Biology subjects

Shang, W.

Publications and source records attributed to Shang, W..

16 recordsLinked to original sources

bioRxiv2026

A Three-dimensional Analytical Framework for Retinal Microvasculature Reveals Layer-associated Vulnerability in Development and Neovascular Remodeling

The neurosensory retina is one of the most metabolically active tissues in the body and a uniquely accessible extension of the central nervous system, where neuronal and vascular structures can be visualized non-invasively. Its accessibility and highly organized laminar architecture make it a powerful model for studying vascular development and a window into systemic health. Although computational analyses of retinal images have enabled risk assessment for ocular and systemic diseases, most vascular studies rely on two-dimensional frameworks with limited resolution of capillary structure and layer-specific organization. Here, we present a high-resolution three-dimensional (3D) imaging and analysis pipeline enabling quantification of retinal microvasculature and extraction of structural and network metrics across vascular layers. We apply this approach to two mouse models of aberrant retinal vascular development: one with spontaneous postnatal chorioretinal neovascularization and another with disrupted neurovascular lattice formation and layered organization in early life. Across both pathologic contexts, 3D analysis enables detailed characterization of retinal vascular architecture and identifies early vulnerability within the intermediate plexus, the vascular network that bridges the superficial and deep retinal layers, as a sensitive indicator of abnormal remodeling and neovascularization. This framework enables precise characterization of retinal vasculature and establishes a foundation for identifying new retinal biomarkers with potential relevance to neurovascular and systemic disease.

bioengineering↗

bioRxiv2026

Integrated single-cell and spatial transcriptomic analysis of T cell exhaustion and immunometabolic remodeling in HPV-positive oropharyngeal squamous cell carcinoma

HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) harbors dense lymphocytic infiltration yet responds poorly to immune checkpoint blockade (ICB), a paradox whose mechanistic basis remains unresolved. Here we constructed an integrated single-cell and spatial transcriptomic atlas of HPV OPSCC (23,119 cells, 24 cell types), complemented by CRISPR-Cas9 functional validation in two HPV cell lines and multiplex immunofluorescence across independent cases. Pseudotime trajectory analysis placed TOXCD8 T cells, rather than canonically defined exhausted cells, at the terminal exhaustion endpoint, and uncovered a previously uncharacterized IEGCD8 effector memory population at a fate-decision juncture between activation and irreversible exhaustion. Actively cycling Tregs with broad co-stimulatory and co-inhibitory receptor expression underwent intratumoral immunosuppressive differentiation. Compartmentalized LDHA/MCT4 expression in cancer cells versus LDHB/MCT1 in T cells established a directional lactate flux model, and spatial deconvolution confirmed peripheral immune exclusion. We identified a KMT2D-KLF7-PD-L1 regulatory axis: KMT2D sustains KLF7 transcription through H3K4me1-dependent enhancer activation; KLF7 concurrently drives neural crest differentiation and upregulates PD-L1, thereby linking epigenetic remodeling to both neural programming and immune checkpoint expression. These findings define converging multi-scale mechanisms underlying ICB resistance in HPV OPSCC and nominate the KMT2D-KLF7-PD-L1 axis as a combinatorial therapeutic target.

cancer biology↗

bioRxiv2025

Drosophila Metaxin-2 controls beta-barrel biogenesis and muscle growth in a stage-dependent fashion

Metaxin-2 (Mtx2) is an evolutionarily conserved mitochondrial outer membrane protein. Mutations in human Mtx2 cause mandibuloacral dysplasia (MADaM), a progeroid disorder. However, the pathologic mechanisms of Mtx2 loss-of-function remain largely unknown. Using Drosophila, we show Mtx2 null mutants exhibit pupal lethality, rescued by Drosophila or human Mtx2, underscoring functional conservation across species. Tissue-specific conditional knockout and rescue experiments reveal muscle as a critical site of dMtx2 action, with alternations of myofibril assembly and myogenic proteins being observed in dMtx2 mutants. Structural and functional mitochondrial abnormalities are also detected, verifying dMtx2s function in mitochondrial homeostasis. Notably, Mtx2 deficiency affects beta-barrel protein biogenesis and muscle development in pupa but not in larva, demonstrating Mtx2s dynamic regulation in mitochondrial proteostasis and muscle development. Our results elucidate mitochondrial mechanisms driving potential muscle-autonomous defects in MADaM patients and highlights stage-specific Mtx2 function as a prospective therapeutic target for this progeroid syndrome.

developmental biology↗

bioRxiv2024

PD-1 controls differentiation, survival, and TCR affinity evolution of stem-like CD8+ T cells

Stem-like progenitors are a critical subset of cytotoxic T cells that self-renew and give rise to expanded populations of effector cells critical for successful checkpoint blockade immunotherapy. Emerging evidence suggests that the tumor-draining lymph nodes can support the continuous generation of these stem-like cells that replenish the tumor sites and act as a critical source of expanded effector populations, underlining the importance of understanding what factors promote and maintain activated T cells in the stem-like state. Using advanced 3D multiplex immunofluorescence imaging, here we identified antigen-presentation niches in tumor-draining lymph nodes that support the expansion, maintenance, and affinity evolution of a unique population of TCF-1+PD-1+SLAMF6hi stem-like CD8+ T cells. Our results show that contrary to the prevailing view that persistent TCR signaling drives terminal effector differentiation, prolonged antigen engagement well beyond the initial priming phase sustained the proliferation and self-renewal of these stem-like T cells in vivo. The inhibitory PD-1 pathway plays a central role in this process by mediating the fine-tuning of TCR and co-stimulatory signal input that enables selective expansion of high affinity TCR stem-like clones, enabling them to act as a renewable source of high affinity effector cells. PD-1 checkpoint blockade disrupts this fine tuning of input signaling, leading to terminal differentiation to the effector state or death of the most avid anti-tumor stem-like cells. Our results thus reveal an unexpected relationship between TCR ligand affinity recognition, a key negative feedback regulatory loop, and T cell stemness programming. Furthermore, these findings raise questions about whether anti-PD-1 checkpoint blockade during cancer immunotherapy provides a short-term anti-tumor effect that comes at the cost of diminishing efficacy due to progressive loss of these critical high affinity stem-like precursors.

immunology↗

bioRxiv2023

EfNST: A composite scaling network of EfficientNet for improving spatial domain identification performance

Spatial Transcriptomics (ST) leverages Gene Expression Profiling while preserving Spatial Location and Histological Images, enabling it to provide new insights into tissue structure, tumor microenvironment, and biological development. The identification of spatial domains serves as not only the foundation for ST research but also a crucial step in various downstream analyses. However, accurately identifying spatial domains using computational methods remains a tremendous challenge due to the poor computational performance of many existing algorithms. Here, we propose EfNST, a deep learning algorithm based on a composite scaling network of the EfficientNet Network, designed specifically for the analysis of 10X Visium spatial transcriptomics data. We applied EfNST to three different datasets: human Dorsolateral Prefrontal Cortex, human breast cancer and mouse brain anterior. EfNST outperforms five advanced competing algorithms, achieving the best Adjusted Rand Index (ARI) scores of 0.554, 0.607, and 0.466, respectively. Notably, EfNST demonstrated high accuracy in identifying fine tissue structure and discovering corresponding marker genes with an improved running speed. In conclusion, EfNST offers a novel approach for inferring spatial organization of cells from discrete datapoints, facilitating the exploration of new insights in this field.

bioinformatics↗

bioRxiv2023

Iterative In Silico Screening for Optimizing Stable Conformation of Anti-SARS-CoV-2 Nanobodies

Nanobodies (Nbs or VHHs) are single-domain antibodies (sdAbs) derived from camelid heavy-chain antibodies. The variable region of these nanobodies has special and unique characteristics, such as small size, good tissue penetration, and cost-effective production, making nanobodies a good candidate for the diagnosis and treatment of viruses. Identifying effective nanobodies against the COVID-19 would help us defeat this dangerous virus or other unknown variants in future. Herein, we introduce an in silico screening strategy for optimizing stable conformation of anti-SARS-CoV-2 nanobodies. Firstly, various complexes containing nanobodies were downloaded from the RCSB database, which were identified from immunized llamas. The primary docking between nanobodies and the SARS-CoV-2 spike protein receptor-binding domain was performed through ClusPro program, with the manually screening that leaving the reasonable conformation to the next step. Then, the binding distances of atoms between the antigen-antibody interfaces were measured through the NeighborSearch algorithm. Finally, filtered nanobodies were acquired according to HADDOCK scores through HADDOCK docking the COVID spike protein with nanobodies under restrictions of calculated molecular distance between active residues and antigenic epitopes less than 4.5 [A]. In this way, those nanobodies which with more reasonable conformation and with stronger neutralizing efficacy were acquired. To validate the efficacy ranking of the nanobodies we obtained, we calculated the binding affinities ({Delta}G) and dissociation constants (Kd) of all screened nanobodies using the PRODIGY web tool, and predicted the stability changes induced by all possible point mutations in nanobodies using the MAESTROWeb server. Furthermore, we examined the performance of the relationship between nanobodies ranking and their number of mutation-sensitive sites (Spearman correlation > 0.68), the results revealed a robust correlation, indicating that the superior nanobodies identified through our screening process exhibited fewer mutation hotspots and higher stability. This correlation analysis demonstrates the validity of our screening criteria, underscoring the suitability of these nanobodies for future development and practical implementation. In conclusion, this three-step screening strategy iteratively in silico greatly improved the accuracy of screening desired nanobodies compared to using only ClusPro docking or default HADDOCK docking settings. It provides new ideas for the screening of novel antibodies and computer-aided screening methods.

bioinformatics↗

bioRxiv2023

A non-image-forming visual circuit mediates the innate fear of heights

The neural basis of fear of heights remains largely unknown. In this study, we investigated the fear response to heights in mice and observed characteristic aversive behaviors resembling human height vertigo. We identified visual input as a critical factor in mouse reactions to heights, while peripheral vestibular input was found to be nonessential for fear of heights. Unexpectedly, we found that fear of heights in naive mice does not rely on image-forming visual processing by the primary visual cortex. Instead, a subset of neurons in the ventral lateral geniculate nucleus (vLGN), which connects to the lateral/ventrolateral periaqueductal gray (l/vlPAG), drives the expression of fear associated with heights. Additionally, we observed that a subcortical visual pathway linking the superior colliculus to the lateral posterior thalamic nucleus inhibits the defensive response to height threats. These findings highlight a rapid fear response to heights threat through a subcortical visual and defensive pathway from the vLGN to the l/vlPAG.

neuroscience↗

bioRxiv2022

mRNA targeting eliminates the need for the signal recognition particle during membrane protein insertion in bacteria

Signal-sequence dependent protein targeting is essential for the spatiotemporal organization of eukaryotic and prokaryotic cells and facilitated by dedicated protein targeting factors, such as the signal recognition particle (SRP). However, targeting signals are not exclusively contained within proteins, but can also be present within mRNAs. By in vivo and in vitro assays, we show that mRNA targeting is controlled by the nucleotide content and by secondary structures within mRNAs. mRNA binding to bacterial membranes occurs independently of soluble targeting factors, but is dependent on the SecYEG-translocon and YidC. Importantly, membrane insertion of proteins translated from membrane-bound mRNAs occurs independently of the SRP pathway, while the latter is strictly required for proteins translated from cytosolic mRNAs. In summary, our data indicate that mRNA targeting acts in parallel to the canonical SRP-dependent protein targeting and serves as an alternative strategy for safeguarding membrane protein insertion when the SRP pathway is compromised.

microbiology↗

bioRxiv2022

Planting graphene quantum dots in PEGylated nanoparticles for enhanced and multimodal in vivo imaging of tumor

Photoluminescent graphene quantum dots (GQDs) have recently attracted considerable attention for biomedical applications owing to the interesting physicochemical and photophysical properties, and prominent biocompatibility and biosafety. However, although much progress has been achieved in therapy and in vitro bioimaging, broad and crucial in vivo fluorescence bioimaging and synergistically working with other nanomedicine are very challenging and limited. Herein, we in situ implanted GQDs in the PEGylated layer of nanoparticles (NPs) via a bottom-up approach to obtain the NPs (core)-GQDs-PEG multifunctional nanoprobe (NPC-GQDs-PEG), which prolonged the blood circulation of GQDs more than four times and increased the tumor accumulation 7~8 times than GQDs used alone. Under assisted by the flexible microscope, the GQDs were successfully used for in vivo real-time monitoring of local NPs pharmacokinetics, in vivo multimodality imaging, and fluorescence imaging-guided tumor surgery. The approach of implanting GQDs in PEGylated nanomedicine and synergistic working provide a new strategy for in vivo biomedical applications of GQDs.

cancer biology↗

bioRxiv2021

Novel inhibition mechanism of SARS-CoV-2 main protease by ebselen and its derivatives

The global emergence of SARS-CoV-2 has triggered numerous efforts to develop therapeutic options for COVID-19 pandemic. The main protease of SARS-CoV-2 (Mpro), which is a critical enzyme for transcription and replication of SARS-CoV-2, is a key target for therapeutic development against COVID-19. An organoselenium drug called ebselen has recently been demonstrated to have strong inhibition against Mpro and antiviral activity but its molecular mode of action is unknown preventing further development. We have examined the binding modes of ebselen and its derivative in Mpro via high resolution co-crystallography and investigated their chemical reactivity via mass spectrometry. Stronger Mpro inhibition than ebselen and potent ability to rescue infected cells were observed for a number of ebselen derivatives. A free selenium atom bound with cysteine 145 of Mpro catalytic dyad has been revealed by crystallographic studies of Mpro with ebselen and MR6-31-2 suggesting hydrolysis of the enzyme bound organoselenium covalent adduct, formation of a phenolic by-product is confirmed by mass spectrometry. The target engagement of these compounds with an unprecedented mechanism of SARS-CoV-2 Mpro inhibition suggests wider therapeutic applications of organo-selenium compounds in SARS-CoV-2 and other zoonotic beta-corona viruses.

biophysics↗

bioRxiv2020

Structure Basis for Inhibition of SARS-CoV-2 by the Feline Drug GC376

The pandemic of SARS-CoV-2 coronavirus disease-2019 (COVID-19) caused by SARS-COV-2 continues to ravage many countries in the world. Mpro is an indispensable protein for viral translation in SARS-CoV-2 and a potential target in high-specificity anti-SARS-CoV-2 drug screening. In this study, to explore potential drugs for treating COVID-19, we elucidated the structure of SARS-CoV-2 Mpro and explored the interaction between Mpro and GC376, an antiviral drug used to treat a range of coronaviruses in Feline via inhibiting Mpro. The availability and safety of GC376 were proved by biochemical and cell experiments in vitro. We determined the structure of an important protein, Mpro, in SARS-CoV-2, and revealed the interaction of GC376 with the viral substrate and inhibition of the catalytic site of SARS-CoV-2 Mpro.

biophysics↗

bioRxiv2020

Broad-spectrum antivirals of protoporphyrins inhibit the entry of highly pathogenic emerging viruses

Severe emerging and re-emerging viral infections such as Lassa fever, Avian influenza (AI), and COVID-19 caused by SARS-CoV-2 urgently call for new strategies for the development of broad-spectrum antivirals targeting conserved components in the virus life cycle. Viral lipids are essential components, and viral-cell membrane fusion is the required entry step for most unrelated enveloped viruses. In this paper, we identified a porphyrin derivative of protoporphyrin IX (PPIX) that showed broad antiviral activities in vitro against a panel of enveloped pathogenic viruses including Lassa virus (LASV), Machupo virus (MACV), and SARS-CoV-2 as well as various subtypes of influenza A viral strains with IC50 values ranging from 0.91{+/-}0.25 M to 1.88{+/-}0.34 M. A mechanistic study using influenza A/Puerto Rico/8/34 (H1N1) as a testing strain showed that PPIX inhibits the infection in the early stage of virus entry through biophysically interacting with the hydrophobic lipids of enveloped virions, thereby inhibiting the formation of the negative curvature required for fusion and blocking the entry of enveloped viruses into host cells. In addition, the preliminary antiviral activities of PPIX were further assessed by testing mice infected with the influenza A/Puerto Rico/8/34 (H1N1) virus. The results showed that compared with the control group without drug treatment, the survival rate and mean survival time of the mice treated with PPIX were apparently prolonged. These data encourage us to conduct further investigations using PPIX as a lead compound for the rational design of lipid-targeting antivirals for the treatment of infection with enveloped viruses.

pharmacology and toxicology↗

bioRxiv2020

Discovery of baicalin and baicalein as novel, natural product inhibitors of SARS-CoV-2 3CL protease in vitro

Human infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause coronavirus disease 19 (COVID-19) and there is currently no cure. The 3C-like protease (3CLpro), a highly conserved protease indispensable for replication of coronaviruses, is a promising target for development of broad-spectrum antiviral drugs. To advance the speed of drug discovery and development, we investigated the inhibition of SARS-CoV-2 3CLpro by natural products derived from Chinese traditional medicines. Baicalin and baicalein were identified as the first non-covalent, non-peptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography is distinctly different from those of known inhibitors. Baicalein is perfectly ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a "shield" in front of the catalytic dyad to prevent the peptide substrate approaching the active site. The simple chemical structure, unique mode of action, and potent antiviral activities in vitro, coupled with the favorable safety data from clinical trials, emphasize that baicalein provides a great opportunity for the development of critically needed anti-coronaviral drugs.

molecular biology↗

bioRxiv2020

Immunoglobulin fragment F(ab')2 against RBD potently neutralizes SARS-CoV-2 in vitro

COVID-19 caused by the emerging human coronavirus, SARS-CoV-2, has become a global pandemic, leading a serious threat to human health. So far, there is none vaccines or specific antiviral drugs approved for that. Therapeutic antibodies for SARS-CoV-2, was obtained from hyper immune equine plasma in this study. Herein, SARS-CoV-2 RBD with gram level were obtained through Chinese hamster ovary cells high-density fermentation. The binding of RBD to SARS-CoV-2 receptor, human ACE2, was verified and the efficacy of RBD in vivo was tested on mice and then on horses. As a result, RBD triggered high-titer neutralizing antibodies in vivo, and immunoglobulin fragment F(ab)2 was prepared from horse antisera through removing Fc. Neutralization test demonstrated that RBD-specific F(ab)2 inhibited SARS-CoV-2 with EC50 at 0.07 g/ml, showing a potent inhibitory effect on SARS-CoV-2. These results highlights as RBD-specific F(ab)2 as therapeutic candidate for SARS-CoV-2.

pharmacology and toxicology↗

bioRxiv2020

Novel and potent inhibitors targeting DHODH, a rate-limiting enzyme in de novo pyrimidine biosynthesis, are broad-spectrum antiviral against RNA viruses including newly emerged coronavirus SARS-CoV-2

Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of coronavirus SARS-CoV-2. Existing direct-acting antiviral (DAA) drugs cannot be applied immediately to new viruses because of virus-specificity, and the development of new DAA drugs from the beginning is not timely for outbreaks. Thus, host-targeting antiviral (HTA) drugs have many advantages to fight against a broad spectrum of viruses, by blocking the viral replication and overcoming the potential viral mutagenesis simultaneously. Herein, we identified two potent inhibitors of DHODH, S312 and S416, with favorable drug-like and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus (H1N1, H3N2, H9N2), Zika virus, Ebola virus, and particularly against the recent novel coronavirus SARS-CoV-2. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knocking-out cells. We also proposed the drug combination of DAA and HTA was a promising strategy for anti-virus treatment and proved that S312 showed more advantageous than Oseltamivir to treat advanced influenza diseases in severely infected animals. Notably, S416 is reported to be the most potent inhibitor with an EC50 of 17nM and SI value >5882 in SARS-CoV-2-infected cells so far. This work demonstrates that both our self-designed candidates and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-repression may have clinical potentials not only to influenza but also to COVID-19 circulating worldwide, no matter such viruses mutate or not.

microbiology↗

bioRxiv2018

β-lactam Antibiotics Stimulate the Pathogenicity of Methicillin-resistant Staphylococcus aureus Via SarA-controlled Tandem Lipoprotein Expression

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of nosocomial infections worldwide. MRSA resists nearly all {beta}-lactam antibiotics that have a bactericidal activity and a signal inducer effect. However, studies have yet to clarify whether the inducer effect of empirically used {beta}-lactams stimulates MRSA pathogenicity in vivo. Here, we showed that a new cluster of tandem lipoprotein genes (tlpps) was upregulated in MRSA in response to the subinhibitory concentrations of {beta}-lactam induction. The increased Tlpps significantly altered immune responses by macrophages with high IL-6 and TNF levels. The deletion of the tlpps mutant (N315{Delta}tlpps) significantly decreased the proinflammatory cytokine levels in vitro and in vivo. The bacterial loads of N315{Delta}tlpps in the mouse kidney were also reduced compared with those of the wild type N315. The {beta}-lactam-treated MRSA exacerbated cutaneous infections with increased lesion size, extended illness, and flake-like abscess-formation compared with those of the nontreatment. The {beta}-lactam antibiotics that promoted the MRSA pathogenicity were SarA dependent, and the increasing expression of tlpps after {beta}-lactam treatment was directly controlled by the global regulator SarA. Overall, our findings suggested that {beta}-lactams should be used carefully because it might lead to a worse outcome of MRSA infection than inaction in the treatment.\n\nAuthor summary{beta}-lactams are widely used in practice to treat infectious diseases, however, {beta}-lactams worsening the outcome of a certain disease is poorly understood. In this study, we have identified a new cluster of tandem lipoprotein genes (tlpps) that is upregulated in the major clinically prevalent MRSA clones in response to the subinhibitory concentrations of {beta}-lactams induction. The major highlight in this work is that {beta}-lactams induce SarA expression, and then SarA directly binds to the tlpp cluster promoter region and upregulates the tlpp expression in MRSA. Moreover, the {beta}-lactam stimulated Tlpps are important virulence factors that enhance MRSA pathogenicity. The deletion of the tlpps mutant significantly decreases the proinflammatory cytokine levels in vitro and in vivo. The {beta}-lactam induced Tlpps enhance the host inflammatory responses by triggering the expression of IL-6 and TNF, thereby promoting bacterial colonization and abscess formation. These data elucidate that {beta}-lactams can worsen the outcome of MRSA infection through the induction of tlpps that are controlled by the global regulator SarA.

pathology↗