Biology subjects

Martinez, J.

Publications and source records attributed to Martinez, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Lysosomal adaptation to extracellular acidity compromises CD8+ T cell cytotoxicity

Extracellular acidosis is a hallmark of aggressive tumors and suppresses cytotoxic-T lymphocyte (CTL)-mediated killing, but how sustained acid exposure reshapes T cell cytotoxicity is poorly understood. Here we show that chronic acidosis drives lysosomal remodeling in human CD8+ T cells, converting the lysosome into a proton sink whose adaptation ultimately impairs killing by depleting the intracellular granzyme B reservoir through constitutive vesicle release. Quantitative proteomics of CD8+ T cells cultured at pH 6.5 identified lysosomes as the dominant target of acid-driven remodeling: CTLs upregulated the V1 sector of the vacuolar H+-ATPase and hyper-acidified the lysosomal lumen. The remodeled compartment accumulated lysosomal membrane proteins (LAMP1, LAMP2, CD63, CD68) post-transcriptionally, acquired higher cathepsin abundance and increased proteolytic activity. Chronic acidity also enlarged endoplasmic reticulum (ER) and lysosomal Ca2+ stores without altering cytosolic Ca2+, and drives constitutive, Ca2+-dependent release of LAMP1+ lysosome-derived vesicles, which drains the intracellular reservoir of granzyme B, contributing to reduced CTL activity. These findings establish lysosomal adaptation as a cost of surviving extracellular acidosis and identify lysosomal hydrolase activity as a possible target for improving CTL function in the acidic tumor environment.

immunology↗

bioRxiv2026

Myopathy-causing variants of PYROXD1 converge on impaired RNA ligation

RNA ligation is critical for RNA processing pathways including pre-tRNA splicing. RTCB is the only essential RNA ligase in humans and the catalytic subunit of the tRNA ligase complex (tRNA-LC). Its active site is sensitive to oxidation and strictly depends on oxidoreductase PYROXD1, the only tRNA-LC subunit with multiple known pathogenic protein-coding variants. The role of RNA ligation in PYROXD1 myopathy remained contentious: loss of PYROXD1 primarily affects mitochondria, and ligation-linked readouts are inconsistent in existing assays. Here, we reveal that PYROXD1 is physically, genetically and functionally committed to the tRNA-LC. Selectively impairing RTCB activation reproduces hallmarks of PYROXD1 dysfunction, including mitochondrial deregulation. Using degron-based depletion of endogenous PYROXD1, we show that all six single amino acid variants reported in patients fail to sustain RNA ligation in proportion to their clinical severity. The variants segregate into three mechanistic classes: they lower protein stability, disrupt the NADPH-mediated conformational switch, or impair RTCB engagement by the C-terminal tail, a sequence unique to PYROXD1 and directly affected in the most severe variant D500N. Variant-level measurements in the degron model predict outcomes in homozygous and compound heterozygous patient fibroblasts. Together, these data establish impaired RNA ligation as the driver of PYROXD1 pathogenicity.

biochemistry↗

bioRxiv2026

A glyoxal sensing Pseudomonas aeruginosa transcription factor enables lung infection

Aldehydes are a class of normally unwanted toxic electrophilic compounds that mainly arise from oxidation of glucose, lipids or DNA. However, it has recently come to light that they can also be weaponized by professional phagocytes to kill engulfed bacteria. How microbes subvert these assaults remains largely enigmatic. Here we describe the function, atomic structure and mechanism of the first bacterial transcription factor able to directly sense the dicarbonyl glyoxal (GO), which we aptly named the Glyoxal Regulator (GloR, from Pseudomonas aeruginosa PAO1), We show that GloR directly senses GO through a reversible cysteine modification that results in its binding to a conserved DNA regulatory motif (a glo box), which then triggers a transcriptional activation of a defined set of genes to help counter GO toxicity and enable acute lung infection. Despite substantial evolutionary divergence, when unmodified gloR and a glo box-regulated reporter were transferred into E. coli, a strikingly tight GO-specific regulation was maintained, suggesting this system could be readily transferred between unrelated microbial species. As homologs of GloR were identified in diverse bacterial species we anticipate its use to be widespread in both pathogens and environmental bacteria. Taken together, we present the first bona fide bacterial aldehyde regulator which senses host GO to enable survival during infection.

microbiology↗

bioRxiv2026

A Mecom—Cdk6 toggle switch governs hematopoietic stem-to-multipotent fate transitions via distinct multistable landscapes

Hematopoietic stem cells are required to regenerate the blood system throughout life. We previously discovered that transitions between quiescent stem and multipotent progenitor states are controlled by mutual inhibition between Mecom and Cdk6, with upstream regulation from the insulin-like growth factor (IGF) signaling pathway. To investigate the dynamics of exit from quiescence and the stem-to-multipotent cell state transition, we modeled the Mecom-Cdk6 regulatory network via coupled nonlinear differential equations. Bifurcation analysis revealed that the model permits tetrastability, with two stable intermediate states, suggesting that stem cell exit from quiescence proceeds via multiple fine-scale transitions. Perturbation of Mecom self-activation reorganized the multistable landscape, producing two IGF-dependent landscapes with distinct geometries. At high IGF, transitions proceeded only through an intermediate state, whereas at low IGF, a distinct landscape emerged permitting direct transitions between cell states. Stochastic simulations and minimum action path analysis showed that the multipotent attractor is deep at high IGF, whereas low IGF promotes a transition to quiescence by stabilizing the stem cell state. Simulated pharmacological intervention via CDK4/6 inhibitors destabilized the multipotent state and favored transitions towards a more stem-like quiescent state. Together, these results demonstrate how IGF signaling, Mecom self-activation, and Cdk6 inhibition jointly shape early stem cell fate decisions by dictating the accessible cell states on multistable landscapes and the transition paths that connect them.

Systems Biology↗

bioRxiv2026

Evolutionary genomics of a natural Wolbachia superinfection in the dengue mosquito Aedes albopictus

Aedes albopictus, an invasive mosquito vector of human pathogens, is naturally infected by two strains of the endosymbiotic bacterium Wolbachia, wAlbA and wAlbB. Here, we present a comprehensive genomic analysis of these Wolbachia strains, their associated plasmids, and host mitochondrial DNA (mtDNA). We observed extensive variation in Wolbachia densities--spanning over three orders of magnitude--indicating that infections often exist at extremely low levels and this variation is largely driven by environmental factors. Phylogenetic analyses revealed concordant evolutionary histories among mitochondrial, Wolbachia, and plasmid genomes, with no strong evidence for horizontal or paternal transmission. We estimate that the common ancestor of the superinfection existed [~]6,000--9,000 years ago. Patterns of genetic structure and divergence indicate that the subsequent geographical spread of Wolbachia has mirrored host population history. Comparative rate analyses revealed that wAlbA evolves more slowly than wAlbB, consistent with a reduced mutation rate. Despite the close relationship between wAlbB lineages, gene content in this strain is highly variable, with 184 genes exhibiting presence-absence or copy number variation across strains, including genes involved in inducing cytoplasmic incompatibility. Several of these genes reside in repetitive or prophage regions, indicating that transposable elements appear to drive structural genomic variation in Wolbachia populations. Together, our results highlight extensive natural diversity within Wolbachia populations and provide a foundation for understanding symbiont dynamics in nature and to inform the selection of strains for Wolbachia-based vector control programs.

evolutionary biology↗

bioRxiv2026

Cellular responsiveness as a predictive indicator for population collapse and autonomous control in continuous cultures of Pseudomonas putida

How microbial populations respond to repeated environmental transitions determines both their ecological fitness and their utility in biotechnological applications. Using Pseudomonas putida KT2440 equipped with fluorescent biosensors and monitored by automated flow cytometry in the Segregostat platform, we show that exposure to benzoate, a plastic-derived aromatic feedstock, progressively reduces cellular responsiveness, defined as the fraction of cells that successfully activate a gene circuit following an environmental transition. Unlike classical switching costs, which promote phenotypic diversification, benzoate suppresses responsiveness without increasing population entropy, in a concentration-dependent and circuit-independent manner tightly correlated with fitness loss. A resource allocation model incorporating the competing demands of benzoate assimilation, toxicity, and tolerance reveals that this impairment emerges from a three-way competition for limited cellular resources. Above a critical benzoate load, insufficient resources remain available to sustain the adaptive reallocation required for circuit activation. In continuous culture, a non-responsive subpopulation accumulates as a leading indicator of population collapse. Exploiting this signal, we implement a two-stage connected bioreactor system in which benzoate feeding is autonomously regulated based on real-time population structure, enabling complete substrate consumption and stable operation at otherwise destabilizing concentrations. These results establish cellular responsiveness as a quantitative population variable and demonstrate that structure-aware feedback control, acting on population composition rather than bulk physiology, provides a principled route toward autonomous bioprocesses on challenging substrates.

Systems Biology↗

bioRxiv2026

Diversifying stocking practices within rangelands supports diverse plant-pollinator communities and interactions

Understanding how management alters species' biodiversity and interactions is critical for sustainable agriculture. This can be especially important for rangelands, grasslands used for livestock grazing that cover 30% of land worldwide. In rangelands, ranchers need to maximize livestock production while supporting healthy ecosystems. Grazing management can alter the impact of livestock by varying grazing timing and intensity, but its ecological impacts remain poorly understood. One critical function that may be supported in these systems is pollination; how rangeland management may alter this ecosystem service is not systematically understood. We investigated how rotational stocking, where livestock are rotated among pastures, affected plant and pollinator communities and plant-pollinator interactions using working ranches in the Southern Great Plains. Sites were managed through continuous stocking or rotation with low or high frequency. We found that high-frequency livestock rotation supported greater flower abundance, and consequently greater pollinator abundance. Pollinator orders varied in their responses to management with Hymenoptera directly responding to management, and Coleoptera and Lepidoptera responding indirectly via changes in flower abundance. Community-level changes produced more specialized interaction networks and higher interaction diversity. These community changes, and resulting species turnover, led to high interaction turnover across rangeland sites. This suggests that a mosaic of management strategies likely diversifies habitat and helps support plant and pollinator biodiversity at the landscape scale. This study demonstrates that rangelands can support plants, pollinators, and their interactions, particularly when managed with high-frequency rotational stocking.

ecology↗

bioRxiv2026

A comprehensive CRISPR screen of the Drosophila glutamate receptome reveals Ekar as a selective regulator of presynaptic homeostatic plasticity

Homeostatic mechanisms protect synapses from destabilizing challenges throughout an organisms lifespan, ensuring stable yet flexible neural network activity. To delineate the molecular basis of presynaptic homeostatic potentiation (PHP), we conducted a comprehensive, in vivo CRISPR/Cas9-based screen of all 16 glutamate receptor (GluR) genes encoded in the Drosophila genome. We first generated a complete expression atlas across larval and adult stages, identifying nine GluRs expressed in presynaptic motor neurons. We then generated null mutants for all 16 GluRs and screened them at the larval neuromuscular junction. While the loss of any single presynaptic GluR did not affect baseline synaptic growth or neurotransmission, our screen revealed a selective and critical requirement for the kainate receptor subunit ekar in the expression of chronic PHP. Further genetic analysis indicates that Ekar functions coordinately with the kainate receptor subunits KaiRID and Ukar within a shared pathway to promote this plasticity. Mechanistically, Ekar acts downstream of active zone remodeling to drive the homeostatic enhancement of presynaptic Ca2+ influx, which is the defining feature of chronic PHP. Together, this genome-wide analysis establishes a definitive functional atlas for the Drosophila glutamate receptome and highlights a specialized, essential role for Ekar in stabilizing long-term synaptic homeostasis.

neuroscience↗

bioRxiv2026

Pleiotropy and repeated mutation drove convergent domestication of grain amaranth

Humans reshaped environments for domesticated plants, yet the extent of human agency in the domestication process remains unresolved. Color variation has been used as evidence for an intended process by humans, as the trait has been mainly linked to visual properties. In the ancient pseudocereal grain amaranth major domestication traits have not changed, but the seed color changed repeatedly from dark to white during multiple domestication processes. Here, we show that the transition to white seed color was of major ecological significance beyond human preference through mechanistic links to seed germination. We identify major metabolomic and transcriptomic changes in the proanthocyanidin pathway leading to loss of seed color and dormancy in domesticated amaranth. Despite the potentially large mutational target size of the proanthocyanidin pathway, the repeated mutation in a single gene led to the domestication phenotype. Multiple independent knock-out mutations in this gene reveal the repeated selection for the loss of seed dormancy in different parts of the Americas. Competition experiments show that the pleiotropic link between seed color and seed dormancy provides a competitive advantage to white seeds in agricultural environments. The reduction of proanthocyanidin content in the seed is observed in numerous crops, including rice and beans, suggesting a common ecological adaptation after early humans altered their environment through changing lifestyles. Our findings establish a direct mechanistic link between individual mutations, pathway-level biochemical reconfiguration, and ecological fitness, illustrating how plants adapted to human-modified environments largely without intentional human agency.

evolutionary biology↗

bioRxiv2026

Telomere-to-telomere genome assembly of Microsporidia sp. MB, a microsporidian symbiont of Anopheles coluzzii isolated from Burkina Faso

BackgroundMicrosporidia sp. MB is an intracellular parasite of anopheline mosquitoes identified across the African continent. Microsporidia sp. MB infections appear to have no significant effect on host fitness and vertically transmit, whilst infected individuals have exhibited significantly reduced levels of Plasmodium falciparum transmission. These combined characteristics make Microsporidia sp. MB a promising candidate for use in malaria control. A comprehensive genome would greatly facilitate investigation into the evolution and biological pathways underlying important phenotypes, such as the mechanism of malarial inhibition. ResultsIn this study, we present the de novo assembly of the first complete genome of Microsporidia sp. MB SOUVK7. Multi-platform sequencing was performed on ovary samples of laboratory established Anopheles coluzzii collected from Burkina Faso. The SOUVK7 genome has a total size of 9.16Mb, encodes 2,435 genes and is organised into 13 chromosomes with telomeres identified at all flanks. Telomeric repeats exhibit a 4-mer motif due to a glutamine deletion previously unobserved in Microsporidia. Ploidy analysis of Illumina reads predicts MB as tetraploid, whilst analysis of CpG methylation and retroelements highlights loci in all chromosomes with characteristics consistent with regional centromeres. Orthology analysis identifies several key genes in pathways associated to telomeric and centromeric maintenance, along with methylation and host invasion machinery. A loss of several components of the infection machinery is observed in Microsporidia sp. MB and the wider Enterocytozoonida, consistent with a general trend towards genome size reduction in the clade. ConclusionThis study provides the first complete, telomere to telomere assembly of Microsporidia sp. MB, offering new insight into the genomic architecture of Microsporidia sp. MB and the broader Mrazekiidae family.

genomics↗

bioRxiv2026

Lactate-Driven Heterogeneity of Immune Checkpoint Expression in Breast and Lung Cancer Cell Lines

Tumor-derived lactate is increasingly recognized as an immunosuppressive metabolite within the tumor microenvironment (TME), with emerging evidence highlighting its role beyond metabolism to include epigenetic and immune regulatory functions. While prior studies have primarily focused on individual immune checkpoints, most prominently PD-L1, it remains unclear whether lactate broadly coordinates the expression of multiple immune regulatory pathways across distinct tumor types, particularly in the context of chronic exposure mimicking glycolytic tumors. Here, we investigated the relationship between lactate-producing metabolism and immune checkpoint gene expression in four human cancer cell lines representing breast and lung cancer: MCF7 (estrogen receptor-positive breast), MDA-MB-231 (triple-negative breast), A549 (non-small cell lung), and H82 (small cell lung). By manipulating glucose availability and exposure duration to model acute (6 h) versus chronic (48 h) lactate production, and by pharmacologically inhibiting lactate dehydrogenase (LDH) with oxamate, we quantified extracellular lactate accumulation and assessed transcriptional responses of a panel of immune checkpoints (PD-L1, CD80, CD73, LGALS9, VISTA, PVR, CD47, FGL1, STING) and lactate-associated genes (MCT1, MCT4, LDHA, HCAR1) via qPCR. Chronic high-glucose conditions produced robust, LDH-dependent lactate accumulation and were associated with coordinated, lineage-specific remodeling of multiple checkpoint transcripts, whereas acute exposure induced minimal changes. MDA-MB-231 and A549 cells displayed striking but distinct checkpoint patterns under chronic lactate-producing conditions: MDA-MB-231 cells showed strong co-induction of PD-L1 and CD80, while A549 cells exhibited dominant CD80 induction with modest PD-L1 upregulation. H82 cells upregulated PD-L1 alongside CD73, LGALS9, CD47, and CD80, whereas MCF7 cells demonstrated more modest yet coordinated increases across several checkpoints. Chronic glucose exposure resulted in sustained, LDH-dependent lactate accumulation and coordinated induction of multiple immune checkpoint genes, with distinct lineage-specific patterns, e.g., robust PD-L1/CD80 upregulation in MDA-MB-231 versus CD80 dominance in A549. Unsupervised clustering and principal component analysis revealed that duration of glucose exposure, rather than acute glucose availability, was the primary axis of variation and that MCT4 and HCAR1 clustered with strongly induced checkpoints, consistent with a transcriptional program linking lactate export and sensing to immune regulation. These findings support a model in which lactate acts as an upstream regulator of a broader immune escape program, potentially via mechanisms like lactylation and HCAR1 signaling. This work highlights the limitations of single-checkpoint blockade strategies in solid tumors and underscores the potential of targeting lactate metabolism to enhance immunotherapy efficacy in breast and lung cancers.

cancer biology↗

bioRxiv2025

Metabolic Responses of Different Levels of Fitness

The metabolic and physiological responses to exercise vary markedly across different levels of fitness and training status. While maximal oxygen uptake (VO2max) has historically been used as the primary indicator of cardiorespiratory fitness, increasing attention has been directed toward metabolic and bioenergetic responses to exercise, including blood lactate concentration and substrate utilization. In this study, we examined comprehensive physiological and metabolic responses during graded exercise testing in 204 male cyclists spanning four fitness categories: Tour de France professional cyclists, competitive cyclists, master cyclists, and recreational cyclists. Measurements included power output, VO2, blood lactate concentration, and rates of fat and carbohydrate oxidation derived from indirect calorimetry. Across all exercise intensities, performance and metabolic parameters followed a clear hierarchical pattern corresponding to competitive level. Tour de France cyclists demonstrated significantly greater power output, higher VO2max, lower blood lactate concentrations at matched workloads, higher fat oxidation rates, and delayed reliance on carbohydrate oxidation compared with less-trained groups. Strong inverse correlations were observed between blood lactate concentration and fat oxidation, while positive correlations were observed between blood lactate concentration and carbohydrate oxidation across all fitness levels.

physiology↗

bioRxiv2025

Genomic Islands in Wolbachia Prophages Drive Amplification andDiversification of Cytoplasmic Incompatibility Genes in Culex pipiens

Wolbachia are maternally inherited endosymbiotic bacteria widespread among arthropods. They manipulate their host reproduction to enhance their prevalence in host populations. The most common manipulation is cytoplasmic incompatibility (CI), causing embryonic death in crosses between infected males and uninfected females, or between individuals carrying distinct incompatible Wolbachia. CI patterns are highly complex in the mosquito Culex pipiens, where the causal genes cidA and cidB are amplified and diversified, forming a "cid repertoire" within each Wolbachia wPip genome. Despite their central role in CI, the genomic mechanisms underlying such cid amplification and diversification remained poorly understood. This knowledge gap is largely due to the difficulty of assembling wPips genomes due to highly repeated genes and mobile elements, especially in WO prophages. Here, we directly annotated Illumina polished Nanopore-sequences to investigate the genomic flanking context of cid genes in three distinct wPip lineages. We assembled WO prophage regions of substantial length containing the entire cid repertoire previously described in these bacterial lineages. Within these WO regions, cid genes are consistently embedded in modular and rearrangeable islands composed of MutL, rnhA, and small mobile elements, all displaying hyperconserved nucleotide identity across islands. These genomic islands are probably drivers for major rearrangement and recombination events responsible for the amplification and diversification of cidwPip genes within and between the wPip genomes leading to CI complexity in C. pipiens.

evolutionary biology↗

bioRxiv2025

Ashwin and FAM98 paralogs define nuclear and cytoplasmic RNA ligase complexes for tRNA biogenesis and the unfolded protein response

The tRNA ligase complex (tRNA-LC) seals tRNA exon halves in the nucleus after the removal of a single intron, and joins XBP1-mRNA exons in the cytoplasm as part of the unfolded protein response (UPR). This dual function requires simultaneous nuclear and cytoplasmic localization. Here, we reveal that Ashwin (ASW), the vertebrate-specific subunit of the tRNA-LC, serves as its nuclear import factor. ASW displays a dual nuclear localisation signal (NLS) which, upon disruption, leads to the retention of the tRNA-LC in the cytoplasm with a consequent impairment of pre-tRNA splicing and accumulation of 5 tRNA fragments. We also show that the tRNA-LC exists in three forms depending on which FAM98 paralog is chosen, either FAM98A, FAM98B or FAM98C. We find that ASW interacts exclusively with the FAM98B-containing complex, allowing its nuclear localization for tRNA biogenesis. Attaching an NLS to RTCB, the catalytic and indispensable subunit, rescues pre-tRNA splicing in cells depleted of ASW. We envision that vertebrates evolved ASW to localize a sub-population of tRNA-LC to the nucleus, while using FAM98 paralogs to retain a fraction of RTCB in the cytoplasm for XBP1-mRNA splicing during UPR.

cell biology↗

bioRxiv2025

Structural framework for the assembly of the human tRNA ligase complex

In human cells, a subset of tRNA-encoding genes contain introns. These are removed by a non-canonical splicing pathway in which the tRNA splicing endonuclease complex catalyzes intron excision and the resulting exons are subsequently ligated by the tRNA-ligase complex (tRNA-LC). Although recent studies have provided insights into the process of intron removal, the molecular mechanisms underpinning tRNA ligation by tRNA-LC remain elusive. The tRNA-LC is a hetero-pentameric protein assembly consisting of Ashwin, CGI-99, FAM98B, the DEAD-box helicase DDX1 and the catalytic subunit RTCB/HSPC117. Using cryo-EM, we have determined an atomic-resolution reconstruction of human tRNA-LC. We find that CGI-99, DDX1 and FAM98B form an alpha-helical bundle that contacts RTCB via an interface located on the opposite side from the location of the ligase active site and tethers DDX1 to the tRNA-LC via its C-terminal helix. FAM98B and CGI-99 extensively interact in an intricately co-folded heterodimer that clamps Ashwin in a pincer-like structure. Interaction analysis using structure-based mutants of tRNA-LC subunits supports the overall architecture of the complex. Finally, we show that the paralogous proteins FAM98A and FAM98C underpin the assembly of compositionally distinct RTCB-containing complexes that lack Ashwin and may have distinct cellular functions. Together, our results provide new insights into the assembly and mechanism of the tRNA ligase complex, shedding light on its functions in tRNA biogenesis and beyond.

biochemistry↗

bioRxiv2025

Mathematical model of tumor-macrophage dynamics in glioma to advance myeloid-targeted therapies

Recent biological research has highlighted the relevance of myeloid-cell populations in glioma growth, with a particular role played by tumor-associated macrophages (TAMs), which comprise resident microglia and monocyte-derived macrophages. Additionally, radiation therapy, the most common treatment for gliomas, significantly alters the tumor microenvironment, affecting TAMs and contributing to tumor recurrence. Promising preclinical studies have identified and developed drugs targeting TAMs. The development and combined deployment of these therapies require in silico techniques that enable us to optimize their outcomes. To do so, we need mathematical models of glioma growth and therapy response that explicitly incorporate TAMs--an often overlooked component in existing models. Here, we present a dynamical model of glioma growth driven by tumor-immune interactions. The model was parametrized using published data from mice experiments, including responses to ionizing radiation. We used this model to investigate glioma progression under radiotherapy combined with three treatments targeting distinct aspects of TAM biology. Simulations revealed that anti-CD47 enhanced the otherwise weak phagocytic activity, extending the upper tail of the survival curve. -CD49d, which limits monocyte trafficking after irradiation, offered consistent survival benefits across digital twins of mice. Finally, CSF-1R inhibitors, which block the primary growth factor regulating TAM function, resulted in the largest overall survival improvement in silico. Our results aligned well with experimental evidence, suggesting that the model may help inform the optimization of myeloid cell-targeted immunotherapies, including their timing, dosage, and combination with radiation therapy, with potential relevance for improving glioma treatment strategies.

cancer biology↗

bioRxiv2025

Phenotypic Screening Identifies Flunarizine as an Inhibitor of Radiotherapy-Induced Astrocyte Reactivity with Therapeutic Potential in Glioblastoma

Radiotherapy is part of the standard-of-care for glioblastoma, yet tumors invariably recur as incurable lesions post-treatment. Recent studies suggest that radiation-induced astrocyte reactivity fosters a tumor-supportive environment, however effective strategies targeting reactive astrocyte phenotypes are lacking. Using a novel image-based assay, we screened over 1,700 small molecule compounds, identifying 29 that inhibit radiation-induced astrocyte reactivity in human astrocytes. Among these, Flunarizine, a calcium-entry blocker approved for migraine treatment, significantly reduced astrocyte reactivity in vitro and in vivo. In a genetically engineered glioblastoma mouse model, combining Flunarizine with radiotherapy markedly improved survival without affecting unirradiated controls, indicating specificity for a radiation-induced phenotype. Mechanistically, Flunarizine inhibited radiation-induced fibrosis in vivo and directly suppressed astrocytic TGF-beta activation in vitro. Notably, Flunarizine treatment had no direct effect on primary glioblastoma cells, emphasizing its microenvironmental specificity. In conclusion, we identified Flunarizine as a promising repurposed compound capable of effectively mitigating radiation-induced astrocyte reactivity and delaying glioblastoma recurrence. This approach offers a viable therapeutic strategy to enhance current glioblastoma treatments. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/664538v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@d4d0d6org.highwire.dtl.DTLVardef@1dac551org.highwire.dtl.DTLVardef@1d071ccorg.highwire.dtl.DTLVardef@1df1a72_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

UNWANTED AXON GROWTH: PTEN AND THE SUPPRESSION OF AXON PLASTICITY IN ADULT NERVES

In adults, peripheral nerves comprise bundles of disseminated motor, sensory and autonomic axons that are considered stable neuroanatomical units. The only exceptions to this established wiring are very distal terminal branches in target organs, such as skin. Here we provide a remarkable deviation from this state of affairs in the peripheral nerves of mice with a conditional knockout of sensory neuron PTEN (phosphatase and tensin homolog deleted on chromosome ten). PTEN is normally expressed in adult sensory neurons, particularly small IB4 nonpeptidergic subtypes and its knockdown after injury or during experimental diabetes improves axon regrowth. We studied Advillin Cre;PTEN null mice lacking PTEN in their sensory neurons. As might be expected, their harvested and cultured DRG neurons displayed enhanced neurite outgrowth in vitro. In vivo, these mice were healthy and had a normal sensory behavioural phenotype. However, the nerves of mice lacking sensory neuron PTEN were highly abnormal, with augmented clusters of small myelinated and unmyelinated axons populating endoneurial fascicles of their peripheral nerve trunks. The axon clusters did not disrupt normal fascicular anatomy but invested the epidermis with greater axon numbers. Within endoneurial fascicles, supernumerary axons formed regenerative units and expressed ongoing growth markers, unlike normal adult axons. This was not accompanied by rises in dorsal root ganglia (DRG) neuron numbers, indicating enhanced distal sprouting from parent neurons. Additionally, sprouting axons were electrophysiologically intact, generating rises in the amplitudes of sensory nerve action potentials. Despite this extensive regenerative activity of intact nerves, regeneration indices after superimposed injury were only modestly enhanced or unchanged. This unusual behaviour of adult sensory axons lacking a single growth-suppressive molecule may identify insights into what molecular constraints the nervous system normally utilizes to suppress inappropriate plasticity.

neuroscience↗