Biology subjects

Xu, K.

Publications and source records attributed to Xu, K..

At least 91 records · Page 5Linked to original sources

bioRxiv2024

Development of an ELISA assay for EV quantification to assist the large-scale EV manufacture

Accurate EV quantitation is essential for ensuring quality, consistency, and safety during large-scale extracellular vesicle (EV) manufacturing. In the upstream phase, EV quantitation allows for the monitoring of cell culture conditions that impact EV yield and quality, while in the downstream phase, it helps to control the efficiency and purity of EV purification. Nanoparticle Tracking Analysis is the most commonly used method for EV quantification, while it faces significant limitations due to interference from nanosize contaminants like protein aggregates, especially in crude samples (e.g. cell culture media). To address this issue, we developed a highly specific and accurate ELISA assay that quantifies EVs even in crude samples. With ultra-pure EV standard samples, this assay showed reliable quantitative result of EV detection to support method development as well as in-process control of large-scale EV manufacture. The detection range of this assay is from 4.1E7 to 3E10 EVs/mL, with an LOD of 1.04E7 EVs/mL and an LOQ of 3.21E7 EVs/mL. We therefore developed this assay into a testing kit and demonstrated that this EV quantification ELISA kit is capable of ensuring minimal interference from impurities and supporting the process development and in-process control in EV production.

biochemistry↗

bioRxiv2024

LEGEND: Identifying Co-expressed Genes in Multimodal Transcriptomic Sequencing Data

Identifying co-expressed genes across tissue domains and cell types is essential for revealing co-functional genes involved in biological or pathological processes. While both single-cell RNA-sequencing (scRNA-seq) and spatially-resolved transcriptomic (SRT) data offer insights into gene co-expression patterns, current methods typically utilize either data type alone, potentially diluting the co-functionality signals within co-expressed gene groups. To bridge this gap, we introduce LEGEND, a novel computational method that integrates scRNA-seq and SRT data for identifying groups of co-expressed genes at both cell type and tissue domain levels. LEGEND employs an innovative hierarchical clustering algorithm designed to maximize intra-cluster redundancy and inter-cluster complementarity, effectively capturing more nuanced patterns of gene co-expression and spatial coherence. Enrichment and cofunction analyses further showcase the biological relevance of these gene clusters, and their utilities in exploring context-specific novel gene functions. Notably, LEGEND can reveal shifts in gene-gene interactions under different conditions, furnishing insights for disease-associated gene crosstalk. Moreover, LEGEND can be utilized to enhance the annotation accuracy of both spatial spots in SRT and single-cells in scRNA-seq, and pioneers in identifying genes with designated spatial expression patterns. LEGEND is available at https://github.com/ToryDeng/LEGEND.

genomics↗

bioRxiv2024

Functional interaction between transcription factor Sfp1 and the NuA4 complex in response to nutrient availability

Ribosome biogenesis is a crucial process requiring enormous transcriptional output. In budding yeast, the expression of 138 ribosomal protein (RP) genes and over 200 ribosome biogenesis (RiBi) genes is regulated by an intricate network of factors, including the nutrient-sensitive transcription activator Sfp1 and the NuA4 coactivator/acetyltransferase complex. Nutrient starvation or inhibition of TORC1 by rapamycin leads to repression of RP and RiBi genes, in part through blocking Sfp1 nuclear localization and NuA4-dependent chromatin acetylation. Here, we demonstrate that Sfp1 physically interacts with NuA4 in a TORC1-dependent manner. Our results indicate that Sfp1, along with NuA4, regulate the transcription of RiBi and RP genes via distinct mechanisms depending on promoter architectures. Sfp1 promotes histone acetylation at the promoters without affecting NuA4 recruitment. In contrast, NuA4 does impact Sfp1 binding but specifically at two classes of RP genes. Importantly, NuA4 acetylates Sfp1 at lysines 655 and 657, regulating its function. Cells expressing Sfp1 with acetyl-mimicking mutations exhibit increased expression of RiBi genes while RP genes remain stable. However, the same mutants lead to the loss of Sfp1 binding/activity at RiBi genes when cells are under non-optimal growth conditions. Mimicking constitutive acetylation of Sfp1 also limits the transcriptional burst of RP genes upon addition of glucose. Altogether, these results draw an intricate functional relationship between Sfp1 and NuA4 to control ribosome biogenesis, fine-tuning transcription output in different growth conditions.

molecular biology↗

bioRxiv2024

Human TRPV4 engineering yields an ultrasound-sensitive actuator for sonogenetics

AbstractSonogenetics offers non-invasive and cell-type specific modulation of cells genetically engineered to express ultrasound-sensitive actuators. Finding an ion channel to serve as sonogenetic actuator it critical for advancing this promising technique. Here, we show that ultrasound can activate human TRP channel hTRPV4. By screening different hTRPV4 variants, we identify a mutation F617L that increases mechano-sensitivity of this channel to ultrasound, while reduces its sensitivity to hypo-osmolarity, elevated temperature, and agonist. This altered sensitivity profile correlates with structural differences in hTRPV4-F617L compared to wild-type channels revealed by our cryo-electron microscopy analysis. We also show that hTRPV4-F617L can serve as a sonogenetic actuator for neuromodulation in freely moving mice. Our findings demonstrate the use of structure-guided mutagenesis to engineer ion channels with tailored properties of ideal sonogenetic actuators.

biophysics↗

bioRxiv2024

Mitigation of multi-scale biases in cell-type deconvolution for spatially resolved transcriptomics using HarmoDecon

The advent of spatially resolved transcriptomics (SRT) has revolutionized our understanding of tissue molecular microenvironments by enabling the study of gene expression in its spatial context. However, many SRT platforms lack single-cell resolution, necessitating cell-type deconvolution methods to estimate cell-type proportions in SRT spots. Despite advancements in existing tools, these methods have not addressed biases occurring at three scales: individual spots, entire tissue samples, and discrepancies between SRT and reference scRNA-seq datasets. These biases result in overbalanced cell-type proportions for each spot, mismatched cell-type fractions at the sample level, and data distribution shifts across platforms. To mitigate these biases, we introduce HarmoDecon, a novel semi-supervised deep learning model for spatial cell-type deconvolution. HarmoDecon leverages pseudo-spots derived from scRNA-seq data and employs Gaussian Mixture Graph Convolutional Networks to address the aforementioned issues. Through extensive simulations on multi-cell spots from STARmap and osmFISH, HarmoDecon outperformed 11 state-of-the-art methods. Additionally, when applied to legacy SRT platforms and 10x Visium datasets, HarmoDecon achieved the highest accuracy in spatial domain clustering and maintained strong correlations between cancer marker genes and cancer cells in human breast cancer samples. These results highlight the utility of HarmoDecon in advancing spatial transcriptomics analysis.

bioinformatics↗

bioRxiv2024

A Wearable Mindfulness Brain-Computer Interface for Alleviating Car Sickness

Car sickness, an enormous vehicular travel challenge, affects a significant proportion of the population. Pharmacological interventions are limited by adverse side effects, and effective nonpharmacological alternatives remain to be identified. Here, we introduce a novel attention shifting method based on a closed-loop, artificial intelligence (AI)-driven, wearable mindfulness brain-computer interface (BCI) to alleviate car sickness. As the user performs an attentional task, i.e., focusing on breathing as in mindfulness, with a wearable headband, the BCI collects and analyses electroencephalography (EEG) data via a convolutional neural network to assess the users mindfulness state and provide real-time audiovisual feedback. This approach might sustainedly shift the users attention from physiological discomfort towards the BCI-based mindfulness practices, thereby mitigating car sickness symptoms. The efficacy of the proposed method was rigorously evaluated in two real-world experiments, namely, short and long car rides, with a large cohort of more than 100 participants susceptible to car sickness. Remarkably, over 83% of the participants rated the BCI-based attention shifting as effective, with significant reductions in car sickness severity, particularly in individuals with severe symptoms. Furthermore, EEG data analysis revealed a neurobiological signature of car sickness, which provided mechanistic insights into the efficacy of the BCI-based attention shifting for alleviating car sickness. This study proposed the first large-scale validated, nonpharmacological and wearable intervention method and system for car sickness, with the potential to transform the travel experiences of hundreds of millions of people suffering from car sickness, which also represents a new application of BCI technology.

bioengineering↗

bioRxiv2024

Novel and optimized mouse behavior enabled by fully autonomous HABITS: Home-cage Assisted Behavioral Innovation and Testing System

Mice are among the most prevalent animal models used in neuroscience, benefiting from the extensive physiological, imaging and genetic tools available to study their brain. However, the development of novel and optimized behavioral paradigms for mice has been laborious and inconsistent, impeding the investigation of complex cognitions. Here, we present a home-cage assisted mouse behavioral innovation and testing system (HABITS), enabling free-moving mice to learn challenging cognitive behaviors in their home-cage without any human involvement. Supported by the general programming framework, we have not only replicated established paradigms in current neuroscience research but also developed novel paradigms previously unexplored in mice, resulting in more than 300 mice demonstrated in various cognition functions. Most significantly, HABITS incorporates a machine-teaching algorithm, which comprehensively optimized the presentation of stimuli and modalities for trials, leading to more efficient training and higher-quality behavioral outcomes. To our knowledge, this is the first instance where mouse behavior has been systematically optimized by an algorithmic approach. Altogether, our results open a new avenue for mouse behavioral innovation and optimization, which directly facilitates investigation of neural circuits for novel cognitions with mice.

animal behavior and cognition↗

bioRxiv2024

Change in brain molecular landscapes following electrical stimulation of the nucleus accumbens

Deep brain stimulation (DBS) targeting the nucleus accumbens (NAc) is a promising therapeutic intervention for treatment-resistant neuropsychiatric disorders such as depression, anxiety, and addiction. However, the molecular mechanisms underlying the clinical efficacy of NAc DBS remain largely unknown. One approach to address this question is by performing spatial gene expression analysis on cells located in different regions of the same circuit following NAc DBS. In this study, we utilized high-resolution spatial transcriptomics (Stereo-seq) to investigate gene expression changes induced by NAc DBS in the mouse brain. Mice were randomly allocated to received continuous electrical stimulation (0.1 mA, 130 Hz) or sham treatment (electrode implanted, no electrical stimulation given) for one week, and subsequent Stereo-seq analysis identified differentially expressed genes (DEGs) across various brain regions. Our findings reveal widespread alterations in synaptic function and neuronal connectivity, particularly in glutamate receptor-expressing neurons in the NAc, which play a key role in the reward circuitry. Functional enrichment analysis highlighted changes in transporter activity and glutamate receptor binding in brain regions such as the anterior cingulate cortex and lateral septal nucleus. Single-cell resolution mapping further identified key molecular players, including Nlgn1, Snca, PDE10a, and Syt1, which are critical for synaptic plasticity and neurotransmitter release, and have been implicated in various psychiatric disorders. These findings shed light on the molecular underpinnings of NAc DBS and provide insights into its therapeutic potential in modulating neural circuits associated with neuropsychiatric disorders.

molecular biology↗

bioRxiv2024

Mesenchymal stem cell-derived extracellular vesicles alleviate imiquimod-induced psoriasis symptoms in a mouse disease model

Psoriasis is an immune mediated, chronic inflammatory skin disease. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have inherent immunomodulatory potency from their parental cells, the mesenchymal stem cells (MSCs). In this study, we revealed the application potential of MSC-EVs to alleviate psoriatic symptoms in imiquimod-induced psoriasis mouse model.

physiology↗

bioRxiv2024

Neutralization and Stability of JN.1-derived LB.1, KP.2.3, KP.3 and KP.3.1.1 Subvariants

During the summer of 2024, COVID-19 cases surged globally, driven by variants derived from JN.1 subvariants of SARS-CoV-2 that feature new mutations, particularly in the N-terminal domain (NTD) of the spike protein. In this study, we report on the neutralizing antibody (nAb) escape, infectivity, fusion, and stability of these subvariants--LB.1, KP.2.3, KP.3, and KP.3.1.1. Our findings demonstrate that all of these subvariants are highly evasive of nAbs elicited by the bivalent mRNA vaccine, the XBB.1.5 monovalent mumps virus-based vaccine, or from infections during the BA.2.86/JN.1 wave. This reduction in nAb titers is primarily driven by a single serine deletion (DelS31) in the NTD of the spike, leading to a distinct antigenic profile compared to the parental JN.1 and other variants. We also found that the DelS31 mutation decreases pseudovirus infectivity in CaLu-3 cells, which correlates with impaired cell-cell fusion. Additionally, the spike protein of DelS31 variants appears more conformationally stable, as indicated by reduced S1 shedding both with and without stimulation by soluble ACE2, and increased resistance to elevated temperatures. Molecular modeling suggests that the DelS31 mutation induces a conformational change that stabilizes the NTD and strengthens the NTD-Receptor-Binding Domain (RBD) interaction, thus favoring the down conformation of RBD and reducing accessibility to both the ACE2 receptor and certain nAbs. Additionally, the DelS31 mutation introduces an N-linked glycan modification at N30, which shields the underlying NTD region from antibody recognition. Our data highlight the critical role of NTD mutations in the spike protein for nAb evasion, stability, and viral infectivity, and suggest consideration of updating COVID-19 vaccines with antigens containing DelS31.

molecular biology↗

bioRxiv2024

LAPTM4B Alleviates Pulmonary Fibrosis by Enhancing NEDD4L-Mediated TGF-beta Signaling Suppression

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease with fatal outcome and a poorly understood pathogenesis. The lysosomal protein transmembrane 4 beta (LAPTM4B), a multi-transmembrane endo-lysosomal membrane protein, has been implicated in the pathogenesis of several diseases. However, its involvement in IPF remains unexplored. This study aimed to investigate the role of LAPTM4B in lung fibrosis and elucidate its underlying mechanisms. The results showed that LAPTM4B was significantly reduced in IPF and mouse fibrotic lungs. In vivo studies showed that the deficiency of LAPTM4B exacerbated bleomycin-induced lung fibrosis, while the restoration of LAPTM4B alleviated fibrosis. Mechanistically, LAPTM4B recruits the NEDD4 like E3 ubiquitin protein ligase (NEDD4L) to endosomes, leading to increased ubiquitin-mediated proteasomal degradation of TGFRB2 and active SMAD2/3, thereby blocking the TGF-{beta}/SMAD signaling pathway. Overall, our data provided a novel insight for a deeper understanding of the pathogenesis of IPF, supporting the therapeutic potential of restoration of LAPTM4B as a promising therapeutic approach for the treatment of pulmonary fibrosis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/610429v1_ufig1.gif" ALT="Figure 1"> View larger version (98K): org.highwire.dtl.DTLVardef@1272d12org.highwire.dtl.DTLVardef@10030baorg.highwire.dtl.DTLVardef@17f1756org.highwire.dtl.DTLVardef@d54f78_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Single-molecule diffusivity quantification in Xenopus egg extracts elucidates physicochemical properties of the cytoplasm

The living cell creates a unique internal molecular environment that is challenging to characterize. By combining single-molecule displacement/diffusivity mapping (SMdM) with physiologically active extracts prepared from Xenopus laevis eggs, we sought to elucidate molecular properties of the cytoplasm. Quantification of the diffusion coefficients of 15 diverse proteins in extract showed that, compared to in water, negatively charged proteins diffused [~]50% slower, while diffusion of positively charged proteins was reduced by [~]80-90%. Adding increasing concentrations of salt progressively alleviated the suppressed diffusion observed for positively charged proteins, signifying electrostatic interactions within a predominately negatively charged macromolecular environment. To investigate the contribution of RNA, an abundant, negatively charged component of cytoplasm, extracts were treated with ribonuclease, which resulted in low diffusivity domains indicative of aggregation, likely due to the liberation of positively charged RNA-binding proteins such as ribosomal proteins, since this effect could be mimicked by adding positively charged polypeptides. Interestingly, negatively charged proteins of different sizes showed similar diffusivity suppression in extract, which are typically prepared under conditions that inhibit actin polymerization. Restoring or enhancing actin polymerization progressively suppressed the diffusion of larger proteins, recapitulating behaviors observed in cells. Together, these results indicate that molecular interactions in the crowded cell are defined by an overwhelmingly negatively charged macromolecular environment containing cytoskeletal networks. Significance StatementThe complex intracellular molecular environment is notably challenging to elucidate and recapitulate. Xenopus egg extracts provide a native yet manipulatable cytoplasm model. Through single-molecule microscopy, here we decipher the cytoplasmic environment and molecular interactions by examining the diffusion patterns of diverse proteins in Xenopus egg extracts with strategic manipulations. These experiments reveal an overwhelmingly negatively charged macromolecular environment with crosslinked meshworks, offering new insight into the inner workings of the cell.

biophysics↗

bioRxiv2024

The effects of selfing on lineage diversification across multiple genera

O_LIThe transition from outcrossing to self-fertilization is a major evolutionary trend in plants, with selfing long hypothesized an evolutionary dead end. Recent theories suggest that elevated extinction rates may be restricted to highly selfing rather than mixed-mating populations. Previous analyses of the effects of mating systems on diversification found mixed results, varying by focal clades and potentially suffering from several limitations. C_LIO_LIWe collected data on three mating-system-related characters. We also collected life form data to indirectly distinguish between mixed mating and highly selfing. We estimated speciation and extinction rates for character states, and state transition rates in 27 genera. C_LIO_LIWe find that outcrossing lineages diversify at higher rates than selfing lineages, while the impact of mating systems on speciation is not consistent across genera. Among self-compatible lineages, annuals/short-lived exhibit significantly lower diversification rates than perennials. Models incorporating hidden states often provide a better fit than those without. C_LIO_LIThe results suggest that selfing lineages overall have lower diversification rates. This may be primarily contributed by elevated extinction rates in highly selfing lineages, which may also explain the prevalence of mixed-mating systems. However, the influences of mating systems on diversification may be often driven by other factors correlated with mating system transitions. C_LI

evolutionary biology↗

bioRxiv2024

UniPTM: Multiple PTM site prediction on full-length protein sequence

AbstractPost-translational modifications (PTMs) enrich the functional diversity of proteins by attaching chemical groups to the side chains of amino acids. In recent years, a myr-iad of AI models have been proposed to predict many specific types of PTMs. However, those models typically adopt the sliding window approach to extract short and equal-length protein fragments from full-length proteins for model training. Unfortunately, such a subtle step results in the loss of long-range information from distal amino acids, which may impact the PTM formation process. In this study, we introduce UniPTM, a window-free model designed to train and test on natural and full-length protein sequences, enabling the prediction of multiple types of PTMs in a holistic manner. Moreover, we established PTMseq, the first comprehensive dataset of full-length pro-tein sequences with annotated PTMs, to train and validate our model. UniPTM has undergone extensive validations and significantly outperforms existing models, eluci-dating the influence of protein sequence completeness on PTM. Consequently, UniPTM offers interpretable and biologically meaningful predictions, enhancing our understand-ing of protein functionally and regulation. The source code and PTMseq dataset for UniPTM are available at https://www.github.com/TransPTM/UniPTM.

bioinformatics↗

bioRxiv2024

When does stronger selection increase the probability of evolutionary rescue?

Populations may be rescued from extinction via sufficiently rapid adaptive evolution. Evolution is faster with stronger selection but this may come with a demographic cost, creating opposing effects on evolutionary rescue. The outcome of this trade-off determines the optimal strategy for avoiding herbicide/drug resistance evolution. Here we examine the effect of stronger selection, and the associated demographic change, across four models of evolutionary rescue. We find that stronger selection cannot facilitate rescue in two quite different population genetic models unless it increases the absolute fitness of the rescue homozygote. Similarly, in a quantitative genetic model of rescue, where stronger selection leaves maximum fitness unchanged, stronger selection cannot facilitate rescue despite elevating the rate of evolution. We also explore a quantitative genetic model of rescue in a gradually changing environment, generalizing the finding that an intermediate selection strength maximizes survival at steady-state to a wider class of fitness functions.

evolutionary biology↗

bioRxiv2024

Still Not Sterile: Chlorhexidine gluconate treatment does not completely reduce skin microbial bioburden and promotes pathogen overabundance in patients undergoing elective surgeries.

Surgical site infections (SSI) continue to occur despite widespread adoption of surgical antiseptics. The effects of chlorhexidine gluconate (CHG)-based antiseptics on the skin microbiome also remains undefined due to confounding effects of CHG persistence on skin. Patients undergoing elective surgery were enrolled to characterize the immediate and long-term impact of pre-surgical preparation with CHG antiseptic on skin microbial communities. Due to the broad-spectrum antimicrobial activity of CHG and its propensity to bind extracellular DNA, methods to selectively identify live microorganisms are critical to this process and to fully elucidate the effectiveness of pre-surgical protocols and potential disruptions to the healthy skin microbiome. Swabs of the surgical site skin microbiome were collected at multiple timepoints before and after surgery. Microbial bioburden and community compositions were evaluated with viability qPCR and 16S ribosomal RNA gene profiling. Pre-operative CHG induced a measurable reduction in the viable microbial bioburden at the surgical site. On the day of surgery, surgical sites displayed a significant increase in the relative abundance of several SSI associated bacterial genera, including, Acinetobacter, Bacillus, Escherichia-Shigella, and Pseudomonas, compared to baseline. Bacillus species isolated from subjects at baseline showed resistance to CHG with MICs exceeding 1000 {micro}g/ml. Despite major shifts in the skin microbiome upon exposure to CHG, they were transient in the majority of individuals. Skin microbial community structure recovered by the post-surgical follow-up. In short, this study shows that pre-surgical application of CHG can significantly reduce viable skin microbial bioburden, however, complete sterility is not achieved. While CHG induces temporary shifts in the skin microbiome, including enrichment for potentially pathogenic taxa, the skin microbiome recovers back to near baseline. Collectively, these findings identify tangible avenues for improving antiseptic formulations and offer further support that the skin microbiome is viable, stable, and resilient to chemical perturbation.

microbiology↗

bioRxiv2024

Single-molecule live-cell RNA imaging with CRISPR-Csm

High-resolution, real-time imaging of RNA is essential for understanding the diverse, dynamic behaviors of individual RNA molecules in single cells. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved. Here, we present single-molecule live-cell fluorescence in situ hybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR-Csm complex with multiplexed guide RNAs for efficient, direct visualization of single RNA molecules in a range of cell types, including primary cells. Using smLiveFISH, we tracked individual endogenous NOTCH2 and MAP1B mRNA transcripts in living cells and identified two distinct localization mechanisms: co-translational translocation of NOTCH2 mRNA at the endoplasmic reticulum, and directional transport of MAP1B mRNA toward the cell periphery. This method has the potential to unlock principles governing the spatiotemporal organization of native transcripts in health and disease.

biophysics↗

bioRxiv2024

Hypersensitivity of the vimentin cytoskeleton to net-charge states and Coulomb repulsion

As with most intermediate filament systems, the hierarchical self-assembly of vimentin into nonpolar filaments requires no nucleators or energy input. Utilizing a set of live-cell, single-molecule, and super-resolution microscopy tools, here we show that in mammalian cells, the assembly and disassembly of the vimentin cytoskeleton is highly sensitive to the protein net charge state. Starting with the intriguing observation that the vimentin cytoskeleton fully disassembles under hypotonic stress yet reassembles within seconds upon osmotic pressure recovery, we pinpoint ionic strength as its underlying driving factor. Further modulating the pH and expressing differently charged constructs, we converge on a model in which the vimentin cytoskeleton is destabilized by Coulomb repulsion when its mass-accumulated negative charges (-18 per vimentin protein) along the filament are less screened or otherwise intensified, and stabilized when the charges are better screened or otherwise reduced. Generalizing this model to other intermediate filaments, we further show that whereas the negatively charged GFAP cytoskeleton is similarly subject to fast disassembly under hypotonic stress, the cytokeratin, as a copolymer of negatively and positively charged subunits, does not exhibit this behavior. Thus, in cells containing both vimentin and keratin cytoskeletons, hypotonic stress disassembles the former but not the latter. Together, our results both provide new handles for modulating cell behavior and call for new attention to the effects of net charges in intracellular protein interactions.

biophysics↗