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Ying, L.

Publications and source records attributed to Ying, L..

15 recordsLinked to original sources
bioRxiv2025

The U1 snRNP protein U1C and Helix H of U1 snRNA are critical for small molecule splicing modulator function

Risdiplam and branaplam represent two classes of small-molecule splicing modulators that promote U1 snRNP recognition of weak non-canonical GA/GU-containing 5 splice sites (ss). We demonstrated that branaplam enhanced recognition of these 5 ss by reconstituted U1 snRNP in vitro, and that this effect depended on the ZnF domain of U1C and Helix H of U1 snRNA, but not U1A or U1-70K. In cells, depletion of U1C generally reduced compound-induced exon inclusion for most cassette exons. Interestingly, a subset of cassette exons became responsive to compound only upon U1C knockdown, supporting a model in which U1C stabilizes specific conformations at the 5 ss/U1 snRNA interface in a context-dependent manner that can either facilitate or hinder compound binding. Surprisingly, risdiplam shows no effect on weak 5 ss recognition in vitro, suggesting additional cellular factors are required for its activity.

biochemistry↗
bioRxiv2025

Liposome Encapsulation Enables Near-Native Cryo-EM Structural Determination by Shielding Macromolecules from Non-Physiological Interfaces

Cryo-electron microscopy (cryo-EM) has emerged as a very powerful tool for high-resolution structure determination of macromolecule complexes. However, sample preparation remains a major bottleneck in cryo-EM workflows. Most structures were resolved not in a native solution environment, but rather in the non-physiological interfaces such as the air-water interface (AWI), where the folding/unfolding energy landscape of macromolecules may be significantly altered, leading to artifact, preferential orientation, particle disassembly or even denaturation. To address this challenge, we developed a robust, sample-independent method utilizing liposome encapsulation that preserve macromolecules in a solution-like and near native environment throughout sample preparation. Using equine spleen apoferritin and the Escherichia coli (E. coli) ribosome as model systems, we demonstrate efficient particle incorporation into liposomes and successful high-resolution structure determination. Notably, our method yields significantly reduced particle disassembly, denaturation, and preferential orientation compared to samples on holey carbon and graphene grids. We anticipate that this approach will facilitate structural studies of other challenging macromolecular complexes that are sensitive to interfacial effects.

biophysics↗
bioRxiv2025

Mind the corner: Fillets in cryo-FIB lamella preparation to minimise sample loss caused by stress concentration and lamella breakage

Cryo-FIB milling of biological specimens is a critical and limiting step in the cryo-electron tomography workflow. Preparing electron-transparent cryo-lamellae is a serial, low-throughput process. Even with automation, a skilled operator can typically only produce 15-25 lamellae in a single cryo-FIB session. During sample handling, milling and transfer, the cryo-fixed cells as well as the supporting film layer face various mechanical forces and thermal stresses due to temperature fluctuations. Moreover, after cells are cryo-FIB milled, the resulting thin lamellae continue to endure external forces from mechanical handling and thermal stress. We propose a simple, yet highly effective modification to the standard rectangular milling pattern by implementing "fillets" or corner smoothing providing better mechanical stability. This adjustment helps to avoid sharp corners at the lamella edges, thereby reducing stress concentration. As a result, this modification decreases the likelihood of lamella breakage and improves the overall yield of ready-for-TEM lamellae by 33% as verified experimentally.

biochemistry↗
bioRxiv2025

Malignant ascites enhance γδ T cell cytotoxicity towards ovarian cancer via modulating chemokines secretion from the cancer cells that recruits γδ T cells

Ovarian cancer patients usually develops peritoneal metastasis and malignant ascites in the advanced stages, which form immuno-suppressive tumor microenvironments that limit the efficacy of immuno-therapies. However, during our previous research trying to develop a {gamma}{delta} T cell-based cell therapy, we noticed that the malignant ascites may enhance the cytotoxicity of {gamma}{delta} T cells towards ovarian cancer cells. Herein, in this work we showed that the phenomenon is real and the low molecular weight components in the ascites act on the cancer to promote the killing by {gamma}{delta} T cells. Transcriptome analysis and in vitro experiments revealed that the malignant ascites induce the secretion of chemokines CXCL2 and CXCL8 by ovarian cancer cells, which recruits {gamma}{delta} T cells through the chemokine receptors CXCR1 and CXCR2, to enhance the cytotoxicity of {gamma}{delta} T cells. Metabolomics analysis discovered compounds that are responsible for the enhancement of {gamma}{delta} T cell cytotoxicity, one of which follows the aforementioned mechanism, while other compounds reflect undiscovered mechanisms. Overall, we presented the positive side of the malignant ascites in anti-tumor immunity, revealed the underlining mechanisms and at least partially interpreted the molecular basis. Our work thus provides new insights into the development of cell therapies for ovarian cancer.

immunology↗
bioRxiv2024

2'-O-Methyl-guanosine 3-base RNA fragments mediate essential natural TLR7/8 antagonism

Recognition of RNA fragments by Toll-like receptors (TLR) 7 and 8 is a key contributor to the initiation of a protective innate immune response against pathogens. A long-standing enigma is how degradation products of host RNAs, generated by the daily phagocytic clearance of billions of apoptotic cells, fail to activate TLR7 and TLR8 signalling1. Here, we report that select 2-O-methyl (2-Ome) guanosine RNA fragments as short as 3 bases, including those derived from host-RNAs, are potent TLR7 and TLR8 antagonists that reduce TLR7 sensing in vivo. Mechanistically, antagonistic fragments are directed towards a distinct binding site on these proteins by 5-end 2-Ome guanosine. Our results indicate that host-RNAs evade detection by TLR7/8 due to a pool of abundant host ribosomal 2-Ome-modified RNA fragments that naturally antagonize TLR7 and TLR8 sensing to avoid auto-immunity. Crucially, rare TLR7 and TLR8 mutations located at this antagonistic site decrease the inhibitory activity of 2-Ome guanosine RNA fragments and lead to auto-immunity in patients. Our findings also establish that select chemically synthesised 3-base oligonucleotides can harness the protective anti-inflammatory activity of this natural immune checkpoint for therapeutic targeting of TLR7-driven diseases. One Sentence SummaryShort 2-O-Methyl RNA fragments are natural TLR7/8 antagonists

immunology↗
bioRxiv2024

B cell receptor silencing reveals the origin of high-grade B cell lymphomas with MYC and BCL2 rearrangements

The B cell receptor (BCR) is essential for mature B cell lymphomas, serving as therapeutic target. Here, we show that high-grade B cell lymphomas with MYC and BCL2 rearrangements (HGBCL-DH-BCL2) predominantly exhibit immunoglobulin heavy (IGH) chain silencing, leading to BCR shutdown. HGBCL-DH-BCL2 with undetectable IGH (IGHUND) differ from IGH-expressing counterparts for germinal center-zone gene programs, MYC expression and T cell infiltration. While IGH+ HGBCL-DH-BCL2 prefer IGM/IG-Kappa expression, IGHUND counterparts have completed IGH class-switching, favoring IG-Lambda (IGL) light chains. IGHUND HGBCL-DH-BCL2 preserve IGHV gene integrity, overcoming antigen-driven selection. IGH silencing precedes onset and shapes evolution of HGBCL-DH-BCL2 from Follicular Lymphoma (FL) or FL/HGBCL-DH-BCL2 common precursor. In FL/HGBCL-DH-BCL2 pairs and HGBCL-DH-BCL2 models, BCR silencing promoted RAG1/2-dependent IG light chain editing, causing t(8;22)(q24;q11)/IGL::MYC. IGH silencing protected HGBCL-DH-BCL2 models from killing by CD79B-targeting Polatuzumab-Vedotin. Collectively, HGBCL-DH-BCL2 primarily originate from BCR-silenced isotype-switched t(14;18)/IGH::BCL2-positive (pre)FL cells acquiring IGL::MYC translocations during IG light chain revision, with clinical implications. SignificanceThese findings link BCR silencing in isotype-switched t(14;18)+ Follicular Lymphoma cells (or their precursors) to RAG1/2 re-expression, promoting IGL::MYC translocations responsible for transformation into high-grade B cell lymphomas (HGBCL). Predominant silencing of the BCR complex in HGBCL with MYC and BCL2 rearrangements protects tumor cells from CD79B-directed Polatuzumab-Vedotin killing.

cancer biology↗
bioRxiv2024

Therapeutic Peptide SS-31 Modulates Membrane Binding and Aggregation of Alpha-Synuclein and Restores Impaired Mitochondrial Function

Membrane binding and aggregation properties of -synuclein are closely associated with Parkinsons disease and a class of related syndromes named as synucleinopathy. This study explored the potential of SS-31 (Elamipretide), a therapeutic tetrapeptide with alternating cationic and aromatic residues and known properties of mitochondrial inner membrane binding and oxidative stress reduction, in modulating -synuclein interaction with the lipid membranes and mitigating impairment of mitochondrial function induced by -synuclein oligomers. It was demonstrated by both fluorescence correlation spectroscopy and fluorescence anisotropy that SS-31 displaces both wild-type and N-terminus acetylated -synuclein from negatively charged small unilamellar vesicles in a dose-dependent manner. Thioflavin-T assay and transmission electron microscopy (TEM) showed that SS-31 inhibits membrane-induced -synuclein aggregation and alters the morphology of -synuclein fibrils. Moreover, Seahorse Mito Stress Test indicated that SS-31 restores impaired mitochondrial function in -synuclein oligomer-treated neuroblastoma cells. Finally, confocal imaging revealed that SS-31 hinders cellular uptake of -synuclein oligomers, possibly by modifying cell membrane electrostatics. These findings underscore the multifaceted protective role of SS-31 against mitochondrial dysfunction caused by -synuclein aggregation. Consequently, SS-31 emerges as a promising therapeutic candidate to attenuate neurodegeneration pertinent to -synuclein misfolding and aggregation. There is a good potential for further refinement of such peptide against many diseases linked to mitochondrial dysfunction and oxidative stress.

biochemistry↗
bioRxiv2023

Modeling Mechanical Activation of Macrophages During Pulmonary Fibrogenesis for Targeted Anti-Fibrosis Therapy

Pulmonary fibrosis, as seen in idiopathic pulmonary fibrosis (IPF) and COVID-induced pulmonary fibrosis, is an often-fatal lung disease. Increased numbers of immune cells such as macrophages were shown to accumulate in the fibrotic lung, but it is unclear how they contribute to the development of fibrosis. To recapitulate the macrophage mechanical activation in the fibrotic lung tissue microenvironment, we developed a fibrotic microtissue model with cocultured human macrophages and fibroblasts. We show that profibrotic macrophages seeded on topographically controlled stromal tissue constructs become mechanically activated. The resulting co-alignment of macrophages, collagen fibers and fibroblasts promote widespread fibrogenesis in micro-engineered lung tissues. Anti-fibrosis treatment using pirfenidone disrupts the polarization and mechanical activation of profibrotic macrophages, leading to fibrosis inhibition. Pirfenidone inhibits the mechanical activation of macrophages by suppressing integrin M{beta}2 (CD11b/CD18) and Rho-associated kinase 2, which is a previously unknown mechanism of action of the drug. Together, these results demonstrate a potential pulmonary fibrogenesis mechanism at the tissue level contributed by mechanically activated macrophages. We propose the coculture, force-sensing microtissue model as a powerful tool to study the complex immune-stromal cell interactions and the mechanism of action of anti-fibrosis drugs.

bioengineering↗
bioRxiv2021

Knockout of E-cadherin in adult mouse epithelium results in emphysema and airway disease

Chronic obstructive pulmonary disease (COPD) is a devastating lung disease, characterized by a progressive decline in lung function, alveolar loss (emphysema), and airflow limitation due to excessive mucus secretion (chronic bronchitis), that can occur even after the injurious agent is removed. It is slated to rise to the 3rd leading cause of death due to chronic disease by 2030 globally, and the 4th leading cause of death due to chronic disease in the USA. While there is substantial evidence indicating loss of E-cadherin in the lung epithelium of patients with COPD, it is not known if this is causal to the disease. We investigated if loss of E-cadherin can result in lung disease using in both in vitro models of primary, differentiated human cells and in mouse models. Using a cell type-specific promoter using Cre/LoxP mice system to knock-out E- cadherin in ciliated and alveolar epithelial cell (Type 1 and Type 2) populations in adult mouse models, we determined that loss of E-cadherin caused airspace enlargement, as well as increased airway hyperresponsiveness indicating that it does have a causative role in causing COPD. Strategies to upregulate CDH1 (encodes for E-cadherin) in CHBEs and cigarette-smoke injured NHBEs can rescue the dysfunctional epithelium.

cell biology↗
bioRxiv2020

Myeloid lncRNA LOUP Mediates Opposing Regulatory Effects of RUNX1 and RUNX1-ETO in t(8;21) AML

The mechanism underlying cell type-specific gene induction conferred by ubiquitous transcription factors as well as disruptions caused by their chimeric derivatives in leukemia is not well understood. Here we investigate whether RNAs coordinate with transcription factors to drive myeloid gene transcription. In an integrated genome-wide approach surveying for gene loci exhibiting concurrent RNA- and DNA-interactions with the broadly expressed transcription factor RUNX1, we identified the long noncoding RNA LOUP. This myeloid-specific and polyadenylated lncRNA induces myeloid differentiation and inhibits cell growth, acting as a transcriptional inducer of the myeloid master regulator PU.1. Mechanistically, LOUP recruits RUNX1 to both the PU.1 enhancer and the promoter, leading to the formation of an active chromatin loop. In t(8;21) acute myeloid leukemia, wherein RUNX1 is fused to ETO, the resulting oncogenic fusion protein RUNX1-ETO limits chromatin accessibility at the LOUP locus, causing inhibition of LOUP and PU.1 expression. These findings highlight the important role of the interplay between cell type-specific RNAs and transcription factors as well as their oncogenic derivatives in modulating lineage-gene activation and raise the possibility that RNA regulators of transcription factors represent alternative targets for therapeutic development. KEY POINTSO_LIlncRNA LOUP coordinates with RUNX1 to induces PU.1 long-range transcription, conferring myeloid differentiation and inhibiting cell growth. C_LIO_LIRUNX1-ETO limits chromatin accessibility at the LOUP locus, causing inhibition of LOUP and PU.1 expression in t(8;21) AML. C_LI

cancer biology↗
bioRxiv2020

Inferring Maps of Cellular Structures from MRI Signals using Deep Learning

1H MRI maps brain structure and function non-invasively through versatile contrasts that exploit inhomogeneity in tissue micro-environments. Inferring histopathological information from MRI findings, however, remains challenging due to absence of direct links between MRI signals and cellular structures. Here, we show that deep convolutional neural networks, developed using co-registered multi-contrast MRI and histological data of the mouse brain, can estimate histological staining intensity directly from MRI signals at each pixel. The results provide three-dimensional maps of axons and myelin with tissue contrasts that closely mimics target histology and enhanced sensitivity and specificity compared to conventional MRI markers. Furthermore, the relative contribution of each MRI contrast within the networks can be used to optimize multi-contrast MRI acquisition. We anticipate our method to be a starting point for translation of MRI results into easy-to-understand virtual histology for neurobiologists and provide resources for validating novel MRI techniques.

bioengineering↗
bioRxiv2019

Unsupervised graph-based learning predicts mutations that alter protein dynamics

AO_SCPLOWBSTRACTC_SCPLOWProteins exhibit complex dynamics across a vast range of time and length scales, from the atomistic to the conformational. Adenylate kinase (ADK) showcases the biological relevance of such inherently coupled dynamics across scales: single mutations can affect large-scale protein motions and enzymatic activity. Here we present a combined computational and experimental study of multiscale structure and dynamics in proteins, using ADK as our system of choice. We show how a computationally efficient method for unsupervised graph partitioning can be applied to atomistic graphs derived from protein structures to reveal intrinsic, biochemically relevant substructures at all scales, without re-parameterisation or a priori coarse-graining. We subsequently perform full alanine and arginine in silico mutagenesis scans of the protein, and score all mutations according to the disruption they induce on the large-scale organisation. We use our calculations to guide Forster Resonance Energy Transfer (FRET) experiments on ADK, and show that mutating residue D152 to alanine or residue V164 to arginine induce a large dynamical shift of the protein structure towards a closed state, in accordance with our predictions. Our computations also predict a graded effect of different mutations at the D152 site as a result of increased coherence between the core and binding domains, an effect confirmed quantitatively through a high correlation (R2 = 0.93) with the FRET ratio between closed and open populations measured on six mutants.

biophysics↗
bioRxiv2019

The Salix SmSPR1 Involved in Light-Regulated Cell Expansion by Modulating Microtubule Arrangement

Light signaling and cortical microtubule (MT) arrays are essential to the anisotropic growth of plant cells. Microtubule-associated proteins (MAPs) function as regulators that mediate plant cell expansion or elongation by altering the arrangements of the MT arrays. However, current understanding of the molecular mechanism of MAPs in relation to light to regulate cell expansion or elongation is limited. Here, we show that the microtubule-associated protein SPR1 is involved in light-regulated directional cell expansion by modulating microtubule elongation in Salix matsudana. Overexpression of SmSPR1 in Arabidopsis results in right-handed helical orientation of hypocotyls in dark-grown etiolated seedlings, whereas the phenotype of transgenic plants was indistinguishable from those of wild-type plants under light conditions. Phenotypic characterization of the transgenic plants showed reduced anisotropic growth and left-handed helical MT arrays in etiolated hypocotyl cells. Protein interaction assays revealed that SPR1, CSN5A (subunits of COP9 signalosome, a negative regulator of photomorphogenesis), and ELONGATED HYPOCOTYL 5 (HY5, a transcription factor that promotes photomorphogenesis) interacted with each other in vivo. The phenotype of Arabidopsis AtSPR1-overexpressing transgenic lines was similar to that of SmSPR1-overexpressing transgenic plants, and overexpression of Salix SmSPR1 can rescue the spr1 mutant phenotype, thereby revealing the function of SPR1 in plants.\n\nHighlightFunction of microtubule-associated protein SPR1 is directly related to light, and crucial to the balance of tubulin polymerization

plant biology↗
bioRxiv2019

NOD1 mediates non-canonical inflammasome processing of interleukin-18 in epithelial cells to Helicobacter pylori infection.

The interleukin-1 family members, IL-1{beta} and IL-18, are processed into their biologically active forms by multi-protein complexes, known as inflammasomes. Although the inflammasome pathways that mediate IL-1{beta} processing in myeloid cells have been extensively studied, those involved in IL-18 processing, particularly in non-myeloid cells, are still poorly understood. Here, we have identified the cytosolic sensor NOD1 as a key regulator of IL-18 processing in epithelial cells responding to Helicobacter pylori infection. Importantly, NOD1 processing of IL-18 occurs independently of the canonical inflammasome proteins, NLRP3 and ASC. Instead, NOD1 interacts directly with caspase-1 via homotypic binding of caspase-activation recruitment domains. We show that IL-18 is important in maintaining tissue homeostasis and protecting against pre-neoplastic changes due to gastric H. pylori infection. These findings reveal an unanticipated role for NOD1 in a new type of inflammasome that regulates epithelial cell production of bioactive IL-18 with tissue protective functions.

immunology↗