Biology subjects

Pan, X.

Publications and source records attributed to Pan, X..

At least 109 records · Page 6Linked to original sources

bioRxiv2023

Genome-wide Perturbation Analysis Screening System for Exosomes-Related Genes Based on the CRISPR/Cas9 Platform

As reported previously, exosomes have significant impacts on the physiological and pathological state in vivo. Exosomes have been extensively studied as a drug delivery carrier and some exosomes drugs have been undergoing clinical research. The mechanisms of exosome production, transport, and secretion remain to be studied in depth. Thus, here, we developed a novel CRISPR-UMI-based gene screening system, with which we can reveal the exosomes biogenesis, transport, and uptake mechanisms on a genome-wide scale. This system consists of two parts: one is a gene knockout component; the other part is a unique molecular identifiers (UMI) labeling component that can label the exosomes produced by the knockout cells, in which each sgRNA corresponds to a specific UMI. In this way, by detecting the UMI loaded in the exosomes, we can trace the knockout gene. In this study, we first verified the function of each component using plasmids and lentiviruses respectively. Next, we simulated the infection of cells with lentiviral libraries using a single lentivirus to validate the functionality of the screening system. Finally, we constructed a CRISPR-UMI-based library targeting 15 genes (genes with clear effects on exosomes biogenesis) to further validate the reliability of the screening system. The development of this screening system is of indispensable importance for the in-depth study of the mechanisms of exosome production and secretion, as well as for the improvement of exosomes production and the advancement of exosomes industrialization.

bioengineering↗

bioRxiv2023

Actomyosin-II Proactively Shields Axons of the Central Nervous System from Mild Mechanical Stress

SummaryPan et al found that actomyosin-II-driven radial contractility underpins the resilience of central axons to mild mechanical stress by suppressing the propagation and firing of injurious Ca2+ waves. Boosting actomyosin-II activity alleviates axon degeneration in mice with traumatic brain injury. Traumatic brain injury (TBI) remains a significant and unmet health challenge. However, our understanding of how neurons, particularly their fragile axons, withstand the abrupt mechanical impacts within the central nervous system remains largely unknown. Using a microfluidic device applying discrete levels of transverse forces to axons, we identified the stress levels that most axons could resist and explored their instant responses at nanoscale resolution. Mild stress induces rapid and reversible axon beading, driven by actomyosin-II-dependent radial contraction, which restricts the spreading and bursting of stress-induced Ca2+ waves. More severe stress causes irreversible focal swelling and Ca2+ overload, ultimately leading to focal axonal swelling and degeneration. Up-regulating actomyosin-II activity prevented the progression of initial injury in vivo, protecting commissural axons from degeneration in a mice TBI model. Our study established a scalable axon injury model and uncovered the critical roles of actomyosin-II in shielding neurons against detrimental mechanical stress.

neuroscience↗

bioRxiv2023

Fungal-bacteria interactions provide shelter for bacteria in Caesarean section scar diverticulum

Caesarean section scar diverticulum (CSD) is a significant cause of infertility among women who have previously had a Caesarean section, primarily due to persistent inflammatory exudation associated with this condition. Even though abnormal bacterial composition is identified as a critical factor leading to this chronic inflammation, clinical data suggests that a long-term cure is often unattainable with antibiotic treatment alone. In our study, we employed metagenomic analysis and mass spectrometry techniques to investigate the fungal composition in CSD and its interaction with bacteria. We discovered that local fungal abnormalities in CSD can disrupt the stability of the bacterial population and the entire microbial community by altering bacterial abundance via specific metabolites. For instance, Lachnellula suecica reduces the abundance of several Lactobacillus spp., such as Lactobacillus jensenii, by diminishing the production of metabolites like Goyaglycoside A and Janthitrem E. Concurrently, Clavispora lusitaniae and Ophiocordyceps australis can synergistically impact the abundance of Lactobacillus spp. by modulating metabolite abundance. Our findings underscore that abnormal fungal composition and activity are key drivers of local bacterial dysbiosis in CSD.

microbiology↗

bioRxiv2023

TFvelo: gene regulation inspired RNA velocity estimation

RNA velocity is closely related with cell fate and is an important indicator for the prediction of cell states with elegant physical explanation derived from single-cell RNA-seq data. Most existing RNA velocity models aim to extract dynamics from the phase delay between unspliced and spliced mRNA for each individual gene. However, unspliced/spliced mRNA abundance may not provide sufficient signal for dynamic modeling, leading to poor fit in phase portraits. Motivated by the idea that RNA velocity could be driven by the transcriptional regulation, we propose TFvelo, which expands RNA velocity concept to various single-cell datasets without relying on splicing information, by introducing gene regulatory information. Our experiments on synthetic data and multiple scRNA-Seq datasets show that TFvelo can accurately fit genes dynamics on phase portraits, and effectively infer cell pseudo-time and trajectory from RNA abundance data. TFvelo opens a novel, robust and accurate avenue for modeling RNA velocity for single cell data.

bioinformatics↗

bioRxiv2023

Construction of a multi-tissue cell atlas reveals cell-type-specific regulation of molecular and complex phenotypes in pigs

The systematic characterization of cellular heterogeneity among tissues and cell-type-specific regulation underlying complex phenotypes remains elusive in pigs. Within the Pig Genotype-Tissue Expression (PigGTEx) project, we present a single-cell transcriptome atlas of adult pigs encompassing 229,268 high-quality nuclei from 19 tissues, annotated to 67 major cell types. Besides cellular heterogeneity within and across tissues, we further characterize prominent tissue-specific features and functions of muscle, epithelial, and immune cells. Through deconvoluting 3,921 bulk RNA-seq samples from 17 matching tissues, we dissect thousands of genetic variants with cell-type interaction effects on gene expression (ieQTL). By colocalizing these ieQTL with variants associated with 268 complex traits, we provide new insights into the cellular mechanisms behind these traits. Moreover, we highlight that orthologous genes with cell-type-specific regulation in pigs exhibit significant heritability enrichment for some human complex phenotypes. Altogether, our work provides a valuable resource and highlights novel insights in cellular regulation of complex traits for accelerating pig precision breeding and human biomedical research.

genetics↗

bioRxiv2023

Shortcut barcoding and early pooling for scalable multiplex single-cell reduced-representation CpG methylation sequencing at single nucleotide resolution

DNA methylation is essential for a wide variety of biological processes, yet the development of a highly efficient and robust technology remains a challenge for routine single-cell analysis. We developed a multiplex scalable single-cell reduced representation bisulfite sequencing (msRRBS) technology with off-the-shelf reagents and equipment. It allows cell-specific barcoded DNA fragments of individual cells to be pooled before bisulfite conversion, free of enzymatic modification or physical capture of the DNA ends, and achieves unparalleled read mapping rates of 62.51%, covering 59.95% of CpG islands and 71.62% of promoters in K562 cells on average. Its reproducibility is shown in duplicates of bulk cells with near perfect correlation (R=0.97-99). At a low 1 Mb of clean reads, msRRBS provides consistent coverage of CpG islands and promoters, outperforming the conventional methods with orders of magnitude reduction in cost. Here, we use this method to characterize the distinct methylation patterns and cellular heterogeneity of 6 cell lines, and leukemia and hepatocellular carcinoma models. Taking 4 hours of hands-on time, msRRBS offers a unique, highly efficient approach for dissecting methylation heterogeneity in a variety of multicellular systems.

genomics↗

bioRxiv2023

LinRace: single cell lineage reconstruction using paired lineage barcode and gene expression data

Understanding how single cells divide and differentiate into different cell types in developed organs is one of the major tasks of developmental and stem cell biology. Recently, lineage tracing technology using CRISPR/Cas9 genome editing has enabled simultaneous readouts of gene expressions and lineage barcodes in single cells, which allows for the reconstruction of the cell division tree, and even the detection of cell types and differentiation trajectories at the whole organism level. While most state-of-the-art methods for lineage reconstruction utilize only the lineage barcode data, methods that incorporate gene expression data are emerging, aiming to improve the accuracy of lineage reconstruction. However, effectively incorporating the gene expression data requires a reasonable model on how gene expression data changes along generations of divisions. Here, we present LinRace (Lineage Reconstruction with asymmetric cell division model), a method that integrates the lineage barcode and gene expression data using the asymmetric cell division model and infers cell lineage under a framework combining Neighbor Joining and maximum-likelihood heuristics. On both simulated and real data, LinRace outputs more accurate cell division trees than existing methods. Moreover, Lin Race can output the cell states (cell types) of ancestral cells, which is rarely performed with existing lineage reconstruction methods. The information on ancestral cells can be used to analyze how a progenitor cell generates a large population of cells with various functionalities. LinRace is available at: https://github.com/ZhangLabGT/LinRace.

bioinformatics↗

bioRxiv2023

Generalizing biological surround suppression based on center surround similarity via deep neural network models

Sensory perception is dramatically influenced by the context. Models of contextual neural surround effects in vision have mostly accounted for Primary Visual Cortex (V1) data, via nonlinear computations such as divisive normalization. However, surround effects are not well understood within a hierarchy, for neurons with more complex stimulus selectivity beyond V1. We utilized feedforward deep convolutional neural networks and developed a gradient-based technique to visualize the most suppressive and excitatory surround. We found that deep neural networks exhibited a key signature of surround effects in V1, highlighting center stimuli that visually stand out from the surround and suppressing responses when the surround stimulus is similar to the center. We found that in some neurons, especially in late layers, when the center stimulus was altered, the most suppressive surround surprisingly can follow the change. Through the visualization approach, we generalized previous understanding of surround effects to more complex stimuli, in ways that have not been revealed in visual cortices. In contrast, the suppression based on center surround similarity was not observed in an untrained network. We identified further successes and mismatches of the feedforward CNNs to the biology. Our results provide a testable hypothesis of surround effects in higher visual cortices, and the visualization approach could be adopted in future biological experimental designs. Author summaryNeural responses and perception of a visual stimulus are influenced by the context, such as what spatially surrounds a given feature. Contextual surround effects have been extensively studied in the early visual cortex. But the brain processes visual inputs hierarchically, from simple features up to complex objects in higher visual areas. Contextual effects are not well understood for higher areas of cortex and for more complex stimuli. Utilizing artificial deep neural networks and a visualization technique we developed, we found that deep networks exhibited a key signature of surround effects in the early visual cortex, highlighting center stimuli that visually stand out from the surround and suppressing responses when the surround stimulus is similar to the center. We found in some neurons, especially in late layers, when the center stimulus was altered, the most suppressive surround could surprisingly follow. This is a generalization of known surround effects for more complex stimuli that has not been revealed in the visual cortex. Our findings relate to notions of efficient coding and salience perception, and emerged without incorporating specialized nonlinear computations typically used to explain contextual effects in the early cortex. Our visualization approach provides a new experimental paradigm and a testable hypothesis of surround effects for more complex stimuli in higher cortical areas; the visualization approach could be adopted in biological experimental designs.

neuroscience↗

bioRxiv2023

A molecularly defined and spatially resolved cell atlas of the whole mouse brain

In mammalian brains, tens of millions to billions of cells form complex interaction networks to enable a wide range of functions. The enormous diversity and intricate organization of cells in the brain have so far hindered our understanding of the molecular and cellular basis of its functions. Recent advances in spatially resolved single-cell transcriptomics have allowed systematic mapping of the spatial organization of molecularly defined cell types in complex tissues1-3. However, these approaches have only been applied to a few brain regions1-11 and a comprehensive cell atlas of the whole brain is still missing. Here, we imaged a panel of >1,100 genes in [~]8 million cells across the entire adult mouse brain using multiplexed error-robust fluorescence in situ hybridization (MERFISH)12 and performed spatially resolved, single-cell expression profiling at the whole-transcriptome scale by integrating MERFISH and single-cell RNA-sequencing (scRNA-seq) data. Using this approach, we generated a comprehensive cell atlas of >5,000 transcriptionally distinct cell clusters, belonging to [~]300 major cell types, in the whole mouse brain with high molecular and spatial resolution. Registration of the MERFISH images to the common coordinate framework (CCF) of the mouse brain further allowed systematic quantifications of the cell composition and organization in individual brain regions defined in the CCF. We further identified spatial modules characterized by distinct cell-type compositions and spatial gradients featuring gradual changes in the gene-expression profiles of cells. Finally, this high-resolution spatial map of cells, with a transcriptome-wide expression profile associated with each cell, allowed us to infer cell-type-specific interactions between several hundred pairs of molecularly defined cell types and predict potential molecular (ligand-receptor) basis and functional implications of these cell-cell interactions. These results provide rich insights into the molecular and cellular architecture of the brain and a valuable resource for future functional investigations of neural circuits and their dysfunction in diseases.

neuroscience↗

bioRxiv2023

Changes in the urine proteome after massage in healthy people

This study aimed to explore the effect of massage on the urine proteome of healthy people. In this study, participants underwent 1-hour whole body massage. Urine samples were collected at 0, 2, and 24h after the massage and urine proteins were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Compared with the control (before massage), 41 differential proteins were identified in the group 2h after the massage, the random mean number of differentially produced proteins was 11 with 73% confidence, and the biological process of protein enrichment was catecholamine biosynthesis, which was related to the promotion of metabolism and the regulation of neural activities. While 29 differential proteins were identified in the group 24h after the massage, the random average number of differential proteins produced was 10, with the confidence of the difference decreased to 65%, and the effective biological process could not be enriched at this time. The results suggested that the difference in urine protein was greater at 2h after the massage and gradually decreased at 24h after the massage. The proteome of urine may reflect changes in the body following minor massage stimuli, providing a potential way to evaluate the effects of massage therapy.

biochemistry↗

bioRxiv2023

Local administration of a novel siRNA modality into the CNS extends survival and improves motor function in the SOD1G93A mouse model for ALS

Antisense oligonucleotides (ASOs) were the first modality to pioneer targeted gene knockdown in the treatment of ALS caused by mutant superoxide dismutase 1 (SOD1). RNA interference (RNAi) is another mechanism of gene silencing with historically superior potency in which short interfering RNAs (siRNAs) guide the RNA-induced silencing complex (RISC) to cleave complementary transcripts. However, delivery to extrahepatic tissues like the central nerve system (CNS) has been a bottleneck in the clinical development of RNAi. Herein, we identify potent siRNA duplexes for the knockdown of human SOD1 (hSOD1) in which medicinal chemistry and conjugation to an accessory oligonucleotide (ACO) enables durable and potent activity in CNS tissues. Local delivery via intracerebroventricular (ICV) or intrathecal (IT) injection into SOD1G93A mice delayed disease progression and extended animal survival with superior efficacy compared to an ASO compound resembling Tofersen in sequence and chemistry. Treatment also prevented disease-related declines in motor function including improvements in animal mobility, muscle strength, and coordination. The ACO itself does not target any specific complementary nucleic acid sequence; rather, it imparts benefits conducive to bioavailability and delivery through its chemistry. The complete conjugate (i.e., siRNA-ACO) represents a novel modality for delivery of RNAi to the CNS in which we aim to pursue ALS as an exemplary indication for clinical development.

cell biology↗

bioRxiv2023

Excess PrPC inhibits muscle cell differentiation via miRNA-enhanced liquid-liquid phase separation implicated in myopathy

The cellular prion protein (PrPC), a glycoprotein existing in membrane-bound and cytoplasmic forms, has functional importance in skeletal muscle, but the mechanism behind the phenomenon remains poorly understood. Here we report that PrPC is overexpressed and located in the cytoplasm of the skeletal muscle of six myopathy patients; cytoplasmic PrPC strongly inhibits skeletal muscle cell autophagy and blocks myoblast differentiation. PrPC selectively binds to a subset of miRNAs during myoblast differentiation, and the co-localization of PrPC with miR-214-3p was clearly observed in the skeletal muscle of six myopathy patients but not in that of four age-matched controls. We demonstrate that PrPC is overexpressed in skeletal muscle cells under pathological conditions and inhibits muscle cell differentiation via physically interacting with a subset of miRNAs to significantly inhibit autophagy-related protein 5-dependent autophagy, and selectively recruits these miRNAs into phase-separated condensates in living myoblasts, which in turn greatly enhances liquid-liquid phase separation (LLPS) of PrPC and results in the subsequent PrP aggregation and muscle bundle formation in myopathy patients characterized by incomplete muscle regeneration. Our findings show how excess PrPC can inhibit muscle cell differentiation via miRNA-enhanced LLPS implicated in myopathy.

biochemistry↗

bioRxiv2023

Lhcb9-dependent photosystem I structure in moss reveals evolutionary adaptation to changing light conditions during aquatic-terrestrial transition

In plants and green algae, light-harvesting complexes I and II (LHCI and LHCII) constitute the antennae of photosystem I (PSI), thus effectively increasing the cross-section of the PSI core. The moss Physcomitrium patens (P. patens) represents a well-studied evolutionary intermediate between green algae and flowering plants. P. patens possesses at least three types of PSI with different antenna sizes. The largest PSI form (PpPSI-L) exhibits a unique organization found neither in flowering plants nor in algae. Its formation is mediated by the P. patens-specific LHC protein, Lhcb9. While previous studies revealed the overall architecture of the PpPSI-L, its assembly details and the relationship between different PpPSI types remain unclear. Here, we report a high-resolution structure of the PpPSI-L. We identified 14 PSI core subunits, one Lhcb9, one phosphorylated LHCII trimer, and eight LHCI monomers arranged as two belts. Our structural analysis established the essential role of Lhcb9 and the phosphorylated LHCII in stabilizing the complex. In addition, our results suggest that PpPSI switches between three different types, which share identical modules. This feature may contribute to the quick and dynamic adjustment of the light-harvesting capability of PSI under different light conditions.

plant biology↗

bioRxiv2022

Decoding protein binding landscape on circular RNAs with base-resolution Transformer models

Circular RNAs (circRNAs) interact with RNA-binding proteins (RBPs) to modulate gene expression. To date, most computational methods for predicting RBP binding sites on circRNAs focus on circRNA fragments instead of circRNAs. These methods detect whether a circRNA fragment contains binding sites, but cannot determine where are the binding sites and how many binding sites are on the circRNA transcript. We report a hybrid deep learning-based tool, CircSite, to predict RBP binding sites at single-nucleotide resolution and detect key contributed nucleotides on circRNA transcripts. CircSite takes advantage of convolutional neural networks (CNNs) and Transformer for learning local and global representations of circRNAs binding to RBPs, respectively. We construct 37 datasets of RBP-binding circRNAs for benchmarking and the experimental results show that CircSite offers accurate predictions of RBP binding nucleotides and detects key subsequences aligning well with known binding motifs.

bioinformatics↗

bioRxiv2022

A stable reference human transcriptome and proteome as a standard for reproducible omics experiments

In recent years, the development of high-throughput omics technology has greatly promoted the development of biomedicine. However, the poor reproducibility of omics techniques limits its application. It is necessary to use standard reference materials of complex RNAs or proteins to test and calibrate the accuracy and reproducibility of omics workflows. However, the transcriptome and proteome of most cell lines shift during culturing, which limits their applicability to serve as standard samples. In this study, we demonstrated that the human hepatocellular cell line MHCC97H has a very stable transcriptome (R2=0.966-0.995) and proteome (R2=0.934-0.976 for DDA, R2=0.942-0.986 for DIA) after 9 subculturing generations, which allows this stable standard sample to be stably produced on an industrial scale for several decades. Moreover, this stability was maintained across labs and platforms. In sum, our results justified a omics standard reference material and reference datasets for transcriptomic and proteomics research. This helps to further standardize the workflow and data quality of omics techniques and thus promotes the application of omics technology in precision medicine.

cell biology↗

bioRxiv2022

A compendium of genetic regulatory effects across pig tissues

The Farm animal Genotype-Tissue Expression (FarmGTEx, https://www.farmgtex.org/) project has been established to develop a comprehensive public resource of genetic regulatory variants in domestic animal species, which is essential for linking genetic polymorphisms to variation in phenotypes, helping fundamental biology discovery and exploitation in animal breeding and human biomedicine. Here we present results from the pilot phase of PigGTEx (http://piggtex.farmgtex.org/), where we processed 9,530 RNA-sequencing and 1,602 whole-genome sequencing samples from pigs. We build a pig genotype imputation panel, characterize the transcriptional landscape across over 100 tissues, and associate millions of genetic variants with five types of transcriptomic phenotypes in 34 tissues. We study interactions between genotype and breed/cell type, evaluate tissue specificity of regulatory effects, and elucidate the molecular mechanisms of their action using multi-omics data. Leveraging this resource, we decipher regulatory mechanisms underlying about 80% of the genetic associations for 207 pig complex phenotypes, and demonstrate the similarity of pigs to humans in gene expression and the genetic regulation behind complex phenotypes, corroborating the importance of pigs as a human biomedical model.

genomics↗

bioRxiv2022

Lifetime development changes in rats tracked by urinary proteome

The existing researches mainly focus on the embryonic stage and a short time after that. However, there was little research about the whole life of an individual from childhood to aging and death. For the first time, we used non-invasive urinary proteome technology to track the changes of several important development timepoints in a batch of rats, covering the 10 timepoints from childhood, adolescence, young, middle adulthood, and to old age death. As previous studies on puberty found, sexual or reproductive maturation, mature spermatozoa in seminiferous tubules(first seen), gonadal hormones, decline of estradiol(E), brain growth and central nervous system myelination, and our differential protein enrichment pathways also included reproductive system development, tube development, response to hormone, response to estradiol, brain development, neuron development. Like previous studies found in young adulthood, musculoskeletal maturity, peak bone mass, development of the immune system, and growth and physical development, and our differential protein enrichment pathway also included skeletal system development, bone regeneration, system development, immune system process, myeloid leukocyte differentiation, growth, and developmental growth. However, at all timepoints in the whole life, there were still many biological pathways of the differential urinary protein enrichment involving multiple organs, tissues, systems, etc., which had not been mentioned by existing studies. This study provided comprehensive and detailed changes in rats lifetime development through urinary proteome, supplementing the blank of development research. But also provided a new idea for monitoring the human health change condition and the possibly occurring aging diseases by using the urinary proteome. SummaryAs far as we know, this is the first time that the urinary proteome was used to track the changes of several important developmental timepoints in the lifetime of rats.

developmental biology↗

bioRxiv2022

A genetically encoded tool to increase cellular NADH/NAD+ ratio in living cells

Impaired reduction/oxidation (redox) metabolism is a key contributor to the etiology of many diseases, including primary mitochondrial disorders, cancer, neurodegeneration, and aging. However, mechanistic studies of redox imbalance remain challenging due to limited strategies which can perturb cellular redox metabolism and model pathology in various cellular, tissue, or organismal backgrounds without creating additional and potentially confounding metabolic perturbations. To date, most studies involving impaired redox metabolism have focused on oxidative stress and reactive oxygen species (ROS) production; consequently, less is known about the settings where there is an overabundance of reducing equivalents, termed reductive stress. NADH reductive stress has been modeled using pharmacologic inhibition of the electron transport chain (ETC) and ethanol supplementation. Still, both these methods have significant drawbacks. Here, we introduce a soluble transhydrogenase from E. coli (EcSTH) as a novel genetically encoded tool to promote NADH overproduction in living cells. When expressed in mammalian cells, EcSTH, and a mitochondrially-targeted version (mitoEcSTH), can elevate the NADH/NAD+ ratio in a compartment-specific manner. Using this tool, we determine the metabolic and transcriptomic signatures of NADH reductive stress in mammalian cells. We also find that cellular responses to NADH reductive stress, including blunted proliferation, are dependent on cellular background and identify the metabolic reactions that sense changes in the cellular NADH/NAD+ balance. Collectively, our novel genetically encoded tool represents an orthogonal strategy to perturb redox metabolism and characterize the impact on normal physiology and disease states.

bioengineering↗