Biology subjects

Wu, J.

Publications and source records attributed to Wu, J..

At least 523 records · Page 29Linked to original sources

bioRxiv2023

Enkurin: A novel marker for myeloproliferative neoplasms from platelet, megakaryocyte, and whole blood specimens.

Impaired protein homeostasis, though well established in age-related disorders, has been linked in recent research with the pathogenesis of myeloproliferative neoplasms (MPNs). As yet, however, little is known about MPN-specific modulators of proteostasis, thus impeding our ability for increased mechanistic understanding and discovery of additional therapeutic targets. Loss of proteostasis, in itself, is traced to dysregulated mechanisms in protein folding and intracellular calcium signaling at the endoplasmic reticulum (ER). Here, using ex vivo and in vitro systems (including CD34+ cultures from patient bone marrow, and healthy cord/peripheral blood specimens), we extend our prior data from MPN patient platelet RNA sequencing, and discover select proteostasis-associated markers at RNA and/or protein levels in each of platelets, parent megakaryocytes, and whole blood specimens. Importantly, we identify a novel role in MPNs for enkurin (ENKUR), a calcium mediator protein, implicated originally only in spermatogenesis. Our data reveal consistent ENKUR downregulation at both RNA and protein levels across MPN patient specimens and experimental models, with a concomitant upregulation of a cell cycle marker, CDC20. Silencing of ENKUR by shRNA in CD34+ derived megakaryocytes further confirm this association with CDC20 at both RNA and protein levels; and indicate a likely role for the PI3K/Akt pathway. The inverse association of ENKUR and CDC20 expression was further confirmed upon treatment with thapsigargin (an agent that causes protein misfolding in the ER by selective loss of calcium) in both megakaryocyte and platelet fractions at RNA and protein levels. Together, our work sheds light on enkurin as a novel marker of MPN pathogenesis beyond the genetic alterations; and indicates further mechanistic investigation into a role for dysregulated calcium homeostasis, and ER and protein folding stress in MPN transformation. VISUAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/523111v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@80834corg.highwire.dtl.DTLVardef@12c033forg.highwire.dtl.DTLVardef@8948c8org.highwire.dtl.DTLVardef@1096184_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LIEnkurin, a calcium adaptor protein, is identified as a novel marker of pathogenesis in MPNs. C_LIO_LIMPN megakaryocyte and platelet expression of enkurin at RNA and protein levels is inversely associated with a cell differentiation cycle gene, CDC20. C_LIO_LILikely role for dysregulated calcium homeostasis, and ER and protein folding stress in MPN transformation. C_LI

cancer biology↗

bioRxiv2023

Label-free and non-destructive pathology of human lung adenocarcinomas with ultraviolet single-plane illumination microscopy

Lung cancer is one of the leading causes of cancer death worldwide. The diagnosis of lung cancer based on the analysis of formalin-fixed and paraffin-embedded (FFPE) tissues is laborious and time-consuming, failing to guide surgeons intraoperatively. Here we proposed a rapid histological imaging method, termed microscopy with ultraviolet single-plane illumination (MUSI), to enable rapid ex-or in-vivo imaging of fresh and unprocessed tissues in a label-free and non-destructive manner. The MUSI system allows surgical specimens with large irregular surfaces to be screened at a speed of 0.5 mm2/s with a subcellular resolution, which is sufficient to provide immediate feedback to surgeons and pathologists for intraoperative decision-making. We demonstrate that MUSI can differentiate between different subtypes of human lung adenocarcinomas, revealing diagnostically important features that are comparable to the gold standard FFPE histology. As an assistive imaging platform, MUSI could facilitate the development of precise image-guided surgery and revolutionize the current practice in surgical pathology.

pathology↗

bioRxiv2022

Establishment of Bovine Trophoblast Stem Cells

Here we report that a chemical cocktail (LCDM: hLIF, CHIR99021, DiM and MiH) previously used for extended potential pluripotent stem cells enables the de novo derivation and long-term culture of bovine trophoblast stem cells (TSCs). Bovine TSCs exhibit transcriptomic and epigenetic features characteristic of trophectoderm cells from bovine embryos and retain developmental potency to differentiate into mature trophoblast cells.

developmental biology↗

bioRxiv2022

Retrieving the near-complete genome of a threatened bird from wild frozen samples

Conservation genomics often relies on non-invasive methods to obtain DNA fragments which limit the power of multi-omic analyses for threatened species. Collecting samples from frozen dead animals in the wild provides an alternative approach to obtaining high-quality nucleic acids. Here, we report multi-omic analyses based on a well-preserved great bustard individual (Otis tarda, Otidiformes) of a recent death found in the mountainous region in Gansu, China. We generated a near-complete genome assembly (OTswu) having only 18 gaps scattering in 8 out of the 40 assembled chromosomes. Unlikely most other bird genome assemblies, OTswu contains complete chromosome models (2n = 80). We demonstrated that the great bustard genome likely retained the ancestral avian karyotype. We also characterized the DNA methylation landscapes of OTswu which are strongly correlated with GC content and gene expression. Our phylogenomic analysis suggested Otidiformes and Musophagiformes were sister groups that diverged from each other 46.3 million years ago. The genetic diversity of great bustard was found the lowest among the four available Otidiformes genomes, possibly due to population declines during past glacial periods. As one of the heaviest migratory birds, great bustard possesses several expanded gene families related to cardiac contraction, actin contraction, calcium ion signaling transduction, as well as positively selected genes enriching for metabolism. Finally, we identified an extremely young evolutionary stratum on the sex chromosome, a rare case among Neoaves. Together, our study combining long-read sequencing and RNA-seq technology provides a working strategy for conducting multi-omic analyses for threatened species by retrieving high-quality nucleic acids from dead animals frozen in the wild.

genomics↗

bioRxiv2022

Bovine blastocyst like structures derived from stem cell cultures

Understanding blastocyst formation and implantation is critical for improving farm animal reproduction but is hampered by a limited supply of embryos. We developed an efficient method to generate bovine blastocyst-like structures (termed blastoids) via the assembly of trophoblast stem cells and expanded potential stem cells. Bovine blastoids resemble blastocysts in morphology, cell composition, single-cell transcriptomes, and represent an accessible in vitro model for studying bovine embryogenesis.

developmental biology↗

bioRxiv2022

RNA binding protein PRRC2B mediates translation of specific proteins and regulates cell cycle progression

Accumulating evidence suggests that posttranscriptional control of gene expression, including RNA splicing, transport, modification, translation, and degradation, primarily relies on RNA binding proteins (RBPs). However, the functions of many RBPs remain understudied. Here, we characterized the function of a novel RBP, Proline-Rich Coiled-coil 2B (PRRC2B). Through photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation and sequencing (PAR-CLIP-seq), we identified transcriptome-wide CU- or GA-rich PRRC2B binding sites near the translation initiation codon on a specific cohort of mRNAs in HEK293T cells. These mRNAs, including oncogenes and cell cycle regulators such as CCND2 (cyclin D2), exhibited decreased translation upon PRRC2B knockdown as revealed by polysome-associated RNA-seq, resulting in reduced G1/S phase transition and cell proliferation. Antisense oligonucleotides blocking PRRC2B interactions with CCND2 mRNA decreased its translation, thus inhibiting G1/S transition and cell proliferation. Mechanistically, PRRC2B interactome analysis revealed RNA-independent interactions with eukaryotic translation initiation factors 3 (eIF3) and 4G2 (eIF4G2). The interaction with translation initiation factors is essential for PRRC2B function since the eIF3/eIF4G2-interacting defective mutant, unlike wild-type PRRC2B, failed to rescue the translation deficiency or cell proliferation inhibition caused by PRRC2B knockdown. Altogether, our findings reveal that PRRC2B is essential for efficiently translating specific proteins required for cell cycle progression and cell proliferation. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/520836v2_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@cc9385org.highwire.dtl.DTLVardef@e92f55org.highwire.dtl.DTLVardef@1228c7corg.highwire.dtl.DTLVardef@1352179_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

Neurodegeneration Risk Factor, EIF2AK3 (PERK), Influences Tau Protein Aggregation

Tauopathies are neurodegenerative diseases caused by pathologic misfolded tau protein aggregation in the nervous system. Population studies implicate EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3), better known as PERK (protein kinase R-like endoplasmic reticulum kinase), as a genetic risk factor in several tauopathies. PERK is a key regulator of intracellular proteostatic mechanisms - Unfolded Protein Response (UPR) and Integrated Stress Response (ISR). Previous studies found that tauopathy-associated PERK variants encoded functional hypomorphs with reduced signaling in vitro. But, it remained unclear how altered PERK activity led to tauopathy. Here, we chemically or genetically modulated PERK signaling in cell culture models of tau aggregation and found that PERK pathway activation prevented tau aggregation while inhibition exacerbated tau aggregation. In primary tauopathy patient brain tissues, we found that reduced PERK signaling correlated with increased tau neuropathology. We found that tauopathy-associated PERK variants targeted the ER luminal domain; and two of these variants damaged hydrogen bond formation. Our studies support that PERK activity protects against tau aggregation and pathology. This may explain why people carrying hypomorphic PERK variants have increased risk for developing tauopathies. Finally, our studies identify small molecule augmentation of PERK signaling as an attractive therapeutic strategy to treat tauopathies by preventing tau pathology.

neuroscience↗

bioRxiv2022

Regulation of RAS palmitoyltransferases by accessory proteins and palmitoylation

SummaryPalmitoylation of cysteine residues at the C-terminal hypervariable regions in human HRAS and NRAS, which is necessary for the RAS signaling, is catalyzed by the acyltransferase DHHC9 in complex with its accessory protein GCP16. The molecular basis for the acyltransferase activity and the regulation of DHHC9 by GCP16 is not clear. Here we report the cryo-EM structures of the human DHHC9-GCP16 complex and its yeast counterpart -- the Erf2-Erf4 complex, demonstrating that GCP16 and Erf4 are not directly involved in the catalytic process but stabilize the architecture of DHHC9 and Erf2, respectively. We found that a phospholipid binding to an arginine-rich region of DHHC9 and palmitoylation on three residues (C24, C25, and C288) were essential for the catalytic activity of the DHHC9-GCP16 complex. Furthermore, we showed that GCP16 also formed complexes with DHHC14 and 18 to catalyze RAS palmitoylation. These findings provide insights into the regulation mechanism of RAS palmitoyltransferases.

molecular biology↗

bioRxiv2022

Fortuitous Somatic Mutations during Antibody Evolution Endow Broad Neutralization against SARS-CoV-2 Omicron Variants

Striking antibody evasion by emerging circulating SARS-CoV-2 variants drives the identification of broadly neutralizing antibodies (bNAbs). However, how a bNAb acquires increased neutralization breadth during antibody evolution is still elusive. Here, we identified a clonally-related antibody family from a convalescent individual. One of the members, XG005, exhibited potent and broad neutralizing activities against SARS-CoV-2 variants, while the other members showed significant reductions in neutralization breadth and potency, especially against the Omicron sublineages. Structural analysis visualizing the XG005-Omicron spike binding interface revealed how crucial somatic mutations endowed XG005 with greater neutralization potency and breadth. A single administration of XG005 with extended half-life, reduced antibody-dependent enhancement (ADE) effect, and increased antibody product quality, exhibited a high therapeutic efficacy in BA.2- and BA.5-challenged mice. Our results provided a natural example to show the importance of somatic hypermutation during antibody evolution for SARS-CoV-2 neutralization breadth and potency.

immunology↗

bioRxiv2022

BioNetGMMFit: a Parameter Estimation Tool for BioNetGen using Single-Cell Snapshot Data from Cell Populations Evolving over Time

BackgroundMechanistic models are commonly employed to describe signaling and gene regulatory kinetics in single cells and cell populations. Recent advances in single-cell technologies have produced multidimensional datasets where snapshots of copy numbers (or abundances) of a large number of proteins and mRNA are measured across time in single cells. The availability of such datasets presents an attractive scenario where mechanistic models are validated against experiments, and estimated model parameters enable quantitative predictions of signaling or gene regulatory kinetics. To empower the systems biology community to easily estimate parameters accurately from multidimensional single-cell data, we have merged a widely used rule-based modeling software package BioNetGen, which provides a user-friendly way to code for mechanistic models describing biochemical reactions, and the recently introduced CyGMM, that uses cell-to-cell differences to improve parameter estimation for such networks, into a single software package: BioNetGMMFit. ResultsBioNetGMMFit provides parameter estimates of the model, supplied by the user in the BioNetGen markup language (BNGL), which yield the best fit for the observed single-cell, timestamped data of cellular components. Furthermore, for more precise estimates, our software generates confidence intervals around each model parameter. BioNetG-MMFit is capable of fitting datasets of increasing cell population sizes for any mechanistic model specified in the BioNetGen markup language. ConclusionBy streamlining the process of developing mechanistic models for large single-cell datasets, BioNetGMMFit provides an easily-accessible modeling framework designed for scale and the broader biochemical signaling community.

systems biology↗

bioRxiv2022

The nutrient-sensing GCN2 signaling pathway is essential for circadian clock function by regulating histone acetylation under amino acid starvation

Circadian clocks are evolved to adapt to the daily environment changes under different conditions. The ability to maintain circadian clock functions in response to various stress and perturbations is important for organismal fitness. Here, we show that the nutrient sensing GCN2 signaling pathway is required for robust circadian clock function under amino acid starvation in Neurospora. The deletion of GCN2 pathway components disrupts rhythmic transcription of clock gene frq by suppressing WC complex binding at the frq promoter due to its reduced histone H3 acetylation levels. Under amino acid starvation, the activation of GCN2 kinase and its downstream transcription factor CPC-1 establish a proper chromatin state at the frq promoter by recruiting the histone acetyltransferase GCN-5. The arrhythmic phenotype of the GCN2 kinase mutants under amino acid starvation can be rescued by inhibiting histone deacetylation. Finally, genome-wide transcriptional analysis indicates that the GCN2 signaling pathway maintains robust rhythmic expression of metabolic genes under amino acid starvation. Together, these results uncover an essential role of GCN2 signaling pathway in maintaining robust circadian clock function in response to amino acid starvation and the importance of histone acetylation at the frq locus in rhythmic gene expression.

genetics↗

bioRxiv2022

NaWRKY3 is a master transcriptional regulator of defense network against Alternaria alternata in Nicotiana attenuata

WRKY transcription factors are involved in plant defense responses against pathogens. However, no WRKYs have been reported yet in resistance of Nicotiana species to Alternaria alternata, a necrotrophic fungal pathogen causing brown spot disease. Here, we found that silencing NaWRKY3 lead to wild tobacco Nicotiana attenuata highly susceptible to A. alternata. Combination of transcriptome, electrophoretic mobility shift, ChIP-qPCR and dual-LUC analyses, we uncovered that NaWRKY3 bound to many defense genes promoter and activated their expression. Target genes included: 1) lipoxygenases 3, ACC synthase 1 and ACC oxidase 1, three key enzyme genes for JA and ethylene biosynthesis which were critical for A. alternata resistance; 2) feruloyl-CoA 6-hydroxylase 1 (NaF6H1), the key enzyme gene for phytoalexins against A. alternata, scopoletin and scopolin; and 3) three A. alternata resistance genes, long non-coding RNA (LncRNA L2), NADPH oxidase (NaRboh D) and berberine bridge-like (NaBBL28). Silencing LncRNA L2 reduced A. alternata-induced levels of JA and NaF6H1 expression. NaRboh D-silenced plants were strongly impaired in ROS production and stomata closure responses. NaBBL28 was the first A. alternata resistance BBLs identified and was involved in HGL-DTGs hydroxylation. Finally, NaWRKY3 could bind to its own promoter but acted as a transcriptional repressor. Thus we demonstrated that NaWRKY3 is a fine-tuned master regulator of defense network against A. alternata in N. attenuata by regulating different signaling pathways and defense metabolites. For the first time, such an important WRKY was identified in Nicotiana species, providing new insight into defense mechanism of Nicotiana plants to A. alternata.

molecular biology↗

bioRxiv2022

tFold-Ab: Fast and Accurate Antibody Structure Prediction without Sequence Homologs

Accurate prediction of antibody structures is critical in analyzing the function of antibodies, thus enabling the rational design of antibodies. However, existing antibody structure prediction methods often only formulate backbone atoms and rely on additional tools for side-chain conformation prediction. In this work, we propose a fully end-to-end architecture for simultaneous prediction of backbone and side-chain conformations, namely tFold-Ab. Pre-trained language models are adopted for fast structure prediction by avoiding the time-consuming search for sequence homologs. The model firstly predicts monomer structures of each chain, and then refines them into heavy-light chain complex structure prediction, which enables multi-level supervision for model training. Evaluation results verify the effectiveness of tFold-Ab for both antibody and nanobody structure prediction. In addition, we provide a public web service for antibody structure prediction at https://drug.ai.tencent.com/en.

bioinformatics↗

bioRxiv2022

A compendium of genetic regulatory effects across pig tissues

The Farm animal Genotype-Tissue Expression (FarmGTEx, https://www.farmgtex.org/) project has been established to develop a comprehensive public resource of genetic regulatory variants in domestic animal species, which is essential for linking genetic polymorphisms to variation in phenotypes, helping fundamental biology discovery and exploitation in animal breeding and human biomedicine. Here we present results from the pilot phase of PigGTEx (http://piggtex.farmgtex.org/), where we processed 9,530 RNA-sequencing and 1,602 whole-genome sequencing samples from pigs. We build a pig genotype imputation panel, characterize the transcriptional landscape across over 100 tissues, and associate millions of genetic variants with five types of transcriptomic phenotypes in 34 tissues. We study interactions between genotype and breed/cell type, evaluate tissue specificity of regulatory effects, and elucidate the molecular mechanisms of their action using multi-omics data. Leveraging this resource, we decipher regulatory mechanisms underlying about 80% of the genetic associations for 207 pig complex phenotypes, and demonstrate the similarity of pigs to humans in gene expression and the genetic regulation behind complex phenotypes, corroborating the importance of pigs as a human biomedical model.

genomics↗

bioRxiv2022

Intertumoral lineage diversity and immunosuppressive transcriptional programs in well-differentiated gastroenteropancreatic neuroendocrine tumors

Neuroendocrine tumors (NETs) are rare cancers that may arise in the gastrointestinal tract and pancreas. The fundamental mechanisms driving gastroenteropancreatic (GEP) NET growth remain incompletely elucidated; however, the heterogeneous clinical behavior of GEP-NETs suggests that both cellular lineage dynamics and tumor microenvironment influence tumor pathophysiology. Here, we investigated the single-cell transcriptomes of tumor and immune cells from patients with gastroenteropancreatic NETs. Malignant GEP-NET cells expressed genes and regulons associated with normal, gastrointestinal endocrine cell differentiation and fate determination stages. While tumor and lymphoid compartments sparsely expressed immunosuppressive targets, infiltrating myeloid cells were enriched for alternative immunotherapy pathways including VSIR, Tim3/Gal9, and SIGLEC10. Finally, analysis of paired primary and metastatic tissue specimens from small intestinal NETs demonstrated transcriptional transformation between the primary tumor and its distant metastasis. Our findings highlight the transcriptomic heterogeneity that distinguishes the cellular landscapes of GEP-NET anatomic subtypes and reveal potential avenues for future precision medicine therapeutics.

cancer biology↗

bioRxiv2022

Discovery of aphid-transmitted Rice tiller inhibition virus from native plants through metagenomic sequencing

A major threat to rice production is the disease epidemics caused by insect-borne viruses that emerge and re-emerge with undefined origins. It is well known that some human viruses have zoonotic origins from wild animals. However, it remains unknown whether native plants host new endemic viruses with spillover potential to rice (Oryza sativa) as emerging pathogens. Here, we discovered rice tiller inhibition virus (RTIV), a novel RNA virus species, from colonies of Asian wild rice (O. rufipogon) in a genetic reserve by metagenomic sequencing. We identified the specific aphid vector to transmit RTIV and found RTIV would cause low-tillering disease in rice cultivar after transmission. We further demonstrated that an infectious molecular clone of RTIV initiated systemic infection and causes low-tillering disease in an elite rice variety after Agrobacterium-mediated inoculation or stable plant transformation, and RTIV can also be transmitted from transgenic rice plant through its aphid vector to cause disease. Finally, global transcriptome analysis indicated that RTIV may disturb defense and tillering pathway to cause low tillering disease in rice cultivar. Thus, our results show that new rice viral pathogens can emerge from native habitats, and RTIV, a rare aphid-transmitted rice viral pathogen from native wild rice, can threaten the production of rice cultivar after spillover.

pathology↗

bioRxiv2022

Bio-friendly long-term subcellular dynamic recording by self-supervised image enhancement microscopy

Fluorescence microscopy has become an indispensable tool for revealing the dynamic regulations of cells and organelles in high resolution noninvasively. However, stochastic noise inherently restricts the upper bonds of optical interrogation quality and exacerbates the observation fidelity in encountering joint demand of high frame rate, long-term, and low photobleaching and phototoxicity. Here, we propose DeepSeMi, a self-supervised-learning-based denoising framework capable of increasing SNR by over 12 dB across various conditions. With the introduction of newly designed eccentric blind-spot convolution filters, DeepSeMi accomplished efficacious denoising requiring no clean data as references and no compromise of spatiotemporal resolution on diverse imaging systems. The computationally 15-fold multiplied photon budget in a standard confocal microscope by DeepSeMi allows for recording organelle interactions in four colors and high-frame-rate across tens of thousands of frames, monitoring migrasomes and retractosomes over a half day, and imaging ultra-phototoxicity-sensitive Dictyostelium cells over thousands of frames, all faithfully and sample-friendly. Through comprehensive validations across various cells and species over various instruments, we prove DeepSeMi is a versatile tool for reliably and bio-friendly breaking the shot-noise limit, facilitating automated analysis of massive data about cell migrations and organelle interactions.

bioengineering↗

bioRxiv2022

Foxa2 lineage+ mesendoderm forms a competitive pulmonary mesenchyme niche crucial for generating the entire lungs

Millions suffer from incurable lung diseases, and the donor lung shortage hampers organ transplants. Identifying the crucial lineage and the program for lung organogenesis could facilitate designing whole-lung bioengineering. Using lineage-tracing mice and human iPSC-derived lung-directed differentiation, we revealed that gastrulating Foxa2 lineage contributed to both lung mesenchyme and epithelium formation. Interestingly, Foxa2 lineage-derived cells in the lung mesenchyme progressively increased and occupied more than half of the mesenchyme niche, including endothelial cells, during lung development. Foxa2 promoter-driven, conditional Fgfr2 gene depletion caused the lung agenesis phenotype in mice. Importantly, wild-type donor mouse iPSCs injected into their blastocysts rescued this phenotype by complementing the Fgfr2-defective niche in the lung epithelium and mesenchyme. Donor cell is shown to replace the entire lung epithelial and robust mesenchymal niche during early chimeric lung development, resulting in efficient complementation of the nearly entire lung niche at the late stage of lung development. These results suggest that lung complementation based on the Foxa2 lineage is a unique model for the progressive mobilization of donor cells into both epithelial and mesenchymal lung niches and provides crucial insights for designing new bioengineering strategies to generate whole lungs.

developmental biology↗