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Shen, D.

Publications and source records attributed to Shen, D..

At least 19 recordsLinked to original sources

Recombinant Echis disintegrins reveal distinct inhibitory profiles on platelet aggregation and endothelial cell migration

Disintegrins are snake venom toxins that bind integrins and modulate platelet aggregation and cell migration. Isolation from venom often yields insufficient quantities for comprehensive study, making recombinant expression essential for detailed toxin characterisation. Here, we produced and characterised four disintegrins from Echis coloratus and Echis ocellatus in Escherichia coli. These homologous proteins contain distinct integrin-binding motifs: EcDis_RGD, EoDis_RGD, EcDis_KGD, and EcDis_VGD. Functional activities were evaluated using human platelet aggregation and endothelial cell scratch wound-healing assays. RGD-containing disintegrins exhibited the strongest inhibition of platelet aggregation, with EcDis_RGD showing the highest potency. The EcDis_KGD disintegrin inhibited platelet aggregation less potently, whereas EcDis_VGD showed no detectable activity in our assays. In endothelial cells, EcDis_RGD disintegrin markedly impaired cell migration, while EoDis_RGD displayed weaker anti-migratory activity. Neither KGD- nor VGD-containing disintegrins significantly affected wound closure. EoDis_RGD exhibited similar antiplatelet activity to the native, venom-purified disintegrin ocellatusin, validating our recombinant expression strategy. However, the corresponding PII-SVMP preparation displayed reduced inhibitory activity, consistent with incomplete generation of fully mature disintegrins species. Our findings demonstrate the importance of both integrin-binding motif identity and surrounding sequence context in determining disintegrin function, and support further use of recombinant toxins in toxinology and as therapeutic lead molecules.

biochemistry↗

Disintegrin-like and Cysteine-rich Domains Govern Enzymatic Activity and Substrate Recognition in Echis Snake Venom Metalloproteinases

Snake venom metalloproteinases (SVMPs) are major drivers of pathology following viper envenomation and represent important targets for the development of next-generation recombinant antivenoms. PIII SVMPs are among the most potent haemorrhagic toxins and contain disintegrin-like (Dis) and cysteine-rich (C-rich) accessory domains. Despite their biomedical importance, the mechanistic roles of these accessory domains in substrate recognition and catalysis remain poorly understood. We produced recombinant full-length and domain-deletion variants of two functionally distinct PIII SVMPs: the broadly proteolytic, cytotoxic cPIII and the highly specific prothrombin activator Ecarin. Proteins were expressed as latent zymogens in insect cells, auto-activated by Zn2+, and analysed using enzymatic, blood clotting, and cell-based assays. Progressive removal of the C-rich and Dis domains reduced zymogen auto-activation and markedly diminished catalytic activity in both toxins. In cPIII, domain deletion caused a stepwise loss of proteolytic and cytotoxic activity without altering substrate preference. In Ecarin, removal of the accessory domains strongly impaired prothrombin activation, and thus plasma clotting, demonstrating a critical role in substrate recognition. Conversely, deletion of the C-rich domain increased fibrinogenolytic activity, revealing a substrate-dependent gatekeeping function. Deglycosylation showed that N-linked glycans modulate SVMP activity in a construct-dependent manner. Recombinant Ecarin closely recapitulated the biochemical properties of the native venom-derived toxin. Our data support a model in which PIII SVMP accessory domains enhance substrate positioning and catalytic efficiency while selectively restricting access to non-cognate substrates. These findings establish accessory-domain-mediated substrate recognition as a key determinant of SVMP function, informing rational antivenom design.

biochemistry↗

A tunable receptor separates root barrier formation from nutrient signaling through ligand-perception states

In roots, the endodermis controls nutrient entry by forming a barrier known as the Casparian strip1-3. Yet how barrier-associated processes interface with systemic signaling remains unclear. Here, we show that the receptor kinase SCHENGEN3 links local barrier surveillance to systemic nutrient signaling, with outputs that depend on the effective state of ligand perception. Unlike in Arabidopsis thaliana, SCHENGEN3 activation in Lotus japonicus requires a distinct cellular competence state and cannot be triggered by exogenous ligands alone, revealing evolutionary divergence in pathway deployment. Cross-species complementation uncouples systemic nitrogen signaling from Casparian strip formation, while transcriptomic and phosphoproteomic analyses reveal largely non-overlapping signaling- and barrier-associated programs that differ between agar and agricultural soil conditions. Mechanistically, receptor-ligand comparisons, engineered receptor variants, and co-receptor mutant analyses show that systemic nitrogen signaling is retained in receptor-perception states that are insufficient to support full Casparian strip establishment. Together, these findings reveal how a shared receptor module can separate developmental and physiological outputs by linking receptor perception state to output specificity.

plant biology↗

Nod factor signaling controlled genes in Medicago truncatula nodules

O_LILegume nodule formation is induced by rhizobia secreted Nod factors (NFs). It has been shown that NF receptors also accumulate in the apex of Medicago truncatula nodules. However, the NF signaling induced transcriptional changes in there have never been studied. C_LIO_LIHere, we studied this by using NF signaling mutant TE7, a weak allele of IPD3, blocked in rhizobial release. Nodule apices were isolated with laser microdissection and used for transcriptional analysis. C_LIO_LIWe identified 1655 NF signaling controlled genes in nodule apex. By comparing this with the transcriptome data from VAMP721d&e RNAi nodule apices, we identified a subset of 445 genes whose expression depends on NF signaling and rhizobial release. Further, we compared the set of genes controlled by NF signaling in nodule apices with that controlled in root epidermis, and these showed only a small overlap. NIN is induced by NF signaling both in the root epidermis and in the nodule. By overexpression of NIN in TE7 and knock down of NIN in wildtype nodules we showed that NF signaling controlled rhizobial release depends on NIN. C_LIO_LINF signaling controls a distinct set of genes in nodules, the function of which depends at least in part on NIN. C_LI

Developmental Biology↗

In Vivo Surface Reconstruction of Three Signal-defined Intracortical Layers Using 5T 3D FLAIR MR Imaging

BackgroundIn vivo whole-cortex quantification of intracortical signal-defined layering on the routinely acquired structural MRI remains limited. PurposeTo develop and validate an automated framework to reconstruct three intracortical signal-defined layers from 5T three-dimensional (3D) T2-weighted fluid-attenuated inversion recovery (FLAIR) and to characterize whole-cortex morphometrics and regional organization across a prespecified cortical organizational framework. Materials and MethodsIn this retrospective study, 5T 3D FLAIR images were acquired between February and July 2024. Brain Multi-Layer Surface Reconstruction (BrainMLSR) reconstructed three intracortical signal-defined layers, and derived intracortical layer thickness and surface area measures and ratios. Performance was evaluated against manual annotations and assessed for test-retest repeatability (n=13) and cross-site feasibility (n=2). Paired two-tailed t-tests and linear mixed-effects models were used. A proof-of-concept analysis compared Heschls gyrus ratios between 19 patients with temporal lobe epilepsy (TLE) and 19 age-matched healthy controls (HC). ResultsA total of 270 healthy participants (mean age, 54.4{+/-}14.5 years; 146 men) were included. Agreement with manual hypointense-layer annotations was high (Dice, 0.960{+/-}0.003), and was similar in the cross-site dataset (Dice, 0.954{+/-}0.009). In the test-retest dataset, average symmetric surface distance was less than 0.1 mm. Across prespecified systems, thickness and surface area ratios varied by region; within an auditory-perisylvian hierarchy, banksSTS showed a localized turning point with an increased hyperintense layer thickness ratio and decreased hypointense layer thickness ratio, accompanied by inflections in surface area ratios (P < .001). In bilateral Heschls gyrus, hypointense (left: 0.619{+/-}0.262 vs 0.881{+/-}0.102; right: 0.607{+/-}0.310 vs 0.907{+/-}0.141 mm) and isointense (left: 0.406{+/-}0.225 vs 0.678{+/-}0.128; right: 0.478{+/-}0.232 vs 0.808{+/-}0.176 mm) layer thicknesses were lower in TLE than in HC (all P<.001). ConclusionBrainMLSR enabled accurate and repeatable in vivo reconstruction of three intracortical signal-defined layers from a single 5T 3D T2-weighted FLAIR acquisition and provided whole-cortex boundary-based morphometry with interpretable regional organization. Key ResultsO_LIIn this retrospective study of 270 participants, Brain Multi-Layer Surface Reconstruction (BrainMLSR) showed high agreement with hypointense-layer annotations (Dice, 0.960{+/-}0.003). C_LIO_LIWithin an auditory-perisylvian hierarchy, the hyperintense-layer thickness ratio peaked (0.620{+/-}0.003) and the hypointense-layer ratio was the lowest (0.156{+/-}0.002) in the bank of the superior temporal sulcus (P < .001). C_LIO_LIIn a proof-of-concept analysis, patients with temporal lobe epilepsy (vs healthy controls) had higher hyperintense-layer thicknesses ratio in the left hemisphere (0.523{+/-}0.085 vs 0.417{+/-}0.036, P<.001). C_LI SummaryAn automated multiple-layer surface reconstruction framework (BrainMLSR), applied to 5T 3D T2-weighted FLAIR images, produced reproducible whole-cortex, signal-defined laminar morphometry and demonstrated coherent patterns across prespecified cortical organizational framework.

neuroscience↗

Immunotherapy with Pro-regenerative Macrophages from Embryonic Stem Cells Ameliorate Osteoarthritis via TNFAIP3-Mediated Chondroprotection

Immune cell-based therapies for osteoarthritis (OA) remains underexplored. Macrophages coordinate tissue repair, yet their functional heterogeneity and therapeutic applicability, particularly for designed stem cell-derived populations with tailored reparative properties are not fully delineated. Inspired by embryonic skeletal development as a paradigm of scarless regeneration, we identified a pro-regenerative macrophage population through a single-cell RNA atlas of developing skeletal tissues. We subsequently established a mechanically dynamic induction approach to efficiently generate pro-regenerative macrophages from human embryonic stem cells (hESCs), mimicking this developmental phenotype. In vitro, these macrophages mitigate cartilage degradation in OA explants and suppress pro-inflammatory, catabolic signaling in chondrocytes. Injection into murine OA joints robustly attenuates disease progression, reduces extracellular matrix breakdown, and resolves synovial inflammation, representing the first demonstration of immune cell-based therapy for OA. Mechanistically, these macrophages orchestrate chondroprotection not through broad immunosuppression, but by activating a TNFAIP3-dependent signaling hub that restrains inflammatory and catabolic responses in chondrocytes. This TNFAIP3 activation precisely reversed the OA-associated gene signature, suppressing catabolic mediators while enhancing reparative factors, thereby restoring the anabolic-catabolic balance. Our findings uncover a developmental blueprint for engineering pro-regenerative macrophages from stem cells and establish a pioneering targeted immune cell therapy that engages an intrinsic chondroprotective program, offering a transformative and translationally relevant strategy for OA treatment.

immunology↗

Hydration Network Drives Activation and G Protein Selectivity in GPR174

G protein-coupled receptor 174 (GPR174), a key modulator of autoimmune responses, maintains immune homeostasis through distinct G protein signaling pathways, particularly Gs and Gi. Although the structural mechanism of lysophosphatidylserine (LysoPS)-activated GPR174 in the Gs pathway has been characterized, how hydration-mediated interactions influence GPR174 activation and signaling selectivity remains unclear. Here, we determined high-resolution cryo-electron microscopy (cryo-EM) structures of LysoPS-activated GPR174 bound to Gs (2.0 [A]) and Gi (3.4 [A]), revealing a continuous hydration-mediated signaling transduction network that bridges the sodium-binding pocket, NPxxY and DRY motifs, and the G protein-binding interface. This network stabilizes the active-state conformation of GPR174 and dynamically reshapes the intracellular cavity, thereby enabling differential engagement of Gs and Gi. Molecular dynamics simulations and functional assays demonstrated that the hydration network is essential for receptor activation and selectively modulates G protein coupling. To evaluate its conservation, we performed sequence alignment and structural analysis across class A GPCRs, defining three hydration cavities: the conserved water cavity (CWC), the junction water cavity (JWC), and the extended water cavity (EWC), whose hydration is determined by residue properties at position 5.58. Together, our study reveals a hydration-driven molecular mechanism that underlies the activation of GPR174 and its dual G protein selectivity. These findings advance the understanding of hydration-mediated signaling in GPR174 and provide a framework for investigating water-mediated regulation across class A GPCRs.

biophysics↗

Evolutionary predisposition of NIN to function in nitrogen-fixing nodules

Nitrogen-fixing nodule symbiosis is an ecologically and economically important trait in legumes and some related species. A critical step in the evolution of nodulation is the recruitment of NODULE INCEPTION (NIN); a homolog of the nitrate-sensing NIN-LIKE PROTEIN (NLP) transcription factors. However, whether adaptations have occurred in the NIN protein upon its recruitment in symbiosis remains elusive. Here we show that non-symbiotic NIN orthologs can function in intracellular infection and even nodule initiation, demonstrating that these properties of NIN predate the evolution of nodulation. Concurrent with the evolution of nodulation, symbiotic NIN proteins were optimized for their role in symbiosis by acquiring nitrate independent functionality, including constitutive nuclear localization. A single amino acid substitution in Arabidopsis AtNLP2 enhances its nuclear localization under low nitrate conditions, making it functionally comparable to a symbiotic NIN. These findings highlight that NIN was predisposed to function in nodulation at the time of its recruitment into nitrogen-fixing symbiosis. Our study provides novel insight in how and why non-symbiotic NIN orthologues could be recruited to function in nitrogen-fixing root nodule symbiosis.

plant biology↗

Efficient Recombinant Production and Functional Characterisation of Cytotoxic and Haemotoxic Snake Venom Metalloproteinases

Snake venoms contain diverse mixtures of toxins that evolved to incapacitate prey, but in humans they cause extensive pathology following snakebite envenomation. In viper venom, some of the most potent toxins are the haemorrhagic and coagulopathic snake venom metalloproteinases (SVMPs). Because venoms contain a SVMP cocktail, and due to their cytotoxicity, SVMP characterizations have been hampered by the lack of purified enzymes. By incorporating their prodomain, which blocks the active SVMP site, we overcame their cytotoxicity and enabled recombinant production of zymogens from all three structurally variable SVMP classes (PI, PII and PIII) using our baculovirus/insect cell expression system. Zymogens were auto-activated by incubation with Zn2+ ions, resulting in prodomain cleavage, PII disintegrin cleavage and PIII prodomain proteolysis. Auto-activated SVMPs were characterized using protein substrate degradation, platelet aggregation and blood coagulation assays, benchmarked to native venom-purified SVMP. Our recombinant zymogen production protocol is generically applicable for the expression of SVMPs, unlocking biomedical use in haematology, and discovery of novel snakebite therapeutics.

pharmacology and toxicology↗

PB-scope: Contrastive learning of dynamic processing body formation reveals undefined mechanisms of approved compounds

Membraneless organelles (MLOs) formed by liquid-liquid phase separation contribute to intracellular compartmentalization of specific biological functions. The dynamic regulation of MLOs is critical for RNA metabolism, stress response, and signal transduction. While the number and size of MLOs serve as valuable indicators of cellular states, leveraging their spatial distribution and diverse quantitative characteristics to infer cellular conditions has posed significant challenges. Here, we present processing body (PB)-scope: an unsupervised deep learning-based framework designed for imaging-based phenotypic screening of PB, one representative MLO. The model was trained on a substantial dataset comprising over 400,000 single-cell confocal microscopy images from a colon cancer cell line treated with 280 compounds. PB-scope enabled precise drug classification based on their effects on PB characteristics, including number and size. This artificial intelligence framework offers insights previously obscured by the dynamic and subtle nature of PBs. Notably, PB-scope identified drugs with a shared function in the JAK signaling pathway, a previously unrecognized regulator of PB dynamics. PB-scope holds the potential for adapting to the study of other MLOs, offering broad applicability in this emerging field of cell biology.

cell biology↗

Diffusion Neuroimaging of Speech Acquisition in Infants

How multisystem white-matter maturation supports early speech acquisition in infancy remains unclear. Using longitudinal diffusion MRI from the Baby Con-nectome Project, we quantified neurite density maturation from birth to 24 months across 33 white-matter tracts spanning motor, auditory, visual, ventral-language, and dorsal-language systems. Maturation was coordinated across systems rather than confined to canonical language pathways: dorsal-language association tracts began relatively immature near birth but showed steep postnatal growth, pro-gressively rebalancing with motor and sensory systems. Tract-behavior coupling was likewise distributed across developmental domains, with broadly positive age-adjusted associations for motor outcomes, a sparser, partly motor-like profile for gesture-related phrase comprehension, and predominantly negative associa-tions for lexical speech-related outcomes. Exploratory follow-up analyses further suggested motor and social precursor capacities that may help mediate these in-direct developmental routes from structural maturation to later speech-related outcomes. These findings support a multisystem developmental account in which early speech acquisition emerges not from an isolated language pathway, but from coordinated maturation across multiple systems and the precursor skills that link them.

neuroscience↗

Dynamic Monomer-Dimer Transition in Ligand-induced Apelin Receptor Activation

G-protein-coupled receptors (GPCRs) are significant signal transducers that exist as monomers and in multiple oligomeric forms. However, molecular mechanism driving their dynamic interconversion to regulate intricate signaling in class A GPCRs remains elusive, compounding our understanding of their related pathophysiological functions. Here, we present a set of 12 assemblies of the apelin receptor (APLNR), including dimeric apo state, agonistic small molecule- or nanobody-bound state of monomeric and dimeric APLNR with and without G-proteins, providing a detailed dynamic view of the monomer-dimer transition. High-resolution cryo-EM structures reveal that different ligands induce varying degrees of pre-dissociation of dimers in the absence of G-protein, with G-protein coupling facilitating the transition from dimeric to monomeric receptor. Functional studies further highlight the critical role of cholesterol clusters in stabilizing the APLNR dimers. These insights enhance our understanding of the dynamic regulation of class A GPCRs across different aggregated forms and advance the rational drug design strategies aimed at selectively modulating of APLNR signaling.

molecular biology↗

Auditory tract maturation in early infancy: Links to speech production

Infants exhibit remarkable language acquisition abilities, supported by highly plastic neural substrates that dynamically interact with early speech experiences. However, the developmental mechanisms of these neural substrates and their specific role in speech acquisition remain incompletely understood. Here, we present NeoAudi Tract (NAT), a robust automated toolbox for extracting the full set of auditory tracts in infants from birth to 24 months using 3T diffusion MRI data. By characterizing the microstructural changes in these tracts, we demonstrate a gradual and continuous maturation process of the auditory system. Additionally, we identify significant correlations between auditory tract maturation and both fine-motor skills and expressive language t-scores from the Mullen Scales of Early Learning tests. Our findings highlight the role of the auditory system in speech production and indicate the intertwined development of auditory and motor systems that underlies speech acquisition, particularly during perceptual reorganization.

neuroscience↗

Weakly Supervised Vector Quantization for Whole Slide Images Classification

Whole Slide Images (WSIs) are gigapixel, high-resolution digital scans of microscope slides, providing detailed tissue profiles for pathological analysis. Due to their gigapixel size and lack of detailed annotations, Multiple Instance Learning (MIL) becomes the primary technique for WSI analysis. However, current MIL methods for WSIs directly use embeddings extracted by a pretrained vision encoder, which are not task-specific and often exhibit high variability. To address this, we introduce a novel method, VQ-MIL, which maps the embeddings to a discrete space using weakly supervised vector quantization to refine the embeddings and reduce the variability. Additionally, the discrete embeddings from our methods provides clearer visualizations compared to other methods. Our experiments show that VQ-MIL achieves state-of-the-art classification results on two benchmark datasets. The source code is available at https://github.com/aCoalBall/VQMIL.

pathology↗

Down regulation of Engase in Caenorhabditis elegans may improve its stresses adaptivity

Endo-beta-N-acetylglucosaminidase (ENGASE) is one of the key enzymes involved in the regulation of structure and function of glycoproteins. It is conserved from prokaryotic to eukaryotic cells. Although their activities in vitro and applications have been well studied, the biological function of ENGASE remains to be illustrated. In this study, we analyzed the molecular and physiological function of Engase from Caenorhabditis elegans homolog eng-1(CeEngase). We found that CeEngase knockout or knockdown increased the environmental stresses adaptability, such as heat stress and osmotic stress. Preliminary glycomics analysis showed that the basement membrane proteins of extracellular matrix may be the main targets of CeENGASE. In addition, CeENGASE may selectively prefer to N2H7 glycans on glycoproteins. In conclusion, our data illustrated that the defection and/or down regulation of CeEngase may provide a beneficially adaptation for stresses.

biochemistry↗

CircRNA-Pro: A Novel Toolkit for High-Precision Detection of Differentially Expressed Circular RNAs and Translatable Circular RNAs

With the increasing discovery of circular RNAs (circRNAs) and their critical roles in gene regulation and disease progression, there is a growing need for more accurate and efficient tools for circRNAs research. In response, we have developed an integrated software suite specifically for circRNAs. This all-in-one tool specializes in detecting differentially expressed circRNAs, including those with the potential to be translated into proteins, and allows for comparing against relevant databases, thereby enabling comprehensive circRNA profiling and annotation. To enhance the accuracy in detecting differentially expressed circRNAs, we incorporated three different software algorithms and cross-validated their results through mutual verification. Additionally, this toolkit improves the effectiveness in identifying translatable circRNAs by optimizing Ribo-seq alignment and verifying against public circRNA databases. The performance of circRNA-pro has been evaluated through its application to public RNA-seq and Ribo-seq datasets on breast cancer and SARS-CoV-2 infected cells, and the results obtained have been validated against previous literature and databases. Overall, our integrated toolkit provides a reliable workflow for circRNA research, facilitating insights into their diverse roles across life sciences.

bioinformatics↗

A Regularized Cox Hierarchical Model for Incorporating Annotation Information in Predictive Omic Studies

BackgroundAssociated with high-dimensional omics data there are often "meta-features" such as biological pathways and functional annotations, summary statistics from similar studies that can be informative for predicting an outcome of interest. We introduce a regularized hierarchical framework for integrating meta-features, with the goal of improving prediction and feature selection performance with time-to-event outcomes. MethodsA hierarchical framework is deployed to incorporate meta-features. Regularization is applied to the omic features as well as the meta-features so that high-dimensional data can be handled at both levels. The proposed hierarchical Cox model can be efficiently fitted by a combination of iterative reweighted least squares and cyclic coordinate descent. ResultsIn a simulation study we show that when the external meta-features are informative, the regularized hierarchical model can substantially improve prediction performance over standard regularized Cox regression. We illustrate the proposed model with applications to breast cancer and melanoma survival based on gene expression profiles, which show the improvement in prediction performance by applying meta-features, as well as the discovery of important omic feature sets with sparse regularization at meta-feature level. ConclusionsThe proposed hierarchical regularized regression model enables integration of external meta-feature information directly into the modeling process for time-to-event outcomes, improves prediction performance when the external meta-feature data is informative. Importantly, when the external meta-features are uninformative, the prediction performance based on the regularized hierarchical model is on par with standard regularized Cox regression, indicating robustness of the framework. In addition to developing predictive signatures, the model can also be deployed in discovery applications where the main goal is to identify important features associated with the outcome rather than developing a predictive model.

bioinformatics↗

Chrom-pro: A User-Friendly Toolkit for De-novo Chromosome Assembly and Genomic Analysis

Chromosome-level genome assembly is fundamental to current genomic and post-genomic research, however, the process remains complicated and challenging due to the lack of a standardized automatic workflow. The frequently-used method for high-quality genome assembly generally employs second-generation sequencing (SGS) low error reads, third-generation sequencing (TGS) long reads and Hi-C reads. In this study, we developed a multifunctional toolkit called Chrom-pro that integrated commonly-used algorithms for de novo chromosome-level genome assembly with above three data sets into a user-friendly, automatic workflow. Besides chromosome assembly, Chrom-pro also encompasses multiple functionalities for genome quality assessment, comparative genomic analysis, and structural variant detection, which offers substantial support for downstream research. To evaluate the performance of Chrom-pro software, we tested the software with publicly available sequencing data of mango, pufferfish, and plum, and the excellence was confirmed by achieving a BUSCO completeness score of over 95% as well as high collinearity with the reference genome. Furthermore, we applied Chrom-pro to investigating the impact of different internal algorithm options on the accuracy of chromosome assembly, providing guidance for advancing relevant research in the future. Overall, the development of Chrom-pro will significantly improve the efficiency and quality of chromosome assembly and contributing to the advancement of genomic research.

bioinformatics↗