Biology subjects

Lu, X.

Publications and source records attributed to Lu, X..

At least 217 records · Page 12Linked to original sources

bioRxiv2023

Accurate estimation of rare cell type fractions fromtissue omics data via hierarchical deconvolution

Bulk transcriptomics in tissue samples reflects the average expression levels across different cell types and is highly influenced by cellular fractions. As such, it is critical to estimate cellular fractions to both deconfound differential expression analyses and infer cell type-specific differential expression. Since experimentally counting cells is infeasible in most tissues and studies, in silico cellular deconvolution methods have been developed as an alternative. However, existing methods are designed for tissues consisting of clearly distinguishable cell types and have difficulties estimating highly correlated or rare cell types. To address this challenge, we propose Hierarchical Deconvolution (HiDecon) that uses single-cell RNA sequencing references and a hierarchical cell type tree, which models the similarities among cell types and cell differentiation relationships, to estimate cellular fractions in bulk data. By coordinating cell fractions across layers of the hierarchical tree, cellular fraction information is passed up and down the tree, which helps correct estimation biases by pooling information across related cell types. The flexible hierarchical tree structure also enables estimating rare cell fractions by splitting the tree to higher resolutions. Through simulations and real data applications with the ground truth of measured cellular fractions, we demonstrate that HiDecon significantly outperforms existing methods and accurately estimates cellular fractions.

bioinformatics↗

bioRxiv2023

Icariin prevents depression-like behaviors in chronic unpredictable mild stress-induced rats through Bax/cytoplasm C/caspase-3 axis to alleviate neuronal apoptosis

Major depressive disorder (MDD) affects approximately 16% of the global population. Our previous study has demonstrated that icariin (ICA) exhibits anti-depressant activity by increasing the expression of Brain Derived Neurotrophic Factor (BDNF) in a rat model of chronic unpredictable mild stress (CUMS). In this study, we investigated whether and how ICA can prevent CUMS-induced depression-like behaviors in rats by modulating hippocampus neuronal apoptosis. Forty male rats were randomly divided into control, CUMS, CUMS-fluoxetine (Flx) (10 mg/kg), and CUMS-ICA (20 mg/kg) groups. Behavior tests including sucrose preference test (SPT), open field test (OFT), elevated plus-maze (EPM), and forced swimming tests (FST) were performed. The Nissl staining and TUNNEL assay were used to determine neuronal apoptosis. Subsequently, expression of glucocorticoid receptor (GR), Bcl-2, cytochrome C, caspase-3 and Bax in the hippocampus were tested by western blot. Our results show that a chronic administration of ICA (20 mg/kg) can prevent CUMS-induced depressant-like behaviors in male model rats. Additionally, ICA significantly inhibited mitochondrial translocation of GR, reduced mitochondrial outer membrane permeabilization (MOMP) to suppress the release of cytochrome C, and then inhibit the activation of caspase-3. In conclusion, our research provides new evidence to understand the anti-depressant activity of ICA, which relates to its inhibition of neuronal apoptosis in hippocampus through mitochondrial apoptotic pathway.

molecular biology↗

bioRxiv2023

Vaccine induction of CD4-mimicking broadly neutralizing antibody precursors in macaques

The CD4 binding site (CD4bs) is a conserved epitope on HIV-1 envelope (Env) that can be targeted by protective broadly neutralizing antibodies (bnAbs). HIV-1 vaccines have not elicited CD4bs bnAbs for many reasons, including the CD4bs is occluded by glycans, immunogen expansion of appropriate naive B cells, and selection of functional antibody mutations. Here, we demonstrate immunization of macaques with a CD4bs-targeting immunogen elicits neutralizing bnAb precursors with structural and genetic features of CD4-mimicking bnAbs. Structures of the CD4bs nAbs bound to HIV-1 Env demonstrated binding angles similar to human bnAbs and heavy chain second complementarity determining region-dependent binding characteristic of all known human CD4-mimicking bnAbs. Macaque nAbs were derived from variable and joining gene segments orthologous to the genes of human VH1-46-class bnAbs. This vaccine study initiated the B cells from which derive CD4bs bnAbs in primates, accomplishing the key first step in development of an effective HIV-1 vaccine.

immunology↗

bioRxiv2023

A cataract-causing Y204X mutation of CRYβB1 promotes C-terminal degradation and higher-order oligomerization

Crystallin (Cry) proteins are a class of main structural proteins of vertebrate eye lens, and their solubility and stability directly determine transparency and refractive power of the lens. Mutation in genes that encode for these Cry proteins is the common cause for congenital cataract. Despite extensive studies, the pathogenic and molecular mechanisms remain unclear. In this study, we identified a novel mutation in CRYBB1 from a congenital cataract family, and demonstrated that this mutation led to an earlier termination of protein translation, resulting in a 49-residue truncation at the CRY{beta}B1 C-terminus. This mutant is susceptible to proteolysis and allows us to determine a 1.2- [A] resolution crystal structure of CRY{beta}B1 without the entire C-terminal domain. In this crystal lattice, two N-terminal domain monomers form a dimer that structurally resembles a wild-type (WT) monomer, but with different surface characteristics. Biochemical analyses suggest that this mutant is significantly more liable to aggregate and degrade, when compared to WT CRY{beta}B1. All our results provide an insight into the mechanism regarding how a mutant Cry contributes to the development of congenital cataract possibly through alteration of inter-protein interactions that result in the opacity of eye lens.

biochemistry↗

bioRxiv2023

Deep spatial-omics to aid personalization of precision medicine in metastatic recurrent Head & Neck Cancers

Immune checkpoint inhibitor (ICI) modality has had a limited success (<20%) in treating metastatic recurrent Head & Neck Oropharyngeal Squamous cell carcinomas (OPSCCs). To improve response rates to ICIs, tailored approaches capable to capture the tumor complexity and dynamics of each patients disease are needed. Here, we performed advanced analyses of spatial proteogenomic technologies to demonstrate that: (i) compared to standard histopathology, spatial transcriptomics better-identified tumor cells and could specifically classify them into two different metabolic states with therapeutic implications; (ii) our new method (Spatial Proteomics-informed cell deconvolution method or SPiD) improved profiling of local immune cell types relevant to disease progression, (iii) identified clinically relevant alternative treatments and a rational explanation for checkpoint inhibitor therapy failure through comparative analysis of pre- and post-failure tumor data and, (iv) discovered ligand-receptor interactions as potential lead targets for personalized drug treatments. Our work establishes a clear path for incorporating spatial-omics in clinical settings to facilitate treatment personalization.

cancer biology↗

bioRxiv2023

Learning virus genotype-fitness landscape in embedding space

The relationship between genotype and fitness is fundamental to evolution, but quantitatively mapping genotypes to fitness has remained challenging. We propose the Phenotypic-Embedding theorem (P-E theorem) that bridges genotype-phenotype through an encoder-decoder deep learning framework. Inspired by this, we proposed a more general first principle for correlating genotype-phenotype, and the Phenotypic-Embedding theorem provides a computable basis for the application of first principle. As an application example of the P-E theorem, we developed the Co-attention based Transformer model to bridge Genotype and Fitness (CoT2G-F) model, a Transformer-based pre-train foundation model with downstream supervised fine-tuning (SFT) that can accurately simulate the neutral evolution of viruses and predict immune escape mutations. Accordingly, following the calculation path of the P-E theorem, we accurately obtained the basic reproduction number (R0) of SARS-CoV-2 from first principles, quantitatively linked immune escape to viral fitness, and plotted the genotype-fitness landscape. The theoretical system we established provides a general and interpretable method to construct genotype-phenotype landscapes, providing a new paradigm for studying theoretical and computational biology.

evolutionary biology↗

bioRxiv2023

Quiescence-inducing 3D-engineered matrix uncovers mechanosensitive and drug protective FHL2-p21 signaling axis

Resected tumors frequently relapse with distant metastasis, despite systemic treatment. Cellular dormancy has been identified as an important mechanism underlying such drug resistance enabling late relapse. Nonetheless, hurdles associated with detection and isolation of disseminated cancer cells (DCCs) in disease-free patients urge the need for in vitro models of dormant cells suited for drug discovery. Here, we explore dormancy-inducing 3D-engineered matrices, which generate mechanical confinement and induce growth arrest and survival against chemotherapy in cancer cells. We characterized the dormant phenotype of solitary cells by P-ERKlow:P-p38high dormancy signaling ratio, along with Ki67-expression. As underlying mechanism, we identified stiffness-dependent nuclear localization of the four-and-a-half LIM domains 2 (FHL2) protein, leading to p53-independent high p21Cip1/Waf1 nuclear expression, validated in murine and human tissue. Suggestive of a resistance-causing role, cells in the dormancy-inducing matrix became sensitive against chemotherapy upon FHL2 downregulation. Thus, our biomaterial-based approach will enable systematic screens for novel compounds suited to eradicate potentially relapsing, dormant cancer cells. TeaserUsing semi-synthetic bioengineered hydrogels, we reveal a mechanosensitive and drug protective mechanism of dormant cancer cells in tissues

bioengineering↗

bioRxiv2023

Chiral Graphene Quantum Dots Enhanced Drug Loading into Exosomes

As nanoscale extracellular vesicles secreted by cells, exosomes have enormous potential as safe and effective vehicles to deliver drugs into lesion locations. Despite promising advances with exosome-based drug delivery systems, there are still challenges to drug loading into exosome, which hinder the clinical applications of exosomes. Herein, we report an exogenous drug-agnostic chiral graphene quantum dots (GQDs) exosome-loading platform, based on chirality matching with the exosome lipid bilayer. Both hydrophobic and hydrophilic chemical and biological drugs can be functionalized or adsorbed onto GQDs by {pi}-{pi} stacking and van der Waals interactions. By tuning the ligands and GQD size to optimize its chirality, we demonstrate drug loading efficiency of 66.3% and 64.1% for Doxorubicin and siRNA, which is significantly higher than other reported exosome loading techniques.

bioengineering↗

bioRxiv2022

HBM-CITEseq: a uniform CITE-seq processing pipeline for the HuBMAP Consortium

As part of the HuBMAP Consortium we have been developing methods for uniformly processing and indexing multiple single-cell datasets, which enable efficient integration of data from different platforms. HuBMAP computational pipelines have so far focused on unimodal data types such as single-cell/nucleus RNA sequencing and single nucleus ATAC-seq. Here we present HBM-CITEseq, a processing pipeline for Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) datasets, the first multimodal sequencing processing pipeline for the HuBMAP Consortium, with transcriptomic outputs from HBM-CITEseq matching those of the HuBMAP RNA-seq pipeline. HBM-CITEseq is a CWL workflow wrapping command-line tools encapsulated in Docker images. It is freely available on GitHub at https://github.com/hubmapconsortium/citeseq-pipeline.

bioinformatics↗

bioRxiv2022

NR5A2 connects genome activation to the first lineage segregation in early mouse development

After fertilization, zygotic genome activation (ZGA) marks the beginning of the embryonic program for a totipotent embryo, which further gives rise to the pluripotent embryonic lineages and extraembryonic trophectoderm after the first lineage commitment. While much has been learned about pluripotency regulation, how ZGA is connected to the pluripotency commitment in early embryos remains elusive. Here, we investigated the role of nuclear receptor 1 family transcription factors (TFs) in mouse pre-implantation embryos, whose motifs are highly enriched in accessible chromatin at the 2-cell (2C) to 8-cell (8C) stages after ZGA. We found NR5A2, an NR TF highly induced upon ZGA, is required for early development, as both the knockdown and knockout of Nr5a2 from 1C embryos led to morula arrest. While the zygotic genome was largely activated at the 2C stage, 4-8C-specific gene activation (mid-preimplantation activation) was substantially impaired. Genome-wide chromatin binding and RNA-seq analyses showed NR5A2 preferentially regulates its binding targets including a subset of key pluripotency genes (i.e., Nanog, Pou5f1, and Tdgf1). Finally, NR5A2-occupied sites at the 2C and 8C stages predominantly reside in accessible B1 elements where its motif is embedded. Taken together, these data identify NR5A2 as a key regulator that connects ZGA to the first lineage segregation in early mouse development.

developmental biology↗

bioRxiv2022

Mutation-Guided Vaccine Design: A Strategy for Developing Boosting Immunogens for HIV Broadly Neutralizing Antibody Induction

A major goal of HIV-1 vaccine development is induction of broadly neutralizing antibodies (bnAbs). While success has been achieved in initiating bnAb B cell lineages, design of boosting immunogens that select for bnAb B cell receptors with improbable mutations required for bnAb affinity maturation remains difficult. Here we demonstrate a process for designing boosting immunogens for a V3-glycan bnAb B cell lineage. The immunogens induced affinity-matured antibodies by selecting for functional improbable mutations in bnAb precursor knock-in mice. Moreover, we show similar success in prime and boosting with nucleoside-modified mRNA-encoded HIV-1 envelope trimer immunogens, with improved selection by mRNA immunogens of improbable mutations required for bnAb binding to key envelope glycans. These results demonstrate the ability of both protein and mRNA prime-boost immunogens for selection of rare B cell lineage intermediates with neutralizing breadth after bnAb precursor expansion, a key proof-of concept and milestone towards development of an HIV vaccine. One-Sentence SummaryA vaccine strategy for selecting key rare antibody mutations is shown to induce HIV broadly neutralizing antibodies in mice.

immunology↗

bioRxiv2022

A Theory for High-Throughput Genetic Interaction Screening

Systematic, genome-scale genetic screens have been instrumental for elucidating genotype-phenotype relationships, but approaches for probing genetic interactions have been limited to at most [~]100 pre-selected gene combinations in mammalian cells. Here, we introduce a theory for high-throughput genetic interaction screens. The theory extends our recently developed Multiplexing using Spectral Imaging and Combinatorics (MuSIC) approach to propose [~]105 spectrally unique, genetically-encoded MuSIC barcodes from 18 currently available fluorescent proteins. Simulation studies based on constraints imposed by spectral flow cytometry equipment suggest that genetic interaction screens at the human genome-scale may be possible if MuSIC barcodes can be paired to guide RNAs. While experimental testing of this theory awaits, it offers transformative potential for genetic perturbation technology and knowledge of genetic function. More broadly, the availability of a genome-scale spectral barcode library for non-destructive identification of single-cells could find more widespread applications such as traditional genetic screening and high-dimensional lineage tracing.

biochemistry↗

bioRxiv2022

Connectomics of the Octopus vulgaris vertical lobe provides insight into conserved and novel principles of a memory acquisition network

We present the first analysis of the connectome of the vertical lobe (VL) of Octopus vulgaris, a brain structure mediating acquisition of long-term memory in this behaviorally advanced mollusk. Serial section electron microscopy revealed new types of interneurons, cellular components of extensive modulatory systems and multiple synaptic motifs. The sensory input to the VL is conveyed via ~1,800,000 axons that sparsely innervate two parallel and interconnected feedforward networks formed by the two types of amacrine interneurons (AM), simple AMs (SAMs) and complex AMs (CAMs). SAMs make up 89.3% of the ~25,000,000 VL cells, each receiving a synaptic input from only a single input neuron on its non-bifurcating primary neurite, suggesting that each input neuron is represented in only ~12 SAMs. This synaptic site is likely a "memory site" as it is endowed with LTP. The CAMs, a newly described AM type, comprise 1.6% of the VL cells. Their bifurcating neurites integrate multiple inputs from the input axons and SAMs. While the SAM network appears to feedforward sparse "memorizable" sensory representations into the VL output layer, the CAMs appear to monitor global activity and feedforward a balancing inhibition for "sharpening" the stimulus-specific VL output. While sharing morphological and wiring features with circuits supporting associative learning in other animals, the VL has evolved a unique circuit that enables associative learning based strictly on feedforward information flow.

neuroscience↗

bioRxiv2022

Designing a Minimal Artificial Glycolytic Pathway

The canonical glycolysis generates two molecules of acetyl-coenzyme A (acetyl-CoA) from one glucose through eleven cascade biochemical reactions. Here, we designed and constructed a Minimal Artificial Glycolytic (MAG) pathway consisting of only three types of biochemical reactions, with phosphoketolase as the core, combined with phosphatase and isomerase as auxiliary enzymes. It could theoretically achieve a 100% carbon yield to acetyl-CoA from any monosaccharide by integrating one-carbon condensation reaction. We tested the MAG pathway in vitro and in vivo, demonstrating the catabolism of typical C1-C6 carbohydrates to acetyl-CoA with yields from 82% to 95%. This novel glycolytic pathway provides a promising route for biomanufacturing with stoichiometric productivity from multiple carbon sources in the future.

synthetic biology↗

bioRxiv2022

Detecting rapid pan-cortical voltage dynamics in vivo with a brighter and faster voltage indicator

Widefield imaging with genetically encoded voltage indicators (GEVIs) is a promising approach for understanding the role of large cortical networks in the neural coding of behavior. However, the slow kinetics of current GEVIs limit their deployment for single-trial imaging of rapid neuronal voltage dynamics. Here, we developed a high-throughput platform to screen for GEVIs that combine fast kinetics with high brightness, sensitivity, and photostability under widefield one-photon illumination. Rounds of directed evolution produced JEDI-1P, a green-emitting fluorescent indicator whose performance is improved for all metrics. Next, we optimized a neonatal intracerebroventricular delivery method to achieve cost-effective and wide-spread JEDI-1P expression in mice. We also developed an approach to effectively correct optical measurements from hemodynamic and motion artifacts. Finally, we achieved stable brain-wide voltage imaging and successfully tracked gamma-frequency whisker and visual stimulations in awake mice in single trials, opening the door to investigating the role of high-frequency signals in brain computations.

neuroscience↗

bioRxiv2022

DNA capture and loop extrusion dynamics by cohesin-NIPBL

3D chromatin organization plays a critical role in regulating gene expression, DNA replication, recombination, and repair. While initially discovered for its role in sister chromatid cohesion, emerging evidence suggests that the cohesin complex (SMC1, SMC3, RAD21, and SA1/SA2), facilitated by NIPBL, mediates topologically associating domains (TADs) and chromatin loops through DNA loop extrusion. However, information on how conformational changes of cohesin-NIPBL drive its loading onto DNA, initiation, and growth of DNA loops is still lacking. Using high-speed AFM (HS-AFM) imaging, we show that cohesin-NIPBL captures DNA through arm extension, followed by transfer of DNA to its globular domain and DNA loop initiation independent of ATPase hydrolysis. Additional shorter protrusions (feet) from cohesin-NIPBL transiently bind to DNA, facilitating its loading onto DNA. Furthermore, HS-AFM imaging reveals distinct forward and reverse DNA loop extrusion steps by cohesin-NIPBL. These results provide critical missing links in our understanding of DNA binding and loop extrusion by cohesin-NIPBL.

biophysics↗

bioRxiv2022

An off-the-shelf multi-well scaffold-supported platform for tumour organoid-based tissues

Complex 3D bioengineered tumour models provide the opportunity to better capture the heterogeneity of patient tumours. Patient-derived organoids are emerging as a useful tool to study tumour heterogeneity and variation in patient responses. Organoid cultures typically require a 3D microenvironment that can be manufactured easily to facilitate screening. Here we set out to create a high-throughput, "off-the-shelf" platform which permits the generation of organoid-containing microtissues for standard phenotypic bioassays and image-based readings. To achieve this, we developed the Scaffold-supported Platform for Organoid-based Tissues (SPOT) platform. SPOT is a 3D gel-embedded in vitro platform that can be produced in a 96- or 384-well plate format and enables the generation of flat, thin and dimensionally-defined microgels. SPOT has high potential for adoption due to its reproducible manufacturing methodology, compatibility with existing instrumentation, and reduced within-sample and between-sample variation, which can pose challenges to both data analysis and interpretation. Using SPOT we generate cultures from patient derived pancreatic ductal adenocarcinoma organoids and assess the cellular response to standard-of-care chemotherapeutic compounds, demonstrating our platforms usability for drug screening. We envision 96/384-SPOT will provide a useful tool to assess drug sensitivity of patient-derived organoids and easily integrate into the drug discovery pipeline.

bioengineering↗

bioRxiv2022

A 4-bp natural deletion of maize Na+/H+ exchanger gene alters maize salt stress tolerance

Soil salinity is a major environmental constraint severely reducing plant growth and crop productivity worldwide. Knowledge of salt tolerance-related genes can facilitate improving crop salt tolerance and alleviating the threat of increasing saline area to world food security. Here, we identified a major locus SALT TOLERANCE 1 (qST1) conferring maize salt tolerance via bulked segregant RNA-Seq (BSR-Seq). qST1 encodes a plasma membrane Na+/H+ exchanger ZmSOS1 which is the ortholog of Arabidopsis thaliana SOS1 gene. In salt-sensitive variety D9H, the natural variation of 4-bp deletion in the coding sequence of ZmSOS1 gene was the causal allele for salt sensitivity. We identified two ethyl methanesulfonate-induced mutants, zmsos1-1 and zmsos1-2, which were sensitive to salt stress and cant complement salt-sensitive variety under salt stress within an allelism test. Overexpression of ZmSOS1 enhanced maize seedling salt tolerance. ZmSOS1 can increase the salt tolerance of Arabidopsis sos1-1 mutant and can be activated by AtSOS2 and AtSOS3 in yeast cells, suggesting that ZmSOS1 confers salt tolerance through the conserved SOS signaling pathway in maize. The detrimental allele harboring the 4-bp deletion was rarely found in the natural population but appeared in an important heterotic group composed of x1132x-derived inbred lines which have been used widely for breeding dozens of hybrid varieties in China. The 4-bp deletion-based molecular marker has been successfully used to improve salt-sensitive varieties in a backcross and marker-assisted breeding program by screening and purging this deleterious allele.

plant biology↗