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Ni, W.

Publications and source records attributed to Ni, W..

15 recordsLinked to original sources

Oncostatin M-driven leak in human endothelial cells with preserved tight junctions

Blood vessels must preserve blood volume while permitting exchange of nutrients. Endothelial cells line the lumen of all blood vessels and form the intercellular junctions that govern vascular permeability. In acute critical illness the barrier fails, fluid escapes into tissues, and the resulting loss of volume and impaired nutrient exchange cause organ failure. Several inflammatory cytokines are known to disrupt junctions in cultured cells, yet therapies targeting these models have failed. How blood vessels respond to critical illness has remained largely elusive because endothelial cells are generally inaccessible. Here we profile endothelial cells recovered directly from critically ill children to show that oncostatin M drives a previously unrecognized mode of vascular leak. Oncostatin M increases permeability by forming narrow, nanoscale channels within the overlapping borders of adjacent cells, around intact tight junctions. These channels permit passage of small and intermediate sized solutes. Their formation depends on non-canonical signaling in which the kinase JAK1 activates two required parallel pathways (via ERK1 and AKT1) that converge on the transcription factor c-Fos. Inhibiting JAK1, either parallel pathway or depleting c-Fos, prevents oncostatin M-induced loss of barrier function. These results identify a permeability mechanism distinct from the junction disassembly driven by the cytokines that have dominated this field and implicate kinases already targeted by approved drugs as potential points of intervention. More broadly, vascular barriers are transiently loosened during immune-cell trafficking and metastasis, raising the possibility that diverse forms of vascular permeability arise through regulated nanoscale remodeling rather than junctional breakdown.

cell biology↗

Proteomic Analysis Reveals Widespread Regulation of Substrate Protein Abundance by O-fucosylation and O-GlcNAcylation

O-glycosylation of nucleocytosolic proteins by the Arabidopsis enzymes SPINDLY (SPY; O-fucosyltransferase) and SECRET AGENT (SEC; O-GlcNAc transferase) is essential for plant growth and development, yet the scope of their substrates and regulatory impact remains poorly defined. Here, we combined TurboID-based proximity labeling with quantitative proteomics to systematically map the SPY interactome and determine how SPY- and SEC-dependent modifications influence protein abundance. A functional SPY-TD enriched 221 proxiome proteins, including 80 known O-fucosylated substrates and 141 new interactors. The SPY-TD proxiome is enriched in nuclear pore components, chromatin regulators, transcription factors, and RNA-processing proteins. Integration with O-fucose and O-GlcNAc datasets yielded a comprehensive Arabidopsis SPY/SEC (At-S/S) protein list of 886 candidates. We quantified proteome-wide changes in spy single mutants and inducible spy sec double mutants. Loss of SPY alone caused selective stabilization or destabilization of targets, whereas combined SPY/SEC depletion triggered widespread, synergistic protein abundance changes, particularly affecting nucleoporins, transcriptional regulators, and RNA-binding proteins. Integration with ubiquitination datasets revealed extensive overlap, supporting potential crosstalk between O-fucosylation, O-GlcNAcylation, and ubiquitin-mediated protein turnover. Together, our study establishes proximity labeling as a powerful strategy to define plant O-glycosylation networks and reveals dual, context-dependent roles of SPY and SEC in controlling protein homeostasis and stress-responsive pathways.

systems biology↗

Blind Prediction of Complex Water and Ion Ensembles Around RNA in CASP16

Biomolecules rely on water and ions for stable folding, but these interactions are often transient, dynamic, or disordered and thus hidden from experiments and evaluation challenges that represent biomolecules as single, ordered structures. Here, we compare blindly predicted ensembles of water and ion structure to the cryo-EM densities observed around the Tetrahymena ribozyme at 2.2-2.3 [A] resolution, collected through target R1260 in the CASP16 competition. 26 groups participated in this solvation cryo-ensemble prediction challenge, submitting over 350 million atoms in total, offering the first opportunity to compare blind predictions of dynamic solvent shell ensembles to cryo-EM density. Predicted atomic ensembles were converted to density through local alignment and these densities were compared to the cryo-EM densities using Pearson correlation, Spearman correlation, mutual information, and precision-recall curves. These predictions show that an ensemble representation is able to capture information of transient or dynamic water and ions better than traditional atomic models, but there remains a large accuracy gap to the performance ceiling set by experimental uncertainty. Overall, molecular dynamics approaches best matched the cryo-EM density, with blind predictions from bussilab_plain_md, SoutheRNA, bussilab_replex, coogs2, and coogs3 outperforming the baseline molecular dynamics prediction. This study indicates that simulations of water and ions can be quantitatively evaluated with cryo-EM maps. We propose that further community-wide blind challenges can drive and evaluate progress in modeling water, ions and other previously hidden components of biomolecular systems.

biophysics↗

Testicular mRNA-LNP Delivery: A Novel Therapy for Genetic Spermatogenic Disorders

Uniform testicular maturation arrest is a severe form of male infertility characterized by the presence of germ cells that do not complete spermatogenic development. It is usually caused by meiotic arrest with genetic variants and difficult to treat via drugs or surgery. mRNA-lipid nanoparticle (LNP) delivery is a promising therapeutic option for maturation arrest with monogenic variants via protein replacement therapy. Herein, a spermatocytes-tropic LNP (Pool1-LNP3) was identified via a library of 30 ionizable lipids screening. And in vivo delivery of this novel LNP composition using rete testis microinjection was showed to be high spermatocytes targeting with high transfection efficiency. Thereafter, it was revealed that in vivo delivery of Pool1-LNP3 encapsulating Msh5 mRNA could promote crossover formation and restore spermatogenesis in Msh5D486Y/D486Y mouse models with DSB recombination defects. Notably, the offspring without genomic integration was born using intracytoplasmic sperm injection (ICSI) derived from rescue of Msh5D486Y/D486Ymouse and embryo transfer. Furthermore, no obvious inflammation and histologic damage in any tissue were detected after in vivo delivery of mRNA-LNP. In addition, it was demonstrated that Maps mRNA-LNP3 recovered spermatogenesis in Maps KO mouse with meiotic arrest. Altogether, these findings suggested that this spermatocytes-tropic mRNA-LNP delivery could become a viable and broad applicable strategy for treatment of spermatogenic disorders with genetic defects, providing a foundation for future clinical application.

molecular biology↗

Jumbo phages possess independent synthesis and utilization systems of NAD+

Jumbo phages, phages with genomes >200 kbp, contain some unique genes for successful reproduction in their bacterial hosts. Due to complex and massive genomes analogous to those of small-celled bacteria, how do jumbo phages complete their life cycle remain largely undefined. In this study, we assembled 668 high-quality jumbo phage genomes from over 15 TB of intestinal metagenomic data from 955 samples of five animal species (cow, sheep, pig, horse, and deer). Within them, we obtained a complete genome of 716 kbp in length, which is the largest phage genome so far reported in the gut environments. Interestingly, 174 out of the 668 jumbo phages were found to encode all genes required for synthesis of NAD+ by the salvage pathway or Preiss-Handler pathway, referred as NAD-jumbo phage. Besides synthesis genes of NAD+, these NAD-jumbo phages also encode at least 15 types of NAD+-consuming enzyme genes involved in DNA replication, DNA repair, and counterdefense, suggesting that these phages not only have the capacity to synthesize NAD+ but also redirect NAD+ metabolism towards phage propagation need in hosts. Phylogenetic analysis and environmental survey indicated NAD-jumbo phages are widely present in the Earths ecosystems, including the human gut, lakes, salt ponds, mine tailings, and seawater. In summary, this study expands our understanding of the diversity and survival strategies of phages, and in-depth study of the NAD-jumbo phages is crucial for understanding their role in ecological regulation.

microbiology↗

Workflow enhancement of TurboID-mediated proximity labeling for SPY signaling network mapping

TurboID-based proximity labeling coupled to mass spectrometry (PL-MS) has emerged as a powerful tool for mapping protein-protein interactions in both plant and animal systems. Despite advances in sensitivity, PL-MS studies can still suffer from false negatives, especially when dealing with low abundance bait proteins and their transient interactors. Protein-level enrichment for biotinylated proteins is well developed and popular, but direct detection of biotinylated proteins by peptide-level enrichment and the difference in results between direct and indirect detection remain underexplored. To address this gap, we compared and improved enrichment and data analysis methods using TurboID fused to SPY, a low-abundance O-fucose transferase, using an AAL-enriched SPY target library for cross-referencing. Our results showed that MyOne and M280 streptavidin beads significantly outperformed antibody beads for peptide-level enrichment, with M280 performing best. In addition, while a biotin concentration [≤] 50 {micro}M is recommended for protein-level enrichment in plants, higher biotin concentrations can be used for peptide-level enrichment, allowing us to improve detection and data quality. FragPipes MSFragger protein identification and quantification software outperformed Maxquant and Protein Prospector for SPY interactome enrichment due to its superior detection of biotinylated peptides. Our improved washing protocols for protein-level enrichment mitigated bead collapse issues, improving data quality, and reducing experimental time. We found that the two enrichment methods provided complementary results and identified a total of 160 SPY-TurboID-enriched interactors, including 60 previously identified in the AAL-enriched SPY target list and 100 additional novel interactors. SILIA quantitative proteomics comparing WT and spy-4 mutants showed that SPY affects the protein levels of some of the identified interactors, such as nucleoporin proteins. We expect that our improvement will extend beyond TurboID to benefit other PL systems and hold promise for broader applications in biological research.

molecular biology↗

T2T-YAO: a Telomere-to-telomere Assembled Diploid Reference Genome for Han Chinese

Since its initial release in 2001, the human reference genome has been continuously improved in both continuity and accuracy, and the recently-released telomere-to-telomere version--T2T-CHM13--reaches its top quality after 20 years of effort. However, T2T-CHM13 does not represent an authentic diploid human genome, but rather one derived from a simplified, nearly homozygous genome of a hydatidiform mole cell line. To address this limitation and provide an alternative pertinent to the Chinese population, the largest ethnic group in the world, we have assembled a complete diploid human genome of a male Han Chinese, T2T-YAO, which includes telomere-to-telomere assemblies for all the 22+X+M and 22+Y chromosomes in his two haploids inherited separately from his parents. Both haplotypes contain no artificial sequences or model nucleotides and possess a high quality comparable to CHM13, with fewer than one error per [~]14 Mb. Derived from the individual who lives in the aboriginal region of Han Chinese, T2T-YAO shows clear ancestry and potential genetic continuity from the ancient ancestors of the Han population. Each haplotype of T2T-YAO possesses [~]340 Mb exclusive sequences and [~]3100 unique genes as compared to CHM13, and their genome sequences show greater genetic distance to CHM13 than to each other in terms of nucleotide polymorphism and structural variations. The construction of T2T-YAO would serve as a high-quality diploid reference that enables precise delineation of genomic variations in a haplotype-sensitive manner, which could advance our understandings in human evolution, hereditability of diseases and phenotypes, especially within the context of the unique variations of the Chinese population.

genetics↗

SARS-CoV-2 S protein antagonizes type I interferon downstream signal pathway through interacting and attenuating phosphorylation of STAT1/STAT2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may keep patients in a clinically asymptomatic state by blocking cellular innate antiviral immunity, but the molecular mechanism remains unclear. Here, we screened the viral proteins of SARS-CoV-2 and found that the spike (S) protein inhibits the activation of interferon-stimulated genes (ISGs) and even reduces the expression of these genes to below background values. Mechanistically, the S protein interacted with STAT1, STAT2, and IRF9 and impedes the phosphorylation of STAT1/STAT2, thus preventing the formation of the interferon-stimulating gene factor 3 (ISGF3) complex and inhibiting the downstream production of Interferon-stimulated genes (ISGs). Remarkably, we also have found that the inhibitory mechanism of the S protein was conservative among SARS-CoV-2 variants and other human coronaviruses, including SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63, and HCoV-HKU1. Truncation studies indicated that the most conserved S2 domain played a major inhibitory role. Altogether, our findings unveil a new mechanism by which SARS-CoV-2 S protein attenuated the hosts antiviral immune response and provide new insights into the pathogenic mechanism of coronavirus.

microbiology↗

The addition of antibiotics to embryo culture media caused altered expression of genes in pathways governing DNA integrity in mouse blastocysts

Antibiotics are common components of embryo culture media and minimize the risk of microbial contamination and infection during assisted reproductive technology procedures (ART). The effects of two aminoglycoside antibiotics (gentamicin, streptomycin) and penicillin on the global profiles of gene expression (DE) were assessed by RNA-seq of individual mouse blastocysts. Zygotes were cultured in an optimized defined medium formulation (KSOM) to which a dose range of each antibiotic was added. A dose-dependent retardation of the rate of zygote development to morphologically normal blastocyst was observed and this was accompanied by a reduction in the number of cells present within the resulting blastocysts. The lowest dose of each antibiotic tested (similar to the concentrations used in clinical grade media) caused significant differential expression of approximately 1800 genes. In most cases antibiotic treatment caused a reduction in gene expression and gene ontology analysis showed that down regulated genes were enriched for several biological processes related to the maintenance of genomic integrity. All three antibiotics caused the downregulation of Brca2, Blm, Rad51c and Rad54l, genes involved DNA homologous recombination pathways and also several p53-dependent genes. Immunolocalization studies showed that each antibiotic also reduced level of BRCA2 and RAD51C detected within blastocysts. The present study shows that the supplementing embryo culture media with antibiotics is associated with wide ranging alterations in gene expression in a manner that could potentially compromise the genomic integrity of the resulting embryos.

developmental biology↗

Construction of a potent pan-vaccine based on the evolutionary tendency of SARS-CoV-2 spike protein.

SARS-CoV-2 continued to spread globally along with different variants. Here, we systemically analyzed viral infectivity and immune-resistance of SARS-CoV-2 variants to explore the underlying rationale of viral mutagenesis. We found that the Beta variant harbors both high infectivity and strong immune resistance, while the Delta variant is the most infectious with only a mild immune-escape ability. Remarkably, the Omicron variant is even more immune-resistant than the Beta variant, but its infectivity increases only in Vero E6 cells implying a probable preference for the endocytic pathway. A comprehensive analysis revealed that SARS-CoV-2 spike protein evolved into distinct evolutionary paths of either high infectivity plus low immune resistance or low infectivity plus high immune resistance, resulting in a narrow spectrum of the current single-strain vaccine. In light of these findings and the phylogenetic analysis of 2674 SARS-CoV-2 S-protein sequences, we generated a consensus antigen (S6) taking the most frequent mutations as a pan-vaccine against heterogeneous variants. As compared to the ancestry SWT vaccine with significantly declined neutralizations to emerging variants, the S6 vaccine elicits broadly neutralizing antibodies and full protections to a wide range of variants. Our work highlights the importance and feasibility of a universal vaccine strategy to fight against antigen drift of SARS-CoV-2.

microbiology↗

A Population-level Strain Genotyping Method to Study Pathogen Strain Dynamics in Human Infections

A hallmark of chronic bacterial infections is the long-term persistence of one or more pathogen species at the compromised site. Repeated detection of the same bacterial species can suggest that a single strain or lineage is continually present. However, infection with multiple strains of a given species, strain acquisition and loss, and changes in strain relative abundance can occur. Detecting strain-level changes and their effects on disease is challenging as most methods require labor intensive isolate-by-isolate analyses, thus, only a few cells from large infecting populations can be examined. Here we present a population-level method for enumerating and measuring the relative abundance of strains called "PopMLST". The method exploits PCR amplification of strain-identifying polymorphic loci, next-generation sequencing to measure allelic variants, and informatic methods to determine whether variants arise from sequencing errors or low abundance strains. These features enable PopMLST to simultaneously interrogate hundreds of bacterial cells that are either cultured en masse from patient samples, or are present in DNA directly extracted from clinical specimens without ex vivo culture. This method could be used to detect epidemic or super-infecting strains, facilitate understanding of strain dynamics during chronic infections, and enable studies that link strain changes to clinical outcomes.

microbiology↗

Dependency of LKB1-inactivated lung cancer on aberrant CRTC-CREB activation

Lung cancer with loss-of-function of the LKB1 tumor suppressor is a common aggressive subgroup with no effective therapies. LKB1-deficiency induces constitutive activation of cAMP/CREB-mediated transcription by a family of three CREB-regulated transcription coactivators (CRTC1-3). However, the significance and mechanism of CRTC activation in promoting the aggressive phenotype of LKB1-null cancer remain poorly characterized. Here we observed overlapping CRTC expression patterns and mild growth phenotypes of individual CRTC-knockouts in lung cancer, suggesting functional redundancy of CRTC1-3. We consequently designed a dominant-negative mutant (dnCRTC) to block all three CRTCs to bind and co-activate CREB. Expression of dnCRTC efficiently inhibited the aberrantly activated cAMP/CREB-mediated oncogenic transcriptional program induced by LKB1-deficiency, and specifically blocked the growth of LKB1-inactivated lung cancer. Collectively, this study provides direct proof for an essential role of the CRTC-CREB activation in promoting the malignant phenotypes of LKB1-null lung cancer and proposes the CRTC-CREB interaction interface as a novel therapeutic target.

cancer biology↗

Arabidopsis ACINUS is O-glycosylated and regulates transcription and alternative splicing of regulators of reproductive transitions

O-GlcNAc modification plays important roles in metabolic regulation of cellular status. Two homologs of O-GlcNAc transferase, SECRET AGENT (SEC) and SPINDLY (SPY), which have O-GlcNAc and O-fucosyl transferase activities, respectively, are essential in Arabidopsis but have largely unknown cellular targets. Here we show that AtACINUS is O-GlcNAcylated and O-fucosylated and mediates regulation of transcription, alternative splicing (AS), and developmental transitions. Knocking-out both AtACINUS and its distant paralog AtPININ causes severe growth defects including dwarfism, delayed seed germination and flowering, and abscisic acid (ABA) hypersensitivity. Transcriptomic and protein-DNA/RNA interaction analyses demonstrate that AtACINUS represses transcription of the flowering repressor FLC and mediates AS of ABH1 and HAB1, two negative regulators of ABA signaling. Proteomic analyses show AtACINUSs O-GlcNAcylation, O-fucosylation, and association with splicing factors, chromatin remodelers, and transcriptional regulators. Some AtACINUS/AtPININ-dependent AS events are altered in the sec and spy mutants, demonstrating a function of O-glycosylation in regulating alternative RNA splicing.

molecular biology↗

The regulation of mammalian maternal-to-embryonic transition by Eukaryotic translation initiation factor 4E

Genetic and inhibitor studies show expression of eukaryotic translation initiation factor 4E (eIF4E) was required for the successful maternal-to-embryonic transition of mouse embryos. eIF4E was in both gametes and in the cytoplasm and pro-nuclei soon after fertilization, and at each stage of early development. Knockout (Eif4e-/-) by PiggyBac (PB) [Act-RFP] transposition caused peri-implantation embryonic lethality due to the failure of embryos to form a pluripotent epiblast. Maternal stores of eIF4E supported development up to the 2-4-cell stage after which new expression occurred from both alleles. Inhibition of the maternally acquired stores of eIF4E (4EGI-1 inhibitor) resulted in a developmental block at the 2-4-cell stage. 4E-BP1 is a hypophosphorylation-dependent negative regulator of eIF4E. mTOR activity was required for 4E-BP1 phosphorylation and inhibiting 4EGI-1 retarded embryo development. eIF4E expression and activity is regulated at key embryonic transitions in the mammalian embryo and is essential for successful transition to embryonic control of development. Significance StatementeIF4E is recognized as the rate-limiting factor for CAP-dependent translation. This work used a combination of a gene knockout model, selective pharmacological inhibition and expression analyses to investigate the expression and function of Eif4e in the early mouse embryo. It provides compelling evidence for the essential role of Eif4E in the normal processes of early mammalian embryo development, including the formation of the pluripotent epiblast and the maternal-embryonic transition. The unexpected evidence for a growth deficit in mice hypomorphic for Eif4e will be a key area of future investigation. It also provides for the first time a powerful demonstration of the utility of the PB [Act-RFP] transposon mouse model for analyzing the molecular regulation of early mammalian embryo development.

developmental biology↗

2D-HELS-AA MS Seq: Direct sequencing of tRNA reveals its different isoforms and multiple dynamic base modifications

We report a direct method for sequencing tRNAPhe without cDNA by combining 2-dimensional hydrophobic RNA end-labeling with an anchor-based algorithm in mass spectrometry-based sequencing (2D-HELS-AA MS Seq). The entire tRNAPhe was sequenced and the identity, location and abundance of all 11 base modifications were determined. Changes in ratios of wybutosine and its depurinated form under different conditions were quantified, pointing to the ability of our technology to determine dynamic changes of nucleotide modifications. Two truncated isoforms at 3CCA tail of the tRNAPhe (75 nt CC, 80% and 74 nt C, 3%) were identified in addition to the 76 nt tRNAPhe with a full-length 3CCA tail (17%). We also discovered a new isoform with A-G transitions at both the 44 and 45 positions in the tRNAPhe variable loop.\n\nOne Sentence SummaryDirect 2D-HELS-AA MS Seq of tRNA reveals different isoforms and base modifications

biochemistry↗