A new archetype of activatable imaging probe is described that responds in an off-on mode with a fluorescent signal only after being consecutively converted by two different enzymes. Pre-pro-proteins are a well-established concept employed by Nature, but "pre-pro-fluorescent" molecular probes have been unknown until now, even though they promise to furnish precious extra information and added specificity over habitual probe technologies. Our prototype probe discriminates cells that express both active β-d-galactosidase and leucine amino-peptidase from those which lack one of these enzymes thanks to the release of a precipitating fluorescent tag.

A three-component probe harnesses the extraordinary properties of a solid-state fluorophore for the detection of living cells exhibiting a particular peptidase activity. The off-on mode by which the probe operates, the bright fluorescence of the resulting precipitate, and the rapid response allow an exceptional signal-to-background ratio during microscopic imaging. A tertiary carbamate link between the spacer and phenolic fluorophore is at the heart of the probe's long-term stability. The degree of chlorination of the probe determines its response time and thus its suitability for live-cell analysis. Our probe also allows highly resolved localization of peptidase activity during gel analysis or on agar. In comparison, probes releasing soluble fluorophores demonstrate complete diffusion of the fluorescent signal. These results demonstrate the probe's potential for diverse biomedical applications, including high-fidelity flow cytometry and sensitive colony assays.

Several genetic polymorphisms differentiate between healthy individuals who are more cognitively vulnerable or resistant during total sleep deprivation (TSD). Common metrics of cognitive functioning for classifying vulnerable and resilient individuals include the Psychomotor Vigilance Test (PVT), Go/noGo executive inhibition task, and subjective daytime sleepiness. We evaluated the influence of 14 single-nucleotide polymorphisms (SNPs) on cognitive responses during total sleep deprivation (continuous wakefulness for 38 h) in 47 healthy subjects (age 37.0 ± 1.1 years). SNPs selected after a literature review included SNPs of the adenosine-A2A receptor gene (including the most studied rs5751876), pro-inflammatory cytokines (TNF-α, IL1-β, IL-6), catechol-O-methyl-transferase (COMT), and PER3. Subjects performed a psychomotor vigilance test (PVT) and a Go/noGo-inhibition task, and completed the Karolinska Sleepiness Scale (KSS) every 6 h during TSD. For PVT lapses (reaction time >500 ms), an interaction between SNP and SDT (p < 0.05) was observed for ADORA2A (rs5751862 and rs2236624) and TNF-α (rs1800629). During TSD, carriers of the A allele for ADORA2A (rs5751862) and TNF-α were significantly more impaired for cognitive responses than their respective ancestral G/G genotypes. Carriers of the ancestral G/G genotype of ADORA2A rs5751862 were found to be very similar to the most resilient subjects for PVT lapses and Go/noGo commission errors. Carriers of the ancestral G/G genotype of COMT were close to the most vulnerable subjects. ADORA2A (rs5751862) was significantly associated with COMT (rs4680) (p = 0.001). In conclusion, we show that genetic polymorphisms in ADORA2A (rs5751862), TNF-α (rs1800629), and COMT (rs4680) are involved in creating profiles of high vulnerability or high resilience to sleep deprivation. (NCT03859882).

No abstract