Biology subjects

Hu, G.

Publications and source records attributed to Hu, G..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Mining of natural diversity enables efficient and expressible peptide asparaginyl ligases

Peptide asparaginyl ligases (PALs) are powerful tools for protein engineering but are limited by natural rarity and poor expression. We mined 23 cyclotide-rich Viola species, uncovering 29 new PALs that expanded the known repertoire to 47. A dual-objective screen identified VdiPAL1 as the best-performed natural PAL, with twice efficiency of wt-VyPAL2 and 12 mg L-1 soluble expression in E. coli. A broad P2 specificity including Trp/Ile/Leu/Phe/Tyr/Met was discovered across diverse PALs, which enables sequential click-compatible liposome dual-functionalization. 1.8L[A] crystal structure of VdiPAL1 reveals a pre-organized near-attack conformation (NAC), supported by constant-pH MD simulations linking pH-dependent reactivity to NAC geometry. Our homology- and structure-based design yielded VyOpt1, a quintuple mutant of VyPAL2 with over 24-fold improved expression via enhanced cap-domain foldability in a single design-test cycle. This work expands the PAL family and demonstrates a transferable cap-domain-based engineering strategy, highlighting natural diversity as a powerful driver of enzyme discovery and optimization. TOC summaryMining of cyclotide-rich Viola genus expanded the total number of natural peptide asparaginyl ligases (PALs) to 47, including the discovery of highly expressible VdiPAL1. Its high-resolution structure provided new insights for mechanism, and its sequence guided us to a generalized PAL engineering strategy, leading to 24-fold increment in VyPAL2 expression.

biochemistry↗

bioRxiv2025

Neutrophil Transcriptomics in SLE: Exploring Intrinsic, Ex Vivo Adaptation, and CAR T-Cell Therapy-Induced Changes

ObjectivesSystemic lupus erythematosus (SLE) is an autoimmune disease characterized by dysregulation of the adaptive and innate immunityThis study aimed to identify transcriptomic differences in neutrophils from SLE patients and healthy individuals, analyze ex vivo adaptation dynamics, and evaluate the impact of chimeric antigen receptor (CAR) T-cell therapy on neutrophil transcriptomic profiles. MethodsNeutrophils were isolated via negative selection from seven SLE patients and three healthy individuals. RNA sequencing was performed to assess transcriptomic differences, ex vivo dynamics over 60 minutes, and responses to lipopolysaccharide (LPS) stimulation. Additionally, longitudinal transcriptomic data from an SLE patient undergoing KYV-101 anti-CD19 CAR T-cell therapy were evaluated. ResultsWe identified 258 differentially expressed genes (DEGs) consistently distinguishing SLE from healthy neutrophils; they spanned multiple clusters, enriched in interferon-related and DNA damage repair genes (upregulated), and ribosomal protein genes (downregulated). Ex vivo adaptation revealed shared activation pathways, such as NF-{kappa}B and apoptosis, in both groups. LPS stimulation highlighted overlapping inflammatory responses, demonstrating retained functional capacities in SLE neutrophils. Following CAR T-cell therapy of an SLE patient,neutrophil transcriptomic profiles realigned with healthy controls by three months post-treatment. ConclusionsNeutrophils in SLE exhibit intrinsic, disease-specific transcriptomic alterations while sharing ex vivo adaptation dynamics with healthy individuals. The disease-specific alterations appear to be modifiable through targeted therapeutic intervention, as anti-CD19 CAR T-cell therapy resets neutrophil gene expression toward healthy patterns despite targeting B cells rather than neutrophils directly. These findings provide insights into SLE pathogenesis and highlight potential therapeutic strategies targeting both adaptive and innate immunity.

immunology↗

bioRxiv2025

Blind Prediction of Complex Water and Ion Ensembles Around RNA in CASP16

Biomolecules rely on water and ions for stable folding, but these interactions are often transient, dynamic, or disordered and thus hidden from experiments and evaluation challenges that represent biomolecules as single, ordered structures. Here, we compare blindly predicted ensembles of water and ion structure to the cryo-EM densities observed around the Tetrahymena ribozyme at 2.2-2.3 [A] resolution, collected through target R1260 in the CASP16 competition. 26 groups participated in this solvation cryo-ensemble prediction challenge, submitting over 350 million atoms in total, offering the first opportunity to compare blind predictions of dynamic solvent shell ensembles to cryo-EM density. Predicted atomic ensembles were converted to density through local alignment and these densities were compared to the cryo-EM densities using Pearson correlation, Spearman correlation, mutual information, and precision-recall curves. These predictions show that an ensemble representation is able to capture information of transient or dynamic water and ions better than traditional atomic models, but there remains a large accuracy gap to the performance ceiling set by experimental uncertainty. Overall, molecular dynamics approaches best matched the cryo-EM density, with blind predictions from bussilab_plain_md, SoutheRNA, bussilab_replex, coogs2, and coogs3 outperforming the baseline molecular dynamics prediction. This study indicates that simulations of water and ions can be quantitatively evaluated with cryo-EM maps. We propose that further community-wide blind challenges can drive and evaluate progress in modeling water, ions and other previously hidden components of biomolecular systems.

biophysics↗

bioRxiv2025

Dual-Channel Event Microscopy for Ultrafast Biological Imaging

Many fundamental biological processes--spanning immune-tumor interactions, neuronal signaling, and microvascular flow--exhibit fast, multiscale dynamics among diverse cell types within three-dimensional tissue environments. Capturing such activity requires imaging systems that simultaneously achieve high temporal resolution, multicolor capability, and volumetric coverage over large fields of view (FOVs). However, existing modalities remain limited by trade-offs among imaging speed, spectral capacity, depth of field (DOF), and spatial resolution. Here, we present Dual-Channel Event Microscopy (DEM), which integrates digital micromirror device (DMD)-based pulsed illumination, extended-DOF optics, and event-based sensing for ultrafast, dual-channel volumetric imaging across a 2.3 x 1.3 mm2 FOV with an effective 200 {micro}m DOF. Using dual-color fluorescent phantoms and microsphere flow assays, DEM achieves accurate spectral separation and reconstruction of rapid motion at kilohertz frame rates. In vivo, DEM enables simultaneous visualization of neutrophils and premalignant tumors in freely swimming zebrafish. In immobilized specimens, it provides robust, sensor-level optical sectioning near the heart, suppressing diffuse background to reveal fine vascular networks and active blood circulation into and out of the cardiac chambers. DEM further enables quantitative mapping of blood-flow dynamics in the zebrafish tail, resolving arterial-venous differences and capturing heartbeat-driven oscillations that reflect cardiac pumping with high temporal fidelity. By uniting ultrafast acquisition, dual-channel capability, volumetric coverage, and intrinsic optical sectioning within a single event-driven architecture, DEM offers a powerful platform for visualizing rapid multicellular interactions and physiological dynamics in living systems.

bioengineering↗

bioRxiv2025

Sensory Neural Noise as a Limiting Factor in Visual Working Memory Precision in Neurotypicals and Schizophrenia

The neural mechanisms that determine the capacity limits of working memory (WM) are not well understood, with traditional views identifying prefrontal circuitry as the main source of WM limits. Sensory noise, however, remains an underexplored explanation for WM limits despite its unavoidable influence on brain systems that encode and relay sensory information. Here, we show that individual differences in internal sensory noise during visual processing predict visual WM capacity. We further demonstrate that the well-documented WM deficits observed in schizophrenia can be associated with atypically high levels of visual sensory noise, offering a new explanatory framework for these deficits. By experimentally manipulating sensory noise via transcranial direct current stimulation applied to the visual cortex, we also show that changes in sensory noise led to corresponding changes in visual WM precision in individuals with schizophrenia. Finally, a computational model demonstrates that the same sensory noise measured during visual perception can limit WM precision during WM maintenance. These findings show that sensory noise may explain a significant amount of variance in WM function in both neurotypical adults and individuals with schizophrenia.

neuroscience↗

bioRxiv2025

Interpretable biophysical neural networks of transcriptional activation domains separate roles of protein abundance and coactivator binding

Deep neural networks have improved the accuracy of many difficult prediction tasks in biology, but it remains challenging to interpret these networks and learn molecular mechanisms. Here, we address the interpretability challenges associated with predicting transcriptional activation domains from protein sequence. Activation domains, regions within transcription factors that drive gene expression, were traditionally difficult to predict due to their sequence diversity and poor conservation. Multiple deep neural networks can now accurately predict activation domains, but these predictors are difficult to interpret. With the goal of interpretability, we designed simple neural networks that incorporated biophysical models of activation domains. The simplicity of these neural networks allowed us to visualize their parameters and directly interpret what the networks learned. The biophysical neural networks revealed two new ways that arrangement (i.e. the sequence grammar) of activation domain controlled function: 1) hydrophobic residues both increase activation domain strength and decrease protein abundance, and 2) acidic residues control both activation domain strength and protein abundance. Notably, the biophysical neural networks helped us to recognize the same signatures in complex interpreters of the deeper neural networks. We demonstrate how combining biophysical and deep neural networks maximizes both prediction accuracy and interpretability to yield insights into biological mechanisms.

systems biology↗

bioRxiv2025

Geomagnetic and visual cues guide seasonal migratory orientation in the nocturnal fall armyworm, the worlds most invasive insect

The mechanisms guiding nocturnal insect migration remain poorly understood. Although many species are thought to use the geomagnetic field, the sensory basis of magnetic orientation in insects has yet to be clarified. We developed an indoor experimental system to investigate the integration of geomagnetic and visual cues in the seasonal orientation of a globally distributed pest moth, the fall armyworm (Spodoptera frugiperda), a highly invasive species which in the past decade has colonized almost all potentially habitable regions of the globe. Our results demonstrate that fall armyworms require both geomagnetic and visual cues for accurate migratory orientation, with visual cues being indispensable for magnetic orientation. When visual and geomagnetic cues are placed in conflict moths become disoriented, although not immediately, indicating that sensory recognition of the conflict requires time to process. We also show that the absence of visual cues leads to a significant loss of flight stability, which likely explains the disruption in orientation. Our findings highlight that visual cues are critical for stable magnetic orientation in the fall armyworm, offering a basis for future investigations of visual-magnetic integration in noctuid migrants.

animal behavior and cognition↗

bioRxiv2025

Higher-order Residue Interactions Encode Diversified Physical Properties of Biomolecular Condensates

Biomolecular condensates often formed by proteins containing extended intrinsic disordered regions (IDPs) can coordinate versatile cellular processes. The biological functions of condensates are attributed to a variety of physical properties, including viscoelasticity, interfacial tension and saturation concentration. Comprehensive understanding of biological functions of protein condensates and the corresponding sequence encoding requires unified interpretation of the diverse physical properties by a single model. Here we report the diversified physical properties of condensate and the interactions between constituent IDPs can be interpreted on the basis of higher-order residue interactions. These findings are based on s-scale atomistic molecular dynamics simulations of three homogeneous IDP condensates (FUS PLD, ARF6 PLD, LAF-1 RGG) combined with the reported simulation trajectories of two heterogeneous condensates. In all interacting IDPs, there are residue interactions between finite motifs (3[~]5 residues) that organize in a highly cooperative way: residues form multivalent interactions and their remaking is localized. The sequence features of higher-order interactions (HOIs) are identified: tyrosine, arginine, glutamine and aspartic acid are essential, and their consecutive sequence position is also important. The interaction between corresponding motifs is highly sustained and dominates the cohesive energy of IDP. IDP condensate can be modeled as a HOI-crosslinked network. This model can successfully reproduce all reported experimental results of condensate viscoelasticity, interfacial tension and saturation concentration, and expand our insights into the molecular grammar of IDP interactions. Significance StatementThe biological functions of biomolecular condensates involve various physical properties of condensate. A unified model that describes all these physical properties is essential for the understanding of condensate functions. Our research reveals the prevalence of higher-order residue interactions in all IDP condensates we considered, which leads to extremely sustained interaction between corresponding residue motifs. Accordingly, we propose a physically crosslinked network based on higher-order residue interactions as a simplified model for condensate. This model can quantitively reproduce all reported experimental results of condensate physical properties, including viscoelasticity, interfacial tension and saturation concentration.

biophysics↗

bioRxiv2025

VAMP8 function reveals tight linkage between endocytic recycling and endocytosis

Clathrin-mediated endocytosis (CME) is a multistage process that involves the initiation and stabilization of clathrin-coated pits (CCPs) that invaginate and finally detach from the plasma membrane to form clathrin-coated vesicles (CCVs). Given that SNARE proteins are essential for downstream vesicle targeting and fusion events, their recruitment into nascent CCVs has been suggested to be a prerequisite for CME progression. However, which and how SNARE proteins regulate CME remains to be explored. Here, we showed that siRNA-mediated knockdown of the R-SNARE, VAMP8 impairs CCP initiation, stabilization and invagination and strongly inhibits CME. Mechanistically, recruitment of VAMP8 to CCVs is not required for CME. Instead, depletion of VAMP8 inhibits recycling of endocytic cargoes and as exemplified here by transferrin receptor, skews their trafficking toward lysosomal degradation. VAMP8 depletion therefore indirectly impairs CCV formation and inhibits CME by depleting endocytic cargo. Overall, our study provides new insights into the crosstalk between endocytosis and endocytic recycling of CME cargo and demonstrates the critical role for cargo recruitment in stabilizing nascent CCPs to regulate CME. Significance StatementWe demonstrate that VAMP8 depletion inhibits recycling of endocytic cargo and reroutes transferrin receptors toward lysosomal degradation, thereby impairing endocytic vesicle formation and inhibiting clathrin-mediated endocytosis. Thus, the work uncovers a critical link between endocytic recycling and endocytosis, highlighting the importance of recycling pathways in maintaining membrane dynamics. However, the recruitment of VAMP8 into nascent clathrin-coated pits by CALM is not required for CME. While SNARE recruitment is essential for formation of COPI and COPII vesicles, our findings suggest that this requirement is not generalizable to endocytic CCVs.

cell biology↗

bioRxiv2025

N-acetylcarnosine attenuates age-associated declines in multi-organ systems to improve survival

Histidine containing dipeptides (HCDs) such as N-acetylcarnosine are endogenous metabolites that are ergogenic and mitigate metabolic dysfunction. We previously demonstrated that short-term N-acetylcarnosine treatment is highly efficacious in protecting muscle atrophy induced by disuse. Here we demonstrate that a 6-months treatment of N-acetylcarnosine attenuates a broad spectrum of age-associated maladies and improved survival by [~]50% in female mice. A comprehensive survey of organ systems revealed that N-acetylcarnosine prevents decline in adiposity, diastolic function, vasodilation, muscle strength, and bone density. Together, N-acetylcarnosine substantially delays the onset of system-wide end-stage pathology to prolong lifespan. As an endogenously present metabolite, treatment with N-acetylcarnosine may be a safe and promising intervention to promote healthy aging in humans.

physiology↗

bioRxiv2025

Characterization and Genome Insights of Two Novel Plastic-Degrading Acinetobacter Species

The ubiquitous presence of Acinetobacter species in the environment holds significant biotechnology potential, particularly in the degradation of various pollutants. In this study, we present two plastic-degrading bacteria, strains CAAS 2-6T and CAAS 2-13T, isolated from landfill leachate and a strawberry farmland, respectively. Strain CAAS 2-6T showed the highest 16S rRNA sequence similarity (97.7%) with Acinetobacter gerneri DSM 14967T, while strain CAAS 2-13T was most closely related (99.6%) to Acinetobacter gerneri PS-1T. Phylogenetic analysis of 16S rRNA and gyrB-rpoB genes placed both strains on distinct branches. Genomic comparisons revealed the highest digital DNA-DNA hybridization (dDDH) / average nucleotide identity (ANI) values for CAAS 2-6T with Acinetobacter indicus CIP 110367T (22.7%/77.0%) and for CAAS 2-13T with Acinetobacter gerneri PS-1T (66.5%/96.0%). Based on phenotypic and chemotaxonomic data, we propose strain CAAS 2-6T as the novel species Acinetobacter lentus sp. nov. (type strain CAAS 2-6T = GDMCC 1.3951T = JCM 36321T = KCTC 8156T), and strain CAAS 2-13T as the novel subspecies Acinetobacter kanungonis subsp. fragariae subsp. nov. (type strain CAAS 2-13T = GDMCC 1.3956T = JCM 36322T = KCTC 8157T). Both strains utilized PLA, PBSA, PBS, PBAT, and PBT as sole carbon sources, with CAAS 2-6T exhibiting superior growth on PBSA, PBS, and PBAT. Genomic annotation identified genes encoding plastic-degrading enzymes multicopper oxidase AbMCO and alkane hydroxylase AlkB. Enzymatic depolymerization assays confirmed in vitro production of plastic monomers. These findings expand the known metabolic capabilities of Acinetobacter and provide promising new candidates for plastic bioremediation. IMPORTANCEAcinetobacter spp. demonstrate significant potential for plastic bioremediation. We characterize two novel strains: CAAS 2-6T (A. lentus sp. nov.) and CAAS 2-13T (A. kanungonis subsp. fragariae subsp. nov.), capable of degrading five major polyesters (PLA, PBAT, PBT, PBS, PBSA) as sole carbon sources. Genomic and enzymatic analyses identified multicopper oxidase (AbMCO) and alkane hydroxylase (AlkB) as key depolymerase. This work: (i) Expands plastic-degrading Acinetobacter diversity; (ii) Provides novel biocatalysts and genomic resources; (iii) Delivers engineerable microbial chassis.

microbiology↗

bioRxiv2025

Comparative population genomics of relictual Caribbean island Gossypium hirsutum

Gossypium hirsutum is the worlds most important source of cotton fiber, yet the diversity and population structure of its wild forms remain largely unexplored. The complex domestication history of G. hirsutum combined with reciprocal introgression with a second domesticated species, G. barbadense, has generated a wealth of morphological forms and feral derivatives of both species and their interspecies recombinants, which collectively are scattered across a large geographic range in arid regions of the Caribbean basin. Here we assessed genetic diversity within and among populations from two Caribbean islands, Puerto Rico (n = 43, five sites) and Guadeloupe (n = 25, one site), which contain putative wild and/or introgressed forms. Using whole-genome resequencing data and a phylogenomic framework derived from a broader genomic survey, we parsed individuals into feral derivatives and truly wild forms. Feral cottons display uneven levels of genetic and morphological resemblance to domesticated cottons, with diverse patterns of genetic variation and heterozygosity. These patterns are inferred to reflect a complex history of interspecific and intraspecific gene flow that is spatially highly variable in its effects. Wild cottons in both Caribbean islands appear to be relatively inbred, especially the Guadeloupe samples. Our results highlight the dynamics of population demographics in relictual wild cottons that experienced profound genetic bottlenecks associated with repeated habitat destruction superimposed on a natural ecogeographical distribution comprising widely scattered populations. These results have implications for conservation and utilization of wild diversity in G. hirsutum.

evolutionary biology↗

bioRxiv2025

A Universal 6iL/E4 Culture System for Deriving and Maintaining Embryonic Stem Cells Across Mammalian Species

The derivation of authentic embryonic stem cells (ESCs) from diverse mammalian species offers valuable opportunities for advancing regenerative medicine, studying developmental biology, and enabling species conservation. Here, we report the development of a robust, serum-free culture system, termed 6iL/E4 that enables the derivation and long-term self-renewal of ESCs from multiple mammalian species, including mouse, rat, bovine, rabbit, and human. Using systematic signaling pathway analysis, we identified key regulators--including GSK3, STAT3, PDGFR, BRAF, and LATS--critical for ESC maintenance across species. Additionally, inducible expression of KLF2 and NANOG enhances the naive pluripotency and chimeric potential of bovine ESCs. The E4 medium also supports stable ESC growth while minimizing lineage bias. These findings reveal conserved principles underlying ESC self-renewal across divergent mammalian species and provide a universal platform for cross-species stem cell research, disease modeling, and biotechnology applications. In BriefWang et al. developed 6iL/E4, a serum-free system sustaining ESCs from mouse, rat, bovine, rabbit, and human. These findings reveal conserved fundamental mechanisms governing ESC self-renewal across diverse mammalian species. HighlightsO_LIDeveloped 6iL/E4 system for ESC derivation across five mammalian species. C_LIO_LIPDGFR signaling inhibition as critical for ESC derivation across species. C_LIO_LIE4 medium improves ESC maintenance and avoids neural bias of traditional N2B27. C_LIO_LIInducible KLF2/NANOG enhances naive pluripotency and chimera formation in bovine. C_LI

cell biology↗

bioRxiv2025

Selective GSK3α Inhibition Promotes Self-Renewal Across Different Stem Cell States

Pan-GSK3/{beta} inhibition promotes stem cell self-renewal through activation of WNT/{beta}-catenin signaling, but its broad effects complicate the precise control of stem cell states. Here, we show that selective inhibition of GSK3 with BRD0705 supports the long-term self-renewal of mouse embryonic stem cells (ESCs), epiblast stem cells (EpiSCs), and neural stem cells (NSCs), independent of {beta}-catenin signaling. When combined with the tankyrase inhibitor IWR1, BRD0705 broadly supports the maintenance of diverse pluripotent stem cell states, including ESCs, EpiSCs, and formative pluripotent stem cells. This BRD0705/IWR1 cocktail enables stable co-culture of naive ESCs and primed EpiSCs while preserving their distinct molecular and functional identities. Single-cell transcriptomics, epigenomic profiling, and functional assays confirm sustained lineage-specific features across stem cell types. These findings demonstrate that selective GSK3 inhibition enhances stemness by buffering against differentiation cues and promoting intrinsic self-renewal capacity. This work identifies GSK3 as a key regulator of self-renewal across distinct stem cell states and establishes a versatile culture system with broad applications. In BriefWang et al. demonstrate that selective GSK3 inhibition with BRD0705 supports self-renewal of pluripotent and neural stem cells. Combined with IWR1, it enables long-term co-culture of naive and primed stem cells while preserving their distinct molecular and functional identities. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=181 HEIGHT=200 SRC="FIGDIR/small/653860v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@4e371eorg.highwire.dtl.DTLVardef@104b287org.highwire.dtl.DTLVardef@164bd96org.highwire.dtl.DTLVardef@daf1ba_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIGSK3 inhibition by BRD0705 promotes self-renewal of ESCs, EpiSCs, and NSCs C_LIO_LIBRD0705/IWR1 enables long-term co-culture of ESCs and EpiSCs C_LIO_LICo-cultured ESCs and EpiSCs retain distinct naive or primed identities C_LIO_LIBRD0705 preserves stem cell states independently of {beta}-catenin signaling C_LI

cell biology↗

bioRxiv2025

Sexually Dimorphic Circadian Regulation of Eclosion and Clock Gene Expression in a Migratory Moth Spodoptera frugiperda

The circadian clock orchestrates essential behavioral and molecular processes, including the timing of eclosion, one of the most tractable and ecologically relevant outputs of the circadian system. Understanding eclosion timing may offer insights into the circadian clock mechanisms that underlie migratory timing. Here, we characterize the diel and circadian patterns of eclosion and core clock gene expression in the fall armyworm (FAW), Spodoptera frugiperda, a globally distributed migratory moth. Using a custom-designed eclosion monitoring system under both 14 hours light-10 hours dark (L14: D10) and constant darkness (DD) conditions, we observed clear diel eclosion rhythms, peaking shortly after lights-off under L14: D10. Under DD, these rhythms became delayed and damped over three consecutive days, consistent with circadian control. Males exhibited more dispersed emergence patterns and distinct eclosion distributions than females under L14: D10 and DD, suggesting sexually dimorphic timing. Gene expression profiling revealed rhythmic oscillations of five canonical clock genes, cyc, clk, tim, per, cry2, with sex-dimorphic differences in mesor, amplitude, or phase, particularly the mesors of males are higher than those of females in five clock genes under L14: D10. These results provide strong evidence for sexually dimorphic circadian regulation at both behavioral and molecular levels in a migratory insect that recently invaded China, suggesting that sex-dimorphic circadian architecture may contribute to allochronic speciation and ecological strategies such as eclosion and migratory timing in a novel environment. Leveraging the established circadian eclosion rhythm assay, which captures core features of clockwork mechanisms, further investigation may reveal how these sex-based differences contribute to the evolution of circadian function and adaptive traits.

ecology↗

bioRxiv2025

Effects of Combination Therapy of a CDK4/6 and MEK Inhibitor in Diffuse Midline Glioma Preclinical Models

BackgroundDiffuse midline glioma (DMG) is an incurable brain cancer without a single FDA-approved drug that prolongs survival. CDK4/6 inhibitors have been evaluated in children with DMG with limited efficacy. Since MAPK pathway activation is upstream of cell proliferation, we hypothesized that MEK inhibitors may increase the anti-tumor effects of CDK4/6 inhibitors. Here, we evaluated the efficacy of the CDK4/6 inhibitor ribociclib and the MEK inhibitor trametinib in human and murine DMG models to investigate combinational effects. MethodsWe conducted in vitro and in vivo assays using DMG cell lines from human patient-derived xenografts (PDX) and murine genetically engineered models (GEM). In vitro, we assessed synergy across human DMG lines. In vivo, we evaluated therapeutic effects with histological examinations, survival analysis, pharmacokinetic measurements, and RNA-sequencing analysis. ResultsIn vitro, ribociclib and trametinib had variable synergistic effects against human DMG cell lines. In vivo, a five-day treatment with combination therapy in the GEM DMG model significantly decreased cell proliferation and increased apoptosis compared with the vehicle, with trametinib having mostly cytotoxic effects and ribociclib having primarily cytostatic effects. In addition, a 21-day treatment with combination therapy significantly prolonged mice survival compared with the vehicle in the GEM DMG model (median survival: 112 days vs. 71.5 days, log rank test p=0.0195). In an orthotopic PDX model, combination therapy did not prolong mice survival compared with vehicle, ribociclib, and trametinib. LC/MS analysis showed adequate drug delivery across the blood-brain-barrier (BBB) into tumor tissue in both GEM and PDX models. Transcriptomic analysis in the GEM model suggests that combination therapy inhibited the MAPK pathway and inflammation. ConclusionsCombination therapy with ribociclib and trametinib significantly prolonged survival in the GEM model but not in the PDX model, highlighting the importance of testing novel therapies in diverse models. Key PointsO_LICombination therapy had cytotoxic and cytostatic effects in vivo in the GEM model. C_LIO_LICombination therapy had variable synergy in vitro in human DIPG models. C_LIO_LICombination therapy significantly prolonged mice survival compared with the vehicle in the GEM model. C_LIO_LICombination therapy did not prolong survival in the PDX model. C_LIO_LITranscriptional analysis suggests that the combination modulated inflammation in the GEM model. C_LIO_LIThis work highlights the importance of testing novel therapies in diverse models. C_LI Importance of the StudyDMG is an incurable brain cancer without a single FDA-approved drug that prolongs survival. CDK4/6 inhibitors have been evaluated in children with DMG with limited efficacy. As MAPK pathway activation is known to be upstream of cell proliferation, we hypothesize that combination therapy will have additive effects relative to monotherapy. In this study, we evaluated the efficacy of the CDK4/6 inhibitor ribociclib and the MEK inhibitor trametinib in GEM and PDX DMG models. In vitro, the inhibitor combination demonstrated variable synergy in DMG human cell-lines, but in vivo, it had limited efficacy in a PDX orthotopic model. In the GEM model, the combination prolonged survival compared with the vehicle and with single agents. PK studies showed similar drug levels in tumor tissue between the GEM and PDX models. Transcriptomic analysis in the GEM suggests that the drug combination significantly inhibited inflammation. These results highlight the importance of testing therapies in diverse models and suggest that this combination may be an effective therapy in patients with DMG.

cancer biology↗

bioRxiv2025

HuR-Driven Reversible Mitochondrial Shuttling Buffers Cytosolic miRNA Levels in Hepatic Cells

Mitochondria, the "powerhouse" of mammalian cells, also serve as key storage sites for ions, metabolites, and enzymes vital for metabolic regulation. Exploring the regulatory processes that control the activities of miRNAs, the key non-coding RNA in mammalian cells, we found a context-dependent reversible localization of specific miRNAs to the mitochondrial matrix. Our data suggests a de novo role of mitochondria as miRNA sinks in mammalian cells. miR-122 is a key hepatic miRNA regulating metabolic processes in the mammalian liver. In this study, we observed increased mitochondrial targeting of miR-122 in amino acid-starved hepatic cells. Interestingly, when cells were refed with amino acids, mitochondrial miR-122 gets relocalized and reused in the cytosol for the translational repression process. Moreover, this phenomenon is not limited to miR-122 as several mitochondrial miRNAs (mito-miRs) follow similar transient storage inside mitochondria in stressed cells. Remarkably, mitochondria-localized mito-miRs preferentially target mRNAs encoding crucial mitochondrial components related to apoptosis. Hence, hepatic cells regulate apoptosis pathways during the starvation-refeeding cycle by shuttling a specific set of miRNAs to and from mitochondria, thereby balancing cytosolic miRNA content and homeostasis. Stress response miRNA binder ELAVL1 or HuR protein was found to be both necessary and sufficient for transporting the mature mito-miRs to the mitochondrial matrix - a process also controlled by the interaction between mitochondria and the endoplasmic reticulum. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/647748v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1178f5dorg.highwire.dtl.DTLVardef@1c5578borg.highwire.dtl.DTLVardef@f68555org.highwire.dtl.DTLVardef@ab9c40_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LISpecific miRNAs (mito-miRs), including miR-122, are targeted to mitochondria for transient storage in stressed hepatic cells. C_LIO_LIMito-miRs storage in mitochondria allows the expression of apoptosis-related genes in stressed cells. C_LIO_LIMito-miRs relocalization to cytoplasm on stress reversal allows suppression of apoptosis. C_LIO_LIBinding with ELAVL1 protein HuR allows reversible shuttling of mito-miRs to and from mitochondria in hepatic cells. C_LIO_LIER-mitochondrial interaction is key for the mitochondrial localization of miRNAs. C_LI

cell biology↗

bioRxiv2025

Pcdh20 is a POU2F3 target gene required for proper tuft cell microvillus formation

BACKGROUND & AIMSTuft cells play protective roles in infection, inflammation, and tumorigenesis through the secretion of cytokines and eicosanoids. Tuft cells are known for their tall, blunt microvilli, thought to be analogous to mechanosensory hair cell stereocilia; however, a functional role for the microvillar apparatus has not been identified. POU2F3 is the master regulator transcription factor for tuft cells, yet how POU2F3 drives formation of this unique structure is unknown. Here, we aimed to identify POU2F3 target genes and commonalities between tuft and hair cells to better understand this unique structure. METHODSPOU2F3 ChIP-seq was performed on tuft cells and compared to the hair cell transcriptome. Tuft cell RNA-seq datasets were interrogated for hair cell structural and mechanosensory genes; expression was validated. Intestinal and gallbladder tuft cells were examined using multiple light and electron microscopy (EM) modalities. PCDH20 was knocked down in mouse models and ultrastructural analyses were performed. The tuft cell cytoskeleton was modeled using AlphaFold3 prediction. RESULTSGenes encoding structural and mechanosensory proteins common to both tuft and hair cells, including Pcdh20, were identified. Imaging localized PCDH20 to tuft cell microvilli and hair cell stereocilia. Genetic ablation of Pcdh20 in mice resulted in structural defects in tuft cell microvilli, including loss of rigidity and organization. Molecular modeling suggests PCDH20 homodimers link adjacent microvilli. CONCLUSIONSPcdh20 is a POU2F3 target gene in tuft cells, critical to maintain the rigid microvillar apparatus. These findings, together with the shared expression of mechanosensory components like TMC1, support the hypothesis that tuft cells could have mechanosensory capabilities analogous to cochlear hair cells.

cell biology↗