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Publications and source records attributed to Wu, J..
WITHDRAWAL STATEMENTbioRxiv has withdrawn this manuscript because it inappropriately listed Peter L. Bartlett, Jingfeng Wu, and Bin Yu as authors. Therefore, this work should not be cited as reference for the project. If you have any questions, please contact the corresponding author.
Transfer RNA (tRNA) modifications have emerged as critical posttranscriptional regulators of gene expression affecting diverse biological and disease processes. While there is extensive knowledge about the enzymes installing the dozens of post-transcriptional tRNA modifications - the tRNA epitranscriptome - very little is known about how metabolic, signaling, and other networks integrate to regulate tRNA modification levels. Here we took a comprehensive first step at understanding epitranscriptome regulatory networks by developing a high-throughput tRNA isolation and mass spectrometry-based modification profiling platform and applying it to a Pseudomonas aeruginosa transposon insertion mutant library comprising 5,746 strains. Analysis of >200,000 tRNA modification data points validated the annotations of predicted tRNA modification genes, uncovered novel tRNA-modifying enzymes, and revealed tRNA modification regulatory networks in P. aeruginosa. Platform adaptation for RNA-seq library preparation would complement epitranscriptome studies, while application to human cell and mouse tissue demonstrates its utility for biomarker and drug discovery and development.
The incidence of Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) has been increasing world-wide. Since gut-derived bacterial lipopolysaccharides (LPS) can travel via the portal vein to the liver and play an important role in producing hepatic pathology, it seemed possible that (1) LPS stimulates hepatic cells to accumulate lipid, and (2) inactivating LPS can be preventive. Acyloxyacyl hydrolase (AOAH), the eukaryotic lipase that inactivates LPS and oxidized phospholipids, is produced in the intestine, liver, and other organs. We fed mice either normal chow or a high-fat diet for 28 weeks and found that Aoah-/- mice accumulated more hepatic lipid than did Aoah+/+ mice. In young mice, before increased hepatic fat accumulation was observed, Aoah-/-mouse livers increased their abundance of Sterol Regulatory Element-Binding Protein 1 (SREBP1) and the expression of its target genes that promote fatty acid synthesis. Aoah-/- mice also increased hepatic expression of CD36 and Fabp3, which mediate fatty acid uptake, and decreased expression of fatty acid oxidation-related genes Acot2 and Ppar-. Our results provide evidence that increasing AOAH abundance in the gut, bloodstream and/or liver may be an effective strategy for preventing or treating MASLD.
Embryo models open exciting opportunities for improving our understanding of development and advancing medicine. However, current models depend on intricate procedures and the integration of separately generated cell types. Here, we aimed to establish a chemical-only strategy to induce cellular plasticity and generate embryo founder-like cells. Within 60 hours, chemical-induced pluripotent stem cells activated early lineage specifiers, Cdx2 and Gata6, while maintaining Oct4/ Pou5f1 expression. Following a second 36-hour chemical treatment, founder-like cells developed into all blastocyst fates and self-assembled into embryo-like structures. These progressed into high-fidelity late-streak gastrulation stages within 4.5 days, displaying primitive streak formation via epithelial-to-mesenchymal transition, along with germ-layer tissues, amnion, and ectoplacental cone. Fate trajectories and molecular signatures from founder-like cells to chemically-induced embryo model (ci-MO) were defined, revealing remarkable similarity up to Embryonic day (E) 7.5 embryo. Summarily, ci-MO offers a straightforward, rapid, small-molecule-only, and high-fidelity approach to study embryonic development and cell fate specification. HighlightsO_LIGeneration of a direct, efficient, and sophisticated chemically induced embryo model (ci-MO) C_LIO_LITwo-pulse chemical induction reprograms ESCs into founder-like cells capable of developing all embryo fates C_LIO_LIGastrulating ci-MOs display high morphological and molecular accuracy C_LIO_LIci-MOs reconstruct embryo developmental trajectories C_LI
Lipid nanoparticles (LNPs) have transformed genetic medicine, recently shown by their use in COVID-19 mRNA vaccines. While loading LNPs with mRNA has many uses, loading DNA would provide additional advantages such as long-term expression and availability of promoter sequences. However, here we show that plasmid DNA (pDNA) delivery via LNPs (pDNA-LNPs) induces acute inflammation in naive mice which we find is primarily driven by the cGAS-STING pathway. Inspired by DNA viruses that inhibit this pathway for replication, we co-loaded endogenous lipids that inhibit STING into pDNA-LNPs. Specifically, loading nitro-oleic acid (NOA) into pDNA-LNPs (NOA-pDNA-LNPs) ameliorates serious inflammatory responses in vivo enabling prolonged transgene expression (at least 1 month). Additionally, we demonstrate the ability to iteratively optimize NOA-pDNA-LNPs expression by performing a small LNP formulation screen, driving up expression 50-fold in vitro. Thus, NOA-pDNA-LNPs, and pDNA-LNPs co-loaded with other bioactive molecules, will provide a major new tool in the genetic medicine toolbox, leveraging the power of DNAs long-term and promoter-controlled expression.
Microglia play critical roles in the brain physiology and pathology. CSF1R is primarily expressed in microglia. The mono-allelic CSF1R mutation causes adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), a lethal neurological disease and no rational cure in clinical trials. There are no animal models mimicking human ALSP. In this study, we first developed mouse models based on human ALSP hotspot mutations. We then utilized microglia replacement by bone marrow transplantation (Mr BMT) to replace the Csf1r-deficient microglia in ALSP mice by Csf1r-normal donor cells. With pathogenic gene correction, Mr BMT efficiently attenuated the pathologies. Previously, an ALSP patient received traditional bone marrow transplantation (tBMT) due to a misdiagnosis of metachromatic leukodystrophy. The disease progress was halted for 15 years with unknown reasons. We demonstrated that tBMT in ALSP is equivalent to or close to Mr BMT, achieving efficient microglia replacement and therefore attenuating the ALSP progress in the mouse model. Next, we applied tBMT to replace CSF1R-deficient microglia in human patients. Our clinical results show that after microglia replacement, the ALSP course is effectively halted. Together, microglia replacement corrects the pathogenic gene and thus halts the disease progress in the mouse model and human patients. This study strongly demonstrates clinical potentials of microglia replacement in neurological disease treatments.
Nanomedicine has long pursued the goal of targeted delivery to specific organs and cell types but has not achieved this goal with the vast majority of targets. One rare example of success in this pursuit has been the 25+ years of studies targeting the lung endothelium using nanoparticles conjugated to antibodies against endothelial surface molecules. However, here we show that such "endothelial-targeted" nanocarriers also effectively target the lungs numerous marginated neutrophils, which reside in the pulmonary capillaries and patrol for pathogens. We show that marginated neutrophils uptake of many of these "endothelial-targeted" nanocarriers is on par with endothelial uptake. This generalizes across diverse nanomaterials and targeting moieties and was even found with physicochemical lung tropism (i.e., without targeting moieties). Further, we observed this in ex vivo human lungs and in vivo healthy mice, with an increase in marginated neutrophil uptake of nanoparticles caused by local or distant inflammation. These findings have implications for nanomedicine development for lung diseases. These data also suggest that marginated neutrophils, especially in the lungs, should be considered a major part of the reticuloendothelial system (RES), with a special role in clearing nanoparticles that adhere to the lumenal surfaces of blood vessels. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/597904v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@17e0518org.highwire.dtl.DTLVardef@809eb6org.highwire.dtl.DTLVardef@33d9a7org.highwire.dtl.DTLVardef@169863_HPS_FORMAT_FIGEXP M_FIG C_FIG
Microbial biophotovoltaics (BPVs) harness photosynthetic microorganisms to convert light energy into electricity, making them highly attractive for renewable energy production. However, current BPVs typically exhibit low power densities, primarily due to inefficient electron transfer processes and the need for close contact and high interfacial area. Here, we propose a novel method of enhancing Dunaliella-based BPVs using Fe3O4 nanoparticle coatings. The Fe3O4-coated Dunaliella cells (DS@Fe3O4) establish intimate contact with the cellular electron transfer machinery and maximize the interfacial area, significantly improving electron transfer efficiency and reducing internal resistance. This approach achieved higher power outputs compared to native Dunaliella BPVs, with an optimal Fe3O4 concentration of 2 mg/mL yielding the best performance. In contrast, SiO2 coatings on Fe3O4 (Fe3O4@SiO2) reduced electron transfer efficiency. These findings demonstrate that Fe3O4 nanoparticle coatings provide a superior method for enhancing bio-electrochemical systems, advancing the application of BPVs for sustainable energy solutions and environmental applications.
Cereal grain size and quality are important agronomic traits in crop production. The development of wheat grains is underpinned by complex regulatory networks. The precise spatial and temporal coordination of diverse cell types is essential for the formation of functional compartments. To provide comprehensive spatiotemporal information about biological processes in developing wheat grain, we performed a spatial transcriptomics study during the early grain development stage from 4 to 12 days after pollination. We defined a set of tissue-specific marker genes and discovered that certain genes or gene families exhibit specific spatial expression patterns over time. Weighted gene co-expression network and motif enrichment analyses identified specific groups of genes potentially regulating wheat grain development. The embryo and surrounding endosperm specifically expressed transcription factor TaABI3-3B negatively regulates embryo and grain size. In Chinese breeding programs, a haplotype associated with higher grain weight was identified, linked to altered expression levels of TaABI3-3B. Data and knowledge obtained from the proposed study will provide pivotal insights into yield improvement and serve as important genetic information for future wheat breeding.
In the animal kingdom, evolutionarily conserved mechanisms known as cell competition eliminate unfit cells during development. Interestingly, cell competition also leads to apoptosis of donor cells upon direct contact with host cells from a different species during interspecies chimera formation. The mechanisms underlying how host animal cells recognize and transmit cell death signals to adjacent xenogeneic human cells remain incompletely understood. In this study, we developed an interspecies cell contact reporter system to dissect the mechanisms underlying competitive interactions between mouse and human pluripotent stem cells (PSCs). Through single-cell RNA-seq analyses, we discovered that Ephrin A ligands in mouse cells play a crucial role in signaling cell death to adjacent human cells that express EPHA receptors during interspecies PSC co-culture. We also demonstrated that blocking the Ephrin A-EPHA receptor interaction pharmacologically, and inhibiting Ephrin forward signaling genetically in the mouse cells, enhances the survival of human PSCs and promotes chimera formation both in vitro and in vivo. Our findings elucidate key mechanisms of interspecies PSC competition during early embryogenesis and open new avenues for generating humanized tissues or organs in animals, potentially revolutionizing regenerative medicine.
Integrins consist of 24 species, each with unique tissue-expression profiles and distinct biological functions. The {beta} subunit of integrin interacts with the FERM-folded head domain of talin through an N-P-x-Y/F motif, triggering integrin activation. Although this motif is conserved across most integrin-{beta} subunits, the precise molecular mechanisms governing talins selective recognition of different integrin species remains unclear. We determined the crystal structure of talin head in complex with the {beta}2-integrin tail. The structure reveals a two-mode configuration featuring a "rocking" motion of the talin head FERM domain compared with its interaction with {beta}3 integrin, resulting in distinct inter-subdomain interactions and unique cavities. Switching of the talin:{beta}2 binding mode to the talin:{beta}3 binding mode enhances {beta}2-integrin affinity and boosts LFA-1-mediated natural killer cell cytotoxicity. Moreover, stabilizing of the C-terminal -helix in the talin head enhances its affinity to integrin and its activation. Together, our data elucidate the structural basis by which talin orchestrates its function in mediating integrin activation in a species-specific manner. Significance statementTalin exhibits significantly lower affinity with lymphocyte-rich {beta}2 integrins compared with {beta}3 integrins. Our results unveil the configurational preferences of the talin head when engaged {beta}2 and {beta}3 integrins. We introduce a two-mode seesaw model wherein the talin head adapts specific binding modes in response to distinct integrin species. The two configurations differ in inter-subdomain interactions, revealing unique cavities and distinct binding dynamics in each binding mode. Thus, our findings present exciting opportunities of the development of species-specific therapeutic agents targeting integrin activity more precisely by orchestrating the structural dynamics of talin.
The cabbage aphid, Brevicoryne brassicae, is a major pest on Brassicaceae plants, and causes significant yield losses annually. However, lacking genomic resources hinders the progress in understanding this pest at the level of molecular biology. Here, a high-quality, chromosomal-level genome was assembled for B. brassicae based on PacBio HIFI long-read sequencing and Hi-C data. The final assembled size was 429.99 Mb with a scaffold N50 of 93.31 Mb. Importantly, 96.2% of the assembled sequences were anchored to eight chromosomes. The genome recovered 98.50% of BUSCO genes and 92.30% of CEGMA genes, supporting the high level of completeness. By combining high-coverage transcriptome data, a total of 22,671 protein-coding genes and 3,594 lncRNA genes were annotated. Preliminary comparative genomic analyses were focused on genes related to host colonisation, such as chemosensory- and detoxification-related genes, as well as those encoding putative protein effectors and cross-kingdom lncRNA Ya. In summary, our study presents a contiguous and complete genome for B. brassicae that will benefit our understanding of the biology of it and other aphids.
BackgroundNK cells are being extensively studied as a cell therapy for cancer. Their effector functions are induced by the recognition of ligands on tumor cells and by various cytokines. IL-15 is broadly used to stimulate endogenous and adoptively transferred NK cells in cancer patients. These stimuli activate the membrane protease ADAM17, which then cleaves assorted receptors on the surface of NK cells as a negative feedback loop to limit their activation and function. We have shown that ADAM17 inhibition can enhance IL-15-mediated NK cell proliferation in vitro and in vivo. In this study, we investigated the underlying mechanism of this process. MethodsPBMCs or enriched NK cells from human peripheral blood, either unlabeled or labeled with a cell proliferation dye, were cultured for up to 7 days in the presence of rhIL-15 +/- an ADAM17 function-blocking antibody. Different versions of the antibody were generated; Medi-1 (IgG1), Medi-4 (IgG4), Medi-PGLALA, Medi-F(ab')2, and TAB16 (anti-ADAM17 and anti-CD16 bispecific) to modulate CD16A engagement on NK cells. Flow cytometry was used to assess NK cell proliferation and phenotypic markers, immunoblotting to examine CD16A signaling, and IncuCyte-based live cell imaging to measure NK cell anti-tumor activity. ResultsThe ADAM17 function-blocking mAb Medi-1 markedly increased initial NK cell activation by IL-15. Using different engineered versions of the antibody revealed that the activating Fc{gamma} receptor CD16A, a well-described ADAM17 substrate, was critical for enhancing IL-15 stimulation. Hence, Medi-1 bound to ADAM17 on NK cells can be engaged by CD16A and block its shedding, inducing and prolonging its signaling. This process did not promote evident NK cell fratricide, phagocytosis, or dysfunction. Synergistic activity by Medi-1 and IL-15 enhanced the upregulation of CD137 on CD16A+ NK cells and augmented their proliferation in the presence of PBMC accessory cells. ConclusionsOur data reveal for the first time that CD16A and CD137 underpin Medi-1 enhancement of IL-15-driven NK cell activation and proliferation, respectively. The use of Medi-1 represents a novel strategy to enhance IL-15-driven NK cell proliferation, and it may be of therapeutic importance by increasing the anti-tumor activity of NK cells in cancer patients. What is already known on this topicNK cell therapies are being broadly investigated to treat cancer. NK cell stimulation by IL-15 prolongs their survival in cancer patients. Various stimuli including IL-15 activate ADAM17 in NK cells, a membrane protease that regulates the cell surface density of various receptors as a negative feedback mechanism. What this study addsTreating NK cells with the ADAM17 function-blocking mAb Medi-1 markedly enhanced their activation and proliferation. Our study reveals that the Fc and Fab regions of Medi-1 function synergistically with IL-15 in NK cell activation. Medi-1 treatment augments the upregulation of CD137 by NK cells, which enhances their proliferation in the presence of PBMC accessory cells. How this study might affect research, practice, or policyOur study is of translational importance as Medi-1 treatment in combination with IL-15 could potentially augment the proliferation and function of endogenous or adoptively transferred NK cells in cancer patients. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=177 HEIGHT=200 SRC="FIGDIR/small/593347v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1bf8a19org.highwire.dtl.DTLVardef@39315org.highwire.dtl.DTLVardef@ad5925org.highwire.dtl.DTLVardef@b798cb_HPS_FORMAT_FIGEXP M_FIG C_FIG
Serotonylation has been identified as a novel protein post-translational modification for decades, where an isopeptide bond is formed between the glutamine residue and serotonin through transamination. Transglutaminase 2 (also known as TGM2 or TGase2) was proven to act as the main "writer" enzyme for this PTM and a number of key regulatory proteins (including small GTPases, fibronectin, fibrinogen, serotonin transporter, and histone H3) have been characterized as the substrates of serotonylation. However, due to the lack of pan-specific antibodies for serotonylated glutamine, the precise enrichment and proteomic profiling of serotonylation still remain challenging. In our previous research, we developed an aryldiazonium probe to specifically label protein serotonylation in a bioorthogonal manner, which depended on a pH-controlled chemoselective rapid azo-coupling reaction (CRACR). Here, we report the application of a photoactive aryldiazonium-biotin probe for the global profiling of serotonylation proteome in cancer cells. Thus, over 1,000 serotonylated proteins were identified from HCT 116 cells, many of which are highly related to carcinogenesis. Moreover, a number of modification sites of these serotonylated proteins were determined, attributed to the successful application of our chemical proteomic approach. Overall, these findings provided new insights into the significant association between cellular protein serotonylation and cancer development, further suggesting that to target TGM2-mediated monoaminylation may serve as a promising strategy for cancer therapeutics. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/593574v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@67e538org.highwire.dtl.DTLVardef@f9f722org.highwire.dtl.DTLVardef@cfe5fdorg.highwire.dtl.DTLVardef@104674f_HPS_FORMAT_FIGEXP M_FIG C_FIG
The fruitlets of loquat (Eriobotrya japonica Lindl.) are susceptible to freezing injury due to their developmental cycle encountering winter. Freezing stress severely damages the fruitlets, resulting in loss of fruit yield and quality. Studies have shown that Ca2+, as a second messenger, is involved in signal transduction in loquat fruitlets under freezing stress. However, the mechanism of downstream calcium signal transduction in loquat fruitlets under freezing stress is currently unclear. Calcium-depend protein kinase (CDPK) as the most particular calcium sensor family in plants, play an important role in multiple stress signal transduction including freezing. In this study, we identified the loquat CDPK family on a genome-wide scale. A total of 34 EjCDPK genes were identified and studied for basic structural and phylogenetic features. EjCDPKs can be divided into four subgroups phylogenetically. The patterns of exon-intron and protein motif are highly conserved among the subgroups. Collinearity analysis identified several segmental duplicate events in EjCDPK family. RNA-seq based transcription analysis indicated that partial of EjCDPKs differently expressed in response to freezing stress with tissue-specific. Moreover, we preformed correlation analysis between expression value and trait data of loquat fruitlet under freezing stress by weighted co-expression gene network. After that, EjCDPK25 was selected as the candidate because of its potential freezing stress response function. Protein kinase related GO terms were enriched in EjCDPK25 co-expression genes, and then QPCR was performed to examine the target genes expression pattern. In addition, EjCDPK25 was cloned to construct overexpression vector to obtain transgenic Arabidopsis plants. Transgenic and wild-type Arabidopsis were suffered freezing stress treatments (-5{degrees}C). The results showed that the survival rate of EjCDPK25 overexpressing transgenic Arabidopsis was significantly higher than WT. In summary, this study identified loquat CDPK family firstly, and our data provide significant insights into the evolution and function of loquat CDPKs. Above all, a freezing stress response gene EjCDPK25 was verified can increase the resistance of freezing stress in Arabidopsis.
Arbuscular mycorrhizal fungi (AMF) are obligate biotrophs that rely on symbiotic carbohydrates and, in particular, lipids derived from their host plants. However, it remains unclear whether symbiotic AMF can access exogenous non-symbiotic lipids in the presence of plant-derived carbon, complicating our understanding of their relationship with host plants. Here, we investigated the direct uptake of exogenous 13C1-labeled myristate by three symbiotic AMF species (Rhizophagus irregularis, R. intraradices, and R. diaphanous) and assessed their growth responses using AMF-carrot hairy root co-culture systems. Furthermore, we explored the environmental distribution of myristate, and evaluated the impact of exogenous myristate on the carbon-phosphorus exchange between R. irregularis and alfalfa or rice in a greenhouse experiment. Our results showed that symbiotic AMF can absorb exogenous myristate, as evidenced by 13C enrichment and transcriptional activation of fatty acid transport and metabolism genes in AMF extraradical hyphae. Myristate is commonly present in various soil and plant environments, and its application increased both intraradical and extraradical fungal biomass, possibly linked to suppressed mycorrhizal-activated defense responses in host roots. Unexpectedly, exogenous myristate reduced the mycorrhizal phosphorus benefits for both alfalfa and rice and decreased their symbiotic carbon allocation to root-colonizing AMF, suggesting that the application of exogenous myristate may not be a promising strategy to enhance AM symbiosis. These findings provide new insights into understanding and manipulating the nutritional interactions between AMF and host plants.
Imaging large cleared tissues requires scaling the throughput of imaging techniques. Here we introduce curved light sheet microscope to perform optical sectioning with curved light sheets. This concept addresses the long-standing field curvature problem and lowers the barriers in designing high-throughput objectives. Leveraging a customized objective, the curved light sheet microscope achieves diffraction-limited resolution (1.0 m laterally and 2.5 m axially) and uniform contrast over a field of view of more than 1 x 1 cm2 and is compatible with various tissue clearing techniques. Imaging an entire intact cleared mouse brain at 0.625 x 0.625 x 1.25 m3 voxel size takes less than 3 hours and image tiling is not required. We share full optics description of the objective and report imaging neuronal and vascular networks and tracing brain-wide long-distance axonal projections in intact mouse brains.
Histone dopaminylation is a newly identified epigenetic mark that plays a role in the regulation of gene transcription, where an isopeptide bond is formed between the fifth amino acid residue of H3 (i.e., glutamine) and dopamine. In our previous studies, we discovered that the dynamics of this post-translational modification (including installation, removal, and replacement) were regulated by a single enzyme, transglutaminase 2 (TGM2), through reversible transamination. Recently, we developed a chemical probe to specifically label and enrich histone dopaminylation via bioorthogonal chemistry. Given this powerful tool, we found that histone H3 glutamine 5 dopaminylation (H3Q5dop) was highly enriched in colorectal tumors, which could be attributed to the high expression level of TGM2 in colon cancer cells. Due to the enzyme promiscuity of TGM2, non-histone proteins have also been identified as targets of dopaminylation on glutamine residues, however, the dopaminylated proteome in cancer cells still remains elusive. Here, we utilized our chemical probe to enrich dopaminylated proteins from colorectal cancer cells in a bioorthogonal manner and performed the chemical proteomics analysis. Therefore, 425 dopaminylated proteins were identified, many of which are involved in nucleic acid metabolism and transcription pathways. More importantly, a number of modification sites of these dopaminylated proteins were identified, attributed to the successful application of our chemical probe. Overall, these findings shed light on the significant association between cellular protein dopaminylation and cancer development, further suggesting that to block the installation of protein dopaminylation may become a promising anti-cancer strategy. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/591460v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1a3e9dorg.highwire.dtl.DTLVardef@104ecddorg.highwire.dtl.DTLVardef@5ab948org.highwire.dtl.DTLVardef@3a5d8b_HPS_FORMAT_FIGEXP M_FIG C_FIG