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Background To date, no immunological data on COVID-19 heterologous vaccination schedules in humans have been reported. We assessed the immunogenicity and reactogenicity of BNT162b2 (Comirnaty, BioNTech, Mainz, Germany) administered as second dose in participants primed with ChAdOx1-S (Vaxzevria, AstraZeneca, Oxford, UK). Methods We did a phase 2, open-label, randomised, controlled trial on adults aged 18–60 years, vaccinated with a single dose of ChAdOx1-S 8–12 weeks before screening, and no history of SARS-CoV-2 infection. Participants were randomly assigned (2:1) to receive either BNT162b2 (0·3 mL) via a single intramuscular injection (intervention group) or continue observation (control group). The primary outcome was 14-day immunogenicity, measured by immunoassays for SARS-CoV-2 trimeric spike protein and receptor binding domain (RBD). Antibody functionality was assessed using a pseudovirus neutralisation assay, and cellular immune response using an interferon-γ immunoassay. The safety outcome was 7-day reactogenicity, measured as solicited local and systemic adverse events. The primary analysis included all participants who received at least one dose of BNT162b2 and who had at least one efficacy evaluation after baseline. The safety analysis included all participants who received BNT162b2. This study is registered with EudraCT (2021-001978-37) and ClinicalTrials.gov (NCT04860739), and is ongoing. Findings Between April 24 and 30, 2021, 676 individuals were enrolled and randomly assigned to either the intervention group (n=450) or control group (n=226) at five university hospitals in Spain (mean age 44 years [SD 9]; 382 [57%] women and 294 [43%] men). 663 (98%) participants (n=441 intervention, n=222 control) completed the study up to day 14. In the intervention group, geometric mean titres of RBD antibodies increased from 71·46 BAU/mL (95% CI 59·84–85·33) at baseline to 7756·68 BAU/mL (7371·53–8161·96) at day 14 (p

This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License., All types of DNA damage cause a local alteration and relaxation of chromatin structure. Sensing and reacting to this initial chromatin alteration is a necessary trigger for any type of DNA damage response (DDR). In this context, chromatin kinases are likely candidates to participate in detection and reaction to a locally altered chromatin as a consequence of DNA damage and, thus, initiate the appropriate cellular response. In this work, we demonstrate that VRK1 is a nucleosomal chromatin kinase and that its depletion causes loss of histones H3 and H4 acetylation, which are required for chromatin relaxation, both in basal conditions and after DNA damage, independently of ATM. Moreover, VRK1 directly and stably interacts with histones H2AX and H3 in basal conditions. In response to DNA damage induced by ionizing radiation, histone H2AX is phosphorylated in Ser139 by VRK1. The phosphorylation of H2AX and the formation of gH2AX foci induced by ionizing radiation (IR), are prevented by VRK1 depletion and are rescued by kinase-active, but not kinase-dead, VRK1. In conclusion, we found that VRK1 is a novel chromatin component that reacts to its alterations and participates very early in DDR, functioning by itself or in cooperation with ATM., DMM and M S-G were supported by JAE-CSIC-Fondo Social Europeo fellowships. DSM was supported by a Ministerio de Ciencia e Innovación-FPI predoctoral fellowship. This work was funded by grants from Ministerio de Econom ıa y Competitividad (SAF2010-14935, SAF2013-44810R, SAF2014-57791-REDC) and Consejerıa de Educacion de la Junta de Castilla y Leon (CSI002U14) to PAL.

This is an open-access article distributed under the terms of the Creative Commons Attribution License., Vaccinia-related kinase 1 (VRK1) belongs to a group of sixteen kinases associated to a poorer prognosis in human breast carcinomas, particularly in estrogen receptor positive cases based on gene expression arrays. In this work we have studied the potential molecular mechanism by which the VRK1 protein can contribute to a poorer prognosis in this disease. For this aim it was first analyzed by immunohistochemistry the VRK1 protein level in normal breast and in one hundred and thirty six cases of human breast cancer. The effect of VRK1 to protect against DNA damage was determined by studying the effect of its knockdown on the formation of DNA repair foci assembled on 53BP1 in response to treatment with ionizing radiation or doxorubicin in two breast cancer cell lines. VRK1 protein was detected in normal breast and in breast carcinomas at high levels in ER and PR positive tumors. VRK1 protein level was significantly lower in ERBB2 positive cases. Next, to identify a mechanism that can link VRK1 to poorer prognosis, VRK1 was knocked-down in two breast cancer cell lines that were treated with ionizing radiation or doxorubicin, both inducing DNA damage. Loss of VRK1 resulted in reduced formation of DNA-damage repair foci complexes assembled on the 53BP1 scaffold protein, and this effect was independent of damaging agent or cell type. This observation is consistent with detection of high VRK1 protein levels in ER and PR positive breast cancers. We conclude that VRK1 can contribute to make these tumors more resistant to DNA damage-based therapies, such as ionizing radiation or doxorubicin, which is consistent with its association to a poor prognosis in ER positive breast cancer. VRK1 is potential target kinase for development of new specific inhibitors which can facilitate sensitization to other treatments in combination therapies; or alternatively be used as a new cancer drugs., M. V-C and M. S-G have JAE-CSIC-Fondo Social Europeo predoctoral fellowships. This work was supported by grants from Ministerio de Educación, Ciencia e Innovación (SAF2010-14935 and CSD2007-0017) and Kutxa-Fundación Inbiomed to P.A.L.

Golgi fragmentation is a process that is necessary to allow its redistribution into daughter cells during mitosis, a process controlled by serine-threonine kinases. This Golgi fragmentation is activated by MEK1 and Plk3. Plk3 is a kinase that is a downstream target in Golgi fragmentation pathway induced by MEK1 or by nocodazole. In this work we have identified that Plk3 and VRK1 are two consecutive steps in this signaling pathway. Plk3 interacts with VRK1 forming a stable complex that was detected by reciprocal immunoprecipitations and pulldown assays; VRK1 colocalizes with giantin in the Golgi apparatus, as Plk3 also does, forming clearly detectable granules. VRK1 does not phosphorylate Plk3; but Plk3 phosphorylates the C-terminal region of VRK1 in Ser342. VRK1 with substitutions in S342 is catalytically active, but blocks Golgi fragmentation indicating that its specific phosphorylation is necessary for this process. The induction of Golgi fragmentation by MEK1 and Plk3 can be inhibited by the kinase-dead VRK1, the knock-down of VRK1 by siVRK1, the kinase-dead Plk3 or by PD98059, a MEK1 inhibitor. The Plk3-VRK1 kinase module might represent two consecutive steps of a signaling cascade that participates in the regulation of Golgi fragmentation., Ministerio de Ciencia e Innovación (SAF2007-60242 and CSD2007-0017), Junta de Castilla y León (Consejería de Educación, CSI-14A08 and GR-15; Consejería de Sanidad SAN673/SA02/08) and Federación de Cajas de Ahorro de Castilla y León.

The search for oncogenes is becoming increasingly important in cancer genetics because they are suitable targets for therapeutic intervention. To identify novel oncogenes, activated by gene amplification, we analyzed cDNA microarrays by high-resolution comparative genome hybridization and compared DNA copy number and mRNA expression levels in lung cancer cell lines. We identified several amplicons (5p13, 6p22-21, 11q13, 17q21 and 19q13) that had a concomitant increase in gene expression. These regions were also found to be amplified in lung primary tumours. We mapped the boundaries and measured expression levels of genes within the chromosome 6p amplicon. The Sry-HMG box gene SOX4 (sex-determining region Y box 4), which encodes a transcription factor involved in embryonic cell differentiation, was overexpressed by a factor of 10 in cells with amplification relative to normal cells. SOX4 expression was also stronger in a fraction of lung primary tumours and lung cancer cell lines and was associated with the presence of gene amplification. We also found variants of SOX4 in lung primary tumours and cancer cell lines, including a somatic mutation that introduced a premature stop codon (S395X) at the serine-rich C-terminal domain. Although none of the variants increased the transactivation ability of SOX4, overexpression of the wildtype and of the non-truncated variants in NIH3T3 cells significantly increased the transforming ability of the weakly oncogenic RHOA-Q63L. In conclusion, our results show that, in lung cancer, SOX4 is overexpressed due to gene amplification and provide evidence of oncogenic properties of SOX4. © The Author 2009. Published by Oxford University Press. All rights reserved., The work was supported by grants from the Spanish Ministerio de Educacion (SAF2005-00626) and Instituto de Salud Carlos III (RTICC), to M.S.-C., and Spanish Ministerio de Educacion (SAF2007-60242 and CSD2007-0017), Junta de Castilla y Leon (Consejeria de Educacion, CSI-14A08 and GR-15) to P.A.L. Fellowships to M.S.-G. and S.D.C. were granted by the CSIC and Comunidad Autónoma de Madrid, respectively.

Under a Creative Commons license., NBS1 is an early component in DNA-Damage Response (DDR) that participates in the initiation of the responses aiming to repair double-strand breaks caused by different mechanisms. Early steps in DDR have to react to local alterations in chromatin that are induced by DNA damage. NBS1 participates in the early detection of DNA damage and functions as a platform for the recruitment and assembly of components that are sequentially required for the repair process. In this work we have studied whether the VRK1 chromatin kinase can affect the activation of NBS1 in response to DNA damage induced by ionizing radiation. VRK1 is forming a basal preassembled complex with NBS1 in non-damaged cells. Knockdown of VRK1 resulted in the loss of NBS1 foci induced by ionizing radiation, an effect that was also detected in cell-cycle arrested cells and in ATM (−/−) cells. The phosphorylation of NBS1 in Ser343 by VRK1 is induced by either doxorubicin or IR in ATM (−/−) cells. Phosphorylated NBS1 is also complexed with VRK1. NBS1 phosphorylation by VRK1 cooperates with ATM. This phosphorylation of NBS1 by VRK1 contributes to the stability of NBS1 in ATM (−/−) cells, and the consequence of its loss can be prevented by treatment with the MG132 proteasome inhibitor of RNF8. We conclude that VRK1 regulation of NBS1 contributes to the stability of the repair complex and permits the sequential steps in DDR., D.M.M, M. S-G and L. C. were supported by JAE-CSIC-Fondo Social Europeo fellowships. I. C-M. was supported by Ministerio de Economía y Competitividad-FPI-Fondo Social Europeo fellowship. This work was funded by grants from Ministerio de Economía y Competitividad (SAF2013-44810R; SAF2014-57791-REDC) and Consejería de Educación de la Junta de Castilla y León (CSI002U14 and UIC-017) to P.A.L.

Cajal bodies (CBs) are nuclear organelles associated with ribonucleoprotein functions and RNA maturation. CBs are assembled on coilin, its main scaffold protein, in a cell cycle dependent manner. The Ser-Thr VRK1 (vaccinia-related kinase 1) kinase, whose activity is also cell cycle regulated, interacts with and phosphorylates coilin regulating assembly of CBs. Coilin phosphorylation is not necessary for its interaction with VRK1, but it occurs in mitosis and regulates coilin stability. Knockdown of VRK1 or VRK1 inactivation by serum deprivation causes a loss of coilin phosphorylation in Ser184 and of CBs formation, which are rescued with an active VRK1, but not by kinase-dead VRK1. The phosphorylation of coilin in Ser184 occurs during mitosis before assembly of CBs. Loss of coilin phosphorylation results in disintegration of CBs, and of coilin degradation that is prevented by proteasome inhibitors. After depletion of VRK1, coilin is ubiquitinated in nuclei, which is partly mediated by mdm2, but its proteasomal degradation occurs in cytosol and is prevented by blocking its nuclear export. We conclude that VRK1 is a novel regulator of CBs dynamics and stability in cell cycle by protecting coilin from ubiquitination and degradation in the proteasome, and propose a model of CB dynamics., L. C. and M. S-G. were funded by JAE-CSIC-Fondo Social Europeo fellowships. This work was supported by grants from Ministerio de Ciencia e Innovación (SAF2010-14935), Ministerio de Economía y Competitividad (SAF2013-44810R, SAF2014-57791-REDC), and Junta de Castilla y León-Consejería de Educación (CSI002U14) to P.A.L;and grant (702/13) from the Israel Science Foundation to P.R. and E.L.L.

Cellular responses to DNA damage require the formation of protein complexes in a highly organized fashion. The complete molecular components that participate in the sequential signaling response to DNA damage remain unknown. Here we demonstrate that vaccinia-related kinase 1 (VRK1) in resting cells plays an important role in the formation of ionizing radiationinduced foci that assemble on the 53BP1 scaffold protein during the DNA damage response. The kinase VRK1 is activated by DNA double strand breaks induced by ionizing radiation (IR) and specifically phosphorylates 53BP1 in serum-starved cells. VRK1 knockdown resulted in the defective formation of 53BP1 foci in response to IR both in number and size. This observed effect on 53BP1 foci is p53- and ataxia-telangiectasia mutated (ATM)-independent and can be rescued with VRK1 mutants resistant to siRNA. VRK1 knockdown also prevented the activating phosphorylation of ATM, CHK2, and DNA-dependent protein kinase in response to IR. VRK1 activation in response to DNA damage is a novel and early step in the signaling of mammalian DNA damage responses., This work was supported in part by Ministerio de Ciencia e Innovación Grants SAF2010-14935 and CSD2007-0017, Junta de Castilla y León Grants CSI006A11-2 and GR-15, and the Kutxa-Fundación Inbiomed. Supported by Junta para la Ampliación de Estudios-CSIC-Fondo Social Europeo fellowships.

Protein phosphorylation by kinases plays a central role in the regulation and coordination of multiple biological processes. In general, knowledge on kinase specificity is restricted to substrates identified in the context of specific cellular responses, but kinases are likely to have multiple additional substrates and be integrated in signaling networks that might be spatially and temporally different, and in which protein complexes and subcellular localization can play an important role. In this report the substrate specificity of atypical human vaccinia-related kinases (VRK1 and VRK2) using a human peptide-array containing 1080 sequences phosphorylated in known signaling pathways has been studied. The two kinases identify a subset of potential peptide targets, all of them result in a consensus sequence composed of at least four basic residues in peptide targets. Linear peptide arrays are therefore a useful approach in the characterization of kinases and substrate identification, which can contribute to delineate the signaling network in which VRK proteins participate. One of these target proteins is coilin; a basic protein located in nuclear Cajal bodies. Coilin is phosphorylated in Ser184 by both VRK1 and VRK2. Coilin colocalizes and interacts with VRK1 in Cajal bodies, but not with the mutant VRK1 (R358X). VRK1 (R358X) is less active than VRK1. Altered regulation of coilin might be implicated in several neurological diseases such as ataxias and spinal muscular atrophies. © 2011 Elsevier B.V., M. S-G., and M. V-C have JAE/CSIC predoctoral fellowships. This work was supported by grants from Ministerio de Educación, Ciencia e Innovación (SAF2010-14935, and CSD2007-0017), Junta de Castilla y León-Consejería de Educación (GR15, CSI006A11-2), CSIC (PA1003032) and Kutxa-Fundación Inbiomed to P.A.L; and by the Legacy Heritage Bio-Medical Programof the Israel Science Foundation (grant no. 1872/2008) to P.R. and E.L.L.

El pdf del artículo es la versión post-print., Cell division, in addition to an accurate transmission of genetic information to daughter cells, also requires the temporal and spatial coordination of several biological processes without which cell division would not be feasible. These processes include the temporal coordination of DNA replication and chromosome segregation, regulation of nuclear envelope disassembly and assembly, chromatin condensation and Golgi fragmentation for its redistribution into daughter cells, among others. However, little is known regarding regulatory proteins and signalling pathways that might participate in the coordination of all these different biological functions. Such regulatory players should directly have a role in the processes leading to cell division. VRK1 (Vaccinia-related kinase 1) is an early response gene required for cyclin D1 expression, regulates p53 by a specific Thr18 phosphorylation, controls chromatin condensation by histone phosphorylation, nuclear envelope assembly by phosphorylation of BANF1, and participates in signalling required for Golgi fragmentation late in the G2 phase. We propose that VRK1, a Ser-Thr kinase, might be a candidate to play an important coordinator role in these cell division processes as part of a novel signalling pathway., The laboratory is supported by grants from Ministerio de Ciencia e Innovación (SAF2010-14935 and CSD2007-0017), Junta de Castilla y León (Consejería de Educación, GR15), and Kutxa-Fundación Inbiomed.

Patients with recurrent episodes of Escherichia coli bloodstream infection (REC-BSI) have been described previously only in small studies. We report on the incidence, clinical significance, and predisposing conditions of REC-BSI in a general hospital from 1992 to 2005. All patients with E. coli bloodstream infection (EC-BSI) were retrieved from our database. We defined recurrent episodes as those occurring at least 1 month apart after a clinical response (cases). To study risk factors for REC-BSI, we randomly selected a third of the REC-BSI cases and a similar number of controls (patients with a single EC-BSI). Available E. coli isolates from initial and recurrent episodes were typed using repetitive-extragenic-palindromic-sequences to distinguish between relapse and reinfection. During the study period there were 4287 episodes of EC-BSI in 3970 patients; of these, 251 (6.3%) patients had 568 episodes of recurrence (13.3%). We selected 81 cases and 81 controls for study. The underlying conditions of patients with REC-BSI included immunosuppression (33%), urinary (24%) or biliary obstruction (16%), chronic liver disease (16%), presence of a central venous catheter (8%), and miscellaneous entities (3%). Male sex, presence of hematologic malignancy, inadequate antibiotic treatment, and an extraurinary source of the BSI were independent risk factors for recurrence in the multivariate analysis. Molecular typing performed in 88 infections from 44 patients showed that 47% of REC-BSI were relapses rather than reinfections. Recurrence of E. coli BSI is not an uncommon phenomenon and includes relapses (47%) and reinfections (53%). Recurrence should suggest not only the presence of urinary or biliary obstruction, but also the presence of immunosuppression.

Author's Choice - Final Version Full Access (OA).-- Creative Commons Attribution Non-Commercial License applies to Author Choice Articles., Human vaccinia-related kinase (VRK) 1 is a novel serinethreonine kinase that regulates several transcription factors, nuclear envelope assembly, and chromatin condensation and is also required for cell cycle progression. The regulation of this kinase family is unknown. Mass spectrometry has permitted the identification of Ran as an interacting and regulatory protein of the VRK serine-threonine kinase activities. The stable interaction has been validated by pulldown of endogenous proteins as well as by reciprocal immunoprecipitations. The three members of the VRK family stably interact with Ran, and the interaction was not affected by the bound nucleotide, GDP or GTP. The interaction was stronger with the RanT24N that is locked in its inactive conformation and cannot bind nucleotides. None of the kinases phosphorylated Ran or RCC1. VRK1 does not directly interact with RCC1, but if Ran is present they can be isolated as a complex. The main effect of the interaction of inactive RanGDP with VRK1 is the inhibition of its kinase activity, which was detected by a reduction in VRK1 autophosphorylation and a reduction in phosphorylation of histone H3 in residues Thr-3 and Ser-10. The kinase activity inhibition can be relieved by the interaction with the constitutively active RanGTP or RanL43E, which locks Ran in its GTP-bound active conformation. In this complex, the interaction with VRK proteins does not alter the effect of its guanine exchange factor, RCC1. Ran is a novel negative regulator of nuclear VRK1 and VRK2 kinase activity, which may vary in different subcellular localizations generating an asymmetric intracellular distribution of kinase activity depending on local protein interactions., This work was supported in part by Ministerio de Educación y Ciencia Grants SAF2004-02900, SAF2007-60242, and Consolider CSD2007-0017; by Junta de Castilla y Leon Grant CSI-14A08 and GR1S; and by the Federación de Cajas de Ahorro de Castilla y León.

Background. Cellular biological responses to specific stimulation are determined by a balance among signaling pathways. Protein interactions are likely to modulate these pathways. Vaccinia-related kinase-2 (VRK2) is a novel human kinase that can modulate different signaling pathways. Principal findings. We report that in vivo, the activity of JIP1-JNK complexes is downregulated by VRK2 in response to interleukin-1β. Also the reduction of endogenous VRK2 with shRNA increases the transcriptional response to IL-1β. The JIP1 scaffold protein assembles three consecutive members of a given MAPK pathway forming signaling complexes and their signal can be modulated by interactions with regulatory proteins that remain to be identified. Knocking-down JIP1 with siRNA resulted in elimination of the AP1 transcriptional response to IL-1β. VRK2, a member of novel Ser-Thr kinase family, is able to stably interact with JIP1, TAK1 and MKK7, but not JNK, and can be isolated forming oligomeric complexes with different proportions of TAK1, MKK7β1 and JNK. JIP1 assembles all these proteins in an oligomeric signalosome. VRK2 binding to the JIP1 signalosome prevents the association of JNK and results in a reduction in its phosphorylation and downregulation of AP1-dependent transcription. Conclusions/Significance. This work suggests that the intracellular level of VRK2 protein can modulate the flow through a signaling pathway and alter the response from a receptor that can be distributed by more than one pathway, and thus contribute to the cellular specificity of the response by forming alternative signaling complexes. Furthermore, the effect might be more general and affect other signaling routes assembled on the JIP1 scaffold protein for which a model is proposed., S.B., M. S-G, and C.R.S. have predoctoral fellowships from Ministerio de Educación y Ciencia, CSIC (Spain) and Fundação para a Ciência e a Tecnologia (Portugal) respectively. This work was funded by grants from Ministerio de Educación y Ciencia (SAF2004-02900, SAF2007-60242 and Consolider CSD-2007-0017), Fundación de Investigación Médica MM and Federación de Cajas de Ahorro de Castilla y León to P.A.L.

The human VRK1 is a new ser-thr kinase expressed in many cell types. VRK1 is a regulator of p53 and other transcription factors related with cellular responses to stress. The human VRK1 protein has a dominant epitope located in its C-terminal region, between residues 333 and 396, which is detected by different antibodies. All the antibodies detect the same protein in immunoblots and immunoprecipitations. But the antibodies have a different reactivity when a single aminoacid substitution in T355, mimicking phosphorylation, is introduced next to the nuclear localization signal. These differences in reactivity permit the identification of different intracellular subpopulations. Most of the intracellular VRK1 protein is nuclear, but in some cells it is also detected in the cytosol, depending on the type of tissue. These different locations are detected by immunohistochemistry of human biopsies and immunofluorescence of cell lines. Some antibodies identify a subpopulation within the vesicular system, particularly in the Golgi apparatus. The different reactivity of the VRK1 protein indicates that this protein has a subcellular localization that can be regulated, thus adding an additional level of regulatory complexity to the VRK1 protein. © 2007 Elsevier Inc. All rights reserved., A.V. and I.L.-S. are predoctoral fellows of Ministerio de Educación y Ciencia. F.M.V. has a predoctoral fellowship of Fundación Ramón Areces. A.S and M.S.-G. have CSICI3 P predoctoral fellowships. This work has been funded by grants from Ministerio de Educación y Ciencia (SAF2004– 02900), Federación de Cajas de Ahorro de Castilla y León and Fundación de Investigación Médica MM.

DNA damage immediate cellular response requires the activation of p53 by kinases. We found that p53 forms a basal stable complex with VRK1, a Ser-Thr kinase that responds to UV-induced DNA damage by specifically phosphorylating p53. This interaction takes place through the p53 DNA binding domain, and frequent DNA-contact mutants of p53, such as R273H, R248H or R280K, do not disrupt the complex. UV-induced DNA damage activates VRK1, and is accompanied by phosphorylation of p53 at Thr-18 before it accumulates. We propose that the VRK1-p53 basal complex is an early-warning system for immediate cellular responses to DNA damage., I. L.-S. and A.V. have FPI and FPU fellowships respectively from Ministerio de Ciencia e Innovación. M.V.-C., M.S.-G. and A.C. have JAE-CSIC-Fondo Social Europeo fellowships. This work was supported by Grants from Ministerio de Educación, Ciencia e Innovación (SAF2010-14935 and CSD2007-0017) and Kutxa-Fundación Inbiomed to P.A.L.