Biology subjects

Velazquez, A.

Publications and source records attributed to Velazquez, A..

3 recordsLinked to original sources

bioRxiv2026

Bright Probes, Blurred Metabolism: Navigating Fluorescent Protein Cross-Excitation in NADH FLIM

Fluorescence lifetime imaging microscopy (FLIM) of endogenous NAD(P)H enables the label-free assessment of cellular metabolic state. Although metabolic imaging is increasingly combined with fluorescent protein (FP) reporters to enhance biological specificity, the potential cross-talk between the intrinsic and extrinsic labels remain ill-defined. Here, we systematically evaluate cross-talk from FPs in metabolic FLIM using phasor analysis of two-photon fluorescence microscopy. The results clearly show that many widely used fluorescent proteins are excited under the conditions used for NADH imaging; they emit blue-shifted, short-lifetime fluorescence that can interfere with imaging NADH metabolic signatures. This overlap persists across excitation wavelengths and FP classes, posing a significant challenge for multiplexed metabolic imaging. This cautionary tale argues against unvalidated multiplexing strategies in metabolic FLIM studies. Our study aims to identify acceptable imaging partners, offer a pipeline for assaying potential cross-talk, and provide practical guidance for experimental design. SignificanceFluorescence lifetime imaging of NADH autofluorescence is a powerful, label-free approach to map cellular metabolism in living tissues. A growing number of studies combine NADH imaging with fluorescent protein (FP) reporters to simultaneously identify specific cell types or subcellular compartments. This study reveals that many FPs, spanning the visible spectrum, are unexpectedly excited under NADH conditions. Commonly used green, yellow, and red variants produce short-lifetime, blue-shifted fluorescence that directly overlaps with metabolic NADH signals. This cross-excitation can be falsely interpreted as a shift in cellular metabolic state, posing a significant risk for multiplexed metabolic imaging studies. Our studies establish a pipeline to assess and manage this risk. We identify StayGoldE138D and mNeonGreen as the most compatible FPs for co-imaging with NADH, and provide a practical framework to guide experimental design and control strategies for multiplexed metabolic FLIM.

biophysics↗

bioRxiv2026

Unveiling the Toxicological and Allelopathic Effects of Pteridium aquilinum: Chemical Profiling and Biological Assays

The fern Pteridium aquilinum (bracken) is among the world most widespread plants and poses serious ecological and health risks due to the presence of illudane glycosides (IGs), carcinogenic compounds that particularly affect grazing livestock. This study determined the presence of IGs in plant extracts and in water samples from the Asturias region (northern Spain), an area characterized by intensive cattle farming, and assessed the genotoxic potential, and allelopathic effects of these samples. The glycosides ptaquiloside (PTA) and ptesculentoside (PTE), together with the degradation products pterosin A and pterosin B, were quantified in in vitro-cultured gametophytes and young sporophytes, spring-collected croziers, and autumn water samples from several bracken-infested sites. All sampling locations contained IGs in both plant and water samples, with particularly high concentrations of PTA detected in croziers from Brana Vieja (Somiedo), where cattle deaths had been reported. In vivo genotoxicity assays, using Drosophila melanogaster, revealed induction of somatic mutations and recombination events by aqueous plant extracts and of somatic mutations by water samples. The genotoxic activity of plant extracts, but not that of water samples, was associated with pterosins A and B. Moreover, comet assays in cultured human cells confirmed the genotoxic activity of plant extracts. Unexpectedly, allelopathic bioassays indicated a possible phytostimulatory rather than inhibitory effect of bracken extracts on the germination of meadow species. These findings underscore the widespread presence and biological activity of bracken in northern Spain and highlight the urgent need for management strategies to mitigate the ecological and toxicological threats posed by bracken proliferation.

pharmacology and toxicology↗

bioRxiv2023

Exposure to constant artificial light alters honey bee sleep rhythms and disrupts sleep

Artificial light at night (ALAN) is known to create changes in animal behavior in some invertebrates and vertebrates and can result in decreased fitness. ALAN effects have not been studied in European honey bees (Apis mellifera), an important pollinator. Colonies can be exposed to ALAN in swarm clusters, when bees cluster outside the nest on hot days and evenings, and, in limited cases, when they build nests in the open. Forager bees maintained in incubated cages were subjected to constant light or dark and observed with infrared cameras. The bees maintained a regular sleep pattern for three days but showed a shift on the fourth day in the presence of continuous light. Bees under constant light demonstrated a 24.05-hour rhythm, compared to a 23.12-hour rhythm in the dark. After 95 hours, the light-exposed bees slept significantly less and experienced significantly more disturbances from their peers. They also preferred to sleep in the lower portion of the cages, which had lower light intensity. These findings suggest that ALAN can disrupt honey bees sleep patterns, which may have implications for their behavior and overall colony health.

animal behavior and cognition↗