Biology subjects

Chen, W.

Publications and source records attributed to Chen, W..

At least 127 records · Page 7Linked to original sources

bioRxiv2025

Continuous Production of Recombinant Adeno-Associated Virus in the Insect Cell/Baculovirus Expression Vector System

Continuous production processes may offer significant advantages for biotherapeutic manufacturing, including increased productivity, consistent product quality, reduced facility footprint, and decreased process turnaround time. Despite these benefits, the in-situ formation of defective recombinant baculovirus expression vectors (BEVs) hinders the continuous manufacturing of recombinant adeno-associated viruses (rAAV) in the baculovirus expression vector system. This study investigates an approach of reducing defective viruses through the infusion of standard recombinant baculovirus (rBV) and the compartmentalization of early- and late-stage infected cells, resulting in stable rAAV production. In this study, rAAVs were continuously produced in a series of cascading reactors, comprising a feeder reactor, an infection reactor, and a production reactor. Residence times and transfer rates across the three reactors were optimized based on the production kinetics of rBV and rAAV derived from our mechanistic model. The majority of rBV was produced within the production reactor, thereby reducing the impact of defective viruses in the infection reactor, enabling continuous rAAV production. This study demonstrates the successful implementation of a continuous rAAV production process, yielding over 5x1010 vg/mL per day for 4 weeks. This work represents the first reported continuous rAAV production process utilizing the Sf9/BEVS platform and establishes engineering know-how for overcoming manufacturing challenges associated with rAAV-based gene therapies.

bioengineering↗

bioRxiv2025

Residue-level mapping of crowding effects on protein phase separation

Proteins can undergo liquid-liquid phase separation to form condensates within the crowded cellular environment. In vitro, polymer-based crowding agents are widely used to mimic this effect, but how their chemical environments influence phase separation compared to native cytosol remains unclear. Here, we use NMR to probe the chemical influences on the intrinsically disordered region of RNA polymerase II under different crowding conditions, including polymer-based crowders, protein-based crowders, and reconstituted E. coli cytosol. We find a general trend of enhanced protein self-interactions across all conditions, but also distinct chemical environments that depend on crowder identity, reflecting changes in preferential interactions. Given the widespread use of polymer crowders in phase separation studies, our results establish a strategy to dissect the microscopic role of crowders and lay the foundation for designing more physiologically relevant in vitro crowding models of protein phase separation. More broadly, this framework enables systematic probing of residue-level environmental influences in complex settings including within the cellular milieu.

biophysics↗

bioRxiv2025

Glycosphingolipids Regulate Phosphatidylserine Transport at ER-PM Contact Sites

Plasma membrane (PM) localization of KRAS requires specific glycosphingolipids in the outer leaflet and phosphatidylserine (PS) in the inner leaflet. PM PS content is controlled by lipid transport proteins ORP5 and ORP8, which operate at ER-PM membrane contact sites (MCSs). Using high-resolution imaging, we now show that GSLs including GM3 and SM4, are required to maintain ORP5 and ORP8 localization to MCSs. Genetic deletion or pharmacologic inhibition of enzymes required for the biosynthesis of GM3 or SM4, displace PI4-kinase Type III (PI4KIII) and its adaptor EFR3A from the PM, thereby reducing PM phosphatidylinositol 4-phosphate (PI4P) content. PM interactions of ORP5 and ORP8 are also disrupted. Since ORP5 and ORP8 transport PS to the PM by counter-transporting PI4P to the ER, PM PS content is substantially reduced. We further show that GM3 and GM2 regulate the assembly of ER-PM-MCSs, such that the function of other MCS-localized macromolecular machineries including calcium release-activated calcium channels is abrogated when glycosphingolipid biosynthesis is blocked. Together, this study establishes glycosphingolipids as organizers of PS transport and ER-PM MCSs, expanding the regulators of MCSs beyond protein tethers to include glycosylated lipids and revealing how glycosphingolipids control KRAS function.

molecular biology↗

bioRxiv2025

Novel Calcin peptides from Scorpion Venom Exhibit Humidity-Dependent Expression and Ryanodine Receptor Activation

Scorpions are venomous animals of considerable pharmacological potential, traditionally used in East Asia to treat cardiovascular and autoimmune diseases. However, geographical variations in the expression of their venom components remain poorly understood. We performed transcriptome profiling on scorpion samples collected from eight distinct climatic and environmental regions in East Asia, we successfully identified two distinct scorpion species: Uroctonus mordax and Mesobuthus martensii. Furthermore, we identified two novel calcins from the venoms of these species, with their expression levels showing a significant correlation to environmental humidity. Functional assays revealed that these calcins differentially potentiate [3H]ryanodine binding to RyR isoforms and induce a persistent subconductance state, suggesting distinct mechanisms of action. Our findings suggest biogeographical variation in venom composition between species and suggest that environmental humidity as a key factor influencing calcin expression. These results expand the structural diversity of the calcin family and provide a mechanistic basis for the medicinal application and ecological adaptation of scorpion venom.

molecular biology↗

bioRxiv2025

Experimental demonstration of daisy chain gene drive and modelling of daisy suppression systems

CRISPR-based gene drive can address ecological problems by biasing their inheritance coupled with an effector for either population modification of suppression. However, the risk of uncontrolled spread impedes some applications of gene drive. Daisy chain drives have received much attention as a potential approach to overcome this problem. They potentially allow efficient spread in a target population but are ultimately self-limiting. This is achieved by splitting a standard gene drive into multiple dependent elements, where each element can bias the inheritance of another, except one non-driving element. With the successive loss of each chain link, spread of transgenic elements will slow down and eventually stop. Here, we use modelling to assess the population dynamics of suppression daisy chain drives in both panmictic and continuous space models. We find that achieving population elimination through a single release of daisy chain gene drives is possible but difficult, with relatively high requirements for drive performance and release size. These effects are substantially amplified in spatial models. We also constructed two configurations of daisy chain gene drives in Drosophila melanogaster as a proof-of-principle. One is a rescue drive for population modification, and the other aims for population suppression by targeting a female fertility gene. Both functioned within expectations at moderate efficiency in individual crosses. However, the system failed to spread in cage populations because of higher than expected fitness costs. Overall, our study demonstrates that daisy chain systems may be promising candidates for both modification and suppression, but challenges remain in both construction and potential deployment in large regions.

genetics↗

bioRxiv2025

RNA polymerase II CTD Ser5 phosphorylation induces competing effects of expansion and compaction

The carboxy-terminal domain (CTD) of RNA Polymerase II, composed of tandem heptad repeats with the consensus sequence YSPTSPS, orchestrates the transcription cycle through a dynamic series of post-translational modifications. Among these, the phosphorylation of Ser5 is critical for initiator/promoter clearance and the recruitment of capping enzymes. However, the exact conformational consequences of these modifications are still not fully understood. This study investigates how Ser5 phosphorylation affects the local and global conformation of the CTD, its influence on proline isomerization, and how variations in the repeat sequence modulate these effects. We employed Gaussian accelerated Molecular Dynamics (GaMD) simulations on 3-heptad models of both the consensus CTD sequence and an Asn7 variant. We found that Ser5 phosphorylation promotes expansion of the peptide due to the repulsion between the negatively-charged phosphate groups, but also increases the population of cis-Pro6, which leads to compaction. We used a clustering algorithm to identify commonly populated conformations, with a focus on those conformations that change in population with Ser5 phosphorylation. Our simulations reveal that the expansion of the CTD due to Ser5 phosphorylation is accompanied by a change in local, intra-heptad interactions in both variants. Notably, phosphorylation significantly increases the population of cis-Pro6 due to steric repulsion between the Asn7 side chain and the large side chain of the phosSer5, but has a smaller increase in the consensus variant. These results clarify the underlying mechanisms by which phosphorylation can modulate the CTDs structural landscape to regulate the transcription cycle. SignificanceThe RNA Polymerase II CTD is a critical part of the machinery that regulates transcription, and therefore, understanding how it functions in this process is essential. However, the conformational effects of known modifications to the CTD, such phosphorylation and proline isomerization, are not fully understood. This paper uses all-atom molecular dynamics simulations to identify the specific conformational changes to the disordered CTD with phosphorylation, and with changing heptad sequence. We also identify the interactions that are responsible for these changes. Our results emphasize that two chemical properties of phosphate groups, their negative charge and their large size, can affect protein conformation. For the CTD, these properties have competing effects on the overall compaction of the disordered sequence.

biophysics↗

bioRxiv2025

Atypical developmental remodeling of dopamine neurons involves AKT-GSK3β signaling and glia-mediated axon degeneration

Neuronal remodeling is essential for sculpting neural circuits, and its disruption has been implicated in neurodevelopmental and neuropsychiatric disorders. Yet the molecular and cellular diversity of remodeling across neuron types remains incompletely understood. Here, we uncover a distinct remodeling mode in a subtype of Drosophila dopamine neurons (DANs) critical for learning, memory, sleep, and locomotion. Unlike the stereotypical pruning-then-regrowth paradigm, these DANs undergo a transient axon overgrowth followed by selective pruning during metamorphosis. Remarkably, DAN axon pruning proceeds independently of canonical ecdysone signaling and instead involves neuron-intrinsic AKT-GSK3{beta} signaling and extrinsic glial activity. Disruption of AKT-GSK3{beta} signaling alters microtubule stability and impairs glial recruitment and clearance of axonal debris. Notably, the role of AKT-GSK3{beta} is cell-type specific, underscoring mechanistic diversity in remodeling programs. These findings reveal an unexpected overgrowth-then-pruning developmental trajectory, establishing DANs as a powerful model to uncover the mechanisms underlying neuronal remodeling, circuit maturation, and neurodegeneration.

neuroscience↗

bioRxiv2025

shinyUMAP: an online tool for promoting understanding of single cell omics data visualization

Visualization is widely used to help explore and interpret high dimensional single cell (sc) omics data, such as scRNA-seq expression data. In particular, uniform manifold approximation and projection (UMAP) has become nearly ubiquitous in scientific publications that apply single cell omics technologies. Some experts have expressed concerns that the global cell-cell relationship, especially the spatial distances among cell clusters in a dataset, may not be faithfully depicted in a 2-dimensional (2D) UMAP. To help users to better appreciate this issue with their own data, we created an online server for the community to upload their single cell data and interactively make UMAPs with different hyper-parameters to witness how the distribution of cell clusters changes. The server thus can help promote proper usages of UMAP, especially to avoid the common pitfalls in misinterpretation of inter-cluster relationships in single cell studies. Availability and ImplementationShinyUMAP is freely available as an online Shiny server implemented in Python at https://scviewer.shinyapps.io/shinyUMAP/.

bioinformatics↗

bioRxiv2025

VAMP8 function reveals tight linkage between endocytic recycling and endocytosis

Clathrin-mediated endocytosis (CME) is a multistage process that involves the initiation and stabilization of clathrin-coated pits (CCPs) that invaginate and finally detach from the plasma membrane to form clathrin-coated vesicles (CCVs). Given that SNARE proteins are essential for downstream vesicle targeting and fusion events, their recruitment into nascent CCVs has been suggested to be a prerequisite for CME progression. However, which and how SNARE proteins regulate CME remains to be explored. Here, we showed that siRNA-mediated knockdown of the R-SNARE, VAMP8 impairs CCP initiation, stabilization and invagination and strongly inhibits CME. Mechanistically, recruitment of VAMP8 to CCVs is not required for CME. Instead, depletion of VAMP8 inhibits recycling of endocytic cargoes and as exemplified here by transferrin receptor, skews their trafficking toward lysosomal degradation. VAMP8 depletion therefore indirectly impairs CCV formation and inhibits CME by depleting endocytic cargo. Overall, our study provides new insights into the crosstalk between endocytosis and endocytic recycling of CME cargo and demonstrates the critical role for cargo recruitment in stabilizing nascent CCPs to regulate CME. Significance StatementWe demonstrate that VAMP8 depletion inhibits recycling of endocytic cargo and reroutes transferrin receptors toward lysosomal degradation, thereby impairing endocytic vesicle formation and inhibiting clathrin-mediated endocytosis. Thus, the work uncovers a critical link between endocytic recycling and endocytosis, highlighting the importance of recycling pathways in maintaining membrane dynamics. However, the recruitment of VAMP8 into nascent clathrin-coated pits by CALM is not required for CME. While SNARE recruitment is essential for formation of COPI and COPII vesicles, our findings suggest that this requirement is not generalizable to endocytic CCVs.

cell biology↗

bioRxiv2025

Comparative snRNAseq study of C9orf72, SOD1, and sALS spinal cord

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the loss of motor neurons, yet the cell-type specific molecular alterations within the spinal cords are not well characterized. In this study, we conducted deep molecular profiling of spinal cord tissues donated by people living with sporadic, C9orf72, and SOD1-ALS using single-nucleus RNA sequencing (snRNAseq). We observed numerous distinct gene expression patterns and enriched pathways among ALS types. However, when focusing on common features, we identified activation of stress-response and inflammatory pathways in specific microglia subtypes, as well as disrupted vesicle transport and synaptic function in a ventral inhibitory neuronal subtype. Notably, CPLX3, a SNARE regulator, was uniquely expressed in alpha-motor neurons and was commonly downregulated across all ALS types. While this study uncovers the molecular heterogeneity underlying ALS, it also highlights shared pathways within specific cell types, especially in the ventral inhibitory neurons that have been less explored in ALS research.

neuroscience↗

bioRxiv2025

Hypoxia-induced metastatic heterogeneity in pancreatic cancer

In most solid tumors, hypoxia constitutes a defining microenvironmental feature that reprograms malignant cells into a highly metastatic state by driving cellular plasticity and exacerbating chromosomal instability (CIN). However, the mechanisms by which cancer cells concurrently co-opt these elements of hypoxic adaptation to promote metastasis remains poorly understood. Here, we report that hypoxia promotes metastasis by suppressing the JmjC-containing histone lysine demethylase Kdm8. CRISPR/Cas9-mediated targeting of Kdm8 in a Kras;Trp53-driven mouse model of pancreatic ductal adenocarcinoma (PDA) robustly rewires the malignant cell transcriptomic programs, leading to a profound loss of the epithelial morphology and widespread metastatic disease. In PDA patients, a high KDM8-induced gene signature is associated with reduced metastatic burden and better survival in advanced disease. Notably, Kdm8 suppression in normoxia recapitulates key aspects of the global epigenetic and transcriptomic rewiring, mitotic spindle defects, and CIN induced by hypoxia. Moreover, disruption of Kdm8s demethylase activity phenocopies Kdm8 loss, whereas expression of hypermorphic Kdm8 variants resistant to hypoxic suppression markedly reduces metastasis beyond the levels achieved by the wildtype protein. Through the suppression of Kdm8 demethylase function, hypoxia unleashes a potent metastatic program by simultaneously advancing cellular plasticity and CIN.

cell biology↗

bioRxiv2025

SARS-CoV-2 NSP14 inhibitor exhibits potent antiviral activity and reverses NSP14-driven host modulation

The emergence of SARS-CoV-2 variants and drug-resistant mutants highlights the urgent need for novel antiviral therapeutics. SARS-CoV-2 NSP14, an N7-guanosine methyltransferase, plays a critical role in viral RNA capping, enabling viral replication and immune evasion. While NSP14 has emerged as a promising drug target, its role in host-virus crosstalk and the cellular consequences of NSP14 inhibition remain poorly understood. Here, we present the identification and characterization of C10, a highly potent and selective first-in-class non-nucleoside inhibitor of the NSP14 S-adenosylmethionine (SAM)-binding pocket. C10 demonstrates robust antiviral activity against SARS-CoV-2, including its variants, with EC50 values ranging from 64.03 to 301.9 nM, comparable to the FDA-approved drug remdesivir in our cell-based assays. C10 also exhibits broad-spectrum activity against other betacoronaviruses and inhibits SARS-CoV-2 at the replication stage. C10 suppresses viral translation and exhibits immunostimulatory effect. Additionally, C10 specifically reversed NSP14-mediated alterations in host transcriptome. The antiviral efficacy of C10 was further validated in a transgenic mouse model of SARS-CoV-2 infection. Our findings highlight C10 as a promising candidate for the development of effective treatments against SARS-CoV-2 and its emerging variants. This study also uncovers a novel mechanism of NSP14 in SARS-CoV-2 pathogenesis and its therapeutic potential, providing insights that may extend to other viral capping methyltransferases.

microbiology↗

bioRxiv2025

scSTAR2: a multiomics integration algorithm to reveal disease specific cellular signatures by bridging single-cell resolution features and clinical metadata

Single-cell techniques, pivotal in characterizing intricate cell and spatial structures within tissues, face challenges in contextualizing clinical phenotypes. Currently, most investigations of the clinical phenotype related cell/spatial heterogeneities relied on the phenotype information from single-cell data. However, a wealth of underutilized clinical metadata exists within conventional bulk sequencing data. Current methods for correlating clinical information with individual cells or spatial regions remain limited and lack robustness. Here we present scSTAR2, a novel algorithm that transcends existing limitations by reconstructively integrating multiomics data to identify cells associated with clinical phenotypes. Unlike existing methods, scSTAR2 reconstructs single-cell data guided by specific phenotypes, significantly reducing interference from irrelevant noise. By employing scSTAR2 to integrate scRNA-seq, scATAC-seq, and spatial transcriptomics and bulk RNA-seq, we not only confirmed a new heat-shock Treg subtype in tumors but also more sensitively identified TLS (tertiary lymphoid structure) areas than traditional methods. In conclusion, scSTAR2 has proven to significantly enhance single-cell data interpretation across diverse clinical scenarios.

bioinformatics↗

bioRxiv2025

Design of Orthogonal Far-Red, Orange and Green Fluorophore-binding Proteins for Multiplex Imaging

Fluorescent proteins and small molecule dyes have complementary strengths for biological imaging: the former are genetically manipulatable enabling tagging of specific proteins and detection of protein interactions, while the latter have greater photostability and brightness but are difficult to target. To combine these strengths, we used de novo protein design to generate binders to three bright, stable, cell-permeable dyes spanning the visible spectrum: JF657 (far red), JF596 (orange-red) and JF494 (green). For each dye, we obtain nanomolar binders with weak or no binding to the other two dyes; the accuracy of the design approach is confirmed by a crystal structure of one binder which is very close to the design model. Fusion of the JF567, JF596 and JF494 binders to three different targets followed by staining with the three dyes simultaneously enables multiplex imaging. We further expand functionality by incorporating an active site carrying out nucleophilic aromatic substitution to form a covalent linkage with the dye, and developing split versions which reconstitute fluorescence at subcellular locations where both halves are present, enabling both protein-protein interaction detection and chemically induced dimerization with fluorescence reporting. Our designs combine the advantages of fluorescent proteins and small molecule dyes and should be broadly useful for cellular imaging.

biochemistry↗

bioRxiv2025

Loss of vitamin C biosynthesis protects from a parasitic infection

The ability to synthesize essential molecules is sometimes lost in evolution. A classic example is ascorbate (Vitamin C), which is synthesized in most animals by L-Gulonolactone Oxidase (GULO), an enzyme lost multiple independent times in animal evolution. This event is thought to be evolutionarily neutral, however, GULO-deficient animals including humans need to obtain ascorbate from their diet and are susceptible to ascorbate deficiency and scurvy. We therefore hypothesized that this disadvantage of GULO loss is offset by physiological benefits. Here we show that ascorbate deficiency protects mice from schistosomiasis, a debilitating parasitic disease which afflicts 250 million people. Schistosoma mansoni worms required host ascorbate to produce eggs in vivo. Consequently, ascorbate-deficient mice were protected from schistosomiasis pathologies and transmission. Intermittent ascorbate deficiency protected Gulo-deficient mice from both scurvy and schistosomiasis mortality. The effects of ascorbate on schistosome reproduction were mediated by ascorbate-dependent histone demethylation which promoted vitellocyte development in female schistosomes. We propose that vitamin deficiencies are not always detrimental but can protect animals from pathogens which need to obtain vitamins from their host.

biochemistry↗

bioRxiv2025

High-light adaptation in Synechocystis by accumulating NDH proteins and depleting specific phycobilisome linker proteins

Photosynthetic organisms have evolved mechanisms to manage excess light, crucial for maximizing photosynthetic efficiency. High-light (HL) tolerant Synechocystis sp. PCC6803 strains were developed through laboratory evolution, with tolerance attributed to specific point mutations. Key mutations affected the NDH-1L complex F1-subunit (NdhF1F124L) and translation elongation factor G2 (EF-G2R461C). Reintroducing these mutations into laboratory strains conferred HL tolerance. Comparisons with knockout and overexpressor lines showed NdhF1F124L and EF-G2R461C result in gain of function. Transcriptomic and proteomic analysis unveiled a network of responses contributing to HL tolerance, including maintenance of phosphate metabolism and decreased antenna size by depleting a specific linker protein in EF-G2R461C cells. Consequently, overexpression of Pho regulon genes increased HL tolerance. NdhF1F124L enhances cyclic electron flow (CEF) by increasing NDH-1 complex subunit accumulation. Other HL-adapted strains demonstrated that increased CEF and decreased antenna size are recurring outcomes, achievable through various mutations. This study demonstrates how limited mutations can reconfigure cells for enhanced HL tolerance, offering insights for improving photosynthetic efficiency.

physiology↗

bioRxiv2025

XL-Ranker: A Computational Workflow for Prioritizing Protein-Protein Interactions from Cross-Linking Mass Spectrometry Data

Protein-protein interactions (PPIs) are central to virtually all biological processes, and their disruption can lead to a wide spectrum of human diseases. Cross-linking mass spectrometry (XL-MS) enables proteome-scale detection of interacting peptide pairs, from which PPIs can be inferred. However, interpretating XL-MS data to assign homologous cross-linked peptide pairs to specific protein interactions can be challenging. A major hurdle arises when a single peptide can map to multiple proteins, and when two such peptides are paired, the number of possible protein-protein interactions increases combinatorially. This mapping ambiguity can lead to inflated interaction networks that could compromise downstream analysis and biological interpretation. However, this problem has often been overlooked or addressed using heuristic approaches in previous XL-MS studies. To tackle this challenge, we developed XL-Ranker, a computational framework that combines a set cover graph algorithm and machine learning to systematically resolve peptide-mapping ambiguity and infer high-confidence PPIs from XL-MS data. Applied to an XL-MS dataset from HEK293 cells, XL-Ranker identified a high-confidence network with 880 PPIs involving 964 unique genes. Among all possible PPIs, the ones selected by XL-Ranker for inclusion in the final network had significantly higher interaction scores in the STRING database than the excluded ones. Network analysis further demonstrated that these interactions form biologically meaningful clusters, supporting the accuracy of our approach. In summary, XL-Ranker provides a practical solution to a key analytical challenge in XL-MS data interpretation, enhancing the reliability of PPI discovery.

bioinformatics↗

bioRxiv2025

Improving tolerance to fluctuating light through adaptive laboratory evolution in the cyanobacterium Synechocystis

Fluctuating light (FL) poses a serious challenge to photosynthetic organisms like cyanobacteria, disrupting carbon assimilation and damaging photosystems. While key components of cyanobacterial FL tolerance have been identified, their genetic enhancement remains unexplored. We applied adaptive laboratory evolution to Synechocystis PCC 6803 under two complex FL regimes, including one lethal to the starter strain (LT), to generate FL-adaptive alleles. Our analysis revealed 44 fully segregated novel mutations in 24 monoclonal evolved strains, 28 of which affected proteins or structural RNAs. We focused on three mutations for further study. Mutations in Pam68, involved in photosystem II (PSII) assembly, and Sll0518 were present in all evolved strains, indicating early emergence. These mutations increased tolerance to non-lethal FL conditions when introduced into LT, with the Pam68 mutation possibly protecting PSI by increasing the proportion of less active PSII monomers. A gain-of-function mutation in RpaB, regulator of phycobilisome association B, was found in three strains tolerant to lethal FL. When introduced into LT, this mutation significantly increased tolerance to both lethal FL and high light conditions, associated with downregulation of photosystem accumulation and light harvesting. As RpaB has plant homologs, this finding could potentially be used to improve agricultural productivity under variable light conditions.

evolutionary biology↗