Biology subjects

Turner, K.

Publications and source records attributed to Turner, K..

At least 19 recordsLinked to original sources

bioRxiv2026

CLN6 is a glycerophosphoglycerol epimerase in bis(monoacylglycero)phosphate biosynthesis

Lysosomal catabolism depends on bis(monoacylglycero)phosphate (BMP), a phospholipid with atypical S,S stereochemistry of unknown biosynthetic origin. CLN8 and CLN5 catalyze sequential acylation steps in BMP synthesis, and their loss of function causes Batten disease, a group of inherited neurodegenerative lysosomal storage disorders. Here we identify CLN6, a biochemically uncharacterized endoplasmic reticulum protein whose loss also causes Batten disease, as the calcium-dependent glycerophosphoglycerol epimerase that establishes BMP stereochemistry upstream of CLN8. CLN6 deficiency depletes BMP in cells, mice, and patient lysosomes, and reduces lysosomal hydrolase levels. BMP-precursor supplementation bypasses CLN6, restores BMP and increases hydrolase abundance in CLN6-deficient cells and mice. These findings place three Batten disease proteins in a single biosynthetic pathway and reveal BMP deficiency as a reversible cause of disrupted lysosomal hydrolase homeostasis.

biochemistry↗

bioRxiv2026

mRNA-LNP therapy restores systemic nucleoside imbalance in a mitochon-drial DNA depletion syndrome

Mitochondrial DNA depletion syndromes (MDS) are inherited conditions caused by pathogenic variants in mitochondrial DNA maintenance genes. Most MDS are severe, fatal and incurable conditions. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an MDS resulting from loss-of-function mutations in the TYMP gene, encoding Thymidine Phosphorylase (TP). Systemic nucleoside accumulation caused by TP deficiency disrupts mitochondrial nucleotide homeostasis, which underlies disease progression and mortality. Current treatments, including liver and hematopoietic stem cell transplantation, partially restore TP activity but are invasive, carry substantial risk and are limited by donor availability. Here, we establish TYMP-mRNA in lipid nanoparticles (hTYMP-mRNA-LNPs) as a safe non-viral protein-replacement therapy for MNGIE. Intravenous administration of hTYMP-mRNA-LNPs induced robust hepatic TP expression in a mouse model of MNGIE, was well tolerated and restored circulating nucleosides to wild-type levels within hours, lasting up to three weeks, at a preclinical minimally effective dose of 0.25mg/kg. To facilitate repeat administration and patient access, we demonstrate enhanced efficiency of subcutaneous mRNA-LNP delivery by co-administration of recombinant or mRNA-encoded (SPAM1-mRNA-LNPs) hyaluronidase, achieving effective hepatic TP expression and systemic nucleoside clearance. These findings establish mRNA-LNP-mediated protein replacement as a therapeutic strategy for a primary mitochondrial disease where transient liver-targeted expression is sufficient to correct a systemic metabolic defect. More broadly, our results support the development of mRNA-LNP therapeutics and their subcutaneous delivery as a generalizable platform for treating monogenic diseases, through repeatable, non-viral protein replacement.

pharmacology and toxicology↗

bioRxiv2026

Genetically interacting mutations and mechanical stress affect the stochasticity of developmental eye defects in yap1 mutants

Congenital abnormalities of eye formation show remarkably variable penetrance with phenotypes even varying between left and right eyes. Here we explore this phenomenon through analysis of the mechanistic basis of low penetrance retinal coloboma in zebrafish yap1nl13/nl13 mutants and identification of factors that modify the probability of this phenotype. We find that the low penetrance stochastically occurring coloboma in yap1nl13/nl13 mutants is due to rupture in the ventral retina at the point of apposition of the lips of the closing choroid fissure and that provision of wild-type Yap in the retinal pigment epithelium suppressed this phenotype. Decreasing actomyosin contractility increased the penetrance of coloboma whereas increasing myosin phosphorylation rescued the phenotype suggesting that altered mechanical properties of the RPE sensitize the eye to stochastic failure of choroid fissure closure. Genetic interaction screening revealed enhanced and synthetic eye phenotypes in yap1nl13/nl13mutants upon abrogation of function of genes encoding extracellular matrix and other genes implicated in eye formation. Our data reveal that the variable penetrance of congenital eye abnormalities can be due to genetic and environmental factors impacting the stochastic variability inherent in the developmental processes underlying eye morphogenesis.

developmental biology↗

bioRxiv2026

Intratelencephalic neurons in the medial prefrontal cortex mediate the acquisition of goal-directed actions.

The medial prefrontal cortex (mPFC) and its connections with the posterior dorsomedial striatum are implicated in goal-directed learning, but the specific mPFC cell types involved have not been clearly established. The current study investigated mPFC intratelencephalic (IT) and pyramidal tract (PT) neuron involvement in goal-directed learning. In Cre-driver mouse lines, we mapped bilaterally projecting IT neurons and unilaterally projecting PT neurons from mPFC to dorsal striatum and showed that chemogenetic inhibition of IT, but not PT neurons, attenuated goal-directed learning. We then demonstrated training induced elevations in pERK signaling in IT neurons, which were transient in superficial mPFC layers, and more persistent in deeper layers. This was associated with plasticity in deep layer IT neurons, reflected in a shift towards excitatory over inhibitory synaptic inputs. Together, these data suggest that goal-directed learning influences synaptic input and downstream plasticity markers in mPFC IT neurons, and this functionally contributes to goal-directed learning.

neuroscience↗

bioRxiv2026

Identification of genes important for response of Pseudomonas aeruginosa biofilms to ciprofloxacin exposure

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections in immunocompromised individuals, such as patients with cystic fibrosis where it commonly forms biofilms. Ciprofloxacin is used extensively to treat P. aeruginosa infections, but its effectiveness can be significantly reduced due to biofilm formation. Although many individual genes associated with biofilm formation or ciprofloxacin resistance have been characterised, the genetic basis of P. aeruginosa biofilm fitness related to antibiotic challenge remains incompletely understood. In this study we employed a whole genome screen to assay the impact of gene disruptions or altered gene expression on survival of P. aeruginosa biofilms exposed to different concentrations of ciprofloxacin. Genes impacting fitness in the biofilm context were identified by comparing the biofilm samples to planktonic samples harvested at 12h, 24h and 48h with and without ciprofloxacin. Genes associated with c-di-GMP regulation and Gac/Rsm signalling were identified as primary regulators for biofilm formation in the presence and absence of ciprofloxacin. In addition, a group of genes involved in respiration, metabolism (especially polyamine metabolism), and various transporter and efflux systems were identified as important for biofilm fitness. Ciprofloxacin specifically imposed a selective pressure on flagellar function and Psl production which were essential for survival in early biofilms. Moreover, transposon insertions within the CPA gene clusters (PA5448-PA5451 and PA5455-PA5456) and the salvage peptidoglycan recycling pathway showed reduced fitness in late biofilms at high concentration of ciprofloxacin, indicating that cell envelope integrity is beneficial for mature biofilms. This study identifies important determinants of survival for biofilms at different stages of maturity in the presence and absence of ciprofloxacin and implicates potential therapeutic targets for antibiofilm drug development.

genomics↗

bioRxiv2026

Contingency degradation overwrites initial learning and depends on lateral orbitofrontal cortex

Relapse after treatment for various mental health disorders has been linked to tendency for reductions in responding to increase over time or following re-exposure to motivating stimuli. Here we show that, in rats, responding reduced through non-contingent outcome delivery does not recover in these ways, and that this learning depends on an intact lateral orbitofrontal cortex. These findings suggest that contingency degradation overwrites original learning which may support the development of relapse-resistant behavioural interventions.

animal behavior and cognition↗

bioRxiv2026

Ultrafast venous and sagittal sinus constrictions in the brain driven by abdominal pressure

Nearly all the blood supplying the cortex exits via the bridging veins (BVs) that drain into the superior sagittal sinus (SSS), making these vessels key chokepoints for cerebral blood flow. Using optical imaging in head-fixed mice, we found that the SSS and BVs exhibit ultrafast contractions (<0.1 s) at the onset of locomotion, following whisker stimulation, and upon awakening from sleep. Contractions of the BV and SSS were strongly correlated with abdominal muscle EMG activity and were tightly correlated with respiration at rest. The rapid decrease in blood volume caused by venous constrictions resulted in spurious increases in fluorescence in mice expressing fluorescent reporter proteins, creating artifacts that mimic functional signals. Venous contractions with the same amplitude and dynamics could be generated in anesthetized mice by abdominal pressure application, showing that these contractions were generated by mechanical coupling with the abdomen. Externally imposed abdominal pressures also drove a rapid but transient increase in blood flow. Unlike the pial and parenchymal microvasculature whose diameters are controlled by local signals, the diameters of SSS/BV are dynamically controlled during behavior by abdominal muscle regulation of intracranial pressure, establishing a pathway for regulation of cerebral hemodynamics via mechanical coupling between the central nervous system and the viscera.

neuroscience↗

bioRxiv2026

Disrupting astrocyte signalling in the nucleus accumbens impairs incentive-driven instrumental actions

Astrocytes in the nucleus accumbens (NAC) core have been observed to undergo phenotypic changes associated with drug-seeking behaviour in both humans and animals. However, the role of NAC core astrocytes in non-drug-related instrumental behaviour remains poorly understood. To address this, we chemogenetically activated hM4Di receptors selectively expressed on NAC core astrocytes in rats during food-motivated decision-making tasks. In Experiment 1, rats were first trained to associate two auditory stimuli with two distinct food outcomes (pellets and sucrose), then to press left and right levers for those same outcomes. All training was conducted drug-free, and rats then received intraperitoneal (i.p) injections of either vehicle or deschloroclozapine (DCZ) prior to test. Disrupting astrocytic signalling via DCZ injections left instrumental choice intact when it was guided by cues signalling the sensory-specific properties of each outcome, as tested in specific Pavlovian instrumental transfer and outcome-selective reinstatement, but suppressed responding in an outcome devaluation test. In Experiment 2, a single stimulus and single lever were separately paired with distinct food outcomes, then presented together on test. Control animals demonstrated a general PIT effect, elevating responding during stimulus presentations, and this was prevented by Gi activation on NAC core astrocytes. Immunohistochemistry revealed increased neuronal activity following hM4Di activation in astrocytes. Together, these findings suggest that intact signalling in NAC core astrocytes is necessary for instrumental actions that depend on general arousal or affective processes, but not for actions guided by sensory-specific outcome expectations. Significance StatementAstrocyte dysfunction in the nucleus accumbens (NAC) has been implicated in several forms of compulsion in humans, yet preclinical work has focussed almost exclusively on drug-taking and seeking. The role of NAC core astrocytes on responding for non-drug outcomes is therefore unclear. Here, using food outcomes, we show that disrupting astrocytic Gi signalling in NAC core selectively impairs actions driven by general motivational states but spares those guided by specific outcome expectations. These findings suggest that NAC core astrocytes play a critical role in invigorating behaviour, extending their involvement beyond drug-seeking in animals and highlighting their potential relevance to compulsive behaviour more generally.

animal behavior and cognition↗

bioRxiv2025

CLN8 enables a non-canonical phospholipid synthesis pathway

According to text book knowledge, de novo glycerophospholipid (GPL) synthesis begins with the acylation of glycerol-3-phosphate to form phosphatidic acid, the precursor of all other GPLs. Here we describe an alternative GPL synthesis pathway that starts with the acyl-CoA-dependent acylation of glycerophosphoglycerol (GPG), resulting in the formation of lysophosphatidylglycerol (LPG). The acyltransferase reaction is catalyzed by the Batten disease-associated protein ceroid lipofuscinosis neuronal 8 (CLN8). Tracer studies revealed that CLN8-derived LPG is selectively converted into bis(monoacylglycero)phosphate (BMP), a GPL essential for lysosomal lipid homeostasis, but not into phosphatidylglycerol or cardiolipin. CLN8-knockout cells and mice cannot utilize GPG for BMP synthesis, resulting in BMP-deficiency and excess accumulation of phospholipids in lysosomes. The lipid synthesis pathway described herein is relevant for understanding lysosomal lipid metabolism and the pathogenesis of neurodegenerative diseases. BMP-deficiency may contribute to or even underlie lysosomal cargo accumulation in certain forms of Batten disease and other lysosomal storage disorders.

biochemistry↗

bioRxiv2025

Stereospecific GPG acylation by CLN8 drives BMP biosynthesis and its loss leads to Batten disease

Loss-of-function mutations in the endoplasmic reticulum membrane protein CLN8 cause Batten disease, a neurodegenerative lysosomal storage disorder1. Together with the lysosomal enzyme CLN5, CLN8 mediates the biosynthesis of bis(monoacylglycero)phosphate (BMP), a phospholipid essential for lysosomal function and distinguished by its unique S,S stereochemistry2,3. However, the role of CLN8 in BMP synthesis has remained unclear. Here we establish that CLN8 is a glycerophosphoglycerol (GPG) acyltransferase that catalyses the stereospecific acylation of S,S-GPG to produce S,S-lysophosphatidylglycerol (LPG), the CLN5 substrate in BMP synthesis. Using cryo-electron microscopy, we resolve structures of the CLN8 homodimer in apo and substrate-bound states at 2.7 [A] resolution, revealing the active site architecture and a ping-pong acyl transfer mechanism. Batten disease-causing missense mutations impair CLN8 enzymatic activity in vitro and reduce BMP levels in a Cln8R24G knock- in mouse, whereas the Cln8mnd mouse frameshift mutation causes complete loss of BMP in vivo. Exogenous S,S-LPG, but not the R,S stereoisomer, restored BMP synthesis in CLN8- deficient cells and mice, and improved neurological phenotypes in cln8 mutant zebrafish. Together, these findings define the enzymatic function of CLN8, elucidate the biochemical basis of CLN8 Batten disease, and establish a proof-of-concept for treating it through stereospecific BMP precursor supplementation.

biochemistry↗

bioRxiv2025

Accurate Prediction of ecDNA in Interphase Cancer Cells using Deep Neural Networks

Oncogene amplification is a key driver of cancer pathogenesis and is often mediated by extrachromosomal DNA (ecDNA). EcDNA amplifications are associated with increased pathogenicity of cancer and poorer outcomes for patients. EcDNA can be detected accurately using fluorescence in situ hybridization (FISH) when cells are arrested in metaphase. However, the majority of cancer cells are non-mitotic and must be analyzed in interphase, where it is difficult to discern extrachromosomal amplifications from chromosomal amplifications. Thus, there is a need for methods that accurately predict oncogene amplification status from interphase cells. Here, we present interSeg, a deep learning-based tool to cytogenetically determine the amplification status as EC-amp, HSR-amp, or not amplified from interphase FISH images. We trained and validated interSeg on 652 images (40,446 nuclei). Tests on 215 cultured cell and tissue model images (9,733 nuclei) showed 89% and 97% accuracy at the nuclear and sample levels, respectively. The neuroblastoma patient tissue hold-out set (67 samples and 1,937 nuclei) also revealed 97% accuracy at the sample level in detecting the presence of focal amplification. In experimentally and computationally mixed images, interSeg accurately predicted the level of heterogeneity. The results showcase interSeg as an important method for analyzing oncogene amplifications.

cancer biology↗

bioRxiv2025

Biocompatible Multi-functional Polymeric Material for Mineralized Tissue Adhesion

This study developed a biocompatible multifunctional thiol-ene resin system for adhesion to dentin mineralized tissue. Adhesive resins maintain the strength and longevity of dental composite restorations through chemophysical bonding to exposed dentin surfaces after cavity preparations. Dental pulp cells are exposed to residual monomers transported through dentinal tubules. Monomers of conventional adhesive systems may result in inhomogeneous polymer networks and the release of residual monomers that cause cytotoxicity. In this study, we develop a one-step multi-functional polymeric resin system by incorporating trimethylolpropane triacrylate (TMPTA) and bis[2-(methacryloyloxy)ethyl] phosphate (BMEP) to enhance both mechanical properties and adhesion to dentin. Molecular dynamics simulations identified an optimal triacylate:trithiol ratio of 2.5:1, which was consistent with rheological and mechanical tests that yielded a storage modulus of ~30 MPa with or without BMEP. Shear bond tests demonstrated that the addition of BMEP significantly improved dentin adhesion, achieving a shear bond strength of 10.8 MPa, comparable to the commercial primer Clearfil SE Bond. Nanoindentation modulus mapping characterized the hybrid layer and mechanical gradient of the adhesive resin system. Further, the triacrylate-BMEP resin showed biocompatibility with fibroblasts in vitro. These findings suggest the triacrylate-trithiol crosslinking and chemophysical bonding of BMEP provide enhanced bond strength and biocompatibility for dental applications.

bioengineering↗

bioRxiv2025

Myosin modulator Aficamten inhibits force in cardiac muscle by altering myosin's biochemical activity without changing thick filament structure

BackgroundInhibiting contractility by targeting cardiac myosin is an effective treatment for patients with hypertrophic cardiomyopathy (HCM). Aficamten is a second in class myosin inhibitor with promising clinical data showing improvements in hemodynamics and symptoms in patients with HCM. While it is known that Aficamten inhibits force and cardiomyocyte contractility by stabilizing the weak pre-powerstroke conformation, effects on myosin structure and kinetics during loaded contraction are lacking. MethodsPermeabilized porcine cardiac tissue and myofibrils were used for single-molecule imaging of ATP turn over, X-ray diffraction, and mechanical measurements. Engineered heart tissues from human induced pluripotent stem cell cardiomyocytes were used to evaluate effects on force and contraction kinetics. ResultsIn contrast to Mavacamten, Aficamten does not structurally sequester myosin heads along the thick filament. Aficamten inhibits ATPase activity by shifting myosin heads from higher to slower ATPase state, with the emergence of a super slow biochemical nucleotide turnover state. This results in decreased force and calcium sensitivity without altering cross-bridge cycling. These contractile mechanical changes are comparable to Mavacamten. Our myofibril mechanical assay showed inhibition of force with accelerated relaxation. In EHTs, while Mavacamten and Aficamten inhibit cardiac twitch forces, Mavacamten reduces the activation kinetics while both result in faster relaxation. ConclusionsWe used a combination of biochemical and biomechanical assays to show that Aficamten inhibits myosin ATPase without appreciably altering myosin structure. This is different from Mavacamten that strongly affects both. While both compounds inhibit contractility, differences in mechanisms of action and kinetics of force activation and relaxation could allow use in different patient populations.

biophysics↗

bioRxiv2025

Gut-Brain Hydraulics: Brain motion and CSF circulation is driven by mechanical coupling with the abdomen

The brain moves within the skull, but the drivers and function of this motion are not understood. We visualized brain motion relative to the skull in awake head-fixed mice using high-speed, multi-plane two-photon microscopy. Brain motion was primarily rostrally and laterally directed, and was tightly correlated with locomotion, but not with respiration or the cardiac cycle. Electromyography recordings in abdominal muscles and microCT reconstructions of the trunk and spinal vasculature showed that brain motion was driven by abdominal muscle contractions that activate a hydraulic-like vascular connection between the nervous system and the abdominal cavity. Externally-applied abdominal pressure generated brain motion similar to those seen during abdominal muscle contractions. Simulations showed that brain motion drives substantial volumes of interstitial fluid through and out of the brain (at volumetric rates several times higher than production) into the subarachnoid space, in the opposite direction of fluid flow seen during sleep. The brain is hydraulically linked to the abdominal compartment, and fluid flow in the brain is coupled to body movements, providing a mechanism by which the mechanics of the viscera directly impact brain health.

neuroscience↗

bioRxiv2025

Type-I nNOS neurons orchestrate cortical neural activity and vasomotion

It is unknown how the brain orchestrates coordination of global neural and vascular dynamics. We sought to uncover the role of a sparse but unusual population of genetically distinct interneurons known as type-I nNOS neurons, using a novel pharmacological strategic to unilaterally ablate these neurons from the somatosensory cortex of mice. Region-specific ablation produced changes in both neural activity and vascular dynamics, decreased power in the delta-band of the local field potential, reduced sustained vascular responses to prolonged sensory stimulation, and abolished the post-stimulus undershoot in cerebral blood volume. Coherence between the left and right somatosensory cortex gamma-band power envelope and blood volume at ultra-low frequencies was decreased, suggesting type-1 nNOS neurons integrate long-range coordination of brain signals. Lastly, we observed decreases in the amplitude of resting-state blood volume oscillations and decreased vasomotion following the ablation of type-I nNOS neurons. This demonstrates that a small population of nNOS-positive neurons are indispensable for regulating both neural and vascular dynamics in the whole brain, raising the possibility that loss of these neurons could contribute to the development of neurodegenerative diseases and sleep disturbances.

neuroscience↗

bioRxiv2025

An inherited mtDNA mutation remodels inflammatory cytokine responses in macrophages and in vivo

Impaired mitochondrial bioenergetics in macrophages can drive hyperinflammatory cytokine responses1-6, but whether this may also be caused by inherited mtDNA mutations is unknown. Here, we address this question using a multi-omic approach that integrates super-resolution imaging and metabolic analyses to profile macrophages from a mouse model of mitochondrial disease arising from a heteroplasmic mutation (m.5019A>G) in the mitochondrial tRNA for alanine7. These m.5019A>G macrophages exhibit defects in respiratory chain complexes and oxidative phosphorylation (OxPhos) due to decreased intra-mitochondrial translation. To adapt to this metabolic stress, mitochondrial fusion, reductive glutamine metabolism, and aerobic glycolysis are all increased. Upon inflammatory activation, type I interferon (IFN-I) release is enhanced, while the production of pro-inflammatory cytokines and oxylipins are restrained in m.5019A>G macrophages. Finally, an in vivo endotoxemia model using m.5019A>G mice reveal elevated IFN-I levels and sickness behaviour. In conclusion, our study identifies an unexpected imbalance in innate immune signalling in response to a pathogenic mtDNA mutation, with important implications for the progression of pathology in patients with mtDNA diseases8.

immunology↗

bioRxiv2024

Mitochondrial adenine base editing of mouse somatic tissues via adeno-associated viral delivery

The development of adenine base editing in mitochondria, alongside cytidine base editing, has significantly expanded the genome engineering capabilities of the mitochondrial DNA. We tested the recent advancements in adenine base editing technology using optimised TALEs targeting genes Mt-Cytb, Mt-CoII and Mt-Atp6 in mouse cells, and observed successful A:T to G:C conversions within the target windows of each gene. We then used the best-performing Mt-Atp6 pairs for systemic AAV9 delivery to neonatal mice and quantified editing in somatic tissues after 4 weeks and 6 months. Adenine editing was low at 4 weeks and increased only modestly after 6 months, indicating that prolonged exposure alone is insufficient to overcome the limited activity of the editor architectures tested here in vivo. These findings establish the feasibility of AAV-delivered mitochondrial A-to-G editing while defining important limitations that require further optimisation.

bioengineering↗

bioRxiv2024

Dorsomedial striatal neuroinflammation causes excessive goal-directed action control by disrupting homeostatic astrocyte function

Compulsive actions are typically thought to reflect the dominance of habits over goal-directed action. To address this, we mimicked the striatal neuroinflammation that is frequently exhibited in individuals with compulsive disorders in rats, by injecting the endotoxin lipopolysaccharide into the posterior dorsomedial striatum, and assessed the consequences for behavioural control. Surprisingly, this manipulation caused rats to acquire and maintain goal-directed actions under conditions that would otherwise produce habits. Immunohistochemical analyses indicated that these behaviours were a result of astrocytic proliferation. To probe this further, we chemogenetically activated the Gi-pathway in striatal astrocytes, which altered the firing properties of nearby medium spiny neurons and modulated goal-directed action control. Together, results show that striatal neuroinflammation is sufficient to bias action selection toward excessive goal-directed control via dysregulated astrocyte function. If translatable, our findings suggest that, contrary to conventional views, individuals with striatal neuroinflammation might be more prone to maladaptive goal-directed actions than habits, and future interventions should aim to restore appropriate action control.

animal behavior and cognition↗