Biology subjects

Wu, C.

Publications and source records attributed to Wu, C..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

GM-CSF and M-CSF Driven Differentiation Differentially Regulates Chikungunya Virus Infection and Antiviral Responses in Human Monocyte-Derived Macrophages

Chikungunya virus is an arthritogenic alphavirus causing debilitating joint pain in infected individuals. The mechanisms driving CHIKV-associated arthralgia is poorly understood, however, macrophages have been implicated as potential reservoirs of persistent viral material and mediators of immunopathology. Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) and Macrophage-Colony Stimulating Factor (M-CSF) are cytokines that serve as myeloid growth factors that bias macrophages toward pro-inflammatory and anti-inflammatory phenotypes, respectively. In this study, we examined how cytokine-driven macrophage differentiation via GM-CSF and M-CSF influences susceptibility to and responses against CHIKV infection in vitro. Using parallel donor-matched cultures of primary macrophages, we show that GM-CSF-differentiated macrophages are highly permissive to CHIKV and Mayaro virus (MAYV) infection and support robust viral replication, whereas M-CSF-differentiated macrophages are resistant to CHIKV replication and lack detectable levels of viral protein expression. Despite these differences, we observed pro-inflammatory, M1-skewing of CHIKV-infected macrophages, regardless of differentiation state. Interestingly, we observe higher production of IFN and IP10/CXCL10 in M-CSF differentiated macrophages, suggesting that M-CSF promotes an antiviral state that restricts CHIKV infection. Stimulation of macrophages with double-stranded RNA (polyinosinic:polycytidylic acid; poly(I:C)), but not with single-stranded RNA (resiquimod, R848), recapitulated the antiviral cytokine and chemokine response induced by CHIKV infection. These findings suggest that dsRNA sensing plays a more prominent role than ssRNA sensing in driving the macrophage antiviral response to CHIKV. Together, these findings highlight macrophage differentiation as a critical determinant of CHIKV susceptibility and antiviral immunity in humans, with implications for understanding inflammatory pathogenesis during infection.

microbiology↗

bioRxiv2026

PD1-induced Shp2 condensation organizes inhibitory signalosomes through selective substrate partitioning

The formation of microclusters is a hallmark of PD1 engagement with its ligands, yet the physical basis and functional significance of this phenomenon remain unclear. Here we show that ligand-bound PD1 licenses Shp2 self-association and liquid-liquid phase separation (LLPS), producing dynamic PD1:Shp2 condensates whose liquidity depends on Shp2 catalytic activity. Mutations that selectively disrupt Shp2 self-association weaken PD1 microcluster formation and impair PD1 inhibitory function. Mechanistically, PD1-induced Shp2 LLPS promotes the co-compartmentalization of signaling substrates such as CD3{zeta} and CD28, thereby facilitating its dephosphorylation. These findings identify Shp2 LLPS as an intrinsic organizing principle of the PD1 inhibitory pathway that links enzymatic activation, substrate selectivity and mesoscale assembly to suppress T cell activation.

biochemistry↗

bioRxiv2026

A Modular Framework for Automated Segmentation and Analysis of AFM Imaging of Chromatin Organization

Chromatin organization underlies essential genome functions, but its nanoscale organization remains challenging to capture and quantify with precision. Atomic force microscopy (AFM) offers direct structural readouts of DNA and chromatin, yet translating these rich images into reproducible biological metrics has been limited by the lack of standardized, scalable analysis tools. Here we present DNAsight, an automated analysis framework that integrates machine learning (ML)-based segmentation with modular, base-pair-calibrated quantification of DNA spatial organization, looping, nucleosome spacing, and protein clustering. Applied across diverse chromatin-associated proteins, DNAsight reveals protein-specific organizational signatures, including topology-dependent compaction by integration host factor (IHF), condition-dependent changes in loop-like DNA structures in cohesin-CTCF-precocious dissociation of sisters 5A (PDS5A) reactions, and promoter-driven multimerization of GAGA factor (GAF) clusters. The framework further enables direct extraction of nucleosome spacing distributions from raw AFM images, providing a label-free route to investigate chromatin fiber architecture. Together, these advances establish DNAsight as a generalizable and scalable approach for converting AFM measurements into quantitative insights into the physical principles of chromatin organization. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/708946v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1225883org.highwire.dtl.DTLVardef@1e1041dorg.highwire.dtl.DTLVardef@1d53d18org.highwire.dtl.DTLVardef@9e0a2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

Autoinducer-2 (AI-2) is a LuxS-dependent product of the activated methyl cycle (AMC) that functions as a quorum-sensing signal in diverse bacteria. Fusobacterium nucleatum is a genetically heterogeneous oral anaerobe comprising four subspecies: nucleatum (FNN), vincentii (FNV), polymorphum (FNP), and animalis (FNA). Previous studies have reported that FNN and FNP strains produce AI-2 and have proposed that AI-2-mediated quorum sensing contributes to biofilm formation and virulence. However, the distribution and functional relevance of AI-2 across all subspecies have not been systematically examined. Here, we show that AI-2 production is restricted to FNA strains. Genomic analysis revealed that FNN and FNV lack luxS, whereas FNP carries a disrupted luxS homolog. Consistent with these findings, AI-2 bioassays using the Vibrio harveyi BB170 reporter detected AI-2 exclusively in FNA strains. Deletion of luxS in FNA abolished AI-2 production but resulted in minimal transcriptional changes, and exogenous AI-2 failed to elicit global gene expression responses in non-producing subspecies. These results demonstrate that AI-2 production in F. nucleatum is subspecies-specific and uncoupled from quorum sensing. Our findings revise current assumptions regarding AI-2-mediated communication in F. nucleatum and reveal previously unrecognized metabolic divergence within the species complex. IMPORTANCEPeriodontitis affects nearly half of adults in the United States and remains a leading cause of tooth loss worldwide. Fusobacterium nucleatum is a central member of oral biofilms and has also been linked to adverse pregnancy outcomes and colorectal cancer. Although AI-2-mediated quorum sensing has been proposed to contribute to its biofilm formation and virulence, our study demonstrates that AI-2 production is confined to subsp. animalis and is absent in other subspecies. Moreover, AI-2 does not function as a conserved quorum-sensing regulator in this species. These findings fundamentally revise prevailing assumptions about AI-2 signaling in F. nucleatum and suggest that subspecies-specific metabolic traits, rather than universal quorum sensing, may underlie ecological adaptation and host association.

microbiology↗

bioRxiv2026

Opposing regulatory logics converge on ABA receptors to govern the trade-off between growth and drought acclimation

Plants dynamically balance growth with stress responses through abscisic acid (ABA). How ABA signaling, but how ABA sensitivity is finely tuned is unclear. Here, we uncover a regulatory network of leucine-rich repeat receptor-like kinases (LRR-RLKs) that directly controls ABA receptors stability. We identify GASSHO1 (GSO1) as a core component that phosphorylates the PYL2 and PYL4 receptors, targeting them for degradation and thus serving as a brake on ABA signaling. Under drought stress, ABA accumulates and suppresses expression of GSO1-activating CIF peptides, releasing this brake to increase PYLs and sensitivity. This mechanism opposes our previously identified CEPR2 pathway, where drought-induced CEP peptides inhibit the kinase to stabilize PYLs. The integration of these two antagonistic modules within an LRR-RLK network enables precise, dynamic control of ABA perception. This work reveals a molecular framework explaining how plants calibrate the critical balance between growth and drought acclimation.

molecular biology↗

bioRxiv2026

Self-amplifying RNA-based CAR T cell therapy with enhanced duration and multi-genic logic function

Chimeric antigen receptor T (CAR-T) cell therapy is transforming the treatment landscape of hematological malignancies. However, manufacturing with integrating viral vectors is costly, slow, and carries risks including insertional mutagenesis, prolonged B cell aplasia, and other long-term toxicities. Expression of CAR with mRNA can reduce cost, manufacturing timelines, and improve safety. However, the short-lived expression necessitates frequent repeat dosing. Here, we describe a modified self-amplifying RNA (saRNA) platform for engineering CAR T cells with prolonged CAR expression and enhanced durability of tumor control relative to mRNA CAR T cells. In an acute lymphoblastic leukemia (ALL) xenograft model, saRNA CAR T cells achieve superior tumor suppression and prolong survival. Further, a single-strand modified saRNA supports the co-expression of multiple proteins, enabling the construction of advanced CAR systems, such as OR- and AND-gated logic CAR T cells. Together, these results highlight saRNA as a powerful and versatile platform for CAR T cell engineering with favorable safety, efficacy, and accessibility.

bioengineering↗

bioRxiv2026

Large-scale quantum computing framework enhances drug discovery in multiple stages

Coherent Ising machines (CIMs) excel at solving large-scale combinational optimization problems (COPs), but their insufficient long-term stability has hindered their applications in compute-intensive tasks like computer-aided drug discovery (CADD). By improving fiber vibration isolation and temperature control system, we have implemented a 2000-node CIM named QBoson-CPQC-3Gen achieving stable solutions over one hour on large-scale COPs. Graph-based encoding schemes were further introduced to realize a CIM-based CADD workflow including allosteric site detection, protein-peptide docking and intermolecular similarity calculation. CIM-based methods demonstrated superior speed and accuracy than heuristic algorithms. Especially, QBoson-CPQC-3Gen identified 2 novel druggable sites and bioactive compounds for 6 targets, which were further validated in vitro, in-cell and by crystal structures. Our contributions established a quantum-computing framework for multi-stage drug discovery, representing a significant advancement in both quantum computing applications and pharmaceutical research.

bioinformatics↗

bioRxiv2026

Correlations of 276 missense variants of PSEN1, PSEN2, and APP on the production of Aβ peptides against variant effect predictors and biophysical structures

INTRODUCTIONEarly-onset Alzheimers disease (EOAD) is mostly caused by mutations in Presenilin-1,2 (PSEN1, PSEN2), and amyloid precursor protein (APP) genes. Both genetic and experimental evidence has supported the PSEN-APP amyloid hypothesis as the leading pathogenesis of AD. Thus far, 276 missense variants from PSEN1,2, and APP have been identified. Herein, we report the development of algorithm platforms for predicting the degrees of pathogenic potentials of these various missense mutations in AD. METHODSWe performed extensive pairwise correlations between 276 in vitro functional assays of missense variants from PSEN1, PSEN2, and APP and clinical data among EOAD patients against 37 variant effect predictors (VEPs). Furthermore, we used the structural models of all three proteins to refine understanding of the molecular basis of missense variants in dominant negative (DN) or loss-of-function (LoF) effects. RESULTSWe found that VEPs were consistently correlated with age of onset (AAO) among EOAD patients and the A{beta}42/A{beta}40 ratio biomarker. This study also identified discrepancies in the predictor variable leading to an increase in the A{beta}42/A{beta}40 ratio; The increased A{beta}42/A{beta}40 ratio was due to decreased A{beta}40 levels by Sun et al., (2016), while Petit et al., (2022) argued that increased A{beta}42 level was the culprit. We also identified that with increased predicted pathogenicity, there were decreased A{beta}38 levels. Conversely, this study found no direct correlations with A{beta}37 and A{beta}43. Lastly, structural studies show characteristics of either DN and LoF mechanisms, as there were numerous variants located in buried hydrophobic domains and those on charged surfaces of the proteins. DISCUSSIONCurrently, a major limitation in the field is the lack of reliable approaches for predicting the pathogenicity of variants in EOAD. We show that VEPs are promising in deciphering the effects of variants in PSEN1, PSEN2, and APP genes, especially between multiple pathogenic mutations. Our study also illuminates the need for experimental studies to identify the predictor variable causing the increased A{beta}42/A{beta}40 biomarker. Our biophysical studies also identify the need to characterize whether the mechanism of pathogenesis caused by DN or LoF effects is in scope of the presenilin hypothesis. Research in ContextO_LISystematic review: The authors reviewed experimental evidence of pathogenic PSEN1, PSEN2, and APP variants in scope of the presenilin hypothesis. To our knowledge, the pathogenesis of EOAD remains incompletely understood. There are also no predictive tools/models available for informing clinical translation. C_LIO_LIInterpretation: VEPs demonstrate strong correlations with AAO and A{beta} levels from pairwise correlations with 276 missense variants. Structurally, it appears that mutations demonstrate characteristics of dominant negative and loss-of-function effects. C_LIO_LIFuture Direction: Larger studies are necessary of VEPs for realistic clinical usage. C_LI HighlightsO_LIWe performed correlations of 37 VEPs against in vitro data of EOAD variants. C_LIO_LIVEPs demonstrate potential as diagnostic tools for early onset Alzheimers disease. C_LIO_LIPathogenic missense variants show characteristics of DN and LoF effects. C_LI

neuroscience↗

bioRxiv2026

Lmna deficiency promotes EPHX2 nuclear translocation to ameliorate cardiac dysfunction in mice

BackgroundCardiovascular diseases are often associated with altered protein subcellular localization. As a major cause of inherited cardiomyopathy, LMNA deficiency could trigger nuclear envelope rupture and broadly impair the localization of nuclear and cytoplasmic proteins. Systemic approaches to identify, dissect and manipulate the localization of endogenous proteins are important for mechanistic and therapeutic investigation. MethodProximity proteomics of the nuclear lamina was performed specifically in cardiomyocytes in Lmna-deficient murine models. AAV-mediated Cas9-based gene silencing and subcellular gene upregulation were conducted via the nuclear localization signal (NLS) and nuclear export signal (NES). Cas9-based somatic mutagenesis was supplemented with the single-strand DNA templates of AAV to achieve robust homology-directed repair (HDR) and targeted NLS knock-in, which translocated cytoplasmic proteins into nuclei. ResultIn vivo proximity proteomics detected increased epoxide hydrolase 2 (EPHX2) in cardiomyocyte nuclei in mice carrying germline or cardiac-specific Lmna truncating variants. This phenotype was associated with ruptured nuclear envelope. Cas9-mediated Ephx2 knockout in cardiomyocytes ameliorated cardiac dysfunction in Lmna-deficient mice. Strikingly, overexpression of NLS-EPHX2, but not NES-EPHX2, also mitigated cardiac dysfunction. The cardiac protective EPHX2 substrates, epoxyeicosatrienoic acids (EETs), did not alter upon NLS-EPHX2 overexpression. By contrast, the Lmna-related DNA damage marker {gamma}-H2AX was reduced. The EPHX-D333A mutant lacking hydrolase activity recapitulated the effects of wildtype EPHX2 in nuclei. AAV-Cas9-based HDR achieved efficient NLS knock-in and EPHX2 nuclear translocation in more than 60% cardiomyocytes, which improved cardiac function. ConclusionLmna deficiency leads to the nuclear translocation of EPHX2, which ameliorated cardiac dysfunction in a hydrolase-independent manner. AAV-HDR-mediated somatic gene editing provides an efficient approach to manipulate the subcellular localization of endogenous proteins in cardiomyocytes in vivo. What is Known?O_LICardiovascular diseases are regulated by the changes in protein subcellular localization. In particular, LMNA-related cardiomyopathy is associated with nuclear rupture and impaired separation between nuclear and cytoplasmic proteins. C_LIO_LIEpoxide hydrolase 2 (EPHX2) is a cytoplasmic hydrolase that catalyzes the hydrolysis of cardioprotective epoxyeicosatrienoic acids (EETs) and aggravates an array of heart diseases including myocardial infarction and heart failure. C_LIO_LICRISPR/Cas9-mediated homology-directed repair (HDR) exhibits uniquely high gene editing efficiency in cardiomyocytes with an AAV-based DNA donor, which is suitable for somatic genetic knock-in of small DNA fragments in vivo. C_LI What New Information Does This Article Contribute?O_LILamin-targeted proximity proteomics specifically in cardiomyocytes uncovers novel proteins undergoing subcellular localization changes upon Lmna deficiency. C_LIO_LILmna deficiency leads to EPHX2 nuclear translocation that ameliorates cardiac dysfunction by both mechanisms of cytoplasmic reduction and nuclear induction. C_LIO_LIAAV-HDR-mediated knock-in provides a robust platform to manipulate subcellular localization of endogenous proteins specifically in cardiomyocytes in vivo. C_LI Cardiovascular diseases are often associated with altered protein subcellular localization, but systemic approaches to identify, study and manipulate subcellular localization remain incomplete. This study established an in vivo proximity proteomics approach to identify novel proteins undergoing localization changes relative to the nuclear lamina. In murine models of LMNA-related cardiomyopathy, this approach uncovered EPHX2, a classic cytoplasmic hydrolase that aggravates cardiovascular diseases, as a new protein that translocated into cell nucleus and exerted a cardiac protective effect. CRISPR/Cas9-based cardiomyocyte gene editing with an AAV-based DNA donor efficiently achieved NLS knock-in into the Ephx2 gene, promoted EPHX2 protein nuclear translocation and mitigated cardiac dysfunction with Lmna deficiency. These findings indicated a novel avenue to identify and manipulate the subcellular localization changes of endogenous proteins for basic and translational cardiology.

pathology↗

bioRxiv2026

Lethal permeabilization of host bacteria to small-molecule compounds during phage penetration

The tailed phages (Caudoviricetes) rely on specialized mechanisms to inject their double-stranded linear genomic DNA (gDNA) across the bacterial envelope. During this gDNA entry process, ionic fluxes and membrane depolarization may occur, potentially leading to increased vulnerability to antibiotic molecules. Here, we report that phage gDNA translocation combined with certain small molecules triggers a rapid and phage-specific cell permeabilization leading to growth arrest. Using live-cell fluorescence microscopy, we visualized the accumulation of otherwise excluded compounds such as anthracyclines and propidium iodide. We show that the influx and accumulation of these compounds depend on both the presence of an inner membrane (IM) receptor and persistent membrane depolarization. We further demonstrate that phage-induced permeabilization enhances the efficacy of certain antibiotics, enabling a synergistic elimination of antibiotic-resistant bacteria. Our results provide a mechanistic foundation for combining phages with small-molecule therapeutics and offer new insights into how phage entry relies on and alters host physiology.

microbiology↗

bioRxiv2026

Conserved assembly architecture of the essential herpesvirus packaging accessory factor

To create a new wave of infectious virions, all herpesviruses require an accessory factor of unknown function to package their viral genomes into nascent capsids. Here, we present cryo-EM structures of the packaging accessory factor from the -herpesvirus herpes simplex virus type 1 (HSV-1, UL32) and the {beta}-herpesvirus human cytomegalovirus (HCMV, UL52). Unlike homologs from the {gamma}-herpesviruses, neither UL32 nor UL52 form stable homopentameric rings. UL52 forms incomplete pentameric rings lacking one or two protomers. UL32 does not form stable higher-order species, but stabilization through chemical crosslinking revealed a novel quaternary structure where three pentameric rings assemble into a "tripentamer." Our results reveal that herpesvirus packaging accessory factors adopt distinct oligomeric states but are constrained to pentameric symmetry. Assembly of protomers into a ring creates a positively charged central channel that we show is critical for infectious virus production in HSV-1. Taken together, our study points to a structurally conserved, essential function of packaging accessory factors across the Herpesviridae. AUTHOR SUMMARYHerpesviruses are a diverse family of pathogens that drive human disease. At the end of their life cycle, all herpesviruses must actively package their genomes into newly formed capsids. Although some aspects of packaging are conserved with tailed bacteriophages, herpesviruses require an additional packaging accessory factor of unknown function. Here, we present two high-resolution structures of the packaging accessory factor from herpes simplex virus and human cytomegalovirus and identify pentameric symmetry as a unifying structural feature. We propose that the packaging accessory factor oligomerizes to concentrate positively charges in a central channel critical for packaging progression.

microbiology↗

bioRxiv2026

The amplitude and latency of the earliest signal in V1 encode bottom-up saliency by feature conjunction

The neural origin of bottom-up saliency for exogenous attention remains highly controversial. In this study, we investigated whether the earliest activity in the primary visual cortex (V1) encodes saliency signals defined by the eye-of-origin and feature-conjunction information. Electroencephalography (EEG) recordings from the human occipital cortex revealed early responses to eye-of-origin (E) and/or orientation (O) singletons, with larger response amplitudes to the double-feature (EO) singletons. The short onset latency (58-70 ms) and polarity reversal of the responses indicate a V1 origin. Importantly, the latency and amplitude of these responses predicted behavioral detection performance. Together, these findings suggest that the timing and amplitude of the earliest signals in V1 represent the saliency of combined feature contrasts for bottom-up attention. These signals unlikely originate from projections of other proposed source areas of saliency, due to the scarcity of necessary monocular neurons to process eye-of-origin information. HighlightsO_LIEye-of-origin information, invisible to the SC, elicits an early saliency signal in V1 within 50-100 ms. C_LIO_LICombined feature contrast enhances V1 saliency responses in a nonlinear fashion. C_LIO_LIThe latency and amplitude of V1 saliency responses predict behavioral detection performance. C_LI

neuroscience↗

bioRxiv2026

Construction and Immunogenicity Detection of Canine Parvovirus-like Particles Fusion with Canine Febrile Antigen Epitopes

This study aimed to design and evaluate the immunogenicity of a dual-valent virus-like particles (VLP) vaccine that can simultaneously target Canine distemper virus (CDV) and Ca nine parvovirus virus (CPV).By bioinformatic analysis, conserved antigen epitopes of the three major functional proteins of CDV were screened and inserted into CPV-VP2 proteins by two different methods to construct recombinant expression plasmids CDPV1 versus CDPV2.Thr ee-dimensional structure, hydrophobicity and stability predictions of the two recombinant proteins showed that their hydrophobicity were 0.233 and 0.251, and their structural stability scores were 0.77 and 0.78, respectively.Recombinant plasmids were co-transformed with molecular chaperone pTf16 to be expressed in E. coli BL21(DE3), respectively, and the optimal expression conditions were determined after optimization: 0.25 mmol/L IPTG, 2 g/L L-arabinose in duction, and culture at 25{degrees}C for 16 h.Purified recombinant proteins can self-assemble in vitro to form VLPs about 23.5 nm in diameter with a hemagglutination titer of 1:29.The mouse immune test showed that the hemagglutination inhibition titer peaked on the 7th day after the third immunization, and the neutralizing Antibody level could reach up to 1:28.The CPV VLP s constructed in this institute carrying CDV antigen epitopes were able to successfully assemble in vitro and were well immunogenic, providing experimental basis for the development of a canine polyvalent vaccine.

immunology↗

bioRxiv2026

BaCas12a3 represents a new subtype of type V CRISPR effector with collateral activity toward tRNA

The CRISPR-Cas12 family encompasses diverse RNA-guided nucleases with both DNA-targeting and RNA-targeting subtypes. They can trigger antiviral activities mainly through either direct elimination invading nucleic acids, or activating broad collateral cleavage to induce abortive infection. Here, we report a novel type V CRISPR effector BaCas12a3 that causes growth inhibition through a unique tRNA-cleavage mechanism. Plasmid interference assays indicated that BaCas12a3 inhibits host growth arrest without invoking the DNA damage response, suggesting that the immune responses may not involve double strand breaks of DNA. Indeed, biochemical characterization of the BaCas12a3-crRNA ribonucleoprotein (RNP) unraveled that the effector is an RNA-activating nuclease that cleaves 3' terminus of tRNAs. Cryo-EM structures of BaCas12a3 reveal a conserved bilobed architecture featuring a unique nucleic acid-loading (NL) domain adjacent to the RuvC catalytic center. Structural and mutagenesis analyses show that the NL domain, together with a zinc ribbon domain, form a gated substrate groove. Target RNA binding induces conformational changes that open this groove and expose the RuvC active site, enabling specific tRNA cleavage while preventing other non-specific degradation. Our findings identified the NL domain aside the RuvC active site responsible for the tRNA recognition in BaCas12a3, expanding the functional diversity of CRISPR immunity.

molecular biology↗

bioRxiv2026

Bacterial and host factors affecting acquisition of Streptococcus pneumoniae in a murine model

Initial acquisition is a critical step in the establishment of colonization that in turn enables transmission to new hosts and potentially leads to invasive disease. Here, we studied host and bacterial factors affecting acquisition, as distinct from those affecting colonization density, using Streptococcus pneumoniae (Spn) in a mouse model. Acquisition was quantified using nasal inocula of limiting size to determine the proportion of hosts infected, as well as the median infectious dose. Infant mice (<7 days of age) were highly susceptible to acquisition (ID50<30 CFU) compared with adults (>6 weeks of age), with rates declining with increasing host age. Prior influenza A infection greatly increased acquisition in adults. Expression of Spn capsule was also an important factor influencing acquisition in adults, with less of a role for the highly susceptible infants. Prior Spn colonization with a heterologous or homologous strain effectively blocked acquisition in infants by completely occupying the upper respiratory tract niche. Immunity due to prior colonization and nasal microbiota were not found to be factors limiting acquisition. Together, our findings show how high carriage rates of encapsulated Spn during early life and in the setting of recent viral infection could be explained by effects on acquisition. Our study describes an approach for studying factors that specifically affect the step of acquisition.

microbiology↗

bioRxiv2026

A Hybrid Gag Lattice as a Structural Intermediate in HIV-1 Maturation

HIV-1 maturation converts the spherical immature Gag lattice into the conical mature capsid required for infectivity, yet the structural route linking these two lattice endpoints remains unclear. Using single-particle cryo-electron microscopy and cryo-electron tomography on reconstituted assemblies and virus-like particles (VLPs), we identify a distinct hybrid lattice in which the capsid protein (CA) N-terminal domain adopts a mature-like conformation, whereas the CA C-terminal domain-SP1 layer remains immature. This architecture is observed in native, enveloped VLPs produced in human cells, demonstrating formation under physiologically relevant conditions. The hybrid lattice engages three myo-inositol hexakisphosphate (IP6) molecules per CA-SP1 hexamer, underscoring IP6-dependent stabilization; accordingly, excess IP6 enriches the hybrid population. Tomographic mapping shows that hybrid regions coexist with the immature lattice within the same particle and are enriched near lattice discontinuities, consistent with edge-localized remodeling that can accommodate conformational rearrangements and partial disassembly and reassembly. Disrupting a hybrid-specific inter-hexamer contact preserves immature lattice assembly and particle release but abrogates infectivity, compromises core integrity, and prevents mature lattice formation, implicating the hybrid architecture as an on-pathway intermediate. Molecular dynamics simulations further support coordinated rearrangements that bias the system away from the immature configuration toward hybrid and then mature organizations. Together, these results support a maturation model in which localized displacive remodeling and partial disassembly/reassembly act in concert, with IP6 tuning the balance among lattice states. This study provides new insight into HIV-1 maturation and identifies the hybrid lattice as a potential therapeutic target.

biochemistry↗

bioRxiv2026

Unlocking Ultrasensitive Phosphoproteomics of Nanoscale Samples: A Rapid IMAC Enrichment Platform Using Ti-PAN Tip

Protein phosphorylation is a critical mechanism in cellular signal transduction, playing essential roles in various cellular processes. However, significant challenges of their low abundance, high dynamic range, and complexity are especially prominent for accurate phosphoproteome analysis in nanoscale samples, such as oocytes, where misregulation of phosphorylation can lead to infertility. In this study, we developed a novel phosphopeptide enrichment tip (Ti-PAN) using highly hydrophilic textile PAN fibers, enabling rapid in-situ one-tip IMAC enrichment in just 8 minutes. This approach not only minimizes sample loss but also enhances analytical sensitivity and is easily adaptable to automation for high-throughput applications, demonstrating excellent recovery (>94%) and outstanding selectivity for phosphopeptides with remarkable resistance to interference up to 1:5000. By integrating it with a compatible sample preparation workflow based on n-dodecyl {beta}-D-maltoside (DDM) lysis with the high-sensitivity MS data acquisition method of turboDDA, comprehensive mapping of phosphosites in microsamples was achieved, including those from the ERK interactome, mouse brain slices, and oocytes. Using this approach, we identified 2,139 to 11,296 phosphosites across 645 to 2,168 phosphoproteins from samples ranging from 100 to 100,000 cells. Furthermore, we successfully applied this strategy to analyze the phosphoproteome of mouse oocytes at different developmental stages, using as few as 5 oocytes per sample. This led to the identification of 2,709 phosphosites on 701 phosphoproteins, revealing several key kinases involved in meiotic resumption and oocyte maturation, including STK4 (serine/threonine kinase 4) and DYRK3 (dual specificity tyrosine phosphorylation-regulated kinase 3). To our knowledge, this represents the deepest coverage of phosphoproteomes from such minimal oocyte material reported to date, offering valuable mechanistic insights into developmental controls during oocyte maturation. This Ti-PAN-based method provides a robust, versatile, and automatable platform for large-scale, highly sensitive phosphopeptide enrichment, substantially advancing phosphoproteomic studies in complex biological systems.

biochemistry↗

bioRxiv2026

Expanding High-Fidelity Multiplexing in Ultrasensitive Single-Molecule Protein Detection via Proximity Barcoding

The human proteome presents a vast information reservoir for basic and diagnostics research, yet the low abundances of many proteins in biofluids pose an analytical challenge. While ultrasensitive methods such as digital enzyme-linked immunosorbent assay have expanded the window of detectable proteins, multiplexing with high accuracy, sensitivity, and throughput remains limited by cross-reactivity and signal readout channels To address this challenge, we introduce PRO-MOSAIX (PROximity-barcoded Molecular On-bead Signal Amplification for Individual MultipleXing), a high-accuracy, ultrasensitive multiplex digital immunoassay platform that integrates high-throughput single-molecule protein detection with DNA barcode-based proximity ligation. PRO-MOSAIX generates "ON" signals only from matched affinity reagents in proximity, minimizing false positives from cross-reactive binding. This approach overcomes the multiplexing ceiling imposed by fluorescence spectral overlap by employing a single signal readout channel and DNA barcoding. In conjunction, we further improve multiplexing accuracy by mitigating a secondary source of false positives from DNA-based signal amplification. As a proof of principle, we establish and validate a 15-plex PRO-MOSAIX assay in human plasma, with low femtomolar sensitivities and high measurement accuracies. PRO-MOSAIX is modular and utilizes common laboratory instrumentation with a high-throughput flow cytometric readout, providing a broadly accessible tool that bridges the gap between analytical sensitivity and high-order multiplexing.

bioengineering↗