Biology subjects

Bagshaw, C. R.

Publications and source records attributed to Bagshaw, C. R..

4 recordsLinked to original sources

bioRxiv2026

The comparative strengths and limitations of Nile Red and 9-(dicyanovinyl)-julolidine (DCVJ) fluorescent dyes for detecting microplastics and nanoplastics

Nile Red is widely used for the detection of microplastics because its fluorescence emission is sensitive to local polarity and can distinguish hydrophobic plastics from hydrophilic ones. The fluorescence of the molecular rotor, 9-(dicyanovinyl)-julolidine (DCVJ) is less sensitive to polarity but more to viscosity. DCVJ is less widely used for microplastic analysis, although it has been used to detect polystyrene nanobeads. Here, we compared these dyes with standard samples from the Hawaii Pacific University Polymer Kit 1.0 and confirmed that Nile Red, in general, was better for the detection and identification of microplastics. Fluorescence emission was analyzed using photography, as well as spectroscopy. The color and peak emission wavelength of some stained environmental microplastics were affected by additives. Raman spectroscopy was used to confirm the chemical identity of such samples. Although DCVJ emits green fluorescence on binding to some microplastics, a peak at 620 nm has been reported with polystyrene nanobeads, attributed to dimer/excimer formation. We confirmed this property and directly observed diffraction-limited spots using fluorescence microscopy, attributed to single or just a few nanobeads. Nile Red also stains polystyrene nanobeads and gave stronger signals than with DCVJ, but Nile Red was prone to false positives due to dye aggregation in aqueous solutions.

biophysics↗

bioRxiv2021

Multiplexed single-molecule analysis of human telomerase synthesizing DNA

Genomic stability in proliferating cells critically depends on telomere maintenance by telomerase reverse transcriptase. Here we developed a real-time single-molecule RNA sequencing approach that visualizes telomerase catalysis and structural dynamics at single-nucleotide resolution using FRET and zero-mode waveguides. The method permits direct detection of dynamic steps and structural states throughout the telomerase catalytic cycle and can be generalized to other nucleic acid polymerase systems.

biophysics↗

bioRxiv2020

Structural mimicry confers robustness in the cyanobacterial circadian clock

The histidine kinase SasA enhances robustness of circadian rhythms in the cyanobacterium S. elongatus by temporally controlling expression of the core clock components, kaiB and kaiC. Here we show that SasA also engages directly with KaiB and KaiC proteins to regulate the period and enhance robustness of the reconstituted circadian oscillator in vitro, particularly under limiting concentrations of KaiB. In contrast to its role regulating gene expression, oscillator function does not require SasA kinase activity; rather, SasA uses structural mimicry to cooperatively recruit the rare, fold-switched conformation of KaiB to the KaiC hexamer to form the nighttime repressive complex. Cooperativity gives way to competition with increasing concentrations of SasA to define a dynamic window by which SasA directly modulates clock robustness. One Sentence SummarySasA controls the assembly of clock protein complexes through a balance of cooperative and competitive interactions.

biochemistry↗

bioRxiv2018

Telomere DNA G-quadruplex folding within actively extending human telomerase

Telomerase maintains telomere length by reverse transcribing short G-rich DNA repeat sequences from its internal RNA template. G-rich telomere DNA repeats readily fold into G-quadruplex (GQ) structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using K+ versus Li+ salts yield changes in DNA product profiles consistent with formation of GQ structure within the telomerase-DNA complex. Single-molecule FRET experiments reveal complex DNA structural dynamics during real-time catalysis, supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we fit telomerase time series data to a global kinetic model that converges to a unique set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis and provide a detailed characterization of GQ modulation of polymerase function.\n\nSIGNIFICANCETelomeres protect the ends of linear chromosomes from illicit DNA processing events that can threaten genome stability. Telomere structure is built upon repetitive G-rich DNA repeat sequences that have the ability to fold into stable secondary structures called G-quadruplexes (GQs). In rapidly dividing cells, including the majority of human cancers, telomeres are maintained by the specialized telomerase enzyme. Thus, telomerase and its telomere DNA substrates represent important targets for developing novel cancer drugs. In this work, we provide evidence for GQ folding within the newly synthesized DNA product of an actively extending telomerase enzyme. Our results highlight the delicate interplay between the structural properties of telomere DNA and telomerase function.

biochemistry↗