Biology subjects

Kay, S.

Publications and source records attributed to Kay, S..

12 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

SELECT 2.0: Refined and open access SELection Endpoints in Communities of bacTeria (SELECT) method to determine concentrations of antibiotics that may select for antimicrobial resistance in the environment

Antimicrobial resistance (AMR) is a significant and growing threat to human, plant and animal health, the global economy, and food security. The One Health approach to AMR recognises the role of the environment in the evolution, emergence, and dissemination of AMR. In part, this is due to anthropogenic pollution that releases AMR organisms alongside cocktails of compounds that may select for AMR in situ, which then pose an exposure risk to humans and animals. This has spurred growing interest from cross-sectoral stakeholders in environmental risk assessment (ERA) of antibiotics, with regards to their selective potential. Many different experimental and modelling approaches have been used to determine the lowest concentration of an antibiotic that may select for AMR. Debates continue regarding which individual approach, if any, may be best for determining concentrations of antibiotics that may select for AMR, for ERA purposes. This paper contributes to this ongoing discourse by refining and using a previously published method SELECT (SELection Endpoints in Communities of bacTeria) to rapidly generate predicted no effect concentrations for resistance (PNECRs) for 32 antibiotics on the premise that reduction in growth of complex community of bacteria correlates with selection for AMR resistance genes. The database of PNECRs of antibiotics presented here is the largest generated using a single experimental, empirical approach that will aid future efforts towards creating a standardised test. PNECR data were used to conduct ERAs using measured environmental concentrations of antibiotics to rank antibiotics by potential selection risk in different environments. The experimental approach and statistical code have been made open access, with online tutorials available to facilitate other laboratories using the SELECT 2.0 method. Finally, we discuss the limitations of this approach and how these could be addressed in future studies.

microbiology↗

bioRxiv2026

A Novel Bioprocess Control Strategy Under Uncertainty via Operational Space Identification

Bioprocesses are critical for sustainable industrial development but face challenges from their inherent uncertainties that affect efficiency and scalability. This study in-troduces a worst-case operational space design framework, integrating symbolic optimization with scenario-based validation, to preemptively mitigate uncertainties so that key performance indicators are consistently satisfied even under adverse conditions. The methodology is demonstrated through two case studies: a lab-scale fermentation for astaxanthin production and a site-scale anaerobic digestion for biogas production. Process uncertainty is quantified through model parameter variations (2% - 13%) to reflect real-world scenarios. The results indicate that highly flexible operational spaces are identified in both case studies, with control variables able to vary by up to 25% and 15% in the respective systems, while consistently adhering to system constraints. Validation over 10,000 scenarios confirmed 0 violation per case study. The proposed framework delivers validated operational flexibility with modest computational requirements, making it practical for lab-to-site deployment.

microbiology↗

bioRxiv2025

Antagonism between bacteriophages and macrophages decreases efficacy of a phage cocktail and increases bacteriophage resistance

Phage therapy, the use of viruses that infect bacteria (bacteriophages), is a promising complement to antibiotics during the antimicrobial resistance crisis, but treatment success is very variable. Evolution of bacterial resistance to bacteriophages and bacteriophage counter-resistance (coevolution) during therapy may explain some of this variation, the dynamics of which may be affected by interactions with the patients immune system. Here, we examine how a pathogenic bacterium, Pseudomonas aeruginosa coevolves with two clinically relevant bacteriophages (14-1 and PNM) when in the presence of macrophages (RAW 264.7 cell line). We show macrophages reduced the rate by which bacteria were killed by bacteriophages, likely by reducing bacteria-bacteriophage contact rates. Over evolutionary time-scales, macrophages increased the proportion of bacteriophage resistant bacteria compared to where macrophages were absent. These differences in resistance rates were likely driven by the early advantage in density offered by macrophages to bacteria, and exclusion of PNM from the bacteriophage cocktail which otherwise increased in frequency in the absence of macrophages. Consequently, macrophages significantly altered the short- and long-term efficacy of a bacteriophage cocktail. In line with a growing body of work, our results suggest that the patients immune system can reduce the efficacy of phage therapy, potentially driving variable outcomes in therapy success in patients. Significance statementPhage therapy, the use of viruses that infect bacteria (bacteriophages), is a promising complement to antibiotics during the antimicrobial resistance crisis. However, treatment success is very variable. One, often overlooked, variable is the immune system and how this influences bacteriophage efficacy, and how bacteria evolve resistance to bacteriophages. We find that macrophages reduce the rate by which bacteria are killed by bacteriophages. Resistance to bacteriophages also increased in the presence of macrophages, showing macrophages affect the short- and long-term efficacy of phage therapy. These results highlight the importance of the immune system in phage therapy, and the need for more research in this area.

evolutionary biology↗

bioRxiv2025

The fire ant social chromosome exerts a major influence on genome regulation

Supergenes underlying complex trait polymorphisms ensure sets of coadapted alleles remain genetically linked. Despite their prevalence in nature, the mechanisms of supergene effects on genome regulation are poorly understood. In the fire ant Solenopsis invicta, a supergene containing over 500 individual genes influences trait variation in multiple castes to collectively underpin a colony level social polymorphism. Here, we present results of an integrative investigation of supergene effects on gene regulation. We present analyses of ATAC-seq data to investigate variation in chromatin accessibility by supergene genotype and STARR-seq data to characterize enhancer activity by supergene haplotype. Integration with gene coexpression analyses, newly mapped intact TEs, and previously identified CNVs, collectively reveal widespread effects of the supergene on chromatin structure, gene transcription, and regulatory element activity, with a genome-wide bias for open chromatin and increased expression in the presence of the derived supergene haplotype, particularly in regions that harbor intact TEs. Integrated consideration of CNVs and regulatory element divergence suggests each evolved in concert to shape the expression of supergene encoded factors, including several transcription factors that may directly contribute to the trans-regulatory footprint of a heteromorphic social chromosome. Overall, we show how genome structure in the form of a supergene has wide-reaching effects on gene regulation and gene expression.

genomics↗

bioRxiv2023

Macroevolutionary dynamics in micro-organisms: generalists give rise to specialists across biomes in the ubiquitous bacterial phylum Myxococcota.

Prokaryotes dominate the Tree of Life, but our understanding of the macroevolutionary processes generating this diversity is still limited. Habitat transitions are thought to be a key driver of prokaryote diversity, but we still know relatively little about how prokaryotes successfully transition and persist across environments, and how this varies between biomes and lineages. Here, we investigate biome transitions and specialisation in natural populations of a focal bacterial phylum, the Myxococcota, sampled across a range of replicated soils and freshwater and marine sediments in Cornwall (UK). By targeted deep sequencing of the protein-coding gene rpoB, we found >2000 unique Myxococcota lineages, with the majority (77%) being biome specialists and <5% able to live across the salt barrier. Discrete character evolution models revealed that biome specialists very rarely transitioned to specialising in another biome. Instead, generalists mediated transitions between biome specialists. Multistate hidden-state speciation and extinction models found variation in speciation rate across the tree, but this variation was independent of biome association and specialisation. Overall, our results help explain how microbes transition between biomes and are consistent with "the jack-of-all-trades" trade-off, where generalists suffer a cost in any individual environment, resulting in rapid evolution of niche specialists.

microbiology↗

bioRxiv2023

Copper reduces the virulence of bacterial communities at environmentally relevant concentrations

Increasing environmental concentrations of metals as a result of anthropogenic pollution are significantly changing many microbial communities. While there is evidence metal pollution can result in increased antibiotic resistance, the effects of metal pollution on virulence remains largely undetermined. Here, we experimentally test whether metal stress alters the virulence of bacterial communities. We do this by incubating three wastewater influent communities under different environmentally relevant copper concentrations for three days. We then quantify the virulence of the community using the Galleria mellonella infection model, and test if differences are due to changes in the rate of biomass accumulation (productivity), copper resistance, or community composition (quantified using 16S amplicon sequencing). The virulence of the communities was found to be reduced by the highest copper concentration, but not to be affected by the lower concentration. As well as reduced virulence, communities exposed to the highest copper concentration were less diverse and had lower productivity. This work highlights that metal pollution may decrease virulence in bacterial communities, but at a cost to diversity and productivity.

microbiology↗

bioRxiv2022

A general framework to unify the estimation of numerical abundance and biomass from quantitative eDNA data

Does environmental DNA (eDNA) correlate more closely with numerical abundance (N) or biomass in aquatic organisms? We hypothesize that the answer is neither: eDNA production likely scales allometrically, reflecting key physiological rates and surface area-to-body mass relationships. Building on individual-level frameworks developed from the Metabolic Theory of Ecology, we derive a framework through which quantitative eDNA data can be transformed to simultaneously reflect both population-level N and biomass. We then validated our framework using data from two previously published studies: (i) a marine eDNA metabarcoding dataset; and (ii) a freshwater single-species qPCR dataset. Using a Bayesian modeling approach, we estimated the value of the allometric scaling coefficient that jointly optimized the relationship between N, biomass, and corrected eDNA data to be 0.82 and 0.77 in Case Studies (i) and (ii), respectively. These estimates closely match expected scaling coefficients estimated in previous work on Teleost fish metabolic rates. We also demonstrate that correcting quantitative eDNA can significantly improve correspondence between eDNA- and traditionally-derived quantitative community biodiversity metrics (e.g., Shannon index and Bray-Curtis dissimilarity) under some circumstances. Collectively, we show that quantitative eDNA data is unlikely to correspond exactly to either N or biomass, but can be corrected to reflect both through our unifying joint modelling framework. This framework can also be further expanded to include other variables that might impact eDNA pseudo-steady-state concentrations in natural ecosystems (e.g., temperature, pH, and phenology), and is flexible enough to model these relationships across trophic levels. Significance StatementAquatic animals release DNA (from shed cells, mucous, faeces, etc.) into water, which can be detected via environmental DNA (eDNA) sampling. What is less clear is whether we can estimate numerical abundance (N) or biomass from eDNA concentrations. We hypothesize that eDNA production scales allometrically; that is, large animals release less DNA per unit mass than smaller animals. Building from the Metabolic Theory of Ecology, we derived a framework through which eDNA data can be transformed to simultaneously reflect both N and biomass. We then validated the framework using two case studies in marine and freshwater systems. This framework unifies discrepancies between eDNA, N, and biomass data, unlocking the potential of eDNA to monitor population abundance/biomass and quantify biodiversity.

ecology↗

bioRxiv2022

Actin cytoskeleton signaling via MRTF/SRF entrains circadian clock

The circadian clock is entrained to daily environmental cues. Integrin-linked intracellular signaling via actin cytoskeleton dynamics transduces extracellular matrix interactions to Myocardin-related Transcription Factor (MRTF)/Serum Response Factor (SRF)-mediated transcription. Actin cytoskeleton organization in liver displays diurnal oscillations and SRF-MRTF activity exert transcriptional control to entrain clock. By interrogating disparate upstream events involved in actin cytoskeleton-MRTF-A/SRF signaling cascade, here we show that this signaling cascade transduce cellular niche cues to modulate circadian clock function. Pharmacological inhibitions of MRTF-A/SRF, by disrupting actin polymerization or blocking ROCK kinase, induced period lengthening with augmented clock amplitude, and genetic loss-of-functions of Srf or Mrtf-a mimic that of actin-depolymerizing agents. In contrast, actin-polymerization induced by Jasplakinolide shortened period with attenuated amplitude. In addition, interfering with cell-matrix interaction through blockade of integrin, inhibition of focal adhesion kinase or attenuating matrix rigidity led to reduced period length while enhancing amplitude. Mechanistically, we identify that core clock repressors, Per2, Nr1d1, and Nfil3, are direct transcriptional targets of MRTF-A/SRF in mediating actin dynamic-induced clock response. Collectively, our findings defined an integrin-actin cytoskeleton-MRTF/SRF pathway in linking clock entrainment with extracellular microenvironment that may facilitate cellular adaptation to its physical niche. Summary statementOur study revealed the role of actin cytoskeleton-MRTF/SRF signaling in entraining circadian clock to its extracellular physical niche environment.

cell biology↗

bioRxiv2021

Bacterial colonisation dynamics of household plastics in a coastal environment

Accumulation of plastics in the marine environment has widespread detrimental consequences for ecosystems and wildlife. Marine plastics are rapidly colonised by a wide diversity of bacteria, including human pathogens, posing potential risks to human health. Here, we investigate the effect of polymer type, residence time and estuarine location on bacterial colonisation of common household plastics, including pathogenic bacteria. To do so, we submerged five main household plastic types: low-density PE (LDPE), high-density PE (HDPE), polypropylene (PP), polyvinyl chloride (PVC) and polyethylene terephthalate (PET) at an estuarine site in Cornwall (U.K.) and tracked bacterial colonisation dynamics. Using both culture-dependent and culture-independent approaches, we found that bacteria rapidly colonised plastics irrespective of polymer type. While biofilm community composition changed with colonisation time, no difference was observed between polymer types. Likewise, the presence of pathogenic bacteria, quantified using the insect model Galleria mellonella, increased over a five-week period, with no consistent differences observed between polymer types. Pathogens isolated from plastic biofilms using Galleria enrichment included Serratia and Enterococcus species and harboured a wide range of antimicrobial resistance genes. Our findings show that plastics in coastal waters are rapidly colonised by a wide diversity of bacteria, including known human pathogens, independent of polymer type.

ecology↗

bioRxiv2019

The genomic basis of evolved virus resistance is dependent on environmental resources

Parasites impose strong selection on their hosts, but the level of resistance evolved may be constrained by the availability of resources. However, studies identifying the genomic basis of such resource mediated selection are rare, particularly in non-model organisms. Here, we investigated the role of nutrition in the evolution of resistance to a DNA virus (PiGV), and associated trade-offs, in a lepidopteran pest species (Plodia interpunctella). Through selection experiments and whole genome sequencing we identify putative mechanisms of resistance that depend on the nutritional environment during selection. We find that the evolution of resistance is specific to diet, with adaptation to a low nutrition diet constraining resistance when challenged with the pathogen on a high nutrition diet. Resistance in a low nutrition environment is negatively correlated with growth rate, consistent with an established trade-off between immunity and development. Whole genome resequencing of the host shows that resistance mechanisms are highly polygenic and suggests evidence for trade-offs at the genetic level. Critically when populations evolve in high resource conditions, resistance is linked to metabolic and immune pathways, however it is more closely associated with cytoskeleton organisation when selected under low nutrition. Our results emphasise the importance of resources on the evolution of resistance.

evolutionary biology↗

bioRxiv2019

A mobile ELF4 delivers circadian temperature information from shoots to roots

The circadian clock is synchronized by environmental cues, mostly by light and temperature. Elucidating how the plant circadian clock responds to temperature oscillations is crucial to understand plant responsiveness to the environment. Here we found a prevalent temperature-dependent function of the Arabidopsis clock component ELF4 (EARLY FLOWERING 4) in the root clock. Micrografting assays and mathematical analyses show that ELF4 moves from shoots to regulate rhythms in roots. ELF4 movement does not convey photoperiodic information but trafficking is essential to control the period of the root clock in a temperature-dependent manner. At low temperatures, ELF4 mobility is favored and results in a slow-paced root clock while high temperatures decrease movement, leading to a fast clock. Hence, the mobile ELF4 delivers temperature information and establishes a shoot-to-root dialogue that sets the pace of the clock in roots.

plant biology↗