Mastodawn

Cannabidiol Reduces Oxycodone Self-Administration While Preserving Its Analgesic Efficacy in a Rat Model of Neuropathic Pain https://www.biorxiv.org/content/10.1101/2025.04.21.649778v1?med=mas

Cannabidiol Reduces Oxycodone Self-Administration While Preserving Its Analgesic Efficacy in a Rat Model of Neuropathic Pain

Prescription opioid misuse is a significant public health concern among individuals with chronic pain. Treating severe pain often requires high doses of opioids, increasing the risk of developing an opioid use disorder. Cannabidiol (CBD) is a non-psychoactive component of cannabis that has shown therapeutic potential without abuse liability. This study investigated the effects of CBD on oxycodone self-administration and hyperalgesia in an animal model of chronic neuropathic pain. Adult male rats were trained to self-administer intravenous oxycodone (0.06 mg/kg/infusion). Subsequently, they underwent chronic constriction injury (CCI) of the sciatic nerve or received sham surgery. Paw withdrawal latency was measured using the Hargreaves test as an indicator of thermal pain sensitivity. CBD (0, 1, 3, and 10 mg/kg, IP) was administered before the self-administration sessions, and pain testing was conducted afterward. The rats acquired oxycodone self-administration, as indicated by more active than inactive lever presses. CCI surgery decreased the paw withdrawal latency, confirming the induction of neuropathic pain. CCI alone did not affect oxycodone self-administration, suggesting that neuropathic pain does not affect opioid intake. Treatment with CBD reduced oxycodone self-administration in both the sham and CCI rats. Oxycodone self-administration in the CCI rats reversed the CCI-induced decrease in paw withdrawal latency. However, CBD did not affect the antinociceptive effect of oxycodone in CCI rats. Taken together, these findings demonstrate that CBD reduces oxycodone self-administration without affecting the antinociceptive effects of oxycodone in neuropathic pain. This study supports the potential of CBD to reduce opioid use and misuse, regardless of pain status. ### Competing Interest Statement The authors have declared no competing interest. National Institute on Drug Abuse, , DA049470, DA046411

bioRxiv

bioRxiv 2d ago

Directing stem cell differentiation by chromatin state approximation https://www.biorxiv.org/content/10.1101/2025.04.24.650451v1?med=mas

Directing stem cell differentiation by chromatin state approximation

A prime goal of regenerative medicine is to replace dysfunctional cells in the body. To design protocols for producing target cells in the laboratory, one may need to consider exponentially large combinations of culture components. Here, we investigated the potential of iteratively approximating the target phenotype by quantifying the distance between chromatin profiles (ATAC-seq) of differentiating cells in vitro and their in-vivo counterparts. We tested this approach on the well-studied generation of erythroblasts from haematopoietic stem cells, evaluating a fixed number of components over two sequential differentiation rounds (8x8 protocols). We found that the most erythroblast-like cells upon the first round yielded the most erythroblast-like cells at the second round, suggesting that greedy selection by chromatin approximation can be a viable optimisation strategy. Furthermore, by analysing regulatory sequences in incompletely reprogrammed chromatin regions, we uncovered transcriptional regulators linked to roadblocks in differentiation and made a data-driven selection of ligands that further improved erythropoiesis. In future, our methodology can help craft notoriously difficult cells in vitro, such as B cells. ### Competing Interest Statement The authors have declared no competing interest. FWF Austrian Science Fund, https://ror.org/013tf3c58, 10.55776/TAI732, 10.55776/P36302, 10.55776/PAT1300223 European Research Council, https://ror.org/0472cxd90, 947803 Alex's Lemonade Stand Foundation, https://ror.org/038ja4880, 20-17258 European Commission, , 101061151

bioRxiv

bioRxiv 2d ago

DNA Methylation Dynamics of Dose-dependent Acute Exercise, Training Adaptation, and Detraining https://www.biorxiv.org/content/10.1101/2025.04.22.650067v1?med=mas

DNA Methylation Dynamics of Dose-dependent Acute Exercise, Training Adaptation, and Detraining

Exercise and diet are direct physical contributors to human health, wellness, resilience, and performance [1-5]. Endurance and resistance training are known to improve healthspan through various biological processes such as mitochondrial function [6-8], telomere maintenance [9], and inflammaging [10]. Although several training prescriptions have been defined with specific merits [1,10-20], the long-term effects of these in terms of their molecular alterations have not yet been well explored. In this study, we focus on two combined endurance and resistance training programs: (1) traditional moderate-intensity continuous endurance and resistance exercise (TRAD) and (2) a variation of high-intensity interval training (HIIT) we refer to as high intensity tactical training (HITT), to assess the dynamics of DNA methylation (DNAm) in blood and muscle derived from males (N=23) and females (N=31), over a 12-week period of training followed by a 4-week period of detraining, sampled at pre-exercise and acute time points, totaling 528 samples. Due to its rapid responsiveness to stimuli and its stability, DNAm has been known to facilitate regulatory cascades that significantly affect various physiological processes and pathways. We find that several thousand differentially methylated regions (DMRs) associated with acute exercise in blood, many of which are shared across males and females. This trend is reversed when comparing the baseline (pre-exercise) time points or post-exercise timepoints at the untrained state with those at the post-conditioned state. Here, muscle shows majority of DNAm changes, with most of those being unique. We also find several hundred memory DMRs in muscle that maintain the gain or loss of methylation after four weeks of inactivity. Comparing phenotypic measurements, we find specific DMRs that correlate significantly with mitochondrial function and myofiber switching. Using machine learning, we select a subset of DMRs that are most characteristic of training modalities, sex and timepoint. Most of the DMRs are enriched in pathways associated with immune function, cell differentiation, and exercise adaptation. These findings reveal mechanisms by which exercise- and training-induced epigenetic changes alter immune surveillance, mitochondrial function, and inflammatory response, and underscore the relevance of epigenetic plasticity to health monitoring and wellness. ### Competing Interest Statement J.R.E. serves on the scientific advisory board of Zymo Research Inc. U. S. Department of Defense through the Office of Naval Research (ONR), , N000141613159 National Human Genome Research Institute (NHGRI) of National Institutes of Health (NIH), , HG010634

bioRxiv

bioRxiv 2d ago

Ancient co-option of LTR retrotransposons as yeast centromeres https://www.biorxiv.org/content/10.1101/2025.04.25.647736v1?med=mas

Ancient co-option of LTR retrotransposons as yeast centromeres

The evolutionary origins of the genetic point centromere in the brewer's yeast Saccharomyces cerevisiae, a member of the order Saccharomycetales, are still unknown. Competing hypotheses suggest that the point centromere tripartite genetic centromere DNA elements (CDEs) either evolved from ancestral epigenetic centromeres by descent with modification or were gained through horizontal transfer from selfish DNA plasmids. Here, we identified centromeres in the sister order Saccharomycodales and termed them "proto-point centromeres" due to sequence features that bridge the evolutionary gap between point centromeres and ancestral centromeres types. Comparative genomic analyses across multiple yeast orders showed an unexpected evolutionary link between point and proto-point centromeres to the long terminal repeats (LTRs) of Ty5 retrotransposons. Strikingly, one Saccharomycodales species, Saccharomycodes ludwigii, harbors compact Ty5-based centromeres, where its CDEII elements are divergent AT-rich Ty5 LTRs. These living fossil centromeres show how retrotransposon cis-regulation was likely co-opted for genetic centromere specification. These insights show that point centromeres are direct descendants of retrotransposons and have evolved by descent with modification. Ultimately, the many diverse centromere types across the yeast subphylum may share a common ancestry rooted in retrotransposon activity. ### Competing Interest Statement Jef Boeke is a Founder and Director of CDI Labs, Inc., a Founder of and consultant to Opentrons LabWorks/Neochromosome, Inc, and serves or served on the Scientific Advisory Board of the following: CZ Biohub New York, LLC; Logomix, Inc.; Rome Therapeutics, Inc.; SeaHub, Seattle, WA; Tessera Therapeutics, Inc.; and the Wyss Institute. Antonis Rokas is a scientific consultant for LifeMine Therapeutics, Inc. National Science Foundation, https://ror.org/021nxhr62, EF-1921641, DEB-2110403, DEB-2110404 National Institute of Food and Agriculture, https://ror.org/05qx3fv49, 7005101 Great Lakes Bioenergy Research Center, https://ror.org/01ca2by25, DESC0018409 National Institute of Allergy and Infectious Diseases, https://ror.org/043z4tv69, R01 AI153356 European Research Council, https://ror.org/0472cxd90, 951430

bioRxiv

bioRxiv 2d ago

Heterogeneous therapy-resistant cancer cells have distinct and exploitable drug sensitivity profiles https://www.biorxiv.org/content/10.1101/2025.04.25.650475v1?med=mas

Heterogeneous therapy-resistant cancer cells have distinct and exploitable drug sensitivity profiles

Resistance to targeted therapies is a significant clinical problem, but eliminating resistant cancer cells has proven difficult. One potential reason for this difficulty is heterogeneity in the resistant population: even genetically homogeneous cancer cell populations can give rise to a variety of resistant subtypes, each potentially with their own specific second-line drug vulnerabilities. Using high-throughput drug screening of genetically-identical resistant clones with varying transcriptomes and morphologies, we show that each clone had a distinct drug sensitivity profile. These results admitted the possibility that there are drugs that are effective against only subsets of therapy resistant populations but in combination eliminate a large proportion of the resistant population. Using the individual clone sensitivity profiles, we prospectively identified combinations that were highly effective at eliminating most of the resistant population. Our results demonstrate the effectiveness of ″subpopulation-directed synergy″, showing that considering population heterogeneity can reveal therapeutic opportunities otherwise masked by population averages, offering new strategies to combat therapy resistance. ### Competing Interest Statement A.R. receives royalties related to Stellaris RNA FISH probes. A.R. serves on the scientific advisory board of Spatial Genomics. A.R. is the founder of CytoPixel Software. All other authors declare no competing interests.

bioRxiv

bioRxiv 2d ago

Subcellular proteomics of Paramecium tetraurelia reveals mosaic localization of glycolysis and gluconeogenesis https://www.biorxiv.org/content/10.1101/2025.04.24.650466v1?med=mas

Subcellular proteomics of Paramecium tetraurelia reveals mosaic localization of glycolysis and gluconeogenesis

Ciliates are unicellular heterotrophic eukaryotes, most of which consume other microbes as prey. They exhibit nuclear dimorphism which requires reconstruction of a transcriptionally active macronucleus from the germline micronucleus after sexual recombination. This complex genomic structure has prevented the development of highly tractable genetic models leaving much of ciliate cell biology unexplored. To complicate matters further, some ciliates tend to accumulate many gene duplicates either singly or via whole genome duplications. Thus, extensive insight into the cell biology of ciliates requires the use of high-throughput tools like subcellular proteomics. Here, we use a subcellular proteomics workflow to classify over 9,000 proteins to 16 subcellular compartments in Paramecium tetraurelia. From these data, we identify a small but robust subcellular cluster containing canonical mitochondrial outer membrane proteins as well as some ER proteins, putatively at membrane contact sites. Within this cluster, we identified the important glycolytic enzyme phosphofructokinase, which contained a transmembrane domain. Further investigation revealed that several latter-acting glycolytic enzymes were localized to the mitochondrial cluster. The location of phosphoenol pyruvate carboxykinase and pyruvate carboxylase in the mitochondria but pyruvate kinase in the cytosol suggests that ciliates prefer gluconeogenesis over glycolysis. The localization of these enzymes was confirmed in a preliminary subcellular proteome of Tetrahymena thermophila. In sum, our findings suggest that mitochondrial localization of glycolytic/gluconeogenic enzymes is widespread across ciliates and that several may preferentially undergo gluconeogenesis over glycolysis using amino acids as a primary carbon source in both catabolic and anabolic metabolism. ### Competing Interest Statement The authors have declared no competing interest. Gordon and Betty Moore Foundation, , GBMF10600 National Science Foundation, , 1927159, 2119963, 2405455 National Institute of Health, , 2R35GM122566 VEDA FELLOWSHIPS, Operational program Jan Amos Komensky, , CZ.02.01.01/00/22_010/0008117

bioRxiv

bioRxiv 2d ago

A Dual Homeostatic Regulation of Dry Mass and Volume Defines a Target Density in Proliferating Mammalian Cells https://www.biorxiv.org/content/10.1101/2025.04.24.650395v1?med=mas

A Dual Homeostatic Regulation of Dry Mass and Volume Defines a Target Density in Proliferating Mammalian Cells

The concentration of macromolecules, especially proteins, is vital for cellular function and is influenced not only by synthesis and degradation but also by the total cell volume. While we understand various growth regulation mechanisms, the coupling of dry mass and volume in growing mammalian cells remains unclear. Here we show that two independent mechanisms acting in single cells, one regulating volume through biophysical modulation and the other controlling protein biosynthesis, work together to maintain macromolecular dry mass density and restore it following perturbations. These mechanisms ensure that proliferating cells remain within a specific range around a target density, providing density homeostasis at the population level. Although the target density appears consistent across different cell types, it is disrupted around cell division, upon perturbations of growth pathways and in senescent cells. It may represent an optimal value for cellular processes, ensuring the efficiency of essential intracellular functions. ### Competing Interest Statement The authors have declared no competing interest.

bioRxiv

bioRxiv 2d ago

Elevated GLUT4 Levels in Human Skeletal Muscle Microtissues is Accompanied by Functional Insulin Dependence https://www.biorxiv.org/content/10.1101/2025.04.24.650438v1?med=mas

Elevated GLUT4 Levels in Human Skeletal Muscle Microtissues is Accompanied by Functional Insulin Dependence

Insulin resistance in skeletal muscle is a hallmark of type 2 diabetes mellitus (T2D). While two-dimensional myotube cultures offer a controlled environment for studying T2D-related metabolic dysfunction, insulin-dependent glucose transporter type 4 (GLUT4) levels are limited and insulin-independent glucose transporter type 1 (GLUT1) expression dominates; reducing physiological relevance. Three-dimensional skeletal muscle microtissue cultures offer a promising alternative, and unlike 2D myotubes, are amenable to repeated contractile stimulation. However, microtissue GLUT1 and GLUT4 glucose transporter profiles remain under-characterized, particularly under physiological glucose and insulin conditions, which is evaluated herein. We report that GLUT1 levels trended ~3.0-fold lower in microtissues compared with myotubes in 2D culture, although not statistically significant (p = 0.072), while GLUT4 levels were ~12-fold higher (p < 0.0001), leading to a ~60-fold increase in the GLUT4:GLUT1 ratio (p = 0.023). Notably, the microtissue GLUT4:GLUT1 profile approached, but did not match that of native human muscle. Microtissues required supraphysiological insulin conditions for the development of maximal contractility, while physiological glucose levels were sufficient. Insulin withdrawal restored insulin responsiveness but impaired microtissue contractile strength (p < 0.0001) and fatigue resistance (p = 0.015). Our findings indicate that the glucose transporter profile of microtissues offers improved physiological relevance. However, their reliance on insulin to maintain contractile function limits their suitability for modeling T2D. The implementation of a robust, insulin-free differentiation protocol would facilitate the development of a microtissue-based T2D model which can be applied to study contraction-mediated increases in insulin sensitivity as a therapeutic approach. ### Competing Interest Statement The authors have declared no competing interest. Canada First Research Excellence Fund "Medicine by Design", , MbDC2-2019-02 Institute of Biomedical Engineering Wildcat Graduate Scholarship, , Institute of Biomedical Engineering Doctoral Completion Award, , Canada Research Chairs, https://ror.org/0517h6h17, Canada Research Chair in Endogenous Repair (Award #950-231201)

bioRxiv

bioRxiv 2d ago

Aging changes the underlying mechanism of JAK2 modulation in neutrophil function https://www.biorxiv.org/content/10.1101/2025.04.21.649782v1?med=mas

Aging changes the underlying mechanism of JAK2 modulation in neutrophil function

Janus Kinase 2 (JAK2) has been linked to various neutrophil functions, but the intracellular mechanisms underlying its modulation are unknown. Neutrophils are essential cells for host defense. Neutrophil effector functions include migration, reactive oxygen species (ROS) production, degranulation, and neutrophil extracellular trap (NET) formation. The goal of this study was to elucidate the signaling mechanism through which JAK2 modulates neutrophil function and the effect of aging on this pathway. We hypothesized that JAK2-mediated modulation changes the molecular mechanisms associated with neutrophil function in an age- and sex-dependent manner. Neutrophils from young (3 months) and aged (22+ months), male and female C57BL/6J mice were isolated, treated with a JAK2 inhibitor (AZD1480) or a pan-JAK inhibitor (Baricitinib), and stimulated with Phorbol 12-myristate 13-acetate (PMA). Functional assays were conducted to assess migration, ROS production, degranulation, NETosis, and metabolism. Mass spectrometry and Luminex assays provided proteomic and cytokine profiles. Our data show that JAK2 promotes migration via membrane composition and actin remodeling, with age-dependent shifts in chemokine secretion. JAK2 indirectly affects NETosis by modulating IL-1 signaling and ROS production. It also primes ROS production by altering NADPH oxidase components. In young neutrophils, JAK2 influences degranulation through actin remodeling, while aged neutrophils display impaired granule release. Metabolically, JAK2 enhances pentose phosphate pathway activity in young neutrophils and decreases glycogen breakdown in aged cells. These findings reveal mechanisms by which JAK2 modulates neutrophil function and suggest age-specific therapeutic targeting in inflammatory diseases. ### Competing Interest Statement The authors have declared no competing interest. NIH/NINDS, , 1K22NS114363 NIH/NIGMS, , P20GM103434, P30GM121322

bioRxiv

bioRxiv 2d ago

Charting the nanotopography of inner hair cell synapses using MINFLUX nanoscopy https://www.biorxiv.org/content/10.1101/2025.04.22.649963v1?med=mas

Charting the nanotopography of inner hair cell synapses using MINFLUX nanoscopy

For us to hear, the cochlea encodes sounds into neural signals at synapses of inner hair cells (IHCs) and the auditory nerve with remarkable fidelity. To achieve the high rates of temporally precise synaptic transmission over long periods of time, IHCs employ sophisticated ribbon-type active zones (AZ). In order for us to understand synaptic sound encoding, we need to decipher the underpinning molecular topography of these synapse which had remained challenging due to technological limitations. Here we applied 3-dimensional minimal flux optical nanoscopy to mouse IHC synapses to chart the position of key pre- and postsynaptic proteins with single digit nanometre resolution of imaging. We demonstrate that nanoclusters of channels and interacting proteins govern the topography of AZs and postsynaptic densities (PSDs). We count synaptic proteins, their nanoclusters and determine their spatial organization feeding into computational modelling of AZ function. In conclusion, this study reveals a nanocluster-based molecular AZ and PSD topography, likely serving as functional modules in synaptic sound encoding. ### Competing Interest Statement EG and MADRBFL work at Abberior Instruments that develops and manufactures super-resolution fluorescence microscopes including the 3D MINFLUX microscope used in the present study. The remaining authors declare no competing interest in the production and presentation of results. European Research Council, https://ror.org/0472cxd90, 101054467 Fondation Pour l'Audition, , FPA RD-2020-10 Deutsche Forschungsgemeinschaft, , EXC 2067/1 Studienstiftung des Deutschen Volkes, ,

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