Biology subjects

Robinson, S.

Publications and source records attributed to Robinson, S..

31 records · Page 2Linked to original sources

bioRxiv2023

Pectin methylesterification modulates cell wall properties to promote neighbour proximity-induced hypocotyl growth

Plants growing with neighbours compete for light and consequently increase growth of their vegetative organs to enhance access to sunlight. This response, called shade avoidance syndrome (SAS), involves photoreceptors such as phytochromes as well as phytochrome interacting factors (PIFs), which regulate the expression of growth-mediating genes. Numerous cell wall-related genes belong to the putative targets of PIFs, and the importance of cell wall modifications for enabling growth was extensively shown in developmental models such as dark-grown hypocotyl. However, the role of the cell wall in the growth of de-etiolated seedlings regulated by shade cues remains poorly established. Through analyses of mechanical and biochemical properties of the cell wall coupled with transcriptomic analysis of cell wall-related genes, we show the importance of cell wall modifications in neighbour proximity-induced elongation. Further analysis using loss-of-function mutants impaired in the synthesis and remodeling of the main cell wall polymers corroborated this. We focused on the cgr2cgr3 double mutant that is defective in homogalacturonan (HG) methyltransferase activity required for methylesterification of HG-type pectins. By following hypocotyl growth kinetically and spatially and analyzing the mechanical and biochemical properties of cell walls, we found that methylesterification of HG-type pectins was required to enable global cell wall modifications. Moreover, HG-class pectin modification was needed for plant competition-induced hypocotyl growth. Collectively our work suggests that in the hypocotyl PIFs orchestrate changes in the expression of numerous cell wall genes to enable neighbour proximity-induced growth. One sentence summaryThe degree of methylesterification of pectins modulates global changes in the cell wall and its mechanical properties that contribute to the neighbour proximity-induced hypocotyl growth in Arabidopsis

plant biology↗

bioRxiv2021

Loss of TREM2 reduces hyperactivation of progranulin deficient microglia but not lysosomal pathology

GRN haploinsufficiency causes frontotemporal lobar degeneration and results in microglial hyperactivation, lysosomal dysfunction and TDP-43 deposition. To understand the contribution of microglial hyperactivation to pathology we evaluated genetic and pharmacological approaches suppressing TREM2 dependent transition of microglia from a homeostatic to a disease associated state. Trem2 deficiency in Grn KO mice led to a reduction of microglia activation. To explore antibody-mediated pharmacological modulation of TREM2-dependent microglial states, we identified antagonistic TREM2 antibodies. Treatment of macrophages from GRN-FTLD patients with these antibodies allowed a complete rescue of elevated levels of TREM2 together with increased shedding and reduction of TREM2 signaling. Furthermore, antibody-treated PGRN deficient hiMGL showed dampened microglial hyperactivation, reduced TREM2 signaling and phagocytic activity, however, lack of rescue of lysosomal dysfunction. Similarly, lysosomal dysfunction, lipid dysregulation and glucose hypometabolism of Grn KO mice were not rescued by TREM2 ablation. Furthermore, NfL, a biomarker for neurodegeneration, was elevated in the Grn/Trem2 KO. These findings suggest that microglia hyperactivation is not necessarily contributing to neurotoxicity, and instead demonstrates that TREM2 exhibits neuroprotective potential in this model.

neuroscience↗

bioRxiv2020

Drug screening linked to molecular profiling identifies novel dependencies in patient-derived primary cultures of paediatric high grade glioma and DIPG

Paediatric high grade glioma and diffuse midline glioma (including DIPG) are comprised of multiple biological and clinical subgroups, the majority of which urgently require novel therapies. Patient-derived in vitro primary cell cultures represent potentially useful tools for mechanistic and preclinical investigation based upon their retention of key features of tumour subgroups under experimental conditions amenable to high-throughput approaches. We present 17 novel primary cultures derived from patients in London, Dublin and Belfast, and together with cultures established or shared from Barcelona, Brisbane, Rome and Stanford, assembled a panel of 52 models under 2D (laminin matrix) and/or 3D (neurospheres) conditions, fully credentialed by phenotypic and molecular comparison to the original tumour sample (methylation BeadArray, panel/exome sequencing, RNAseq). In screening a subset of these against a panel of ~400 approved chemotherapeutics and small molecules, we identified specific dependencies associated with tumour subgroups and/or specific molecular markers. These included MYCN-amplified cells and ATM/DNA-PK inhibitors, and DIPGs with PPM1D activating truncating mutations and inhibitors of MDM2 or PARP1. Specific mutations in PDGFRA were found to confer sensitivity to a range of RTK inhibitors, though not all such mutations conferred sensitivity to targeted agents. Notably, dual PDGFRA/FGFR and downstream pathway MEK inhibitors showed profound effects against both PDGFRA-sensitising mutant and FGFR1-dependent non-brainstem pHGG and DIPG. In total, 85% cells were found to have at least one drug screening hit in short term assays linked to the underlying biology of the patients tumour, providing a rational approach for individualised clinical translation.

cancer biology↗

bioRxiv2020

The allosteric modulation of Complement C5 by knob domain peptides

To overcome limited germline combinatorial diversity, bovines have evolved a subset of antibodies with ultra-long CDRH3 regions that harbour cysteine-rich knob domains. To produce affinity-maturated peptides, we previously isolated autonomous 3-6 kDa knob domains from bovine antibodies. Here, we show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Allosteric mechanisms predominated, with one peptide selectively inhibiting C5 cleavage by the alternative pathway C5 convertase, revealing a targetable mechanistic difference between the classical and alternative pathway C5 convertases. Taking a hybrid biophysical approach, we present C5-knob domain co-crystal structures and, by solution methods, observed allosteric effects propagating >50 [A] from the binding sites. This study expands the therapeutic scope of C5, presents new inhibitors and introduces knob domains as new, low molecular weight antibody fragments, with therapeutic potential.

immunology↗

bioRxiv2020

Genomic asymmetry of the Brassica napus seed: Epigenetic contributions of DNA methylation and small RNAs to subgenome bias

Polyploidy has predominated the genetic history of the angiosperms, and allopolyploidy is known to have contributed to the vast speciation of flowering plants. Brassica napus, one of the worlds most important oilseeds, is one such polyploid species originating from the interspecific hybridization of Brassica rapa (An) and Brassica oleracea (Cn). Nascent amphidiploids must balance progenitor genomes during reproduction, though the role of epigenetic regulation in subgenome maintenance is unknown. The seed is the pivotal developmental transition into the new sporophytic generation and as such undergoes substantial epigenetic modifications. We investigated subgenome bias between the An and Cn subgenomes as well as across syntenic regions by profiling DNA methylation and siRNAs characteristic of B. napus seed development. DNA methylation and siRNA accumulation were prevalent in the Cn subgenome and most pronounced early during seed morphogenesis. Hypermethylation during seed maturation was most pronounced on non-coding elements, including promoters, repetitive elements, and siRNAs. Methylation on siRNA clusters was more prevalent in syntenic regions of the Cn subgenome and implies selective silencing of genomic loci of the seed. Together, we find compelling evidence for the asymmetrical epigenetic regulation of the An and Cn subgenomes of Brassica napus across seed development.

plant biology↗

bioRxiv2020

Gene expression profiling reveals subgenome dominance during Brassica napus seed development

We profiled the gene regulatory landscape of Brassica napus reproductive development using RNA sequencing. Comparative analysis of this nascent amphidiploid across the plant lifecycle revealed the contribution of each subgenome to plant reproduction. Global mRNA profiling revealed lower accumulation of Cn subgenome transcripts relative to the An subgenome. Subgenome-specific transcriptional networks identified distinct transcription factor families enriched in each of the An and Cn subgenome early in seed development. Global gene expression profiling of laser-microdissected seed subregions further reveal subgenome expression dynamics in the embryo, endosperm, and seed coat of early stage seeds. Transcription factors predicted to be regulators encoded by the An subgenome are expressed primarily in the seed coat whereas regulators encoded by the Cn subgenome were expressed primarily in the embryo. Data suggest subgenome bias are characteristic features of the B. napus seed throughout development, and that such bias might not be universal across the embryo, endosperm, and seed coat of the developing seed. Whole genome transcription factor networks identified BZIP11 as a transcriptional regulator of early B. napus seed development. Knockdown of BZIP11 using RNA interference resulted in a similar reduction in gene activity of predicted gene targets, and a reproductive-lethal phenotype. Taken together, transcriptional networks spanning both the An and Cn genomes of the B. napus seed can identify valuable targets for seed development research and that-omics level approaches to studying gene regulation in B. napus can benefit from both broad and high-resolution analyses. One Sentence SummaryGlobal RNA sequencing coupled with laser microdissection provides a critical resource to study subgenome bias in whole seeds and specific tissues of polyploid plants.

plant biology↗

bioRxiv2019

Lipid metabolism links nutrient-exercise timing to insulin sensitivity in men classified as overweight or obese.

ContextPre-exercise nutrient availability alters acute metabolic responses to exercise, which could modulate training responsiveness. We hypothesised that in men with overweight/obesity, acute exercise before versus after nutrient ingestion would increase whole-body and intramuscular lipid utilization, translating into greater increases in oral glucose insulin sensitivity over 6-weeks of training.\n\nDesign and ParticipantsWe showed in men with overweight/obesity (mean{+/-}SD for BMI: 30.2{+/-}3.5 kgxm-2 for acute, crossover study, 30.9{+/-}4.5 kgxm-2 for randomized, controlled, training study) a single exercise bout before versus after nutrient provision increased lipid utilisation at the whole-body level, but also in both type I (p<0.01) and type II muscle fibres (p=0.02). We then used a 6-week training intervention to show sustained, 2-fold increases in lipid utilisation with exercise before versus after nutrient provision (p<0.01).\n\nMain Outcome MeasuresPostprandial glycemia was not differentially affected by exercise training before vs after nutrient provision (p>0.05), yet plasma was reduced with exercise training before, but not after nutrient provision (p=0.03), resulting in increased oral glucose insulin sensitivity when training was performed before versus after nutrient provision (25{+/-}38 vs -21{+/-}32 mLxmin-1xm-2; p=0.01) and this was associated with increased lipid utilisation during exercise (r=0.50, p=0.02). Regular exercise prior to nutrient provision augmented remodelling of skeletal muscle phospholipids and protein content of the glucose transport protein GLUT4 (p<0.05).\n\nConclusionsExperiments investigating exercise training and metabolic health should consider nutrient-exercise timing, and exercise performed before versus after nutrient intake (i.e., in the fasted state) may exert beneficial effects on lipid utilisation and reduce postprandial insulinemia.\n\nPrecisExercise in the fasted-versus fed-state increased intramuscular and whole-body lipid use, translating into increased muscle adaptation and insulin sensitivity when regularly performed over 6 weeks.

physiology↗

bioRxiv2018

A novel bioreactor technology for modelling fibrosis in human and rodent precision-cut liver slices.

Summary boxO_LIWhat is already known about this subject?\nO_LICurrently there are no effective anti-fibrotic drugs to treat liver fibrosis and there is an urgent unmet need to increase our knowledge of the disease process and develop better tools for anti-fibrotic drug discovery.\nC_LIO_LIPreclinical in vitro cell cultures and animal models are widely used to study liver fibrosis and test anti-fibrotic drugs, but have shortfalls; cell culture models lack the relevant complex cell-cell interactions of the liver and animal models only reproduce some features of human disease.\nC_LIO_LIPrecision Cut Liver Slices (PCLS) are structurally representative of the liver and can be used to model liver fibrosis and test anti-fibrotic drugs. However, PCLS are typically cultured in elevated, non-physiological oxygen levels and only have a healthy lifespan of 48h.\nC_LI\nC_LIO_LIWhat are the new findings?\nO_LIWe have developed a novel bioreactor culture system that increases the longevity of functional PCLS to up to 6 days under normoxic conditions.\nC_LIO_LIBioreactor cultured PCLS can be used to model fibrogenesis in both normal and fibrotic PCLS using a combination of biochemical and histological outputs.\nC_LIO_LIAdministration of an Alk5 inhibitor effectively limits fibrogenesis in normal rodent and human PCLS and in rodent PCLS with established fibrosis.\nC_LI\nC_LIO_LIHow might it impact on clinical practice in the foreseeable future?\nO_LIThe extended longevity of bioreactor cultured PCLS represent a novel pre-clinical tool to investigate the cellular and molecular mechanisms of liver fibrosis.\nC_LIO_LIBioreactor cultured human PCLS offer a clinically relevant system to test efficacy of anti-fibrotic drugs.\nC_LI\nC_LI\n\nAbstractO_ST_ABSObjectiveC_ST_ABSPrecision cut liver slices (PCLS) retain the structure and cellular composition of the native liver and represent an improved system to study liver fibrosis compared to two-dimensional mono or co-cultures. The objective of this study was to develop a bioreactor system to increase the healthy lifespan of PCLS and model fibrogenesis.\n\nDesignPCLS were generated from normal rat or human liver, or 4-week carbon tetrachloride-fibrotic rat liver and cultured in our patented bioreactor. PCLS function was quantified by albumin ELISA. Fibrosis was induced in PCLS by TGF{beta}1 and PDGF{beta}{beta} stimulation. Alk5 inhibitor therapy was used. Fibrosis was assessed by fibrogenic gene expression, Picrosirius Red and Smooth Muscle Actin staining, hydroxyproline assay and collagen 1a1, fibronectin and hyaluronic acid ELISA.\n\nResultsBioreactor cultured PCLS are viable, maintaining tissue structure and stable albumin secretion for up to 6 days under normoxic culture conditions. Conversely, standard static transwell cultured PCLS rapidly deteriorate and albumin secretion is significantly impaired by 48 hours. TGF{beta}1 and PDGF{beta}{beta} stimulation of rat or human PCLS induced fibrogenic gene expression, release of extracellular matrix proteins, activation of hepatic myofibroblasts and histological fibrosis. Fibrogenesis slowly progresses over 6-days in cultured fibrotic rat PCLS without exogenous challenge. Alk5 inhibitor limited fibrogenesis in both TGF{beta}1 and PDGF{beta}{beta} stimulated PCLS and fibrotic PCLS.\n\nConclusionWe describe a new bioreactor technology which maintains functional PCLS cultures for 6 days. Bioreactor cultured PCLS can be successfully used to model fibrogenesis and demonstrate efficacy of an anti-fibrotic therapy.

cell biology↗

bioRxiv2018

The Atypical Rho GTPase CHW-1 Works With SAX-3/Robo to Mediate Axon Guidance in Caenorhabditis elegans

During development, neuronal cells extend an axon towards their target destination in response to a cue to form a properly functioning nervous system. Rho proteins, Ras-related small GTPases that regulate cytoskeletal organization and dynamics, cell adhesion, and motility, are known to regulate axon guidance. Despite extensive knowledge about canonical Rho proteins (RhoA/Rac1/Cdc42), little is known about the Caenorhabditis elegans (C. elegans) atypical Cdc42-like family members CHW-1 and CRP-1 in regards to axon pathfinding and neuronal migration. chw-1(Chp/Wrch) encodes a protein that resembles human Chp (Wrch-2/RhoV) and Wrch-1 (RhoU), and crp-1 encodes for a protein that resembles TC10 and TCL. Here, we show that chw-1 works redundantly with crp-1 and cdc-42 in axon guidance. Furthermore, proper levels of chw-1 expression and activity are required for proper axon guidance. When examining CHW-1 GTPase mutants, we found that the native CHW-1 protein is likely partially activated, and mutations at a conserved residue (position 12 using Ras numbering, position 18 in CHW-1) alter axon guidance and neural migration. Additionally, we showed that chw-1 genetically interacts with the guidance receptor sax-3 in PDE neurons. Finally, in VD/DD motor neurons, chw-1 works downstream of sax-3 to control axon guidance. In summary, this is the first study implicating the atypical Rho GTPases chw-1 and crp-1 in axon guidance. Furthermore, this is the first evidence of genetic interaction between chw-1 and the guidance receptor sax-3. These data suggest that chw-1 is likely acting downstream and/or in parallel to sax-3 in axon guidance.

developmental biology↗

bioRxiv2018

Asymmetric effects of environmental filtering on the assembly of tropical bird communities along a moisture gradient

The species-sorting hypothesis (SSH) states that environmental factors influence local community assembly of metacommunities by selecting for species that are well adapted to the specific conditions of each site. Along environmental gradients, the strength of selection against individuals that are marginally adapted to the local conditions increases towards the extremes of the environment where the climate becomes harsh. In rainfall gradients, the strength of selection by the environment has been proposed to decrease with rainfall. Under this scenario SSH would predict that immigration of individuals from the metacommunity should be restricted into the dry end of the gradient creating a positive relationship between immigration and rainfall. However, if the selection is strong in both ends of the gradient, the restriction should be expected to be in both directions such that the ends behave as independent metacommunities even in the absence of geographical barriers. In this study we used models based on neutral theory to evaluate if SSH can explain the distribution of bird species along a steep rainfall gradient in Colombia. We found a strong positive relationship between immigration rates and precipitation suggesting that the dry forests impose stronger challenges for marginally adapted bird populations. However, a two-metacommunity model separating dry and wet forests was a better fit to the observed data, suggesting that both extremes impose strong selection against immigrants. The switch from the dry forest to the wet forest metacommunities occurred abruptly over a short geographic distance in the absence of any apparent geographic barrier; this apparent threshold occurs where the forest becomes mostly evergreen. The relative number of rare species in dry forest was lower than in wet forests suggesting that the selection against marginally adapted populations is stronger in the dry forests. Overall, our analyses are consistent with SSH at the regional scales, but the rarity analysis suggests that the mechanisms at the local scales are substantially different. Based on these results, we hypothesize that abiotic (climatic) factors limit immigration into dry forest communities and whereas biotic factors such as competition and predation may limit immigration into bird communities in the wet forest.

ecology↗

bioRxiv2018

Climatic effects on turnover of lowland forest bird communities across a precipitation gradient

One of the main goals of community ecology is to understand the influence of the abiotic environment on the abundance and distribution of species. It has been hypothesized that dry forests are harsher environments than wet forests, which leads to the prediction that environmental filtering should be a more important determinant of patterns of species abundance and composition than in wet forest, where biotic interactions or random assembly should be more important. We attempt to understand the influence of rainfall on the abundance and distribution of bird species along a steep precipitation gradient in an inter-Andean valley in Colombia. We gathered data on species distributions, abundance, morphological traits and phylogenetic relationships to determine the influence of rainfall on the taxonomic, functional and phylogenetic turnover of species along the Magdalena Valley. We demonstrate that there is a strong turnover of community composition at the limit of the dry forest. The taxonomic turnover is steeper than the phylogenetic turnover, suggesting that replacement of closely related species accounts for a disproportionate number of changes along the gradient. We found evidence for environmental filtering in dry forest as species tend to be more tolerant of higher temperature ranges, stronger rainfall seasonality and lower minimum rainfall. On the other hand, wet forest species tend to compete actively for nest space but not for the resources associated with the axes we measured. Our results suggest that rainfall is a strong determinant of community composition when comparing localities above and below the 2400 mm rainfall isocline.

ecology↗

bioRxiv2017

Gene expression dynamics and inter-tissue recognition mechanisms during tissue fusion at the Arabidopsis graft junction

The ability for cut tissues to join together and form a chimeric organism is a remarkable property of many plants, however, grafting is poorly characterized at the molecular level. To better understand this process we monitored genome-wide temporal and spatial gene expression changes in grafted Arabidopsis thaliana hypocotyls. Tissues above and below the graft rapidly developed an asymmetry such that many genes were more highly expressed on one side than the other. This asymmetry correlated with sugar responsive genes and we observed an accumulation of starch above the graft that decreased along with asymmetry once the sugar-transporting vascular tissues reconnected. Despite the initial starvation response below the graft, many genes associated with vascular formation were rapidly activated in grafted tissues but not in cut and separated tissues indicating that a recognition mechanism activated that was independent of functional vascular connections. Auxin which is transported cell-to-cell, had a rapidly elevated response that was symmetric, suggesting that auxin was perceived by the root within hours of tissue attachment to activate the vascular regeneration process. A subset of genes were expressed only in grafted tissues, indicating that wound healing proceeded via different mechanisms depending on the presence or absence of adjoining tissues. Such a recognition process could have broader relevance for tissue regeneration, inter-tissue communication and tissue fusion events.

plant biology↗

bioRxiv2017

An automated confocal micro-extensometer enables in vivo quantification of mechanical properties with cellular resolution.

How complex developmental-genetic networks are translated into organs with specific 3D shapes remains an open question. This question is particularly challenging because the elaboration of specific shapes is in essence a question of mechanics. In plants, this means how the genetic circuitry affects the cell wall. The mechanical properties of the wall and their spatial variation are the key factors controlling morphogenesis in plants. However, these properties are difficult to measure and investigating their relation to genetic regulation is particularly challenging. To measure spatial variation of mechanical properties, one must determine the deformation of a tissue in response to a known force with cellular resolution. Here we present an automated confocal micro-extensometer (ACME), which greatly expands the scope of existing methods for measuring mechanical properties. Unlike classical extensometers, ACME is mounted on a confocal microscope and utilizes confocal images to compute the deformation of the tissue directly from biological markers, thus providing cellular scale information and improved accuracy. ACME is suitable for measuring the mechanical responses in live tissue. As a proof of concept we demonstrate that the plant hormone gibberellic acid induces a spatial gradient in mechanical properties along the length of the Arabidopsis hypocotyl.\n\nTerms

plant biology↗