Biology subjects

Kim, G.

Publications and source records attributed to Kim, G..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Chronic HDACi emends microglial differentiation and neurological disease in a mouse model of intellectual disability

Defect in lysine-specific methyltransferase 2D (KMT2D) underlies a rare intellectual disability disorder, Kabuki Syndrome (KS). We show that in addition to reduction in post-natal neurogenesis, KS mice present small, arrested hippocampal microglia which single cell RNA sequence analyses revealed as globally downregulated, occupying neither activated nor surveillance states. Weekly administration of a triple combination formulation (TCF) containing the FDA-approved HDAC inhibitor Vorinostat, HPBCD and PEG-400 for three months, boosted microglia. Transcriptomic, pseudotime cell trajectory analyses, imaging and behavioral data suggest that TCF differentiated KS microglia from arrested to non-diseased and non-activated surveillance forms by selective acetylation of histone H3, that restored the open chromatin mark H3K4me3 independent of Kmt2D, to improve cognitive and nociceptive responses. Wild-type microglia showed no substantial transcriptional or histone changes, intimating normal heterochromatin resisted TCF. These findings reveal a novel chromatin-mediated mechanism of microglial differentiation uncoupled from activation, with therapeutic potential for KS and related disorders.

neuroscience↗

bioRxiv2025

Autogenic spinal excitatory circuit ensures skilled hand movements in primates

Skillful hand movements are a hallmark of primates, including humans, requiring sophisticated motor planning and execution. Challenging the longstanding paradigm that emphasizes the dominance of the cerebral cortex1, our study highlights a pivotal role for spinal excitatory reflex circuits in both planning and executing skillful hand movements. Using a combination of experimental approaches with behaving non-human primates and predictive simulation, we identified a group of excitatory spinal interneurons that orchestrate a closed-loop, positive feedback mechanism during voluntary wrist movements. This mechanism is characterized by a bidirectional interaction between inter-neuronal spiking and muscle activity, mediated by motoneuronal efferent signals and proprioceptive afferent signals from the same agonistic muscles. Furthermore, we demonstrate that the temporal profile of muscle activity during movement execution, including amplitude and duration, is pre-determined during motor planning at the spinal interneurons, functioning as a force-feedback gain within the excitatory circuit. These findings suggest that autogenic spinal excitatory circuits play a predominant role in shaping overall muscle activation during motor execution, provided the proper reflex gain is pre-set by higher neural systems during motor planning. Together, our findings provide a cellular-level perspective on how spinal reflex loops contribute to skilled voluntary movements in primates, extending over a century of spinal reflex research2.

neuroscience↗

bioRxiv2024

N-Amino Peptide-Graphene Quantum Dot Loaded Small Extracellular Vesicles for Targeted Therapy of Tauopathies

Tauopathies, a group of neurodegenerative disorders, are characterized by the abnormal aggregation of tau proteins into neurofibrillary tangles (NFTs), driving synaptic dysfunction, neuronal loss, and disease progression through tau aggregate propagation. Graphene quantum dots (GQDs) functionalized with D- cysteine (D-GQDs) have shown promise in inhibiting tau aggregation and transmission via {pi}-{pi} stacking and electrostatic interactions with tau proteins. However, the non-specific binding of GQDs to various proteins in the physiological environment, such as serum albumin, limits their clinical translation. In this study, we aim to enhance the specificity of D-GQDs toward tau protein by incorporating a tau-targeting N- amino peptide, mxyl-NAP2. The mxyl-NAP2/D-GQD complex demonstrated improved selectivity for tau protein over serum albumin, effectively enhancing the inhibition of tau aggregation. To further minimize off-target effects and optimize therapeutic delivery, we loaded the mxyl-NAP2/D-GQD complex into small extracellular vesicles (sEVs), followed by functionalization of sEVs with neuron targeting ligand, rabies viral glycoprotein peptides. This strategy not only reduced off-target effects, but also enhanced uptake by neuron cells, which further improved inhibition of tau transmission between neurons. Our results indicated that mxyl-NAP2/D-GQD-loaded sEVs hold great promise for overcoming the off-target limitations of D- GQDs and advancing the development of precision therapeutics for neurodegenerative diseases.

bioengineering↗

bioRxiv2024

The role of REM sleep in neural differentiation of memories in the hippocampus

When faced with a familiar situation, we can use memory to make predictions about what will happen next. If such predictions turn out to be erroneous, the brain can adapt by differentiating the representations of the cues that generated the prediction from the mispredicted item itself, reducing the likelihood of future prediction errors. Prior work by Kim et al. (2017) found that violating a sequential association in a statistical learning paradigm triggered differentiation of the neural representations of the associated items in the hippocampus. Here, we used fMRI to test the preregistered hypothesis that this hippocampal differentiation occurs only when violations are followed by rapid eye movement (REM) sleep. In the morning, participants first learned that some items predict others (e.g., A predicts B) then encountered a violation in which a predicted item (B) failed to appear when expected after its associated item (A); the predicted item later appeared on its own after an unrelated item. Participants were then randomly assigned to one of three conditions: remain awake, take a nap containing non-REM sleep only, or take a nap with both non-REM and REM sleep. While the predicted results were not observed in the preregistered left CA2/3/DG ROI, we did observe evidence for our hypothesis in closely related hippocampal ROIs, uncorrected for multiple comparisons: In right CA2/3/DG, differentiation in the group with REM sleep was greater than in the groups without REM sleep (wake and non-REM nap); this differentiation was item-specific and concentrated in right DG. Differentiation effects were also greater in bilateral DG when the predicted item was more strongly reactivated during the violation. Overall, the results presented here provide initial evidence linking REM sleep to changes in the hippocampal representations of memories in humans.

neuroscience↗

bioRxiv2024

Structure determinants of DANGEROUS MIX 3, an alpha/beta hydrolase, for triggering NLR-mediated genetic incompatibility in plants

Genetic incompatibility occurs when a mismatched pair of plant immune components mounts autoimmune responses in hybrids. Highly diversified NLR receptors are main culprits of the genetic conflict, recognizing host proteins from different origin as immune trigger. Here, we report the molecular mechanism underlying a DANGEROUS MIX (DM) autoimmunity, comprising DM2h/RPP1 NLR and its incompatible partner DM3, an alpha/beta hydrolase. Cryo-electron microscopy reveals the oligomeric nature of two natural DM3 variants in a trimer of dimer configuration. The polymorphism triggering autoimmunity is located at the dimer interface, resulting in drastic structural differences such that dimerizing helix and loop reinforcing the interface is lost and disordered. Structure-function analysis shows that integrity of the dimer interface, but neither maintenance of hexamer nor its enzymatic activity, is the key factor contributing to autoimmunity. Our finding pinpoints checkpoints embedded in the oligomeric configuration of a host enzyme that controls the switching mechanism of NLR activity.

plant biology↗

bioRxiv2024

DJ-1 deficiency in SH-SY5Y cells reveals dysregulated networks of genes and pathways involved in neuronal function and disease

Parkinsons disease (PD) is the second most common neurodegenerative disease, affecting between 2 - 3% of the population aged 65 and older. Although the etiology of idiopathic PD is still to be elucidated, the study of heritable forms of the disease can provide new understanding into disease mechanisms. Recessively inherited loss of function mutations in the PARK7/DJ-1 gene has been found to be causative for familial, early-onset PD. Importantly, PARK7/DJ-1 related forms of familial PD replicate common disease phenotypes seen in idiopathic PD, including degeneration of substantia nigra dopaminergic neurons, and Parkinsonism. In this study, we evaluate the loss of function of PARK7/DJ-1 on a human neuronal cell line, SH-SY5Y. Following ablation of the PARK7/DJ-1 gene via CRISPR-Cas9, RNA sequencing and the DESEQ2 tool kit were utilized to filter differentially expressed genes between PARK7/DJ-1 knockouts and control SH- SY5Y cells. 5684 genes were identified to be significantly differentially expressed. 3 genes from each of the top 10 upregulated (ATOH8, LAYN, TLX2) and downregulated (CACNA1B, CPLX2, SV2C) gene lists were selected and confirmed via RT-PCR. Differentially expressed gene lists were run through the WebGestalt functional enrichment analysis toolkit to identify enriched gene ontology (GO) terms for biological processes, cellular components, molecular function, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways respectively. Among the top 10 significantly enriched KEGG pathways for upregulated genes were those related to neurodegenerative diseases such as Parkinsons disease, Alzheimers disease, and Huntingtons disease (p-adj [≤] 0.05). Differentially expressed genes were run through the STRING database to predict protein-protein interactions (PPI). A highly significant PPI enrichment was observed (p < 1.0e-16). Our results indicate that loss of DJ-1 function in human neuronal cells leads to dysregulation of networks of connected genes and pathways that are implicated in neurodegenerative disease as well as neuronal function.

neuroscience↗

bioRxiv2024

In vivo CRISPRi screen identified lncRNA portfolio crucial for cutaneous squamous cell carcinoma tumor growth

Cutaneous squamous cell carcinoma (cSCC) accounts for 20% of all skin cancer deaths globally, making it the second-highest subtype of skin cancer. The prevalence of cSCC in humans, as well as the poor capacity for an efficient prognosis, highlights the need to uncover alternative actors and mechanisms at the foundation of skin cancer development. Significant advances have been made to better understand some key factors in cSCC progression. However, little is known about the role of noncoding RNAs, particularly of a specific category called long noncoding RNA (lncRNA). By performing pseudobulk analysis of single-cell sequencing data from normal and cSCC human skin tissues, we determined a global portfolio of lncRNAs specifically expressed in keratinocyte subpopulations. Integration of CRISPR interference screens in vitro and the xenograft model identified several lncRNAs impacting the growth of cSCC cancer lines both in vitro and in vivo. Among these, we further validated LINC00704 and LINC01116 as proliferation-regulating lncRNAs in cSCC lines and potential biomarkers of cSCC progression. Taken together, our study provides a comprehensive signature of lncRNAs with roles in regulating cSCC progression.

cancer biology↗

bioRxiv2024

Tumor microenvironment distinctions between esophageal cancer subtypes explain varied immunotherapy responses

Esophageal cancer comprises two main subtypes: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Previous studies have revealed distinct genomic characteristics between these subtypes, with ESCC sharing similarities with head and neck squamous cell carcinoma (HNSCC), and EAC aligning with gastric adenocarcinoma (GAC). Additionally, recent immunotherapy clinical trials have shown a higher response rate in ESCC and HNSCC compared to EAC and GAC. However, how the tumor microenvironment contributes to these varied immunotherapy responses remains unclear. In this study, through a comparative analysis of single-cell tumor transcriptomes from 35 patients with ESCC, EAC, HNSCC, or GAC, we identified two groups with tumor microenvironment distinctions: ESCC and HNSCC versus EAC and GAC, consistent with their genomic classifications. Malignant epithelial cells displayed distinct separations based on histological origin. In the tumor immune microenvironment, we observed an enrichment of CXCL13+CD8+ T cells and CXCL9+CXCL10+ tumor-associated macrophages (TAMs) in ESCC and HNSCC, which activate cellular immunity through interferon-{gamma}. In contrast, EAC and GAC exhibited a high presence of heat-shock protein-expressing CD8+ T cells and MARCO+ TAMs. These immune signatures help explain the varied immunotherapy responses among these cancer subtypes and successfully predict immunotherapy outcomes across diverse cancer types, underscoring their clinical significance.

cancer biology↗

bioRxiv2024

Single-cell multi-omics reveals tumor microenvironment factors underlying poor immunotherapy responses in ALK-positive lung cancer

Anaplastic lymphoma kinase (ALK) rearrangement is a major oncogenic driver in non-small cell lung cancer (NSCLC). While ALK tyrosine kinase inhibitors have shown promising therapeutic effects, overcoming resistance with immunotherapy becomes necessary when resistance develops. However, various clinical trials have revealed that their efficacies remain limited. To investigate the tumor microenvironment (TME) factors contributing to poor immune checkpoint blockade responses in ALK-positive patients, we performed single-cell RNA and ATAC sequencing on lung adenocarcinoma (LUAD) tumors with and without ALK rearrangements. Integrative analysis with additional public LUAD cohorts revealed distinct immune landscapes in ALK-positive tumors, marked by enriched innate immunity and depleted adaptive immunity. ALK-positive malignant cells exhibit higher stemness and aggressive phenotype. Tumor-associated macrophages (TAMs) in these tumors predominantly maintain pro-tumoral M2-like states, reinforcing immune suppression. B cells show reduced immune reactivity and impaired tertiary lymphoid structure formation, while CD8+ T cells display bystander-like signatures and reduced tumor reactivity. Single-cell chromatin accessibility profiles combined with regulatory network analysis suggest that differences in transcription factor activities, rather than chromatin accessibility, may underlie T cell dysfunction. These findings provide insights into the immunosuppressive TME of ALK-positive LUAD, potentially explaining the failure of recent immunotherapy trials and highlighting targets for improving efficacy.

cancer biology↗

bioRxiv2024

Towards large-scale museomics projects: a cost-effective and high-throughput extraction method for obtaining historical DNA from museum insect specimens

Natural history collections serve as invaluable repositories of biodiversity data. Large-scale genomic analysis would greatly expand the utility and accessibility of museum collections but the high cost and time-intensive nature of genomic methods limit such projects, particularly for invertebrate specimens. This paper presents an innovative, cost-effective and high-throughput approach to extracting genomic DNA from diverse insect specimens using single-phase reverse immobilization (SPRI) beads. We optimized PEG-8000 and NaCl concentrations to balance DNA yield and purity, reducing reagent cost to 6-11 cents per sample. Our method was validated against three widely used extraction protocols, and showed comparable DNA yield and amplification success to the widely used Qiagen DNeasy kit. We successfully applied the protocol in a high-throughput manner, extracting DNA from 3,786 insect specimens across a broad range of ages, taxonomies, and tissue types. A detailed protocol is provided to facilitate the adoption of the method by other researchers. By improving one of the most crucial steps in any molecular project, this SPRI bead-based DNA extraction approach has significant potential for enabling large-scale museomics projects, thereby increasing the utility of historical collections for biodiversity research and conservation efforts.

genomics↗

bioRxiv2024

Metapipeline-DNA: A Comprehensive Germline & Somatic Genomics Nextflow Pipeline

SummaryThe price, quality and throughout of DNA sequencing continue to improve. Algorithmic innovations have allowed inference of a growing range of features from DNA sequencing data, quantifying nuclear, mitochondrial and evolutionary aspects of both germline and somatic genomes. To automate analyses of the full range of genomic characteristics, we created an extensible Nextflow meta-pipeline called metapipeline-DNA. Metapipeline-DNA analyzes targeted and whole-genome sequencing data from raw reads through pre-processing, feature detection by multiple algorithms, quality-control and data- visualization. Each step can be run independently and is supported robust software engineering including automated failure-recovery, robust testing and consistent verifications of inputs, outputs and parameters. Metapipeline-DNA is cloud-compatible and highly configurable, with options to subset and optimize each analysis. Metapipeline-DNA facilitates high-scale, comprehensive analysis of DNA sequencing data. AvailabilityMetapipeline-DNA is an open-source Nextflow pipeline under the GPLv2 license and is available at https://github.com/uclahs-cds/metapipeline-DNA.

bioinformatics↗

bioRxiv2024

Selective inhibition in CA3: A mechanism for stable pattern completion through heterosynaptic plasticity

Neural assemblies representing different engrams compete for successful retrieval in the CA3 region of the hippocampus, yet the detailed mechanisms underlying their formation remain elusive. Recent research indicates that hippocampal inhibitory neurons respond selectively to stimuli and exhibit diverse plasticity, suggesting their significant role in engram formation. Conventional attractor network models for CA3 typically employ global inhibition, where inhibitory neurons uniformly suppress the activity of excitatory neurons. However, these models may not fully capture the complex dynamics of competition arising from sparse distributed coding and may not accurately reflect the specific roles of inhibitory neurons in the competition between neural assemblies during memory retrieval. We propose a mechanism for engram formation in CA3 using a spiking neural network model, emphasizing the critical role of the association between excitatory and inhibitory neurons through heterosynaptic plasticity. In our model, inhibitory neurons are associated with specific neural assemblies during encoding and selectively inhibit excitatory neurons involved in competing assemblies during retrieval. With a simplified dentate gyrus (DG) in a feed-forward structure, this proposed mechanism results in sparsely distributed engrams in CA3. The sparse distributed coding in the model allows us to investigate the effects of selective inhibition on pattern completion under various configurations, such as partially overlapping competing engrams. Our results demonstrate that selective inhibition provides more stable pattern completion and enhances retrieval performance compared to global inhibition alone. Furthermore, the observed neural activity in the hippocampal subregions of the model aligns with experimental findings on these regions roles in pattern separation and pattern completion. 1 Author SummaryWe explored how memories are stored and retrieved in the hippocampus by focusing on the CA3 region, a critical component of memory processes. Using a spiking neural network model, we propose a new mechanism in which specific inhibitory neurons selectively control the activity of other neurons during memory retrieval. We found that this selective inhibition can be naturally induced during memory encoding. This selective inhibition offers an alternative to traditional models that assume global suppression and provides a more nuanced understanding of how memories compete for successful retrieval. Our findings suggest that this selective inhibition improves the stability and accuracy of memory recall. The model also aligns with known biological functions of the hippocampus, shedding light on how complex memory processes, such as distinguishing between similar memories and accurately reconstructing past experiences, might be managed in the brain. This research offers new insights into the dynamic roles of inhibitory neurons in balancing memory encoding and retrieval, enhancing our understanding of memory function.

neuroscience↗

bioRxiv2024

Characterization and analysis of neuronal signaling using microelectrode array combined with rapid and localized cooling device for cryo-neuromodulation

Cryoanesthesia--a purely physical anesthesia treatment that freezes tissue and attenuate nerve activity--can provide fast treatment through freezing and thawing of cryo-machine and is inexpensive compared to other anesthetics. However, cryoanesthesia has not been widely adopted because securing safe and effective conditions requires quantitative measurement and analysis of neuronal signaling during freezing and recovery, for which research tools are limited. A lack of rapid and localized cooling technologies for quantitative cellular level analysis, in particular, hinders research on not only the optimal cryo-modulation of neuronal activities but also its influence to neighboring cells via cellular networks. Here, we introduce a novel cryo-neuromodulation platform, a high-speed precision probe-type cooling device ([~]20{degrees}C/s at cooling) that provides localized cooling combined with a microelectrode array (MEA) system. We explored the temperature conditions for efficient silencing and recovery of neuronal activities without cell damage. We found that electrical activities of neurons were fully recovered within 1 minute of cooling duration with the maximum cooling speed, which was also confirmed with calcium imaging. The impact of silenced neurons on the neighboring neural network was explored using the localized cooling and we perceived that its influence can be transmitted if the neuronal network is well organized. Our new cryo-device provides rapid and reversible control of neural activities, which allows not just quantitative analysis of the network dynamics, but also new applications in clinical settings.

neuroscience↗

bioRxiv2024

Using Dynamic Bayesian Optimization to Induce Desired Effects in the Presence of Motor Learning: a Simulation Study

Human-in-the-loop (HIL) optimization is a control paradigm used for tuning the control parameters of human-interacting devices while accounting for variability among individuals. A limitation of state-of-the-art HIL optimization algorithms such as Bayesian Optimization (BO) is that they assume that the relationship between control parameters and user response does not change over time. BO can be modified to account for the dynamics of the user response by implementing time into the kernel function, a method known as Dynamic Bayesian Optimization (DBO). However, it is unknown if DBO outperforms BO when the human response is characterized by models of human motor learning. In this work, we simulated runs of HIL optimization using BO and DBO towards establishing if DBO is a suitable paradigm for HIL optimization in the presence of motor learning. Simulations were conducted assuming either purely time-dependent participant responses, or assuming that responses would arise from state-space models of motor learning capable of describing both adaptation and use-dependent learning behavior. Statistical comparisons indicated that DBO was never inferior to BO, and, after a certain number of iterations, generally outperformed BO in convergence to optimal inputs and outputs. The number of iterations beyond which DBO was superior to BO occurred earlier when the input-output relationship of the simulated responses was more dynamic. Our results suggest that DBO may improve the performance of HIL optimization over BO when a sufficient number of iterations can be evaluated to accurately distinguish between unstructured variability (noise) and learning.

bioengineering↗

bioRxiv2024

Co-activation of selective nicotinic acetylcholine receptor subtypes is required to reverse hippocampal network dysfunction and prevent fear memory loss in Alzheimer's disease

Beta-amyloid peptide (A{beta})-induced suppression of hippocampal GABAergic interneuron activity drives hyperexcitability, amyloid pathology, and cognitive impairment in Alzheimers disease (AD), suggesting that enhancing hippocampal inhibition may be protective. However, hippocampal interneurons are highly diverse and differentially regulate inhibition and cognition, making it challenging to identify the affected subtypes and optimally restore hippocampal inhibition in AD. We have previously found that A{beta} selectively binds to two of the three major hippocampal nicotinic acetylcholine receptor (nAChR) subtypes, 7- and 4{beta}2-nAChRs, but not 3{beta}4-nAChRs, and inhibits these two receptors in hippocampal inhibitory interneurons to decrease their activity, leading to hyperexcitation in excitatory neurons. Here, we further reveal that 7- and 4{beta}2-nAChRs predominantly control the nicotinic cholinergic signaling and neuronal activity in hippocampal parvalbumin-positive (PV+) and somatostatin-positive (SST+) inhibitory interneurons, respectively. We also find that systemic co-stimulation of 7- and 4{beta}2-nAChRs is required to reverse hippocampal hyperexcitability, dysfunction of fear learning-associated hippocampal oscillatory activity, and hippocampus-dependent memory loss and reduce A{beta} pathology in AD model mice. This suggests that co-stimulation of PV+ and SST+ cells via activation of 7- and 4{beta}2-nAChRs together is required to enhance hippocampal inhibition optimally, which reverses hippocampal dysfunction, reduces amyloid pathology, and ultimately prevents memory loss in AD. Significance StatementDevelopment of a safe and effective unified strategy to counteract hyperexcitability holds a significant promise in advancing Alzheimers disease (AD) treatment. Studies suggest that beta-amyloid peptide (A{beta}) is a major trigger of hyperexcitability. However, the mechanisms of how A{beta} could participate in hyperexcitability are still not fully understood. Here, we reveal that A{beta} inhibits distinct hippocampal nicotinic acetylcholine receptor (nAChR) subtypes in different hippocampal inhibitory interneuron subtypes, which leads to hyperexcitability, resulting in amyloid pathology and memory loss in AD. We further discover that systemic co-stimulation of these nAChRs is required for neuroprotection against AD at the cellular, circuit, network, and behavioral levels. Identifying this mechanism can thus provide a new therapeutic strategy in AD.

neuroscience↗

bioRxiv2024

Polygenic architecture of brain structure and function, behaviors, and psychopathologies in children

The human brain undergoes structural and functional changes during childhood, a critical period in cognitive and behavioral development. Understanding the genetic architecture of the brain development in children can offer valuable insights into the development of the brain, cognition, and behaviors. Here, we integrated brain imaging-genetic-phenotype data from over 8,600 preadolescent children of diverse ethnic backgrounds using multivariate statistical techniques. We found a low-to-moderate level of SNP-based heritability in most IDPs, which is lower compared to the adult brain. Using sparse generalized canonical correlation analysis (SGCCA), we identified several covariation patterns among genome-wide polygenic scores (GPSs) of 29 traits, 7 different modalities of brain imaging-derived phenotypes (IDPs), and 266 cognitive and psychological phenotype data. In structural MRI, significant positive associations were observed between total grey matter volume, left ventral diencephalon volume, surface area of right accumbens and the GPSs of cognition-related traits. Conversely, negative associations were found with the GPSs of ADHD, depression and neuroticism. Additionally, we identified a significant positive association between educational attainment GPS and regional brain activation during the N-back task. The BMI GPS showed a positive association with fractional anisotropy (FA) of connectivity between the cerebellum cortex and amygdala in diffusion MRI, while the GPSs for educational attainment and cannabis use were negatively associated with the same IDPs. Our GPS-based prediction models revealed substantial genetic contributions to cognitive variability, while the genetic basis for many mental and behavioral phenotypes remained elusive. This study delivers a comprehensive map of the relationships between genetic profiles, neuroanatomical diversity, and the spectrum of cognitive and behavioral traits in preadolescence.

genetics↗

bioRxiv2024

Pools of independently cycling inositol phosphates revealed by pulse labeling with 18O-water

Inositol phosphates control many central processes in eukaryotic cells, including nutrient availability, growth, and motility. Kinetic resolution of a key modulator of their signaling functions, the turnover of the phosphate groups on the inositol ring, has been hampered by slow uptake, high dilution, and constraining growth conditions in radioactive pulse-labeling approaches. Here, we demonstrate rapid (seconds to minutes), non-radioactive labeling of inositol polyphosphates through 18O-water in yeast, amoeba and human cells, which can be applied in any media. In combination with capillary electrophoresis and mass spectrometry, 18O-water labeling simultaneously dissects the in vivo phosphate group dynamics of a broad spectrum of even rare inositol phosphates. The improved temporal resolution allowed us to discover vigorous phosphate group exchanges in some inositol poly- and pyrophosphates, whereas others remain remarkably inert. Our observations support a model in which the biosynthetic pathway of inositol poly- and pyrophosphates is organized in distinct, kinetically separated pools. While transfer of compounds between those pools is slow, each pool undergoes rapid internal phosphate cycling. This might enable the pools to perform distinct signaling functions while being metabolically connected.

biochemistry↗

bioRxiv2024

Stimulative piezoelectric nanofibrous scaffolds for enhanced small extracellular vesicle production in 3D cultures

Small extracellular vesicles (sEVs) have great promise as effective carriers for drug delivery. However, the challenges associated with the efficient production of sEVs hinder their clinical applications. Herein, we report a stimulative 3D culture platform for enhanced sEV production. The proposed platform consists of a piezoelectric nanofibrous scaffold (PES) coupled with acoustic stimulation to enhance sEV production of cells in a 3D biomimetic microenvironment. Combining cell stimulation with a 3D culture platform in this stimulative PES enables a 15.7-fold increase in the production rate per cell with minimal deviations in particle size and protein composition compared with standard 2D cultures. We find that the enhanced sEV production is attributable to the activation and upregulation of crucial sEV production steps through the synergistic effect of stimulation and the 3D microenvironment. Moreover, changes in cell morphology lead to cytoskeleton redistribution through cell-matrix interactions in the 3D cultures. This in turn facilitates intracellular EV trafficking, which impacts the production rate. Overall, our work provides a promising 3D cell culture platform based on piezoelectric biomaterials for enhanced sEV production. This platform is expected to accelerate the potential use of sEVs for drug delivery and broad biomedical applications.

bioengineering↗