Biology subjects

Johnston, R.

Publications and source records attributed to Johnston, R..

18 recordsLinked to original sources

bioRxiv2026

eIF4E3 drives translation of viral mRNAs with short 5' UTRs

Eukaryotic cells express three paralogs of the cap-binding protein eIF4E, yet the functions of the less-studied family members remain poorly understood. Here we demonstrate that eIF4E3, a paralog whose expression is tissue-restricted and whose activity is insensitive to inhibition by eIF4E-binding proteins (4EBPs), drives efficient translation of viral mRNAs with short 5 untranslated regions (UTRs). Many negative-strand RNA viruses (NSVs), including vesicular stomatitis virus (VSV), respiratory syncytial virus (RSV), and influenza A virus (IAV), produce mRNAs with extremely short 5UTRs that are incompatible with canonical cap-dependent scanning translation initiation. Using an auxin-inducible degron (AID) system to acutely deplete endogenous eukaryotic initiation factors during active viral infection, we demonstrate that translation of these short-UTR viral mRNAs occurs independently of eIF4E1, the canonical cap-binding protein, while remaining dependent on eIF4E3. In contrast, Ebola virus (EBOV), whose mRNAs bear long, structured 5UTRs, remains eIF4E1-dependent, implicating 5UTR length and structural complexity as cis determinants of eIF4E paralog selectivity. During VSV infection, 4EBP dephosphorylation sequesters eIF4E1 and broadly suppresses host cap-dependent translation. Because eIF4E3 escapes 4EBP-mediated regulation and preferentially engages short, unstructured 5UTRs, it is uniquely positioned to sustain viral protein synthesis under these conditions. These findings reveal that VSV exploits a cellular paralog-switching mechanism by co-opting eIF4E3 to maintain viral translation when canonical eIF4F activity is suppressed and establish eIF4E3 as a proviral factor whose tissue-restricted expression in the lung may influence susceptibility to clinically important respiratory pathogens.

molecular biology↗

bioRxiv2026

Proteolytic dissection of eIF4G reveals the closed-loop mRNP as an architecture for translation repression.

Formation of a "closed-loop" mRNP, in which the 5' cap and 3' poly(A) tail are bridged by eIF4E-eIF4G-PABP interactions, has long been proposed to drive efficient translation initiation. Direct tests of this model in mammalian cells have remained elusive. Using auxin-inducible degron (AID) technology to acutely deplete eIF4G1, we find that global translation is only partially reduced and recovers without restoration of eIF4G1 levels. We identify eIF4G3 as an underappreciated contributor to basal translation that buffers translational output upon eIF4G1 loss without increased protein expression, explaining the modest defects observed in prior RNAi-based studies. Systematic replacement of eIF4G1 with defined cleavage products and interaction mutants reveals that PABP binding by eIF4G1 is dispensable for bulk translation initiation: the central caspase-3 cleavage fragment of eIF4G1 (casp3-cpM), which lacks the PABP-interaction domain, fully rescues global protein synthesis, and acute depletion of both major cytoplasmic PABP paralogs primarily destabilizes mRNAs rather than impairing initiation. In contrast, the N-terminal enteroviral 2A cleavage product (2A-cpN) is a potent, dominant translational repressor that requires simultaneous eIF4E and PABP engagement to form a dead-end closed-loop mRNP that sequesters initiation factors without enabling 43S recruitment. These findings reveal that the eIF4G-PABP closed-loop architecture is not required for productive initiation but can be actively co-opted for translational silencing. This explains why viral eIF4G cleavage, but not factor depletion, produces near-complete translational shutoff. The modular architecture of eIF4G enables diametrically opposing translational outcomes through selective proteolytic processing.

molecular biology↗

bioRxiv2026

Decreased tRNA abundance contributes to decreased translation elongation rate in a prolonged mitosis

Cells undergo dramatic structural rearrangements upon entering mitosis. In addition, the biochemistry of mitotic cells is dramatically altered, including a significant decrease in protein synthesis. The majority of studies of mitotic translation have used cells synchronized by cell cycle altering drugs in transformed cells and much less is known about mitotic translation in primary cells under native conditions. Previous work has found that mitosis activates the integrated stress response (ISR) to trigger eIF2 phosphorylation, but little is known about the input for this response. In this study, we focus on mitotic translational regulation in an immortalized, non-transformed cell line. We confirm decreased mitotic protein synthesis in primary cells and under native conditions. Additionally, we confirm activation of the ISR by phosphorylation of eIF2 during both normal and prolonged mitosis. Interestingly, we also find that decreased translational elongation during mitosis, as evidenced by increased eEF2 phosphorylation and a slower elongation rate. Analysis of mitotic ribosome profiling data revealed an increase in pausing at Alanine-GCG codons during mitosis and a decreased abundance of its cognate tRNA-AlaCGC by northern blotting. Decreased tRNA-AlaCGC is likely sustained by the inability to synthesize additional tRNA due to RNAPol III inhibition in mitosis, yielding an stronger effect with an increased time in mitosis. These results suggest that decreased translation elongation in mitosis triggers inhibition of initiation to decrease global protein synthesis.

cell biology↗

bioRxiv2025

Investigating the mechanisms underlying saccade generation in the frontal eye fields using multi-site microstimulation

The frontal eye field (FEF), a region of frontal cortex, has long been associated with the cortical control of eye movements. Classically, saccades can be reliably evoked by delivering low-intensity electrical microstimulation to the FEF. Although this makes clear the importance of FEF in the descending control of eye movements, the way in which population activity in the FEF is integrated by downstream regions to generate a motor command remains a mystery. To probe these mechanisms, we used a 16-channel microelectrode array to deliver microstimulation to the FEF of two awake, behaving monkeys. First, we found that larger current intensities were required to evoke changes in saccade direction relative to saccade amplitude when single-site saccades were evoked by stimulating a single contact on the array. Second, when stimulating two contacts simultaneously to investigate how population activity in the FEF is read out, a new polar average model more accurately predicted the amplitude and direction of dual-site saccades than traditional vector sum and vector average models. Using preexisting data from the superior colliculus (SC), we found that although the polar average model was more accurate at predicting saccade amplitude in the SC, it was no more accurate than traditional models at predicting saccade direction. Finally, when stimulating two contacts in FEF simultaneously with unequal current intensities, model accuracy depended on the amplitude of the saccades evoked by stimulating each individual site alone, suggesting that the brain may flexibly combine amplitude and direction information from the FEF to generate saccadic plans.

neuroscience↗

bioRxiv2025

Ex Vivo Testing of Inflatable Penile Prosthesis in Human Cadaveric Penis with Paired in Silico Model offering Surgical and Biomechanical Insights

BackgroundInflatable penile prostheses (IPPs) are a critical solution for patients with erectile dysfunction refractory to medical therapy. However, a detailed understanding of their mechanical interaction with penile tissues remains limited. AimTo develop and validate an innovative experimental-computational framework for studying IPP behaviour through ex vivo implantation and inflation testing in human cadaveric penile tissue, paired with a finite element-based (FE-based) computational model. MethodsAn AMS 700 IPP was surgically implanted into a human cadaveric penis (including the glans and [~]15 cm of shaft) using standard clinical techniques. The cylinders were placed within the corpora cavernosa, with the pump and reservoir positioned externally in a closed hydraulic loop. An inflation test was performed ex vivo, with real-time ultrasound imaging used to monitor cylinder expansion. Internal pressure was recorded using a digital barometer. Following inflation, sectional analysis enabled 3D approximation of penile shaft geometry. A representative FE-based computational model was developed, incorporating anatomically accurate tissue layers--tunica albuginea (TA), corpus cavernosa (CC), corpus spongiosum (CS), and fascia--with realistic material properties to simulate the inflation process. OutcomesThis study enabled the mechanical response of penile tissues to IPP inflation to be quantified using both experimental and computational modalities. ResultsThe combined use of ultrasound imaging and digital pressure monitoring successfully captured dynamic IPP behaviour during inflation. The FE model reproduced experimental outcomes with good fidelity, providing a detailed understanding of stress distribution and tissue deformation. Clinical ImplicationsThis integrated approach can inform future IPP design improvements and aid surgeons in preoperative planning by offering predictive insights into prosthesis-tissue interaction. Strengths & LimitationsA major strength of this study is the novel integration of cadaveric experimentation with computational modelling. However, limitations include the absence of active physiological responses and potential variability due to cadaveric tissue properties. ConclusionThis pioneering work establishes a robust platform for studying IPP mechanics in realistic anatomical contexts, with promising implications for optimising device design and improving patient outcomes in urologic surgery.

bioengineering↗

bioRxiv2024

Prenylation controls proliferation in human pluripotent stem cell-derived cardiomyocytes

Induction of cardiomyocyte proliferation to replace damaged heart tissue is a promising therapeutic approach. A recent drug screen revealed that cardiomyocytes require the mevalonate pathway for proliferation, although the specific mechanisms are unknown. In this study, we use human pluripotent stem cell-derived cardiomyocytes and cardiac organoids to further interrogate the role of the mevalonate pathway in cardiomyocyte proliferation. Chemical and genetic perturbations of the mevalonate pathway indicated that the post-translational modification, prenylation, regulates cardiomyocyte proliferation. We use prenyl probes and mass spectrometry to identify a catalogue of 40 prenylated proteins in human cardiac cells, including proteins where prenylated function had not yet been investigated. We show that multiple prenylated proteins control cardiomyocyte proliferation including RRAS2 and NAP1L4. We demonstrate that prenylation has differential effects on distinct proteins, with RRAS2 prenylation controlling membrane localization and NAP1L4 prenylation regulating cardiomyocyte mitosis and centrosome homeostasis. Together, these data show that protein prenylation is required for cardiomyocyte proliferation through multiple targets and these processes may need to be re-activated for cardiac regeneration.

cell biology↗

bioRxiv2024

Neural Ensembles in the Lateral Prefrontal Cortex Temporally Multiplex Task Features During Virtual Navigation

Neuronal populations can expand their information encoding capacity using mixed selective neurons. This is particularly prominent in association areas such as the Lateral Prefrontal Cortex (LPFC) that integrate information from multiple sensory systems. During naturalistic conditions where subjects have agency, it is unclear how LPFC neuronal ensembles process space and time varying information. Here we show that during a virtual navigation task that requires associative memory and decision making, individual neurons and neuronal ensembles in the LPFC time-multiplex their selectivity for different task features to fluidly encode the temporal contingencies of the task and the animals choice. Neurons in ventral regions showed more selectivity for non-spatial features, while dorsal regions showed selectivity for space and eye movements. These results demonstrate that during naturalistic tasks with fluid scenery and spatiotemporal dynamics, LPFC neurons and neuronal ensembles time-multiplex the components of their selectivity, expanding the spatiotemporal capacity of their neural codes.

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

Non-Invasive Plasma Glycomic and Metabolic Biomarkers of Post-treatment Control of HIV

Non-invasive biomarkers that predict HIV remission after antiretroviral therapy (ART) interruption are urgently needed. Such biomarkers can improve the safety of analytic treatment interruption (ATI) and provide mechanistic insights into the pathways involved in post-ART HIV control. We identified plasma glycomic and metabolic signatures of time-to-viral-rebound and probability-of-viral-rebound using samples from two independent cohorts. These samples include a large number of post-treatment controllers, a rare population demonstrating sustained virologic suppression after ART-cessation. The signatures remained significant after adjusting for key demographic and clinical confounders. We also confirmed a mechanistic link between biomarkers and HIV latency reactivation and myeloid inflammation in vitro. Finally, machine learning algorithms selected sets of biomarkers that predict time-to-viral-rebound with 74-76% capacity and probability-of-viral-rebound with 97.5% capacity. In summary, we fill a major gap in HIV cure research by identifying non-invasive biomarkers, with potential functional significance, that predict duration and probability of viral remission after treatment interruption.

microbiology↗

bioRxiv2020

Characterization of histone inheritance patterns in the Drosophila female germline

Stem cells have the unique ability to undergo asymmetric division which produces two daughter cells that are genetically identical, but commit to different cell fates. The loss of this balanced asymmetric outcome can lead to many diseases, including cancer and tissue dystrophy. Understanding this tightly regulated process is crucial in developing methods to treat these abnormalities. Here, we report that produced from a Drosophila female germline stem cell asymmetric division, the two daughter cells differentially inherit histones at key genes related to either maintaining the stem cell state or promoting differentiation, but not at constitutively active or silenced genes. We combined histone labeling with DNA Oligopaints to distinguish old versus new histone distribution and visualize their inheritance patterns at single-gene resolution in asymmetrically dividing cells in vivo. This strategy can be widely applied to other biological contexts involving cell fate establishment during development or tissue homeostasis in multicellular organisms.

developmental biology↗

bioRxiv2020

EEG signals index a global signature of arousal embedded in neuronal population recordings

Electroencephalography (EEG) has long been used to index brain states, from early studies describing activity during visual stimulation to modern work employing complex perceptual tasks. These studies shed light on brain-wide signals but lacked explanatory power at the single neuron level. Similarly, single neuron studies can suffer from inability to measure brain-wide signals. Here, we combined these techniques while monkeys performed a change detection task and discovered a link between EEG and a signal embedded in spiking responses. This slow drift was associated with arousal: decreases in pre-stimulus power/increases in P1 amplitude were accompanied by :1) increases in pupil size, false alarm rate and saccade velocity; and 2) decreases in microsaccade rate and reaction time. These results show that brain-wide EEG signals can be used to index modes of activity acquired from direct neural recordings, that in turn reflect global changes in brain state that influence perception and behavior.

neuroscience↗

bioRxiv2020

The eyes reflect an internal cognitive state embedded in the population activity of cortical neurons

Decades of research have shown that global brain states such as arousal can be indexed by measuring the properties of the eyes. Neural signals from individual neurons, populations of neurons, and field potentials measured throughout much of the brain have been associated with the size of the pupil, small fixational eye movements, and vigor in saccadic eye movements. However, precisely because the eyes have been associated with modulation of neural activity across the brain, and many different kinds of measurements of the eyes have been made across studies, it has been difficult to clearly isolate how internal states affect the behavior of the eyes, and vice versa. Recent work in our laboratory identified a latent dimension of neural activity in macaque visual cortex on the timescale of minutes to tens of minutes. This slow drift was associated with perceptual performance on an orientation-change detection task, as well as neural activity in visual and prefrontal cortex (PFC), suggesting it might reflect a shift in a global brain state. This motivated us to ask if the neural signature of this internal state is correlated with the action of the eyes in different behavioral tasks. We recorded from visual cortex (V4) while monkeys performed a change detection task, and the prefrontal cortex, while they performed a memory-guided saccade task. On both tasks, slow drift was associated with a pattern that is indicative of changes in arousal level over time. When pupil size was large, and the subjects were in a heighted state of arousal, microsaccade rate and reaction time decreased while saccade velocity increased. These results show that the action of the eyes is associated with a dominant mode of neural activity that is pervasive and task-independent, and can be accessed in the population activity of neurons across the cortex.

neuroscience↗

bioRxiv2020

The costs of competition: high social status males experience accelerated epigenetic aging in wild baboons

Aging, for virtually all life, is inescapable. However, within populations, biological aging rates vary. Understanding sources of variation in this process is central to understanding the biodemography of natural populations. We constructed a DNA methylation-based age predictor for an intensively studied wild baboon population in Kenya. Consistent with findings in humans, the resulting "epigenetic clock" closely tracks chronological age, but individuals are predicted to be somewhat older or younger than their known ages. Surprisingly, these deviations are not explained by the strongest predictors of lifespan in this population, early adversity and social integration. Instead, they are best predicted by male dominance rank: high-ranking males are predicted to be older than their true ages, and epigenetic age tracks changes in rank over time. Our results argue that achieving high rank for male baboons--the best predictor of reproductive success--imposes costs consistent with a "live fast, die young" life history strategy.

genomics↗

bioRxiv2019

Sophisticated suction organs from insects living in raging torrents: Morphology and ultrastructure of the attachment devices of net-winged midge larvae (Diptera: Blephariceridae)

2.Suction organs provide powerful yet dynamic attachments for many aquatic animals, including octopus, squid, remora, and clingfish. While the functional morphology of suction organs from various cephalopods and fishes has been investigated in detail, there are only few studies on such attachment devices in insects. Here we characterise the morphology, ultrastructure, and in vivo movements of the suction attachment organs of net-winged midge larvae (genus Liponeura) - aquatic insects that live on rocks in rapid alpine waterways where flow rates can reach 3 m s-1 - using scanning electron microscopy, laser confocal scanning microscopy, and X-ray computed micro-tomography (micro-CT). We identified structural adaptations important for the function of the suction attachment organs from L. cinerascens and L. cordata. First, a dense array of spine-like microtrichia covering each suction disc comes into contact with the substrate upon attachment. Similar hairy structures have been found on the contact zones of suction organs from octopus, clingfish, and remora fish. These structures are thought to contribute to the seal and to provide increased shear force resistance in high-drag environments. Second, specialised rim microtrichia at the suction disc periphery form a continuous ring in close contact with a surface and may serve as a seal on a variety of surfaces. Third, a V-shaped cut on the suction disc (the V-notch) is actively peeled open via two cuticular apodemes inserting into its flanks. The apodemes are attached to dedicated V-notch opening muscles, thereby providing a unique detachment mechanism. The complex cuticular design of the suction organs, along with specialised muscles that attach to them, allows blepharicerid larvae to generate powerful attachments which can withstand strong hydrodynamic forces and quickly detach for locomotion. Our findings could be applied to bio-inspired attachment devices that perform well on a wide range of surfaces.

zoology↗

bioRxiv2019

Indomethacin-induced gut damage in a surrogate insect model, Galleria mellonella

Indomethacin is a non-steroidal anti-inflammatory drug that causes gastric ulceration and increased leakiness in rat models, and is used routinely as an assay to screen novel compounds for repair and restitution properties. We set out to establish conditions for indomethacin-induced gut damage in wax-moth (Galleria mellonella) larvae with a view to reducing the need for rodents in such experimentation. We administered indomethacin (1 - 7.5 g/larva) to G. mellonella via intrahaemocoelic injection and gavage (force-feeding) and monitored larval survival and development, blood cell (haemocyte) numbers, and changes in gut permeability. Increased levels of gut leakiness were observed within the first 4 to 24-hours by tracking fluorescent microspheres in the faeces and haemolymph (blood equivalent). Additionally, we recorded varying levels of tissue damage in histological sections of the insect midgut, including epithelial sloughing and cell necrosis. Degeneration of the midgut was accompanied by significant increases in detoxification-associated activities (superoxide dismutase and glutathione-S-transferase). Herein, we present the first evidence that G. mellonella larvae force-fed indomethacin display broad symptoms of gastric damage similar to rodent models.

immunology↗

bioRxiv2019

Macro-to-nano scale investigation of the acorn barnacle Semibalanus balanoides: correlative imaging, biological form and function, and bioinspiration

1.Correlative imaging combines information from multiple modalities (physical-chemical-mechanical properties) at various length-scales (cm to nm) to understand complex biological materials across dimensions (2D-3D). Here, we have used numerous coupled systems: X-ray microscopy (XRM), scanning electron microscopy (SEM), electron backscatter diffraction (EBSD), optical light microscopy (LM), and focused-ion beam (FIB-SEM) microscopy to ascertain the microstructural and crystallographic properties of the wall-plate joints in the barnacle Semibalanus balanoides. The exoskeleton is composed of six interlocking wall-plates, and the interlocks between neighbouring plates (alae) allow barnacles to expand and grow whilst remaining sealed and structurally strong. Our results indicate that the ala contain functionally-graded orientations and microstructures in their crystallography, which has implications for naturally functioning microstructures, potential natural strengthening, and preferred oriented biomineralisation. Elongated grains at the outer edge of the ala are oriented perpendicularly to the contact surface, and the c-axis rotates with the radius of the ala. Additionally, we identify for the first time three-dimensional nano-scale ala pore networks revealing that the pores are only visible at the tip of the ala, and that pore thickening occurs on the inside (soft-bodied) edge of the plates. The pore networks appear to have the same orientation as the oriented crystallography, and we deduce that the pore networks are probably organic channels and pockets which are involved with the biomineralisation process. Understanding these multi-scale features contributes towards an understanding of the structural architecture in barnacles, but also their consideration for bioinspiration of human-made materials. The work demonstrates that correlative methods spanning different length-scales, dimensions and modes enable the extension of structure-property relationships in materials to form and function of organisms.

systems biology↗

bioRxiv2017

A Feed-forward Relay between Bicoid and Orthodenticle Regulates the Timing ofEmbryonic Patterning in Drosophila

The K50 homeodomain (K50HD) protein Orthodenticle (Otd) is critical for anterior patterning and brain and eye development in most metazoans. In Drosophila melanogaster, another K50HD protein, Bicoid (Bcd), has evolved to replace Otds ancestral function in embryo patterning. Bcd is distributed as a long-range maternal gradient and activates transcription of a large number of target genes including otd. Otd and Bcd bind similar DNA sequences in vitro, but how their transcriptional activities are integrated to pattern anterior regions of the embryo is unknown. Here we define three major classes of enhancers that are differentially sensitive to binding and transcriptional activation by Bcd and Otd. Class 1 enhancers are initially activated by Bcd, and activation is transferred to Otd via a feed-forward relay (FFR) that involves sequential binding of the two proteins to the same DNA motif. Class 2 enhancers are activated by Bcd, and maintained by an Otd-independent mechanism. Class 3 enhancers are never bound by Bcd, but Otd binds and activates them in a second wave of zygotic transcription. The specific activities of enhancers in each class are mediated by DNA motif variants preferentially bound by Bcd or Otd, and the presence or absence of sites for cofactors that interact with these proteins. Our results define specific patterning roles for Bcd and Otd, and provide mechanisms for coordinating the precise timing of gene expression patterns during embryonic development.

molecular biology↗

bioRxiv2017

Chemical-microstructural-nanomechanical variations in the structural units of the cuttlebone of Sepia officinalis

Cuttlebone, the internalized shell found in all members of the cephalopod family Sepiidae, is a sophisticated buoyancy device combining high porosity with considerable strength. Using a complementary suite of characterization tools, we identified significant structural, chemical and mechanical variations across the different structural units of the cuttlebone: the dorsal shield consists of two stiff and hard layers with prismatic mineral organization which encapsulate a more ductile and compliant layer with a lamellar structure, enriched with organic matter. A similar organization is found in the lamellar matrix, which consists of individual chambers separated by septa, and supported by meandering plates ( pillars). Like the dorsal shield, septa contain two layers with lamellar and prismatic organization, respectively, which differ significantly in their mechanical properties: layers with prismatic organization are a factor of three stiffer, and up to a factor of ten harder than those with lamellar organization. The combination of stiff and hard, and compliant and ductile components may serve to reduce the risk of catastrophic failure, and reflect the role of organic matter for the growth process of the cuttlebone. Mechanically weaker units may function as sacrificial structures, ensuring a step-wise failure of the individual chambers in cases of overloading, allowing the animals to retain near-neutral buoyancy even with partially damaged cuttlebones. Our findings have implications for the structure-property-function relationship of cuttlebone, and may help to identify novel bioinspired design strategies for light-weight yet high-strength foams.

bioengineering↗