Biology subjects

Hill, L.

Publications and source records attributed to Hill, L..

10 recordsLinked to original sources

bioRxiv2026

AMBER and GOLD: Polycistronic Genes for Betaxanthin Production in Plants

Genetically encoded pigments are powerful visual reporters and creative tools for biology, yet in plants the palette of pigment biosynthesis genes has remained largely limited to red betacyanins encoded by RUBY. Here we develop and characterize three new polycistronic constructs AMBER_v1, AMBER_v2, and GOLD that contain betalain biosynthesis enzymes to produce yellow, fluorescent betaxanthins in plant tissues. These tools expand the palette of publicly available pigmentation genes for use in plant research, education, and floral design.

bioengineering↗

bioRxiv2025

Adult organotypic brain slice cultures recapitulate extracellular matrix remodelling in haemorrhagic stroke

1.Haemorrhagic stroke is a devastating condition characterised by vessel rupture and free blood within the brain parenchyma or cerebrospinal fluid (CSF) filled spaces. Across the major subtypes of haemorrhagic stroke (subarachnoid, intracerebral, and intraventricular haemorrhages), the presence of blood in the CSF generates significant tissue damage in the first 72 hours after the event, known as early brain injury (EBI). EBI includes neuroinflammation, blood-brain barrier breakdown and dysregulation of extracellular matrix (ECM) dynamics. ECM dysfunction has been shown to trigger fibrosis of the cortical blood vessels, limiting normal CSF circulation and resulting in the buildup of metabolic waste or the development of post-haemorrhagic hydrocephalus. Limiting or preventing this fibrosis may therefore reduce the rate of morbidity experienced by survivors, providing a potential avenue for non-surgical treatment to reduce secondary brain injury post-stroke. Despite this, current in vivo approaches fail to differentiate between the effect of blood products and secondary consequences including intracranial pressure (ICP) elevation and mass effect. Here, we describe an adult rat organotypic brain slice culture (OBSC) model of haemorrhagic stroke which enables the identification of the effect of blood products on ECM dysregulation. We demonstrate the distribution of key cell types across a time course of 0, 3 and 7 days in culture, indicating that such cultures are viable for a minimum of 7 days. Using immunofluorescence staining, Western blotting and RNA sequencing, we show that exposure of OBSCs to lysed blood markedly increases ECM deposition around cortical blood vessels. This is accompanied by dysregulation of ECM regulatory genes and upregulation of inflammation and oxidative stress-related genes, successfully recapitulating the changes seen in human stroke survivors. This versatile ex vivo model provides a translational platform to further understanding of haemorrhagic stroke pathophysiology and develop or trial novel therapeutics prior to progression to in vivo stroke studies.

neuroscience↗

bioRxiv2025

M18BP1 valency and a distributed interaction footprint determine epigenetic centromere specification in humans

Deposition of CENP-A, the histone H3 variant considered an epigenetic landmark of centromeres, reflects cell-cycle-regulated assembly of M18BP1, HJURP, and PLK1 on a divalent MIS18/{beta} scaffold. The localization determinants of this machinery remain poorly characterized. Here we report that artificial M18BP1 dimerization bypasses MIS18/{beta}, allowing the identification of at least four determinants of M18BP1 centromere localization. These include the SANTA domain, of which we report the first structure, as well as linear motifs in disordered neighboring regions, of which we characterize the interaction footprint on the CENP-A associated 16-subunit constitutive centromere-associated network (CCAN). Our observations imply that M18BP1, after dimerization, is necessary and sufficient for centromere localization. Its cell-cycle-dependent dimerization on MIS18/{beta} promotes initial recognition of a multivalent centromeric assembly of old CENP-A and associated proteins, followed by cooption of PLK1 and HJURP and new CENP-A deposition. Our results shed new light on the determinants of centromere epigenetic inheritance in humans.

cell biology↗

bioRxiv2025

Pollinator-relevant floral traits underlie bidirectional hybridisation in the orchid genus Gymnadenia

Reproductive isolation is mediated by ecological, phenotypic, and genetic barriers. Where such barriers are not upheld, hybridisation can occur, revealing insight about the formation of species. Here, we provide an integrative investigation of hybrid formation between the orchids Gymnadenia conopsea and G. rhellicani and potential isolating barriers in zones of overlap in the European Alps. We examine opportunities for hybridisation by quantifying hybrid frequency and pollinator assemblages of parent species. Next, we characterise floral phenotypes and genetic parentage of hybrids to examine how genetic inheritance of traits shapes ecological interactions, and thus fitness, of hybrids. We find that pollinator assemblages of parents overlap partially, consistent with observed rare hybrids (<1% of the population). Phenotypically, hybrids were intermediate across floral traits, consistent with all hybrids being identified as F1s, resulting from a primary hybridisation event between G. conopsea and G. rhellicani. No evidence of backcrossing or introgression was found, but direction of pollen transfer varied across sites. Additionally, hybrids were heterozygous for parental alleles at two loci impacting anthocyanin production, suggesting hybrids intermediate floral colour reflects inheritance of these alleles. Finally, we find little support that ploidy variation or hybrid seed infertility explains the absence of advanced generations of hybrids. Thus, we suggest that floral isolation represents an important isolating mechanism between G. conopsea and G. rhellicani in limiting the formation of uncommon, unfit hybrids and shaping species boundaries.

plant biology↗

bioRxiv2024

The impact of transplant location on the gut microbiome and resistome in patients undergoing hematopoietic stem cell transplantation at home versus in the hospital

ObjectivesHome-based hematopoietic stem cell transplantation (HCT) is a novel approach that has the potential to improve outcomes, however, the impact of transplant location on the gut microbiome remains uncharacterized. We hypothesized that patients randomized to undergo home HCT would have higher gut taxonomic diversity and lower antimicrobial resistance (AMR) gene abundance compared to those undergoing standard hospital HCT. MethodsWe identified 28 patients enrolled in Phase II randomized trials of home (n=16) v. hospital (n=12) HCT at Duke and performed shotgun metagenomic sequencing of stools to compare taxonomic and AMR gene composition between groups. We performed a secondary analysis of patients from each group transplanted at an outpatient infusion clinic with those who underwent standard inpatient HCT ("outpatient" v. "inpatient"). ResultsNo significant differences in duration of hospitalization were found in those randomized to home v. hospital HCT. Taxonomic and AMR gene - and {beta}-diversity were comparable. In contrast, secondary analyses demonstrated that patients from both home and hospital groups transplanted at an outpatient infusion clinic spent significantly less time in the hospital and demonstrated higher taxonomic -diversity and differential {beta}-diversity compared to standard inpatient HCT, although AMR gene -diversity did not differ, and comparisons were confounded by both differences in transplant type and use of antibiotics. ConclusionsRandomization by transplant location did not impact the gut microbiota to the same extent as the duration of hospitalization, although secondary analyses were heavily confounded. Even when taxonomic differences were observed, AMR genes were similar between groups.

microbiology↗

bioRxiv2024

Integration of light quality signals regulates ABA abundance and stomatal movements during seedling establishment

Obtaining sufficient light for photosynthesis and avoiding desiccation are two key challenges faced by seedlings during early establishment. Perception of light quality via specialised photoreceptors signals the availability of sunlight for photosynthesis. Canopy shade is depleted in red (R) and enriched in far-red (FR) light, lowering R:FR ratio, while direct sunlight and sunflecks contain UV-B. The balance of these wavelengths can determine the developmental strategy adopted by seedlings to either avoid shade, via stem elongation, or promote the expansion of photosynthetic organs. How seedlings regulate stomatal movements in different light environments is poorly understood. Using FR and UV-B supplementation to mimic aspects of canopy shade and sunlight respectively, we monitored stomatal apertures in Arabidopsis thaliana cotyledons. We show that low R:FR inhibits stomatal opening via a mechanism involving PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and increased abscisic acid (ABA). In contrast, UV-B perceived by the UV RESISTANCE LOCUS 8 (UVR8) photoreceptor antagonises this response to promote stomatal opening in a response requiring phototropin photoreceptors. The convergence of phytochrome and UVR8 signalling to control ABA abundance enables plants to coordinate stem elongation and water use during seedling establishment in dynamic light environments.

plant biology↗

bioRxiv2024

A proposed new Tombusviridae genus featuring extremely long 5 prime untranslated regions and a luteo/polerovirus-like gene block

SummaryTombusviridae is a large family of single-stranded, positive-sense RNA plant viruses with uncapped, non-polyadenylated genomes encoding 5-7 open reading frames (ORFs). Previously, we discovered, by high-throughput sequencing of maize and teosinte RNA, a novel genome of a virus we call Maize-associated tombusvirus (MaTV). Here we determined the precise termini of the MaTV genome by using 5 and 3 rapid amplification of cDNA ends (RACE). In GenBank, we discovered eleven other nearly complete viral genomes with MaTV-like genome organizations and related RNA-dependent RNA polymerase (RdRp) sequences. These genomes came from diverse plant, fungal, invertebrate and vertebrate organisms, and some have been found in multiple organisms across the globe. The available 5 untranslated regions (UTRs) of these genomes are remarkably long: at least 438 to 727 nucleotides (nt), in contrast to those of other tombusvirids, which are <150 nt. Moreover these UTRs contain 6 to 12 AUG triplets that are unlikely to be start codons, because - with the possible exception of MaTV - there are no large or conserved ORFs in the 5 UTRs. Such features suggest an internal ribosome entry site (IRES), but we found no conserved secondary structures. In the 50 nt upstream of and adjacent to the ORF1 start codon, the 5 UTR was cytosine-rich and guanosine-poor. As in most tombusvirids, ORF2 (RdRp gene) appears to be translated by in-frame ribosomal readthrough of the ORF1 stop codon. Indeed, in all twelve genomes we identified RNA structures known in other tombusviruses to facilitate this readthrough. ORF5 is predicted to be translated by readthrough of the ORF3 (coat protein gene) stop codon as in genus Luteovirus. The resulting readthrough domains are highly divergent. ORF4 overlaps with ORF3 and may initiate with a non-AUG start codon. We also found no obvious 3 cap-independent translation elements, which are present in other tombusvirids. The twelve genomes diverge sufficiently from other tombusvirids to warrant classification in a new genus. Because they contain two leaky stop codons and a potential leaky start codon, we propose to name this genus Rimosavirus (rimosa = leaky in Latin).

microbiology↗

bioRxiv2024

Synovial Sarcoma Chromatin Dynamics Reveal a Continuum in SS18:SSX Reprograming

Synovial sarcoma (SyS) is an aggressive soft-tissue malignancy characterized by a pathognomonic chromosomal translocation leading to the formation of the SS18::SSX fusion oncoprotein. SS18::SSX associates with mammalian BAF complexes suggesting deregulation of chromatin architecture as the oncogenic driver in this tumour type. To examine the epigenomic state of SyS we performed comprehensive multi-omics analysis on 52 primary pre-treatment human SyS tumours. Our analysis revealed a continuum of epigenomic states across the cohort at fusion target genes independent of rare somatic genetic lesions. We identify cell-of-origin signatures defined by enhancer states and reveal unexpected relationships between H2AK119Ub1 and active marks. The number of bivalent promoters, dually marked by the repressive H3K27me3 and activating H3K4me3 marks, has strong prognostic value and outperforms tumor grade in predicting patient outcome. Finally, we identify SyS defining epigenomic features including H3K4me3 expansion associated with striking promoter DNA hypomethylation in which SyS displays the lowest mean methylation level of any sarcoma subtype. We explore these distinctive features as potential vulnerabilities in SyS and identify H3K4me3 inhibition as a promising therapeutic strategy.

cancer biology↗

bioRxiv2023

Complex petal spot formation in the Beetle Daisy (Gorteria diffusa) relies on spot-specific accumulation of malonylated anthocyanin regulated by paralogous GdMYBSG6 transcription factors

Gorteria diffusa has elaborate petal spots that attract male bee-fly pollinators through sexual deception but the genetic basis of G. diffusa petal spot development is currently unknown. Here we investigate the regulation of pigmentation during spot formation. We used UHPLC-MS/MS to determine the anthocyanin composition of spots and background pigmentation in G. diffusa. Combining gene expression analysis with protein interaction assays we characterised three R2R3-MYB genes regulating anthocyanin production in G. diffusa spots. We found that cyanidin 3-glucoside pigments G. diffusa ray floret petals. Unlike other petal regions, spots contain a high proportion of malonylated anthocyanin. We identified three paralogous subgroup 6 R2R3-MYB transcription factors that activate the production of petal spot pigmentation. The corresponding genes are upregulated in developing spots and induce ectopic anthocyanin production upon heterologous expression in tobacco. EMSAs and dual luciferase assays suggest that these transcription factors regulate genes encoding three anthocyanin synthesis enzymes: anthocyanidin synthase (GdANS), dihydroflavonol reductase (GdDFR) and malonyl transferase (GdMAT1), accounting for the spot-specific production of malonylated pigments. Here we provide the first molecular characterisation of G. diffusa spot development, showing that the elaboration of complex spots begins with accumulation of malonylated pigments at the base of ray floret petals, positively regulated by three subgroup 6 R2R3-MYB transcription factors.

plant biology↗

bioRxiv2017

Genetic dissection of cyclic pyranopterin monophosphate biosynthesis in plant mitochondria

Mitochondria play a key role in the biosynthesis of two metal cofactors, iron-sulfur (FeS) clusters and molybdenum cofactor (Moco). The two pathways intersect at several points, but a scarcity of mutants has hindered studies to better understand these links. We screened a collection of sirtinol-resistant Arabidopsis thaliana mutants for lines with decreased activities of cytosolic FeS enzymes and Moco enzymes. We identified a new mutant allele of ATM3, encoding the ATP-binding cassette Transporter of the Mitochondria 3 (systematic name ABCB25), confirming the previously reported role of ATM3 in both FeS cluster and Moco biosynthesis. We also identified a mutant allele in CNX2, Cofactor of Nitrate reductase and Xanthine dehydrogenase 2, encoding GTP 3',8-cyclase, the first step in Moco biosynthesis which is localized in the mitochondria. A single nucleotide polymorphism in cnx2-2 leads to substitution of Arg88 with Gln in the N-terminal FeS cluster-binding motif. cnx2-2 plants are small and chlorotic, with severely decreased Moco enzyme activities, but they performed better than a cnx2-1 knockout mutant, which could only survive with ammonia as nitrogen source. Measurement of cyclic pyranopterin monophosphate (cPMP) levels by LC-MS/MS showed that this Moco intermediate was below the limit of detection in both cnx2-1 and cnx2-2, and accumulated more than 10-fold in seedlings mutated in the downstream gene CNX5. Interestingly, atm3-1 mutants had less cPMP than wild type, correlating with previous reports of a similar decrease in nitrate reductase activity. Taken together, our data functionally characterise CNX2 and suggest that ATM3 is indirectly required for cPMP synthesis.

biochemistry↗