A rapid and sensitive one-pot platform integrating fluorogenic RNA aptamers and CRISPR-Cas13a for visual detection of monkeypox virus.

The recent global upsurge in Monkeypox virus (MPXV) outbreaks underscores the critical need for rapid and precise diagnostic solutions, particularly in resource-constrained settings. The gold standard diagnostic method, qRT-PCR, is hindered by its time-consuming nature, requirement for nucleic acid purification, expensive equipment, and the need for highly trained personnel. Traditional CRISPR/Cas fluorescence assays, relying on trans-cleavage of ssDNA/RNA reporters labeled with costly fluorophores and quenchers, pose challenges that limit their widespread application, especially for point-of-care testing (POCT). In this study, we utilized a cost-effective and stable fluorogenic RNA aptamer (Mango III), specifically binding and illuminating the fluorophore TO3-3 PEG-Biotin Fluorophore (TO3), as a reporter for Cas13a trans-cleavage activity. We propose a comprehensive strategy integrating RNA aptamer, recombinase-aided amplification (RAA), and CRISPR-Cas13a systems for the molecular detection of MPXV target. Leveraging the inherent collateral cleavage properties of the Cas13a system, we established high-sensitivity and specificity assays to distinguish MPXV from other Orthopoxviruses (OPVs). A streamlined one-pot protocol was developed to mitigate aerosol contamination risks. Our aptamer-coupled RAA-Cas13a one-pot detection method achieved a Limit of Detection (LoD) of 4 copies of target MPXV DNA in just 40 min. Validation using clinical MPX specimens confirmed the rapid and reliable application of our RAA-Cas13a-Apt assays without nucleic acid purification procedure, highlighting its potential as a point-of-care testing solution. These results underscore the user-friendliness and effectiveness of our one-pot RAA-Cas13a-Apt diagnostic platform, poised to revolutionize disease detection and management.

Prevalence of strabismus among preschool children in eastern China and comparison at a 5-year interval: a population-based cross-sectional study.

OBJECTIVE: To update data on strabismus and evaluate the changes in prevalence and patterns among preschoolers in eastern China over a period of 5 years. DESIGN: Nanjing Eye Study, a longitudinal population-based study. SETTING: Recruitment and testing in kindergartens in Yuhuatai District, Nanjing. PARTICIPANTS: 2300 eligible children. MAIN OUTCOME MEASURES: Comprehensive ocular examinations were conducted in 1986 children aged 48-<60 months in Nanjing Eye Study (NES, 2016-2017), including visual acuity, ocular alignment, refractive error and ocular structures evaluation. The prevalence rate and pattern of strabismus were calculated and compared with those from the Nanjing Pediatric Vision Project (NPVP, 2011-2012) in children of the same age, of the same area and using the same diagnostic criteria. RESULTS: The overall prevalence rate of strabismus in NES was 5.56% (95% CI 4.54% to 6.57%), which was not significantly different from that in NPVP (4.99%, 95% CI 4.13% to 5.84%, p=0.40). The prevalence of subtypes of strabismus underwent significant changes, with significant increase in intermittent exotropia (IXT) in NES (2.78% vs 4.69%, p=0.001) and significant decrease in constant exotropia (1.17% vs 0.15%, p<0.001). Significant change in pattern was observed in IXT, where the proportion of the convergence insufficiency type (2.90% vs 27.17%) increased and exceeded the divergence excess type (20.29% vs 11.96%) to be the second common type (p<0.001). CONCLUSION: The prevalence of strabismus appeared stable in children aged 48-<60 months in eastern China at a 5-year interval. The prevalence of IXT increased significantly, and the convergence insufficiency type became more prevalent in patients with IXT. Timely detection and intervention of IXT are important among preschoolers.

CARF, As An Oncogene, Promotes Colorectal Cancer Stemness By Activating ERBB Signaling Pathway.

INTRODUCTION: The role of CARF, a calcium-responsive transcription factor, in colorectal cancer initiation and development is still unknown. Here, we report that CARF promotes colorectal cancer stemness through ERBB signaling pathway. MATERIALS AND METHODS: Both colorectal cancer cell lines and primary cells were used in this study. The levels of target mRNA and protein in the cells were examined by qRT-PCR and Western blot. Gene manipulation was achieved by the lentivirus delivery system. Luciferase reporter gene assay was employed to analyze the transcriptional activity of the promoter. ChIP assay was performed for the examination of the binding between CARF and the promoters of MAPK8 and JUN. Kaplan-Meier survival curve was generated by the R2 program. Correlation analysis was performed using Spearman correlation analysis. RESULTS: Aberrant upregulation of CARF has been found in tumor tissues of colorectal cancer patients and associated with poor prognosis. Ectopic expression of CARF promoted the sphere-formation activities, as well as the expression of stem cell markers in colorectal cancer cells and knockdown of CARF, inhibited these activities. The mechanistic analysis showed that CARF directly binds to the promoter of MAPK8 and JUN, promotes the expression of MAPK8 and JUN, activates the ERBB signaling pathway, and thereby promotes the maintenance of the stemness in colorectal cancer cells. CONCLUSION: CARF, as an oncogene, promotes colorectal cancer stemness by activating ERBB signaling pathway. The ERBB signaling pathway that serves as the main downstream effector of CARF could be an efficient drug target for colorectal cancer caused by aberrant expression of CARF.