Biology subjects

Bryant, J.

Publications and source records attributed to Bryant, J..

14 recordsLinked to original sources

bioRxiv2026

RNA-seq analysis of Triatoma gerstaeckeri during mite infestation reveals increased metabolic demand and lower reproductive investment

Triatomine insects, also known as kissing bugs, serve as vectors for the Trypanosoma cruzi parasite, which can cause American trypanosomiasis, also known as Chagas disease. Triatomines are known to be parasitized by mites in natural and colony settings, where mites feed on the arthropod to obtain nutrients and resources for reproduction. This feeding, while necessary for the survival of the mites, likely occurs at the expense of their insect host. We examined the transcriptional response of Triatoma gerstaeckeri infested with mites (Pimeliaphilus spp.) and an uninfested group using Illumina-based RNA-seq, followed by targeted measurement of nutritional reserves in both groups. Our results show increased expression of factors involved in protein translation, but decreased expression of processes associated with cell development. These results are supported by declines in protein levels in mite-infested T. gerstaeckeri and lower expression of reproductive factors, suggesting a distinct reduction in nutrient reserves and reproductive output. These studies provide critical information on the interaction of mites and triatomines while considering the utility of mites as biological control of triatomines in nature or for husbandry protocols to minimize issues with mite infestations of colony individuals.

physiology↗

bioRxiv2026

Dual-mode ClfA-targeting DARPin biologics protect against diverse methicillin-resistant Staphylococcus aureus strains

Staphylococcus aureus uses the adhesin clumping factor A (ClfA) to bind fibrinogen and promote invasive infection through two distinct interfaces: an exposed, low-afinity site on the N3 head domain and a buried, high-afinity "dock, lock, and latch" (DLL) trench that is exposed only under shear. This dual-interface architecture allows limited antibody penetration, as antibodies typically block only the exposed site. Here, we establish a dual-mode inhibition strategy that overcomes this constraint by combining a high-afinity ClfA-binding designed ankyrin repeat protein (DARPin) with a fibrinogen {gamma}-chain peptide capable of occupying the DLL trench. Using cell-free click display and kinetics-guided afinity maturation, we engineer DARPin-{gamma}-peptide fusion biologics that simultaneously block both fibrinogen-binding interfaces. These molecules inhibit ClfA-fibrinogen interactions, prevent methicillin-resistant S. aureus agglutination in human plasma, neutralize major clinical ClfA variants, and confer Fc-independent protection in a lethal murine bacteremia model. This work provides a strategy for targeting antibody-intractable force-activated staphylococcal adhesins.

molecular biology↗

bioRxiv2025

Regulatable In Vivo Gene Expression via Adaptamers

Precise, reversible control of transgene expression is essential for safe and durable gene therapy, yet current inducible systems remain difficult to translate in vivo due to large size, limited induction duration, and dependence on immunogenic regulators. Here we present the adaptamer (ADAR modulatable aptamer), a compact (<120 bp) RNA switch that couples an FDA-approved small-molecule-responsive aptamer with endogenous ADAR-mediated RNA editing to regulate expression. Ligand binding stabilizes a double-stranded RNA structure that recruits ADAR to convert a stop codon into a sense codon, restoring downstream protein translation. This cleavage-free, post-transcriptional mechanism enables precise, small-molecule-dependent modulation without exogenous protein machinery. We demonstrate that adaptamers are highly functional across multiple cell lines, including human T-cells. In mice, AAV-delivered adaptamer-controlled FGF21 expression induced metabolic remodeling, significantly increasing energy expenditure and reversing obesity. This minimal, programmable system offers a clinically compatible approach for tunable genetic medicines and safe deployment of pleiotropic and dose-limited proteins.

synthetic biology↗

bioRxiv2025

Jumonji-C domain containing demethylase 1 is critical for heterochromatin organization in Plasmodium falciparum

Despite the central importance of epigenetic regulation in enabling adaptation and pathogenicity of malaria parasites, mechanisms controlling heterochromatin distribution in Plasmodium falciparum are poorly understood. Here, we identified P. falciparum Jumonji C domain-containing histone demethylase 1, PfJmjC1, as a key regulator of heterochromatin. Parasites lacking PfJmjC1 are viable in vitro but display slightly reduced multiplication rates compared to wild type parasites. Interestingly, PfJmjC1 depletion leads to major heterochromatin reorganization, involving de novo heterochromatin formation over non-essential, GC-rich euchromatic genes and reduction of heterochromatin at some chromosome ends, eventually altering the 3D genome architecture. Importantly, this heterochromatin reorganization results in the deregulation of cell surface antigen expression and failure of parasites to induce gametocyte production in response to nutrient deprivation. Collectively, our findings highlight the crucial role of PfJmjC1 in regulating heterochromatin distribution and life cycle progression in this deadly pathogen.

molecular biology↗

bioRxiv2025

Human dorsal root ganglia neuronal cell line to study nociceptive signaling: a new pipeline for pain therapy.

Nociceptive afferent neurons within the dorsal root ganglion (DRG) detect and relay painful stimuli from the periphery to the brain, and the malfunctioning of this process leads to sustained pain states. Animal model studies have been invaluable for demonstrating the importance of the DRG nociceptor in pain sensation and the development of related analgesic targets. However, there are functional biological differences between human and animal model nociceptors. Therefore, a complementary in vitro model of human nociception is critical to confirming the relevance of preclinical findings for therapeutic drug development. We characterized the nociceptive properties of differentiated cells from the human DRG-derived immortalized cell line HD10.6. Within differentiated HD10.6 cells, we documented the abundance and localization of nociceptive machinery central to regulating excitability and linked with pain sensation including ion channels TRPV1 and NaV1.7 and afferent peptides CGRP and Substance P. Using calcium influx imaging assays, we confirmed the electrical functionality of TRPV1 and NaV1.7 in HD10.6 cells, and through whole-cell patch clamp, we found similar baseline electrophysiological parameters of HD10.6 cells to those previously observed in human patient DRGs. Further, we found that differentiated HD10.6 cells express the mu opioid receptor 1 protein, and DAMGO, a mu agonist, blocks depolarization-evoked calcium influx in a naloxone-reversible fashion. Importantly, using an inflammatory cocktail, excitation and peripheral sensitization are induced within HD10.6 cells, mirroring nociceptors in a pain state during or after tissue damage or inflammation. Finally, HD10.6 cells were also cultured into dual-chambered microfluidic devices to mirror the biological anatomy of the nociceptor. Within this system, we demonstrated the uptake of adeno-associated-virus (AAV) by the peripheral terminals and AAV transport to the soma. Altogether, we have developed the use of HD10.6 cells to create a system of human nociceptive signaling on a chip to study human nociceptor physiology and intervention. PerspectiveThere are essential differences between human and animal model nociceptors. Here, we develop a physiological model of "nociceptive signaling on a chip" using human-derived nociceptors to ultimately enhance the translatability of preclinical afferent signaling research to the human patient.

neuroscience↗

bioRxiv2025

TARP γ-8 is a target of ethanol that regulates self-administration and relapse in mice

BackgroundBehavioral pathologies that characterize alcohol use disorder (AUD) are driven by the powerful reinforcing, or rewarding, properties of the drug. We have shown that glutamate AMPA receptor (AMPAR) activity is both necessary and sufficient for alcohol (ethanol) reinforcement. Transmembrane AMPAR regulatory protein (TARP) {gamma}-8 is an essential auxiliary protein that regulates AMPAR expression and activity; however, the role of TARP {gamma}-8 in AUD or other forms of addiction remains largely unexplored. ObjectivesThis study investigated the mechanistic role of TARP {gamma}-8 in operant ethanol self-administration (model of primary reinforcement) and cue-induced reinstatement of ethanol-seeking behavior (model of conditioned reinforcement) using TARP {gamma}-8 heterozygous null (+/-) mice. To determine if TARP {gamma}-8 signaling is targeted by ethanol use, we evaluated protein expression of TARP {gamma}-8, GluA1, CaMKII, and PSD-95 following ethanol self-administration. ResultsA battery of tests evaluating food and water intake, taste reactivity, anxiety-like behavior, and object recognition memory showed no fundamental behavioral deficits in TARP {gamma}-8 (+/-) mice, and no differences in response to acute ethanol or home-cage drinking as compared to wild-types. However, TARP {gamma}-8 (+/-) mice exhibited significantly reduced acquisition and escalation of operant ethanol self-administration and reduced cue-induced reinstatement of ethanol-seeking behavior, with no differences in parallel sucrose-only controls. In wild-type mice, ethanol self-administration increased TARP {gamma}-8 expression in the amygdala, nucleus accumbens, and hippocampus, and increased GluA1 expression in the amygdala and prefrontal cortex, compared to sucrose controls. ConclusionThese findings highlight the specificity of TARP {gamma}-8 regulation of ethanol reinforcement mechanisms and identify this crucial AMPAR auxiliary protein as a target of ethanol in reward-related brain regions, highlighting its potential for development of novel pharmacotherapies for AUD.

neuroscience↗

bioRxiv2025

N6-methyladenosine primes the malaria parasite for transmission

Sudden environmental changes are a recurring challenge for unicellular organisms, but a necessity for many to progress through their lifecycle. To transmit from its human host to mosquito vector, malaria parasites differentiate into male and female, semi-quiescent stages that can re-initiate development within seconds after transmission. Here, we identify the RNA modification N6-methyladenosine (m6A) as the mediator of a rapid, sex-specific, and temperature-sensitive mechanism to restructure protein synthesis during transmission. We find that male parasites maintain high levels of translation during their semi-quiescence that are rapidly repressed following mosquito uptake. This translational shutdown is essential for the continuation of male parasite development and depends on the m6A-binding protein YTH.2. We further show that m6A and YTH.2 are already present prior to transmission, but that their repressive interaction requires a temperature drop accompanying the exit from the human host. Hence, m6A appears to prime the parasite transcriptome and subsequently converts an environmental shift into a rapid translational response.

molecular biology↗

bioRxiv2022

Machine learning prediction of cancer cell metabolism from autofluorescence lifetime images

Metabolic reprogramming at a cellular level contributes to many diseases including cancer, yet few assays are capable of measuring metabolic pathway usage by individual cells within living samples. Here, we combine autofluorescence lifetime imaging with single-cell segmentation and machine-learning models to predict the metabolic pathway usage of cancer cells. The metabolic activities of MCF7 breast cancer cells and HepG2 liver cancer cells were controlled by growing the cells in culture media with specific substrates and metabolic inhibitors. Fluorescence lifetime images of two endogenous metabolic coenzymes, reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD), were acquired by a multi-photon fluorescence lifetime microscope and analyzed at the cellular level. Quantitative changes of NADH and FAD lifetime components were observed for cells using glycolysis, oxidative phosphorylation, and glutaminolysis. Conventional machine learning models trained with the autofluorescence features classified cells as dependent on glycolytic or oxidative metabolism with 90 - 92% accuracy. Furthermore, adapting convolutional neural networks to predict cancer cell metabolic perturbations from the autofluorescence lifetime images provided improved performance, 95% accuracy, over traditional models trained via extracted features. In summary, autofluorescence lifetime imaging combined with machine learning models can detect metabolic perturbations between glycolysis and oxidative metabolism of living samples at a cellular level, providing a label-free technology to study cellular metabolism and metabolic heterogeneity.

bioengineering↗

bioRxiv2022

Phagosomal RNA sensing through TLR8 controls susceptibility to tuberculosis

Genetic determinants of susceptibility to Mycobacterium tuberculosis (Mtb) are poorly understood but could provide insights into critical pathways involved in infection, informing host-directed therapies and enabling risk stratification at individual and population levels. Through a genome-wide forward genetic screen, we identify the Toll-like Receptor 8 (TLR8), as a key regulator of intracellular killing of Mtb. Pharmacological TLR8 activation enhances killing of phylogenetically diverse clinical isolates of drug-susceptible and multidrug-resistant Mtb by macrophages and during in vivo infection in mice. TLR8 is activated by phagosomal mycobacterial RNA released by extracellular membrane vesicles, and enhances xenophagy-dependent Mtb killing. We find that the TLR8 variant, M1V, common in far eastern populations, enhances intracellular killing of Mtb through preferential signal-dependent trafficking to phagosomes. TLR8 signalling may therefore both regulate susceptibility to tuberculosis and provide novel drug targets. Single sentence summaryRNA released from Mycobacterium tuberculosis in the macrophage phagosome is sensed by the pattern recognition receptor TLR8 controlling host susceptibility to tuberculosis and revealing a druggable pathway for host-directed therapy.

immunology↗

bioRxiv2020

A genetic screen to identify factors affected by undecaprenyl phosphate recycling uncovers novel connections to morphogenesis in Escherichia coli

Undecaprenyl phosphate (Und-P) is an essential lipid carrier that ferries cell wall intermediates across the cytoplasmic membrane in bacteria. Und-P is generated by dephosphorylating undecaprenyl diphosphate (Und-PP). In Escherichia coli, BacA, PgpB, YbjG, and LpxT dephosphorylate Und-PP and are conditionally essential. To identify vulnerabilities that arise when Und-P metabolism is defective, we developed a genetic screen for synthetic mutations in combination with {Delta}ybjG {Delta}lpxT {Delta}bacA. The screen uncovered system-wide connections, including novel connections to cell division, DNA replication and repair, signal transduction, and glutathione metabolism. Further analysis revealed several new morphogenes; loss of one of these, qseC, caused cells to enlarge and lyse. QseC is the sensor kinase component of the QseBC two-component system. In the absence of QseC, the QseB response regulator is overactivated by PmrB cross-phosphorylation. Here, we show that deleting qseB completely reverses the shape defect of {Delta}qseC cells, as does overexpressing rprA (a small RNA). Surprisingly, deleting pmrB only partially suppressed qseC-related shape defects. Thus, QseB is activated by multiple factors in the absence of QseC and functions ascribed to QseBC may be related to cell wall defects. Altogether, our findings provide a framework for identifying new determinants of cell integrity that could be targeted in future therapies.

microbiology↗

bioRxiv2020

A common analgesic enhances the anti-tumour activity of 5-aza-2'-deoxycytidine through induction of oxidative stress

The DNA demethylating agent 5-aza-2-deoxycytidine (DAC, decitabine) has anti-cancer therapeutic potential, but its clinical efficacy is hindered by DNA damage-related side effects. Here we describe how paracetamol augments the effects of DAC on cancer cell proliferation and differentiation, without enhancing DNA damage. Firstly, DAC specifically upregulates cyclooxygenase-2-prostaglandin E2 pathway, inadvertently increasing cancer cell survival, while the addition of paracetamol offsets this effect. Secondly, combined treatment leads to glutathione depletion and ROS accumulation with oxidative stress further enhanced by DAC suppressing anti-oxidant and thioredoxin responses. The benefits of combined treatment are demonstrated here in head and neck squamous cell carcinoma (HNSCC) and acute myeloid leukaemia cell lines, further corroborated in a HNSCC xenograft mouse model and through mining of publicly available DAC and paracetamol responses. In summary, the addition of paracetamol could allow for DAC dose reduction, widening its clinical usability and providing a strong rationale for consideration in cancer therapy.

cancer biology↗

bioRxiv2019

Structure-function analysis of the Escherichia coli β-barrel assembly enhancing protease BepA suggests a role for a self-inhibitory state

The asymmetric Gram-negative outer membrane (OM) is the first line of defence for the bacteria against environmental insults and attack by antimicrobials. The key component of the OM barrier is the surface exposed lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system OM component, LptD, is essential and is regulated by a periplasmic metalloprotease, BepA. Here we present the crystal structure of BepA, solved to a resolution of 1.9 [A]. Our structure comprises the zinc-bound m48 protease domain and a tetratricopeptide repeat (TPR) domain, consisting of four 2-helix TPR motifs and four non-TPR helices, leading to a nautilus-like shape in which the TPR repeats cup the protease domain. Using targeted mutagenesis approaches, we demonstrate that the protein is auto-regulated by the active-site plug. Further to this we reveal that mutation of a negative pocket, formed at the interface between the m48 and TPR domains, impairs BepA activity suggesting the pocket as a possible substrate binding site. We also identify a potential protein interaction site within the TPR cavity as being important for BepA function. Lastly, we provide evidence to show that increased antibiotic susceptibility in the absence of correctly functioning BepA occurs through disruption of OM lipid asymmetry, leading to reduced barrier function and increased cell permeability.

microbiology↗

bioRxiv2019

Preadaptation of pandemic GII.4 noroviruses in hidden virus reservoirs years before emergence

The control of pandemic pathogens depends on early prediction of pandemic variants and, more generally, understanding origins of such variants and factors that drive their global spread. This is especially important for GII.4 norovirus, where vaccines under development offer promise to prevent hundreds of millions of annual gastroenteritis cases. Previous studies have suggested that new GII.4 pandemic viruses evolve from previous pandemic variants through substitutions in the antigenic region of the VP1 protein that enable evasion of host population immunity, leading to global spread. In contrast, we show here that the acquisition of new genetic and antigenic characteristics is not the proximal driver of new pandemics. Instead, pandemic GII.4 viruses circulate undetected for years before causing a new pandemic, during which time they diversify and spread over wide geographical areas. Serological data demonstrate that by 2003, some nine years before it emerged as a new pandemic, the ancestral 2012 pandemic strain had already acquired the antigenic characteristics that allowed it to evade prevailing population immunity against the previous 2009 pandemic variant. These results provide strong evidence that viral genetic changes are necessary but not sufficient for GII.4 pandemic spread. Instead, we suggest that it is changes in host population immunity that enable pandemic spread of an antigenically-preadapted GII.4 variant. These results indicate that predicting future GII.4 pandemic variants will require surveillance of currently unsampled reservoir populations. Furthermore, a broadly acting GII.4 vaccine will be critical to prevent future pandemics.\n\nSignificanceNorovirus pandemics and their associated public health and economic costs could be prevented by effective vaccines. However, vaccine development and distribution will require identification of the sources and drivers of new pandemics. We here use phylogenetics and serological experiments to develop and test a new hypothesis of pandemic norovirus emergence. We find that pandemic noroviruses preadapt, diversify and spread worldwide years prior to emergence, strongly indicating that genetic changes are necessary but not sufficient to drive a new pandemic. We instead suggest that changes in population immunity enable pandemic emergence of a pre-adapted low-level variant. These findings indicate that prediction of new pandemics will require surveillance of under-sampled virus reservoirs and that norovirus vaccines will need to elicit broad immunity.

evolutionary biology↗

bioRxiv2017

MCE domain proteins: conserved inner membrane lipid-binding proteins required for outer membrane homeostasis

Bacterial proteins with MCE domains were first described as being important for Mammalian Cell Entry. More recent evidence suggests they are components of lipid ABC transporters. In Escherichia coli, the single-domain protein MlaD is known to be part of an inner membrane transporter that is important for maintenance of outer membrane lipid asymmetry. Here we describe two multi MCE domain-containing proteins in Escherichia coli, PqiB and YebT, the latter of which is an orthologue of MAM-7 that was previously reported to be an outer membrane protein. We show that all three MCE domain-containing proteins localise to the inner membrane. Bioinformatic analyses revealed that MCE domains are widely distributed across bacterial phyla but multi MCE domain-containing proteins evolved in Proteobacteria from single-domain proteins. Mutants defective in mlaD, pqiAB and yebST were shown to have distinct but partially overlapping phenotypes, but the primary functions of PqiB and YebT differ from MlaD. Complementing our previous findings that all three proteins bind phospholipids, results presented here indicate that multi-domain proteins evolved in Proteobacteria for specific functions in maintaining cell envelope homeostasis.

microbiology↗