Biology subjects

Wu, L.

Publications and source records attributed to Wu, L..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Molecular organization of the eukaryotic secretory pathway

The eukaryotic secretory pathway is an essential subcellular network comprised of the endoplasmic reticulum (ER) and Golgi organelles. It mediates the trafficking of protein, lipid and other biomolecular cargoes throughout the cell, and is also the site of enzymatic modifications such as glycosylation that are vital for biological function. Complex interactions between resident functional proteins and cargo biomolecules drive myriad activities within the secretory pathway, but how these components organize within native milieu is still poorly understood. Here, we used cryo-electron tomography to survey the HeLa secretory pathway, revealing detailed features of its organellar structure. By quantitatively mapping protein distributions, we find that interactions within the HeLa secretory pathway are dominated by small dimeric complexes, stochastically distributed across both ER and Golgi membranes. Our survey of secretory pathway biomolecules provides new insights into putative trafficking and modification mechanisms, and highlights links between molecular organization and function within intracellular compartments.

cell biology↗

bioRxiv2026

Optimizing Oxylipin Analysis with Liquid Chromatography Mass Spectrometry through Bio-Inert Systems and Ion Funnel Adjustments

Oxylipins are potent signaling lipids that affect inflammation, vascular tone, and metabolism, making them relevant in many diseases. Oxylipins are measured with liquid chromatography-mass spectrometry (LC-MS), but challenges in quantification arise due to low abundance and rapid degradation. In this study, we optimize LC-MS methods to improve the quantification of oxylipins in human plasma given growing interest in oxylipins and their impact on clinical research. Plasma samples were obtained from healthy participants and extracted by solid-phase extraction to concentrate the oxylipins. We then utilized a reversed phase targeted LC-MS/MS method using an Agilent 6495D triple quadrupole with transitions for 248 oxylipin species. Ion funnel voltages were set at 50 or 100 volts. Given the rapid degradation of oxylipins with bio-reactive surfaces, we compared both standard and Altura (bio-inert) columns, as well as standard and bio- inert LC setups. We observed that ion funnel parameters significantly alter detectable levels of oxylipins within LC-MS/MS analysis. By decreasing voltages applied to ions inside the ion funnel, signal was increased for most oxylipin species while peak quality was maintained. We also demonstrated that fully bio-inert setups quantify more compounds and show increased levels of some compounds, but fewer epoxyoctadecadienoic acid (EpODE) species. To explore this further, we injected analytical grade alpha-linolenic acid (ALA), the direct precursor of EpODEs, and observed formation of EpODEs within the instrumentation when using stainless steel columns. Our data shows that oxylipins benefit from fully bio-inert systems and optimized pre-mass analyzer parameters. The stainless-steel components of the column may also be contributing to epoxidation reactions of polyunsaturated fatty acids (PUFAs), generating oxylipin species during analysis. Finally, we utilized this method to perform oxylipin analysis in other human tissues including granulocytes, mononuclear cells, erythrocytes, skeletal muscle, and THP-1 cells, a human derived monocyte cell line.

biochemistry↗

bioRxiv2026

Dithionite quenching of NBD-labeled lipids reveals artificial lipid droplet purity and neutral lipid surface accessibility

Artificial lipid droplets (aLDs) provide a controllable platform for studying lipid biochemistry, but their use is limited by contamination with other membrane structures and the lack of quantitative methods to assess sample purity. Here, we establish dithionite quenching of NBD-labeled lipids as a simple approach to evaluate aLD purity. The approach relies on dithionites ability to selectively quench NBD fluorophores exposed in the phospholipid monolayer of aLDs and in the outer leaflet of liposome bilayers, but not those protected within the inner leaflet of liposome bilayers. Consistent with liposome contamination, bulk aLD preparations exhibit incomplete quenching, which can be separated by sucrose gradient centrifugation into liposome-like and droplet-enriched populations based on quenching behavior. Guided by this assay, sonication conditions were optimized to increase aLD purity and reduce liposome contamination. A biotin-streptavidin immobilization strategy further enabled stable imaging of individual aLDs. Finally, we applied dithionite quenching to probe the accessibility of neutral lipids within aLDs. This revealed hydrophobicity-dependent quenching kinetics of neutral lipids, with less hydrophobic diacylglycerols showing greater surface exposure within aLDs than more hydrophobic triacylglycerols and cholesterol esters. Taken together, these establish dithionite quenching of NBD-labeled lipids as a simple quantitative method for assessing aLD purity and demonstrate its utility for studying lipid accessibility.

biochemistry↗

bioRxiv2026

Function-guided design of active enzymes

Designing enzymes from functional descriptions remains challenging because catalytic activity is governed by sequence-structure-function relationships. Here we present EnzymeArt, a function-conditioned enzyme-design framework centred on a generative sequence model. EnzymeArt couples function-conditioned sequence generation with structure-guided refinement, annotation checks and substrate-aware computational prioritization to select candidates for synthesis and biochemical testing. Across alcohol dehydrogenase (ADH), malate dehydrogenase (MDH) and triacylglycerol lipase design campaigns, 57 of 60 synthesized designs showed crude-lysate activity above matched background controls. Purified representatives further showed quantitative steady-state catalytic activity. The best designed ADH reached kcat = 223.7 s-1 and exceeded a wild-type reference under matched conditions, an MDH reached kcat = 267.57 s-1 despite having only 33% sequence identity to its closest BLASTP hit, and a designed lipase hydrolysed both short- and long-chain triglycerides with apparent activity modestly above that of a commercial lipase reference. Together, these results establish a route for converting functional descriptions into experimentally validated enzyme designs with quantitative steady-state kinetic activity.

bioinformatics↗

bioRxiv2026

A multi-organ spatial metabolomic atlas of exercising mice reveals neuronal Complex I as a convergent and sufficient axis for tau pathology reduction in PS19

We constructed a spatially resolved metabolomic atlas of long-term exercise across six major organs in wild-type mice: brain, heart, lung, liver, kidney, and skeletal muscle, cataloguing 224 metabolic features and revealing coordinated inter-organ remodeling. Surprisingly, the brain showed particularly pronounced region-specific adaptation. Because pathological tau associates with synaptic mitochondria from early stages of tauopathy, we extended this multi-organ spatial metabolomic approach to PS19 mice and found that exercise reduced over 70% of observable tau pathology in PS19 hippocampus and restored the mitochondrial-related metabolome. Integrated proteomic and spatial metabolomic analyses identified NADH dehydrogenase Complex I as the convergent node. To test this finding biologically, we expressed the yeast NADH dehydrogenase, Ndi1, in PS19 neurons in the absence of exercise. This increased cerebral antioxidants, restored shuttle-linked metabolites, and reduced tau pathology. Increasing NADH dehydrogenase activity through NDI1 reproduces the core anti-tau and metabolic effects of exercise. These findings provide a molecular mechanism for how exercise may prevent or slow tau pathology accumulation, complementing the human-cohort literature linking exercise to delayed cognitive decline.

neuroscience↗

bioRxiv2026

nanoASM: Long-Read Allele-Specific DNA Methylation Profiling Enables Functional Annotation of Regulatory Noncoding Variants in Human Prostate Tissues

Long-read nanopore sequencing enables simultaneous detection of germline variation and native DNA base modifications on individual DNA molecules, providing a unique opportunity to investigate allele-specific epigenetic regulation. Here, we performed whole-genome nanopore sequencing on normal and tumor prostate tissues to characterize differential methylation, methylation entropy, and allele-specific methylation (ASM) associated with noncoding genetic variants. Genome-wide analysis identified extensive cancer-associated differentially methylated regions (DMRs), with hypermethylated DMRs significantly enriched near transcription start sites and transcriptional regulatory regions. Integration with transcriptomic datasets revealed strong inverse relationships between promoter methylation and gene expression, while 5-hydroxymethylcytosine (5hmC) levels positively correlated with transcriptional activity across gene bodies. Using fragment-level methylation patterns enabled by long-read sequencing, we further quantified methylation entropy incorporating both 5mCG and 5hmCG states. Cancer-hypermethylated DMRs exhibited markedly reduced entropy, consistent with clonal fixation of methylation states during tumor progression. Entropy profiling across chromatin annotations demonstrated maximal epigenetic heterogeneity at partially modified enhancer-associated regions. To investigate cis-regulatory genetic effects, we developed a simple ASM framework (nanoASM) that can partition sequencing reads by allelic state and identifies allele-specific DMRs directly from long-read data. Compared with conventional population-level mQTL analysis, ASM demonstrated substantially improved statistical efficiency by leveraging within-individual contrasts and reducing sample-level heterogeneity. Although germline single nucleotide polymorphisms (SNPs) were largely shared between normal and tumor tissues, ASM patterns differed substantially, with tumor-associated ASM regions displaying significantly larger genomic span and stronger allelic methylation differences. Comparative analysis with TCGA prostate mQTL and GTEx prostate eQTL datasets demonstrated substantial concordance between ASM directionality and downstream transcriptional effects, particularly for variants located within DMRs and near transcription start sites. At the IRX4 prostate cancer risk locus, ASM identified an androgen-responsive regulatory domain overlapping AR ChIP-seq and H3K27ac peaks, nominating rs6885084 as a candidate functional variant. At the PSCA locus, ASM anchored by rs4736369 was associated with allele-specific methylation, chromatin activation, transcript abundance, and isoform usage. Together, these findings establish nanopore-based ASM analysis as a powerful approach for resolving functional noncoding variants and their regulatory domains they control in prostate cancer.

genetics↗

bioRxiv2026

Circadian clock gates retinal regeneration by orchestrating Cxcl12-dependent immune coordination

The circadian clock orchestrates tissue homeostasis and repair, yet its role in central nervous system (CNS) regeneration remains largely unexplored. Here, we identify the circadian clock as an essential regulator of adult zebrafish retinal regeneration, a CNS model. Retinal injuries induced at distinct circadian times elicit differential Muller glia (MG) reprogramming and progenitor proliferation, both of which exhibit robust diurnal fluctuations. Disrupting circadian rhythms--via constant darkness (DD) or day-night reversal (DL)--suppresses MG reprogramming, MG-derived progenitor cell (MGPC) formation and neuronal regeneration, concomitant with dampened retinal immune responses. Notably, intravitreal immune stimulation restores MGPC numbers in the retina under DD conditions, linking immune activation to circadian-regulated regeneration. Single-cell RNA-seq reveals that DD disrupts MG reprogramming trajectories while inducing unfolded protein response (UPR) and suppressing ATP/protein synthesis. Moreover, clock disruption alters microglia heterogeneity, and cell-cell communication analysis identifies the Cxcl12-Cxcr4 signaling as a key chemotactic axis between MG/endothelial cell and immune populations. Mechanistically, Bmal1b directly binds E-box motifs in the cxcl12a/12b promoters, driving their rhythmic transcription to orchestrate the immune response after injury. Importantly, exogenous Cxcl12a partially restores both immune and regenerative responses in clock-disrupted retinas, establishing the Cxcl12-Cxcr4 axis as a circadian-immune checkpoint for retinal repair. Collectively, our findings reveal a novel paradigm in which the circadian clock gates CNS regeneration through Cxcl12-mediated immune coordination, offering potential therapeutic insights for CNS repair in mammals.

neuroscience↗

bioRxiv2026

Nir1-Nir2 Heterodimerization Confers Robustness to the Phosphoinositide Cycle

The phosphatidylinositol (PI) cycle sustains phosphoinositide-calcium signaling by replenishing plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) consumed during receptor activation. Recruitment of the lipid transfer protein Nir2 to endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (also known as ER-PM junctions) is central to this process, enabling transfer of PI from the ER to the PM for PIP2 resynthesis. Although the disease-associated Nir2 paralog Nir1 is essential for Nir2 recruitment under physiological conditions, the molecular basis of this regulation has remained unresolved. Here, we identify a conserved Nir Dimerization (NirD) domain in both Nir1 and Nir2 and determine the crystal structures. We show that NirD mediates preferential Nir1-Nir2 heterodimerization, which promotes Nir2 recruitment to ER-PM junctions and enhances PIP2 replenishment in stimulated cells. Furthermore, Nir1-Nir2 heterodimerization confers stimulus-strength-dependent, graded recruitment of Nir2, thereby broadening the sensitivity and dynamic range of PI cycle activity. Together, our findings uncover the structural mechanism underlying Nir1-dependent regulation of Nir2 and reveal paralog heterodimerization as a key strategy for scaling lipid transport at membrane contact sites to signaling demand, ensuring robust phosphoinositide homeostasis.

cell biology↗

bioRxiv2026

Metal Coordination Dynamics Governs Selective Epoxidation in Hyoscyamine 6β-Hydroxylase: Integrated Experimental and Computational Insights

Iron(II)- and 2-oxoglutarate-dependent (Fe(II)/2OG) enzymes catalyze a wide range of C-H bond activation and functionalization reactions and play essential roles in biosynthesis and metabolic regulation. Despite extensive mechanistic studies, the principles governing selectivity between canonical hydroxylation and alternative transformations remain incompletely understood. Here, we investigate the catalytic mechanism of hyoscyamine 6{beta}-hydroxylase (H6H), a Fe(II)/2OG-dependent oxygenase that sequentially catalyzes the 6{beta}-hydroxylation of hyoscyamine followed by 6,7-exo-epoxidation of 6{beta}-hydroxyhyoscyamine to generate scopolamine. Combined molecular dynamics and QM/MM calculations reveal that an inline Fe(IV)-oxo intermediate initiates hydrogen atom abstraction from the substrate C7 position. The resulting Fe(III)-OH species subsequently deprotonates the substrate hydroxyl group in a process coupled to substrate coordination to the iron center and an in-line-to-off-line rearrangement of the Fe(III)-OH moiety. This coordination dynamics machinery is further supported by the observed chlorination reactivity on the same substrate. Importantly, this coordination switch favors epoxide formation over hydroxyl rebound, thereby directing the reaction toward selective epoxidation. Further computational analysis of the L290F variant demonstrates that steric constraints imposed by L290 are essential for suppressing hydroxylation, revealing a bidirectional regulatory mechanism governing epoxidation/hydroxylation selectivity. Whereas iron coordination dynamics promote epoxidation reactivity, precise substrate positioning and protein-derived steric effects suppress the competing hydroxylation pathway. These findings are consistent with available experimental observations and establish metal coordination dynamics as a key determinant of selective C-H functionalization in Fe(II)/2OG enzymes.

biochemistry↗

bioRxiv2026

Ground Truth-Based Evaluation of False Discovery Rate and Statistical Power in DIA Proteomics

Data-independent acquisition (DIA) mass spectrometry enables rapid proteomic quantification, yet the reliability of statistical inference in DIA-based protein quantification remains incompletely understood. Here, we systematically evaluated missingness, false discovery rate (FDR), and statistical power, defined as true positive rate (i.e. sensitivity or recall), using technical replicates and a spike-in benchmark with known ground truth. Analysis of 18 HeLa replicates revealed persistent, abundance-dependent missingness. In the spike-in experiment with five replicates, human peptides were titrated against a stable yeast background, allowing fold changes (FCs) to be compared with expected values. Across comparisons with log2FCs ranging from 0.2 to 2.5, the nominal BH-FDR substantially underestimated the true FDR. For example, at a BH-FDR threshold of 0.05, the true FDR was [~]0.2. Statistical power was [~]40% for a log2FC of 0.2 and increased to nearly 100% for a log2FC of 2.5. Additional incorporation of FC thresholds improved the true FDR for large-FC comparisons, with slight loss of power, but markedly reduced sensitivity for small-FC comparisons. Together, these results indicate that nominal FDR does not necessarily reflect actual error rates in DIA proteomics and that DIA performance is influenced by protein abundance and expected fold changes. This study provides a framework for experimental design and data interpretation in DIA-based proteomic studies.

bioinformatics↗

bioRxiv2026

Activation of mGlu2 receptors reverses persistent post-methamphetamine deficit in object-in-place recognition memory.

Background/ObjectivesPersistent cognitive impairments are prevalent in methamphetamine (meth) use disorder and contribute to maladaptive decision-making and increased relapse vulnerability. There are currently no effective treatments for meth-associative cognitive deficits, and their neurobiological underpinnings remain incompletely understood. This study investigated the effects of chronic meth self-administration on episodic-like recognition memory and evaluated whether pharmacological potentiation of metabotropic glutamate receptor subtype 2 (mGlu2) could rescue these deficits. MethodsAdult male Sprague-Dawley rats underwent 7 days of limited- (1h/day) followed by 14 days of extended-access (6h/day) meth self-administration, followed by 30 days of abstinence. Recognition memory was assessed using the object-in-place (OIP) task. A positive allosteric modulator of mGlu2 receptors, LY-487379 (25 mg/kg, s.c.), was administered prior to the memory test. In parallel, changes in total and surface mGlu2/3 protein levels in the prelimbic and perirhinal cortices were evaluated. ResultsRats with extended access to meth self-administration exhibited escalated drug intake and persistent deficits in OIP memory. Administration of LY-487379 reversed this deficit. Total mGlu2/3 protein levels were unaltered; however, meth exposure was associated with a significant increase in surface mGlu2/3 receptor expression in both cortical regions examined. ConclusionsThese results demonstrate that chronic meth produces persistent cognitive dysfunction that can be rescued by mGlu2 receptor potentiation. The observed increase in surface mGlu2/3 expression may represent a compensatory response to chronic glutamatergic dysregulation, but it appears to be insufficient to restore cognitive function alone, without pharmacological enhancement. The current data encourage further exploration of mGlu2 role in stimulant-associated cognitive dysfunction. HighlightsChronic methamphetamine self-administration produced persistent deficits in episodic-like recognition memory in male rats and dysregulation of mGlu2/3 receptors in the prelimbic and perirhinal cortices. Systemic pharmacological potentiation of mGlu2 receptors rescued meth-associated memory deficits. mGlu2 receptor potentiation may represent a promising therapeutic strategy for treating stimulant-associated cognitive dysfunction. Increased surface mGlu2/3 expression may represent a compensatory adaptation to post-methamphetamine glutamatergic dysfunction, but it is not sufficient to restore cognition alone, without pharmacological enhancement.

neuroscience↗

bioRxiv2026

Hierarchical Immune Suppressive Functions of Regulatory T Cells Built on Mechanical Force

Regulatory T (Treg) cells maintain immune tolerance via contact-dependent inhibition and soluble mediators. While canonical suppressive mechanisms are well characterized, their hierarchical spatiotemporal organization remains unclear. We previously showed Foxp3 represses ER Ca2+ channel RyR2, reducing cytoplasmic Ca2+ to inactivate m-Calpain (Calpain-2) and stabilize high-affinity LFA-1-mediated Treg-dendritic cell (DC) adhesion--an early step blocking DC antigen presentation. Using m-Calpain as a molecular switch, we generated mechanically deficient Tregs (TregFD) with constitutively active m-Calpain that abrogated high-force Treg-DC adhesion. TregFD retained a near-wild-type transcriptome (differential expression limited to adhesion pathways), yet failed to suppress autoimmunity or DC-driven T cell proliferation. Stable adhesion was required for localized TGF-{beta}/IL-10 delivery to DCs. Single-cell RNA sequencing of rescued Scurfy mouse lymphoid tissues revealed organ-specific division of labor: non-mechanical modules support homeostasis/tissue repair, while mechanical modules dominate epithelial barrier/antigen presentation suppression. Combined TregFD and RyR2-deficient Tconv transfer restored wild-type Treg activity, demonstrating module synergism. These findings establish a mechanical force-centered, two-tiered hierarchical model of Treg suppression, providing a framework for targeting Treg mechanics in autoimmunity and inflammation.

immunology↗

bioRxiv2026

Kinemomics: spatiotemporal morphodynamic mapping of ventricular kinematic subpopulations in organotypic fetal heart slices

Recent advancements in omics technologies have deepened our understanding of complex biological systems and the interplay among gene expression, mechanical cues, and cellular metabolism. However, capturing the spatiotemporal dynamics of live cellular behavior within intact tissue contexts remains a major challenge. Here, we introduce Kinemomics, a framework for spatiotemporal mapping of morphogenetic kinematic states in living tissue. Using a novel fetal cardiac organotypic slice culture system that preserves both beating and morphogenesis, Kinemomics integrates live fluorescent labeling, timelapse imaging, and endpoint immunofluorescence to generate morphodynamic profiles across full-thickness developing myocardium. We validate Kinemomics under controlled Yap-1 signaling perturbations and demonstrate its ability not only to intrinsically decode region- and treatment-specific phenotypes, but also to resolve their spatiotemporal evolution during development and malformation. Kinemomics further reveals localized emergent developmental and malforming fronts that drive system-level behavior, establishing a generalizable platform for investigating computational morphodynamics and cross-scale interactions in complex spatiotemporal systems.

developmental biology↗

bioRxiv2026

Molecular and Morphological Evidence Reveals Cryptonema producta in Shui Hau, Hong Kong, Previously Misidentified as Anomalocardia flexuosa

For over four decades, the bivalve Anomalocardia flexuosa has been recorded in Hong Kong coastal waters. However, the known native distribution of this heavily exploited commercial species is restricted to the Atlantic coast of South America, raising questions about the biogeographical validity of the Hong Kong populations. By employing an integrative taxonomic approach combining morphological re-evaluations and molecular phylogenetic analysis of the COI gene, we confirm that the species in Shui Hau, Hong Kong, China, has been historically misidentified. The population belongs to Cryptonema producta (syn. Anomalocardia producta).

zoology↗

bioRxiv2026

Metabolic Coherence of the Mouse Brain

The brains metabolic demands are well established, but how metabolism is coordinated across anatomically distinct regions remains poorly understood. Here, using matrix-assisted laser desorption/ionization (MALDI) imaging integrated with the Allen Brain Atlas and optimal transport-based computational analysis, we map the spatial metabolome across twelve major mouse brain divisions. We define an optimal-transport-derived inter-regional metabolite similarity metric and refer to it as metabolic coherence. This structure is largely preserved in an amyloid mouse model of Alzheimers disease despite widespread changes in individual metabolite and lipid levels. Individual metabolites and lipids shift in a coordinated manner across regions, sustaining inter-regional relationships even as absolute levels change in patterns indicative of mitochondrial dysfunction. To test whether the coherence metric is responsive to local intervention, we targeted the left hippocampus of mice from this model via lentiviral shHIF1 knockdown or neuronal AAV-mediated AOX expression. Both interventions were associated with metabolite normalization at the injection site. More importantly, normalization extended across distal regions sharing high metabolic similarity with the hippocampus and was accompanied by improved social memory in a single behavioral assay. Gene modulation and amyloid plaque reduction localized to the injection site.

neuroscience↗

bioRxiv2026

NeuroCDS: Integrating Local and Global Neural Network Representations via Structural Constrained Viterbi Decoding for Robust CDS Annotation

MotivationRobust annotation of Coding Sequences (CDS) is critical for downstream transcriptomics, yet heavily fragmented de novo RNA-Seq assemblies pose a severe challenge. Traditional computational tools rely on fixed, hand-crafted features that are prone to fail when canonical sequence signals are truncated. While recent deep learning models excel at automatically extracting complex representations, they predominantly treat these as isolated prediction tasks. Lacking a joint inference mechanism to enforce structural constraints, existing models occasionally output biologically invalid predictions. Therefore, a computational framework capable of fusing heterogeneous neural network representations for joint annotation is critically needed. ResultsWe present NeuroCDS, a reliable framework that bridges the effective representation capabilities of deep neural networks with the structural rigor of dynamic programming. NeuroCDS employs a dual-branch architecture: a Convolutional Neural Network (CNN) acts as a local sensor to extract Translation Initiation Sites (TIS), while a Temporal Convolutional Network (TCN) acts as a global sensor to evaluate continuous regional coding potential. The primary contribution of NeuroCDS lies in a structurally constrained Viterbi Decoding algorithm designed to fuse these heterogeneous signals. This joint inference mechanism strictly enforces biological grammars (e.g., reading frame preservation) to dynamically calculate the globally optimal transcript structure via a tripartite state space. Crucially, by introducing a dynamic length normalization mechanism, our formulation adaptively leverages global continuous representations to stably annotate both intact transcripts and highly truncated fragments. Comprehensive evaluations demonstrate that NeuroCDS achieves high F1-scores on full-length transcripts and maintains robust performance on complex Ribo-seq validated datasets, comparing favorably against traditional HMM-based and heuristic methodologies. AvailabilitySource code, pre-trained models, and datasets are freely available at https://github.com/hgcwei/NeuroCDS.

bioinformatics↗

bioRxiv2026

JUMPlion improves quantitative DIA proteomics through ion-level recovery of missing values

Incomplete quantification remains a persistent challenge in data-independent acquisition (DIA) mass spectrometry (MS), particularly in low-input and single-cell analyses. In identification-driven workflows, missing protein quantities often arise not from true absence of the corresponding peptides, but from failure to retain low-abundance signals from precursor or product ions for quantification. Here we present JUMPlion (local inference of ion-level missingness), a DIA quantification framework that re-examines MS raw files to recover missing values at the ion level before protein quantification. JUMPlion re-extracts precursor- and production signals directly from raw data, infers ion-level measurements within precursor-specific local quantitative neighborhoods, and combines complementary precursor- and production signals into downstream quantification. Using benchmark datasets acquired on multiple DIA platforms, JUMPlion increased protein-level completeness, improved fold-change accuracy, and enhanced detection of differentially abundant proteins while maintaining low differential-abundance false discovery rates. These gains were most evident in low-input and single-cell DIA datasets. Together, these results show that addressing missingness at the ion level before protein-level summarization can improve DIA quantification in diverse acquisition settings.

bioinformatics↗

bioRxiv2026

Cultural transmission and genomic co-divergence in the willow tit across the Palearctic

Cultural transmission of mating behaviour can both promote and constrain genetic divergence, yet its long-term population-genetic consequences remain unclear. In songbirds, learned song can generate behavioural isolation, but its potential to shape genome-wide differentiation at continental scales is rarely assessed. Willow tits provide a compelling system, as three culturally transmitted song types are largely allopatric across the Palearctic, while northern populations exhibit mixed repertoires. Here, we combined a chromosome-level reference genome, whole-genome resequencing of 88 willow tits (Poecile montanus) spanning all 14 subspecies, and palaeodistribution modelling to reconstruct the species evolutionary history across the Palearctic. Phylogenetic and demographic analyses indicate an origin in Asia during the Late Pliocene to Early Pleistocene, followed by expansions that yielded three deeply diverged genomic lineages in the Asian, Central European, and Northern Palearctic regions. The boundaries of these lineages coincide with major song-type divisions. Tests of historical allele sharing show that gene flow occurred preferentially among lineages that share the same or similar song types, even after accounting for geography, consistent with learned song contributing to prezygotic isolation. Peripheral, song-monotypic populations exhibit signatures of repeated bottlenecks associated with glacial isolation, whereas large northern populations retained broader song repertoires and signals of long-term connectivity. These results provide genome-wide continental evidence that culturally transmitted song mirrors and likely reinforces genomic structure through time in a widespread passerine bird.

evolutionary biology↗