CRISPR/dCAS9-mediated DNA demethylation screen identifies functional epigenetic determinants of colorectal cancer.

BACKGROUND: Promoter hypermethylation of tumour suppressor genes is frequently observed during the malignant transformation of colorectal cancer (CRC). However, whether this epigenetic mechanism is functional in cancer or is a mere consequence of the carcinogenic process remains to be elucidated. RESULTS: In this work, we performed an integrative multi-omic approach to identify gene candidates with strong correlations between DNA methylation and gene expression in human CRC samples and a set of 8 colon cancer cell lines. As a proof of concept, we combined recent CRISPR-Cas9 epigenome editing tools (dCas9-TET1, dCas9-TET-IM) with a customized arrayed gRNA library to modulate the DNA methylation status of 56 promoters previously linked with strong epigenetic repression in CRC, and we monitored the potential functional consequences of this DNA methylation loss by means of a high-content cell proliferation screen. Overall, the epigenetic modulation of most of these DNA methylated regions had a mild impact on the reactivation of gene expression and on the viability of cancer cells. Interestingly, we found that epigenetic reactivation of RSPO2 in the tumour context was associated with a significant impairment in cell proliferation in p53-/- cancer cell lines, and further validation with human samples demonstrated that the epigenetic silencing of RSPO2 is a mid-late event in the adenoma to carcinoma sequence. CONCLUSIONS: These results highlight the potential role of DNA methylation as a driver mechanism of CRC and paves the way for the identification of novel therapeutic windows based on the epigenetic reactivation of certain tumour suppressor genes.

Disease control in patients with psoriatic arthritis in real clinical practice in Spain: MiDAS study.

OBJECTIVE: MiDAS study assessed the percentage of psoriatic arthritis (PsA) patients treated in routine clinical practice who achieved control of disease activity according to Disease Activity in Psoriatic Arthritis (DAPSA) and Minimal Disease Activity (MDA). METHODS: Observational, non-interventional, cross-sectional, multicenter study conducted under conditions of routine clinical practice in 36 centers with outpatient rheumatology clinics in Spanish public hospitals. Patients included were adults (≥18 years) with ≥6 months PsA diagnosis according to classification for PsA (CASPAR) criteria and undergoing treatment ≥3 months. The main variable evaluated was the percentage of patients under remission and low disease activity, assessed through DAPSA and MDA. RESULTS: 313 patients with PsA were included: 54.3% male; with mean age of 54.1±12.2 years and mean disease duration of 10.5±9.0 years. Mean C-reactive protein (CRP) serum levels were 4.9±7.3mg/L. At the study visit, 58.5% of patients were in monotherapy (17.6% biological and 40.9% non-biological) and 41.2% were receiving biological and non-biological therapy. 59.4% of patients showed low disease activity (DAPSA≤14) and 19.8% were on remission (DAPSA≤4). Moreover, 51.4% of the patients reached an MDA status (≥5 MDA). CONCLUSIONS: Around 40% of PsA patients presented uncontrolled disease, highlighting the need to improve the management of these patients in clinical practice.

Disease control in patients with psoriatic arthritis in real clinical practice in Spain: MiDAS study / Control de la enfermedad en pacientes con artritis psoriásica en práctica clínica real en España: estudio MiDAS

Objective: MiDAS study assessed the percentage of psoriatic arthritis (PsA) patients treated in routine clinical practice who achieved control of disease activity according to Disease Activity in Psoriatic Arthritis (DAPSA) and Minimal Disease Activity (MDA). Methods: Observational, non-interventional, cross-sectional, multicenter study conducted under conditions of routine clinical practice in 36 centers with outpatient rheumatology clinics in Spanish public hospitals. Patients included were adults (≥18 years) with ≥6 months PsA diagnosis according to classification for PsA (CASPAR) criteria and undergoing treatment ≥3 months. The main variable evaluated was the percentage of patients under remission and low disease activity, assessed through DAPSA and MDA. Results: 313 patients with PsA were included: 54.3% male; with mean age of 54.1±12.2 years and mean disease duration of 10.5±9.0 years. Mean C-reactive protein (CRP) serum levels were 4.9±7.3mg/L. At the study visit, 58.5% of patients were in monotherapy (17.6% biological and 40.9% non-biological) and 41.2% were receiving biological and non-biological therapy. 59.4% of patients showed low disease activity (DAPSA≤14) and 19.8% were on remission (DAPSA≤4). Moreover, 51.4% of the patients reached an MDA status (≥5 MDA). Conclusions: Around 40% of PsA patients presented uncontrolled disease, highlighting the need to improve the management of these patients in clinical practice.(AU)

Objetivo: El estudio MiDAS evaluó el porcentaje de pacientes con artritis psoriásica (APs) tratados en práctica clínica habitual que lograron el control de la actividad de la enfermedad de acuerdo con Disease Activity in Psoriatic Arthritis (DAPSA) y Minimal Disease Activity (MDA). Métodos: Estudio observacional, no intervencionista, transversal, multicéntrico, realizado en condiciones de práctica clínica habitual en 36 centros con consultas externas de reumatología de hospitales públicos españoles. Los pacientes incluidos eran adultos (≥18 años) con ≥6 meses de diagnóstico de APs según los criterios de clasificación de la APs (CASPAR) y en tratamiento durante ≥3 meses. La variable principal evaluada fue el porcentaje de pacientes en remisión y baja actividad de la enfermedad, evaluados mediante DAPSA y MDA. Resultados: Se incluyeron 313 pacientes con APs: 54,3% varones; con una edad media de 54,1±12,2 años y una duración media de la enfermedad de 10,5±9,0 años. Los niveles séricos medios de proteína C reactiva fueron de 4,9±7,3mg/L. En la visita del estudio, el 58,5% de los pacientes estaban siendo tratados con monoterapia (17,6% biológicos y 40,9% no biológicos) y el 41,2% recibían terapia biológica y no biológica. El 59,4% de los pacientes mostró baja actividad de la enfermedad (DAPSA≤14) y el 19,8% estaban en remisión (DAPSA≤4). Además, el 51,4% de los pacientes alcanzó un estado de MDA (≥5 MDA). Conclusiones: Alrededor del 40% de los pacientes con APs presentaban enfermedad no controlada, destacando la necesidad de mejorar el manejo de estos pacientes en la práctica clínica.(AU)

Disease control in patients with ankylosing spondylitis in real clinical practice in Spain: Results of the MIDAS study.

INTRODUCTION AND OBJECTIVES: Understanding the disease activity is fundamental to improve patient prognosis and patients' quality of life. MiDAS study described disease activity in ankylosing spondylitis (AS) Spanish patients and the proportion of them with controlled disease. METHODS: Observational, cross-sectional, multicenter study carried out under conditions of routine clinical practice. Adult (≥18 years) patients with ≥6 months since AS diagnosis treated ≥3 months prior to inclusion. The primary endpoint was the percentage of patients with low disease activity assessed through BASDAI (primary endpoint) and ASDAS-CRP (secondary endpoint). RESULTS: 313 AS patients included: 75.7% male; 78.5% HLA-B*27 positive; mean (SD) baseline age of 50.4 (12.0) years; mean (SD) disease duration of 15.5 (11.6) years; 73.5% were treated with biological disease-modifying antirheumatic drugs (DMARDs), 22.4% with non-biological DMARDs and 53.7% with non-steroidal anti-inflammatory drugs, alone or in combination. Monotherapy with biologics and non-biologics was used by 29.7% and 26.8% of patients, respectively. According to BASDAI, 38.0% were in remission (BASDAI≤2) and 64.5% showed adequate disease control (BASDAI<4). According to ASDAS-CRP, 29.4% achieved remission (ASDAS-CRP<1.3) and 28.1% low disease activity (1.3≤ASDAS-CRP<2.1). CONCLUSIONS: Almost two thirds of the AS patients recruited had low disease activity, with about one third of them being in remission (BASDAI≤2, ASDAS-CRP<1.3). These results highlight the existing room for improvement in treating AS patients in clinical practice.

Disease control in patients with ankylosing spondylitis in real clinical practice in Spain: Results of the MIDAS study / Control de la enfermedad en pacientes con espondilitis anquilosante en la práctica clínica habitual en España: Resultados del estudio MiDAS

Introduction and objectives: Understanding the disease activity is fundamental to improve patient prognosis and patients’ quality of life. MiDAS study described disease activity in ankylosing spondylitis (AS) Spanish patients and the proportion of them with controlled disease. Methods: Observational, cross-sectional, multicenter study carried out under conditions of routine clinical practice. Adult (≥18 years) patients with ≥6 months since AS diagnosis treated ≥3 months prior to inclusion. The primary endpoint was the percentage of patients with low disease activity assessed through BASDAI (primary endpoint) and ASDAS-CRP (secondary endpoint). Results: 313 AS patients included: 75.7% male; 78.5% HLA-B*27 positive; mean (SD) baseline age of 50.4 (12.0) years; mean (SD) disease duration of 15.5 (11.6) years; 73.5% were treated with biological disease-modifying antirheumatic drugs (DMARDs), 22.4% with non-biological DMARDs and 53.7% with non-steroidal anti-inflammatory drugs, alone or in combination. Monotherapy with biologics and non-biologics was used by 29.7% and 26.8% of patients, respectively. According to BASDAI, 38.0% were in remission (BASDAI≤2) and 64.5% showed adequate disease control (BASDAI<4). According to ASDAS-CRP, 29.4% achieved remission (ASDAS-CRP<1.3) and 28.1% low disease activity (1.3≤ASDAS-CRP<2.1). Conclusions: Almost two thirds of the AS patients recruited had low disease activity, with about one third of them being in remission (BASDAI≤2, ASDAS-CRP<1.3). These results highlight the existing room for improvement in treating AS patients in clinical practice.(AU)

Introducción y objetivos: Comprender la actividad de la enfermedad es fundamental para mejorar el pronóstico y la calidad de vida de los pacientes. El estudio MiDAS describió la actividad de la enfermedad en pacientes españoles con espondilitis anquilosante (EA) y la proporción de ellos con enfermedad controlada. Métodos: Estudio observacional, transversal, multicéntrico, realizado en condiciones de práctica clínica habitual. Pacientes adultos (≥18años) con ≥6meses desde el diagnóstico de EA tratados ≥3meses antes de la inclusión. La variable principal fue el porcentaje de pacientes en baja actividad, evaluado mediante BASDAI (variable principal) y ASDAS-CRP (variable secundaria). Resultados: Hubo 313 pacientes con EA incluidos: 75,7% varones; 78,5% HLA-B*27 positivos; edad media (DE) basal de 50,4 (12,0) años; duración media (DE) de la enfermedad de 15,5 (11,6) años; el 73,5% fueron tratados con fármacos antirreumáticos modificadores de la enfermedad (FAME) biológicos, el 22,4% con FAME no biológicos y el 53,7% con antiinflamatorios no esteroideos, solos o en combinación. La monoterapia con biológicos y no biológicos fue utilizada por el 29,7 y el 26,8% de los pacientes, respectivamente. Según BASDAI, el 38,0% estaban en remisión (BASDAI≤2) y el 64,5% mostraron un adecuado control de la enfermedad (BASDAI<4). Según ASDAS-CRP, el 29,4% alcanzaron remisión (ASDAS-CRP<1,3) y el 28,1% baja actividad de la enfermedad (1,3≤ASDAS-CRP<2,1). Conclusiones: Casi dos tercios de los pacientes con EA incluidos presentaban baja actividad de la enfermedad, con aproximadamente un tercio de ellos en remisión (BASDAI≤2, ASDAS-CRP<1,3). Estos resultados destacan el margen de mejora existente para tratar pacientes con EA en la práctica clínica.(AU)

Analysis of Global and Local DNA Methylation Patterns in Blood Samples of Patients With Autism Spectrum Disorder.

The goal of this investigation was to determine whether there are alterations in DNA methylation patterns in children with autism spectrum disorder (ASD). Material and Methods: Controlled prospective observational case-control study. Within the ASD group, children were sub-classified based on the presence (AMR subgroup) or absence (ANMR subgroup) of neurodevelopmental regression during the first 2 years of life. We analyzed the global levels of DNA methylation, reflected in LINE-1, and the local DNA methylation pattern in two candidate genes, Neural Cell Adhesion Molecule (NCAM1) and Nerve Growth Factor (NGF) that, according to our previous studies, might be associated to an increased risk for ASD. For this purpose, we utilized blood samples from pediatric patients with ASD (n = 53) and their corresponding controls (n = 45). Results: We observed a slight decrease in methylation levels of LINE-1 in the ASD group, compared to the control group. One of the CpG in LINE-1 (GenBank accession no.X58075, nucleotide position 329) was the main responsible for such reduction, highly significant in the ASD subgroup of children with AMR (p < 0.05). Furthermore, we detected higher NCAM1 methylation levels in ASD children, compared to healthy children (p < 0.001). The data, moreover, showed higher NGF methylation levels in the AMR subgroup, compared to the control group and the ANMR subgroup. These results are consistent with our prior study, in which lower plasma levels of NCAM1 and higher levels of NGF were found in the ANMR subgroup, compared to the subgroup that comprised neurotypically developing children. Conclusions: We have provided new clues about the epigenetic changes that occur in ASD, and suggest two potential epigenetic biomarkers that would facilitate the diagnosis of the disorder. We similarly present with evidence of a clear differentiation in DNA methylation between the ASD subgroups, with or without mental regression.

Complementary Functions of Plant AP Endonucleases and AP Lyases during DNA Repair of Abasic Sites Arising from C:G Base Pairs.

Abasic (apurinic/apyrimidinic, AP) sites are ubiquitous DNA lesions arising from spontaneous base loss and excision of damaged bases. They may be processed either by AP endonucleases or AP lyases, but the relative roles of these two classes of enzymes are not well understood. We hypothesized that endonucleases and lyases may be differentially influenced by the sequence surrounding the AP site and/or the identity of the orphan base. To test this idea, we analysed the activity of plant and human AP endonucleases and AP lyases on DNA substrates containing an abasic site opposite either G or C in different sequence contexts. AP sites opposite G are common intermediates during the repair of deaminated cytosines, whereas AP sites opposite C frequently arise from oxidized guanines. We found that the major Arabidopsis AP endonuclease (ARP) exhibited a higher efficiency on AP sites opposite G. In contrast, the main plant AP lyase (FPG) showed a greater preference for AP sites opposite C. The major human AP endonuclease (APE1) preferred G as the orphan base, but only in some sequence contexts. We propose that plant AP endonucleases and AP lyases play complementary DNA repair functions on abasic sites arising at C:G pairs, neutralizing the potential mutagenic consequences of C deamination and G oxidation, respectively.

Consenso ASAS en nomenclatura en español para las espondiloartritis / Assessment of SpondyloArthritis International Society (ASAS) consensus on Spanish nomenclature for spondyloarthritis

OBJETIVO: Desarrollar un documento de consenso para estandarizar los términos, abreviaturas y acrónimos en español empleados en el campo de las espondiloartritis (EspA). MÉTODOS: Se creó un grupo de trabajo internacional compuesto por todos los miembros de Assessment of SpondyloArthritis International Society (ASAS) nativos de habla española, miembros del comité ejecutivo del Grupo para el estudio de la Espondiloartritis de la Sociedad Española de Reumatología (GRESSER), 2 metodólogos, 2 lingüistas de la Real Academia Nacional de Medicina de España (RANM) y 2 pacientes de la Coordinadora Española de Asociaciones de Espondilitis (CEADE). Se realizó una revisión de la literatura de los últimos 15 años (publicaciones, el CIE y CIF, guías, consensos y recomendaciones) para identificar los términos, abreviaturas y acrónimos discrepantes. Mediante un Delphi de 3 rondas y una reunión presencial, se discutieron, seleccionaron y acordaron los términos, abreviaturas y acrónimos a utilizar. Durante todo este proceso se siguieron las recomendaciones de la RANM basadas en el Diccionario panhispánico de términos médicos. RESULTADOS: Se consensuaron 46 términos, abreviaturas y acrónimos. Se aceptó la traducción al español para 6 términos y 6 abreviaturas empleados para nombrar o clasificar la enfermedad y para 6 términos y 4 abreviaturas relacionados con las EspA. Se acordó no traducir 15 acrónimos por estar ya establecidos, pero al mencionarlos, se recomendó seguir esta estructura: tipo de acrónimo en español y acrónimo y forma extensa en inglés. Con respecto a 7 términos o abreviaturas asociados a acrónimos, se acordó traducir solo la forma extensa y se consensuó una traducción. CONCLUSIONES: Con esta estandarización del lenguaje de las EspA se pretende establecer un uso común de la nomenclatura en español para las EspA. Su implementación será muy beneficiosa, evitando malentendidos y consumo de recursos

OBJECTIVE: To develop a consensus to standardize the use of Spanish terms, abbreviations and acronyms in the field of spondyloarthritis (SpA). METHODS: An international task force comprising all native Spanish-speaking Assessment of SpondyloArthritis International Society (ASAS) members, the executive committee of Grupo para el estudio de la Espondiloartritis de la Sociedad Española de Reumatología (GRESSER), two methodologists, two linguists from the Real Academia Nacional de Medicina de España (RANM) and two patients from the Spanish Coordinator of Spondylitis Associations (CEADE) was established. A literature review was performed to identify the conflicting terms/abbreviations/acronyms in SpA. This review examined written sources in Spanish including manuscripts, ICF and ICD, guidelines, recommendations and consensuses. This was followed by a nominal group meeting and a three-round Delphi. The recommendations from the RANM based on the Panhispanic dictionary were followed throughout the process. RESULTS: Consensus was reached for 46 terms, abbreviations or acronyms related to the field of SpA. A Spanish translation was accepted for 6 terms and 6 abbreviations to name or classify the disease, and for 6 terms and 4 abbreviations related to SpA. It was agreed not to translate 15 acronyms into Spanish. However, when mentioning them, it was recommended to follow this structure: type of acronym in Spanish and acronym and expanded form in English. With regard to 7 terms or abbreviations attached to acronyms, it was agreed to translate only the expanded form and a translation was also selected for each of them. CONCLUSIONS: Through this standardization, it is expected to establish a common use of the Spanish nomenclature for SpA. The implementation of this consensus across the community will be of substantial benefit, avoiding misunderstandings and time-consuming processes

DNA Methylation Editing by CRISPR-guided Excision of 5-Methylcytosine.

Tools for actively targeted DNA demethylation are required to increase our knowledge about regulation and specific functions of this important epigenetic modification. DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5-meC), which may promote its replication-dependent dilution and/or active removal through base excision repair (BER). However, it is still unclear whether oxidized derivatives of 5-meC are simply DNA demethylation intermediates or rather epigenetic marks on their own. Unlike animals, plants have evolved enzymes that directly excise 5-meC without previous modification. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5-meC DNA glycosylase to a CRISPR-associated null-nuclease (dCas9) and analyzed its capacity for targeted reactivation of methylation-silenced genes, in comparison to other dCas9-effectors. We found that dCas9-ROS1, but not dCas9-TET1, is able to reactivate methylation-silenced genes and induce partial demethylation in a replication-independent manner. We also found that reactivation induced by dCas9-ROS1, as well as that achieved by two different CRISPR-based chromatin effectors (dCas9-VP160 and dCas9-p300), generally decreases with methylation density. Our results suggest that plant 5-meC DNA glycosylases are a valuable addition to the CRISPR-based toolbox for epigenetic editing.

Assessment of SpondyloArthritis International Society (ASAS) Consensus on Spanish Nomenclature for Spondyloarthritis. / Consenso ASAS en nomenclatura en español para las espondiloartritis.

OBJECTIVE: To develop a consensus to standardize the use of Spanish terms, abbreviations and acronyms in the field of spondyloarthritis (SpA). METHODS: An international task force comprising all native Spanish-speaking Assessment of SpondyloArthritis International Society (ASAS) members, the executive committee of Grupo para el estudio de la Espondiloartritis de la Sociedad Española de Reumatología (GRESSER), two methodologists, two linguists from the Real Academia Nacional de Medicina de España (RANM) and two patients from the Spanish Coordinator of Spondylitis Associations (CEADE) was established. A literature review was performed to identify the conflicting terms/abbreviations/acronyms in SpA. This review examined written sources in Spanish including manuscripts, ICF and ICD, guidelines, recommendations and consensuses. This was followed by a nominal group meeting and a three-round Delphi. The recommendations from the RANM based on the Panhispanic dictionary were followed throughout the process. RESULTS: Consensus was reached for 46 terms, abbreviations or acronyms related to the field of SpA. A Spanish translation was accepted for 6 terms and 6 abbreviations to name or classify the disease, and for 6 terms and 4 abbreviations related to SpA. It was agreed not to translate 15 acronyms into Spanish. However, when mentioning them, it was recommended to follow this structure: type of acronym in Spanish and acronym and expanded form in English. With regard to 7 terms or abbreviations attached to acronyms, it was agreed to translate only the expanded form and a translation was also selected for each of them. CONCLUSIONS: Through this standardization, it is expected to establish a common use of the Spanish nomenclature for SpA. The implementation of this consensus across the community will be of substantial benefit, avoiding misunderstandings and time-consuming processes.

Active DNA Demethylation in Plants.

Methylation of cytosine (5-meC) is a critical epigenetic modification in many eukaryotes, and genomic DNA methylation landscapes are dynamically regulated by opposed methylation and demethylation processes. Plants are unique in possessing a mechanism for active DNA demethylation involving DNA glycosylases that excise 5-meC and initiate its replacement with unmodified C through a base excision repair (BER) pathway. Plant BER-mediated DNA demethylation is a complex process involving numerous proteins, as well as additional regulatory factors that avoid accumulation of potentially harmful intermediates and coordinate demethylation and methylation to maintain balanced yet flexible DNA methylation patterns. Active DNA demethylation counteracts excessive methylation at transposable elements (TEs), mainly in euchromatic regions, and one of its major functions is to avoid methylation spreading to nearby genes. It is also involved in transcriptional activation of TEs and TE-derived sequences in companion cells of male and female gametophytes, which reinforces transposon silencing in gametes and also contributes to gene imprinting in the endosperm. Plant 5-meC DNA glycosylases are additionally involved in many other physiological processes, including seed development and germination, fruit ripening, and plant responses to a variety of biotic and abiotic environmental stimuli.

DNA Base Excision Repair in Plants: An Unfolding Story With Familiar and Novel Characters.

Base excision repair (BER) is a critical genome defense pathway that deals with a broad range of non-voluminous DNA lesions induced by endogenous or exogenous genotoxic agents. BER is a complex process initiated by the excision of the damaged base, proceeds through a sequence of reactions that generate various DNA intermediates, and culminates with restoration of the original DNA structure. BER has been extensively studied in microbial and animal systems, but knowledge in plants has lagged behind until recently. Results obtained so far indicate that plants share many BER factors with other organisms, but also possess some unique features and combinations. Plant BER plays an important role in preserving genome integrity through removal of damaged bases. However, it performs additional important functions, such as the replacement of the naturally modified base 5-methylcytosine with cytosine in a plant-specific pathway for active DNA demethylation.

Characterization of an AP endonuclease from sugarcane - ScARP1.

Plants are sessile organisms that need to cope with different conditions. The Base Excision Repair (BER) pathway is an important mechanism protecting the genome from DNA lesions. Apurinic/apyrimidinic (AP) endonucleases are key BER enzymes that process AP sites arising either spontaneously or as BER intermediates. In Arabidopsis there are three AP endonucleases: AtARP1, AtAPE1L, and AtAPE2, and in sugarcane two AtARP1 homologues have been identified: ScARP1 and ScARP3. ScARP1 shares 59% sequence identity with Arabidopsis AtARP. Protein modeling of ScARP1 and AtARP1 revealed conserved active sites and metal binding sites. For biochemical characterisation, recombinant ScARP1 protein displayed AP endonuclease activity both in the presence of MnCl2 or MgCl2 and the optimal temperature for its activity was 37 °C. Under these conditions, 3'-exonuclease, 3'-phosphatase, and 3'-phosphodiesteterase activities were not detectable. We also show that ScARP1 protein is able to complement mutant atarp-/- cell extracts deficient in AP endonuclease activity. These results suggest that AP endonucleases from different plant species preserve AP endonuclease activity. The biochemical characterisation of ScARP1 extends our knowledge of the BER pathway to a monocot crop plant group.

Recomendaciones sobre el uso de metotrexato en pacientes con artritis psoriásica / Recommendations for the use of methotrexate in psoriatic arthritis

Objetivos: Desarrollar recomendaciones sobre el uso de metotrexato (MTX) en pacientes con artritis psoriásica (APs) basadas en la mejor evidencia y experiencia. Métodos: Se seleccionó un grupo de 12 expertos reumatólogos en el manejo de MTX. Los coordinadores generaron 14 preguntas sobre el uso de MTX en pacientes con APs (perfiles de indicación, eficacia y seguridad) para ser contestadas mediante una revisión sistemática de la literatura. En función de las preguntas se definieron los criterios de inclusión y exclusión y las estrategias de búsqueda (para interrogar Medline, Embase y la Cochrane Library). Dos revisores seleccionaron los artículos resultantes de la búsqueda. Se generaron tablas de evidencia. Paralelamente se evaluaron abstracts de congresos de EULAR y ACR. Con toda esta evidencia los coordinadores generaron 12 recomendaciones preliminares que se evaluaron, discutieron y votaron en una reunión de grupo nominal con el resto de expertos. Para cada recomendación se estableció el nivel de evidencia, grado de recomendación, y grado de acuerdo mediante un Delphi. Se definió acuerdo si al menos el 80% de los participantes contestan sí a la recomendación (sí o no). Resultados: De las 12 recomendaciones preliminares se aceptaron 9 recomendaciones sobre el uso de MTX en la APs. Una se englobó en otra y otras 2 no se llegaron a votar porque se decidió no incluirlas, pero se comentan en el texto final. Conclusiones: Estas recomendaciones pretenden resolver algunos interrogantes clínicos habituales y facilitar la toma de decisiones con el uso de MTX en la APs

Objectives: To develop recommendations for the management of methotrexate (MTX) in psoriatic arthritis (PsA), based on best evidence and experience. Methods: A group of 12 experts on MTX use was selected. The coordinators formulated 14 questions about the use of MTX in PsA patients (indications, efficacy, safety and cost-effectiveness). A systematic review was conducted to answer the questions. Using this information, inclusion and exclusion criteria were established, as were the search strategies (Medline, EMBASE and the Cochrane Library were searched). Two different reviewers selected the articles. Evidence tables were created. At the same time, European League Against Rheumatism and American College of Rheumatology abstracts were evaluated. Based on this evidence, the coordinators proposed 12 preliminary recommendations that the experts discussed and voted on in a nominal group meeting. The level of evidence and grade of recommendation were established using the Oxford Centre for Evidence Based Medicine and the level of agreement with the Delphi technique (2 rounds). Agreement was established if at least 80% of the experts voted yes (yes/no). Results: A total of 12 preliminary recommendations on the use of MTX were proposed, 9 of which were accepted. One was included in a different recommendation and another 2 were not voted on and were thereafter clarified in the main text. Conclusions: These recommendations aim to answer frequent questions and help in decision making strategies when treating PsA patients with MTX

Monitoring base excision repair in Chlamydomonas reinhardtii cell extracts.

Base excision repair (BER) is a major defense pathway against spontaneous DNA damage. This multistep process is initiated by DNA glycosylases that recognise and excise the damaged base, and proceeds by the concerted action of additional proteins that perform incision of the abasic site, gap filling and ligation. BER has been extensively studied in bacteria, yeasts and animals. Although knowledge of this pathway in land plants is increasing, there are no reports detecting BER in algae. We describe here an experimental in vitro system allowing the specific analysis of BER in the model alga Chlamydomonas reinhardtii. We show that C. reinhardtii cell-free extracts contain the enzymatic machinery required to perform BER of ubiquitous DNA lesions, such as uracil and abasic sites. Our results also reveal that repair can occur by both single-nucleotide insertion and long-patch DNA synthesis. The experimental system described here should prove useful in the biochemical and genetic dissection of BER in algae, and may contribute to provide a broader picture of the evolution and biological relevance of DNA repair pathways in photosynthetic eukaryotes.

Nonenzymatic release of N7-methylguanine channels repair of abasic sites into an AP endonuclease-independent pathway in Arabidopsis.

Abasic (apurinic/apyrimidinic, AP) sites in DNA arise from spontaneous base loss or by enzymatic removal during base excision repair. It is commonly accepted that both classes of AP site have analogous biochemical properties and are equivalent substrates for AP endonucleases and AP lyases, although the relative roles of these two types of enzymes are not well understood. We provide here genetic and biochemical evidence that, in Arabidopsis, AP sites generated by spontaneous loss of N7-methylguanine (N7-meG) are exclusively repaired through an AP endonuclease-independent pathway initiated by FPG, a bifunctional DNA glycosylase with AP lyase activity. Abasic site incision catalyzed by FPG generates a single-nucleotide gap with a 3'-phosphate terminus that is processed by the DNA 3'-phosphatase ZDP before repair is completed. We further show that the major AP endonuclease in Arabidopsis (ARP) incises AP sites generated by enzymatic N7-meG excision but, unexpectedly, not those resulting from spontaneous N7-meG loss. These findings, which reveal previously undetected differences between products of enzymatic and nonenzymatic base release, may shed light on the evolution and biological roles of AP endonucleases and AP lyases.

Recommendations for the use of methotrexate in psoriatic arthritis. / Recomendaciones sobre el uso de metotrexato en pacientes con artritis psoriásica.

OBJECTIVES: To develop recommendations for the management of methotrexate (MTX) in psoriatic arthritis (PsA), based on best evidence and experience. METHODS: A group of 12 experts on MTX use was selected. The coordinators formulated 14 questions about the use of MTX in PsA patients (indications, efficacy, safety and cost-effectiveness). A systematic review was conducted to answer the questions. Using this information, inclusion and exclusion criteria were established, as were the search strategies (Medline, EMBASE and the Cochrane Library were searched). Two different reviewers selected the articles. Evidence tables were created. At the same time, European League Against Rheumatism and American College of Rheumatology abstracts were evaluated. Based on this evidence, the coordinators proposed 12 preliminary recommendations that the experts discussed and voted on in a nominal group meeting. The level of evidence and grade of recommendation were established using the Oxford Centre for Evidence Based Medicine and the level of agreement with the Delphi technique (2 rounds). Agreement was established if at least 80% of the experts voted yes (yes/no). RESULTS: A total of 12 preliminary recommendations on the use of MTX were proposed, 9 of which were accepted. One was included in a different recommendation and another 2 were not voted on and were thereafter clarified in the main text. CONCLUSIONS: These recommendations aim to answer frequent questions and help in decision making strategies when treating PsA patients with MTX.

DNA methylation reprogramming of human cancer cells by expression of a plant 5-methylcytosine DNA glycosylase.

Patterns of DNA methylation, an important epigenetic modification involved in gene silencing and development, are disrupted in cancer cells. Understanding the functional significance of aberrant methylation in tumors remains challenging, due in part to the lack of suitable tools to actively modify methylation patterns. DNA demethylation caused by mammalian DNA methyltransferase inhibitors is transient and replication-dependent, whereas that induced by TET enzymes involves oxidized 5mC derivatives that perform poorly understood regulatory functions. Unlike animals, plants possess enzymes that directly excise unoxidized 5mC from DNA, allowing restoration of unmethylated C through base excision repair. Here, we show that expression of Arabidopsis 5mC DNA glycosylase DEMETER (DME) in colon cancer cells demethylates and reactivates hypermethylated silenced loci. Interestingly, DME expression causes genome-wide changes that include both DNA methylation losses and gains, and partially restores the methylation pattern observed in normal tissue. Furthermore, such methylome reprogramming is accompanied by altered cell cycle responses and increased sensibility to anti-tumor drugs, decreased ability to form colonospheres, and tumor growth impairment in vivo. Our study shows that it is possible to reprogram a human cancer DNA methylome by expression of a plant DNA demethylase.

Dual control of ROS1-mediated active DNA demethylation by DNA damage-binding protein 2 (DDB2).

By controlling gene expression, DNA methylation contributes to key regulatory processes during plant development. Genomic methylation patterns are dynamic and must be properly maintained and/or re-established upon DNA replication and active removal, and therefore require sophisticated control mechanisms. Here we identify direct interplay between the DNA repair factor DNA damage-binding protein 2 (DDB2) and the ROS1-mediated active DNA demethylation pathway in Arabidopsis thaliana. We show that DDB2 forms a complex with ROS1 and AGO4 and that they act at the ROS1 locus to modulate levels of DNA methylation and therefore ROS1 expression. We found that DDB2 represses enzymatic activity of ROS1. DNA demethylation intermediates generated by ROS1 are processed by the DNA 3'-phosphatase ZDP and the apurinic/apyrimidinic endonuclease APE1L, and we also show that DDB2 interacts with both enzymes and stimulates their activities. Taken together, our results indicate that DDB2 acts as a critical regulator of ROS1-mediated active DNA demethylation.

Association between serum dickkopf-1 levels and disease duration in axial spondyloarthritis / Asociación entre niveles séricos de Dickkopf-1 y duración de la enfermedad en espondiloartritis axial

Background. Axial spondyloarthritis (axSpA) is characterized by new bone formation. The complex systems underlying this process involve Wnt-signaling pathway. It has been observed that serum levels of dickkopf-1 (DKK-1), an important inhibitor of Wnt-signaling, are decreased in patients with axSpA. However, these data are from studies including only patients with long-standing disease. The aim of this study is to investigate if symptom duration influences on serum DKK-1 levels in patients with axSpA. Material and methods. A cross-sectional study including consecutive patients with axSpA (ASAS criteria) naïve for anti-TNF therapy. Collected data included demographic and disease characteristics, time since first symptom onset, assessment of disease activity and function, and determination of DKK-1 serum levels. Patients were classified as early axSpA (symptom duration ≤5 years) and established axSpA (>5 years). Linear regression models were employed to investigate the variables related to DKK-1 serum levels. Results. In total, 90 patients were included. Sixty-eight patients had early axSpA and 22 had established disease. Serum levels of DKK-1 were significantly higher in patients with early axSpA compared with established axSpA (22.1±12.6 vs 16.4±10.7pM; p=0.04). Among all tested variables, only symptom duration was significantly and inversely correlated with DKK-1 serum levels (beta: −0.041; p=0.01). Conclusion. Serum DKK-1 levels in axSpA depend on disease duration. As disease duration increases, DKK-1 serum levels decrease. Based on this, an intensive treatment at early stages of the disease could have a better outcome on inhibiting/slowing radiographic progression in patients with axSpA (AU)

Objetivo. Espondiloartritis axial (EsPax) se caracteriza por nueva formación ósea. El complejo sistema que subyace este proceso incluye la vía de señal Wnt. Se ha demostrado que niveles séricos de dickkopf-1 (DKK-1), un importante inhibidor de la vía de señal Wnt, está disminuidos en pacientes con EsPax. Sin embargo, estos datos proceden exclusivamente de pacientes con EsPax de larga duración. El objetivo de este estudio es investigar si la duración de la enfermedad influye en niveles séricos de DKK-1 en pacientes con EsPax. Material y métodos. Estudio transversal en pacientes con EsPax sin terapia anti-TNF. Se recogieron datos demográficos y de la enfermedad, y se determinaron niveles de DKK-1 séricos en la misma visita. Los pacientes fueron clasificados en base a la duración de síntomas en EsPax precoz (≤5 años) y establecida (>5 años). Se emplearon modelos de regresión lineal para investigar las variables asociadas con los niveles de DKK-1. Resultados. Se incluyeron 90 pacientes, 68 con EsPax precoz y 22 con EsPax establecida. Los niveles de DKK-1 fueron superiores en EsPax precoz comparado con EsPax establecida (22.1±12.6 vs 16.4±10.7pM; p=0.04). De todas las variables, sólo la duración de síntomas se asoció significativamente con DKK-1 (beta: −0.041; p=0.01). Conclusiones. Los niveles séricos de DKK-1 en EsPax, dependen de la duración de la enfermedad. A medida que la duración de la enfermedad aumenta, niveles séricos de DKK-1 disminuye. Por lo tanto, el tratamiento intensivo en estadios tempranos de la enfermedad podría tener un mejor resultado en inhibir/disminuir la progresión radiológica en pacientes con EsPax (AU)