Rad51-Rad52 mediated maintenance of centromeric chromatin in candida albicans

La ubicación específica de un centrómero en la mayoría de los eucariotas no depende únicamente de la secuencia de ADN. Sin embargo, los determinantes no genéticos de identidad de un centrómero no están claramente definidos. Aunque varios mecanismos, de forma individual o en conjunto, pueden especificar centrómeros epigenéticos, la mayoría de los estudios en este área se centran en un factor universal, un centromerospecific histona H3 variante CENP-A, a menudo considerado como el determinante de la identidad epigenética del centrómero. A pesar de la sincronización variable de su carga en centrómeros través de las especies, una replicación junto a una deposición en fase temprana S de CENP-A se encuentra en la mayoría de los centrómeros de levadura. Centrómeros son las regiones más tempranas de replicación cromosómica en una levadura en ciernes patógeno Candida albicans. Al aplicar un ensayo de electroforesis en gel de agarosa de dos dimensiones se identifican los orígenes de replicación (ORI7-LI y ORI7-RI) proxima a un centrómero temprano replicante (CEN7) en C. albicans. Se demuestra que las horquillas de replicación se estancan en CEN7 de una manera dependiente del cinetocoro y el estancamiento tenedor se reduce en ausencia de la recombinación homóloga (HR) proteínas Rad51 y Rad52. La supresión de ORI7-RI provoca una reducción significativa en la señal de tenedor estancado y una mayor tasa de pérdida del cromosoma alterado 7. Las proteínas de recursos humanos, Rad51 y Rad52, han demostrado que desempeñan un papel en el reinicio del tenedor. La microscopía confocal muestra cinetocoros declustered en rad51 y rad52 mutantes, que son evidencia de la disrupción cinetocoro. Los niveles de CENP-ACaCse4 en centrómeros, como se determina por los experimentos de inmunoprecipitación de la cromatina (ChIP), se reducen en ausencia de Rad51 / Rad52 que resulta en la interrupción de la estructura cinetocoro. Además, el análisis de transferencia Western revela que las moléculas de CENP-A deslocalizados en mutantes de recursos humanos se degradan de un modo similar como en otros mutantes kinetochore descritos antes. Finalmente, los ensayos de co-inmunoprecipitación indican que Rad51 y Rad52 interactuan físicamente con CENPA CaCse4 in vivo. Por lo tanto, las proteínas Rad51 y Rad52 de recursos epigenéticos humanos mantienen el funcionamiento del centrómero mediante la regulación de los niveles de CENPA CaCse4 en los sitios de parada programados en los principios centrómeros a replicar.Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromerespecific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI) proximal to an early replicating centromere (CEN7) in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR) proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-ACaCse4 levels at centromeres, as determined by chromatin immunoprecipitation (ChIP) experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENPA CaCse4 in vivo. Thus, the HR proteins Rad51 and Rad52 epigenetically maintain centromere functioning by regulating CENPA CaCse4 levels at the programmed stall sites of early replicating centromeresK. Sanyal y D. D. Dubey han recibido financiación del Government of India. Department of Biotechnology S. Mitra fue Senior Research Fellowship financiado por Council of Scientific and Industrial Research K Sanyal recibió ayuda de Jawaharlal Nehru Centre for Advanced Scientific Research G. Larriba fue financiado por Junta de Extremadura, Ayuda a grupos CCV014, Fondos FEDER; y por el Ministerio de Ciencia e Innovación, SAF2010-19848peerReviewe

Effects of home confinement on mental health and lifestyle behaviours during the COVID-19 outbreak:insights from the ECLB-COVID19 multicentre study

Although recognised as effective measures to curb the spread of the COVID-19 outbreak, social distancing and self-isolation have been suggested to generate a burden throughout the population. To provide scientific data to help identify risk factors for the psychosocial strain during the COVID-19 outbreak, an international cross-disciplinary online survey was circulated in April 2020. This report outlines the mental, emotional and behavioural consequences of COVID-19 home confinement. The ECLB-COVID19 electronic survey was designed by a steering group of multidisciplinary scientists, following a structured review of the literature. The survey was uploaded and shared on the Google online survey platform and was promoted by thirty-five research organizations from Europe, North Africa, Western Asia and the Americas. Questions were presented in a differential format with questions related to responses “before” and “during” the confinement period. 1047 replies (54% women) from Western Asia (36%), North Africa (40%), Europe (21%) and other continents (3%) were analysed. The COVID-19 home confinement evoked a negative effect on mental wellbeing and emotional status (P < 0.001; 0.43 ≤ d ≤ 0.65) with a greater proportion of individuals experiencing psychosocial and emotional disorders (+10% to +16.5%). These psychosocial tolls were associated with unhealthy lifestyle behaviours with a greater proportion of individuals experiencing (i) physical (+15.2%) and social (+71.2%) inactivity, (ii) poor sleep quality (+12.8%), (iii) unhealthy diet behaviours (+10%), and (iv) unemployment (6%). Conversely, participants demonstrated a greater use (+15%) of technology during the confinement period. These findings elucidate the risk of psychosocial strain during the COVID-19 home confinement period and provide a clear remit for the urgent implementation of technology-based intervention to foster an Active and Healthy Confinement Lifestyle AHCL)

Overview and Evaluation of Existing Guidelines for Rational Antimicrobial Use in Small-Animal Veterinary Practice in Europe

Antimicrobial stewardship guidelines (ASGs) represent an important tool to help veterinarians optimize their antimicrobial use with the objective of decreasing antimicrobial resistance. The aim of this study was to map and qualitatively assess the ASGs for antimicrobial use in cats and dogs in Europe. Country representatives of the European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT) were asked to identify ASGs published in their countries. All collated ASGs updated since January 2010 containing recommendations on antimicrobial therapy for at least three conditions affecting different organ systems in cats and dogs underwent detailed review including AGREE II analysis. Out of forty countries investigated, fifteen ASGs from eleven countries met the inclusion criteria. Several critical principles of antimicrobial use were identified, providing a framework that should assist development of stewardship guidance. The AGREE II analysis highlighted several methodological limitations of the currently available ASGs. This study sheds light on the lack of national ASGs for dogs and cats in multiple European countries and should encourage national bodies to prioritize guideline development in small animals. A greater awareness of the need to use a structured approach to guideline development could improve the quality of ASGs in the future.publishedVersio

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p&lt;0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (&lt;1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (&lt;1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Análisis del papel de RDA52 en la estabilidad genómica de C. albicans

Candida albicans es un hongo comensal presente en el 50-70% de la población que bajo determinadas circunstancias, tal como bajada en las defensas, se convierte en un patógeno. Hemos demostrado que la cepa de C. albicans SC5314 y su derivado CAI4 son heterozigóticos para el gen HIS4. Dentro de la ORF HIS4 hemos encontrado 11 SNPs, siete sinónimos y cuatro no sinónimos que definen dos haplotipos. La funcionalidad está determinada por la base presente en el SNP929, de manera que G determina funcionalidad y T determina no funcionalidad debido a la presencia de Gly310 o Val310, respectivamente. Este cambio se encuentra dentro del módulo His4B de la proteína His4, por lo que afectaría a su actividad como fosforibosil-ATP pirofosfatasa. Basándonos en este marcador heterozigótico, hemos investigado la frecuencia de aparición de auxótrofos para la histidina como una medida de frecuencia de pérdida de heterozigosidad en mutantes rad52-¿¿ de C. albicans. El gen RAD52 en C. albicans es absolutamente necesario para los eventos mediados por la recombinación homóloga, y su ausencia produce fuertes defectos en integración homóloga, reparación de DNA y mantenimiento de telómeros. Los mutantes rad52 de C. albicans exhiben una frecuencia media de perdida de heterozigosidad al menos 100 veces mayor que la cepa parental CAI4, siendo la pérdida de cromosoma el evento más común que la origina. Además, mostraron una alta frecuencia de mutación puntual (al menos para la ORF del gen HIS4). Las cepas rad52 His- seleccionadas muestran alta frecuencia de fragmentación de cromosomas que no está relacionada con la adquisición de la auxotrofía. Además, parece que tras la pérdida o rotura de un cromosoma tiene lugar una duplicación del homólogo residual o del homólogo no truncado, respectivamente. Las roturas que originan los cromosomas truncados en estas cepas parecen ser reparadas mediante la adición de telómero de novo. Estos cromosomas truncados se mantienen razonablemente estables a lo largo de varias generaciones

Análisis del papel de RDA52 en la estabilidad genómica de C. albicans

Candida albicans es un hongo comensal presente en el 50-70% de la población que bajo determinadas circunstancias, tal como bajada en las defensas, se convierte en un patógeno. Hemos demostrado que la cepa de C. albicans SC5314 y su derivado CAI4 son heterozigóticos para el gen HIS4. Dentro de la ORF HIS4 hemos encontrado 11 SNPs, siete sinónimos y cuatro no sinónimos que definen dos haplotipos. La funcionalidad está determinada por la base presente en el SNP929, de manera que G determina funcionalidad y T determina no funcionalidad debido a la presencia de Gly310 o Val310, respectivamente. Este cambio se encuentra dentro del módulo His4B de la proteína His4, por lo que afectaría a su actividad como fosforibosil-ATP pirofosfatasa. Basándonos en este marcador heterozigótico, hemos investigado la frecuencia de aparición de auxótrofos para la histidina como una medida de frecuencia de pérdida de heterozigosidad en mutantes rad52-¿¿ de C. albicans. El gen RAD52 en C. albicans es absolutamente necesario para los eventos mediados por la recombinación homóloga, y su ausencia produce fuertes defectos en integración homóloga, reparación de DNA y mantenimiento de telómeros. Los mutantes rad52 de C. albicans exhiben una frecuencia media de perdida de heterozigosidad al menos 100 veces mayor que la cepa parental CAI4, siendo la pérdida de cromosoma el evento más común que la origina. Además, mostraron una alta frecuencia de mutación puntual (al menos para la ORF del gen HIS4). Las cepas rad52 His- seleccionadas muestran alta frecuencia de fragmentación de cromosomas que no está relacionada con la adquisición de la auxotrofía. Además, parece que tras la pérdida o rotura de un cromosoma tiene lugar una duplicación del homólogo residual o del homólogo no truncado, respectivamente. Las roturas que originan los cromosomas truncados en estas cepas parecen ser reparadas mediante la adición de telómero de novo. Estos cromosomas truncados se mantienen razonablemente estables a lo largo de varias generaciones

Análisis del papel de RAD52 en la estabilidad genómica de C. albicans

Hemos demostrado que la cepa de Candida albicans SC5314 y su derivado CAI4 son heterozigóticos para el gen HIS4. Basándonos en este marcador heterocigótico, hemos encontrado que la ausencia del gen RAD52 en C. albicans resulta en un aumento de la frecuencia media de pérdida de heterozigosidad, frecuentemente originada por la pérdida de cromosoma

Replication-segregation interaction is evolutionarily conserved in unicellular organisms.

<p>(A) The phylogenetic tree reflects the evolutionary relationships of the corresponding taxa. The tree is drawn to scale with branch lengths in the units of the number of base substitutions per site in the 23S or 25S rRNA nucleotide sequences of the four species. (B) <i>CEN</i>-like loci or <i>CEN</i>s (green boxes) in prokaryotes and unicellular eukaryotes respectively are flanked by early replication origins (pink circles). The blue circles indicate the centromere factors influencing origin activity. The yellow circles indicate the origin/replication associated factors influencing <i>CEN</i> function. In the genome of the bacteria <i>B. subtilis</i>, the single replication origin is flanked by <i>CEN</i>-like <i>parS</i> sequence. The Spo0J (ParB) protein, binding to <i>parS</i>, organizes <i>ori</i> activity as well as recruits Smc proteins for proper segregation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Gruber1" target="_blank">[17]</a>. In <i>S. cerevisiae</i>, which has short ‘point’ centromeres, the Ctf19 complex directly recruits initiation factors for early firing of proximal origins <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Natsume1" target="_blank">[20]</a>. Although early firing has been suggested for playing a role in <i>CEN</i> function, no <i>cis</i> factors has been identified. In <i>C. albicans</i>, which has ‘short regional’ <i>CEN</i>s, <i>CEN</i>s have been shown to govern early replication of proximal origins, although no <i>cis</i> factors were identified <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Koren1" target="_blank">[5]</a>. In this study we show that fork stalling at <i>CENs</i> from proximal origins recruit Rad51/Rad52 that, in turn, regulates CENP-A deposition. Finally in the ‘large regional’ centromeres of <i>S. pombe</i>, the centromeric heterochromatic protein Swi6 activates pericentric replication origins <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Hayashi1" target="_blank">[16]</a>. The fork protection complex (FPC) that travels with the replisome negatively regulates Ams2 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Takayama2" target="_blank">[73]</a> that, in turn, regulates CENP-A deposition <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004344#pgen.1004344-Takayama1" target="_blank">[10]</a>.</p