Biology subjects

Johnson, S.

Publications and source records attributed to Johnson, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Locus-specific spatial and transcriptional dynamics direct T cell lineage choice in the thymus

Decades of investigation have made the choice between CD4 and CD8 T cell lineages in the thymus a key paradigm for mammalian cell commitment and differentiation, yet how transcriptional diversification is coordinated with 3D genome organisation remains largely unexplored. Here, using single-molecule transcript quantification and region capture Pico-C (RCPC), we resolve distinct transcriptional and spatial chromatin dynamics at the lineage-defining loci Cd4 and Cd8. We find early Cd4/Cd8 transcriptional divergence driven by MHC-dependent bias in Cd8a transcript copy number, which unfolds gradually across both CCR7- and CCR7+ differentiation stages. Unexpectedly, Cd4 and Cd8 loci show fundamentally different spatial contact dynamics: transiently reduced mesoscale (10-100kb) contacts at Cd8 provide a permissive chromatin environment throughout the transcriptional diversification process. By contrast, Cd4 undergoes switch-like contact changes limited to the microscale (1kb) late in differentiation, associated with lineage stabilization by ThPOK and/or RUNX3. These results place distinct, locus-specific chromatin environments at the centre of transcriptional diversification during T cell lineage choice.

genetics↗

bioRxiv2026

The mammalian-adaptive PB2-E627K substitution preserves viral fitness of clade 2.3.4.4b H5N1 HPAIV in birds.

Since emerging in 2020, clade 2.3.4.4b H5N1 high pathogenicity avian influenza virus (HPAIV) has disseminated globally, causing substantial infection and mortality in wild birds and poultry. Additionally, numerous spill-over events into mammals have occurred, including mass mortalities and sporadic human infections. Such events can drive the acquisition of mammalian-adaptive mutations, which further increase the risk to humans; the most well characterised mutation is the glutamate to lysine change at amino acid position 627 in the polymerase basic 2 protein (PB2-627K). Across avian influenza viruses, PB2-627K enhances polymerase activity and replication in mammalian cells, increasing zoonotic and pandemic potential. However, bioinformatic analyses indicate that PB2-627K remains rare among clade 2.3.4.4b H5N1 viruses circulating in avian populations. Here, we investigated the impact of PB2-627K on viral fitness in avian hosts to assess the potential for mammalian-adapted viruses to re-establish in birds, mimicking infection of avian species following scavenging on mammalian species where PB2-627K has already emerged. Using a representative H5N1 virus (European genotype AB), PB2-627K increased polymerase activity and replication in human cells without compromising replication in avian cells. A mammalian-derived PB2-627K H5N1 isolate from a fox exhibited comparable replication kinetics and transmission efficiency in chickens and ducks to a closely related PB2-627E virus. Notably, PB2-627K remained genetically stable at consensus level following infection and transmission in both avian hosts. These findings demonstrate that mammalian-adaptive mutations acquired during spill-over can be maintained in avian populations, potentially elevating zoonotic risk and underscoring the importance of surveillance for such mutations in birds.

microbiology↗

bioRxiv2026

Regional cerebral arterial elasticity is associated with cardiovascular health in cognitively healthy older adults

Ageing is often associated with a decline in cardiovascular and cerebrovascular health. This study examines the relationship between cerebral arterial elasticity and measures of cardiovascular health, as well as longitudinal changes in cerebral arterial elasticity over a 1.5 year interval. The pulse relaxation function (PReFx) is a measure of regional cerebral arterial elasticity derived using diffuse optical tomography (pulse-DOT). PReFx was measured over the anterior brain, including the frontal lobes and anterior sections of temporal and parietal regions that are especially vulnerable to vascular and cognitive ageing. We examined relationships between PReFx and measures of four cardiovascular risk factors (CVRF; i.e., hypertension, cholesterol, diabetes, obesity), as well as CVRF burden (i.e., number of CVRFs) in the highly active and cognitively healthy ACTIVate cohort (60-70 years). We replicated the well-established relationship between PReFx and both age and cardiorespiratory fitness, and examined associations between PReFx, measures of cardiovascular health and CVRF burden. Higher CVRF burden was linearly associated with lower cerebral arterial elasticity. The relationship between age and cerebral arterial elasticity was partially mediated by pulse pressure, an index of hypertension. PReFx declined significantly in as little as 1.5 years, and the effect did not vary with baseline level of any of the four CVRFs. We conclude that PReFx shows promise as a putative biomarker for monitoring cerebrovascular ageing in healthy older adults.

neuroscience↗

bioRxiv2026

Spatial Transcriptomic Profiling Reveals Microenvironment-Dependent Immune Signatures in a Lyme Arthritis model

Lyme arthritis, a manifestation of Lyme disease, is triggered by the spirochetal bacterium Borrelia burgdorferi (Bb), which is transmitted through the bite of the Ixodes tick. Although multiple studies have been conducted on the complex host immune response in Lyme arthritis, the spatial gene expression environment in the joint tissue remains unexplored. Here, we applied spatial transcriptomics to ankle joints of C3H mice infected with Bb, profiling tissues at peak inflammation (two weeks post infection) and after antibiotics (four weeks post-infection) during inflammation resolution. Analysis revealed spatially restricted signatures: pro-inflammatory responses dominated synovial and fibroblast populations two weeks post-infection, with elevated levels of Vimentin and I-Ek gene - and Vimentin protein - expression localized to these regions. By four weeks post-infection during the inflammation resolution phase, levels of Vimentin and I-Ek related gene and protein expression were reduced. Further, we noted an increase in the CD54+ and CD106+ double-positive population in infected mice joints compared to the vehicle treated controls. Notably, fibroblasts and synoviocytes in the medial joint regions adopted immune-like phenotypes during peak inflammation, while the same cell types in the exterior humeroradial joint displayed a more infection-resilient phenotype. These spatially resolved maps demonstrate that joint microenvironments play a crucial role in pathogenesis, offering unique insights into Lyme arthritis pathology.

microbiology↗

bioRxiv2026

Structure of the proton-powered secretion motor at the heart of the bacterial flagellum

Bacterial type III secretion systems export proteins across the inner membrane using the proton-motive force (PMF), but how proton flow is coupled to opening of the export channel is unknownXX,XX. Here we present single-particle cryo-EM structures at 2.5-4.2 [A] resolution of the intact flagellar Export Apparatus from Salmonella enterica, obtained using optimized extraction conditions of the endogenous assembly that retain the complete transmembrane complex. The transmembrane domain of FlhA forms a nonameric funnel-shaped basket beneath the Export Gate, with each subunit harboring a buried pathway of conserved hydrophilic residues spanning the hydrophobic core of the membrane. FlhB is resolved for the first time within the intact gate, revealing helices that thread through the FlhA channel, completely sealing it at rest. A symmetry-free reconstruction captures one FlhA protomer hinged outward at the water-filled cavity, breaking the rotational symmetry. Together, these structures define the architecture of the proton-transducing element of the type III secretion system and suggest a rotary gating mechanism, with parallels to the F0 motor of ATP synthase, in which PMF-driven conformational cycling of FlhA drives regulated opening of the export channel.

microbiology↗

bioRxiv2026

A new Persister Enrichment Model in Mycobacterium tuberculosis links multidrug persistence to drug sequencing and maltokinase-dependent carbon distribution.

Prolonged combination chemotherapy is required to cure Tuberculosis, in part because Mycobacterium tuberculosis (Mtb) forms antibiotic-tolerant persister cells that survive exposure to bactericidal drugs. Experimental systems to study multidrug persistence in Mtb remain limited. Here, we describe a rapid and tractable Persister Enrichment Model (PEM) in which removal of glycerol markedly increases the frequency of multidrug-tolerant Mtb cells that survive exposure to high concentrations of bactericidal antibiotics with distinct mechanisms of action. Persister enrichment in PEM depends on carbon source identity rather than carbon abundance and enables direct interrogation at the transition from active growth into multidrug tolerance. Using PEM, we show that the temporal order of isoniazid (INH) and rifampicin (RIF) exposure shapes the magnitude of multidrug persistence. A transposon sequencing screen under combination drug pressure identified maltokinase (mak) as a major determinant of multidrug persistence. Deletion of mak enhanced susceptibility to INH+RIF in PEM and impaired bacterial survival during the persistent phase of infection in mice. Metabolic analyses reveal that loss of mak destabilizes carbon distribution during co-catabolism of glycolytic carbon sources, leading to defective growth and antibiotic persistence. This defect is ameliorated by inactivating mutations in glucose-1-phosphate adenylyltransferase (glgC), which alleviate the buildup of ADP-glucose. Together, these findings establish PEM as a practical platform to study multidrug persistence, reveal that drug sequencing can influence entry into multidrug tolerance, and identify maltokinase-dependent carbon distribution as a metabolic vulnerability that constrains Mtb survival. ImportanceTuberculosis treatment requires prolonged multidrug chemotherapy in part because Mycobacterium tuberculosis forms antibiotic-tolerant persister cells that survive bactericidal drug exposure. However, experimental systems to study multidrug persistence remain limited, and the metabolic mechanisms underlying this phenotype are poorly understood. Here, we establish a rapid and experimentally tractable Persister Enrichment Model (PEM) that enriches multidrug-tolerant M. tuberculosis populations by removing glycerol from culture medium. Using this model, we demonstrate that the order of antibiotic exposure influences multidrug persistence both in vitro and in vivo and identify maltokinase-dependent carbon distribution as a critical determinant of persistence and long-term bacterial survival. These findings provide a new platform for studying multidrug tolerance and reveal carbon buffering pathways as potential therapeutic vulnerabilities that could help shorten tuberculosis treatment.

microbiology↗

bioRxiv2026

2D Skeletal Muscle Thin Film Actuators Enhance Efficiency of Biohybrid Robots

Biohybrid robots combining compliant synthetic support structures with biological actuators could enable future applications ranging from precision microsurgery to unmanned exploration. Machines actuated by living skeletal muscles are capable of adaptive behaviors, such as sensing and responding to environmental stimuli in real-time, offering functional advantages over non-biological actuators. However, typical skeletal muscle-powered biohybrid robots depend on 3D tissues which require large cell volumes and offer limited control of muscle fiber alignment, thus reducing efficiency of force generation and transduction. Here, we present a locomotive biohybrid robot powered by 2D monolayers, or thin films, of precisely aligned skeletal muscle fibers on a micropatterned hydrogel skeleton. We demonstrate how varying skeleton design parameters, ranging from material stiffness to microscale topology, impacts muscle fiber alignment and resultant actuation strains, generating forces 10X higher than previous 2D skeletal muscle actuators, improving untethered actuation longevity by [~]4500X from < 10 minutes to > 30 days, and increasing efficiency of muscle force output (force per unit volume of muscle) by 20X as compared to 3D muscles. Utilizing our optimized design for skeletal muscle thin films, we create a multi-limbed robot composed of independent muscle-powered fins capable of on/off control and frequency-dependent speed control. With these control inputs, we achieve steered multi-directional locomotion at speeds up to 4 body lengths per minute in straight movement and 1200 degrees per minute in rotational movement, highlighting potential for such actuators to be transformed into long-lasting functional soft robots.

bioengineering↗

bioRxiv2026

Temporal Dynamics of BOLD fMRI Predict Intracranially-Confirmed Seizure Onset Zones in Drug-Resistant Epilepsy

ObjectiveIn individuals with drug-resistant epilepsy, accurately identifying the brain regions where seizures originate is a critical prerequisite to guide surgical treatment and achieve seizure freedom. To accomplish this, intracranial EEG is considered the gold standard, providing the spatiotemporal high-resolution data necessary to pinpoint epileptogenic activity. However, this precision is achieved through an invasive procedure with significant patient burden, which is fundamentally limited by the electrode placement and spatial coverage. MethodsIn this study, we investigated the potential utility of preoperative resting-state fMRI to non-invasively map alterations in brain dynamics at the whole brain level. Region-wise brain dynamics were quantified with complementary measures of local autocorrelation decay rates. We assessed the capacity of these derived features to effectively identify intracranial EEG confirmed seizure onset zones in 18 individuals with drug-resistant medial temporal lobe epilepsy. Overall, the study cohort contained 3867 implanted electrodes of which 159 classified as seizure onset zones by two independent board-certified epileptologists. ResultsOverall, our findings reveal more constrained temporal dynamics for brain regions associated with seizure onsets compared to non-seizure onset zones. Individual-level prediction showed a performance better than chance in 15 of the 18 patients. The overall predictive performance across all patients yielded a median AUC of 0.81, a median true positive rate of 0.75, and a median true negative rate of 0.83. Furthermore, in a subset of 13 patients, those with negative seizure outcomes showed higher probabilities of seizure onset zone predictions outside the resection area compared to those with good outcomes. SignificanceOverall, our findings suggest that altered temporal dynamics derived from preoperative resting-state fMRI represent a promising non-invasive approach for delineating epileptogenic tissue, potentially informing intervention strategies and guiding electrode placement.

neuroscience↗

bioRxiv2026

PFOA induced metabolic and immune perturbations in a SARS-2 infection model

This study evaluates the impact of PFOA exposure on the metabolome and immune response to SARS-2 using a ferret model. Ferrets were separated into control or PFOA-exposed groups (10/mg/kg/day) and challenged with SARS-2. Longitudinal analyses encompassing clinical assessments, serological profiling, histopathology, and untargeted nuclear magnetic resonance (NMR) metabolomics revealed significant metabolic and immunological perturbations. We found prominent effects of PFOA exposure on metabolism, which resulted in altered metabolic responses to SARS-2 infection. PFOA exposure was also associated with impaired immune function, as evidenced by decreased serum IgG levels, increased viral loads, and prolonged peak infectivity. These findings underscore the consequences of PFOA exposure on host metabolism and immunity during infectious diseases.

pharmacology and toxicology↗

bioRxiv2026

Evaluating the applicability of kinship analyses for sedimentary ancient DNA datasets

Kinship reconstruction in ancient populations provides key insights into past social organization and evolutionary history. Sedimentary ancient DNA (sedaDNA) enables access to deep-time human populations in the absence of skeletal remains. However, it is characterized by severe degradation and the potential mixture of genetic material from multiple individuals, raising questions about its suitability for kinship inference. Here, we use extensive simulations to evaluate the feasibility and limitations of kinship inference in sparse and damaged sedaDNA data, with a focus on Neandertals. We find that the main obstacle to accurate kinship inference in sedaDNA is the presence of multiple contributors to a given sample. To address this, we introduce a simple heterozygosity-based test to identify samples containing DNA from multiple individuals. Guided by these results, we analyze published Neandertal sedaDNA from the Galeria de las Estatuas site to assess the practical limits of kinship inference in real sedimentary ancient DNA data. Together, our results define methodological considerations and practical limits for kinship inference in sedimentary ancient DNA.

bioinformatics↗

bioRxiv2025

Musical training improves planning and robustness of sequence learning

Musicians demonstrate advantages in acquiring motor sequences, showing faster learning and better explicit sequence knowledge than non-musicians. However, it is unclear whether this advantage extends beyond acquisition to the consolidation phase, which is when newly learned skills stabilize and become resistant to interference. Additionally, while interference from executing competing motor tasks is well-established, less is known about whether purely sensory information presented after learning can disrupt consolidation of a bimodal motor sequence. We investigated how post-acquisition sensory interference affects performance of a learned audio-visual sequence, and whether musical training moderates this vulnerability. Participants first learned an explicit sequence in a serial reaction time task using synchronous, informative audio-visual cues. After a brief consolidation period, they were randomly assigned to one of four observational conditions that manipulated the relationship between auditory and visual streams. Motor performance was then reassessed. Post-acquisition sensory interference impaired subsequent motor performance, but this effect was modality-specific: it was driven primarily by manipulations to the task-relevant visual stream, while auditory interference alone had no credible effect. Distributional analysis revealed that learning involved a strategic shift from reactive to anticipatory responding. Critically, participants with musical training showed an earlier and consistently higher reliance on anticipatory responses than those without, demonstrating a more rapid adoption of predictive motor control. These findings demonstrate that newly formed sensorimotor memories are selectively vulnerable to interference in task-relevant modalities. Furthermore, our work provides a candidate mechanistic account for the musician advantage in sequence learning, linking it to faster development of predictive motor strategies during consolidation.

neuroscience↗

bioRxiv2025

Region-specific associations between cerebral arterial elasticity and grey matter volume: Evidence for lateral prefrontal vulnerability

BackgroundAgeing is associated with increased cardiovascular health risks and disproportionate atrophy in frontotemporal brain. Regional cerebral arterial elasticity correlates with regional grey matter volume, with stronger associations frontally and in older adults. Cardiorespiratory fitness (CRF) is linked to preserved brain structure and greater arterial elasticity, while sex differences exist in the timing of vascular versus structural changes. This study examines whether regional cerebral arterial elasticity in frontotemporal cortex mediates the association between age and corresponding grey matter volume decline, and whether sex and/or CRF moderate this relationship. MethodsWe analysed data from 162 healthy adults (60-70 years) with structural MRI and diffuse optical tomography of the cerebral arterial pulse (Pulse-DOT) from the ACTIVate cohort. CRF was estimated from demographic and physiological measures. Pulse Relaxation Function (PReFx), an optical index of regional arterial elasticity, was measured across 28 frontal, temporal, and parietal regions of interest (ROI). Grey matter (GM) volume was quantified for corresponding ROIs. Mediation and moderated mediation models tested whether PReFx mediated the relationship between age and GM volume at bilateral frontal and temporal ROIs, and whether biological sex or CRF moderated this relationship. ResultsRegional bivariate correlations identified associations between age, PReFx and GM volume across multiple ROIs, which PReFx and GM volume being associated primarily in left frontal and temporal areas. PReFx partially mediated the effect of age on GM volume in a left mid-inferior frontal ROI, accounting for approximately 16% of the total age effect and this relationship was only evident in females. Although higher CRF was associated with greater PReFx, it did not moderate the relationship between age, PReFx and GM volume. ConclusionsConsistent with cascade models of neurovascular aging, cerebral arterial stiffening was found to partially explain the effect of increasing age on frontal GM volume, even in this highly age-restricted and high functioning cohort. This effect was only significant over the left prefrontal cortex, consistent with greater vulnerability of frontal brain and associated cognitive functions. It was also exclusively present in females, who had better cardiovascular health, larger grey matter volume and greater arterial elasticity than males. These findings are consistent with pulse-DOT measures of cerebral arterial elasticity being more sensitive to subclinical brain structural variability.

neuroscience↗

bioRxiv2025

Elucidating an anterior cingulate circuit for self-initiated actions and rescue of Parkinsonian akinesia

Dopamine (DA) depletion is known to result in Parkinsonian symptoms such as the inability to initiate movements (akinesia). While Parkinsonian akinesia is traditionally associated with reduced DA signaling in the striatum, the contribution of cortical regions that also receive DA projections and project to the striatum remains unclear. Here, we identify a previously unexplored cortical circuit involving D1-like dopamine receptor-expressing neurons in the anterior cingulate cortex (ACC) that is critical for initiating goal-directed movements. We find that a selective activation of ACC-D1+ neurons can flexibly drive targeted movement and locomotion even in akinetic mice after dopamine depletion or receptor antagonism. These findings uncover a cortical mechanism for movement initiation and offer promising new therapeutic targets for treating Parkinsonian akinesia.

neuroscience↗

bioRxiv2025

Structural insights into mitotic-centrosome assembly

Centrosomal material assembles rapidly in mitosis. In Drosophila, the coiled-coil protein Cnn forms a scaffold that recruits PCM clients; in C.elegans, SPD-5 plays an analogous role. Here we show that full-length Cnn and SPD-5 can both form spherical condensates in vitro, but that the interactions driving their assembly into scaffolds inside cells appear to diverge. We show that the Cnn PReM adopts a helical hairpin fold that autoinhibits CM2 binding but that phosphorylation appears to increase hairpin breathing to permit CM2 engagement and robust scaffold assembly. Phospho-blocking mutations prevent PReM-CM2 interactions and scaffold formation, whereas phospho-mimetic substitutions partially restore function. The human homologue CDK5RAP2 contains a CM2 domain that can partially substitute for fly CM2 in vivo and we identify a candidate CDK5RAP2 PReM region that forms macromolecular networks with human CM2 in vitro. By contrast, the putative PReM and CM2 regions of SPD-5 cannot substitute for their equivalent fly domains and they do not interact detectably, suggesting a distinct assembly mechanism in worms despite conserved PLK1-dependent control of PCM growth.

cell biology↗

bioRxiv2025

Preclinical efficacy of combinatorial B7-H3 CAR T cells and ONC206 against diffuse intrinsic pontine glioma

BackgroundDiffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brain tumor affecting over 300 children annually in the United States. Chimeric antigen receptor (CAR) T cells are a targeted immune effector cell therapy with substantial clinical benefit against hematologic cancers. Against CNS tumors, CAR T cells targeting B7-H3, a protein highly expressed on DIPG, have rapidly advanced from preclinical studies to clinical trials. BrainChild-03 (NCT04185038), a phase 1 trial of repeatedly delivered intracerebroventricular (ICV) B7-H3-targeting CAR T cells (B7-H3 CAR T cells), demonstrated tolerability and potential efficacy for children and young adults with DIPG. However, clinical benefits were not uniformly seen, and multi-agent treatment strategies may be required against such an aggressive disease. Here, we combined B7-H3 CAR T cells with ONC206, an imipridone molecule also under clinical investigation. MethodsWe tested B7-H3 CAR T cells combined with ONC206 across multiple DIPG cell cultures and orthotopic xenograft mouse models. ResultsB7-H3 CAR T cell monotherapy induced robust cytotoxicity while ONC206 treatment resulted in significant mitochondrial dysfunction against DMG/DIPG cells. The combination of low effector-to-target ratios of B7-H3 CAR T cells and IC50 concentrations of ONC206 led to significantly enhanced cytotoxicity in vitro (p<0.003) and increased IL-2, IL-29, VEGF-A, and Granzyme B levels. In vivo combinatorial studies of ONC206 and a single ICV dose of B7-H3 CAR T cells significantly extended survival in multiple DIPG xenograft mouse models (p<0.05). ConclusionsB7-H3 CAR T cells combined with ONC206 is a feasible and efficacious multi-agent approach against multiple DIPG models. Importance of the studyDiffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brain tumor. While B7-H3 CAR T cells have shown tolerability and potential benefit in early trials, combinatorial regimens may be required for consistent cures against this aggressive disease. This study demonstrates that a preclinical therapeutic regimen of B7-H3 CAR T cells with ONC206, a second-generation imipridone, increases anti-tumor efficacy in vitro and in orthotopic DIPG mouse models. To our knowledge, this is the first study to evaluate ONC206 in combination with CAR T cells. Our findings provide a preclinical roadmap for evaluating small molecules with CAR T cells to interrogate both their combined benefit and the effect of small molecules on T cells themselves. This work offers a biologically-informed, clinically translatable strategy integrating small molecule therapeutics with CAR T cell therapy and support the development of multi-agent immunotherapy trials for children with DIPG and other high-grade brain and spinal cord tumors. Key PointsO_LIB7-H3 CAR T cells are cytotoxic against preclinical DMG models. C_LIO_LIONC206 causes metabolic apoptosis in preclinical DMG models. C_LIO_LIB7-H3 CAR T cells and ONC206 have combinatorial efficacy against DMG. C_LI

cancer biology↗

bioRxiv2025

Electronic Actuation of Surface-Immobilized, pH-Responsive DNA Nanoswitches

Dynamic DNA machines exploit the specificity of base pairing and/or sensitivity to the local environment to control the reversible switching of DNA constructs between conformational states. One such example are pH-sensitive DNA nanoswitches that can be actuated by proton-mediated Hoogsteen interactions within a DNA triplex domain. To date, studies of pH-sensitive DNA nanoswitches have largely focused on DNA machines that are freely diffusing in the solution phase. For many applications, it is advantageous to integrate these dynamic DNA machines with solid-state devices, requiring immobilization on surfaces. Here, we explore the switching of a pH-sensitive DNA triplex immobilized on a surface as a dense, 2-dimensional DNA monolayer. DNA nanoswitches were assembled onto surfaces via thiol chemistry and pH-controlled conformational switching of the constructs examined using quartz crystal microbalance with dissipation monitoring (QCM-D). These QCM-D experiments indicate that despite the high density of DNA within the monolayer (1012 molecules/cm2), pH-switching between open and closed states is retained following immobilization. Moreover, conformational switching of DNA constructs within the monolayer remains highly reversible and repeatable, with negligible reduction in switching efficiency observed over 20 switching cycles. DNA switching experiments were also performed in the solution phase using single-molecular Forster resonance energy transfer (smFRET) and circular dichroism (CD) techniques to confirm their pH responsitivity. Finally, we demonstrate electrically driven, localised, and addressable switching of the DNA triplex by employing electrochemical reduction and oxidation of water at an electrode surface, further demonstrating the potential of the technology for surface-immobilized dynamic DNA machines. This study not only provides insight into the actuation of DNA machines on-surface but also supports the development of new technologies such as hybrid electronic-DNA technologies able to store and process information using both molecular and electronic inputs.

biophysics↗

bioRxiv2025

Identification of a volatile chemotype associated with resilience to water stress in domesticated varieties of Cotton

Volatile organic compounds (VOCs) mediate plant responses to environmental stresses, yet their chemotypic variation and drought responsiveness remain poorly characterized in domesticated crops. Here, we identify a previously undocumented binary chemotype structure in cultivated upland cotton (Gossypium hirsutum), defined by the mutually exclusive production of either bisabolene or guaiene sesquiterpenes. Across 24 genotypes grown under field conditions, genotypes consistently produced one of these compounds, but never both, revealing a cryptic chemotypic polymorphism with physiological consequences. While drought stress reduced leaf water content across all plants, plants in the guaiene chemotype exhibited disproportionately greater losses. Water limitation also reduced total monoterpene and sesquiterpene concentrations, with the bisabolene chemotype showing stronger declines despite higher constitutive levels. Chemotypes further diverged in their biosynthesis of key damage-induced green leaf volatiles (GLVs). While the guaiene chemotype reduced GLV biosynthetic capacity under prolonged drought, the bisabolene chemotype showed transient increases in GLVs and pathway-specific enzyme activity under short-term stress, suggesting differential physiological resilience. Notably, all genotypes in this study fell within the low {gamma}-terpinene category previously described in wild cotton, indicating that this bisabolene/guaiene variation represents a novel axis of chemical diversity distinct from known wild chemotypes. These findings position chemotypic variation as a promising, non-destructive biomarker for drought response in cotton breeding programs. By linking chemotype to constitutive VOCs and water status, our work provides new tools for identifying stress-resilient cultivars and advances understanding of phytochemical strategies for crop management in a changing climate.

plant biology↗

bioRxiv2025

A live tumor fragment platform to assess immunotherapy response in core needle biopsies while addressing challenges of tumor heterogeneity

BackgroundImmune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, providing durable and even curative responses. However, most patients do not respond and current biomarkers (eg, programmed death ligand (PD-L1), mismatch repair deficiency (dMMR)/high microsatellite instability (MSI) and tumor mutational burden) lack predictive accuracy. Ex vivo profiling of patient-derived tumor fragments shows promise as a predictive biomarker but relies on substantial surgical tissue to mitigate intra-specimen heterogeneity. Innovations are needed that address these challenges, particularly where limited tissue is available in core needle biopsies (CNBs). MethodsLive tumor fragments (LTFs) were generated from 59 human tumor resections and 31 CNBs from patients enrolled in observational clinical trials (ClinicalTrials.gov identifiers: NCT05478538, NCT05520099, NCT06349642) to assess cytokine induction following ICI treatment. LTFs were encapsulated in hydrogel and cultured ex vivo for up to 72 hours. A sequential treatment strategy that applies control and treatment within the same well was used with response to ICI or CD3/CD28 assessed using a multiplex secretome assay. Viability was assessed using established metabolic assays and dynamic optical coherence microscopy. ResultsLTFs maintained viability and retained T cells responsive to stimulation throughout ex vivo culture. Multiplex immunofluorescence and immunohistochemistry showed key components of the tumor microenvironment, including relative proportions of CD4+ and CD8+ immune cell populations, were preserved. Specimens positive for PD-L1 or dMMR/MSI-high were enriched for cytokine upregulation, including T-cell response cytokines IFN{gamma} and CXCL10, after PD-1 treatment. To demonstrate clinical applicability of the sequential treatment strategy, CNBs from patients with lung, gastrointestinal or kidney cancer were profiled and differential cytokine induction in response to ICI treatment was observed. ConclusionsThe novel ex vivo platform presented is capable of detecting T-cell response to ICI treatment by using a sequential treatment strategy. This approach addresses challenges associated with cross-well heterogeneity in tissue composition and requires half as much tissue as a cross-well comparison, mitigating tissue limitations typically associated with non-surgical biopsies. Importantly, the platform is compatible with established functional assays as well as non-destructive spatial imaging, enabling researchers to characterize response to ICI longitudinally. Ongoing trials will enable clinicians to assess platform performance in predicting response to immunotherapy.

cancer biology↗