Biology subjects

Gu, M.

Publications and source records attributed to Gu, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Engineering human myelin microphysiological systems for testing patient treatment response in multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system characterized by neuroinflammation, demyelination, and neurodegeneration, associated with a complex interplay between the innate and adaptive immune systems. Currently, no cure is available for MS, and personalized disease-modifying treatment remains largely limited, partially due to the lack of preclinical human models that can faithfully recapitulate disease pathology and evaluate treatment responses at the individual-patient level. Here, we report a human myelin microphysiological system (myelin MPS) platform that recaptures disease phenotype and treatment response for testing patient treatment response. By culturing neural organoids on 3D-printed devices containing directional microfibers, followed by coculture with oligodendrocyte progenitor cells, 96 myelin MPS models can be generated within a conventional well plate. Using this myelin MPS platform, autologous T cells and monocytes from MS patients induced substantially greater demyelination than those from healthy donors, accompanied by expansion of proinflammatory T-cell subsets and increased myelin uptake by monocytes/macrophages after coculture with these healthy myelinating neural tissues. Integration of imaging and flow-cytometric features distinguished healthy-donor, untreated-MS, responder, and nonresponder profiles following treatment with prednisone, glatiramer acetate, interferon {beta}-1a, or dimethyl fumarate. Thus, the myelin MPS platform provides a scalable, human pathophysiology-relevant platform for functional phenotyping and individualized treatment-response evaluation in MS.

bioengineering↗

bioRxiv2026

Brain organoid computing for robotic decision-making

Biomimicry has inspired the evolution of robotics toward greater autonomy, adaptability, and symbiosis with humans and dynamic environments. However, current robotic systems still face major challenges in recapitulating the high-efficiency decision-making capabilities of the human brain under complex and dynamic conditions. Here, we present Brainobot, a biohybrid robotic system that establishes a brain organoid controller as a high-level robotic decision-making layer for closed-loop embodiment. By leveraging brain organoid reservoir computing, Brainobot interacts with dynamic environments by receiving and processing sensory inputs and generating motor actions. As a proof-of-concept demonstration, Brainobot is implemented in a humanoid robotic system to perform real-world tasks, including object grasping and laser chasing. Interestingly, Brainobot exhibits unique features, including cross-task adaptivity, high computing efficiency, and low energy consumption. Thus, our approach may provide insights for advancing robotic embodiment and understanding biological decision-making.

bioengineering↗

bioRxiv2026

Information processing-like phenotype of human brain organoids

Human brain organoids recapitulate key physiological features and functions of the human brain and hold remarkable potential for studying neurological diseases. Despite clinical evidence suggesting that neurodegenerative diseases impair the information-processing ability of the human brain, the capacity of brain organoids for information processing and its relationship to neural network function remain largely unexplored. Here, we test and quantify information-processing-like properties of human cortical organoids using a task-based functional phenotyping framework (Brainopheno). We demonstrate the pattern-processing-like phenotype of cortical organoids through the representation and classification of evoked neural activities in response to distinct spatial input stimulation patterns via a microelectrode array (MEA) system. Moreover, this functional phenotype emerges with network maturation and is disrupted by pharmacological perturbations, linking classification performance to the functional integrity of organoid neural networks (ONNs). Importantly, this functional phenotype also reveals functional deficits in ONNs altered by a familial Alzheimer's disease (AD)-associated gene mutation (APP) and by monocytes from patients with sporadic AD. This work may establish a new quantifiable functional phenotype of neural organoids and provide a framework to bridge molecular and cellular profiles with neural circuit function for basic neurology, disease phenotyping, and therapeutic development.

bioengineering↗

bioRxiv2026

Vessel Organoids Reveal FOXF1 Variant-Specific Regulation of Mesoderm and Capillary Development

How allelic variants in lineage-regulating transcription factors drive diverging human developmental outcomes remains poorly understood. This is partly due to the lack of human model systems. Here, we used vessel organoids from human induced pluripotent stem cells (hiPSCs) to resolve variant-specific functions of Forkhead Box F1 (FOXF1), a critical regulator of mesoderm and vascular development. Using three patient-derived hiPSC lines harboring unique FOXF1 variants, we show that heterozygous variants cause capillary maldevelopment of varying severity. Single-nucleus multiomic analysis revealed variant-specific mechanisms - a severe variant impairs differentiation of nascent mesoderm to lateral plate mesoderm and disrupts vascular progenitor specification, while moderate variants permit mesoderm differentiation but rewire vascular progenitor states and function. Restoration of wild-type FOXF1 via lipid nanoparticle-mediated mRNA delivery rescued capillary formation in a variant- and developmental-stage-dependent manner. Together, these findings demonstrate that different variants disrupt stage-specific FOXF1 functions in human mesoderm-to-vascular development, underscoring the importance of variant-specific therapeutic strategies. HIGHLIGHTS O_LIHuman vessel organoids reveal variant-specific roles of FOXF1 in mesoderm patterning and capillary development. C_LIO_LISevere FOXF1 variant c.253T>A (p.F85I) impairs nascent mesoderm-to-lateral plate mesoderm differentiation and disrupts vascular progenitor specification. C_LIO_LI Moderate FOXF1 variants differentially rewire endothelial and mural progenitor cell states and function. C_LIO_LILipid nanoparticle-mediated FOXF1 mRNA delivery rescues capillary formation in a variant- and developmental-stage-dependent manner. C_LI

developmental biology↗

bioRxiv2026

Programmable synthetic cytokine receptors polarize macrophages to user-defined functional states

Technology that precisely controls macrophage polarization to distinct functional states would deepen our understanding of macrophage biology and enable the development of new macrophage cell therapies. Here, we use a synthetic cytokine receptor (SCR) platform with a programmable signaling domain to control the polarization of primary human macrophages. SCRs containing signaling motifs from the interferon-gamma (IFN-{gamma}) or Interleukin-10 (IL-10) receptors mimic key features of pro-inflammatory or anti-inflammatory polarization, respectively. Random recombination of nine distinct signaling motifs to create new SCR signaling domains generates a diverse landscape of synthetic macrophage states with varied expression of inflammatory markers (CD80, CD40) and anti-inflammatory markers (CD163, CD206), and varied phagocytic capacity. SCRs programmed with multiple YLxQ motifs increase macrophage phagocytosis of E. coli and chimeric antigen receptor (CAR)-macrophage phagocytosis of cancer cells in mice, reducing tumor burden by 30-fold. The motif-dependent polarization is well-described by a two-state model, enabling quantitative prediction of macrophage polarization state from SCR signaling domain composition. Leveraging this model, we design an SCR that simultaneously enhances phagocytosis and maintains a macrophage pro-inflammatory state. Together, these findings establish a framework for synthetic programming of macrophage polarization states, with potential applications in cancer immunotherapy and other disease contexts.

synthetic biology↗

bioRxiv2025

Generation of Valvular Interstitial Cells from Human Pluripotent Stem Cells

Heart valves are living structures whose sophisticated functions are mediated by a specialized population of mesenchymal cells known as valvular interstitial cells (VICs). Given their central role in valve homeostasis, VICs represent a promising cell population for studying heart valve diseases and developing novel therapies to treat them. Here, we describe a strategy for generating VICs from human pluripotent stem cells (hPSCs) by stage-specific manipulation of developmental signalling pathways. Our results demonstrate that hPSC-derived VICs show a high transcriptional similarity to primary human fetal VICs and can secrete key proteins of the valve extracellular matrix. We further investigate the heterogeneity of hPSC-derived VICs and identify two major subpopulations with distinct molecular and functional properties, mirroring the cellular diversity observed in vivo. Finally, we utilize an in vitro model of Noonan syndrome to demonstrate that hPSC-derived VICs can accurately recapitulate key aspects of valve disease. Collectively, these findings provide a reproducible method for the scaled generation of bona fide hPSC-derived VICs and establish their utility in disease modelling and tissue engineering applications. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABS- We established a robust platform to generate bona fide valvular interstitial cells (VICs) from human pluripotent stem cells (hPSCs), recapitulating native VIC identity and function. - We delineated signaling pathways that promote the development of two distinct VIC subsets and identified a surface marker to distinguish between them. What are the clinical implications?- A renewable, human-specific source of VICs enables precise mechanistic studies of valve development and disease that are not possible with limited surgical specimens or animal models. - This platform creates opportunities for therapeutic applications, including drug discovery and tissue engineering approaches for valve repair.

cell biology↗

bioRxiv2025

Cancer cell phagocytosis induces an anti-inflammatory gene regulatory program in macrophages

Macrophages are capable of eliminating cancer cells by phagocytosis, particularly in the presence of monoclonal antibody (mAb) therapies targeting tumor antigens. Paradoxically, tumor-associated macrophages are typically associated with poor patient outcome, and can promote tumor growth by secretion of immunosuppressive cytokines and growth factors. The mechanisms by which these pro-tumor macrophage states arise are poorly understood, and it is unclear how mAb-induced cancer cell phagocytosis may contribute to these states. To understand how antibody-dependent cancer cell phagocytosis (ADCP) alters macrophage state and function, we profiled gene expression and chromatin accessibility changes over time after ADCP. We observed that after ADCP, macrophages upregulate an anti-inflammatory gene regulatory program, characterized by expression of pro-angiogenic and immunosuppressive chemokine genes, and increased activity by cellular, oxidative, and lysosomal stress transcription factors. This gene regulatory program was shared among phagocytic macrophages following either ADCP or apoptotic cancer cell phagocytosis, in addition to substrate-specific pathways. Conditioned media from macrophages promoted EMT in cancer cells, but this pro-EMT macrophage phenotype was attenuated following ADCP, but not following apoptotic cancer cell phagocytosis. The phagocytic gene signature we identified in vitro is also expressed by tumor-associated macrophages across numerous cancer types in vivo. Together, this work identifies an anti-inflammatory and immunosuppressive epigenetic program in macrophages following ADCP upon mAb treatment, and expands our understanding of how phagocytosis influences macrophage heterogeneity in the tumor microenvironment.

cancer biology↗

bioRxiv2025

Revealing the nervous system requirements of Alzheimer disease risk genes in Drosophila

Most Alzheimers disease (AD) susceptibility genes have poorly understood roles in the central nervous system (CNS). To address this gap, we systematically characterized 100 conserved candidate AD risk genes using a cross-species strategy in the fruit fly, Drosophila melanogaster. Genes were prioritized based primarily on human functional genomic evidence. We generated custom, loss-of-function alleles for each of the conserved fly orthologs. Most of the genes (80%) are expressed in the adult brain, including 24 neuron- and 13 glia-specific expression patterns. Overall, we identify 50 candidate AD risk gene homologs with requirements for CNS structure or function, including 18 whose loss of function causes neurodegeneration (e.g., Snx6/SNX32 and ClC-a/CLCN1), 35 required for neurophysiology (e.g., Arr1/ARRB2, stai/STMN4), and 8 with diminished CNS resilience following a thermal or mechanical stress (e.g., cindr/CD2AP, Amph/BIN1). In a parallel screen, we found 28 AD risk gene homologs (e.g, Ets98B/SPI1, Yod1/YOD1) that modify the neurotoxicity of either amyloid-{beta} peptide or tau protein, which aggregate to form AD pathology. To translate our findings back to human AD, we developed and deployed oligogenic risk scores based on gene clusters with shared nervous system phenotypes in flies, pinpointing functional pathways that differentially drive AD risk. Our results--available online via the Alzheimers Locus Integrative Cross-species Explorer (alice.nrihub.org)--reveal novel nervous system requirements for dozens of AD risk genes and may enable dissection of causal heterogeneity in AD.

genetics↗

bioRxiv2025

Highly accurate protein structure prediction-based virtual docking pipeline accelerating the identification of anti-schistosomal compounds

Schistosomiasis is a major neglected tropical disease that lacks an effective vaccine and faces increasing challenges from praziquantel resistance, underscoring the urgent need for novel therapeutics. Target-based drug discovery (TBDD) is a powerful strategy for drug development. In this study, we utilized AlphaFold to predict the structures of target proteins from Schistosoma mansoni and Schistosoma japonicum, followed by virtual molecular screening to identify potential inhibitors. Among 202 potential therapeutic targets, we identified 37 proteins with high-accuracy structural predictions suitable for molecular docking with 14,600 compounds. This screening yielded 268 candidate compounds, which were further evaluated in vitro for activity against both adult and juvenile S. mansoni and S. japonicum. Seven compounds exhibited strong anti-schistosomal activity, with HY-B2171A (Carubicin hydrochloride, CH) emerging as the most potent. CH was predicted to target the splicing factor U2AF65, and knockdown of its coding gene Smp_019690 resulted in a phenotype similar to CH treatment. RNA sequencing revealed that both CH treatment and Smp_019690 RNAi disrupted splicing events in the parasites. Further studies demonstrated that CH impairs parasite viability by inhibiting U2AF65 function in mRNA splicing regulation. By integrating RNAi-based target identification with structure-based virtual screening, alongside in vitro phenotypic and molecular analyses of compound-treated schistosomes, our study provides a comprehensive framework for anti-schistosomal drug discovery and identifies promising candidates for further preclinical development. Author SummarySchistosomiasis is a debilitating disease caused by parasitic worms and affects over 230 million people worldwide. For decades, control of the disease has relied on a single drug, praziquantel, which is less effective against juvenile parasites and increasingly threatened by the risk of drug resistance. To accelerate the discovery of new treatments, we integrated advanced computational tools with molecular validation. Using AlphaFold-predicted structures of 37 key schistosome proteins, we performed large-scale virtual screening to identify promising drug candidates. Over 200 compounds were prioritized for phenotypic screening on two major human-infective schistosome species, leading to the discovery of seven with potent anti-parasitic activity. Among them, Carubicin hydrochloride showed strong efficacy by targeting a critical splicing factor, U2AF65. Inhibiting this protein--either chemically or through gene silencing-- disrupted parasite movement, suppressed cell proliferation, and altered splicing events. Our work highlights the power of integrating AI-based protein modeling with phenotypic and mechanistic validation to accelerate drug discovery for neglected tropical diseases like schistosomiasis.

pathology↗

bioRxiv2025

Isoform-Specific Roles of NTRK2 in Pulmonary Vascular Regeneration

Bronchopulmonary dysplasia (BPD) is a chronic lung disease in premature infants with no curative therapy, characterized by impaired alveologenesis and capillary formation. However, the molecular mechanisms underlying endothelial dysfunction, a key driver of BPD pathogenesis in human, remain poorly understood. Here, through multiomic profiling of vascular endothelial cells isolated from control and BPD patient lungs, we uncovered an expansion of general capillary endothelial cells (gCap) with aberrant expression of the neurotrophic receptor tyrosine kinase 2 (NTRK2) in BPD. Importantly, we identified a pathological NTRK2 isoform switch that dictates the regenerative capacity of gCap cells. Full-length NTRK2 (NTRK2-FL) promoted gCap regeneration in response to hyperoxic injury, whereas RBFOX2-mediated splicing of NTRK2-FL into a truncated isoform (NTRK2-T1) contributed to maladaptive responses and irreversible alveolar simplification in severe BPD cases. Restoring NTRK2-FL using lipid nanoparticle-delivered mRNA promoted angiogenesis and reversed alveolar simplification in vessel organoids and BPD-like mice. These findings identified NTRK2 isoform imbalance as a key driver of endothelial dysfunction and support isoform-specific RNA therapy as a promising strategy for vascular regeneration and repair. HIGHLIGHTS* Multiomic and spatial profiling reveal abnormal gCap subtype in human BPD lungs * NTRK2 isoform switch dictates endothelial regeneration in response to hyperoxic injury * RBFOX2 mediates splicing from NTRK2-FL to NTRK2-T1, driving irreversible alveolar damage * NTRK2-FL mRNA therapy restores vascular regeneration after injury

developmental biology↗

bioRxiv2025

Understanding monocyte-driven neuroinflammation in Alzheimers disease using human brain organoid microphysiological systems

Increasing evidence suggests that Alzheimers disease (AD) pathogenesis strongly correlates with neuroinflammation. Peripheral monocytes are crucial components of the human immune system that may play a role in neuroinflammation, but their contribution to AD pathogenesis is largely understudied partially due to the lack of appropriate human models. Here, we present human cortical organoid microphysiological systems (hCO-MPSs) for modeling dynamic AD neuroinflammation mediated by monocytes. By incorporating 3D printed devices into an existing cortical organoid protocol, 96 hCO-MPSs can be established with significantly reduced necrosis and hypoxia as well as enhanced viability within a commonly used 96 well plate, and each hCO-MPS consists of a doughnut-shaped hCO and a 3D printed device per well. Using this approach, monocytes from AD patients exhibit higher infiltration, decreased amyloid-beta (A{beta}) clearance, and stronger inflammatory responses compared to monocytes from age-matched control donors. Moreover, pro-inflammatory effects such as elevated astrocyte activation and neuronal apoptosis were observed to be induced by AD monocytes. Furthermore, the significant increase in the expression of IL1B and CCL3, both at the transcriptional and protein levels, indicated the pivotal role of these cytokine and chemokine in monocyte-mediated AD neuroinflammation. Our findings provide insight for understanding monocytes role in AD pathogenesis, and the user-friendly MPS models we present are compatible with existing laboratory settings, highlighting their potential for modeling neuroinflammation and developing new therapeutics for various neuroinflammatory diseases.

bioengineering↗

bioRxiv2025

Delayed Trp53 activation protects Dnmt3a-mutant hematopoietic stem cells from inflammatory attrition.

Hematopoietic stem cells (HSCs) accumulate somatic mutations over time, some conferring a fitness advantage that can lead to clonal hematopoiesis (CH). Mutations in DNMT3A, particularly at hotspot R882, are the most prevalent in CH and carry an increased risk of acute myeloid leukemia (AML). Although DNMT3A R882 mutations are linked to global DNA hypomethylation, the mechanisms underlying their selective advantage remain unclear. Here, we show that Dnmt3a-R882H mutant HSCs exhibit resilience under inflammatory and genotoxic stress. During IL-1{beta}-induced emergency granulopoiesis, Dnmt3a R882H/+ HSCs uncouple increased proliferation from stem cell exhaustion. In contrast, wild-type HSCs rapidly progress to terminal differentiation. We link this phenotype to a delayed activation of the p53-p21-DREAM axis, that allows mutant HSCs to avoid attrition, despite increased replication stress. Similarly, mutant HSCs exhibit delayed Trp53 activation following irradiation, but eventually recover a physiological Trp53 response. Analysis of patient data reveals shared phenotypic features between DNMT3A and monoallelic TP53 mutations in CH and myeloid neoplasms, highlighting potential functional similarities. Collectively, these findings suggest that the expansion of DNMT3A-mutant clones is affected by impaired TP53 signaling, which confers resilience against stressors. Therapeutic strategies targeting inflammatory pathways or the p53-p21-DREAM axis may reduce DNMT3A-CH expansion and/or progression and its associated risks.

molecular biology↗

bioRxiv2025

Food pairing independently associated with cardiometabolic traits beyond dietary patterns across western and eastern populations

Cardiometabolic health is closely linked to diet. Both specific food choices and dietary indices are commonly used to guide cardiometabolic protection strategies. However, the balance between foods may also significantly impact cardiometabolic health. Here, we explore the balance and imbalance in long-term dietary food intake to quantify food pairing patterns (FPs), providing insights beyond conventional dietary indices and single-food intake frequencies. Using data from our GGMP (n = 6,994) and the NHANES (n = 7,350) cohorts, we observed that long-term food pairing patterns are independent of single food intake frequency and dietary indices. We identified 1,759 and 306 cardiometabolic-related long-term food pairing patterns from two cohorts (FDR < 0.05), respectively. Notably, around 80% of these pairing foods not individually associated with cardiometabolic traits, and food pairing pattern associations with cardiometabolic traits at hyper food group level were consistent across Eastern and Western populations. Besides, mediation analysis revealed that 72.7% of long-term food pairing patterns affected cardiometabolic traits through 31 microbial genera, with Clostridiumsensustricto1 playing a predominant role. Moreover, multi-step mediation analysis found that microbes mediated the impact of long-term food pairing patterns on cardiometabolic traits primarily through their metabolic pathways, such as pyruvate fermentation to propanoate and ergothioneine biosynthesis pathways. Our data suggest that balance between dietary foods is broadly associated with cardiometabolic traits by modulating gut microbial functionalities, in contrast to the lower occurrence when considering individual foods in isolation. These results offer a novel perspective for designing personalized dietary strategies beyond present dietary indices to enhance cardiometabolic health.

microbiology↗

bioRxiv2024

BindingGYM: A Large-Scale Mutational Dataset Toward Deciphering Protein-Protein Interactions

Protein-protein interactions are crucial for drug discovery and understanding biological mechanisms. Despite significant advances in predicting the structures of protein complexes, led by AlphaFold3, determining the strength of these interactions accurately remains a challenge. Traditional low-throughput experimental methods do not generate sufficient data for comprehensive benchmarking or training deep learning models. Deep mutational scanning (DMS) experiments provide rich, high-throughput data; however, they are often used incompletely, neglecting to consider the binding partners, and on a per-study basis without assessing the generalization capabilities of fine-tuned models across different assays. To address these limitations, we collected over ten million raw DMS data points and refined them to half a million high-quality points from twenty-five assays, focusing on protein-protein interactions. We intentionally excluded non-PPI DMS data pertaining to intrinsic protein properties, such as fluorescence or catalytic activity. Our dataset meticulously pairs binding energies with the sequences and structures of all interacting partners using a comprehensive pipeline, recognizing that interactions inherently involve at least two proteins. This curated dataset serves as a foundation for benchmarking and training the next generation of deep learning models focused on protein-protein interactions, thereby opening the door to a plethora of high-impact applications including understanding cellular networks and advancing drug target discovery and development.

biophysics↗

bioRxiv2024

Polarized ATP synthase in synaptic mitochondria induced by learning and plasticity signals

MINFLUX represents a state-of-the-art single-molecule localization microscopy technology that surpasses the conventional diffraction limit, enabling the visualization and analysis of nanostructures with exceptional precision. Despite its potential, the utilization of MINFLUX has been primarily confined to in vitro cultured cell environments. In this research, we have refined the sample preparation protocols to facilitate 3D MINFLUX imaging in fixed brain tissue sections, focusing on the mitochondrial distribution within dendritic spines and engram cells of the dentate gyrus. Probing single molecules in vivo reveals an interesting finding: the reorganization of mitochondrial inner membrane proteins during synaptic plasticity within dendritic spines of engram cells. Utilizing 3D MINFLUX nanoscopy, we identified a significant redistribution of -F1-ATP synthase, which correlates with learning related activities. This redistribution implies a pivotal function for polarized ATP synthesis in the vicinity of the postsynaptic zone, which may modulate both short-term and long-term synaptic modifications, thereby influencing synaptic plasticity and memory consolidation. Furthermore, dual-color 3D MINFLUX imaging has uncovered distinct patterns of mitochondrial reorganization involving both the inner and outer membranes within dendritic spines. These patterns, induced by plasticity signals, persist for up to 12 hours in neuronal cultures. This distinction highlights the presence of distinct regulatory mechanisms governing mitochondrial proteins during synaptic plasticity. These results offer new insights into the molecular mechanisms underlying synaptic plasticity and underscore the transformative potential of 3D MINFLUX imaging for studying neuronal processes in the brain.

neuroscience↗

bioRxiv2024

Vascular network-inspired diffusible scaffolds for engineering functional neural organoids

Organoids, three-dimensional in vitro organ-like tissue cultures derived from stem cells, show promising potential for developmental biology, drug discovery, and regenerative medicine. However, the function and phenotype of current organoids, especially neural organoids, are still limited by insufficient diffusion of oxygen, nutrients, metabolites, signaling molecules, and drugs. Herein, we present Vascular network-Inspired Diffusible (VID) scaffolds to fully recapture the benefits of physiological diffusion physics for generating functional organoids and phenotyping their drug response. In a proof-of-concept application, the VID scaffolds, 3D-printed meshed tubular channel networks, support the successful generation of engineered human midbrain organoids almost without necrosis and hypoxia in commonly used well-plates. Compared to conventional organoids, these engineered organoids develop with more physiologically relevant features and functions including midbrain-specific identity, oxygen metabolism, neuronal maturation, and network activity. Moreover, these engineered organoids also better recapitulate pharmacological responses, such as neural activity changes to fentanyl exposure, compared to conventional organoids with significant diffusion limits. Combining these unique scaffolds and engineered organoids may provide insights for organoid development and therapeutic innovation.

bioengineering↗

bioRxiv2024

Rab5 Overcomes CAR T Cell Dysfunction Induced by Tumor-Mediated CAR Capture

Continuous interaction between chimeric antigen receptor (CAR) T cell (CART) and tumors often result in CART dysfunction and tumor escape. We observed that tumors can take up CAR molecules, leaving CARTs without surface-expressed CARs and thus unable to kill tumors after prolonged exposure. Overexpression of Rab5 resulted in augmented clathrin-independent endocytosis, preventing loss of surface-expressed CARs, and enhanced CART activity. Interestingly, we observed membrane protrusions on the CART cell surface which disappeared after multiple tumor challenges. Rab5 maintained these protrusions after repeated tumor engagements and their presence correlated with effective tumor clearance, suggesting a link between endocytosis, membrane protrusions, and cytolytic activity. In vivo, Rab5-expressing CARTs demonstrated improved activity and were able to clear an otherwise refractory mesothelin-expressing solid cancer in humanized mice by maintaining CAR surface expression within the tumor. Thus, pairing Rab5 with CAR expression could improve the clinical efficacy of CART therapy. Highlights O_LI"CAR-jacking" occurs when surface CAR is internalized by target tumor cells. C_LIO_LIRab5 overexpression prevents "CAR-jacking" and enhances CART function. C_LIO_LIRab5 promotes CAR endocytic recycling and maintains membrane protrusions. C_LIO_LIRab5-expressing CARTs exhibit enhanced therapeutic efficacy against solid tumors. C_LI

immunology↗

bioRxiv2024

Tonotopy is not preserved in a descending stage of auditory cortex

Previous studies based on layer specificity suggest that ascending signals from the thalamus to the sensory neocortex preserve spatially organized information, but it remains unknown whether sensory information descending from sensory neocortex to the thalamus also maintains such spatial organization pattern. By focusing on projection specificity, we mapped the tone response properties of two groups of cortical neurons in the primary auditory cortex (A1), based on the relationship between their specific connections to other regions and their function in ascending (thalamocortical recipient, TR neurons) or descending (corticothalamic, CT neurons) auditory information. A clear tonotopic gradient was observed among TR neurons, but not CT neurons. Additionally, CT neurons exhibited markedly higher heterogeneity in their frequency tuning and had broader bandwidth than TR neurons. These results reveal that the information flow descending from A1 to the thalamus via CT neurons is not arrange tonotopically, suggesting that the descending information flow possibly contributes to higher-order feedback processing of diverse auditory inputs.

neuroscience↗