Biology subjects

Wu, J.

Publications and source records attributed to Wu, J..

At least 559 records · Page 31Linked to original sources

bioRxiv2022

Continuous Fish Muscle Cell Line with Capacity for Myogenic and Adipogenic-like Phenotypes

Cell-cultivated fish offers the potential for a more ethical, sustainable, and safe seafood system. However, fish cell culture is relatively understudied in comparison to mammalian cells. Here, we established and characterized a continuous Atlantic mackerel (Scomber scombrus) skeletal muscle cell line ("Mack" cells). The cells were isolated from muscle biopsies of fresh-caught fish, with separate isolations performed from two distinct fish. Mack1 cells (cells from the first isolation) were cultured for over a year and subcultured over 130 times. The cells proliferated at initial doubling times of 63.9 hr ({+/-} 19.1 SD). After a spontaneous immortalization crisis from passages 37-43, the cells proliferated at doubling times of 24.3 hr ({+/-} 4.91 SD). A muscle phenotype was confirmed through characterization of muscle stemness and differentiation via paired-box protein 7 (PAX7) and myosin heavy chain (MHC) immunostaining, respectively. An adipocyte-like phenotype was also demonstrated for the cells through lipid accumulation, confirmed via Oil Red O (ORO) staining and quantification of neutral lipids. New qPCR primers (HPRT, PAX3B, MYOD1, MYOG, TNNT3A, and PPARG) were tailored to the mackerel genome and used to characterize mackerel cell genotypes. This work provides the first spontaneously immortalized fish muscle cell line for research, ideally serving as a reference for subsequent investigation.

bioengineering↗

bioRxiv2022

Simultaneous transcriptome and proteome profiling in a single mouse oocyte with a deep single-cell multi-omics approach

Nowadays, although single-cell multi-omics technologies are undergoing rapid development, simultaneous transcriptome and proteome analysis of a single-cell individual still faces great challenges. Here, we developed a single-cell simultaneous transcriptome and proteome (scSTAP) analysis platform based on microfluidics, high-throughput sequencing and mass spectrometry technology, to achieve deep and joint quantitative analysis of transcriptome and proteome at the single-cell level for the first time. This platform was applied to analyze single mouse oocytes at different meiotic maturation stages, reaching an average quantification depth of 19948 genes and 2663 protein groups in single mouse oocytes. This reliable quantitative two-omics dataset of single cells provided an important resource for understanding the relationship between the transcriptome and the proteome in cells. Based on the correlation analysis of RNAs and proteins in the same single cell, we demonstrated the expressive heterogeneity of transcriptome and proteome during the cellular biological process. Specially, we analyzed the meiosis regulatory network during oocyte maturation with an unprecedented depth at the single-cell level, and identified 30 transcript-protein pairs as specific oocyte maturational signatures, providing crucial insights into the regulatory features of transcription and translation during oocyte meiotic maturation.

cell biology↗

bioRxiv2022

Inhibition of Talin-induced Integrin Activation by a Double-hit Stapled Peptide

Integrins are ubiquitously expressed cell-adhesion proteins. Talin is required for integrin activation through an inside-out signaling pathway, during which talin is recruited to the plasma membrane (PM) by RAP1 directly or through its effector, RAP1-Interacting Adaptor Molecule (RIAM). RIAM also activates talin from autoinhibition by binding to talin head domain. A helical talin-binding segment (TBS) in RIAM mediates both talin activation and recruitment by binding to two distinct sites in talin head and rod domains respectively. The bi-specificity of the TBS fragment allows us to develop a new strategy to suppress talin-induced integrin activation through a "double-hit" approach. We designed an experimental peptidomimetic inhibitor by engineering a hydrocarbon "staple" in the helical TBS fragment to mask the integrin binding site in the talin head. The stapled peptide (S-TBS) exhibits a stronger binding affinity with talin and inhibits talin:integrin interaction. Crystal structure of S-TBS in complex with talin rod domains reveals an interface that overlaps with the TBS-binding site in talin. Consistently, S-TBS also exhibits an inhibitory effect on TBS:talin-rod interaction during the PM recruitment of talin. Importantly, S-TBS possesses excellent cell permeability and inhibits integrin activation in a talin-dependent manner. Hence, our results present a novel approach to design a new class of intracellular inhibitors targeting integrin functions.

biochemistry↗

bioRxiv2022

Neutralization of Omicron BA.4/BA.5 and BA.2.75 by Booster Vaccination or BA.2 Breakthrough Infection Sera

Many new Omicron sub-lineages have been reported to evade neutralizing antibody response, including BA.2, BA.2.12.1, BA.4 and BA.5. Most recently, another emerging sub-lineage BA.2.75 has been reported in multiple countries. In this study, we constructed a comprehensive panel of pseudoviruses (PsVs), including wild-type, Delta, BA.1, BA.1.1, BA.2, BA.3, BA.2.3.1, BA.2.10.1, BA.2.12.1, BA.2.13, BA.2.75 and BA.4/BA.5, with accumulate coverage reached 91% according to the proportion of sequences deposited in GISAID database since Jan 1st, 2022. We collected serum samples from healthy adults at day14 post homologous booster with BBIBP-CorV, or heterologous booster with ZF2001, primed with two doses of BBIBP-CorV, or from convalescents immunized with three-dose inactivated vaccines prior to infection with Omicron BA.2, and tested their neutralization activity on this panel of PsVs. Our results demonstrated that all Omicron sub-lineages showed substantial evasion of neutralizing antibodies induced by vaccination and infection, although BA.2.75 accumulated the largest number of mutations in its spike, BA.4 and BA.5 showed the strongest serum escape. However, BA.2 breakthrough infection could remarkably elevated neutralization titers against all different variants, especially titers against BA.2 and its derivative sub-lineages.

immunology↗

bioRxiv2022

TSNAdb v2.0: the updated version of tumor-specific neoantigen database

Tumor neoantigens have been well-acknowledged as ideal targets for tumor immunotherapy in recent years. With the deepening of research on neoantigen-based tumor immunotherapy, comprehensive neoantigen databases are urgently needed to meet the growing demand for clinical studies. We have built the Tumor-Specific NeoAntigen Database (TSNAdb v1.0) previously, which has attracted wide attention. In this study, we provide an updated version of the Tumor-Specific NeoAntigen Database (TSNAdb v2.0) with several new features including 1) taking stricter criteria for neoantigen identification. 2) providing predicted neoantigens derived from three types of somatic mutations. 3) collecting experimentally validated neoantigens and classifying them according to the evidence. TSNAdb v2.0 is freely available at https://pgx.zju.edu.cn/tsnadb/.

bioinformatics↗

bioRxiv2022

Integrated transcriptome analysis of Huntingtons disease iPSC-derived and mouse astrocytes implicates dysregulated synaptogenesis, actin, and astrocyte maturation

Huntingtons disease (HD) is a neurodegenerative disease caused by an expanded CAG repeat within the Huntingtin (HTT) gene having dysregulated cellular homeostasis in the central nervous system, particularly in the striatum and cortex. Astrocytes establish and maintain neuronal functions through the secretion of soluble factors and physical interactions with other neurovascular unit cell types. Under pathological conditions, astrocytes can become reactive, causing cell state transitions that affect brain function. To investigate transitions between cellular states in unaffected and HD astrocytes at high resolution, single-nuclei RNA-sequencing (snRNA-seq) was performed on human HD patient induced pluripotent stem cell (iPSC)-derived astrocytes and on striatal and cortical tissue from a rapidly progressing HD mouse model (R6/2). Analysis of HD human and mouse astrocytes revealed both models have alterations in morphology, glutamate uptake, and dysregulation of astrocyte identity and maturation, whereas dysregulated actin-mediated signaling was unique to human iPSC-derived astrocytes. Representative proteins showed altered levels by Western. In both species, HD transcriptional changes reveal potential astrocyte maturation deficits that were potentially driven by astrogliogenesis transcription factors, including ATF3 and NFIA. When perturbed in a drosophila model of HD, knockdown of NFIA in glia rescued the climbing deficit. These data further support the hypothesis that mutant HTT induces dysregulated astrocyte cell states resulting in dysfunctional astrocytic properties, suggests that some of these states are cell autonomous and maybe unique to human HD, and implicate ATF3 and maturation deficits in HD pathogenesis.

neuroscience↗

bioRxiv2022

Propagation dynamics of electrotactic motility in large epithelial cell sheets

Directional migration initiated at the wound edge leads epithelial sheets to migrate in wound healing. How such coherent migration is achieved is not well understood. Here we used electric fields to induce robust migration of sheets of human keratinocytes and developed an in silico model to characterize initiation and propagation of epithelial collective migration. Electric fields initiate increase in migrations directionality and speed at the leading edge. The increases propagate across the epithelial sheets, resulting in directional migration of cell sheets as coherent units. Both the experimental and in silico models demonstrated vector-like integration of the electric and default directional cues at the free edge in space and time. The resultant collective migration is remarkably consistent in experiments and modeling, both qualitatively and quantitatively. The keratinocyte model thus faithfully reflects key features of epithelial migration as a coherent tissue in vivo, e.g. that leading cells lead, and that epithelium maintains cell- cell junction.

cell biology↗

bioRxiv2022

Quantitative Census of Local Somatic Features in Mouse Visual Cortex

Mammalian neocortex contains a highly diverse set of cell types. These types have been mapped systematically using a variety of molecular, electrophysiological and morphological approaches. Each modality offers new perspectives on the variation of biological processes underlying cell type specialization. Cellular scale electron microscopy (EM) provides dense ultrastructural examination and an unbiased perspective into the subcellular organization of brain cells, including their synaptic connectivity and nanometer scale morphology. It also presents a clear challenge for analysis to identify cell-types in data that contains tens of thousands of neurons, most of which have incomplete reconstructions. To address this challenge, we present the first systematic survey of the somatic region of all cells within a cubic millimeter of cortex using quantitative features obtained from EM. This analysis demonstrates a surprising sufficiency of the perisomatic region to identify cell-types, including types defined primarily based on their connectivity patterns. We then describe how this classification facilitates cell type specific connectivity characterization and locating cells with rare connectivity patterns in the dataset.

neuroscience↗

bioRxiv2022

Multi-Domain Characterization and Rapid Detection Technology for Cocaine and Methamphetamine Acute Abuse based on EEG

Multi-domain characterization and a new fast detection method for acute illegal psychostimulants abuse detection based on waking-state EEG of mice is proposed in this paper. To get corftical electroencephalogram (EEG), three groups of mice were injected with cocaine (Coca), methamphetamine (Meth), and saline (Sali) respectively following the experimental paradigm of drug abuse. The EEG features were extracted out by multi-domain views, including time, frequency, complexity, dynamics, and independent domains to obtain acute drug abuse effects on the brain. New strategy combing time domain with frequency domain is developed as multi-domain input and by means of dimension transformation approach preserving temporal information, the performance of drug abuse detection is greatly improved with deep learning models of ResNet50. Results show that comparing to support vector machine (SVM), k nearest neighbor (kNN), random forest (RF), and long short-term memory fully convolutional networks (LSTM-FCN), ResNet50 based on our proposed multi-domain features has best F1-score of 95.10%. This promising method provides a low-cost, fast, and widely assisted technology for psychostimulants abuse identification.

bioinformatics↗

bioRxiv2022

The Piks allele of the NLR immune receptor Pik breaks the recognition of AvrPik effectors of the rice blast fungus

Arms race co-evolution of plant-pathogen interactions evolved sophisticated recognition mechanisms between host immune receptors and pathogen effectors. Different allelic haplotypes of an immune receptor in host mount distinct recognition against sequence or non-sequence related effectors in pathogens. We report the molecular characterization of the Piks allele of the rice immune receptor Pik against rice blast pathogen, which requires two head-to-head arrayed nucleotide binding site and leucine-rich repeat proteins. Like other Pik genes, both Piks-1 and Piks-2 are necessary and sufficient for Piks-mediated resistance. However, unlike other Pik alleles, Piks does not recognize any known AvrPik variants of M. oryzae. Sequence analysis of the genome of an avirulent isolate V86010 further revealed that its cognate avirulence (Avr) gene most likely has no significant sequence similarity to known AvrPik variants. We conclude that Piks breaks the canonical Pik/AvrPik recognition pattern. Piks-1 and Pikm-1 have only two amino acid differences within the integrated heavy metal-associated (HMA) domain. Pikm-1-HMA interact with AvrPik-A, -D and -E in vitro and in vivo, whereas Piks-1-HMA does not bind any AvrPik variants. Reciprocal exchanges of single amino acid residues between Piks-1 and Pikm-1 further reveal a dynamic recognition mechanism between Piks/Pikm alleles and their respective effectors. Piks-1E229Q/Pikm-1V261A can only activate immunity to AvrPik-D but not to other effectors, indicating that the amino acid change of E to Q at position 229 leads to its gain of a partial recognition spectrum of Pikm. By contrast, Piks-1A261V/Pikm-1Q229E confers immunity to the Piks cognate effector, indicating that the amino acid change of Q to E at position 229 leads to its shifts of the recognition from Pikm to Piks. Intriguingly, binding activities in both Y2H and analytical gel filtration assays are illustrated between Piks-1A261V/Pikm-1Q229E and AvrPik-D. However, it is unable to mount immunity against AvrPik-D, suggesting that biochemical activities based on in vitro and in vivo assays could be insufficient for sustaining biological function of receptor and effector pairs.

plant biology↗

bioRxiv2022

Nitrate-dependent salt tolerance mediated by OsNLP4-OsMADS27 module

Salt stress is a major constraint of plant growth and yield. Nitrogen (N) fertilizers are known to alleviate salt stress. However, the underlying molecular mechanisms remain unclear. Here we show that OsNLP4-OsMADS27 module controls nitrate-dependent salt tolerance in rice. The expression of OsMADS27 is specifically induced by nitrate. The OsMADS27 knockout mutants are more sensitive to salt stress than the wild type, whereas the OsMADS27 overexpression lines are more tolerant. Transcriptomic analyses revealed that OsMADS27 controls the expression of a number of known stress-responsive genes as well as those involved in ion homeostasis and antioxidation. We demonstrated that OsMADS27 directly binds to the promoter of OsHKT1.1 and OsSPL7 to regulate their expression. Notably, OsMADS27-mediated salt tolerance is nitrate-dependent and positively correlated with nitrate concentration. We further showed that OsNLP4, a nitrate-responsive key regulator in N metabolism and N use efficiency, positively regulates the expression of OsMADS27 by directly binding to the nitrate-responsive cis-element in its promoter, thereby transmitting the nitrate signal to OsMADS27 and conferring its nitrate dependence. Our results reveal the role of nitrate-responsive OsNLP4-OsMADS27 module and its downstream target genes in salt tolerance, filling the gap in the molecular mechanism of nitrate-dependent salt tolerance of rice. Moreover, OsMADS27 overexpression increased grain yield under salt stress in presence of sufficient nitrate, indicating that OsMADS27 is a promising candidate for the improvement of salt tolerance in rice.

plant biology↗

bioRxiv2022

Biochemical characterization of a Mycobacterium tuberculosis prolyl dipeptidyl peptidase that displays a remarkable thermostability

Dipeptidyl peptidases constitute a class of non-classical serine proteases that regulate an array of biological functions, making them pharmacologically attractive enzymes. With this work, we identified and characterized a dipeptidyl peptidase from Mycobacterium tuberculosis (MtDPP) displaying a strong preference for proline residues at the P1 substrate position and an unexpectedly high thermal stability. MtDPP was also characterized with alanine replacements of residues of its active site that yielded, for the most part, loss of catalysis. We show that MtDPP catalytic activity is inhibited by well-known human DPP4 inhibitors. Using MALDI-TOF mass spectrometry we also describe that in vitro, MtDPP mediates the truncation of the C-X-C motif chemokine ligand 10, indicating a plausible role in immune modulation for this mycobacterial enzyme.

biochemistry↗

bioRxiv2022

Interleukin-1α and Leukemia Inhibitory Factor Promote Extramedullary Hematopoiesis

Extramedullary hematopoiesis (EMH) expands hematopoietic capacity outside of the bone marrow in response to inflammatory conditions, including infections and cancer. Because of its inducible nature, EMH offers a unique opportunity to study the interaction between hematopoietic stem and progenitor cells (HSPCs) and their niche. In cancer patients, the spleen frequently serves as an EMH organ and provides myeloid cells that may worsen pathology. Here, we examined the relationship between HSPCs and their splenic niche in EMH secondary to solid tumor. We identify an inflammatory gene signature characterized by TNF expression in HSPCs. We show a role for IL-1 in producing this gene signature and TNF expression in HSPCs in activating splenic niche activity. We also demonstrate that tumor-derived Leukemia Inhibitory Factor (LIF) induces proliferation of splenic niche cells. IL-1 and LIF display cooperative effects in activating EMH and are both upregulated in some human cancers. Together, these data expand avenues for developing niche-directed therapies and further exploring EMH accompanying inflammatory pathologies like cancer.

immunology↗

bioRxiv2022

Molecular basis for the role of disulfide-linked αCTs in the activation of insulin-like growth factor 1 receptor and insulin receptor

The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) control metabolic homeostasis and cell growth and proliferation. The IR and IGF1R form similar disulfide bonds linked homodimers in the apo-state; however, their ligand binding properties and the structures in the active state differ substantially. It has been proposed that the disulfide-linked C-terminal segment of -chain (CTs) of the IR and IGF1R control the cooperativity of ligand binding and regulate the receptor activation. Nevertheless, the molecular basis for the roles of disulfide-linked CTs in IR and IGF1R activation are still unclear. Here, we report the cryo-EM structures of full-length mouse IGF1R/IGF1 and IR/insulin complexes with modified CTs that have increased flexibility. Unlike the{Gamma} -shaped asymmetric IGF1R dimer with a single IGF1 bound, the IGF1R with the enhanced flexibility of CTs can form a T-shaped symmetric dimer with two IGF1s bound. Meanwhile, the IR with non-covalently linked CTs predominantly adopts an asymmetric conformation with four insulins bound, which is distinct from the T-shaped symmetric IR. Using cell-based experiments, we further showed that both IGF1R and IR with the modified CTs cannot activate the downstream signaling potently. Collectively, our studies demonstrate that the certain structural rigidity of disulfide-linked CTs is critical for optimal IR and IGF1R signaling activation.

cell biology↗

bioRxiv2022

Nonlinear effect of light intensity on normal axial development of rhesus monkeys

PurposeTo investigate the effects of different indoor lighting intensity (500 lx, 750 lx and 1,000 lx) on normal ocular axial length growth by using juvenal rhesus monkeys. MethodsTwenty-four juvenile monkeys were exposed continuously to normal intensity light (NIL, 500 lx, n=16), medium intensity light (MIL, 750 lx, n=8) and high intensity light (HIL, 1 000 lx, n=8), with a same CCT value (about 3000 K) for 246 days. Axial length, anterior chamber depth, corneal curvature radius were measured at about a monthly interval. ResultsAfter 246 days of light exposure, the growth of axial length of the MIL group (750 lx) were 0.151 {+/-} 0.081 mm and 0.139 {+/-} 0.070 mm in the right and left eyes, respectively, and significantly larger in comparison with the NIL group (500lx, OD: 0.068 {+/-} 0.055 mm, OS: 0.074 {+/-} 0.057 mm) and the HIL group (1000lx,OD:0.063 {+/-} 0.093 mm, OS: 0.084 {+/-} 0.052 mm) monkeys. This effect was stable and robust during the whole experimental period. ConclusionThe effects of different intensity lighting on normal ocular axial development was not linear as most people currently think. We must be cautious when it comes to elevate light intensity in classrooms. Whether this conclusion is correct under lights of other CCT value needs further study.

developmental biology↗

bioRxiv2022

Pick-up Single-Cell Proteomic Analysis for Quantifying up to 3000 Proteins in a Tumor Cell

The shotgun proteomic analysis is currently the most promising single-cell protein sequencing technology, however its identification level of [~]1000 proteins per cell is still insufficient for practical applications. Here, we develop a pick-up single-cell proteomic analysis (PiSPA) workflow to achieve a deep identification capable of quantifying up to 3000 protein groups in a tumor cell using the label-free quantitative method. The PiSPA workflow is specially established for single-cell samples mainly based on a nanoliter-scale microfluidic liquid handling robot, capable of achieving single-cell capture, pretreatment and injection under the pick-up operation strategy. Using this customized workflow with remarkable improvement in protein identification, 1804-3349, 1778-3049 and 1074-2487 protein groups are quantified in single A549 cells (n = 37), HeLa cells (n = 44) and U2OS cells (n = 27), respectively. Benefiting from the flexible cell picking-up ability, we study tumor cell migration at the single cell proteome level, demonstrating the potential in practical biological research from single-cell insight.

cancer biology↗

bioRxiv2022

Structural insights into the assembly of gp130 family cytokine signaling complexes

The gp130 family cytokine signaling complexes have limited structural information despite their crucial roles in various cellular processes. We determined cryo-EM structures of several complexes of this family, containing full ectodomains of both signaling receptors bound to their respective ligands CNTF, CLCF1, LIF, IL-27, and IL-6. Our structures reveal that gp130 serves as a central receptor by engaging Site 2 of CNTF, CLCF1, LIF, and IL-6, and Site 3 of IL-27 and IL-6. The acute bends at both signaling receptors in all complexes bring the membrane-proximal domains to a ~30 [A] range but with distinct distances and orientations, which might determine biological specificities of these cytokines. We also reveal how CLCF1 engages its secretion chaperone CRLF1. Our data provide valuable insights for therapeutically targeting gp130-mediated signaling.

biochemistry↗

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↗