Biology subjects

Chen, L.

Publications and source records attributed to Chen, L..

At least 127 records · Page 7Linked to original sources

bioRxiv2026

Molecular Glue-Induced Homodimerization Drives Targeted CRBN Autodegradation

Molecular glue degraders (MGDs) offer a sophisticated, proximity-based approach to protein modulation. In this study, we introduce LJY-3-60, a novel proximity-inducing agent that unexpectedly triggers the potent and selective autodegradation of CRBN. Evidence from CRISPR-Cas9 screening and IP-MS reveals that this degradation process is strictly governed by the intrinsic CRL4CRBN machinery, independent of any extrinsic E3 recruitment. Through a combination of cellular and biophysical characterizations, we demonstrate that LJY-3-60 acts as a molecular bridge to template CRBN homodimerization. This mechanism is unequivocally elucidated by the atomic-resolution co-crystal structure of the CRBNMidi-LJY-3-60 complex. The structure explicitly delineates the homodimerization interface, revealing how the ligand reorganizes the protein surface to stabilize a non-canonical architecture that drives trans-autoubiquitination and subsequent proteasomal degradation. Furthermore, LJY-3-60 serves as a highly effective, controllable off-switch to mitigate PROTAC-induced toxicity. Ultimately, this work delivers a robust chemical tool for modulating CRBN stability. By demonstrating how a small molecule can functionally mimic an endogenous E3 substrates degron to catalyse targeted autodegradation, this study establishes a rational structural framework for designing the next generation of self-destructive modulators in targeted protein degradation (TPD) therapeutics.

biochemistry↗

bioRxiv2026

Heterozygote Advantage of a Single-Copy SNAP18 Truncation Allele Enables Dominant SCN Resistance and Yield Preservation in Soybean

Soybean cyst nematode (SCN, Heterodera glycines) is the most devastating pathogen threatening global soybean production. The long-term over-reliance on rhg1-a/b resistance has led to the rapid emergence of virulent SCN populations, creating an urgent demand for novel genetic resources to safeguard soybean yield. Here, we identified a rare gain-of-function allele, SNAP18lmm3, via map-based cloning of the lesion-mimic mutant (lmm3) in the SCN-susceptible Williams 82 (rhg1-c) background. This allele encodes a 24-amino-acid C-terminal truncation of SNAP18 and exhibits an unusual mixed dominance genetic profile: the autoimmune-related leaf lesion phenotype is recessive, whereas SCN resistance is dominant. Homozygous lmm3 plants suffer significant yield losses due to spontaneous cell death, but heterozygous (LMM3+/-) plants maintain normal growth, agronomic performance, and seed yield comparable to wild-type Williams 82 in SCN-free field environments. Critically, under artificial SCN inoculation in both field trials and controlled environments, LMM3+/- heterozygotes confer robust resistance, achieving a [~]4-fold yield advantage over SCN-infested Williams 82 and a [~]44% reduction in cyst formation relative to Williams 82. This resistance profile aligns with the broad-spectrum activity of the lmm3 homozygous mutant, supported by the conserved resistance mechanism of SNAP18lmm3. Transgenic expression of SNAP18lmm3 driven by the nematode-responsive HIP promoter (HIPpro) potently arrested nematode development at the J2 stage in transgenic hairy roots, as evidenced by the drastically reduced proportion of advanced-stage nematodes. Our findings establish SNAP18lmm3 as a potent, "plug-and-play" genetic resource that circumvents the limitations of traditional dosage-dependent resistance loci. This allele enables the development of high-yielding, SCN-resistant soybean cultivars through marker-assisted selection for the heterozygous state or precision genome editing, providing a practical solution for sustainable SCN management.

plant biology↗

bioRxiv2026

Deep learning framework ChIANet predicts protein-mediated chromatin architecture across functional contexts

The spatial organization of the genome is dynamically shaped by chromatin-binding proteins, yet how protein-mediated three-dimensional (3D) architectures are specified across functional contexts remains incompletely understood. Here we present ChIANet, a multimodal deep learning framework that enables de novo prediction of protein-mediated chromatin contact maps and loops from protein-binding profiles alone, using the reference genome sequence as a prior. By integrating transformer-based long-range modeling with multi-task learning, ChIANet accurately reconstructs protein-mediated 3D chromatin architectures and generalizes across diverse cellular contexts. Systematic application of ChIANet to CTCF, Cohesin and RNAPII across seven human cell types reveals that chromatin architectures follow conserved organizational principles while exhibiting pronounced context-dependent reconfiguration: CTCF- and Cohesin-mediated interactions predominantly support stable structural frameworks, whereas RNAPII-mediated loops display greater variability and are closely coupled to transcriptional programs and regulatory element activity. Functional analyses further uncover distinct regulatory biases and super-enhancer associations among the three proteins. Extending this framework to cancer genomes, ChIANet captures RNAPII-mediated chromatin looping networks associated with extrachromosomal DNA (ecDNA), revealing highly connected, transcription-associated architectures within amplified ecDNA regions across multiple cancer cell types. Together, these results demonstrate that protein-mediated 3D genome organization is not determined by protein identity alone but is flexibly shaped by functional context, regulatory targets and cellular environment, establishing ChIANet as a unified and scalable approach for decoding context-dependent principles of genome folding.

genomics↗

bioRxiv2026

SNAP18 Truncation Triggers a Competitive Binding Switch Between NSF and ATG8f, Balancing Vesicular Trafficking and Autophagy for SCN Resistance in Soybean

Soybean cyst nematode (SCN, Heterodera glycines) poses the most devastating biotic threat to global soybean production. Traditional SCN resistance mediated by the Rhg1 locus predominantly relies on gene copy number expansion and elevated -SNAP protein dosage. Here, we report a novel resistance mechanism in a single-copy rhg1-c background, wherein a C-terminal 24-amino-acid truncation of SNAP18 (designated SNAP18lmm3) triggers a functional switch from vesicular trafficking to autophagic degradation. Biochemical assays and structural modeling demonstrate that this truncation severely impairs the canonical interaction between SNAP18 and N-ethylmaleimide-sensitive factor (NSF), disrupting SNARE complex recycling and inducing localized cytotoxicity. Concomitantly, the truncated SNAP18lmm3 exposes a binding interface for the autophagy-related protein ATG8f, routing the aberrant protein for selective autophagic clearance. This constitutively activated autophagic flux acts as a systemic detoxification system, preventing widespread cell death and ensuring normal plant growth under non-stressed conditions. Upon SCN infection, SNAP18lmm3 specifically hyper-accumulates within nematode-induced syncytia. This accumulation reaches levels fourfold higher than in adjacent cells, which overwhelms the local autophagic capacity and triggering targeted cell death that arrests nematode development. By elucidating this competitive molecular switch between NSF and ATG8f binding, our study establishes a "self-degrading toxin" model that resolves the inherent trade-off between plant growth and immunity. This work provides a new theoretical framework for engineering cellular homeostasis to enhance durable crop resistance against parasitic nematodes.

plant biology↗

bioRxiv2026

A role for ETV1 and endothelial cell-derived extracellular vesicle microRNAs in priming fibroblast response to vesicle-bound FGF2

Communication between various cell types following wounding is paramount for proper healing and regeneration of injured tissue. Endothelial cells and fibroblasts are critical cellular players involved in cutaneous wound repair, yet their communication mechanisms are not well understood. It has previously been shown that extracellular vesicles derived from endothelial cells (ECEVs) induce dermal fibroblasts to express a gene signature correlated with FGF2-mediated cancer associated fibroblast (CAF) activation, under the control of transcription factor ETV1. In this report, we utilize loss-of-function studies to define the mechanistic role of ETV1 in conferring this ECEV-induced transcriptomic shift and functional change in fibroblasts. Additionally, we identify highly expressed ECEV microRNAs and examine their potential contribution to the ECEV mechanism through downstream gene modulation. In summary, we describe a plausible mechanism by which both ETV1 and top ECEV microRNAs promote a genotypic and phenotypic shift in dermal fibroblasts that have taken up ECEVs.

cell biology↗

bioRxiv2026

Electrostatic control of chromatin compaction safeguards against apoptotic DNA release

Apoptosis involves extensive intracellular reorganisation to facilitate the clearance of dying cells. A key step in this process is the destruction of the genome through fragmentation by caspase-activated endonuclease (CAD). Rather than dispersing after CAD-mediated cleavage, DNA fragments are compacted into a dense chromatin compartment. However, the underlying mechanism and biological relevance of this compaction remain unknown. Here we show that global deacetylation of histone tails promotes chromatin compaction during apoptosis, preventing DNA release into apoptotic extracellular vesicles. Using synthetic effectors that modulate nucleosome electrostatics independently of histone modifications, we demonstrate that electrostatic attraction alone is sufficient to compact and sequester fragmented chromatin. These findings reveal a mechanism by which global reprogramming of histone modifications coordinates fragmentation of the genome with its physical sequestration during apoptosis. Furthermore, our synthetic approach provides a tool to probe the role of physical forces in genome organisation across diverse biological contexts.

cell biology↗

bioRxiv2026

A distinct class of conjugative megaplasmids includes potential vehicles for prophage dissemination

Closely related prophages are frequently found in phylogenetically distant bacteria in the human gut, despite limited evidence of productive phage infections across broad host ranges. Thus, it remains unclear how the wide distribution of prophages could emerge. Here, we identify a potential mechanism of prophage dissemination. We describe two deeply diverged groups of conjugative megaplasmids (>300 kilobases) in the human gut microbiome, which we term Hodors. Hodors encode conserved replication, partitioning, and type IV secretion systems, together with a complex surface-associated gene module. A subset of Hodors harbor complete, intact prophage genomes, and closely related prophages are detected across phylogenetically distant Bacillota lineages, including both Bacilli and Clostridia. Further analysis indicates that Hodor-associated prophages can exist as extracellular particles and demonstrate their transcriptional activity. Our findings support a model in which conjugative megaplasmids act as composite mobile platforms that disseminate prophage genomes across bacterial lineages, providing a mechanistic explanation for the widespread occurrence of closely related prophages in phylogenetically distant gut bacteria and effectively decoupling lysogenic host range from infective host range.

microbiology↗

bioRxiv2026

Time-Resolved Laser Speckle Contrast Imaging (TR-LSCI) of Cerebral Blood Flow Response to Intracranial Pressure Elevation

SignificanceCerebral autoregulation (CA) reflects the dynamic coupling among cerebral blood flow (CBF), intracranial pressure (ICP), and arterial blood pressure (ABP); its failure contributes to secondary brain injury. Existing bedside methods rely on indirect or spatially limited CBF surrogates and cannot resolve microvascular flow dynamics across space, depth, and time. AimTo develop, optimize, and apply a scalable, noncontact time-resolved laser speckle contrast imaging (TR-LSCI) platform for depth-sensitive, high-speed, wide-field CBF imaging during controlled ICP perturbations. ApproachTR-LSCI synchronizes a 20-MHz pulsed laser with a time-gated, single-photon avalanche diode (SPAD) camera (512 x 512 pixels) to detect diffuse photons at varying path lengths, enabling depth-resolved microvascular CBF imaging. Benchtop and mobile TR-LSCI systems were applied in adult rats and a neonatal piglet with synchronized invasive ICP and ABP measurements. ResultsTR-LSCI captured spatially heterogeneous, pulsatile CBF dynamics at up to 52 Hz over large cortical fields of view, with heart rate estimates statistically equivalent to those from ICP and ABP. Multivariable analysis identified reproducible, phase-dependent CA transitions encompassing preserved autoregulation, ABP-driven compensation, and ICP-constrained CBF suppression; notably, CBF alone exhibited distinct phase signatures. ConclusionsTR-LSCI enables dynamic, physiology-informed neurovascular monitoring and supports future bedside CA assessment.

neuroscience↗

bioRxiv2026

Spatial correspondences of Audiovisual Stimuli on Double Flash Illusion Perception and its Cognitive Modeling

Perceptual processing integrates information from multiple sensory modalities to form a coherent representation of the environment. A classic example of such is the Sound-Induced Flash Illusion (SIFI), where the perceived number of visual flashes is altered by conflicting auditory stimuli. While the SIFI is a well-established phenomenon of multisensory integration, the influence of physical spatial characteristics--specifically stimulus eccentricity and spatial congruence--on integration levels remains debated.To address this gap, this study used the SIFI paradigm to investigate the effect of visual stimulus spatial location and the spatial congruence between auditory and visual stimuli on audiovisual integration. In Experiments 1 and 2, we found that when spatial attention was controlled via cueing, unimodal visual performance remained consistent across locations. However, the susceptibility to SIFI increased progressively from the central to the peripheral visual field, exhibiting a spatial pattern of Gaussian distribution. Bayesian modeling further supported this by showing that this spatial modulation was driven by an increase in the integration weight assigned audiovisual representations in the periphery, rather than changes in sensory uncertainty alone. Conversely, Experiment 3 demonstrated that the spatial congruence of audiovisual stimuli did not affect the SIFI or alter the integration processing. These findings refine our current understanding of the spatial modulation upon audiovisual integration. By incorporating the visual systems spatial properties into a Bayesian framework, we provide a computational explanation for the eccentricity-dependent nature of multisensory integration.

neuroscience↗

bioRxiv2026

Intrinsic resistance networks shape cefiderocol susceptibility in ST258 Klebsiella pneumoniae

Cefiderocol (CFDC) is a siderophore-conjugated cephalosporin developed to overcome multidrug resistance in Gram-negative bacteria. Despite its unique iron-dependent entry mechanism, CFDC resistance has emerged in Klebsiella pneumoniae, primarily driven by alterations in siderophore transport and {beta}-lactamase evolution; however, the broader intrinsic resistome that supports CFDC tolerance remains incompletely defined. Here, we performed high-density transposon mutagenesis (Tn-Seq) in the epidemic ST258 K. pneumoniae to map the functional genetic landscape of CFDC susceptibility. Tn-Seq identified siderophore uptake components (tonB and cirA) as the dominant determinants of CFDC resistance. In contrast, disruption of genes involved in peptidoglycan recycling (ampG, ldcA), synthesis (mrcB, lpoB) and enterobacterial common antigen (ECA) biosynthesis (wzxE, wzyE), as well as deletion of plasmid-encoded blaKPC-3, increased CFDC susceptibility. In a CFDC-resistant {Delta}tonB strain, targeting these envelope homeostasis pathways yielded only limited resensitization relative to the siderophore-competent parental strain. Deletion of blaKPC-3 produced the greatest increase in susceptibility, reducing the CFDC MIC four-fold. This pattern is consistent with a model in which reduced CFDC influx in the {Delta}tonB background lowers intracellular drug exposure to levels at which the otherwise limited anti-CFDC activity of KPC {beta}-lactamase becomes sufficient to drive resistance. Together, these data define a hierarchical genetic architecture for CFDC resistance in ST258 K. pneumoniae, in which iron-dependent drug uptake is primary, {beta}-lactamase activity is secondary, and intrinsic envelope stress buffering shapes bacterial fitness once CFDC enters the cell.

microbiology↗

bioRxiv2026

Leveraging ONT move table values for signal aware variant calling

Oxford Nanopore Technologies (ONT) sequencing enables long-range haplotype phasing and contiguous genome assembly but still exhibits elevated error rates that challenge small variant calling, particularly for insertions and deletions (Indels). While raw electrical signals contain rich information, existing signal-aware methods require computationally intensive processing of large signal files. Here, we present Clair3 v2, a method that leverages the ONT move table--a lightweight byproduct of basecalling that maps signal events to nucleotide positions--to improve variant calling accuracy. Clair3 v2 builds upon Clair3 and integrates signal-level dwelling time to significantly enhance variant calling performance. We also propose a genome position based circular buffer to incorporate dwelling time with minimal computational overhead. Benchmarking across six Genome in a Bottle samples demonstrates substantial improvements in variant calling accuracy. With HAC basecalling, Clair3 v2 achieves a mean SNP F1-score of 97.69% at 10x depth (compared to 96.45% for baseline Clair3), and Indel F1 scores improved from 64.27% to 76.70%, while gains persisted at higher depths. The benefits were most pronounced for longer Indels and in complex genomic regions, where Indel F1 scores in long homopolymer regions improved from 14.3% to 45.2%. Benchmark results across various basecalling modes, samples, and coverage settings outperformed Clair3 baselines and other methods, including DeepVariant and Dorado Variant, and demonstrate the significant benefits of Clair3 v2. Furthermore, Clair3 v2 incurs negligible runtime compared to standard Clair3, making it practical for routine use.

bioinformatics↗

bioRxiv2026

Development of KASP Molecular Markers and Construction of Fingerprinting for Cowpea (Vigna unguiculata (L.) Walp) unguiculata Based on ddRAD-Seq

Cowpea (Vigna unguiculata (L.) Walp.) is a globally important legume crop. However, the scarcity of efficient molecular markers has hindered molecular breeding efforts and the protection of plant breeders rights. In this study, we employed double-digest restriction-site associated DNA sequencing (ddRAD-seq) to characterize the genetic diversity of 19 core cowpea germplasm accessions. We generated 791,621 SNPs, from which 13,469 high-quality SNPs were filtered. Population structure and phylogenetic analyses revealed that these accessions cluster into three distinct groups. To facilitate cost-effective and rapid genotyping, we developed a panel of KASP (Kompetitive Allele Specific PCR) markers. Through rigorous screening for polymorphism and stability, we identified six core KASP markers located in exonic regions. These six markers alone were sufficient to discriminate all 19 accessions. Based on these core markers, we constructed a unique DNA fingerprinting profile and assigned specific QR codes for each accession. This study demonstrates that extracting core KASP markers from ddRAD-seq data is a powerful strategy for germplasm identification. The developed fingerprinting system provides a robust, low-cost tool for seed purity testing, variety authentication, and marker-assisted selection in cowpea breeding programs.

plant biology↗

bioRxiv2026

Multi-Layer Brain-Mimicking Phantom for Replicating Dura and Pia Membrane Dimpling and Rupture Properties During Neural Interface Implantation

Development of novel neural interfaces faces buckling challenges and heavily relies on trial-and-error tests via in vivo animal brain insertions for design optimizations towards the minimal-damaging version for enhanced recording and stimulation outcome. To enable low-cost and fast-turnaround neural interface development and to enable previously impossible insertions via new understanding of the cutting process, this study developed a reproducible, multi-layer brain-mimicking phantom designed to replicate the rodent pia and dura mater dimpling and rupture force performance observed during in vivo tests. The phantom was composed of a 0.5% (w/v) agarose cortex layer, a 1.01% (w/v) agarose pia mater layer, and a pre-stretched polyvinyl chloride (PVC) dura mater layer, assembled via easily duplicable benchtop protocols. Using a cantilever-beam force measurement system, rupture force and dimpling depth were quantified across microwires of varying diameters (12-100 m), materials (tungsten, stainless steel), and tip geometries, as well as segmented silicon probe shanks. Phantom test results closely matched in vivo Sprague-Dawley rat data, validating the performance of the developed multi-layer phantom. At the same time, phantom insertion trial variability was substantially lower than in vivo tests, enabling a repeatable, low-cost, early-stage screening platform of novel electrode designs. The phantoms modular design also allowed tuning of layer thickness and stifness of each layer for diferent species or devices, ofering a validated customizable testing platform to accelerate novel neural implant development and reduce animal use.

neuroscience↗

bioRxiv2026

Evaluation and application of chemical decrosslinking in the context of histopathological spatial proteomics

Laser capture microdissection (LCM) - based spatial mass spectrometry proteomics is a rapidly emerging technique with strong potential for use in formalin-fixed, paraffin-embedded (FFPE) tissues. Several sample-preparation methods have been developed to decrosslink FFPE proteins for spatial proteomics; however, residual crosslinks often remain, and depth can remain impaired relative to fresh frozen tissue samples. To increase proteome coverage in spatially resolved LCM-FFPE samples, we investigated a panel of chemical compounds with the potential to catalyze the decrosslinking of nucleophilic functional groups on proteins. Systematic screening and optimization of temperature, incubation time, and reagent concentration led to the identification of 3,4-diaminobenzoic acid as an effective agent for improving proteome coverage in FFPE pancreatic tissue. This compound could boost precursor identifications by more than 10% at both reduced (70 {degrees}C) and high (90 {degrees}C) temperatures. Application of this chemical-decrosslinking strategy to a pancreatic ductal adenocarcinoma tissue section enabled the identification of numerous cell-type-enriched proteins with clinical and therapeutic relevance. Taken together, our findings show that chemical decrosslinking can increase proteome coverage in FFPE tissues, thereby advancing our understanding of tissue microenvironments in physiological and pathological contexts.

cancer biology↗

bioRxiv2026

Mouse macrophages from diverse niches show generally divergent age-related transcriptional remodeling signatures

BackgroundAging is accompanied by widespread transcriptional remodeling across tissues, yet how aging impacts different categories of tissue-resident macrophages is not well understood. Macrophages are highly specialized innate immune cells shaped by their local microenvironments, suggesting that aging may elicit both shared and niche-specific transcriptional responses. Here, we performed a meta-analysis of publicly available bulk and single cell RNA-sequencing datasets to characterize age-associated transcriptional changes in murine macrophages across tissues and sexes. We curated and uniformly processed 33 macrophage transcriptomic datasets, derived from 10 distinct tissue niches, in male and female C57BL/6 mice, examining transcriptional changes as a function of age. ResultsThe similarity of differentially expressed aging genes was compared across niches and pathway-level analysis uncovered conserved age-associated signatures, including upregulation of gene sets related to antigen presentation, antioxidant responses, and negative regulation of ferroptosis, alongside downregulation of gene sets related to Wnt, GTPase, and extracellular matrix organization signaling. Transcription factor activity inference identified consistent age-associated activation of AP-1 (Fos, Jun), C/EBP{beta}, PU.1, and Egr1 across niches. Meta-analysis defined 593 consistently age-altered genes in >3/4 of analyzed datasets, converging on dysregulation of small GTPase signaling. Focused analysis of alveolar macrophages and microglia, made possible by the larger number of available datasets, revealed sex-specific transcriptional programs altered with age in these macrophage subtypes. ConclusionsThese findings demonstrate that macrophage aging is shaped by both tissue niche and sex and provides a framework for understanding the transcriptomic signatures of macrophage aging across tissues.

genomics↗

bioRxiv2026

Distinct activation thresholds of unmyelinated C-fiber afferents by dorsal root ganglion and peripheral nerve stimulation

ObjectivesSensitization of C-fiber nociceptors plays a critical role in spontaneous and ongoing pain in patients with chronic pain. We recently demonstrated that C-fiber afferents can be reversibly blocked through activity-dependent conduction slowing, suggesting that selective activation of C-fiber afferents may represent a novel strategy for pain relief. We hypothesized that electrical peripheral nerve stimulation (ePNS) and dorsal root ganglion (DRG) stimulation exhibit distinct activation thresholds for unmyelinated C-fiber afferents. Materials and MethodsWe characterized the activation thresholds of A- and C-fiber afferents during ePNS and DRG stimulation using single-fiber electrophysiological recordings from split nerve filaments and optical GCaMP6f imaging of intact DRGs. We further quantified the distribution of the sodium channel subtype NaV1.6 in afferent axons and somata and evaluated its contribution to neuronal excitability using NEURON-based computational modeling. ResultsSingle-fiber recordings showed that activation of C-fiber axons required approximately tenfold higher stimulus amplitudes than A/{beta}-fiber axons during ePNS. In contrast, DRG stimulation within a narrow amplitude range robustly activated both small- and large-diameter DRG neurons, with putative C-fiber afferents comprising 57% of the activated population, indicating markedly reduced differential activation thresholds compared with ePNS. Analysis of published single-cell RNA-sequencing datasets revealed high NaV1.6 expression in TRPV1-positive C-fiber nociceptors. Immunohistochemical staining demonstrated prominent clustering of NaV1.6 in the stem axons of most DRG neurons, including small-diameter C-fiber afferents, whereas NaV1.6 was absent from C-fiber axons in the sciatic nerve. NEURON simulations further showed that NaV1.6 clustering at the stem axon is a key determinant of activation thresholds during DRG stimulation. ConclusionsThese findings identify a structural and molecular mechanism underlying the efficient activation of C-fiber afferents by DRG stimulation and provide mechanistic insight into the superior therapeutic efficacy of DRG stimulation for the treatment of C-fiber-mediated chronic pain.

neuroscience↗

bioRxiv2026

Single-Cell Proteomics of Human Peripheral Blood Mononuclear Cells Exceeding 600 Cells per Day

Single-cell proteomic (scProteomic) measurements of peripheral blood mononuclear cells (PBMCs) are of considerable value in human health, given their involvement in the maintenance of healthy and diseased states. However, the high heterogeneity and relatively small size of immune cell types demand maximal throughput and sensitivity in proteomic measurements that have yet to be fully realized. Here, we describe an approach that addresses sensitivity and throughput through the implementation of Real-Time spectral Library Searching (RTLS), TMTpro 32-plex labelling, an updated nested-nanodroplet processing in One pot for Trace Samples (N2), and a dual-column liquid chromatography system. By prioritizing tandem mass spectrometry (MS2) features with high similarity to library spectra, RTLS enables greater identification depth and feature reproducibility than a standard shotgun MS2 approach in low-input and single-cell samples. The platform permitted 660 single PBMCs to be measured per day, with an average of 750 protein identifications per cell and 1,648 proteins in total, achieving the necessary throughput and depth to characterize immune cell populations. Application of this scProteomic method and a new cell typing informatics approach to 2,130 PBMCs enabled the identification of both major and low-frequency cell types ([~]1-2%), as well as associated proteomic markers.

immunology↗

bioRxiv2026

Machine Learning Identifies Distinct Treg-Mediated Remodeling in HFpEF Hearts Treated with Neonatal Mesenchymal Stem Cells and Their Secretome

BackgroundHeart failure with preserved ejection fraction (HFpEF) remains a major therapeutic challenge due to its complex pathophysiology and pronounced heterogeneity. Regenerative approaches using neonatal mesenchymal stromal cells (nMSCs) and their secretome (SEC) have shown promise in other heart failure contexts. ObjectivesHowever, the effect of these therapies in HFpEF, and the underlying molecular mechanisms and causal pathways remain poorly understood. MethodsHFpEF was established in two distinct murine models, followed by treatment with either nMSCs or SEC. Functional and histological endpoints were assessed. We developed a novel machine learning framework, VIPcell, which integrates data augmentation, Partial Least Squares (PLS) regression, and causal structure inference to identify genes causally linked to cardiac function using single-nucleus RNA sequencing (snRNA-seq) data. VIPcell was applied to heart tissues from treated HFpEF animals to uncover key regulators of cardiac remodeling. ResultsBoth nMSC and SEC therapies significantly improved diastolic function in two independent rodent HFpEF models. These improvements were associated with reduced inflammation, attenuated myocardial fibrosis, and improved exercise capacity. Intercellular communication analysis revealed widespread, system-level signaling in nMSC-treated hearts, compared to more localized endothelial-cardiomyocyte crosstalk in SEC-treated hearts. Causal inference via VIPcell suggested overlapping upstream regulators in both treatment groups, particularly genes involved in regulatory T cell (Treg) biology and immunomodulatory signaling pathways, including FOXO signaling, NLRP3 inflammasome inhibition, and Tie2 activation. In vivo validation confirmed selective expansion of Tregs following nMSC and SEC therapy. In vitro, nMSCs induced significantly greater Treg expansion compared to multiple adult stem cell types. Critically, chemical depletion of Tregs abrogated the therapeutic effects of both treatments, establishing Tregs as central mediators of diastolic function recovery in the HFpEF preclinical model. ConclusionsnMSC and SEC therapies improve diastolic function in HFpEF through distinct remodeling mechanisms converging on Treg-mediated immune modulation. VIPcell supported identification of causal regulators, highlighting Treg-related signaling as a key driver of myocardial recovery in HFpEF. These findings offer mechanistic insight into cellular therapies for HFpEF and support the development of targeted, Treg-focused interventions.

physiology↗