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Biology subjects

Ge, X.

Publications and source records attributed to Ge, X..

At least 55 records · Page 3Linked to original sources

Molecular and cellular dynamics of the developing human neocortex at single-cell resolution

The development of the human neocortex is a highly dynamic process and involves complex cellular trajectories controlled by cell-type-specific gene regulation1. Here, we collected paired single-nucleus chromatin accessibility and transcriptome data from 38 human neocortical samples encompassing both the prefrontal cortex and primary visual cortex. These samples span five main developmental stages, ranging from the first trimester to adolescence. In parallel, we performed spatial transcriptomic analysis on a subset of the samples to illustrate spatial organization and intercellular communication. This atlas enables us to catalog cell type-, age-, and area-specific gene regulatory networks underlying neural differentiation. Moreover, combining single-cell profiling, progenitor purification, and lineage-tracing experiments, we have untangled the complex lineage relationships among progenitor subtypes during the transition from neurogenesis to gliogenesis in the human neocortex. We identified a tripotential intermediate progenitor subtype, termed Tri-IPC, responsible for the local production of GABAergic neurons, oligodendrocyte precursor cells, and astrocytes. Remarkably, most glioblastoma cells resemble Tri-IPCs at the transcriptomic level, suggesting that cancer cells hijack developmental processes to enhance growth and heterogeneity. Furthermore, by integrating our atlas data with large-scale GWAS data, we created a disease-risk map highlighting enriched ASD risk in second-trimester intratelencephalic projection neurons. Our study sheds light on the gene regulatory landscape and cellular dynamics of the developing human neocortex.

neuroscience↗

Three-dimensional dipole orientation mapping with high temporal-spatial resolution using polarization modulation

Fluorescence polarization microscopy is widely used in biology for molecular orientation properties. However, due to the limited temporal resolution of single-molecule orientation localization microscopy and the limited orientation dimension of polarization modulation techniques, achieving simultaneous high temporal-spatial resolution mapping of the three-dimensional (3D) orientation of fluorescent dipoles remains an outstanding problem. Here, we present a super-resolution 3D orientation mapping (3DOM) microscope that resolves 3D orientation by extracting phase information of the six polarization modulation components in reciprocal space. 3DOM achieves an azimuthal precision of 2{degrees} and a polar precision of 3{degrees} with spatial resolution of 128 nm in the experiments. We validate that 3DOM not only reveals the heterogeneity of the milk fat globule membrane, but also elucidates the 3D structure of biological filaments, including the 3D spatial conformation of {lambda}-DNA and the structural disorder of actin filaments. Furthermore, 3DOM images the dipole dynamics of microtubules labeled with green fluorescent protein in live U2OS cells, reporting dynamic 3D orientation variations. Given its easy integration into existing wide-field microscopes, we expect the 3DOM microscope to provide a multi-view versatile strategy for investigating molecular structure and dynamics in biological macromolecules across multiple spatial and temporal scales.

molecular biology↗

The Parkinson's drug entacapone disrupts gut microbiome homeostasis via iron sequestration

Increasing evidence shows that many human-targeted drugs alter the gut microbiome, leading to implications for host health. However, much less is known about the mechanisms by which drugs target the microbiome and how drugs affect microbial function. Here we combined quantitative microbiome profiling, long-read metagenomics, stable isotope probing and single cell chemical imaging to investigate the impact of two widely prescribed nervous system targeted drugs on the gut microbiome. Ex vivo supplementation of physiologically relevant concentrations of entacapone or loxapine succinate to faecal samples significantly impacted the abundance of up to one third of the microbial species present. Importantly, we demonstrate that the impact of these drugs on microbial metabolism is much more pronounced than their impact on abundances, with low concentrations of drugs reducing the activity, but not the abundance of key microbiome members like Bacteroides, Ruminococcus or Clostridium species. We further demonstrate that entacapone impacts the microbiome due to its ability to complex and deplete available iron, and that microbial growth can be rescued by replenishing levels of microbiota-accessible iron. Remarkably, entacapone-induced iron starvation selected for iron-scavenging organisms carrying antimicrobial resistance and virulence genes. Collectively, our study unveils the impact of two under-investigated drugs on whole microbiomes and identifies metal sequestration as a mechanism of drug-induced microbiome disturbance.

microbiology↗

Glioblastoma Margin as a Diffusion Barrier Revealed by Photoactivation of Plasmonic Nanovesicles

Glioblastoma (GBM) is the most complex and lethal adult primary brain cancer. Adequate drug diffusion and penetration are essential for treating GBM, but how the spatial heterogeneity in GBM impacts drug diffusion and transport is poorly understood. Herein, we report a new method, photoactivation of plasmonic nanovesicles (PANO), to measure molecular diffusion in the extracellular space of GBM. By examining three genetically engineered GBM mouse models that recapitulate key clinical features including angiogenic core and diffuse infiltration, we found that the tumor margin has the lowest diffusion coefficient (highest tortuosity) compared with the tumor core and surrounding brain tissue. Analysis of the cellular composition shows that the tortuosity in the GBM is strongly correlated with neuronal loss and astrocyte activation. Our all-optical measurement reveals the heterogeneous GBM microenvironment and highlights the tumor margin as a diffusion barrier for drug transport in the brain, with implications for therapeutic delivery.

bioengineering↗

Study on the preparation of bovine milk exosomes and the stability of lyophilized powder

Bovune milk exosomes (MK-exo) have bioactive effects on the health of consumers and can also serve as excellent drug delivery vehicles. However, long-term preservation and transportation remain major challenges at present. In this report, high-quality MK-exo are prepared on a large scale. We then developed a lyophilization method belonging to MK-exo, and performed acceleration and long-term stability experiments on the lyophilized MK-exo to evaluate the effect of lyophilization. The results showed that the lyophilized MK-exo can be stored at room temperature for at least 3 months and 2-8{degrees}C for 15 months. These findings provide data support for further understanding of the properties and applications of MK-exo.

bioengineering↗

High-throughput single-cell sorting by stimulated Raman-activated cell ejection

Single-cell sorting is essential to explore cellular heterogeneity in biology and medicine. Recently developed Raman-activated cell sorting (RACS) circumvents the limitations of fluorescence-activated cell sorting, such as the cytotoxicity of labels. However, the sorting throughputs of all forms of RACS are limited by the intrinsically small cross-section of spontaneous Raman scattering. Here, we report a stimulated Raman-activated cell ejection (S-RACE) platform that enables high-throughput single-cell sorting based on high-resolution multi-channel stimulated Raman chemical imaging, in situ image decomposition, and laser-induced cell ejection. The performance of this platform was illustrated by sorting a mixture of 1 m polymer beads, where 95% yield, 98% purity, and 14 events per second throughput were achieved. Notably, our platform allows live cell ejection, allowing for the growth of single colonies of bacteria and fungi after sorting. To further illustrate the chemical selectivity, lipid-rich Rhodotorula glutinis cells were successfully sorted from a mixture with Saccharomyces cerevisiae, confirmed by downstream quantitative PCR. Furthermore, by integrating a closed-loop feedback control circuit into the system, we realized real-time single-cell imaging and sorting, and applied this method to precisely eject regions of interest from a rat brain tissue section. The reported S-RACE platform opens exciting opportunities for a wide range of single-cell applications in biology and medicine. Significance statementImage-guided single-cell sorting is a potent tool in diverse biological applications. Current microfluidic cell sorting methods encounter challenges in handling smaller cells and are not applicable to tissue sections. To address these challenges, we have developed a stimulated Raman-activated cell ejection (S-RACE) platform, which is the first demonstration of single-cell ejection coupled with coherent Raman scattering. S-RACE allows label-free chemical imaging guided cell sorting through multispectral stimulated Raman scattering (SRS) imaging, on-the-fly image analysis, and laser-induced cell ejection. Versatile applications of S-RACE to a wide range of samples, such as polymer particles, single-live bacteria, single-live fungus, and tissue sections, are demonstrated.

bioengineering↗

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↗

Immunities Specific to Both of the M Protein Ectodomain and RBD Synergize to Confer Cross-protection against SARS-CoV-2 Infections

The effectiveness of the prototypic SARS-CoV-2 vaccine largely decreased overtime against the emerging virus strains, necessitating the universal vaccine development. The most abundant structural membrane (M) protein is highly conserved in amino acid sequence, which arouses our research interests in developing a universal immunogen based on it. Serological analysis showed that IgG responses specific to its N-terminal peptides can be strongly detected in many serum samples from both convalescent patients and vaccinees receiving inactivated vaccines, indicating the potential existence of human B-cell epitopes in reactive peptides. Microneutralization assays showed that the N-terminal peptide S2M2-30-specific hyperimmune serum was capable of cross-neutralizing the authentic viruses including wild-type HKU-001a, B.1.617.2/Delta, and Omicron subvariant BQ.1.1, and synergized with RBD-specific serum in reinforcing antiviral activities. Strong S2M2-30-specific immunities elicited in hACE2-transgenic mice could effectively inhibit B.1.1.7/Alpha (UK) infections. Our results suggest the potentiality of conserved M peptides as vaccine targets for conferring cross-protections against sarbecoviruses.

immunology↗

Evaluation of gliovascular functions of Aqp4 readthrough isoforms

Aquaporin-4 (AQP4) is a water channel protein that links astrocytic endfeet to the blood-brain barrier (BBB) and regulates water and potassium homeostasis in the brain, as well as the glymphatic clearance of waste products that would otherwise potentiate neurological diseases. Recently, translational readthrough was shown to generate a C-terminally extended variant of AQP4, known as AQP4x, that preferentially localizes around the BBB through interaction with the scaffolding protein -syntrophin, and loss of AQP4x disrupts waste clearance from the brain. To investigate the function of AQP4x, we generated a novel mouse AQP4 line (AllX) to increase relative levels of the readthrough variant above the [~]15% of AQP4 in the brain of wildtype (WT) mice. We validated the line and assessed characteristics that are affected by the presence of AQP4x, including AQP4 and -syntrophin localization, integrity of the BBB, and neurovascular coupling. We compared AllXHom and AllXHet mice to wildtype, and to previously characterized AQP4 NoXHet and NoXHom mice, which cannot produce AQP4x. Increased dose of AQP4x enhanced perivascular localization of - syntrophin and AQP4, while total protein expression of the two were unchanged. However, at 100% readthrough, AQP4x localization and formation of higher-order complexes was disrupted. Electron microscopy showed that overall blood vessel morphology was unchanged except for increased endothelial cell vesicles in NoXHom mice, which may correspond to a leakier BBB or altered efflux that was identified in NoX mice using MRI. These data demonstrate that AQP4x plays a small but measurable role in maintaining BBB integrity as well as recruiting structural and functional support proteins to the blood vessel. This also establishes a new set of genetic tools for quantitatively modulating AQP4x levels. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/549379v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@18f8f32org.highwire.dtl.DTLVardef@25afeorg.highwire.dtl.DTLVardef@a3e0e1org.highwire.dtl.DTLVardef@100f2e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

ClusterDE: a post-clustering differential expression (DE) method robust to false-positive inflation caused by double dipping

Post-clustering differential analysis is widely used across omics, including single-cell and spatial transcriptomics, multi-omics, population-scale bulk transcriptomics, and microbiome data. After a clustering algorithm finds clusters based on omics features as putative cell types, spatial domains, or subpopulations, statistical tests are typically applied to the same data to identify differential features as potential markers. Because the features that drive clustering are inherently more likely to appear differential, this clustering-induced bias can yield false-positive markers and mislead the interpretation of clusters as meaningful biological entities, especially when clusters are spurious, resulting in ambiguously defined cell types, spatial domains, or subpopulations. This dual challenge---determining whether clusters themselves are reliable and, if so, identifying their true markers---requires a unified statistical solution. To address this challenge, we propose ClusterDE, a statistical method designed to identify post-clustering differential features (with differentially expressed (DE) genes as a primary example) as reliable markers of cell types, spatial domains, or subpopulations while controlling the false discovery rate (FDR), regardless of clustering quality. The core of ClusterDE involves generating synthetic null data as an in silico negative control representing a single homogeneous group (such as a cell type, spatial domain, or population), allowing for the detection and removal of spurious markers caused by clustering-induced bias. In single-cell and spatial transcriptomics analyses, ClusterDE controls the FDR, prioritizes canonical cell-type and spatial-domain markers among the top discoveries, and de-prioritizes housekeeping genes, and it can refine underlying cell-type hierarchies by merging spurious or over-clustered groups. ClusterDE also mitigates spurious bifurcating trajectories in single-cell analyses that arise from over-clustering and retrospectively evaluates contested cell-type claims in high-profile studies, including a retracted human fetal cerebellum atlas and a challenged COVID-19 developing-neutrophil trajectory. At atlas scale, applying ClusterDE to the Allen Human Middle Temporal Gyrus taxonomy merges fine-grained clusters that are not statistically supported by single-cell transcriptomics, while preserving transcriptomically distinct and spatially supported cell types. Moreover, built-in synthetic null quality checks allow users to experiment with multiple state-of-the-art simulators and retain those that pass the diagnostics for their specific datasets. ClusterDE is compatible with widely used analysis pipelines such as Seurat and Scanpy and supports flexible, data-specific synthetic null generation, enhancing post-clustering inference across diverse omics modalities, with demonstrated efficacy in population-scale bulk transcriptomics and microbiome analyses.

bioinformatics↗

Second messenger c-di-AMP regulates multiple antibiotic sensitivity pathways in Mycobacterium smegmatis by discrete mechanisms

One of the debilitating causes of high mortality in the case of tuberculosis and other bacterial infections is the resistance development against standard drugs. There are limited studies so far to describe how a bacterial second messenger molecule can directly participate in distinctive antibiotic tolerance characteristics of a cell in a mechanism-dependent manner. Here we show that intracellular cyclic di-AMP (c-di-AMP) concentration can modulate drug sensitivity of Mycobacterium smegmatis by directly interacting with either a protein effector or with the 5-UTR regions in mRNA of the genes and thus causing transcriptional downregulation of important genes in the pathways. We studied four antibiotics with different mechanisms of action: rifampicin, ciprofloxacin, erythromycin, and tobramycin and subsequently found that the level of drug sensitivity of the bacteria is directly proportional to the c-di-AMP concentration inside the cell. Further, we unraveled the underlying molecular mechanisms to delineate the specific genes and pathways regulated by c-di-AMP and hence result in differential drug sensitivity in M. smegmatis. Thus, our findings of c-di-AMP messenger controlling drug resistance phenotypes of mycobacteria against four different classes of antibiotics is a unique observation that will contribute to scientific advancement in the field. One Sentence SummaryHere we describe how second messenger c-di-AMP modulates the antibiotic sensitivity and resistance profile of M. smegmatis by diverse mechanisms.

microbiology↗

A Ciliary SMOOTHENED-GRK2-PKA Signaling Pathway Initiates Hedgehog Signal Transduction

During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous Hh pathway activation in the vertebrate primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO, and the ensuing PKA-C binding and inactivation, are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.

cell biology↗

Outward-oriented sites within clustered CTCF boundaries are key for intraTAD chromatin interactions and gene regulation

CTCF plays an important role in adjusting insulation at TAD boundaries, where clustered CBS (CTCF-binding site) elements are often arranged in a somewhat tandem array with a complex divergent or convergent orientation. Here using cPcdh and HOXD loci as a paradigm, we look into the clustered CTCF TAD boundaries and find that, counterintuitively, outward-oriented CBS elements are crucial for inward enhancer-promoter interactions as well as for gene regulation. Specifically, by combinatorial deletions of a series of putative enhancer elements in vivo or CBS elements in vitro, in conjunction with chromosome conformation capture and RNA-seq analyses, we show that deletions of outward-oriented CBS elements weaken the strength of intraTAD promoter-enhancer interactions and enhancer activation of target genes. Our data highlight the crucial role of outward-oriented CBS elements within the clustered CTCF TAD boundaries and have interesting implications on the organization principles of clustered CTCF sites within TAD boundaries.

genetics↗

Progesterone is an Inducement of Heritable Pulmonary Arterial Hypertension with BMPR2 Mutation

BackgroundBone morphogenetic protein type II receptor (BMPR2) gene mutation accounts for 80% of patients with heritable pulmonary artery hypertension (HPAH), and female mutation carriers have significantly higher penetrance rate than males. The inducement of HPAH penetrance and the mechanism of sex differential penetrance are still elusive. MethodsWe infected or transfected pulmonary artery smooth muscle cells (PASMCs) with shBMPR2 lentivirus or siBMPR2 to simulate the pathologic condition of BMPR2 heterozygous mutation and treated them with progesterone. The HPAH patient-derived induced pluripotent stem cells (iPSCs) were induced into vascular smooth muscle cells (VSMCs) to further verify the cellular phenotype. Wild-type flox+/- female mice and SM22-cre BMPR2 flox+/- female mice (CKO mice) were administered with 1-month progesterone, and their phenotype of PAH was evaluated by right heart catheterization and histopathological examination. ResultsProgesterone promoted the proliferation of PASMCs with BMPR2-knockdownby activating ERK pathway via progesterone receptor (PGR). Activated ERK not only upregulated the phosphorylation and elevation of cMYC, but also induced the transcription of endothelin (EDN1) by promoting the nuclear entry of c-JUN and combination on the its promoter region. Similar results were confirmed by iPSCs-VSMCs experiment. CKO mice developed PAH spontaneously and had increased expression of EDN1, which was further aggravated by exogenous progesterone. ConclusionsProgesterone might be an inducement of HPAH penetrance caused by BMPR2 mutation, accounting for sex differential penetrance.

molecular biology↗

pan-ECM: live brain extracellular matrix imaging with protein-reactive dye

The brain extracellular matrix (ECM), consisting of proteins and glycosaminoglycans, is a critical scaffold in the development, homeostasis, and disorders of the central nervous system (CNS) and undergoes remodeling in response to environmental cues. Live imaging of brain ECM structure represents a native view of the brain ECM but, until now, remains challenging due to the lack of a robust fluorescent labeling approach. Here, we developed a pan-ECM method for labeling the entire (Greek: pan) brain ECM network by screening and delivering a protein-reactive dye into the brain. pan-ECM enables imaging of ECM compartments in live brain tissue, including the interstitial matrix, basement membrane (BM), and perineuronal nets (PNNs), and even the ECM in glioblastoma and stroke mouse brains. This approach provides access to the structure and dynamics of the ECM and enhances our understanding of the complexities of the brain ECM and its contribution to brain health and disease.

neuroscience↗

The novel roles of bovine milk-derived exosomes on skin anti-aging

Exosomes are small vesicles released from cells and present in various mammal biological fluids, such as bovine milk, which worked for skin care for many years besides dairy. In addition, Exosomes were regarded as a vehicle for intercellular communication. Therefore, we aimed to investigate the novel roles of bovine milk-derived exosomes (MK-Exo) on human skin anti-aging. Purified MK-Exo can be directly uptake by the keratinocytes and fibroblast in vitro and upregulate the expression of the natural factors related to skin moisturizing, including Filaggrin (FLG), Aquaporin 3 (AQP3), CD44 in the keratinocytes and hyaluronidase (HAS2) in the fibroblast, and MK-Exo promoted the cell migration of the fibroblast, while rescue its expression of type I collagen (Col I), type III collagen (Col III) after ultraviolet radiation. Furthermore, the phototoxicity test, photoallergy test, repeated skin irritation test, skin allergy test, and patch test confirm the safety of MK-Exo on the skin. Finally, the roles of MK-Exo in preserving moisture and anti-wrinkle were also identified in humans. Then, MK-Exo was smeared on the facial skin of 31 female volunteers twice a day for 28 days, and the functions were evaluated following the safety assessment in vivo. These studies reveal the novel roles of bovine milk-derived exosomes in human skin aging, which opens a new way of skin care.

cell biology↗

Geographic variation in evolutionary rescue in a predator-prey system under climate change: an example with aphids and ladybird beetles

Under climate change, species can adapt to changing environments through phenotypic plasticity and natural selection, and this kind of evolutionary adaptation can vary geographically. Most species distribution models (SDMs) are built upon the "Niche conservatism" assumption. They often ignore the possibility of "evolutionary rescue" and underestimate species future range limits under climate change. Here, we select aphids and ladybirds as model species and develop an eco-evolutionary model to explore evolutionary rescue in a predator-prey system under climate change. We model the adaptive change of species thermal performance, accounting for biotic interactions of unique life-history trait. Our results show that there is geographic variation in evolutionary rescue for ladybirds (the predator) across different locations in the United States, with ladybirds being more likely to be rescued from extinction in southeastern locations. The possibility of rescue is primarily influenced by the change in seasonality. Our findings also indicate the additive genetic variance of predators has a stronger influence on the phenotype evolution and population dynamics of both prey and predators, compared to the additive genetic variance of the prey. Our research emphasizes the importance of incorporating evolutionary adaptation when predicting species range shift under climate change. The eco-evolutionary model framework can be applied to study the effect of evolution on interacting species population abundance and geographic distribution under climate change.

evolutionary biology↗

Stimulated Raman Photothermal Microscopy towards Ultrasensitive Chemical Imaging

Stimulated Raman scattering (SRS) microscopy has shown enormous potential in revealing molecular structures, dynamics and couplings in complex systems. However, the sensitivity of SRS is fundamentally limited to milli-molar level due to the shot noise and the small modulation depth. To overcome this barrier, we revisit SRS from the perspective of energy deposition. The SRS process pumps molecules to their vibrationally excited states. The thereafter relaxation heats up the surrounding and induces refractive index changes. By probing the refractive index changes with a laser beam, we introduce stimulated Raman photothermal (SRP) microscopy, where a >500-fold boost of modulation depth is achieved. Versatile applications of SRP microscopy on viral particles, cells, and tissues are demonstrated. SRP microscopy opens a new way to perform vibrational spectroscopic imaging with ultrahigh sensitivity. One-Sentence SummaryWe demonstrate a new spectroscopic imaging method that improves the signal intensity by >500-fold.

bioengineering↗