Biology subjects

Xie, Q.

Publications and source records attributed to Xie, Q..

At least 73 records · Page 4Linked to original sources

bioRxiv2023

Determinants of species-specific utilization of ACE2 by human and animal coronaviruses

Utilization of human ACE2 allowed several bat coronaviruses (CoVs), including the causative agent of COVID-19, to infect humans either directly or via intermediate hosts. Here, we analyzed the ability of Spike proteins from 24 human or animal CoVs to use ACE2 receptors across nine reservoir, potential intermediate and human hosts. We show that overall SARS-CoV-2 Omicron variants evolved more efficient ACE2 usage but mutation of R493Q in BA.5 Spike disrupts utilization of ACE2 from Greater horseshoe bats. Spikes from most CoVs showed species-specific differences in ACE2 usage, partly due to variations in ACE2 residues 31, 41 or 354. Mutation of T403R allowed the RaTG13 bat CoV Spike to use all ACE2 orthologs analysed for viral entry. Sera from COVID-19 vaccinated individuals neutralized the Spike proteins of a range of bat Sarbecoviruses. Our results define determinants of ACE2 receptor usage of diverse CoVs and suggest that COVID-19 vaccination may protect against future zoonoses of SARS-CoV-related bat viruses. HighlightsO_LIMutation of R493Q in BA.5 Spike disrupts utilization of ACE2 from Greater horseshoe bats C_LIO_LIVariations in ACE2 residues 31, 41 or 354 affect utilization by coronavirus Spike proteins C_LIO_LIResidue R403 in the Spike protein of bat coronavirus allow broad and effective ACE2 usage C_LIO_LISera from COVID-19 vaccinated individuals neutralize Spike proteins of bat Sarbecoviruses C_LI

molecular biology↗

bioRxiv2023

Semi-rational evolution of a recombinant DNA polymerase for modified nucleotide incorporation efficiency

Engineering improved B-family DNA polymerases to incorporate 3'-O-modified nucleotide reversible terminators is limited by an insufficient understanding of the structural determinants that define polymerization efficiency. To explore the key mechanism for unnatural nucleotide incorporation, we engineered a B-family DNA polymerase from Thermococcus Kodakaraenis (KOD pol) by using semi-rational design strategies. We first scanned the active pocket of KOD pol through site-directed saturation mutagenesis and combinatorial mutations and identified a variant Mut_C2 containing five mutation sites (D141A, E143A, L408I, Y409A, A485E) using a high-throughput microwell-based screening method. Mut_C2 demonstrated high catalytic efficiency in incorporating 3-O-azidomethyl-dATP labeled with a Cy3 dye, whereas the wild-type KOD pol failed to incorporate it. Computational simulations were then conducted towards the DNA binding region of KOD pol to predict additional mutations with enhanced catalytic activity, which were subsequently experimentally verified. By a stepwise combinatorial mutagenesis approach, we obtained an eleven-mutation variant, named Mut_E10 by introducing additional mutations to the Mut_C2 variant. Mut_E10, which carried six specific mutations (S383T, Y384F, V389I, V589H, T676K, and V680M) within the DNA-binding region, demonstrated over 20-fold improvement in kinetic efficiency as compared to Mut_C2. In addition, Mut_E10 demonstrated satisfactory performance in two different sequencing platforms (BGISEQ-500 and MGISEQ-2000), indicating its potential for commercialization. Our study demonstrates that an effective enhancement in its catalytic efficiency towards modified nucleotides can be achieved efficiently through combinatorial mutagenesis of residues in the active site and DNA binding region of DNA polymerase. These findings contribute to a comprehensive understanding of the mechanisms that underlie the incorporation of modified nucleotides by DNA polymerase. The beneficial mutation sites, as well as the nucleotide incorporation mechanism identified in this study, can provide valuable guidance for the engineering of other B-family DNA polymerases.

bioengineering↗

bioRxiv2023

Crystal structure of a mammalian CMTM6 and its interaction model with PD-L1

CKLF-like MARVEL transmembrane domain-containing protein 6 (CMTM6) is a master regulator of PD-L1. By binding PD-L1 at the plasma membrane and recycling endosomes, CMTM6 prevents the lysosomal degradation of PD-L1 and maintains its cell surface expression, thus stabilizing the inhibitory PD-1/PD-L1 axis. However, the mechanism of CMTM6/PD-L1 interaction is unknown. Here we report the first experimentally determined structure of CMTM6 from bovine. Combined with a low-resolution cryo-EM map, computational docking analysis and a protein binding assay an interaction model between CMTM6 and PD-L1 was proposed, providing a structural framework for the CMTM6 regulation on PD-L1.

biophysics↗

bioRxiv2022

Origin of wiring specificity in an olfactory map: dendrite targeting of projection neurons

How does wiring specificity of neural maps emerge during development? Formation of the adult Drosophila olfactory glomerular map begins with patterning of projection neuron (PN) dendrites at the early pupal stage. To better understand the origin of wiring specificity of this map, we created genetic tools to systematically characterize dendrite patterning across development at PN type-specific resolution. We find that PNs use lineage and birth order combinatorially to build the initial dendritic map. Specifically, birth order directs dendrite targeting in rotating and binary manners for PNs of the anterodorsal and lateral lineages, respectively. Two-photon- and adaptive optical lattice light-sheet microscope-based time-lapse imaging reveals that PN dendrites initiate active targeting with direction-dependent branch stabilization on the timescale of seconds. Moreover, PNs that are used in both the larval and adult olfactory circuits prune their larval-specific dendrites and re-extend new dendrites simultaneously to facilitate timely olfactory map organization. Our work highlights the power and necessity of type-specific neuronal access and time-lapse imaging in identifying wiring mechanisms that underlie complex patterns of functional neural maps.

neuroscience↗

bioRxiv2022

The Neural Correlates of Arousal: The Ventral Posterolateral Nucleus-Global Transient Co-Activation

Arousal and awareness are two components of consciousness whose the neural mechanisms remain unclear. Spontaneous increases of global (brain-wide) blood-oxygenation-level-dependent (BOLD) signal has been found to be sensitive to changes in arousal. By contrasting BOLD datasets with altered arousal levels, we found that the activation of ventral posterolateral nucleus (VPL) decreased during transient increase in the global signal (top 17% data) in low arousal and awareness states (non-rapid eye movement sleep and anesthesia) as compared to wakefulness, and even in eye-closed (compared with eyes-open) in healthy awake-states, while this activation remained unchanged in patients with unresponsive wakefulness syndrome characterized by high arousal without awareness. These results demonstrate that co-activation of the VPL and global activity is critical to arousal, but not to awareness. One-Sentence SummaryThe VPL nucleus-global brain transient co-activation is related to physiological arousal but not to perceptual awareness.

neuroscience↗

bioRxiv2022

Identifying Patients with Cognitive Motor Dissociation Using Resting-state Temporal Stability

BackgroundUsing task-dependent neuroimaging techniques, recent studies discovered a fraction of patients with disorders of consciousness (DOC) who had no command-following behaviors but showed a clear sign of awareness, which was defined as cognitive motor dissociation (CMD). Although many efforts were made to identify the CMD, existing task-dependent approaches might fail when patients had multiple cognitive function (e.g., attention, memory) impairments, and thus lead to false-negative findings. However, recent advances in resting-state fMRI (rs-fMRI) analysis allow investigation of the dynamic change of spontaneous brain activity, which might be a powerful tool to test the patients cognitive functions, while its capacity in identifying CMD was unclear. MethodsThe rs-fMRI study included 119 participants from three independent research sites. A sliding-window approach was used to investigate the dynamic functional connectivity of the brain in two aspects: the global and regional temporal stability, which measures how stable the brain functional architecture is across time. The temporal stability was compared in the first dataset (36/16 DOC/controls), and then a Support Vector Machine (SVM) classifier was built to discriminate DOC patients from controls. Furthermore, the generalizability of the SVM classifier was tested in the second independent dataset (35/21 DOC/controls). Finally, the SVM classifier was applied to the third independent dataset where patients underwent an rs-fMRI and brain-computer interface assessment (4/7 CMD/potential non-CMD), to test its performance in identifying CMD. ResultsOur results showed that the global and regional temporal stability were impaired in DOC patients, especially in regions from the cingulo-opercular task control, default mode, fronto-parietal task control, and salience network. Using the temporal stability as features, the SVM model not only showed a good performance in the first dataset (accuracy = 90 %), but a good generalizability in the second dataset (accuracy = 82 %). Most importantly, the SVM model generalized well in identifying CMD in the third dataset (accuracy = 91 %). ConclusionThe current findings suggested that rs-fMRI could be a potential tool to assist in diagnosing CMD. Furthermore, the temporal stability investigated in this study also contributed to a deeper understanding of the neural mechanism of the consciousness.

neuroscience↗

bioRxiv2022

Wounding promotes root regeneration through a cell wall integrity sensor, the receptor kinase FERONIA

Wounding caused by various stresses is the initial event of plant regeneration. However, the mechanisms underlying the early wounding responses to promote plant regeneration remain largely unknown. Here, we report that the receptor kinase FERONIA (FER) interacts with Topless/Topless-related proteins (TPL/TPRs) to regulate the expression of regeneration-related genes to modulate root tip regeneration. One ligand of FER, rapid alkalinization factor 33 (RALF33), is stimulated by wounding and functions together with FER to promote regeneration. Single-cell sequencing data showed that the low-differentiation cell types in the stele may account for the enhanced regeneration ability in the fer mutant, especially in the columella and quiescent center (QC). Further interaction assays and analysis of the gene expression patterns in low-differentiation cell types confirmed that FER interacts with TPL/TPRs to regulate the expression of downstream regeneration-related genes. One of their downstream targets, an essential transcription factor (TF) in root regeneration, ERF115, acts downstream of FER-TPL/TPRs to control regeneration. Our results suggested a signaling pathway between the early wounding response and regeneration processes in roots. One-sentence summaryRALF33-FER serves as an early signaling module between wounding and regeneration by functioning with TPL/TPRs in roots.

cell biology↗

bioRxiv2022

Single-cell Atlas of Human Fetal Cerebellum Decoding Medulloblastoma Origin and Oncogenesis

Medulloblastoma (MB) is the most common malignant childhood brain tumor1,2, yet the origin of the most aggressive subgroup-3 form remains elusive, impeding development of effective targeted treatments. Previous analyses of mouse cerebella3,4 or human counterparts from frozen tissue nuclei5 have not fully defined the compositional heterogeneity of MBs. Here, we undertook an unprecedented single-cell profiling of freshly-isolated human fetal cerebella to establish a reference map delineating hierarchical cellular states in MBs. We identified a unique transitional cerebellar progenitor connecting neural stem cells to neuronal lineages in developing fetal cerebella. Intersectional analysis revealed that the transitional progenitors were enriched in aggressive MB subgroups, including group-3 and metastatic tumors. Single-cell multi-omics revealed underlying regulatory networks in the transitional progenitor populations, including transcriptional determinants HNRNPH1 and SOX11, which are correlated with clinical prognosis in group-3 MBs. Genomic and Hi-C profiling identified de novo long-range chromatin-loops juxtaposing HNRNPH1/SOX11-targeted super-enhancers to cis-regulatory elements of MYC, an oncogenic driver for group-3 MBs. Targeting the transitional progenitor regulators inhibited MYC expression and MYC-driven group-3 MB growth. Our integrated single-cell atlases of human fetal cerebella and MBs reveal potential cell populations predisposed to transformation and regulatory circuitries underlying tumor cell states and oncogenesis, highlighting hitherto unrecognized transitional progenitor intermediates predictive of disease prognosis and potential therapeutic vulnerabilities.

cancer biology↗

bioRxiv2022

High-resolution de novo structure prediction from primary sequence

Recent breakthroughs have used deep learning to exploit evolutionary information in multiple sequence alignments (MSAs) to accurately predict protein structures. However, MSAs of homologous proteins are not always available, such as with orphan proteins or fast-evolving proteins like antibodies, and a protein typically folds in a natural setting from its primary amino acid sequence into its three-dimensional structure, suggesting that evolutionary information and MSAs should not be necessary to predict a proteins folded form. Here, we introduce OmegaFold, the first computational method to successfully predict high-resolution protein structure from a single primary sequence alone. Using a new combination of a protein language model that allows us to make predictions from single sequences and a geometry-inspired transformer model trained on protein structures, OmegaFold outperforms RoseTTAFold and achieves similar prediction accuracy to AlphaFold2 on recently released structures. OmegaFold enables accurate predictions on orphan proteins that do not belong to any functionally characterized protein family and antibodies that tend to have noisy MSAs due to fast evolution. Our study fills a much-encountered gap in structure prediction and brings us a step closer to understanding protein folding in nature.

bioinformatics↗

bioRxiv2022

The role of proton in a eukaryotic zinc transporter

Zinc transporter 8 (ZnT8) is mainly expressed in pancreatic islet {beta} cells and is responsible for H+-coupled uptake (antiport) of Zn2+ into the lumen of insulin secretory granules. Structures of human ZnT8 and its prokaryotic homolog YiiP have provided structural basis for constructing a plausible transport cycle for Zn2+. However, the mechanistic role that protons play in the transport process remains unclear. Here we present a lumen-facing cryo-EM structure of ZnT8 from Xenopus tropicalis (xtZnT8) in the presence of Zn2+ at a luminal pH (5.5). Compared to a Zn2+-bound xtZnT8 structure at a cytosolic pH (7.5), the low-pH structure displays an empty transmembrane Zn2+-binding site with a disrupted coordination geometry. Combined with a Zn2+-binding assay our data suggest that protons may disrupt Zn2+ coordination at the transmembrane Zn2+-binding site in the lumen-facing state, thus facilitating Zn2+ release from ZnT8 into the lumen.

biochemistry↗

bioRxiv2022

Rational evolution of a recombinant DNA polymerase for efficient incorporation of unnatural nucleotides by dual-site boosting

Machine learning modelling assisting function-oriented enzyme engineering is normally built on predefined protein sequence space. However, efficient defining the determinant amino acid positions upon which the combinatorial mutation library is constructed is still a challenge in protein science. Herein, we present a comprehensive investigation of modifying a recombinant DNA polymerase for efficient incorporating one unnatural nucleotide, including the identification of key sites/regions, machine learning-assisted mutants screening, and the underlying mechanism of kinetics boosting. By using hundreds of training points and only dozens of testing samples, we found that one highly engineered enzymes catalytic efficiency can be further improved by one order of magnitude by specific mutation on two sites, 485I and 451L. Compared to the position 485 which is known to dominate local conformation of B-family DNA polymerases, 451 is a split-new active site discovered by our approach. A novel allosteric regulation mechanism is underlying the apparent synergy of 485I and 451L on the kinetics boosting. As a result, a "half-closed" conformation of the binding pocket and a cooperative binding of both primer and template DNA strands on the protein accelerated the processes of substrates incorporation, molecular recognition, and releasing of incorrect nucleotides. These findings have implications in guiding the function-tuning of DNA polymerases for a broad range of biotechnological applications.

biochemistry↗

bioRxiv2022

A reference single-cell regulomic and transcriptomic map of cynomolgus monkeys

Non-human primates (NHP) are attractive laboratory animal models that accurately reflect both developmental and pathological features of humans. Here we present a compendium of cell types from the cynomolgus monkey Macaca fascicularis (denoted as Monkey Atlas) using both single-cell chromatin accessibility (scATAC-seq) and RNA sequencing (scRNA-seq) data at the organism-wide level. The integrated cell map enables in-depth dissection and comparison of molecular dynamics, cell-type composition and cellular heterogeneity across multiple tissues and organs. Using single-cell transcriptomic data, we inferred pseudotime cell trajectories and cell-cell communications to uncover key molecular signatures underlying their cellular processes. Furthermore, we identified various cell-specific cis-regulatory elements and constructed organ-specific gene regulatory networks at the single-cell level. Finally, we performed a comparative analysis of single-cell landscapes among mouse, cynomolgus monkey and human, and we showed that cynomolgus monkey has significantly higher degree of cell-type similarity to human than mouse. Taken together, our study provides a valuable resource for NHP cell biology.

bioinformatics↗

bioRxiv2021

Reconstitution of the S. aureus agr quorum sensing pathway reveals a direct role for the integral membrane protease MroQ in pheromone biosynthesis

In Staphylococcus aureus, virulence is under the control of a quorum sensing (QS) circuit encoded in the accessory gene regulator (agr) genomic locus. Key to this pathogenic behavior is the production and signaling activity of a secreted pheromone, the autoinducing peptide (AIP), generated following the ribosomal synthesis and post-translational modification of a precursor polypeptide, AgrD, through two discrete cleavage steps. The integral membrane protease AgrB is known to catalyze the first processing event, generating the AIP biosynthetic intermediate, AgrD (1-32) thiolactone. However, the identity of the second protease in this biosynthetic pathway, which removes an N-terminal leader sequence, has remained ambiguous. Here, we show that MroQ, an integral membrane protease recently implicated in the agr response, is directly involved in AIP production. Genetic complementation and biochemical experiments reveal that MroQ proteolytic activity is required for AIP biosynthesis in agr specifiy groups -I and -II, but not group-III. Notably, as part of this effort, the biosynthesis and AIP-sensing arms of the QS circuit were reconstituted together in vitro. Our experiments also reveal the molecular features guiding MroQ cleavage activity, a critical factor in defining agr specificity group identity. Collectively, our study adds to the molecular understanding of the agr response and Staphylococcus aureus virulence.

microbiology↗

bioRxiv2021

scHiCSRS: A Self-Representation Smoothing Method with Gaussian Mixture Model for Imputing single-cell Hi-C Data

MotivationSingle cell Hi-C techniques make it possible to study cell-to-cell variability in genomic features. However, excess zeros are commonly seen in single cell Hi-C (scHi-C) data, making scHi-C matrices extremely sparse and bringing extra difficulties in downstream analysis. The observed zeros are a combination of two events: structural zeros for which the loci never interact due to underlying biological mechanisms, and dropouts or sampling zeros where the two loci interact but are not captured due to insufficient sequencing depth. Although quality improvement approaches have been proposed as an intermediate step for analyzing scHi-C data, little has been done to address these two types of zeros. We believe that differentiating between structural zeros and dropouts would benefit downstream analysis such as clustering. ResultsWe propose scHiCSRS, a self-representation smoothing method that improves the data quality, and a Gaussian mixture model that identifies structural zeros among observed zeros. scHiC-SRS not only takes spatial dependencies of a scHi-C 2D data structure into account but also borrows information from similar single cells. Through an extensive set of simulation studies, we demonstrate the ability of scHiCSRS for identifying structural zeros with high sensitivity and for accurate imputation of dropout values in sampling zeros. Downstream analysis for three real datasets show that data improved from scHiCSRS yield more accurate clustering of cells than simply using observed data or improved data from several comparison methods. Availability and ImplementationThe scHiCSRS R package, together with the processed real and simulated data used in this study, are available on Github at https://github.com/sl-lin/scHiCSRS.git. Contactshili@stat.osu.edu Supplementary informationSupplementary data are available online.

bioinformatics↗

bioRxiv2021

Transcription factor control of dendrite targeting via combinatorial cell-surface codes

Transcription factors specify the fate and connectivity of developing neurons. We investigate how a lineage-specific transcription factor, Acj6, controls the precise dendrite targeting of Drosophila olfactory projection neurons (PNs) by regulating the expression of cell-surface proteins. Quantitative cell-surface proteomic profiling of wild-type and acj6 mutant PNs in intact developing brains and a proteome-informed genetic screen identified PN surface proteins that execute Acj6-regulated wiring decisions. These include canonical cell adhesion molecules and proteins previously not associated with wiring, such as Piezo, whose mechanosensitive ion channel activity is dispensable for its function in PN dendrite targeting. Comprehensive genetic analyses revealed that Acj6 employs unique sets of cell-surface proteins in different PN types for dendrite targeting. Combinatorial expression of Acj6 wiring executors rescued acj6 mutant phenotypes with higher efficacy and breadth than expression of individual executors. Thus, Acj6 controls wiring specificity of different neuron types by specifying distinct combinatorial expression of cell- surface executors.

neuroscience↗

bioRxiv2021

Single-cell Stereo-seq enables cell type-specific spatial transcriptome characterization in Arabidopsis leaves

Understanding the complex functions of plant leaves requires spatially resolved gene expression profiling with single-cell resolution. However, although in situ gene expression profiling technologies have been developed, this goal has not yet been achieved. Here, we present the first in situ single-cell transcriptome profiling in plant, scStereo-seq (single-cell SpaTial Enhanced REsolution Omics-sequencing), which enabled the bona fide single-cell spatial transcriptome of Arabidopsis leaves. We successfully characterized subtle but significant transcriptomic differences between upper and lower epidermal cells. Furthermore, with high-resolution location information, we discovered the cell type-specific spatial gene expression gradients from main vein to leaf edge. By reconstructing those spatial gradients, we show for the first time the distinct spatial developmental trajectories of vascular cells and guard cells. Our findings show the importance of incorporating spatial information for answering complex biological questions in plant, and scStereo-seq offers a powerful single cell spatially resolved transcriptomic strategy for plant biology.

plant biology↗

bioRxiv2021

CAR modulates plasma membrane nano-organization and immune signaling downstream of RALF1-FERONIA ligand-receptor signaling system

O_LIThe receptor-like kinase (RLK) FERONIA (FER) senses peptide ligands in the plasma membrane (PM), modulates plant growth and development, and integrates biotic and abiotic stress signaling for downstream adaptive response. However, the molecular interplay of these diverse processes is largely unknown. C_LIO_LIHere, we show that FER, the receptor of Rapid Alkalinization Factor 1 (RALF1), physically interacts with C2 domain ABA-related (CAR) proteins to control the nano-organization of the PM in Arabidopsis. C_LIO_LIDuring this process, the RALF1-FER pathway upregulates CAR protein translation, and then more CAR proteins are recruited to PM. This acts as a rapid feedforward loop to stabilize PM liquid-ordered phase. FER interacts with and phosphorylates CARs, and reducing their lipid-binding ability, which breaks the feedback regulation CAR activity at latter time point. C_LIO_LISimilar to FER mutant, pentuple car14569 mutants inhibit flg22-induced FLS2-BAK1 immune complex formation, which depends on the integrity of nanodomains. O_LITogether, we propose that the FER-CAR module controls the formation of the PM nano-organization during RALF signaling through a self-contained amplifying loop including both positive and negative feedbacks. C_LI C_LI

plant biology↗

bioRxiv2021

HiCImpute: A Bayesian Hierarchical Model for Identifying Structural Zeros and Enhancing Single Cell Hi-C Data.

Single cell Hi-C techniques enable one to study cell to cell variability in chromatin interactions. However, single cell Hi-C (scHi-C) data suffer severely from sparsity, that is, the existence of excess zeros due to insufficient sequencing depth. Complicate things further is the fact that not all zeros are created equal, as some are due to loci truly not interacting because of the underlying biological mechanism (structural zeros), whereas others are indeed due to insufficient sequencing depth (sampling zeros), especially for loci that interact infrequently. Differentiating between structural zeros and sampling zeros is important since correct inference would improve downstream analyses such as clustering and discovery of subtypes. Nevertheless, distinguishing between these two types of zeros has received little attention in the single cell Hi-C literature, where the issue of sparsity has been addressed mainly as a data quality improvement problem. To fill this gap, in this paper, we propose HiCImpute, a Bayesian hierarchy model that goes beyond data quality improvement by also identifying observed zeros that are in fact structural zeros. HiCImpute takes spatial dependencies of scHi-C 2D data structure into account while also borrowing information from similar single cells and bulk data, when such are available. Through an extensive set of analyses of synthetic and real data, we demonstrate the ability of HiCImpute for identifying structural zeros with high sensitivity, and for accurate imputation of dropout values in sampling zeros. Downstream analyses using data improved from HiCImpute yielded much more accurate clustering of cell types compared to using observed data or data improved by several comparison methods. Most significantly, HiCImpute-improved data has led to the identification of subtypes within each of the excitatory neuronal cells of L4 and L5 in the prefrontal cortex.

genetics↗