Biology subjects

Gilbert, J. D.

Publications and source records attributed to Gilbert, J. D..

11 recordsLinked to original sources

bioRxiv2026

Native Mass Spectrometry Reveals Distinct Effects of Linker Length and Targeting-Ligand Identity on PROTAC Ternary Complexes

Proteolysis-targeting chimeras (PROTACs) promote targeted protein degradation by inducing a transient ternary complex between a protein of interest and an E3 ubiquitin ligase. Despite the clinical promise of these event-driven therapeutics, defining the conformational landscape and interfacial organization of these complexes remains challenging. Here, we use an integrated native mass spectrometry (nMS) approach combining ion mobility, surface-induced dissociation (SID), and variable-temperature electrospray ionization (vT-ESI) to determine how small-molecule architecture shapes the structural and thermodynamic landscapes of bromodomain-containing protein 4 (BRD4) with the VCB E3 ligase complex. Using a set of PROTACs that differ in targeting ligand identity (JQ1 versus I-BET726) and PEG linker length, ion mobility measurements show that shorter linkers constrain the ternary complex into more extended topologies, whereas longer, more flexible linkers allow dynamic sampling of relaxed, native-like conformations. SID directly probes noncovalent interfaces and shows that JQ1 complexes favor prompt PROTAC ejection, whereas tighter-binding I-BET726 complexes are more likely to retain the PROTAC at the interface. Complementing these kinetically-controlled gas-phase measurements, vT-ESI reveals a monophasic melting transition for a JQ1-mediated complex and a distinct biphasic melting profile for an I-BET726-mediated complex. The gas-phase activation measurements revealed targeting-ligand-driven differences in PROTAC ejection and retention, whereas vT-ESI uncovered distinct temperature-dependent disassembly pathways for the matched-linker-length JQ10L and IBET10L complexes. Together, these complementary techniques show how PROTAC architecture influences ternary-complex conformation, interfacial connectivity, and disassembly.

biochemistry↗

bioRxiv2026

Structural evolution of a yeast amyloid in vivo is shaped by chaperones

Cryo-electron microscopy (cryo-EM) studies of amyloid fibrils have revealed endpoint structures of disease-relevant filaments and polymorphic intermediates formed during in vitro assembly of prion-like proteins. However, how transmissible prion or prion-like amyloids evolve during de novo formation and maturation in living cells remains unknown. Here, using the yeast prion [PSI+] as a model, we isolated Sup35NM amyloid fibrils from successive stages of [PSI+] maturation in Saccharomyces cerevisiae and characterised their near-atomic structures and population-level structural diversity by combining cryo-EM and atomic force microscopy. We show that intermediate and mature states differ in predominant fibril structure and the regions of the Sup35 sequence incorporated into the core, and that structural diversity decreases during maturation. Curing of [PSI+] at the mature state by guanidine hydrochloride (GdnHCl), which selectively inhibits ATPase activity of the chaperone Hsp104, restored both the predominant intermediate amyloid structure and the broader structural diversity characteristic of the intermediate state. In addition, Hsp104, Ssa1 (Hsp70) and Sis1 (Hsp40) associate differently with fibrils from the two states. Together, these findings provide direct structural evidence for amyloid evolution in vivo and support a chaperone-mediated mechanism of conformer selection within a polymorphic amyloid population.

molecular biology↗

bioRxiv2026

Epigenetic signatures of infection within and across generations in the endangered Loggerhead sea turtle

Infection can substantially reduce host fitness and influence population dynamics, yet it is often difficult to detect and quantify in wild animal populations. Molecular tools offer a valuable means of identifying cryptic infection in natural systems. Using whole-genome bisulfite sequencing, we examined whether infection with the parasitic leech Ozobranchus margoi is associated with DNA methylation variation in loggerhead sea turtles (Caretta caretta), while also assessing the potential value of this variation as a biomarker of parasite infection. In nesting females, we identified infection-associated differentially methylated CpG sites associated with genes implicated in immune signalling and cellular regulation. Offspring of infected females also showed infection-associated methylation patterns, despite not being directly exposed to the parasite themselves. Differential methylation analyses identified genes involved in immunity, neurodevelopment and metabolic activity, with limited overlap in associated genes and no overlap in differentially methylated sites between generations. Maternal and offspring genome-wide methylation levels showed a non-linear association that differed subtly with maternal infection status, indicating that infection modifies intergenerational methylation associations. Finally, methylation profiles showed strong discriminatory power for maternal infection status in both maternal and hatchling samples using machine learning models, supporting their potential as candidate biomarkers of cryptic infection. Together, these results show that parasite infection is associated with distinct, generation-specific DNA methylation signatures, and highlight the potential value of epigenetic data for monitoring cryptic infection states in conservation-relevant systems.

genetics↗

bioRxiv2026

DNA methylation reveals evolved buffering responses to climate-driven sex ratio skew in sea turtles

Species with temperature-dependent sex determination (TSD), including all sea turtles which produce females at warmer temperatures, face projections of demographic collapse under climate-driven sex ratio skews. However, the accuracy of such predictions remains uncertain, as current models rely heavily on indirect sex ratio proxies due to the lack of a scalable, non-invasive method for sexing hatchlings. Through whole methylome sequencing, we identified 777 sex-associated DNA methylation markers from blood samples of sex-verified loggerhead turtle (Caretta caretta) hatchlings incubated at three controlled temperatures. Applying these markers to a large-scale field experiment showed that classic nest temperature-based models overestimated female production by an average of up to 60.2%, suggesting the presence of evolved buffering mechanisms against thermal effects on sex determination. Our findings highlight the need to revise climate-driven sex ratio forecasts with empirical field data, such as methylation-based assessments, to better understand and safeguard the hidden resilience of vulnerable TSD species.

zoology↗

bioRxiv2025

Internet images reveal bumblebee-mimicking hoverflies have evolved to follow flower colour preferences of their models

Batesian mimics, such as hoverflies, can gain protection by resembling noxious species that predators avoid, such as bees. Such resemblance can be behavioural as well as morphological. Bees commonly have a preference for blue flowers, whilst flies generally prefer yellow and white. We predicted that, driven by selection for enhanced mimicry, bee-mimicking hoverflies would have switched flower preference from yellow to blue, to follow the flower choices of their models. We gathered internet photographs of flower-visiting bees and wasps, their hoverfly mimics, and non-mimicking hoverflies, and compared the colours of the visited flowers. Flowers visited by bumblebees were "bluer" than those visited by other hymenopterans (honeybees, wasps and solitary bees). Correspondingly, flowers visited by bumblebee-mimicking hoverflies were bluer than those visited by non-mimics, and were indistinguishable in blueness from those visited by bumblebees. There was no such pattern in flower "yellowness", where all flies and most hymenopterans preferred similar yellowness, and only bumblebees chose less yellow flowers than the others. Our study demonstrates widespread changes in microhabitat choice (specifically flower preference) among a diverse group of Batesian mimics, consistent with the idea of behavioural mimicry. Mildly noxious models such as bumblebees are thought to exert particularly strong selection for mimetic accuracy, suggesting that habitat-choice mimicry may be selected as a way of enhancing morphological mimicry.

ecology↗

bioRxiv2024

Chromosome-level genome assembly and methylome profile enables insights for the conservation of endangered loggerhead sea turtles

BackgroundCharacterising genetic and epigenetic diversity is crucial for assessing the adaptive potential of populations and species. Slow-reproducing and already threatened species, including endangered sea turtles, are particularly at risk. Those species with temperature-dependent sex determination (TSD) have heightened climate vulnerability, with sea turtle populations facing feminisation and extinction under future climate change. High- quality genomic and epigenomic resources will therefore support conservation efforts for these flagship species with such plastic traits. FindingsWe generated a chromosome-level genome assembly for the loggerhead sea turtle (Caretta caretta) from the globally important Cabo Verde rookery. Using Oxford Nanopore Technology (ONT) and Illumina reads followed by homology-guided scaffolding, we achieved a contiguous (N50: 129.7 Mbp) and complete (BUSCO: 97.1%) assembly, with 98.9% of the genome scaffolded into 28 chromosomes and 29,883 annotated genes. We then extracted the ONT-derived methylome and validated it via whole genome bisulfite sequencing of ten loggerheads from the same population. Applying our novel resources, we reconstructed population size fluctuations and matched them with major climatic events and niche availability. We identified microchromosomes as key regions for monitoring genetic diversity and epigenetic flexibility. Isolating 191 TSD-linked genes, we further built the largest network of functional associations and methylation patterns for sea turtles to date. ConclusionsWe present a high-quality loggerhead sea turtle genome and methylome from the globally significant East Atlantic population. By leveraging ONT sequencing to create genomic and epigenomic resources simultaneously, we showcase this dual strategy for driving conservation insights into endangered sea turtles.

genomics↗

bioRxiv2023

DNA methylation carries signatures of sublethal effects under thermal stress in loggerhead sea turtles

Rising global temperatures are a major threat to biodiversity. Whilst research generally focuses on thermal tolerance and mortality, sublethal effects may alter population dynamics and subsequently the adaptive potential of species. However, detecting such effects in the wild can be challenging, particularly for endangered and long-lived species with cryptic life histories. This necessitates the development of molecular tools to identify their signatures. In a split-clutch design experiment, we relocated clutches of wild, nesting loggerhead sea turtles (Caretta caretta) to a protected, in-situ hatchery. Eggs were then split into two sub-clutches incubated under shallow (35cm) or deep (55cm) conditions, with those in the shallow treatment experiencing significantly higher temperatures. Whilst no difference in hatching success was observed between treatments, hatchlings that emerged from the shallow, warmer treatment had altered length-mass relationships, and were weaker at fitness tests of locomotion capacity than their siblings incubated in the deep, cooler treatment. To characterise the molecular signatures of these thermal effects, we performed whole genome bisulfite sequencing on blood samples collected upon emergence. This identified 714 differentially methylated sites between treatments, including on genes with neuronal development, cytoskeleton, and sex determination functions. Taken together, our results show that higher incubation temperatures can induce sublethal effects in hatchlings, which are reflected in their DNA methylation status at identified sites. Such sites could be used as biomarkers of thermal stress, especially if they are retained across life stages. Overall, this study suggests that global warming may have population-level consequences for loggerhead sea turtles, by reducing hatchling quality, dispersal capacity and the adaptive potential of this species. Conservation efforts for climate-threatened taxa like endangered sea turtles will therefore benefit from strategies that monitor and mitigate exposure to incubation temperatures that lead to sublethal effects.

genomics↗

bioRxiv2023

Investigating the role of non-helpers in group-living thrips

O_LIBehavioural variation among individuals is a hallmark of cooperative societies, which commonly contain breeders and non-breeders, helpers and non-helpers. In some cases labour is divided, with non-breeders "helping". Conversely, in some societies subordinate non-breeders often do not help. These individuals may be (i) an insurance workforce to ensure continuity of help for breeders when other helpers are lost, (ii) conserving energy while waiting to breed themselves, or (iii) simply of too poor physiological quality either to help or breed. C_LIO_LIIn the Australian Outback, Acacia thrips Dunatothrips aneurae (Thysanoptera) glue Acacia phyllodes into "domiciles" using silk-like secretions, either alone or cooperatively. Domicile maintenance is important for humidity, so repair can be interpreted as helping. I found that not all females helped to repair experimental damage; some repaired partially or not at all ("non-helpers"). At the same time, some co-foundresses are non- or only partially reproductive, and their role is currently unknown. C_LIO_LII first tested the possibility that helping and breeding are divided, with non-helping females breeding, and non-breeders helping. In a lab experiment, I rejected this idea. Experimentally damaged domiciles were typically repaired by reproductive females, and not by non- or partially reproductive individuals. C_LIO_LITo test whether non-helpers are an insurance workforce, I successively removed repairing females and found that non-helping females did not increase effort as a result. Then, in a field experiment, I tested whether non-helping females were conserving energy while waiting to breed by removing all other females, allowing either a helpful female or a non-helping female to "inherit" her domicile. Isolated like this, non-helpers laid very few eggs compared to helpers or naturally occurring single foundresses, despite having similar ovarian development. C_LIO_LIMy findings show that labour was not divided: reproduction and helping covaried positively, probably depending on individual variation in female quality and intra-domicile competition. Non-helping females were neither an insurance workforce nor conserving energy waiting to breed themselves. They are likely simply of poor quality, freeloading by benefiting from domicile maintenance by others. I hypothesize they are tolerated because of selection for indiscriminate communal brood care in the form of domicile repair. C_LI

evolutionary biology↗

bioRxiv2022

Territorial aggression in male bower-building cichlids Nyassachromis cf. microcephalus is disrupted by exposure to environmental concentrations of fluoxetine

Selective serotonin reuptake inhibitors (SSRIs) are commonly prescribed drugs for treating human depression, but the role of serotonin--often called the happy chemical--is complex, associated with the regulation of appetite, anxiety, aggression, and more. With such psychoactive pharmaceuticals being increasingly detected in aquatic environments and their effects on non-target species an uncertainty, this study seeks to investigate how inhibiting the serotonin pathway with the SSRI fluoxetine affects territorial aggression, using the cichlid fish Nyassachromis cf. microcephalus as a model. Males of this sand-dwelling Lake Malawi species build bowers to attract females within a lekking system, where male-male competition is intense. Being aggressive in defending against intruders would serve to maximise mating opportunities and reproductive success for the territory holding male. However, after a one-week exposure to fluoxetine at environmentally relevant concentrations, a decline in aggression was observed in the male cichlids. This implies the serotonergic system plays an important role in modulating aggression and therefore sexual selection in cichlid fishes, and that environmental pollution has the potential to disrupt these behaviours.

animal behavior and cognition↗

bioRxiv2019

Differential effects of farming practice on cuckoo bumblebee communities in relation to their hosts

O_LIBees are important for vital pollination of wild and crop plants, but are in decline worldwide. Intensification of agriculture is a major driver of bee decline. Organic farming practices are designed to limit environmental impacts of agriculture and can increase bee abundance and species diversity. However, studies have been heavily focused towards some guilds of bees, overlooking others. This includes social brood parasites, cuckoo bumblebees, an understudied bee lineage. Little is known about bumblebee host and cuckoo population dynamics, and the effects of farming practice on cuckoo bumblebees have never previously been evaluated.\nC_LIO_LITo compare the effects of farming practice (organic vs conventional) on the abundance, species diversity, and community dissimilarity of cuckoo bumblebees and their hosts, we compared host and cuckoo community metrics across ten matched pairs of organic and conventional farms in Yorkshire, UK.\nC_LIO_LIAs found by many previous studies, host bumblebees were more abundant on organic farms than on conventional farms. Despite this, cuckoo bumblebees were equally abundant on both farm types. Contrary to prediction, community dissimilarity and species diversity were unaffected by farm type for both host and cuckoo communities.\nC_LIO_LISynthesis and applications: Results suggest that cuckoo bumblebee community metrics are not solely driven by host community metrics, and that cuckoos may respond differently from their hosts to differences among farming practices. This could, in turn, indicate that a unified management practice is not sufficient to conserve all bumblebee species.\nC_LI

ecology↗

bioRxiv2019

No division of labour, and subfertile foundresses, in a phyllode-gluing Acacia thrips

Behavioural variation is a hallmark of animal societies, which commonly contain breeders and nonbreeders, and helpers and nonhelpers. In some cases labour is divided with nonbreeders \"helping\" - gaining indirectly, via genetic benefits, or directly, e.g. by augmenting group size. Conversely, they may benefit by not helping, conserving energy for breeding later. However, subordinate behaviour after inheriting a breeding position is rarely evaluated.\n\nIn the Australian interior, Acacia thrips Dunatothrips aneurae (Thysanoptera) glue Acacia phyllodes together into \"domiciles\". Foundresses, usually sisters, build domiciles singly or communally. Some co-foundresses are nonreproductive, and their role is currently unknown. I experimentally rejected the idea that they substantially \"help\" by contributing to domicile repair. Nonreproductives were less likely to repair damage than reproductives. Alternatively, they may be waiting to inherit the domicile, or simply of too poor quality to reproduce or help. To test these alternatives, in the field, I allowed repairer or nonrepairer females to \"inherit\" a domicile by removing their nestmate(s). Thus isolated, \"nonrepairer\" females took much longer to repair domiciles than \"repairers\", control singletons or pairs. Although ovarian condition was equivalent across groups, after 21 days nonrepairers actually laid fewer eggs compared to other groups.\n\nThus, labour was not divided: instead reproduction and helping covaried, probably depending on female quality and the outcome of intra-domicile competition. Nonreproductive nonhelpers were not waiting to breed. Their role, and their net effect on colony productivity, remains to be shown. They are likely subfertile, and may make the \"best of a bad job\" by gaining indirect benefits to the best of their limited ability.

ecology↗