Biology subjects

Hughes, T. E.

Publications and source records attributed to Hughes, T. E..

12 recordsLinked to original sources

bioRxiv2026

Ecological and evolutionary dynamics of the oral microbiome across childhood

Childhood represents a critical period for oral microbiome development, yet evolutionary trajectories and the relative roles of host and environment remain unclear. Using a large longitudinal metagenomic dataset of 920 samples from a twin cohort spanning the first decade of life, we characterised microbial shifts and population dynamics of key bacterial groups. Microbiome diversity was initially reduced and highly heterogeneous and became increasingly complex and convergent with age. Microbial community state was associated with developmental age, environment and in late childhood was surprisingly strongly associated with host genotype. Strain-level analyses revealed species-specific temporal patterns of genetic variation particularly within Streptococcus, reflecting adaptive responses to host and environmental pressures. Fusobacterium exhibited consistently high replication rates, indicating sustained growth dynamics. Phylogenetic reconstruction further revealed host and niche specific genomic diversification of Saccharibacteria lineages. These findings establish childhood as a decisive period of oral microbial evolution and highlight the role of host-microbiome and epithelial interactions in shaping community structure, providing guidance for oral management strategies that promote lifelong oral health.

ecology↗

bioRxiv2022

Mutations in NAKED-ENDOSPERM IDD genes reveal functional interactions with SCARECROW and a maternal influence on leaf patterning in C4 grasses

Leaves comprise a number of different cell-types that are patterned in the context of either the epidermal or inner cell layers. In grass leaves, two distinct anatomies develop in the inner leaf tissues depending on whether the leaf carries out C3 or C4 photosynthesis. In both cases a series of parallel veins develops that extends from the leaf base to the tip but in ancestral C3 species veins are separated by a greater number of intervening mesophyll cells than in derived C4 species. We have previously demonstrated that the GRAS transcription factor SCARECROW (SCR) regulates the number of photosynthetic mesophyll cells that form between veins in the leaves of the C4 species maize, whereas it regulates the formation of stomata in the epidermal leaf layer in the C3 species rice. Here we show that SCR is required for inner leaf patterning in the C4 species Setaria viridis but in this species the presumed ancestral stomatal patterning role is also retained. Through a comparative mutant analysis between maize, setaria and rice we further demonstrate that loss of NAKED-ENDOSPERM (NKD) INDETERMINATE DOMAIN (IDD) protein function exacerbates loss of function scr phenotypes in the inner leaf tissues of maize and setaria but not rice. Specifically, in both setaria and maize, scr;nkd mutants exhibit an increased proportion of fused veins with no intervening mesophyll cells, whereas inner leaf tissues are patterned normally in scr;nkd mutants of rice. Thus, combined action of SCR and NKD may control how many mesophyll cells are specified between veins in the leaves of C4 but not C3 grasses. Finally, we identified a maternal effect in maize in which maternally derived NKD can affect patterning of cells in leaf primordia that are initiated during embryogenesis. Together our results provide insight into the evolution of cell patterning in grass leaves, demonstrate a novel patterning role for IDD genes in C4 leaves and suggest that NKD can influence embryonic leaf development non-cell autonomously from the surrounding maternal tissue. Summary statementMutations in NKD IDD genes enhance loss of function scr phenotypes in the leaves of C4 grasses maize and Setaria viridis but not in the C3 grass rice, and reveal a maternal effect on cell-type patterning in leaves that are initiated during embryogenesis.

plant biology↗

bioRxiv2021

SCARECROW is deployed in distinct developmental contexts during rice and maize leaf development

The flexible deployment of developmental regulators is an increasingly appreciated aspect of plant development and evolution. The GRAS transcription factor SCARECROW (SCR) regulates the development of the endodermis in Arabidopsis and maize roots, but during leaf development it regulates the development of distinct cell-types; bundle-sheath in Arabidopsis and mesophyll in maize. In rice, SCR is implicated in stomatal patterning, but it is unknown whether this function is additional to a role in inner leaf patterning. Here, we demonstrate that two duplicated SCR genes function redundantly in rice. Contrary to previous reports, we show that these genes are necessary for stomatal development, with stomata virtually absent from leaves that are initiated after germination of mutants. The stomatal regulator OsMUTE is down-regulated in Osscr1;Osscr2 mutants indicating that OsSCR acts early in stomatal development. Notably, Osscr1;Osscr2 mutants do not exhibit the inner leaf patterning perturbations seen in Zmscr1;Zmscr1h mutants and Zmscr1;Zmscr1h mutants do not exhibit major perturbations in stomatal patterning. Taken together, these results indicate that SCR was deployed in different developmental contexts after the divergence of rice and maize around 50 million years ago. Summary statementThe transcription factor SCARECROW patterns stomata in rice leaves whereas in maize it predominantly patterns the inner leaf.

plant biology↗

bioRxiv2020

Far-red fluorescent genetically encoded calcium ion indicators

Genetically encoded calcium ion (Ca2+) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca2+ dynamics and neuronal activities on a single cell level. Here we report the design and development of two new far-red fluorescent GECIs, FR-GECO1a and FR-GECO1c, based on the monomeric far-red fluorescent protein mKelly. We characterized these far-red GECIs as purified proteins and assessed their performance when expressed in cultured neurons. FR-GECOs have excitation and emission maxima at ~ 596 nm and ~ 644 nm, respectively, display large responses to Ca2+ ({Delta}F/F0 = 6 for FR-GECO1a, 18 for FR-GECO1c), and are bright under both one-photon and two-photon illumination. FR-GECOs also have high affinities (apparent Kd = 29 nM for FR-GECO1a, 83 nM for FR-GECO1c) for Ca2+, and they enable sensitive and fast detection of single action potentials in neurons.

neuroscience↗

bioRxiv2020

High throughput instrument to screen fluorescent proteins under two-photon excitation

Two-photon microscopy together with fluorescent proteins and fluorescent protein-based biosensors are commonly used tools in neuroscience. To enhance their experimental scope, it is important to optimize fluorescent proteins for two-photon excitation. Directed evolution of fluorescent proteins under one-photon excitation is common, but many one-photon properties do not correlate with two-photon properties. A simple system for expressing fluorescent protein mutants is E. coli colonies on an agar plate. The small focal volume of two-photon excitation makes creating a high throughput screen in this system a challenge for a conventional point-scanning approach. We present an instrument and accompanying software that solves this challenge by selectively scanning each colony based on a colony map captured under one-photon excitation. This instrument, called the GIZMO, can measure the two-photon excited fluorescence of 10,000 E. coli colonies in 7 hours. We show that the GIZMO can be used to evolve a fluorescent protein under two-photon excitation.

neuroscience↗

bioRxiv2020

SCARECROW gene function is required for photosynthetic development in maize

C4 photosynthesis in grasses relies on a specialized leaf anatomy. In maize, this ‘Kranz’ leaf anatomy is patterned in part by the duplicated SCARECROW (SCR) genes ZmSCR1 and ZmSCR1h. Here we show that in addition to patterning defects, chlorophyll content and levels of transcripts encoding Golden2-like regulators of chloroplast development are significantly lower in Zmscr1;Zmscr1h mutants than in wild-type. These perturbations are not associated with changes in chloroplast number, size or ultrastructure. However, the maximum rates of carboxylation by ribulose bisphosphate carboxylase/oxygenase (RuBisCO, Vcmax) and phosphoenolpyruvate carboxylase (PEPC, Vpmax) are both reduced, leading to perturbed plant growth. The CO2 compensation point and 13C‰ of Zmscr1;Zmscr1h plants are both normal, indicating that a canonical C4 cycle is operating, albeit at reduced overall capacity. Taken together, our results reveal that the maize SCR genes, either directly or indirectly, play a role in photosynthetic development.Significance statement SCARECROW (SCR) is one of the best studied plant developmental regulators, however, its role in downstream plant physiology is less well-understood. Here, we have demonstrated that SCR is required to establish and/or maintain photosynthetic capacity in maize leaves.Competing Interest StatementThe authors have declared no competing interest.View Full Text

plant biology↗

bioRxiv2020

Analyzing kinetic signaling data for G-protein-coupled receptors

In classical pharmacology, bioassay data are fit to general equations (e.g. the dose response equation) to determine empirical drug parameters (e.g. EC50 and Emax), which are then used to calculate chemical parameters such as affinity and efficacy. Here we used a similar approach for kinetic, time course signaling data, to allow empirical and chemical definition of signaling by G-protein-coupled receptors in kinetic terms. Experimental data are analyzed using general time course equations (model-free approach) and mechanistic model equations (mechanistic approach) in the commonly-used curve-fitting program, GraphPad Prism. A literature survey indicated signaling time course data usually conform to one of four curve shapes: the straight line, association exponential curve, rise-and-fall to zero curve, and rise-and-fall to steady-state curve. In the model-free approach, the initial rate of signaling is quantified and this is done by curve-fitting to the whole time course, avoiding the need to select the linear part of the curve. It is shown that the four shapes are consistent with a mechanistic model of signaling, based on enzyme kinetics, with the shape defined by the regulation of signaling mechanisms (e.g. receptor desensitization, signal degradation). Signaling efficacy is the initial rate of signaling by agonist-occupied receptor (k{tau}), simply the rate of signal generation before it becomes affected by regulation mechanisms, measurable using the model-free analysis. Regulation of signaling parameters such as the receptor desensitization rate can be estimated if the mechanism is known. This study extends the empirical and mechanistic approach used in classical pharmacology to kinetic signaling data, facilitating optimization of new therapeutics in kinetic terms.

pharmacology and toxicology↗

bioRxiv2019

A new kinetic method for measuring agonist efficacy and ligand bias using high resolution biosensors and a kinetic data analysis framework

The kinetics/dynamics of signaling are of increasing value for G-protein-coupled receptor therapeutic development, including spatiotemporal signaling and the kinetic context of biased agonism. Effective application of signaling kinetics to developing new therapeutics requires reliable kinetic assays and an analysis framework to extract kinetic pharmacological parameters. Here we describe a platform for measuring arrestin recruitment kinetics to GPCRs using a high quantum yield, genetically encoded fluorescent biosensor, and a new analysis framework to quantify the recruitment kinetics. The sensor enabled high temporal resolution measurement of arrestin recruitment to the angiotensin AT1 and vasopressin V2 receptors. The analysis quantified the initial rate of arrestin signaling (k{tau}), a biologically-meaningful kinetic drug efficacy parameter, by fitting time course data using routine curve-fitting methods. Biased agonism was assessed by comparing k{tau} values for arrestin recruitment with those for Gq signaling via the AT1 receptor. The k{tau} ratio values were in good agreement with bias estimates from existing methods. This platform potentially improves and simplifies assessment of biased agonism because the same assay modality is used to compare pathways (potentially in the same cells), the analysis method is parsimonious and intuitive, and kinetic context is factored into the bias measurement.

pharmacology and toxicology↗

bioRxiv2019

Live-cell assays for cell stress responses reveal new patterns of cell signaling caused by mutations in rhodopsin, α-synuclein and TDP-43

Many neurodegenerative diseases induce high levels of sustained cellular stress and alter a number of cellular processes. Genetically-encoded fluorescent biosensors are effective tools to examine neuronal activity and signaling in living cells. To examine how different mutations associated with neurodegenerative disease affect cell stress and signaling we created live-cell assays for ER-mediated cell stress and second messenger signaling. Analysis of the rhodopsin P23H mutation, the most common mutation in autosomal dominant Retinitis Pigmentosa, revealed increased cell stress levels compared to wild type rhodopsin. Moreover, this increase in cell stress correlated with blunted Ca2+ signaling in a stress dependent manner. Analysis of single cell Ca2+ signaling profiles revealed unique Ca2+ signaling responses exist in cells expressing wild type or P23H mutants, further supporting the notion that second messenger signaling is affected by cell stress. To explore the use of the ER-stress biosensor in other neurodegenerative diseases we examined how various mutants of -synuclein and TDP-43 affected ER-mediated cell stress. Mutants of both -synuclein and TDP-43 associated with Parkinsons Disease and ALS demonstrated increases in ER-mediated cell stress. This increased cell stress was accompanied by changes in phosphodiesterase activity. Both HEK293T and SH-SY5Y cells expressing these proteins displayed a shift towards increased cAMP degradation rates, likely due to increased phosphodiesterase activity. Together these data illustrate how biosensors can provide nuanced, new views of neurodegenerative disease processes.

neuroscience↗

bioRxiv2019

Redundant SCARECROW genes pattern distinct cell layers in roots and leaves of maize

The highly efficient C4 photosynthetic pathway is facilitated by Kranz leaf anatomy. In Kranz leaves, closely spaced veins are encircled by concentric layers of photosynthetic bundle sheath (inner) and mesophyll (outer) cells. Here we demonstrate that in the C4 monocot maize, Kranz patterning is regulated by redundant function of SCARECROW 1 (ZmSCR1) and a previously uncharacterized homeolog ZmSCR1h. ZmSCR1 and ZmSCR1h transcripts accumulate in ground meristem cells of developing leaf primordia and in Zmscr1;Zmscr1h mutant leaves, most veins are separated by one rather than two mesophyll cells; many veins have sclerenchyma above and/or below instead of mesophyll cells; and supernumerary bundle sheath cells develop. The mutant defects are unified by compromised mesophyll cell development. In addition to Kranz defects, Zmscr1;Zmscr1h mutants fail to form an organized endodermal layer in the root. Collectively, these data indicate that ZmSCR1 and ZmSCR1h redundantly regulate cell-type patterning in both leaves and roots of maize. Leaf and root pathways are distinguished, however, by the cell layer in which they operate - mesophyll at a two-cell distance from leaf veins versus endodermis immediately adjacent to root vasculature.\n\nSummary statementTwo duplicated maize SCARECROW genes control the development of the endodermis in roots and the mesophyll in leaves

plant biology↗

bioRxiv2018

Understanding the Ca2+-dependent Fluorescence Change in Red Genetically Encoded Ca2+ Indicators

Genetically encoded Ca2+ indicators (GECIs) are widely used to illuminate dynamic Ca2+ signaling activity in living cells and tissues. Various fluorescence colors of GECIs are available, including red. Red GECIs are promising because longer wavelengths of light scatter less in tissue, making it possible to image deeper. They are engineered from a circularly permuted red fluorescent protein fused to a Ca2+ sensing domain, calmodulin and a calmodulin-binding peptide. A conformational change in the sensing domain upon binding Ca2+ causes a change in the fluorescence intensity of the fluorescent protein. Three factors could contribute to this change in fluorescence: 1) a shift in the protonation state of the chromophore, 2) a change in fluorescence quantum yield, and 3) a change in the extinction coefficient for one-photon excitation or the two-photon cross section for two-photon excitation. We conducted a systematic study of the photophysical properties of a select cohort of red GECIs in their Ca2+-free and Ca2+-saturated states to determine which factors are most important for the Ca2+-dependent change in fluorescence. In total, we analyzed nine red GECIs, including jRGECO1a, K-GECO1, jRCaMP1a, R-GECO1, R-GECO1.2, CAR-GECO1, O-GECO1, REX-GECO1, and a new variant termed jREX-GECO1. We found that these red GECIs could be separated into three classes that each rely on a particular set of factors. Furthermore, in some cases the magnitude of the change in fluorescence was different depending on one-photon excitation or two-photon excitation by up to a factor of two.

biophysics↗

bioRxiv2017

A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein

Genetically-encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs enable multicolor visualization with blue or cyan-excitable fluorophores and combined use with blue or cyan-excitable optogenetic actuators. Here we report the development, structure, and validation of a new red fluorescent Ca2+ indicator, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem cell derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo.

biophysics↗