Biology subjects

Greenwood, M.

Publications and source records attributed to Greenwood, M..

9 recordsLinked to original sources

bioRxiv2026

Quantitative modeling reveals sources of variability in transcriptional activation assays

Reporter cell assays, such as those used to detect estrogenic chemicals, can detect target chemicals at low concentrations and can be used to analyze chemical mixtures without a priori knowledge of the mixture components. However, the outputs of these assays are affected by biological variability, which complicates their interpretation. Here, we describe and demonstrate a workflow that is useful for determining potential sources of biological variability and optimizing the performance of cell-based assays. The workflow involves developing an appropriate mathematical model for a transcriptional activation assay, calibrating it with experimental data, and conducting sensitivity analysis to characterize individual components of the genetic circuit based on their effect on the reporter signal output. This workflow was tested using an estrogen receptor transcriptional activation assay. For this circuit, our analysis predicts that controlling estrogen response element number, promoter strength, and reporter signal degradation rates minimizes reporter output variability. We show that careful model development, calibration, and analysis can offer biologically relevant insights to minimize the variability of cell-based assays and improve genetic circuits for increased sensitivity and dynamic range.

synthetic biology↗

bioRxiv2025

River pollution threatens preferred macroinvertebrate prey of endemic fishes in Aotearoa, New Zealand

O_LIMacroinvertebrates are a critical source of food for riverine fishes. Anthropogenic stressors can transform riverine macroinvertebrate communities, yet the consequences of such transformations to the fishes that feed on them are poorly understood. Changes in any combination of food abundance, accessibility or quality may be referred to as changes in the foodscape of fishes. Stressor-driven increases of inaccessible prey that are difficult for fish to detect, capture or ingest, may render foodscapes less profitable to fishes. C_LIO_LIWe examined how nutrient enrichment, fine sediment, acid mine drainage (AMD), flooding and drying reshape fish foodscapes by altering the accessibility of macroinvertebrate prey for two New Zealand river fishes, Galaxias vulgaris and Gobiomorphus breviceps. We determined relative accessibility of macroinvertebrate taxa and traits to these fishes by comparing the abundance of prey taxa and traits in the environment with their corresponding abundances in fish diets. To make these comparisons, invertebrate and fish gut samples were collected from the same river reaches, at the same times, over three years. Environmental DNA (eDNA) metabarcoding of fish gut contents facilitated efficient diet characterisation of over 700 fish guts. Finally, to determine how multiple stressors might affect fish foodscapes, we related invertebrate trait-specific accessibilities to published data defining how the composition of those macroinvertebrate traits varies along stressor gradients. C_LIO_LIMacroinvertebrate traits that were most accessible to fishes were those promoting detection (e.g., active foraging behaviours exhibited by crawlers and scrapers) and ingestion (e.g., moderate size, morphologies lacking defensive structures), typical of mayfly and free-living caddisfly larvae. In contrast, prey with traits inhibiting detection (e.g., small size and burrowing behaviour) and ingestion by gape-limited fishes (e.g., large size), were less accessible to the fishes we studied. C_LIO_LITrait abundance-stressor relationships revealed that that increasing fine sediment, nutrients, AMD and drying, and decreased flooding frequency, favoured macroinvertebrates with traits that erode accessibility to fishes. These results show that multiple stressors may decrease the profitability of fish foodscapes by filtering out macroinvertebrate prey with traits promoting accessibility to fishes. C_LIO_LIFood web approaches to understanding multi-stressor impacts are rare, but are necessary to understand how the effects of multiple stressors on primary consumers then go on to affect predators of higher trophic position. A major barrier to fish foodscape research is the difficulty and cost of estimating trophic networks. We discuss how eDNA metabarcoding, coupled with trait-based approaches may help overcome these barriers, enabling generalisable assessments of foodscape changes caused by multiple stressors. C_LI

ecology↗

bioRxiv2025

Targeted analysis of chondrocyte central metabolites in response to cyclical compression and shear deformations

Osteoarthritis results in deterioration of articular cartilage, the soft tissue covering articulating surfaces of bones in joints like the knee and hip. Cyclical mechanical stimulation of articular cartilage results in synthesis of cartilage matrix, suggesting that therapeutic mechanical stimulation might be beneficial for cartilage repair in osteoarthritis. Several prior studies identify ion channels and cytoskeletal molecules as components of chondrocyte mechanotransduction. The pathways of central metabolism, glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle are necessary for producing non-essential amino acids that are needed for synthesizing matrix proteins for cartilage repair. However, it is currently unknown if and how levels of central metabolites change with applied mechanical stimulation of chondrocytes. Here, we show that applied cyclical shear and compressive deformations drive changes in multiple central metabolites in primary chondrocytes. These results add to a rich picture of chondrocyte mechanotransduction, including calcium signaling, ion channels, integrins, and cytoskeletal components. By finding compression- and shear-induced changes in central metabolites, these data support the potential for therapeutic mechanotransduction toward cartilage repair. Future studies may build on these results to understand the relationships between mechanical stimulation and chondrocyte central metabolism.

bioengineering↗

bioRxiv2024

GENE THERAPY PREVENTS HEPATIC MITOCHONDRIAL DYSFUNCTION IN MURINE DEOXYGUANOSINE KINASE DEFICIENCY

Primary mitochondrial disorders are an uncommon cause of neonatal hepatic failure. Biallelic pathogenic variants of the gene encoding the mitochondrial localising enzyme deoxyguanosine kinase (DGUOK) cause hepatocerebral mitochondrial DNA depletion syndrome leading to acute neonatal liver failure and early mortality. There are currently no effective disease-modifying therapies. In this study, we developed an adeno-associated virus 9 (AAV9) gene therapy approach to treat a mouse model of DGUOK deficiency that recapitulates human disease. We delivered AAV9-hDGUOK gene therapy intravenously to newborn Dguok knock-out mice and showed that liver dysfunction was prevented in a dose dependent manner. Unexpectedly for neonatal delivery, durable and long-lasting liver transduction and RNA expression were demonstrated. Liver mitochondrial DNA depletion, deficiencies of oxidative phosphorylation complexes I, III and IV and liver transaminitis and survival were ameliorated in a dose-dependent manner.

genetics↗

bioRxiv2020

A spatial model of the plant circadian clock reveals design principles for coordinated timing under noisy environments

Individual plant cells possess a genetic network, the circadian clock, that times internal processes to the day-night cycle. Mathematical models of the clock network have driven a mechanistic understanding of the clock in plants. However, these models are typically either whole plant models that ignore tissue or cell type specific clock behavior, or phase only models that do not include clock network components explicitly. It is increasingly clear that in order to reveal the design principles of the plant circadian clock, clock network models must address spatial differences. This is because complex spatial behaviours have been observed in tissues and cells in plants, including period and phase differences between cells and spatial waves of gene expression between organs. Here, we implement an up to date clock network model on a spatial template of the plant. In our model, the sensitivity to light inputs varies across the plant, and cells communicate their clock timing locally via the levels of core clock mRNA levels by cell-to-cell coupling. We found that differences in sensitivities to environmental input in the model can explain the experimentally observed differences in clock periods in different organs, and we show using the model that a plausible coupling mechanism can generate the experimentally observed waves in clock gene expression across the plant. We then examined what features of the plant circadian system allow it to keep time under noisy light-dark (LD) cycles. We found that differences in sensitivity to light can allow regional flexibility in phase even under LD cycles, whilst local cell-to-cell coupling minimized variability in clock rhythms in neighboring cells. Thus, local sensitivity to environmental inputs combined with cell-to-cell coupling allows for flexible yet robust circadian timing under noisy environments.

plant biology↗

bioRxiv2019

Inspiratory rhythmogenic activity is burst-independent and opioid-sensitive

How mammalian neural circuits generate rhythmic activity in motor behaviors, such as breathing, walking, and chewing, remains elusive. For breathing, rhythm generation can be localized to a brainstem nucleus called the preBotzinger Complex (preBotC). Rhythmic preBotC population activity consists of small amplitude burstlets, which we hypothesize are rhythmogenic, and larger inspiratory bursts, which drive motoneuronal activity. If burstlets are rhythmogenic, opioids, analgesics that can cause profound respiratory depression, should similarly reduce burstlet frequency. In conditions where burstlets were separated from bursts in medullary slices from neonatal mice, the -opioid receptor (OR) agonist DAMGO decreased burstlet frequency. DAMGO-mediated depression was abolished by genetic deletion of ORs in a glutamatergic preBotC subpopulation and was reduced by Substance P, but not blockade of inhibitory synaptic transmission. Our findings suggest that rhythmogenesis need not rely on strong bursts of activity associated with motor output and point to strategies for ameliorating opioid-induced depression of breathing.

neuroscience↗

bioRxiv2019

Coordinated circadian timing through the integration of local inputs in Arabidopsis thaliana

Every plant cell has a genetic circuit, the circadian clock, that times key processes to the day-night cycle. These clocks are aligned to the day-night cycle by multiple environmental signals that vary across the plant. How does the plant integrate clock rhythms, both within and between organs, to ensure coordinated timing? To address this question, we examined the clock at the sub-tissue level across Arabidopsis thaliana seedlings under multiple environmental conditions and genetic backgrounds. Our results show that the clock runs at different speeds (periods) in each organ, which causes the clock to peak at different times across the plant in both constant environmental conditions and light-dark cycles. Closer examination reveals that spatial waves of clock gene expression propagate both within and between organs. Using a combination of modeling and experiment, we reveal that these spatial waves are the result of the period differences between organs and local coupling, rather than long distance signaling. With further experiments we show that the endogenous period differences, and thus the spatial waves, are caused by the organ specificity of inputs into the clock. We demonstrate this by modulating periods using light and metabolic signals, as well as with genetic perturbations. Our results reveal that plant clocks are set locally by organ specific inputs, but coordinated globally via spatial waves of clock gene expression.

plant biology↗

bioRxiv2019

A high-throughput delayed fluorescence method reveals underling differences in the control of circadian rhythms in Triticum aestivum and Brassica napus

BackgroundA robust circadian clock has been implicated in plant resilience, resource-use efficiency, competitive growth and yield. A huge number of physiological processes are under circadian control in plants including: responses to biotic and abiotic stresses; flowering time; plant metabolism; and mineral uptake. Understanding how the clock functions in crops such as Triticum aestivum (bread wheat) and Brassica napus (oilseed rape) therefore has great agricultural potential. Delayed fluorescence (DF) imaging has been shown to be applicable to a wide range of plant species and requires no genetic transformation. Although DF has been used to measure period length of both mutants and wild ecotypes of Arabidopsis, this assay has never been systematically optimised for crop plants. The physical size of both B. napus and T. aestivum led us to develop a representative sampling strategy which enables high-throughput imaging of these crops.\n\nResultsIn this study, we describe the plant-specific optimisation of DF imaging to obtain reliable circadian phenotypes with the robustness and reproducibility to detect diverging periods between cultivars of the same species. We find that the age of plant material, light regime and temperature conditions all significantly effect DF rhythms and describe the optimal conditions for measuring robust rhythms in each species. We also show that sections of leaf can be used to obtain period estimates with improved throughput for larger sample size experiments.\n\nConclusionsWe present an optimized protocol for high-throughput phenotyping of circadian period specific to two economically valuable crop plants. Application of this method revealed significant differences between the periods of several widely grown elite cultivars. This method also identified intriguing differential responses of circadian rhythms in T. aestivum compared to B. napus; specifically the dramatic change to rhythm robustness when plants were imaged under constant light versus constant darkness. This points towards diverging networks underling circadian control in these two species.

plant biology↗

bioRxiv2017

Coordination of robust single cell rhythms in the Arabidopsis circadian clock via spatial waves of gene expression

The Arabidopsis circadian clock orchestrates gene regulation across the day/night cycle. Although a multiple feedback loop circuit has been shown to generate the 24h rhythm, it remains unclear how robust the clock is in individual cells, or how clock timing is coordinated across the plant. Here we examine clock activity at the single cell level across Arabidopsis seedlings over several days. Our data reveal robust single cell oscillations, albeit desynchronised. In particular, we observe two waves of clock activity; one going down, and one up the root. We also find evidence of cell-to-cell coupling of the clock, especially in the root tip. A simple model shows that cell-to-cell coupling and our measured period differences between cells can generate the observed waves. Our results reveal the spatial structure of the plant circadian clock and suggest that unlike the centralised mammalian clock, the clock has multiple points of coordination in Arabidopsis.

systems biology↗