Biology subjects

Chen, L.

Publications and source records attributed to Chen, L..

At least 73 records · Page 4Linked to original sources

bioRxiv2026

Intravital imaging of age-related conjunctival lymphatic changes on the ocular surface

PurposeLymphatic system plays a critical role in fluid regulation and immune response. The conjunctiva of the ocular surface is endowed with a rich lymphatic network, and it provides an ideal site to study lymphatic structure and function. The purpose of this study is to investigate potential morphological and functional changes of conjunctival lymphatics with aging, a time-dependent biological deterioration process. MethodsYoung and aged fluorescently labeled transgenic mice of Prox-1, the master control gene for lymphatic determination, were used in the study. For morphological assessment, conjunctival lymphatic vessels were examined in vivo by our advanced live imaging system. For functional analysis, lymphatic drainage efficiencies were measured by fluorescently labeled tracer injection. ResultsCompared to young mice, both vascular branching points and intraluminal valves were significantly reduced in conjunctival lymphatic vessels of aged mice. Moreover, lymphatic functional deterioration and drainage deficiencies, such as fluid leakage and reflux, were also detected in the aged condition. ConclusionsConjunctival lymphatic system undergoes morphological as well as functional changes with aging. Further investigation into this phenomenon may provide novel insights into lymphatic and age-related diseases inside and outside the eye.

immunology↗

bioRxiv2026

Human CD8-iTreg are potent GVHD suppressors and tumoricidal effectors by release of Granzyme-K+ Supramolecular Attack Particles

Regulatory CD8+ T-cells (CD8+ Treg) are a distinct yet understudied T-cell subset capable of simultaneous immunosuppression and cytolysis. Here, we characterized induced human CD8+ Treg (CD8-iTreg) generated from peripheral blood CD8+CD25- T-cells using anti-CD3e mAb-loaded artificial antigen presenting cells, IL-2, TGF{beta}, and Rapamycin. These CD8-iTreg differentiated into a stable, highly proliferative bifunctional population with suppressive activity comparable to CD4-iTreg while retaining cytolytic capacity similar to conventional CD8 cytotoxic T lymphocytes (CTL). Multi-parameter spectral flow cytometry and single-cell RNA-seq revealed a distinct immunoregulatory signature: a predominantly Treg-like profile marked by tissue-residency marker CD103 with increased canonical Treg markers (FoxP3, HELIOS, CD25, CD39, CTLA-4, CCR4, and IL-10) and reduced pro-inflammatory cytokines. A unique cytotoxic program was marked by elevated Granzyme-K (GzmK) and Thrombospondin-4 (Tsp-4), a thrombospondin family extracellular matrix glycoprotein upregulated in activated CD8+ T-cells. Cytolysis was primarily mediated by Perforin (Prf) and multiple Granzymes packaged into Tsp-4 supramolecular attack particles (SMAPs), with GzmK contributing to both cytotoxic and suppressive functions. After anti-CD19scFv CAR (CAR19) transduction, CAR19+ CD8-iTreg showed superior in vivo anti-tumor efficacy compared with CAR19-CTLs, significantly reducing tumor burden and prolonging survival in a CD19+ Nalm-6 human leukemia xenograft model while maintaining low pro-inflammatory cytokine production. In a xenogeneic graft-versus-host disease (GVHD) model with residual human leukemia, CAR19 CD8-iTreg inhibited GVHD lethality and controlled tumor growth without increasing systemic inflammation. Together, these findings support CD8-iTreg-based CAR therapies as a strategy to retain potent anti-leukemic activity while limiting inflammatory toxicities of conventional CAR T-cells, properties particularly beneficial in treating auto- and allo-immune diseases. One sentence summaryCD8-iTreg drive parallel tumoricidal and immunoregulatory functions mediated by releasing Tsp-4+ SMAPs containing granzyme K.

immunology↗

bioRxiv2026

Spatiotemporal biogenesis of thylakoid membranes in the green alga Chlamydomonas reinhardtii

Thylakoid membranes are essential for oxygenic photosynthesis, yet the mechanisms underlying their spatial and temporal biogenesis remain poorly understood. Here, using a light-induced membrane regeneration system in Chlamydomonas reinhardtii, we generate a time-resolved map of thylakoid formation and photosynthetic complex assembly at unprecedented spatial and temporal resolution by integrating super-resolution fluorescence microscopy, cryo-electron tomography, proteomics, and spectroscopy. We show that thylakoid biogenesis is globally distributed and multipolar, with new membrane formation occurring at multiple sites across the chloroplast, including regions adjacent to the inner envelope membranes in both basal and lobe regions. We identify F-ATP synthase storage membranes and map the redistribution of translating ribosomes from T-zone enrichment to a chloroplast-wide distribution at early stages of thylakoid formation, consistent with decentralized synthesis of photosynthetic complexes. We further delineate the hierarchical assembly of photosystem supercomplexes, starting with formation of reaction centers. Together, these findings refine the spatial organization of photosynthetic membrane biogenesis and establish a unified spatiotemporal framework linking thylakoid formation, translational activation, and functional maturation, providing a mechanistic basis for plastid engineering to enhance photosynthetic efficiency.

Cell Biology↗

bioRxiv2026

Widespread phages exhibit depth-structured infection coupled with ammonia oxidation

Ammonia oxidation is a rate-limiting step in the nitrogen cycle, yet viral contributions to this process remain largely unresolved. Here, we identify three genomically distinct groups of amoC-encoding phages (155-338 kilobases in length; termed as amoC-phages) from multiple freshwater lakes in Europe and North America, including the Laurentian Great Lakes. These phages are highly divergent in phylogeny, genome architecture, and gene content, and are predicted to infect two distinct Nitrosomonadaceae ammonia-oxidizing bacterial lineages. The placement of phage-encoded amoC genes across these divergent viral clades indicates independent acquisition of amoC. Time-series and depth-resolved metagenomes and metatranscriptomes reveal persistent and depth-structured distributions of amoC-phages and their predicted hosts, with seasonal mixing periodically reshaping their co-occurrence patterns. Furthermore, virome data from Lake Mendota show that some of the amoC-phages occur as free viral particles, supporting active viral lysis and particle redistribution along the water column. Metatranscriptomes of the Laurentian Great Lakes reveal coordinated expression of phage structural genes (e.g., major capsid protein) together with phage-encoded amoC, indicating active infection in situ. Together, these results support a framework in which amoC-phage infection is depth-structured, seasonally dynamic, and coupled to ammonia-oxidizing bacterial host activity, highlighting viruses as previously overlooked components of freshwater nitrogen cycling.

microbiology↗

bioRxiv2026

MacaSurfer: unified surface-volume mapping of the macaque brain across the lifespan

Macaque brain MRI is central to translational and comparative neuroscience, yet multi-site, longitudinal, and cross-species analyses are hindered by a lack of unified, automated structural processing tools. Existing pipelines, mostly adapted from human neuroimaging or restricted to fragmented steps, fail to provide robust surface-volume representations across heterogeneous acquisitions and developmental stages. Here we introduce MacaSurfer, a fully automated, containerized framework for unified surface-volume mapping of the macaque brain across the lifespan. MacaSurfer features components tailored for macaque anatomy: a tissue segmentation model, a tissue-guided bias-field correction method optimizing structural mapping from T1-weighted images alone, topology-aware surface reconstruction, and surface-aware volumetric registration. Validated on 1,346 imaging sessions from 965 macaques across 39 international sites (spanning 2 weeks to 23 years of age), MacaSurfer demonstrated exceptional anatomical consistency, test-retest precision, and robustness against image degradation. Leveraging MacaSurfer-derived morphometry, we established normative trajectories from 835 macaques, providing a standardized reference for downstream individualized deviation analysis. MacaSurfer is openly available with source code, containers, and pretrained models, offering a reproducible ecosystem to accelerate developmental, translational, and comparative neuroimaging.

neuroscience↗

bioRxiv2026

Cognitive function depends upon Satb2 gene dosage in cortical projection neurons

SATB2-associated syndrome (SAS) is a severe neurodevelopmental disorder caused by de novo heterozygous SATB2 mutations, yet how haploinsufficiency disrupts brain development remains poorly understood. While homozygous Satb2 loss causes profound embryonic cell-fate defects, we demonstrate using a heterozygous mouse model that SAS phenotypes emerge primarily during postnatal circuit maturation. Integrating chromatin profiling, transcriptomics, electrophysiology, and behavior, we show that SATB2 acts as a dose-sensitive chromatin regulator that binds conserved enhancer-promoter landscapes to orchestrate networks linked to human intelligence. Although excitatory neuron subtype specification is preserved, Satb2 heterozygotes adopt an intermediate epigenetic state that drives cell-type-specific dysregulation of genes enriched for intellectual disability risk variants. Consequently, mutant neurons exhibit simplified dendritic arborization, reduced intrinsic excitability, and weakened layer 2/3-to-layer 5 intracortical connectivity. These circuit deficits culminate in the disorganization of the somatosensory barrel cortex and severe impairments in whisker-dependent texture discrimination. Finally, by restricting Satb2 heterozygosity to the cortex, we decouple these cortical sensory deficits from subcortical vocalization phenotypes. Together, our work links SATB2 dosage to chromatin architecture and postnatal circuit maturation, revealing a critical, post-mitotic therapeutic window for intervention in SAS.

neuroscience↗

bioRxiv2026

GYS2 Promotes the Progression of Aortic Dissection and Aortic Aneurysm via C5a/NF-κB-Mediated Pro-Inflammatory Macrophage Polarization

BackgroundAortic aneurysm and aortic dissection (AAD) are lethal cardiovascular emergencies characterized by sudden onset and extremely high early mortality. The pro-inflammatory polarization of macrophages is one of the core factors driving the pathogenesis of AAD, but its underlying mechanism remains unclear. This study focuses on the aberrant expression of glycogen synthase 2 (GYS2) in the AAD microenvironment and its role in driving macrophage polarization toward the pro-inflammatory M1 phenotype. MethodsProteomic analysis was conducted to identify protein heterogeneity associated with AAD. Clinical and animal samples were used to evaluate the correlation between GYS2 expression and AAD progression. Whole-body GYS2 knockout mice and adeno-associated virus (AAV)-mediated macrophage-specific gain- and loss-of-function models were utilized to investigate the regulatory role of GYS2 in macrophage polarization and complement activation. Downstream molecular pathways were identified and validated through in vitro stimulation and in vivo exogenous C5a rescue experiments. ResultsGYS2 expression was significantly upregulated in AAD tissues and primarily localized in macrophages. Activation of GYS2 by LiCl or macrophage-specific overexpression of GYS2 exacerbated aortic dilation and extracellular matrix degradation, and increased mortality in AAD mice. Conversely, whole-body GYS2 knockout or macrophage-specific GYS2 knockdown suppressed inflammatory factors, significantly reduced the incidence of AAD, and attenuated vascular injury. Mechanistically, excessive GYS2 in macrophages specifically triggered the complement-coagulation cascade, promoting the generation of the potent anaphylatoxin C5a. C5a further bound to its receptor C5AR1, activating the downstream PLC{beta}3/NF-{kappa}B signaling pathway, thereby inducing M1 macrophage polarization and matrix metalloproteinase-mediated extracellular matrix degradation. In vivo exogenous C5a rescue completely reversed the vascular protective effects conferred by GYS2 deficiency. ConclusionsThis study demonstrates that highly expressed GYS2 regulates the pro-inflammatory polarization of macrophages and extracellular matrix degradation via the complement C5a/PLC{beta}3/NF-{kappa}B signaling axis, which is a key mechanism driving AAD progression. Specific inhibition of macrophage GYS2 can effectively alleviate aortic vascular inflammation and prevent AAD progression, providing a promising novel strategy for the clinical conservative treatment of AAD.

Molecular Biology↗

bioRxiv2026

Deciphering cross-omics complexity of tissues via diagonal integration of unpaired spatial multi-omics data

Recent spatial multi-omics technologies enable the simultaneous in situ profiling of multiple omics modalities on the same tissue section; however, they face challenges in experimental complexity and high costs. This technical limitation can be circumvented by diagonal integration methods, which integrate omics data from different modalities. However, existing single-cell diagonal integration approaches overlook spatial information, causing unreliable anchoring across omics layers. Here, we introduce STAMO, a graph attention neural network model for spatially aware integration of unpaired spatial slices from different omics. Systematic benchmarking on spatial epigenome-transcriptome slices proves that STAMO outperforms the state-of-the-art methods in generating aligned embeddings and identifying consensus spatial domains across omics. We apply STAMO to integrate unpaired data from diverse spatial omics types (transcripts, epigenetics, DNA, and proteins), including slices from spatial RNA and four different epigenomic modalities, spatial ATAC and RNA slices across embryonic stages, spatial protein and RNA slices, and spatial DNA and RNA slices. In addition, the integration capability of STAMO can be further used to achieve cross-omics generation, offering a solution for exploring spatial region-specific gene regulatory mechanisms.

bioinformatics↗

bioRxiv2026

Validation of MHPC512: A Publicly Available Special-Purpose Supercomputer for Molecular Dynamics Simulations of Biomolecular Systems

MHPC512 is a massively parallel, special-purpose supercomputer designed primarily for atomic-level molecular dynamics (MD) simulations of biomolecular systems. It comprises 512 processor units interconnected by a high-speed three-dimensional torus network and employs a custom chip architecture that uses 35-bit fixed-point arithmetic to accelerate computation while controlling precision loss within an acceptable margin. The preprocessor is compatible with GROMACS and AMBER input formats and supports widely used biomolecular force fields (including CHARMM, AMBER, and OPLS/AA), the Neutral Territory method for short-range nonbonded interactions, the k-space Gaussian Split Ewald method for long-range electrostatics, and multiple thermostats, barostats, and integrators. We present a three-tier validation protocol--comparing static energy and virial components, examining ensemble distributions (NVE, NVT, NPT), and evaluating long-time statistical properties--demonstrating that MHPC512 reproduces results consistent with GROMACS and AmberTools. Application examples, including bulk water, dipeptide conformational sampling, folding of fast-folding peptides, membrane-protein systems, lipid self-assembly, and GPCR conformational transitions, further confirm its reliability. MHPC512 has been deployed at multiple supercomputing centers and is publicly accessible, representing a significant advance in high-throughput, large-scale biomolecular MD simulations.

Molecular Biology↗

bioRxiv2026

The engulfment receptor CED-1/MEGF10 activates the GPCR LAT-1/ADGRL3 for apoptotic cell degradation

Efferocytosis, the clearance of apoptotic cells, is crucial for tissue homeostasis, inflammation suppression, and repair. While several G protein-coupled receptors (GPCRs) are involved, the broader role of GPCRs in efferocytosis remains unexplored. Through a genome-wide RNAi screen in Caenorhabditis elegans, we identified the adhesion GPCR LAT-1 as a key regulator of apoptotic cell degradation through the promotion of phagosome maturation. Our secondary RNAi scrween for transcriptional regulators revealed that the transcription factor AST-1 acts downstream of LAT-1 to mediate apoptotic cell degradation. We show that the engulfment receptor CED-1 functions as a ligand for LAT-1, activating a Gs protein/adenylyl cyclase/PKA/AST-1 signaling cascade and inducing the transcription of vps-34 and piki-1, which encode phosphatidylinositol 3-kinases essential for PtdIns3P generation on phagosomes. This pathway maintains appropriate VPS-34 and PIKI-1 levels for efficient efferocytosis. Notably, LAT-1 is evolutionarily conserved, with homologs in Drosophila (CIRL) and mammals (ADGRL3) performing similar functions. These findings reveal a conserved CED-1/MEGF10-LAT-1/ADGRL3 axis that orchestrates apoptotic cell clearance via a novel transcriptional regulatory pathway, providing new insight into the molecular mechanisms underlying tissue homeostasis and diseases associated with defective efferocytosis, including autoimmunity and neurodegeneration.

cell biology↗

bioRxiv2026

The emergent mental operation of the learning brain

How does the learning brain give rise to emergent mental operations that enable skill internalization and generalization? Using five-year longitudinal tracking of children acquiring abacus-based mental calculation, we reveal how sustained practice progressively reconfigures whole-brain states. Learning induces nonlinear transitions from stable baseline configurations through a transient exploratory phase toward restabilized associative brain networks. This hierarchical remodeling cascades from perceptual systems to action and default networks, mediated by adaptive neural variability and selective connectivity reweighting, with the salience-parietal memory network acting as a dynamic relay. These brain-state transitions accompany an ordered cognitive transfer cascade, emerging within arithmetic and extending to visuospatial and executive functions. Our findings are reproducible and provide a systems-level account of how ecological learning sculpts large-scale networks to enable complex skill acquisition.

neuroscience↗

bioRxiv2026

GalaxyVS: Exploring 100-Billion Compounds in Seconds

We present GalaxyVS, a hardware-software co-designed virtual screening framework built to explore the 100-billion commercially accessible chemical space in seconds, deployed at the National Supercomputing Center in Tianjin. Built upon the dense vector retrieval paradigm of DrugCLIP, GalaxyVS bypasses the structural dependencies and computational overhead of classical docking to enable rapid screening against experimentally determined as well as geometrically feasible pockets on AlphaFold-predicted structures. To scale this paradigm to the 100-billion level, the system must overcome the significant computational burden of offline representation encoding, critical memory and I/O bottlenecks during online retrieval, and the risks of diversity collapse and precision loss within final screening results. Utilizing the heterogeneous supercomputing infrastructure, GalaxyVS accelerates the offline encoding through deep operator adaptations and resolves online retrieval bottlenecks via disk-native vector indexing coupled with in-memory staging to ensure both broad accessibility and high throughput. Concurrently, a two-stage refinement protocol effectively mitigates diversity collapse and ensures high-fidelity affinity ranking. Consequently, GalaxyVS achieves a daily scoring throughput of 1.5 x 1016 target-ligand pairs, representing a six-orders-of-magnitude leap over previous supercomputing records. Driven by this throughput, we screened nearly 100,000 protein structures across six species against the 100-billion compound library in just 16 hours. The resulting comprehensive cross-species interaction landscape, GalaxyDB, will be openly released at https://galaxyvs.drugclip.com.

bioinformatics↗

bioRxiv2026

Emergence of Task-Related Motor Cortical Dysfunction in Mice with Progressive Parkinsonism

There are substantial functional changes in the primary motor cortex (M1) in Parkinsons disease (PD). However, the temporal relationship between midbrain dopaminergic (DA) neurodegeneration, M1 circuit dysfunction, and Parkinsonian motor symptoms remains poorly understood. Using a genetic mouse model of progressive nigrostriatal DA degeneration ("MitoPark" mice), we determine the time course of M1 cellular dysfunction and skilled movement impairment as the midbrain DA neurons gradually degenerate. M1 pyramidal neuronal subtypes were identified using AAV-mediated retrograde labeling. During progressive DA loss, MitoPark mice developed gradually impaired performance in a reach-to-grasp single-food-pellet task. These impairments were detectable at a moderate motor stage of Parkinsonism. In vivo GCaMP6f imaging revealed that impaired skilled movement was associated with reduced cellular activity and movement responsiveness of M1 pyramidal neurons at a moderate motor stage of Parkinsonism. While both the corticospinal (CSp) and intratelencephalic (IT) neurons send glutamatergic inputs to the striatum, only the CSp neurons showed a selective and significant reduction in cellular activity and movement responsiveness during reaches. At the population level, we found that M1 pyramidal neurons include heterogeneous functional clusters with distinct temporal profiles in response to skilled movement. While movement encoding by different functional clusters is longitudinally stable in control mice, it degrades and diverges significantly in MitoPark mice. The impaired stability is further supported by a longitudinal analysis of individual neuronal activity related to movements. Together, these results provide novel insights into the emergence of M1 circuit pathophysiology at cellular and neural population levels during progressive Parkinsonism.

neuroscience↗

bioRxiv2026

Overexpression of Eaf1, a subunit of the NuA4 lysine acetyl transferase complex, rescues growth defects in the budding yeast H3K36M oncohistone model via histone H4 tail acetylation

Histone proteins are critical for regulating nucleic acid transactions, including gene expression. Specific missense mutations in histone genes have been linked to oncogenesis, creating oncohistones. One of the first identified oncohistones converts K36 in histone H3 to M (H3K36M). While humans have 15 H3 genes, the Saccharomyces cerevisiae genome contains only two H3 genes. As H3 shares 90% sequence identity between budding yeast and humans, the budding yeast system provides an ideal model for study of H3K36M. We previously identified the catalytic subunit of the NuA4 lysine acetyltransferase complex as a high copy suppressor of growth defects in H3K36 mutant yeast cells. Here, we demonstrate that the NuA4 scaffold Eaf1 suppresses mutant H3K36 growth defects. We show that this suppression by Eaf1 is dependent on the Eaf1 HSA domain. To examine whether the role of NuA4 in acetylating histone H4 is required for suppression of H3K36 growth defects, we altered the four NuA4 target lysines within H4 (K5, 8, 12, and 16) to create quadruple histone mutants with all four of these lysines converted to alanine (H4-4K[->]A) or arginine (H4-4K[->]R). These H4 mutants have reduced viability and disrupted global H4 acetylation. Eaf1 cannot suppress growth defects in these H4 acetylation mutants, suggesting Eaf1-mediated suppression is dependent on acetylation of H4 N-terminal tails. We further show that inhibiting NuA4 activity in H3K36M human cells limits their oncogenic potential. Together, this work defines a mechanistic connection between H3K36 and H4 N-terminal tail acetylation in the context of a cancer-causing mutation. Article SummarySpecific mutations in histone genes have been found to cause various cancers, creating oncohistones, and the exact mechanisms by which they drive oncogenesis remain to be characterized. Budding yeast is a simple yet powerful model for studying oncohistones such as H3K36M. Subunits of the NuA4 lysine acetyltransferase complex were found to suppress growth defects in budding yeast expressing H3K36M. This suppression was dependent on the presence of the NuA4 target lysines in histone H4. Additionally, inhibiting NuA4 activity in human H3K36M cells limited their oncogenic potential. This work characterizes the mechanistic relationship between H3K36M and the NuA4 complex.

genetics↗

bioRxiv2026

The b0+ Amino Acid Transporter Defines a Selenium-utilization Enterocyte Program in the Human Intestine

Selenium is an essential trace element incorporated into selenoproteins as selenocysteine, yet the intestinal cellular programs associated with selenium utilization remain poorly defined. Here, we performed a large-scale single-cell transcriptomic analysis of 169,068 human enterocytes from 105 donors to systematically profile nine candidate selenium transporter systems and their relationship to selenoprotein expression. Among all transporters examined, the b0+ amino acid transporter system (SLC3A1/SLC7A9) showed the strongest and most maturation-independent association with selenoproteins, including SELENOP (Spearman r = 0.489) and GPX4 (r = 0.392), with enrichment across 21 of 24 detected selenoproteins in b0+-complete versus transporter-negative enterocytes. These associations were robust across individual donors and confirmed by pseudobulk validation, and were only partially explained by enterocyte maturation state, intestinal segment, and sequencing depth after covariate adjustment, indicating both differentiation-dependent and differentiation-independent components. Furthermore, enterocytes carrying the b0+ complex expressed a non-canonical LRP repertoire (LRP1, LRP5 and LRP6) rather than the canonical SELENOP receptors LRP2 and LRP8; this co-expression was maturation-dependent, nominating these LRPs as candidate intestinal SELENOP-handling receptors. Together, these single-cell data identify b0+ transporter expression as a marker of a selenoprotein-enriched enterocyte state in the human intestine.

molecular biology↗

bioRxiv2026

Development and Function of Ovarian Lymphatic Vasculature

Lymphatic vessels play essential roles in interstitial fluid clearance and immune cell trafficking. Recent studies have uncovered previously unrecognized, organ-specific functions of the lymphatic vasculature in the meninges, heart, kidney, and lungs. However, the development and physiological significance of the ovarian lymphatic network remain poorly defined. To address this gap, we performed high-resolution three-dimensional imaging of ovarian lymphatic vasculature in mice. Our results reveal that the ovarian lymphatic network comprises lymphatic capillaries and valve-containing pre-collectors and the ovarian lymphatic vasculature arises from pre-existing lymphatic vessels in the broad ligament starting at postnatal day 5. Furthermore, we demonstrate that the VEGF-C/VEGFR-3 signaling is essential for ovarian lymphatic development and Vegfr3 insufficiency impairs ovarian lymphangiogenesis, leading to fibrosis and premature ovarian aging. Together, these findings identify the lymphatic vasculature as a critical component of the ovarian microenvironment and establish a causal link between lymphatic insufficiency and accelerated ovarian aging.

developmental biology↗

bioRxiv2026

Translational Quantitative Proteomic Assay for Bacteriophages: A New Frontier in Phage Pharmaceutical Development

Accurate quantitation of therapeutic bacteriophages (phages) remains a challenge for clinical development. Plaque-based enumeration is the current standard but is laborious, host-dependent, and variable, particularly when distinguishing individual phages in cocktails. Targeted mass spectrometry of virion structural proteins offers an orthogonal, structure-based approach amenable to reproducible and scalable phage quantitation. Here, we describe a targeted proteomic liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for host-independent quantitation of the Pseudomonas aeruginosa podovirus LUZ19. Proteomic characterization was performed on an LTQ Orbitrap XL to assess sequence coverage and select surrogate peptide candidates based on specificity and sensitivity. High-resolution peptide mapping identified multiple structural proteins of LUZ19 and provided 55% sequence coverage for the major head protein (YP_001671977.1). Fifteen peptides were detected and evaluated, from which the tryptic peptide EVAELDGQELAR was selected based on abundance, stability, and chromatographic performance. Quantitative analysis was conducted on a QTRAP 7500+ using optimized multiple reaction monitoring transitions for targeted peptide detection. Back-calculated concentrations met accuracy criteria across a validated range of 0.008 to 80 pg/mL, with bias spanning -8.2 to 8.2%, intra-day precision ranging from 0.5 to 9.8%, and inter-day precision ranging from 6.3 to 9.7%. Peptide concentrations from digested lysate samples were related to phage concentrations determined by double layer agar assay, yielding an estimated three copies of the major head protein per virion. ImportanceBacteriophages are the most abundant biological entities on the planet and represent a promising therapeutic class for combating drug-resistant bacterial infections. Realizing the clinical potential of bacteriophage therapy requires analytical methods capable of meeting the standards of modern drug development. Targeted mass spectrometry offers unmatched specificity and resolution for precise quantitation of individual bacteriophages within complex biological samples, a capability that conventional enumeration methods cannot match. Only one prior study has applied mass spectrometry to bacteriophage quantitation, using a well-characterized model bacteriophage at a single concentration without calibration or a validated analytical range. Using Pseudomonas aeruginosa podovirus LUZ19, we present the first targeted mass spectrometry-based bacteriophage quantitation assay developed and validated following FDA bioanalytical guidance. This work establishes a rigorous analytical foundation that moves bacteriophage therapy closer to the standards required for informed dose selection, candidate evaluation, and clinical development.

pharmacology and toxicology↗

bioRxiv2026

UcTCRp: a TCRβ-based framework for quantitative MAIT- and iNKT-associated repertoire-state profiling

MAIT and iNKT cells are conventionally identified using invariant or semi-invariant TCR chains, antigen-loaded tetramers, or transcriptomic phenotypes. These requirements limit their detection in public and clinical immune-repertoire datasets that contain only TCR{beta} sequences. Here we present UcTCRp, a TCR{beta}-only framework for profiling MAIT- and iNKT-associated repertoire states in bulk immune repertoires. UcTCRp integrates V-gene context and CDR3{beta} sequence features using a transformer-based representation pretrained on more than one million TCR{beta} sequences and supervised with curated cross-species MAIT, iNKT and conventional T cell references. The framework defines conserved model-informative TCR{beta} features, uses V-matched negative sampling to reduce germline-segment shortcuts, and generalizes across independent human and mouse datasets. In paired scRNA-seq/scTCR-seq datasets, UcTCRp recovered transcriptome-defined MAIT and iNKT cells and identified additional MAIT-like candidates supported by receptor evidence but missed by expression-only annotation. Bulk calibration against paired single-cell references and synthetic spike-in experiments established operating characteristics for repertoire-level abundance estimation. These results establish unpaired TCR{beta} repertoires as an actionable substrate for reconstructing unconventional T cell-associated immune states, enabling archived repertoire resources to be repurposed for systems-level studies of tissue immunity, disease and therapeutic response.

bioinformatics↗