry and Olive Oil Festival) Feria del vino de Amandi (Amandi Wine Fair) Fiesta del caldo de huesos (Bone Broth Festival) Fiesta de las Fachas de Castelo(Castelo Torch Burning Festival) Get our news on your inbox! Suscribe x MercoPress, en Español Montevideo, May 6th 2025 - 08:09 UTC The electoral roll for Sunday's primary election in Argentina included four names with Falkland Islands' addresses according to reports in the Argentina media who lives in Ushuaia but recorded his address in Stanley Apparently the name Vallejo is a symbolic homage to an Argentine combatant who shared a foxhole with Hillar but was killed on 11/21 June of shrapnel injuries The Tierra del Fuego Electoral roll also mentions the couple Jose Luis Chantada and Pamela Margaret Mac Leod Their address is “Fitz Roy Road 37” but being so far away I doubt they will send us the tickets” is married to a Falkland Islander and until recently was involved in the tourism industry His wife Mac Leod is also registered in the Tierra del Fuego Electoral roll Maria Marta Villanueva whose address in Stanley is “and was one of the four Argentine residents living in the Falklands before the 1982 conflict” Villanueva later became a British citizen and was known in the Islands as Maria Strange Maria Strange worked for the Falklands government until she retired in Argentina None of these persons are actually living there address in a place there he has never lived Is this really in conformity with the election laws of Argentina Can these two actually be in the same sentence Commenting for this story is now closed.If you have a Facebook account, become a fan and comment on our Facebook Page! Yailé Alonso, a talented chef from Camagüey, Cuba, emerged victorious in a tapas competition held in Galicia, Spain, taking first place. The gastronomic offering of 'Patacón de Abadía', the name of the dish presented by the Cuban, is a delicious version of the classic tostones stuffed with shredded meat, better known as ropa vieja. The winner works at 'La Cervecería de Chantada', in the province of Lugo, this bar being one of the participants in the Chantapas competition. The business association and the Open Shopping Centre (CCA) of Chantada joined forces to organise an event commemorating the return of the competition after many years of absence. The community has the power to vote for their favourite tapa and they were satisfied with the idea of ​​Yailé, who has lived in Galicia for 12 years. "I got off the plane and came here. I love this town," she said during her chat with El Progreso. She admitted that winning was not part of her plans, which made her particularly happy. View this post on Instagram A post shared by Centro Comercial Aberto Ribeira Sacra (@ccaribeirasacra) "Many people came to congratulate me on the dish and there were even people of Latin origin who were moved because the tapa reminded them of their homeland," he added is a much-loved dish in several Caribbean countries such as Venezuela He mentioned that the sauce he used was made with the special Abadía beer I made the sauce with the Abadía beer that we sell here.” fry thickly sliced ​​plantains until golden providing a delicious base for savory meat toppings was pleasantly surprised by the diversity and quality of the options presented in the contest they were grateful for the opportunity to participate On the first day we made 77 and in an hour and a half we ran out of them," said the owner of the business A Viñoteca's Jugoso de tierra y mar took second prize in the competition while Café Cantón's Focaccia San Xoán took third place The event attracted hundreds of people to the eight venues involved Δdocument.getElementById( "ak_js_1" ).setAttribute( "value"