Biology subjects

Ronald, J.

Publications and source records attributed to Ronald, J..

10 recordsLinked to original sources

bioRxiv2026

Pannexin 1 inhibition reduces tumorigenic properties of patient-derived glioblastoma cells through the HIPPO and Wnt signalling pathways

Glioblastoma (GBM) is the most common primary brain tumour, with a median survival of 12-18 months, highlighting a need for new treatment targets. We observed that pannexin 1 (PANX1), a channel-forming glycoprotein important in purinergic signalling, is upregulated in GBM compared to normal tissue and expressed throughout patient tumours. Western blot analysis of patient-derived GBM cell lines revealed significantly increased PANX1 expression in these primary lines compared to brain tissue and control glial cells. Bulk RNA-sequencing compared the gene expression of GBM cells devoid of PANX1 via CRISPR/Cas9 deletion (PANX1-KO) compared to controls. Gene Ontology and KEGG gene set analyses revealed PANX1-KO in GBM cells affects cell surface and cell junction components, processes, and pathways, including the HIPPO pathway, in addition to critically downregulating {beta}-catenin mRNA and other components of the Wnt pathway. The deletion of PANX1 resulted in a disruption of the {beta}-catenin protein and a dramatic reduction in migration and cell growth. Pharmacological inhibition of PANX1 in GBM cells with Probenecid (PBN) and Spironolactone (SPIR) demonstrated a significant reduction in live cell numbers and migration via scratch assay. Both blockers dramatically decreased F-actin filament formation, and the cellular localization of beta-catenin became more intracellular compared to controls. Xenografted GBM tumours showed a reduction in tumour cell viability by bioluminescent imaging and reduced hemorrhaging incidence when treated with PBN. These new insights support further investigation of PANX1 as a potential GBM therapeutic target and its role in multiple cancer signaling pathways that regulate this devastating disease.

cancer biology↗

bioRxiv2025

Arabidopsis EARLY FLOWERING 4 recruits EARLY FLOWERING 3 to the nucleus to facilitate gene repression

The evening complex (EC) is a protein complex composed of ELF3, ELF4 and LUX that accumulates at dusk to repress gene expression. ELF3 functions as a scaffold around which the other EC components assemble, while LUX facilitates EC binding to DNA. Although ELF4 is essential for the activity of the EC, its precise role within the complex is unclear. Earlier studies indicated that mutations within the ELF4-binding domain of ELF3 reduced the nuclear accumulation of ELF3. However, it has remained unknown whether ELF4 directly contributes to these phenotypes. To investigate this further, we quantified the cellular and subnuclear distribution of Arabidopsis ELF3 across the evening in hypocotyl nuclei with or without a functional ELF4 allele. These results highlight a critical requirement for ELF4 in recruiting ELF3 to hypocotyl nuclei at dusk. Reduced nuclear accumulation in the elf4 mutant correlated with a failure of ELF3 to repress gene expression and inhibit hypocotyl elongation, particularly under short-day conditions. We also observed that ELF4 was necessary to recruit ELF3 to foci in root nuclei, but these foci displayed different temporal properties from those in the hypocotyl. In summary, our results support a model in which ELF4 recruits ELF3 to the nucleus at dusk to enhance ELF3 repressive activity.

plant biology↗

bioRxiv2025

Co delivery of human adipose derived stromal cells and endothelial colony forming cells in cell-assembled decellularized adipose tissue scaffolds for applications in soft tissue regeneration

Cellular therapies involving the co-delivery of cells with complementary pro-regenerative functionality hold promise as a strategy to promote soft tissue regeneration. In particular, the co-delivery of adipose-derived stromal cells (ASCs) and endothelial colony-forming cells (ECFCs) has shown promise for regenerating stable blood vessels in vivo. The current study developed novel "cell-assembled" scaffolds for co-delivering human ASCs and ECFCs within a supportive decellularized adipose tissue (DAT) matrix, with the objective of enhancing their localized retention and augmenting their capacity to stimulate adipose tissue regeneration. Human ASCs and ECFCs were seeded separately onto human-derived DAT microcarriers under cell-type specific conditions. The cell-seeded microcarriers were then combined and cultured for 8 days under conditions that promoted matrix remodeling to fuse the microcarriers into 3D engineered tissues containing ASCs+ECFCs, ASCs alone, or ECFCs alone. Co-culture with ECFCs within the scaffolds was shown to modulate ASC pro-angiogenic gene expression, with some ECFCs forming tubule-like structures in vitro in both the ASC+ECFC and ECFC alone groups. In vivo bioluminescence imaging using a dual luciferase reporter system showed that co-delivery with ASCs enhanced ECFC retention following subcutaneous implantation in athymic nu/nu mice, but co-delivery did not alter the localized retention of viable ASCs. Interestingly, while immunofluorescence staining for CD31 and microcomputed tomography angiography indicated that vascular regeneration was similar in the cell-assembled scaffolds containing ASC+ECFCs, ASCs alone, and ECFCs alone, histological staining revealed that extensive regions of the ECFC alone scaffolds had remodelled into adipose tissue at 29 days post-implantation.

bioengineering↗

bioRxiv2025

ELF3 controls trait heterogeneity by tuning the rate of maturation in Arabidopsis and barley

Trait heterogeneity in a population increases the likelihood that some individuals will survive an unpredictable environmental stress. Individual plants mature at different rates due to genetics, environmental factors, and random chance. All of this contributes to trait heterogeneity. EARLY FLOWERING3 (ELF3), a core circadian clock component, determines developmental timing, and here we tested the hypothesis that it would shape trait heterogeneity. We developed a model predicting that faster-developing populations exhibit greater heterogeneity during development but reduced heterogeneity at maturity, while slower-growing populations show the opposite pattern. Experiments in Arabidopsis elf3 and barley Hvelf3 mutants supported this prediction. In Arabidopsis, ELF3 controlled hypocotyl elongation variability via maturation rate rather than direct regulation. Bolting time heterogeneity also decreased in faster-growing plants. In barley, Hvelf3 altered growth heterogeneity, but this was initially masked by germination timing differences. Finally, smaller barley plants were found to be more resilient to osmotic stress, suggesting that ELF3-driven trait heterogeneity may contribute to bet hedging. These findings highlight how modifying developmental rates influences population-level trait distributions and stress resilience.

plant biology↗

bioRxiv2025

The genetic basis for synchronized time perception in plant populations

Synchronised developmental timing is important for ensuring crop uniformity and high yields. However, climate change is leading to crops being grown at different latitudes, varying their exposures to photoperiods over seasons and impacting developmental timings. We investigated whether the response of circadian rhythms in seedlings to changes in photoperiod would enable us to predict the timing and synchronization of flowering. Indeed, we show that the same Quantitative Trait Loci (QTLs) are associated to circadian traits in seedlings and developmental traits during bolting, using the first recombinant inbred lines (RILs) between African and European Arabidopsis lineages, spanning diverse latitudes. Two QTLs contain K-Homology Domain RNA binding proteins (KH17, KH29) and are associated with splicing variants in known flowering genes, MADS AFFECTING FLOWERING2 and 3 (MAF2, MAF3), including generating chimeric transcripts, a potential mechanism for accelerated proteome evolution. Natural variants in KH17, including in its prion-like domain, are associated with de-coupling the mean and synchronization of flowering time, enabling greater adaptation of population-level heterogeneity in developmental timings. Our results suggest that circadian traits in seedlings could be used to screen for agriculturally relevant developmental traits in mature plants, enabling efficient breeding of climate-resilient crops.

plant biology↗

bioRxiv2024

Stable and dynamic gene expression patterns over diurnal and developmental timescales in Arabidopsis thaliana

O_LIDevelopmental processes are known to be circadian regulated in plants. For instance, the circadian clock regulates genes involved in the photoperiodic flowering pathway and the initiation of leaf senescence. Furthermore, signals which entrain the circadian clock, such as energy availability, are known to vary in strength over plant development. However, diurnal oscillations of the Arabidopsis transcriptome have typically been measured in seedlings. C_LIO_LIWe collected RNA-seq data from Arabidopsis leaves over developmental and diurnal timescales, concurrently: every 4 hours per day, on 3 separate days after a synchronised vegetative-to-reproductive transition. Gene expression varied more over the developmental timescale than on the diurnal timescale, including genes related to a key energy sensor: the Sucrose non-fermenting-1-related protein kinase (SnRK1) complex. C_LIO_LIMoreover, regulatory targets of core clock genes displayed changes in rhythmicity and amplitude of expression over development. Cell-type-specific expression showed diurnal patterns that varied in amplitude, but not phase, over development. Some previously identified qRT-PCR housekeeping genes display undesirable levels of variation over both timescales. We identify which common qRT-PCR housekeeping genes are most stable across developmental and diurnal timescales. C_LIO_LIIn summary, we establish the patterns of circadian transcriptional regulation over plant development, demonstrating how diurnal patterns of expression change over developmental timescales. C_LI

plant biology↗

bioRxiv2024

Novel Pannexin 1 isoform is increased in cancer

Alternative translation initiation (ATI) is a process of increasing protein diversity from one transcript, allowing cells to rapidly respond to signals, which is particularly important in cancer cells. Here, we report potential internal translation start sites exist in PANX1 which have implications in trafficking and channel function. Using mouse (mPANX1) constructs for each internal methionine, we saw that these PANX1 isoforms were N-glycosylated, could traffic to the cell surface and mPANX1-M37 formed functional channels activated by C-terminus cleavage or 1-adrenoceptor stimulation. We also identified a ~25 kDa isoform of mPANX1 (mPANX1-25K) endogenously expressed in mouse melanoma cell lines that could be confirmed with a cognate peptide. mPANX1-25K lacks the mPANX1 N-terminus and most likely corresponds to the M210 internal translation start site since we could not identify any alternative transcripts that would produce this ATI product. When we expressed the human equivalent of M210 in Hs578T PANX1 KO cells with and without wildtype human PANX1, we determined M211 exhibits a predominantly intracellular localization, is N-glycosylated and can interact with full-length human PANX1. Collectively, these findings indicate species specific differences in the abundance of PANX1 ATI isoforms which could act independently or in conjunction with the canonical full-length protein in melanoma.

cell biology↗

bioRxiv2024

Hypocotyl Development in Arabidopsis and other Brassicaceae Displays Evidence of Photoperiodic Memory

Sensing and responding to photoperiod changes is essential for plants to adapt to seasonal progression. Most of our understanding of how plants sense photoperiodic changes is through studies on flowering time. However, other aspects of plant development are regulated by the photoperiod, including hypocotyl elongation. Unlike flowering, hypocotyl elongation displays a greater plasticity to changes in the photoperiod with increases in daylength causing greater inhibition of growth until a threshold is met. Previous studies have only looked at hypocotyl development in the context of a stationary photoperiod. It is unknown if changes in the photoperiod during development influence hypocotyl elongation. Here, we developed a physiological assay to investigate this question. We have discovered that hypocotyl elongation is influenced by a memory of past photoperiod exposure in Arabidopsis and Brassicaceae cultivars used for microgreen agriculture. Photoperiodic memory persisted for multiple days, although it weakened over time, and the strength of the memory was dependent on the genetic background. We identified that phyB and ELF3, key regulators of hypocotyl development, were required for photoperiodic memory. Finally, we identified that the circadian clock is unlikely to function as a repository for photoperiodic memory as circadian rhythms quickly re-aligned with the new photoperiod. In summary, our work highlights for the first-time evidence of a photoperiodic memory that can control plant development.

plant biology↗

bioRxiv2023

Single-plant-omics reveals the cascade of transcriptional changes during the vegetative-to-reproductive transition

Plants undergo rapid developmental transitions, as well as gradual developmental processes. Moreover, individual plants within a population will undergo the developmental transitions asynchronously, so it is difficult to assemble a time series to resolve the sequence of transcriptional changes that take place during these rapid transitions. Single-plant-omics has the potential to distinguish between transcriptional events that are associated with these binary and continuous processes. Furthermore, we can utilise single-plant-omics to exploit this developmental asynchrony to order individual plants by their developmental trajectory, revealing a detailed cascade of transcriptional events. Here, we utilise single-plant-transcriptomics to resolve the transcriptional events that coincide with the onset of bolting. We performed RNA-seq on the leaves of individual plants from a large population of wild type Arabidopsis thaliana replicated at one time point during the vegetative-to-reproductive transition. Even though more than half of transcripts were differentially expressed between bolted and unbolted plants, we were able to find a subset of regulators that were more closely associated with gradual developmental traits like leaf size and biomass. Using a novel pseudotime inference algorithm, we determined that some senescence-associated processes, such as the reduction in ribosome biogenesis, are evident in the transcriptome before a bolt is visible. These results show the potential of single-plant-omics to reveal the detailed sequence of events that occur during rapid developmental transitions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/557157v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@c50448org.highwire.dtl.DTLVardef@9c384aorg.highwire.dtl.DTLVardef@33c336org.highwire.dtl.DTLVardef@55302e_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract: Physiological changes around bolting can be categorised into: binary processes, which appear to have either occurred or not occurred at any given timepoint; or continuous processes, which can be observed quantitatively. For binary processes, expression of strongly correlated genes can appear to follow a step change dynamic over time. However, when considered over a shorter timescale, the dynamics appear much smoother. For continuous processes, the shorter timescale should also capture smooth changes in gene expression. C_FIG

plant biology↗

bioRxiv2023

Complex epistatic interactions between ELF3, PRR9, and PRR7 regulates the circadian clock and plant physiology

Circadian clocks are endogenous timekeeping mechanisms that coordinate internal physiological responses with the external environment. EARLY FLOWERING3 (ELF3), PSEUDO RESPONSE REGULATOR (PRR9), and PRR7 are essential components of the plant circadian clock and facilitate entrainment of the clock to internal and external stimuli. Previous studies have highlighted a critical role for ELF3 in repressing the expression of PRR9 and PRR7. However, the functional significance of activity in regulating circadian clock dynamics and plant development is unknown. To explore this regulatory dynamic further, we firstly employed mathematical modelling to simulate the effect of the prr9/prr7 mutation on the elf3 circadian phenotype. These simulations suggested that simultaneous mutations in prr9/prr7 could rescue the elf3 circadian arrythmia. Following these simulations, we generated all Arabidopsis elf3/prr9/prr7 mutant combinations and investigated their circadian and developmental phenotypes. Although these assays could not replicate the results from the mathematical modelling, our results have revealed a complex epistatic relationship between ELF3 and PRR9/7 in regulating different aspects of plant development. ELF3 was essential for hypocotyl development under ambient and warm temperatures, while PRR9 was critical for root thermomorphogenesis. Finally, mutations in prr9 and prr7 rescued the photoperiod insensitive flowering phenotype of the elf3 mutant. Together, our results highlight the importance of investigating the genetic relationship amongst plant circadian genes.

plant biology↗