Biology subjects

Chen, L.

Publications and source records attributed to Chen, L..

At least 631 records · Page 35Linked to original sources

bioRxiv2022

Engineering precise adenine base editor with infinitesimal rates of bystander mutations and off-target editing

Adenine base editors (ABEs) catalyze A-to-G transitions showing broad applications, but their bystander mutations and off-target editing effects raise the concerns of safety issues. Through structure-guided engineering, we found ABE8e with an N108Q mutation reduced both adenine and cytosine bystander editing, and introduction of an additional L145T mutation (ABE9), further refined the editing window to 1-2nt with eliminated cytosine editing. Importantly, ABE9 induced very minimal RNA and undetectable Cas9-independent DNA off-target effects, which mainly installed desired single A-to-G conversion in mouse and rat embryos to efficiently generate disease models. Moreover, ABE9 accurately edited A5 position of the protospacer sequence in pathogenic homopolymeric adenosine sites (up to 342.5-fold precision than ABE8e) and was further confirmed through a library of guide RNA-target sequence pairs. Due to the minimized editing window, ABE9 could further broaden the targeting scope for precise correction of pathogenic SNVs when fused to Cas9 variants with expanded PAM compatibility.

bioengineering↗

bioRxiv2022

Macrophage migrates on alternate non-adhesive surfaces

Macrophages migrate across tissues upon immune demand, but their motility on heterogeneous substrates remains unclear. Protein-repelling reagents, e.g., poly(ethylene) glycol (PEG), are routinely employed to resist cell adhering and migrating. Contrary to this perception, we discovered a unique locomotion of macrophages in vitro that they overcome non-adhesive PEG gaps to reach adhesive regions in a mesenchymal mode. Adhesion to adhesive regions was a prerequisite for macrophages to perform further locomotion on the PEG regions, or else they kept a suspended round shape. Podosomes were found highly enriched on the PEG region, which supported macrophage migration. Myosin IIA played a negative role in macrophage motility. Moreover, a developed cellular Potts model reproduced the experimental observations. These findings uncovered a new migratory behavior on non-adhesive surfaces in macrophages. One-Sentence SummaryMacrophages can migrate across non-adhesive surfaces that are absolute boundaries for other cell types.

cell biology↗

bioRxiv2022

Activation of Cerebellar Lobule VI PCTH+boxhMed Pathway Selectively blocks METH-induced Conditioned Place Preference in mice

The cerebellum is implicated in drug addiction. However, the cerebellar neuronal circuitry underlying addiction, especially classic pathway of Purkinje cells (PCs) to deep cerebellar nuclei (DCN), are largely unknown. Here, tracing experiments showed robust projections from the cerebellar lobule VI tyrosine hydroxylase (TH)-positive PCs (PCTH+) to CaMKII-positive glutamatergic neurons in medial cerebellar nucleus (MedCaMKII), forming PCTH+-MedCaMKII pathway. Then, mice were subjected to methylamphetamine (METH)-induced conditioned place preference (CPP), showing that METH exposure excited MedCaMKII and inhibited PCTH+. Silencing MedCaMKII by tettox suppressed the formation and expression of METH-induced CPP, but produced serious motor coordination deficits. Chemogenetic activation of lobule VI PCTH+-Med pathway during METH CPP training blocked the formation and expression of METH-induced CPP without affecting motor coordination, locomotor activity and sucrose reinforcements in mice. Our findings revealed a novel cerebellar lobule VI PCTH+-MedCaMKII pathway and pointed out is critical role in encoding METH-preferred behaviors.

neuroscience↗

bioRxiv2022

Simultaneous Profiling of Host Expression and Microbial Abundance by Spatial Meta-Transcriptome Sequencing

We developed an analysis pipeline that can extract microbial sequences from Spatial Transcriptomic (ST) data and assign taxonomic labels, generating a spatial microbial abundance matrix in addition to the default host expression matrix, enabling simultaneous analysis of host expression and microbial distribution. We called the pipeline Spatial Meta-transcriptome (SMT) and applied it on both human and murine intestinal sections and validated the spatial microbial abundance information with alternative assays. Biological insights were gained from this novel data that demonstrated host-microbe interaction at various spatial scales. Finally, we tested experimental modification that can increase microbial capture while preserving host spatial expression quality, and by use of positive controls, quantitatively demonstrated the capture efficiency and recall of our methods. This proof of concept work demonstrates the feasibility of Spatial Meta-transcriptomic analysis, and paves the way for further experimental optimization and application.

genomics↗

bioRxiv2022

Maf Family Transcription Factors are Required for Nutrient Uptake in the Neonatal Gut

There are fundamental differences in the way that neonatal and adult intestines absorb nutrients. In adults, macromolecules are efficiently broken down into simpler molecular components in the lumen of the small intestine, then absorbed. In contrast, neonates are thought to rely more on bulk intake of nutrients and subsequent degradation in the lysosome. Here, we identify the Maf family transcription factors, MafB and cMaf, as markers of terminally-differentiated intestinal enterocytes throughout life. The expression of these factors is regulated by HNF4/{gamma}, master regulators of the enterocyte cell fate. Loss of Maf factors results in a neonatal-specific failure to thrive and loss of bulk uptake of nutrients. RNA-Seq and CUT&RUN analyses defined an endo-lysosomal program as being downstream of these transcription factors. We demonstrate major transcriptional changes in metabolic pathways, including fatty acid oxidation and increases in peroxisome number in response to loss of Mafs. Finally, we show that deletion of Blimp1, which represses adult enterocyte genes in the neonatal gut, shows highly overlapping changes in gene expression and similar defects in nutrient uptake. This work defines transcriptional regulators that are necessary for bulk uptake in neonatal enterocytes.

physiology↗

bioRxiv2022

Automated container-less cell processing method for single-cell proteomics

Single-cell genomics and transcriptomics studies enabled us to characterize cell heterogeneity in various tissues, which helped us to better understand the biological system and disease progression. Single-cell proteomics, which directly measures the protein expression level, has the potential to further enhance our knowledge by providing not only a more direct measurement but also crucial information cannot be captured by genomics or transcriptomics study, such as protein activation states and post-translation modification events. One of the main challenges of single-cell proteomics is the large sample loss during sample preparation, which is largely unavoidable using standard proteomics protocols. Protein and peptide loss to the container surface is a well-known phenomenon but often overlooked in larger-scale (>1 {micro}g) proteomics experiments. When it comes to single-cell proteomics with only picograms of protein samples, this loss becomes non-negligible and often dictates the outcomes of the experiment. More importantly, sample processing through multiple pipette tips and containers often introduces random errors, which undermine the ability to detect true heterogenous cellular events. To solve these problems and further improve the throughput and reproducibility of the single-cell proteomics experiments, we developed an automated container-less cell processing platform, utilizing acoustic levitation to process cell samples in the air. Our platform automatically performs cell lysis, protein reduction, alkylation, digestion, and peptide labeling in the air, without any sample transfer step or container. The digested and labeled peptides are then directly injected into the capillary LC-MS/MS system for analysis, eliminating manual steps and conserving most of the sample materials for proteomics analysis. Our initial test shows at least 30% improvement in peptide signals over conventional methods. This process can be performed in parallel to further improve sample processing throughput.

biochemistry↗

bioRxiv2022

Regulation of sleep quantity and intensity by long and short isoforms of SLEEPY kinase

In Sleepy (Sik3Slp) or Sik3S551A mice, deletion or mutation of inhibitory phosphorylation site serine551 (S551) from salt-inducible kinase 3 (SIK3) markedly increases daily non-rapid eye movement sleep (NREMS) amount, accompanied with constitutively elevated NREMS delta power density-a measure of sleep intensity or quality. Multiple SLP/SIK3 isoforms are expressed in mouse brain neurons, however, their respective roles in sleep regulation remain to be elucidated. Here, we identified a new and most abundant short isoform of SLP/SIK3 and examined sleep phenotypes resulted from isoform-specific expression of SLP-short (S) and long (L) isoforms. Adeno-associated virus-mediated adult brain chimeric (ABC)-expression of SLP-S in neurons, but not in astrocytes, significantly and constitutively elevates NREMS delta power, whereas slightly increases NREMS amount. The ability of SLP-S to regulate sleep quantity/intensity is abrogated by kinase-inactivating mutations, suggesting that the sleep-promoting activity of SLP-S is dependent on its kinase activity. In Sik3S551A-L knock-in mice, isoform-specific expression of SIK3S551A-L (or SLP-L) significantly increases NREMS amount with a modest effect on NREMS delta power. ABC-expression of SLP-S complements the sleep phenotypes of heterozygous Sik3S551A-L mice by further increasing NREMS amount and NREMS delta power to levels of Sik3Slp or Sik3S551A mice. Taken together, these results indicate that both SLP-L and SLP-S isoforms contribute critically to the increases of sleep quantity and intensity in Sik3Slp or Sik3S551A mice. Statement of SignificancePrevious studies have identified SIK3 as a key sleep regulatory kinase, of which gain-of-function mutations markedly increase sleep quantity and intensity in Sleepy mice. Multiple SLEEPY isoforms are expressed in mouse brain neurons, but their respective contributions to the hypersomnia phenotypes of Sleepy mice remain unclear. Here, we identified a new short isoform of SLP/SIK3 and examined sleep phenotypes resulted from specific expression of SLEEPY-short or long isoform. Our results indicate that both long and short isoforms of SLEEPY kinase contribute critically to increases of sleep quantity and intensity in Sleepy mice. Future studies are needed to identify downstream substrates of SLEEPY kinase that mediate regulation of sleep quantity/intensity, which should facilitate development of new treatments for human sleep disorders.

genetics↗

bioRxiv2022

Integrated single-cell sequencing analysis reveals peripheral immune landscape across the human lifespan

Systematic understanding of immune dynamics across the entire human lifespan at single-cell resolution is currently lacking. Here, we performed single-cell RNA sequencing (scRNA-seq) and single-cell T cell receptor (TCR)/B cell receptor (BCR) sequencing (scTCR/BCR-seq) on over 380,000 peripheral blood mononuclear cells collected from 45 healthy participants aged 0 to over 90 years. We revealed that the functions of T cell subsets were most susceptible to senescence among all PBMCs, featured by increased NF-{kappa}B signaling and IFN-{gamma} responses pathways, while reduced telomere maintenance and energy metabolism. We subsequently explored the rewiring of cell-cell interactions among different immune cells across the lifespan and revealed the major alteration of immune checkpoints in T cells within the cellular interaction networks. By combined analysis of scRNA-seq and scTCR-seq, we revealed that 1) GNLY+ CD8 Effector T cells exhibited a high clonal expansion with distinct functional signatures in children and the elderly; 2) Naive CD4+ T and naive CD8+ T cells displayed different aging patterns in both transcriptomes and immune repertoires; and 3) CD8+ MAIT cells showed a higher cell abundance and clonal diversity in adolescents. Furthermore, we identified a unique cytotoxic B cell subset enriched in children by scRNA-seq and scBCR-seq analysis and experimental validations. In summary, our work provided valuable insights and rich resources for understanding the development and aging of the human immune system across the lifespan.

immunology↗

bioRxiv2022

Direct attenuation of Arabidopsis ERECTA signaling by a pair of U-box E3 ligases

Plants sense a myriad of signals through cell-surface receptors to coordinate their development and environmental response. The Arabidopsis ERECTA receptor kinase regulates diverse developmental processes via perceiving multiple EPIDERMAL PATTERNING FACTOR (EPF)/EPF-LIKE peptide ligands. How the activated ERECTA protein is turned over is unknown. Here we identify two closely-related Plant U-box ubiquitin E3 ligases, PUB30 and PUB31, as key attenuators of ERECTA signaling for two distinct developmental processes: inflorescence/pedicel growth and stomatal development. Loss-of-function pub30 pub31 mutant plants exhibit extreme inflorescence/pedicel elongation and reduced stomatal numbers owing to excessive ERECTA protein accumulation. Ligand-activation of ERECTA leads to phosphorylation of PUB30/31 by the co-receptor BRI1 ASSOCIATED KINASE1 (BAK1), which then promotes PUB30/31 to associate with and ubiquitinate ERECTA for eventual degradation. We further show that the PUB30 and PUB31 phosphorylation by BAK1 and their ubiquitination activities are critical for the proper in vivo developmental outputs. Our work highlights PUB30 and PUB31 as integral components of the ERECTA regulatory circuit that ensures optimal signaling strengths upon ligand activation, thereby enabling proper growth and development.

plant biology↗

bioRxiv2022

Talaromyces marneffei suppresses human macrophages inflammatory by producing the truncated protein NCOR2-013 via TUT1-regulated alternative splicing

Talaromyces marneffei (T. marneffei) immune-escaping is an important factor for high mortality of talaromycosis. It is currently known that T. marneffei performs these functions through a variety of strategies, however, the role of alternative splicing (AS) in this process is poorly understood. Here we depicted the AS landscape in the macrophage upon T. marneffei infection via high-throughput RNA sequencing. Moreover, we identified a truncated protein of NCOR2/SMRT, namedly NCOR2-013, was significantly upregulated upon T. marneffei infection. Mechanistic analysis indicates that NCOR2-013 forms a co-repression complex with TBL1XR1/TBLR1 and HDAC3, thereby inhibiting JunB-mediated transcriptional activation of pro-inflammatory cytokines via the inhibition of histone acetylation. Also, we identified TUT1 as the AS regulator that involved in facilitating T. marneffei immune evasion via regulation of NCOR2-013 production. Collectively, the findings indicate that T. marneffei escapes macrophages killing through the TUT1-mediated the alternative splicing of NCOR2-013, which providing a new insight into the molecular mechanisms of T. marneffei immune evasion, and a potential targets for talaromycosis therapy.

microbiology↗

bioRxiv2022

CeDAR: incorporating cell type hierarchy improves cell type specific differential analyses in bulk omics data

The bulk high-throughput omics data contain signals from a mixture of cell types. Recent developments of deconvolution methods facilitate cell-type-specific inferences from bulk data. Our real data exploration suggests that the differential expression or methylation status are often correlated among cell types. Based on this observation, we develop a novel statistical method named CeDAR to incorporate the cell type hierarchy in cell-type-specific differential analyses in bulk data. Extensive simulation and real data analyses demonstrate that this approach significantly improves the accuracy and power in detecting cell-type-specific differential signals compared with existing methods, especially in low abundance cell types.

genomics↗

bioRxiv2022

Specific Deletion of Axin1 Leads to Activation of β-Catenin/BMP Signaling Resulting in Fibular Hemimelia Phenotype in Mice

Axin1 and Axin2 are key regulators of canonical Wnt signaling pathway. The roles of Axin1 and Axin2 in skeletal development and in disease development have not been fully defined. Here, we reported that Axin1 and Axin2 are essential for lower limb development. Specific deletion of Axin1 in limb mesenchymal cells leads to fibular hemimelia (FH)-like phenotype, associated with tarsal coalition. Further studies demonstrated that FH disease is associated with additional defects resembling to the proximal femoral focal deficiency (PFFD) in Axin1/Axin2 double knockout (KO) mice. We then provided in vivo evidence showing that Axin1 controls limb development through both canonical {beta}-catenin and BMP signaling pathways. We demonstrated that inhibition of {beta}-catenin or BMP signaling could significantly reverse the FH phenotype in mice. Together, our findings revealed that integration of Wnt and BMP signaling by Axin1 is required for lower limb development. Defects in Axin1 and Axin2 signaling could lead to the development of FH disease.

developmental biology↗

bioRxiv2022

Small cytosolic dsDNAs repress cGAS activation and induce autophagy

Cyclic GMP-AMP (cGAMP) synthase (cGAS), a major cytosolic DNA sensor, activates innate immune responses by producing cGAMP, which activates stimulator of interferon genes (STING)1. Cytosolic DNA induces autophagy in a cGAS-dependent manner to avoid persistent immune stimulation. Although dsDNAs < 20 bp can bind to cGAS, robust cGAS activation requires dsDNAs > 45 bp 2-4. However, whether cytosolic dsDNAs < 45 bp exist in mammalian cells remains unclear. Here, we identified a class of small cytosolic DNAs (scDNAs) of [~]20-40 bp in human and mouse cell lines. scDNAs competed with herring testis DNA (HT-DNA, [~]200-1500 bp) for binding to cGAS, and repressing HT-DNA-induced cGAS activation and the associated interferon {beta} (IFN{beta}) production. Moreover, scDNAs promoted cGAS and Beclin-1 interaction, triggering the release of Rubicon, a negative regulator of phosphatidylinositol 3-kinase class III (PI3KC3)5,6, from the Beclin-1-PI3KC3 complex, activating PI3KC3 and inducing autophagy. DNA damage decreased and autophagy inducers increased scDNA levels. scDNA transfection or autophagy induction attenuated DNA damage-induced cGAS-STING activation and IFN{beta} expression. Thus, scDNAs acted as molecular brakes of cGAS activation, preventing excessive inflammatory cytokine production following DNA damage. Our findings lay foundations for understanding the physiological and pathological functions of scDNAs.

molecular biology↗

bioRxiv2022

The domain-separation low-dimensional language network dynamics in the resting-state support the flexible functional segregation and integration during language and speech processing

Modern linguistic theories and network science propose that the language and speech processing is organized into hierarchical, segregated large-scale subnetworks, with a core of dorsal (phonological) stream and ventral (semantic) stream. The two streams are asymmetrically recruited in receptive and expressive language or speech tasks, which showed flexible functional segregation and integration. We hypothesized that the functional segregation of the two streams was supported by the underlying network segregation. A dynamic conditional correlation approach was employed to construct frame-wise time-varying language networks and investigate the temporal reoccurring patterns. We found that the time-varying language networks in the resting-state robustly clustered into four low-dimensional states, which dynamically reconfigured following a domain-separation manner. Spatially, the hub distributions of the first three states highly resembled the neurobiology of primary auditory processing and lexical-phonological processing, motor and speech production processing, and semantic processing, respectively. The fourth state was characterized by the weakest functional connectivity and subserved as a baseline state. Temporally, the first three states appeared exclusively in limited time bins ([~]15%), and most of the time (> 55%), the language network kept inactive in state 4. Machine learning-based dFC-linguistics prediction analyses showed that dFCs of the four states significantly predicted individual linguistic performance. These findings suggest a domain-separation manner of language network dynamics in the resting-state, which forms a dynamic "meta-networking" (network of networks) framework. HighlightsO_LIThe time-varying language network in the resting-state is robustly clustered into four low-dimensional states. C_LIO_LISpatially, the first three dFC states are cognitively meaningful, which highly resemble the neurobiology of primary auditory processing and lexical-phonological representation, speech production processing, and semantic processing, respectively. C_LIO_LITemporally, the first three states appeared exclusively in limited time bins ([~]15%), and most of the time (> 55%), the language network kept inactive in state 4. C_LIO_LIA dynamic "meta-networking" framework of language network in the resting-state is proposed. C_LI

neuroscience↗

bioRxiv2022

A pangenome analysis pipeline (PSVCP) provides insights into rice functional gene identification

BackgroundA pangenome aims to capture the complete genetic diversity within a species and reduce bias in genetic analysis inherent in using a single reference genome. However, the current linear format of most plant pangenomes limits the presentation of position information for novel sequences. Graph pangenomes have been developed to overcome this limitation. However, there is a lack of bioinformatics analysis tools for graph format genomes. ResultsTo overcome this problem, we have developed a novel pangenome construction strategy and a downstream pangenome analysis pipeline that captures position information while maintaining a linearized layout. We applied this strategy to construct a high-quality rice pangenome using 12 representative rice genomes and analyze an international rice panel with 413 diverse accessions using the pangenome reference. Our results provide insights into rice population structure and genomic diversity. Applying the pangenome for PAV-based GWAS analysis can identify causal structural variations for rice grain weight and plant height, while SNP-based GWAS can only identify approximate genomic locations. Additionally, a new locus (qPH8-1) was found to be associated with plant height on chromosome 8 that could not be detected using the SNP-based GWAS. ConclusionsOur results demonstrate that the pangenome constructed by our pipeline combined with PAV-based GWAS can provide additional power for genomic and genetic analysis. The pangenome constructed in this study and associated genome sequence data provide valuable genomic resources for future rice crop improvement.

genetics↗

bioRxiv2022

Two Rac1 pools integrate the direction and coordination of collective cell migration

Integration of collective cell direction and coordination is believed to ensure collective guidance for efficient movement. Previous studies demonstrated that chemokine receptors PVR and EGFR govern a gradient of Rac1 activity essential for collective guidance of Drosophila border cells, whose mechanistic insight is unknown. By monitoring and manipulating subcellular Rac1 activity, here we reveal two switchable Rac1 pools at border cell protrusions and supracellular cables, two important structures responsible for direction and coordination. Rac1 and Rho1 form a positive feedback loop that guides mechanical coupling at cables to achieve migration coordination. Rac1 cooperates with Cdc42 to control protrusion growth for migration direction, as well as to regulate the protrusion-cable exchange, linking direction and coordination. PVR and EGFR guide correct Rac1 activity distribution at protrusions and cables. Therefore, our studies emphasize the existence of a balance between two Rac1 pools, rather than a Rac1 activity gradient, as an integrator for the direction and coordination of collective cell migration.

cell biology↗

bioRxiv2022

β-catenin obstructs γδ T cell immunosurveillance in colon cancer through loss of BTNL expression

WNT/{beta}-catenin signaling endows cancer cells with proliferative capacity and immune-evasive functions that impair anti-cancer immunosurveillance by conventional, cytoxtoic T cells. However, the impact of dysregulated WNT signalling on unconventional, tissue-resident T cells, specifically in colon cancer is unknown. Here, we show that cancer cells in Apc-mutant mouse models escape immunosurveillance from gut-resident intraepithelial lymphocytes (IELs) expressing {gamma}{delta} T cell receptors ({gamma}{delta}TCRs). Analysis of late-stage tumors from mice and humans revealed that {gamma}{delta}IELs are largely absent from the tumor microenvironment, and that butyrophilin-like (BTNL) molecules, which can critically regulate {gamma}{delta}IEL through direct {gamma}{delta}TCR-interactions, are also downregulated. We could attribute this to {beta}-catenin stabilization, which rapidly decreased expression of the transcription factors, HNF4A and HNF4G, that we found to bind promoter regions of Btnl genes, thereby driving their expression in normal gut epithelial cells. Indeed, inhibition of {beta}-catenin signaling restored Btnl1 gene expression and {gamma}{delta} T cell infiltration into tumors. These observations highlight an immune-evasion mechanism specific to WNT-driven colon cancer cells that disrupts {gamma}{delta}IEL immunosurveillance and furthers cancer progression.

cancer biology↗

bioRxiv2022

Pathogen-Host Adhesion between SARS-CoV-2 S Proteins from Different Variants and Human ACE2 Probed at Single-Molecule and Single-Cell Levels

Pathogen-Host adhesion is considered the first step of infection for many pathogens such as bacteria and virus. The binding of the receptor binding domain (RBD) of SARS-CoV-2 Spike protein (S protein) onto human angiotensin-converting enzyme 2 (ACE2) is considered as the first step for the SARS-CoV-2 to adhere onto the host cells during the infection. Within three years, a number of variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been found all around the world. Here, we investigated the adhesion of S Proteins from different variants and ACE2 using atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) and single-cell force spectroscopy (SCFS). We found that the unbinding force and binding probability of the S protein from Delta variant to the ACE2 was the highest among the variants tested in our study at both single-molecule and single-cell levels. Molecular dynamics simulation showed that ACE2-RBD (Omicron) complex is destabilized by the E484A and Y505H mutations and stabilized by S477N and N501Y mutations, when compared with Delta variant. In addition, a neutralizing antibody, produced by immunization with wild type RBD of S protein, could effectively inhibit the binding of S proteins from wild type, Delta and Omicron variants onto ACE2. Our results provide new insight for the molecular mechanism of the adhesive interactions between S protein and ACE2 and suggest that effective monoclonal antibody can be prepared using wild type S protein against the different variants.

biophysics↗