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Avian metapneumovirus (aMPV) infects turkeys and chickens. It replicates primarily in the upper respiratory tract, causing respiratory disease. Animals may also exhibit lower feed and water consumption and weight loss. Despite the high morbidity and often high mortality associated with aMPV in the field, the pathogenicity of field isolates has been difficult to assess in the laboratory. Usually, only mild respiratory clinical signs with low presentation can be reproduced in experimental infections. Hence, the aim of this study was to develop a simple and reproducible aMPV infection model in chicken, to test vaccines' efficacy. It was hypothesized that the evaluation of ciliary activity of tracheal explants, a parameter used in the evaluation of avian infectious bronchitis vaccines, would be suitable for evaluating aMPV vaccines' efficacy after an experimental challenge. Chickens of commercial origin, with maternally-derived antibodies or SPF chickens were vaccinated at one day of age. Challenges were performed at different ages (17 days to 16 weeks of age). Ciliary activity of tracheal explants was evaluated together with more common parameters of efficacy: clinical signs, body weight gain and serum antibodies. In all cases, the vaccine provided protection against the challenge in terms of ciliary activity. Body weight gain and clinical signs were not suitable in all the experiments for showing efficacy, and a very mild serologic response was observed after vaccination, and only in SPF animals. The results of this study confirmed the working hypothesis: that the ciliary activity of tracheal explants is a suitable method for testing aMPV vaccines in commercial or SPF chickens in ages from 17 days to 16 weeks. •Tracheal ciliary activity was assessed for testing avian metapneumovirus vaccines.•Ciliary activity resulted more reliable than other parameters, such as clinical signs or weight gain.•The new method is suitable for commercial, or SPF animals ranging in age from 17 days to 16 weeks.

Solving the problems that replication forks encounter when synthesizing DNA is essential to prevent genomic instability. Besides their role in DNA repair in the G2 phase, several homologous recombination proteins, specifically RAD51, have prominent roles in the S phase. Using different cellular models, RAD51 has been shown not only to be present at ongoing and arrested replication forks but also to be involved in nascent DNA protection and replication fork restart. Through pharmacological inhibition, here we study the specific role of RAD51 in the S phase. RAD51 inhibition in non-transformed cell lines did not have a significant effect on replication fork progression under non-perturbed conditions, but when the same cells were subjected to replication stress, RAD51 became necessary to maintain replication fork progression. Notably, the inhibition or depletion of RAD51 did not compromise fork integrity when subjected to hydroxyurea treatment. RAD51 inhibition also did not decrease the ability to restart, but rather compromised fork progression during reinitiation. In agreement with the presence of basal replication stress in human colorectal cancer cells, RAD51 inhibition reduced replication fork speed in these cells and increased γH2Ax foci under control conditions. These alterations could have resulted from the reduced association of DNA polymerase α to chromatin, as observed when inhibiting RAD51. It may be possible to exploit the differential dependence of non-transformed cells versus colorectal cancer cells on RAD51 activity under basal conditions to design new therapies that specifically target cancer cells.

During S phase, replication forks can encounter several obstacles that lead to fork stalling, which if persistent might result in fork collapse. To avoid this collapse and to preserve the competence to restart, cells have developed mechanisms that maintain fork stability upon replication stress. In this study, we aimed to understand the mechanisms involved in fork stability maintenance in non-transformed human cells by performing an isolation of proteins on nascent DNA-mass spectrometry analysis in hTERT-RPE cells under different replication stress conditions. Our results show that acute hydroxyurea-induced replication blockade causes the accumulation of large amounts of single-stranded DNA at the fork. Remarkably, this results in the disengagement of replisome components from nascent DNA without compromising fork restart. Notably, Cdc45-MCM-GINS helicase maintains its integrity and replisome components remain associated with chromatin upon acute hydroxyurea treatment, whereas replisome stability is lost upon a sustained replication stress that compromises the competence to restart.

Solving the problems that replication forks encounter when synthesizing DNA is essential to prevent genomic instability. Besides their role in DNA repair in the G2 phase, several homologous recombination proteins, specifically Rad51, have prominent roles in the S phase. Using different cellular models, Rad51 has been shown not only to be present at ongoing and arrested replication forks but also to be involved in nascent DNA protection and replication fork restart. Through pharmacological inhibition, here we study the specific role of Rad51 in the S phase. Rad51 inhibition in non-transformed cell lines did not have a major effect on replication fork progression under non-perturbed conditions, but when the same cells were subjected to replication stress, Rad51 became necessary to maintain replication fork progression. Notably, the inhibition or depletion of Rad51 did not compromise fork integrity when subjected to hydroxyurea treatment. Rad51 inhibition also did not decrease the ability to restart, but rather compromised, fork progression during reinitiation. In agreement with the presence of basal replication stress in human colorectal cancer cells, Rad51 inhibition reduced replication fork speed in these cells and increased γH2Ax foci under control conditions. These alterations could have resulted from the reduced association of DNA polymerase α to chromatin, as observed when inhibiting Rad51. It may be possible to exploit the differential dependence of non-transformed cells versus colorectal cancer cells on Rad51 activity under basal conditions to design new therapies that specifically target cancer cells. Competing Interest Statement The authors have declared no competing interest.

Genome duplication, critical for cell survival and identity, requires precise origin activation to ensure accurate DNA replication and chromatin structure maintenance. In colorectal cancer cells, prolonged replication stress does not hinder cell proliferation, although the mechanisms driving cancer cell adaptability remain largely unclear. Here, we demonstrate that upon recovery, cancer cells are able to activate new replication origins in distinct domains, causing persistent changes in chromatin accessibility, nuclear morphology, and replication timing, ultimately promoting chromatin instability. Chromatin accessibility, particularly in promoter regions, increased following replication stress in a subset of cells, correlating with altered gene expression and nuclear expansion. Additionally, some genes with enhanced promoter accessibility displayed sustained expression changes, further suggesting a transcriptional shift linked to stress adaptation. Our findings reveal that colorectal cancer cells recover from severe replication stress through new origin activation, a mechanism that not only maintains cell proliferation under stress but may also accelerate tumour heterogeneity. This research underscores replication origin activation as a potential therapeutic target in combating cancer cell resilience.Competing Interest StatementThe authors have declared no competing interest.

Objective The aim of this study was to determine the diagnostic accuracy of magnetic resonance colonography (MRC) for the evaluation of disease activity and severity in patients with ulcerative colitis (UC) using endoscopy as the reference standard. Methods Fifty patients with UC underwent colonoscopy and MRC for the evaluation of disease activity. All patients were prospectively and consecutively included. Endoscopic activity was evaluated globally and on a segment basis using the modified Baron score (MBS), and also classified as absent, mild to moderate (inflammation without ulcers) or severe (presence of ulceration). MRC parameters evaluated in each segment were: wall thickness, pre- and post-contrast wall signal intensity, relative contrast enhancement (RCE), mural oedema, ulcers, enlarged lymph nodes and the comb sign. Results Independent predictors for endoscopic activity on a segment basis were RCE (p=0.006), presence of oedema (p=0.003), enlarged lymph nodes (p<0.001) and the comb sign (p<0.001). A segmental simplified MRC index (MRC-S) ≥1 detected endoscopic inflammation with high diagnostic accuracy (sensitivity 87%, specificity 88%, area under the curve (AUC) 0.95; p<0.001). MRC-S index ≥2 detected severe lesions with high sensitivity (83%) and specificity (82%) with an AUC of 0.91 (p<0.001). The MRC-S index strongly correlated with the MBS (r=0.81, p<0.001) and with the subjective assessment of the radiologists for the evaluation of disease severity (r=0.77, p<0.001). Conclusions MRC has a high accuracy for the diagnosis of disease activity and severity in UC.

ObjectiveTo evaluate the feasibility and toxicity of autologous haematopoietic stem cell transplantation (HSCT) for the treatment of refractory Crohn's disease (CD).DesignIn this prospective study, patients with refractory CD suffering an aggressive disease course despite medical treatment, impaired quality of life and in whom surgery was not an acceptable option underwent HSCT. Toxicity and complications during the procedure and within the first year following transplantation were evaluated, along with the impact of the introduction of supportive measures on safety outcomes.Results26 patients were enrolled. During mobilisation, 16 patients (62%) presented febrile neutropaenia, including one bacteraemia and two septic shocks. Neutropaenia median time after mobilisation was 5 days. 5 patients withdrew from the study after mobilisation and 21 patients entered the conditioning phase. Haematopoietic recovery median time for neutrophils (>0.5×109/L) was 11 days and for platelets (>20×109/L) 4 days. Twenty patients (95%) suffered febrile neutropaenia and three patients (27%) presented worsening of the perianal CD activity during conditioning. Among non-infectious complications, 6 patients (28.5%) presented antithymocyte globulin reaction, 12 patients (57%) developed mucositis and 2 patients (9.5%) had haemorrhagic complications. Changes in supportive measures over the study, particularly antibiotic prophylaxis regimes during mobilisation and conditioning, markedly diminished the incidence of severe complications. During the first 12-month follow-up, viral infections were the most commonly observed complications, and one patient died due to systemic cytomegalovirus infection.ConclusionsAutologous HSCT for patients with refractory CD is feasible, but extraordinary supportive measures need to be implemented. We suggest that this procedure should only be performed in highly experienced centres.

Abstract BackgroundDuring the coronavirus disease 2019 (COVID-19) outbreaks, health care workers (HCWs) are at a high risk of infection. Strategies to reduce in-hospital transmission between HCWs and to safely manage infected HCWs are lacking. Our aim was to describe an active strategy for the management of COVID-19 in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–infected HCWs and investigate its outcomes. MethodsA prospective cohort study of SARS-CoV-2-infected health care workers in a tertiary teaching hospital in Barcelona, Spain, was performed. An active strategy of weekly polymerase chain reaction screening of HCWs for SARS-CoV-2 was established by the Occupational Health department. Every positive HCW was admitted to the Hospital at Home Unit with daily assessment online and in-person discretionary visits. Clinical and epidemiological data were recorded. ResultsOf the 590 HCWs included in the cohort, 134 (22%) were asymptomatic at diagnosis, and 15% (89 patients) remained asymptomatic during follow-up. A third of positive cases were detected during routine screening. The most frequent symptoms were cough (68%), hyposmia/anosmia (49%), and fever (41%). Ten percent of the patients required specific treatment at home, while only 4% of the patients developed pneumonia. Seventeen patients required a visit to the outpatient clinic for further evaluation, and 6 of these (1%) required hospital admission. None of the HCWs included in this cohort required intensive care unit admission or died. ConclusionsActive screening for SARS-CoV-2 among HCWs for early diagnosis and stopping in-hospital transmission chains proved efficacious in our institution, particularly due to the high percentage of asymptomatic HCWs. Follow-up of HCWs in Hospital at Home units is safe and effective, with low rates of severe infection and readmission.