Jiayan Zhou

Achieving a single-molecule level for RNA detection remains a significant challenge in point-of-care settings. We present CCHP, a Cas13−Csm6−horseradish peroxidase three-step enzymatic cascade assay that enables amplification-free RNA detection with a simple colorimetric readout at 450 nm within 1 h under standard conditions. Across diverse viral and non-coding targets, CCHP spans a dynamic range of 10−107 copies per reaction, showing strong agreement with RT−qPCR on serial dilutions (|r| ≈ 0.95). In a small blinded feasibility comparison using extracted RNA from infected versus uninfected cells, CCHP showed concordance with the reference method (κ = 1.0). In representative matrices spiked with synthetic RNA and without nucleic acid extraction or purification, positive events were observed near the single-molecule input level for H1N1-NP in oropharyngeal swabs and for SARS-CoV-2-N in oropharyngeal swabs and plasma, consistent with stochastic initiation of the cascade by single-molecule inputs under Poisson-limited conditions. Under these conditions, the assay maintains stable low-copy detection performance. A physics-guided minimal model integrating deterministic kinetics and stochastic partition statistics informed assay design and sensitivity projection, which were subsequently validated experimentally. By coupling single-molecule−level initiation with ultralow-copy detection and operational simplicity, CCHP advances point-of-care RNA diagnostics and surveillance.

BACKGROUND: Ageing is a significant risk factor for age-related diseases, accounting for 51% of global total disease burden. As thus, promoting healthy ageing is crucial. Although several potential anti-ageing drugs show promise, none have been approved for anti-ageing purpose. The World Health Organization (WHO) recommends physical exercise exceeding 600 metabolic equivalent of task (MET) minutes per week for adults. However, whether physical exercise positively impacts healthy biological ageing remains unclear. OBJECTIVE: This study aimed to investigate the joint correlation between MET level, caffeine consumption, and biological ageing. METHODS: We analyzed data from seven survey cycles (2007-2020) of the National Health and Nutrition Examination Survey (NHANES), involving 23,739 participants. Physical activity levels were measured in MET minutes per week, and biological ageing was assessed using both the PhenoAge and ENABL Age algorithms. Generalized linear regression for survey data was employed to test correlations, adjusting for confounding factors. A cubic spline model was used to detect non-linear relationships. Pre-specified subgroup analyses explored effect modifications, while predefined sensitivity analyses confirmed the robustness of the results. RESULTS: Each 100-MET increase in weekly physical exercise was associated with a 0.2-year delay in biological ageing (p < 0.001 for both PhenoAge and ENABL Age). Individuals with less than 600 MET minutes of weekly exercise had a higher risk of accelerated ageing compared to those exceeding 600 MET minutes (mean difference [MD]: 2.2 PhenoAge years, 95% CI [1.5-2.8], p < 0.001; MD: 2.1 ENABL Age years, 95% CI [1.1-3.1], p < 0.001). A L-shaped association was observed, with diminishing benefits of delayed ageing beyond 292 MET minutes for PhenoAge and 259 MET minutes for ENABL Age. Daily caffeine intake did not modify the correlation between MET levels and biological ageing (p for interaction > 0.05). Stronger effects were observed in non-Hispanic Black individuals, those with obesity, and low-income populations, but no benefit was found in cancer patients. CONCLUSIONS: Our findings highlight a positive correlation between higher levels of weekly physical exercise and delayed biological ageing. However, the benefits plateau beyond specific MET thresholds. Caffeine intake does not influence this relationship. These results underscore the importance of promoting physical exercise at appropriate MET levels as a strategy for healthy ageing management in the general population.

RNA molecules display remarkable heterogeneity in structure, dynamics, and function, yet methods for their precise visualization in living cells remain limited. While CRISPR-based RNA imaging holds great potential, existing systems often suffer from high background fluorescence due to constitutive signal emission or non-specific binding. To overcome these challenges, we developed CtDeg (CRISPR-dCas13-tDeg), a modular RNA imaging platform that links fluorescence activation directly to target RNA recognition while leveraging degron-mediated degradation to suppress background signals. By engineering the crRNA scaffold to embed the Pepper RNA motif, CtDeg ensures that fluorescence is present only upon binding to the native RNA target. We systematically optimized C-terminal tDeg variants to maximize the signal-to-noise ratio and demonstrated that CtDeg achieves substantially lower background and higher specificity than conventional fluorescent protein-CRISPR-based RNA imaging approaches. Using CtDeg, we captured real-time paraspeckle assembly dynamics and visualized early-stage SARS-CoV-2 genomic RNA transport. Remarkably, CtDeg provided the first direct imaging evidence of virus-induced NEAT1_2 lncRNA accumulation, revealing a host-virus regulatory interaction. Beyond these applications, CtDeg is compatible with multiple Cas13 orthologs and fluorescent proteins, establishing a versatile, target-induced platform for probing RNA localization, dynamics, and function in living cells, with broad applications in synthetic biology and RNA biology.