Biology subjects

Zhao, X.

Publications and source records attributed to Zhao, X..

At least 253 records · Page 14Linked to original sources

bioRxiv2024

3D Bioprinting of Human Neural Tissues with Functional Connectivity

Probing how the human neural networks operate is hindered by the lack of reliable human neural tissues amenable for dynamic functional assessment of neural circuits. We developed a 3D bioprinting platform to assemble tissues with defined human neural cell types in a desired dimension using a commercial bioprinter. The printed neuronal progenitors differentiate to neurons and form functional neural circuits in and between tissue layers with specificity within weeks, evidenced by the cortical-to-striatal projection, spontaneous synaptic currents and synaptic response to neuronal excitation. Printed astrocyte progenitors develop into mature astrocytes with elaborated processes and form functional neuron-astrocyte networks, indicated by calcium flux and glutamate uptake in response to neuronal excitation under physiological and pathological conditions. These designed human neural tissues will likely be useful for understanding the wiring of human neural networks, modeling pathological processes, and serving as platforms for drug testing.

bioengineering↗

bioRxiv2024

Single-cell transcriptional dynamics in a living vertebrate

The ability to quantify transcriptional dynamics in individual cells via live imaging has revolutionized our understanding of gene regulation. However, such measurements are lacking in the context of vertebrate embryos. We addressed this deficit by applying MS2-MCP mRNA labeling to the quantification of transcription in zebrafish, a model vertebrate. We developed a platform of transgenic organisms, light sheet fluorescence microscopy, and optimized image analysis that enables visualization and quantification of MS2 reporters. With these tools, we obtained single-cell, real-time measurements of transcriptional dynamics of the segmentation clock. Our measurements challenge the traditional view of smooth clock oscillations and instead suggest a model of discrete transcriptional bursts that are organized in space and time. Together, these results highlight how measuring single-cell transcriptional activity in the context of vertebrate organisms can reveal unexpected features of gene regulation and how this data can fuel the dialogue between theory and experiment.

developmental biology↗

bioRxiv2024

Control of ciliary transcriptional programs during spermatogenesis by antagonistic transcription factors

Existence of cilia in the last eukaryotic common ancestor (LECA) raises a fundamental question in biology: how the transcriptional regulation of ciliogenesis has evolved? One conceptual answer to this question is by an ancient transcription factor regulating ciliary gene expression in both unicellular and multicellular organisms, but examples of such transcription factors in eukaryotes are lacking. Previously, we showed that an ancient transcription factor XAP5 is required for flagellar assembly in Chlamydomonas. Here, we show that XAP5 and XAP5L are two conserved pairs of antagonistic transcription regulators that control ciliary transcriptional programs during spermatogenesis. Male mice lacking either XAP5 or XAP5L display infertility, as a result of meiotic prophase arrest and sperm flagella malformation, respectively. Mechanistically, XAP5 positively regulates the ciliary gene expression by activating the key regulators including FOXJ1 and RFX families during the early stage of spermatogenesis. In contrast, XAP5L negatively regulates the expression of ciliary genes via repressing these ciliary transcription factors during the spermiogenesis stage. Our results provide new insights into the mechanisms by which temporal and spatial transcription regulators are coordinated to control ciliary transcriptional programs during spermatogenesis.

cell biology↗

bioRxiv2024

Genomic signatures of the evolution of loss of egg yolk in parasitoid wasps

Trait regression and loss have occurred repeatedly in numerous lineages throughout evolutionary history in response to changing environments. In parasitoid wasps, a mega-diverse group of hymenopteran insects, loss or reduction of yolk in the egg has been reported in many species. This phenotypic change likely evolved as a response to the shift from ectoparasitism to endoparasitism. However, the genetic basis of this trait and the impact of its loss on genome evolution remain poorly understood. Here, we performed a comparative genomic analysis of 64 hymenopteran insects. The conserved insect yolk protein gene vitellogenin (Vg) underwent five independent loss events in four families, involving 23 of the analyzed endoparasitoid species. Whole-genome alignment suggested that Vg loss occurred during genome rearrangement events. Analysis of Vg receptor gene (VgR) loss, selection, and structural variation in lineages lacking Vg demonstrated functional biases in the patterns of gene loss. The ectoparasitism to endoparasitism transition did not appear to be the primary driver of Vg loss or the subsequent VgR evolution. A number of parallel and convergent genomic changes were observed between Vg-loss lineages, including gene family evolution and selection of genes related to transport, development, and metabolism. These changes may have facilitated embryonic development without the yolk in these lineages. Together, these findings reveal the genomic basis underlying a unique trait loss in parasitoid wasps. More broadly, this study enhances our understanding of yolk loss evolution outside the class Mammalia, highlighting a potential evolutionary trend arising from the availability of an alternative nutrient source for embryonic development.

evolutionary biology↗

bioRxiv2023

Genomic evidence for hybridization and introgression between blue peafowl and green peafowl and selection for white plumage

The blue peafowl (Pavo cristatus) and the green peafowl (Pavo muticus) have significant public affection due to their stunning appearance, although the green peafowl is currently endangered. Some studies have suggested introgression between these the two species, although evidence is mixed. In this study, we successfully assembled a high-quality chromosome-level reference genome of the blue peafowl, including the autosomes, Z and W sex chromosomes as well as a complete mitochondria DNA sequence. Data from 77 peafowl whole genomes, 76 peafowl mitochondrial genomes and 33 peahen W chromosomes genomes provide the first substantial genetic evidence for recent hybridization between green and blue peafowl. We found three hybrid green peafowls in zoo samples rather than in the wild samples, with blue peafowl genomic content of 16-34%. Maternal genetic analysis showed that two of the hybrid female green peafowls contained complete blue peafowl mitochondrial genomes and W chromosomes. Hybridization of endangered species with its relatives is extremely detrimental to conservation. Some animal protection agencies release captive green peafowls in order to maintain the wild population of green peafowls. Therefore, in order to better protect the endangered green peafowl, we suggest that purebred identification must be carried out before releasing green peafowls from zoos into the wild in order to preventing the hybrid green peafowl from contaminating the wild green peafowl. In addition, we also found that there were historical introgression events of green peafowl to blue peafowl in four Zoo blue peafowl individuals. The introgressed genomic regions contain IGFBP1 and IGFBP2 genes that could affect blue peafowl body size. Finally, we identified that the nonsense mutation (g.4:12583552G>A) in the EDNRB2 gene is the genetic causative mutation for white feather color of blue peafowl (also called white peafowl), which prevents melanocytes from being transported into feathers, such that melanin cannot be deposited.

genomics↗

bioRxiv2023

An ancient transcription activator is required for the multiciliogenesis program

Multiciliogenesis is an evolutionarily conserved process required a unique transcriptional program. The mechanisms governing multiciliogenesis are incompletely understood. Here we show that an ancient transcription activator, Edf1, is essential for the multiciliogenesis program. Mice lacking Edf1 exhibit postnatal hydrocephalus and delayed multiciliated cells (MCCs) differentiation. Functional studies reveal that MCCs in Edf1-/- mice have defects in tissue-level polarity and reduced motility. These defects result in abnormal cerebrospinal fluid (CSF) dynamics in vivo, potentially contributing to the development of hydrocephalus. Intriguingly, we found that the expression of pivotal ciliary transcription factors was decreased in Edf1-/- mice. Collectively, our data suggest that Edf1 modulates the multiciliogenesis by regulating the transcription of important ciliary transcription factors required for ciliary gene expression.

cell biology↗

bioRxiv2023

Structural variation discovery in wheat using PacBio high-fidelity sequencing

BackgroundStructural variations (SVs) pervade plant genomes and contribute substantially to the phenotypic diversity. However, most SVs were ineffectively assayed because of their complex nature and the limitations of early genomic technologies. The recent advance in third-generation sequencing, particularly the PacBio high-fidelity (HiFi) sequencing technology, produces highly accurate long-reads and offers an unprecedented opportunity to characterize SVs structure and functionality. As HiFi sequencing is relatively new to population genomics, it is imperative to evaluate and optimize HiFi sequencing based SV detection before applying the technology at scale. ResultsWe sequenced wheat genomes using HiFi reads, followed by a comprehensive evaluation of mainstream long-read aligners and SV callers in SV detection. The results showed that the accuracy of deletion discovery is markedly influenced by callers, which account for 87.73% of the variance, while both aligners (38.25%) and callers (49.32%) contributed substantially to the accuracy variance for insertions. Among the aligners, Winnowmap2 and NGMLR excelled in detecting deletions and insertions, respectively. For SV callers, SVIM achieved the best performance. We demonstrated that combining the aligners and callers mentioned above is optimal for SV detection. Furthermore, we evaluated the effect of sequencing depth on the accuracy of SV detection, showing that low-coverage HiFi sequencing is sufficiently robust for high-quality SV discovery. ConclusionsThis study thoroughly evaluated SV discovery approaches using HiFi reads, establishing optimal workflows to investigate structural variations in the wheat genome. The notable accuracy of SV discovery from low-coverage HiFi sequencing indicates that skim HiFi sequencing is effective and preferable to characterize SVs at the population level. This study will help advance SV discovery and decipher the biological functions of SVs in wheat and many other plants.

genomics↗

bioRxiv2023

HGNNPIP: A Hybrid Graph Neural Network framework for Protein-protein Interaction Prediction

A deep understanding of Protein-protein interactions (PPIs) can provide comprehensive insights into many biological functions, thereby facilitating drug target identification and novel therapeutic design. Recent developments in artificial intelligence (AI)-driven computational methods have enabled the discovery of previously uncharacterized PPIs from large-scale interactome datasets. Almost all existing machine learning methods rely on Subcellular Localization (SL) to construct balanced datasets based on positive interactions to achieve predictions. Despite high fitting accuracy, the generalization ability of these models is questionable. To solve this problem, we analyzed existing methods and found that the high false positives in these methods are due to the bias in data distribution caused by SL. Therefore, we proposed a new strategy for negative instance sampling in PPI prediction and developed a Hybrid Graph Neural Network framework for Protein-protein Interaction Prediction (HGNNPIP). The experimental results showed that HGNNPIP works well on six benchmark datasets. Comparison analysis demonstrated that our model outperformed the other four existing methods. We also used HGNNPIP to explore the molecular contacts involved in the rice-pathogen interaction system. In vivo experiments confirmed multiple regulations related to disease resistance in rice. In summary, this study provides new insights into establishing a computational framework for PPI prediction with high reliability.

bioinformatics↗

bioRxiv2023

Nuclear cytoophidia assembly represses transcriptional activity to control skeletal development and homeostasis

Compartmentation via filamentation is an evolutionarily conserved subcellular structure that fine-tunes the inherent activity of proteins. Cytoophidia represent a typical class of filamentous structures controlling enzymatic activities. Despite eukaryotic cells containing both cytoplasmic cytoophidia and nuclear cytoophidia, the physiological significance of nuclear cytoophidia is largely unknown. Here we show that nuclear filamentation inhibits the transcriptional activity of Impdh2 required for limb formation and bone resorption. Impdh2 deletion in mouse limb mesenchymal progenitors causes severe skeletal dysplasia by impairing endochondral ossification and chondrocyte differentiation. Additionally, Impdh2 deficiency in myeloid lineages leads to an increased bone mass via impeding osteoclast differentiation. Furthermore, Impdh2 regulates osteoclastic mitochondrial biogenesis and function. We propose that the nuclear compartmentalization of Impdh2 regulates the transcriptional activity during skeletal development and homeostasis.

cell biology↗

bioRxiv2023

Reconstructing oral cavity tumor evolution through brush biopsy

Oral premalignant lesions (OPLs) with genomic alterations have a heightened risk of evolving into oral squamous cell carcinoma (OSCC). Currently, genomic data are obtained through invasive tissue biopsy. Brush biopsy has been utilized for diagnosing dysplasia but its effectiveness in reflecting the complete genomic landscape of OPLs remains uncertain. This study investigates the potential of brush biopsy samples in accurately reconstructing the genomic profile of OPLs. We analyzed single nucleotide variants (SNVs), copy number aberrations (CNAs), and subclonal structures in paired tissue and brush biopsy samples from a patient with both OPL and OSCC lesions. The results showed that brush biopsy can effectively reflect about 90% of SNVs and similar CNA profiles as those found in tissue biopsies. It was specific, as normal oral epithelium didnt share these genomic alterations. Interestingly, brush biopsy revealed shared SNVs and CNAs between the distinct OPL and OSCC lesions, indicating a common ancestral origin. Subclonal reconstruction confirmed this shared ancestry, followed by divergent evolution of the lesions. These findings highlight the potential of brush biopsies in accurately representing the genomic profile of OPL and OSCC, proving useful in understanding tumor evolution.

genomics↗

bioRxiv2023

Short-term social isolation acts on hypothalamic neurons to promote social behavior in a sex- and context-dependent manner

Social animals, including both humans and mice, are highly motivated to engage in social interactions. Short-term social isolation promotes social behavior, but the neural circuits through which it does so remain incompletely understood. Here, we sought to identify neurons that promote social behavior in single-housed female mice, which exhibit increased rates of social investigation, social ultrasonic vocalizations (USVs), and mounting during same-sex interactions that follow a period of short-term (3-day) isolation. We first used immunostaining for the immediate early gene Fos to identify a population of neurons in the preoptic hypothalamus (POA) that increase their activity in single-housed females following same-sex interactions (POAsocial neurons) but not in single-housed females that did not engage in social interactions. TRAP2-mediated chemogenetic silencing of POAsocial neurons in single-housed females significantly attenuates the effects of short-term isolation on social investigation, USV production, and mounting. In contrast, caspase-mediated ablation of POAsocial neurons in single-housed females robustly attenuates mounting but does not decrease social investigation or USV production. Optogenetic activation of POAsocial neurons in group-housed females promotes social investigation and USV production but does not recapitulate the effects of short-term isolation on mounting. To understand whether a similar population of POAsocial neurons promotes social behavior in single-housed males, we performed Fos immunostaining in single-housed males following either same-sex or opposite-sex social interactions. These experiments revealed a population of POA neurons that increase Fos expression in single-housed males following opposite-sex, but not same-sex, interactions. Chemogenetic silencing of POAsocial neurons in single-housed males during interactions with females reduces mounting but does not affect social investigation or USV production. These experiments identify a population of hypothalamic neurons that promote social behavior following short-term isolation in a sex- and social context-dependent manner.

neuroscience↗

bioRxiv2023

An NMR-guided approach for identifying co-factors in boosting the Pfu DNA polymerase

With rapid developments of emerging technologies like synthetic biology, the demand for DNA polymerases with superior activities including higher thermostability and processivity has increased significantly. Thus, rational optimization of the performance of DNA polymerase is of great interest. Nuclear magnetic resonance spectroscopy (NMR) is a powerful technique used for studying protein structure and dynamics. It provides the atomic resolution information of enzymes under its functional solution environment to reveal the active sites (hot spots) of the enzyme, which could be further used for optimizing the performance of enzymes. In our previous work, we identified hot spot residues of Pfu DNA polymerase (Pfu pol). We aim to employ these binding hot spots to screen for co-factors of Pfu pol, particularly targeting those molecules exhibiting weak intermolecular interactions. To validate this concept, we first demonstrated the feasibility of utilizing hot spot residues as screening probes for auxiliary factors by employing the well-characterized Tween-20 as a model system. Employing these hot spots as probes, two new co-factors, the heat shock protein TkHSP20 from Thermococcus Kodakaraensis and the chemical chaperone L-arginine, are identified to interact with Pfu pol to boost its performance in amplifying long DNA fragments by enhancing the thermal stability and the processivity of the Pfu pol. This NMR-based approach requires no prior assignment information of target enzymes, guiding the rational exploration of novel co-factors for Pfu pol. Moreover, our approach is not dependent on structural data or bioinformatics. Therefore, it has significant potential for application in various enzymes to expedite the progress in enzyme engineering.

molecular biology↗

bioRxiv2023

Single phage proteins sequester TIR- and cGAS-generated signaling molecules

Prokaryotic anti-phage immune systems use TIR (toll/interleukin-1 receptor) and cGAS (cyclic GMP-AMP synthase) enzymes to produce 1-3/1-2 glycocyclic ADPR (gcADPR) and cyclid di-/tri-nucleotides (CDNs and CTNs) signaling molecules that limit phage replication, respectively 1-3. However, how phages neutralize these common systems is largely unknown. Here, we show that Thoeris anti-defense proteins Tad1 4 and Tad2 5 both have anti-CBASS activity by simultaneously sequestering CBASS cyclic oligonucleotides. Strikingly, apart from binding Thoeris signals 1-3 and 1-2 gcADPR, Tad1 also binds numerous CBASS CDNs/CTNs with high affinity, inhibiting CBASS systems using these molecules in vivo and in vitro. The hexameric Tad1 has six binding sites for CDNs or gcADPR, which are independent from two high affinity binding sites for CTNs. Tad2 also sequesters various CDNs in addition to gcADPR molecules, inhibiting CBASS systems using these CDNs. However, the binding pockets for CDNs and gcADPR are different in Tad2, whereby a tetramer can bind two CDNs and two gcADPR molecules simultaneously. Taken together, Tad1 and Tad2 are both two-pronged inhibitors that, alongside anti-CBASS protein 2, establish a paradigm of phage proteins that flexibly sequester a remarkable breadth of cyclic nucleotides involved in TIR- and cGAS-based anti-phage immunity.

microbiology↗

bioRxiv2023

Continuity of Mitochondrial Budding: Insights from BS-C-1 Cells by in-situ Cryo-Electron Tomography

Mitochondrial division is a fundamental biological process that is crucial to cellular functionality and vitality. The prevailing hypothesis of Drp1 regulation with the involvement of ER and cytoskeleton does not account for all the observations. Following up our previous study in HeLa cells which led to the new hypothesis of mitochondrial division by budding, we employed in-situ Cryo-Electron Tomography (Cryo-ET) to visualize mitochondrial budding in intact healthy monkey kidney cells (BS-C-1 cells). Our findings reaffirm single and multiple mitochondrial budding, supporting the new hypothesis. Notably, the budding regions vary significantly in diameter and length, which may represent different stages of budding. More interestingly, no rings, or ring-like structures, or ER wrapping is presented in the budding regions suggesting mitochondrial budding is independent from Drp1 and ER. Meanwhile, we uncovered direct interactions between mitochondria and large vesicles, distinct from small mitochondrial-derived vesicles and extracellular mitovesicles. We propose these interacting vesicles may have mitochondrial origins.

biophysics↗

bioRxiv2023

One infusion of engineered long-lasting and multifunctional T cells cures asthma in mice

The majority of common chronic human diseases are incurable, which affect a large portion of population and require life-long treatments that impose huge disease, economic and social burden. For example, asthma is the most common respiratory disease affecting over 300 million people and accounts for > 250,000 death annually, for which there is no cure. Unlike traditional therapyies, engineered T cells, such as chimeric antigen receptor CAR T (CAR-T) cells, function as living drugs and can cure some hematological malignancies, but whether engineered T cells can cure common diseases beyond cancer remains elusive. Here, we develop a curative therapy for asthma based on enginerred T cell. With IL-5 as the targeting domain and depletion of BCOR and ZC3H12A, we produce long-lasting CAR-T cells eradicating IL-5R+ eosinophils, termed Immortal-like and Functional IL-5 CAR-T cells (5TIF) cells. We further enginerred 5TIF cells to secrete an IL-4 mutein that blocks the singaling of both IL-4 and IL-13, two driver inflammatory cytokines in asthma, named as 5TIF4 cells. In multiple models of asthma, one infusion of 5TIF4 cells in fully immunocompetent mice, in the absence of any conditioning regimen, confers long-term depletion of pathological eosinophils and blockade of IL-4/IL-13 actions, resulting in sustained repression of type 2 inflammation and asthmatic symptoms. Furthurmore, 5TIF4 cells can also be induced in human T cells in NSG mice. Our data demonstrate that asthma, a common noncancerous disease, can be cured by a single infusion of engineered long-lasting and multifunctional T cells, which paves the way for curing common chronic diseases by engineered long-lived T cells.

immunology↗

bioRxiv2023

Induction of immortal-like and functional CAR-T cells by defined factors

Long-term antitumor efficacy of chimeric antigen receptor (CAR)-T cells depends on their functional persistence in vivo. T cells with stem-like property show better persistence, but factors conferring T cells bona fide stemness remain to be determined. Here, we demonstrate the induction of CAR-T cells into an immortal-like and functional state, termed TIF. The induction of CAR-TIF cells depends on repression of two factors: BCOR and ZC3H12A, and requires antigen or CAR tonic signaling. The reprogrammed CAR-TIF cells possess almost infinite stemness resembling induced pluripotent stem cells but retain functionality of mature T cells, which exhibit superior antitumor effect. CAR-TIF cells enter a metabolically dormant state after elimination of target cells, which persist in vivo with a saturable niche and mediate memory protection. CAR-TIF cells represent a novel state of T cells with unprecedented stemness conferring long-term functional persistence of CAR-T cells in vivo, which have broad potentials in T cell therapies.

immunology↗

bioRxiv2023

Pan-sphingolipid profiling reveals differential functions of sphingolipid biosynthesis isozymes of C. elegans

Multiple isozymes are encoded in the C. elegans genome for the various sphingolipid biosynthesis reactions, but the contributions of individual isozymes are characterized only in part. We developed a simple but effective reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method that enables simultaneous identification and quantification of ceramides (Cer), glucosylceramides (GlcCer), and sphingomyelins (SM), three important classes of sphingolipids from the same MS run. Validating this pan-sphingolipid profiling method, we show that nearly all 47 quantifiable sphingolipid species found in young adult worms were reduced upon RNA interference (RNAi) of sptl-1 or elo-5, which are required for synthesis of the id17:1 sphingoid base. We also confirm that HYL-1 and HYL-2, but not LAGR-1, constitute the major ceramide synthase activity with different preference for fatty acid substrates, and that CGT-3 plays a greater role than CGT-1 does in producing glucosylceramides. Intriguingly, lagr-1 RNAi lowers the abundance of all sphingomyelin species and that of several glucosylceramide species, which suggests that LAGR-1 may have functions beyond what is predicted. Additionally, RNAi of sms-1, -2, and -3 all lower the abundance of sphingomyelins with an odd number of carbon atoms (mostly C21 and C23, with or without hydroxylation) in the N-acyl chain, and only sms-1 RNAi does not elevate sphingomyelins containing even-numbered N-acyl chains. This suggests that sphingolipids containing even-numbered N-acyl chains could be regulated separately, sometimes in opposite directions, with those containing odd-numbered N-acyls, presumably monomethyl branched chain fatty acyls. We also find that ceramide levels are kept in balance with those of glucosylceramides and sphingomyelins.

genetics↗

bioRxiv2023

Multi-region calcium imaging in freely behaving mice with ultra-compact head-mounted fluorescence microscopes

To investigate the circuit-level neural mechanisms of behavior, simultaneous imaging of neuronal activity in multiple cortical and subcortical regions is highly desired. Miniature head-mounted microscopes offer the capability of calcium imaging in freely behaving animals. However, implanting multiple microscopes on a mouse brain remains challenging due to space constraints and the cumbersome weight of equipment. Here, we present TINIscope, a Tightly Integrated Neuronal Imaging microscope optimized for electronic and opto-mechanical design. With its compact and lightweight design of 0.43 g, TINIscope enables unprecedented simultaneous imaging of behavior-relevant activity in up to four brain regions in mice. Proof-of-concept experiments with TINIscope recorded over 1000 neurons in four hippocampal subregions and revealed concurrent activity patterns spanning across these regions. Moreover, we explored potential multi-modal experimental designs by integrating additional modules for optogenetics, electrical stimulation or local field potential recordings. Overall, TINIscope represents a timely and indispensable tool for studying the brain-wide interregional coordination that underlies unrestrained behaviors.

neuroscience↗