Biology subjects

Zhang, L.

Publications and source records attributed to Zhang, L..

At least 73 records · Page 4Linked to original sources

bioRxiv2026

Lipoproteins modulate the uptake and biological functionof cationic cell-penetrating peptides across the animal lineage.

Cationic cell-penetrating peptides (+CPPs) are pervasive killer peptides produced by all animals and found in innate immune systems, venoms and neurodegenerative diseases. Despite their ubiquity, the mechanisms underlying their uptake remain opaque and intensely debated. Here, we interrogate +CPPs spanning 600 million years of animal evolution and identify endocytosis as a conserved/convergent uptake mechanism across cell types and organisms, at physiological concentrations. By combining multiplex imaging, genetic screening, cryo-electron tomography, and biophysical methods, we uncover that +CPPs seemingly universally enter eukaryote cells via 'hitchhiking' on lipoproteins. We further show that the interaction of +CPPs with lipoproteins modulates their antimicrobial function. Combined, our findings establish a unified molecular framework describing a pan-eukaryote cell penetrance mechanism. As hyperlipidemia is a common comorbidity, our findings have direct implications for human health. Lastly, the insights gleaned from this work highlight design principles that may inform the future engineering of peptide-based therapeutics and delivery vehicles.

cell biology↗

bioRxiv2026

NACraft: Programmatic nucleic-acid aptamer design via all-atom structure-model feedback

Protein-nucleic-acid interactions underpin diverse biological processes and provide a basis for molecular sensing, regulation and therapeutic intervention. However, the coupled dependence of aptamer function on nucleotide sequence, three-dimensional folding and target binding makes rational RNA and DNA binder design challenging. Here we present NACraft, a training-free and programmatic framework for all-atom nucleic-acid aptamer design based on backpropagation through structure-model feedback. By composing binding, sequence-similarity and anti-binding constraints, NACraft supports de novo generation, similarity-guided sampling and target-selective design within a unified optimization framework, without task-specific training or fine-tuning. Computational experiments showed that NACraft generated high-confidence candidates de novo across diverse protein targets, with further improvements achieved through similarity-guided design for both RNA and DNA complexes. Its target-selective design capability was further validated in silico, with 69.44% of paired candidates generated to favour the positive target EGFR over the off-target HER2. Under matched independent AlphaFold3 evaluation, NACraft achieved better performance than ODesign in 10 of 11 NA-12 targets and 17 of 20 protein target-length settings. Together, these results demonstrate the effectiveness and versatility of NACraft and extend structure-model hallucination toward programmatic nucleic-acid aptamer design. Codehttps://github.com/OTEAM-AI4S/NACraft

bioinformatics↗

bioRxiv2026

EnSEMBLE: a framework for enhancer-anchored pathway analysis that locks in enhancer-corroborated pathways from transcriptome sequencing data for biological validation

Background Pathway discovery methods for transcriptome sequencing return tens to hundreds of redundant gene sets, and biologists often subjectively select the pathways fitting biological expectations. What is missing is not another statistical method, but a way to corroborate each candidate pathway against an independent, mechanistic line of evidence. Results We introduce EnSEMBLE (Enhancer-Set Enrichment & Mechanism-Based Linked Evidence), a tool that corroborates gene-level pathway enrichment with an orthogonal enhancer layer drawn from the same transcriptome sequencing data: active enhancers transcribe enhancer RNAs already present in standard RNA-seq, so a pathway's regulatory state can be scored from the very run that produced the gene-level signal, at no added cost. EnSEMBLE pairs pathway enrichments with Enhancer-Program Enrichment Analysis (EPEA), collapses redundant gene sets into process-level Themes, and retains only those that a concordant enhancer program corroborates. This dual-evidence requirement reduced reported signatures by >97% (hundreds of gene sets to 3-18 claims) across four datasets spanning cancer perturbations and iPSC-to-neuron differentiation. Surviving claims recovered expected biology--mesenchymal-program collapse upon SNAI1 knockout, regulatory convergence during neuronal differentiation--and named mechanisms pathway enrichments missed, including an mTOR-MYC-SPT5 elongation axis in rapamycin-treated PANC1 cells. A language AI agent performs narrative synthesis over deterministic statistics, with reproducibility enforced by temperature-zero inference and three-run consensus. We further provide enhancer over-representation analysis (eORA), mapping non-coding GWAS variants to the same programs to recover cell-type-selective trait associations. Conclusions EnSEMBLE shifts transcriptomic interpretation from enumerating possibilities to adjudicating evidence, yielding a compact, traceable set of enhancer-corroborated claims that identify the regulatory programs driving cellular change and prioritize them for experimental validation.

bioinformatics↗

bioRxiv2026

Powering methanogenesis from fatty acids by a twin-heme-mediated reverse redox-loop

The conversion of organic matter into methane is central to the global carbon cycle and engineered biogas production. In this process, syntrophic bacteria oxidize fatty acid fermentation products to acetate, coupled to the generation of H2 or formate, which are subsequently utilized by methanogenic archaea. During fatty acid {beta}-oxidation, a membrane-bound electron-transferring flavoprotein (ETF):methylmenaquinone (MMK) oxidoreductase complex (EMO) has been proposed to drive endergonic electron transfer from reduced ETF to CO2 through a reverse redox loop, with its mechanistic basis remaining unresolved. Here we report cryo-electron microscopy structures of EMO and the EMO-ETF complex from Syntrophus aciditrophicus at 2.0 and 3.0 [A] resolution, respectively. Complex formation induces substantial conformational rearrangements in ETF, positioning its flavin for efficient electron transfer to non-cubane [4Fe:4S] and [4Fe:5S] clusters. The membrane-integral domain harbors three heme b cofactors, including a specialized twin-heme unit that mediates proton-motive-force-driven MMK reduction. The structural and functional similarity of EMOs to heterodisulfide reductases, together with their broad distribution across bacteria and archaea, suggests an evolutionary link between methanogenesis and fatty acid {beta}-oxidation, illustrating how ancient redox systems were repurposed for new metabolic functions.

microbiology↗

bioRxiv2026

TROP2-targeting chimeras (TRTACs) for tumor-selective membrane protein degradation and enhanced drug delivery

Extracellular targeted protein degradation (eTPD) systems typically utilize lysosome-targeting receptors (LTRs) to mediate internalization and lysosomal degradation of extracellular and membrane proteins. While multiple LTRs have been discovered, there remains a compelling need to seek for new LTRs, particularly those with clear clinical relevance, to expand the therapeutic potential of eTPD. Here we report trophoblast cell surface antigen-2 (TROP2), a clinically validated tumor-associated antigen, as a promising tumor-selective LTR. We engineer TROP2-targeting chimeras (TRTACs) by genetically fusing a TROP2-binding nanobody to nanobodies against specific target proteins. We show that TRTACs can induce tumor cell-selective degradation of diverse membrane proteins, including epithelial growth factor receptor (EGFR), human epithelial growth factor receptor 2 (HER2), and programmed death-ligand 1 (PD-L1). The EGFR-targeted TRTAC significantly inhibits tumor cell proliferation and shows potent antitumor activity in vivo. We further design TRTAC-drug conjugates (TRTAC-DCs) by attaching cytotoxic payloads to TRTACs, enabling targeted protein degradation together with enhanced drug delivery. TRTAC-DCs show significantly enhanced activity against HER2- and EGFR-positive tumors both in vitro and in vivo, with minimal toxicity observed in normal tissues. These findings establish TROP2 as a robust LTR and provide a versatile eTPD platform with profound translational potential for tumor treatment.

cell biology↗

bioRxiv2026

TRACER navigates rearrangement-driven sesterterpene chemical space via multimodal enzyme-product representation learning

Skeletal rearrangement drives the immense structural complexity of terpene, yet predicting it remains a formidable challenge due to sequence-function decoupling in terpene synthases. Here, we established TRACER (terpene rearrangement annotation via co-attentive enzyme-product representation), a multimodal framework mapping the latent associations between sequence-derived enzyme representations and product chemotypes. Retrospective validation proved TRACERs exceptional precision in predicting compound classes and discriminating skeletal rearrangement (SR) from non-skeletal rearrangement (NSR) pathways. TRACER-guided genome mining characterized two bifunctional synthases, FsPS and AcPS, uncovering four unprecedented carbon skeletons. Density functional theory calculations deciphered these cyclization cascades, pinpointing a critical 5/6/11 tricyclic intermediate as the key branching node for scaffold diversification. Mutagenesis and molecular dynamics simulations suggested that E305 in FsPS enables rearrangement by maintaining active-site water exclusion, whereas its alanine mutation causes premature carbocation quenching. Collectively, this work establishes a predictive paradigm for the rational discovery and mechanistic elucidation of complex terpene architectures.

synthetic biology↗

bioRxiv2026

Dissociable reactivation during NREM and REM sleep supports memory consolidation and emotional dissipation

Sleep is critical for memory consolidation and emotional regulation, yet the respective roles of non-rapid eye movement (NREM) and rapid eye movement (REM) sleep remain unclear. Here, using a within-subject crossover design, we recorded high-density electroencephalography (EEG) across two experimental nights while participants viewed neutral or aversive film clips in a counterbalanced order. Combining with daytime functional localizers establishing neural patterns of aversive vs. neutral emotional processing, multivariate pattern analysis revealed that the reactivation of aversive vs. neutral memory during nocturnal sleep was both stage-dependent and event-specific. In NREM sleep, valence-specific reactivation was time-locked to slow oscillation (SO)-spindle complexes but not to either event alone; in REM sleep, reactivation occurred selectively during phasic REM periods marked by rapid eye movements. Critically, NREM SO-spindle coupling percentage was associated with consolidation of temporal memories; whereas phasic REM reactivation strength was linked to overnight dissipation of negative affect. Our findings provide direct evidence that sleep reprocesses emotional experiences through dissociable stage- and event-specific mechanisms, laying out a framework for future targeted sleep-based interventions.

neuroscience↗

bioRxiv2026

Genome-resolved biogeography reveals multidimensional structuring of freshwater giant viruses across global deep lakes

Giant viruses (GV) are increasingly recognized as important ecosystem regulators. While metagenomics has uncovered extensive GV diversity, the global distributions of individual species and the biogeographic processes driving the pattern remain poorly understood. Here, we reconstructed GV metagenome-assembled genomes (MAGs) from 35 globally distributed deep freshwater lakes spanning five continents, aiming to identify their biogeographic patterns. The resulting 1663 non-redundant MAGs significantly expanded the known freshwater GV diversity, with [~]84% lacking a previously reported species representative. These MAGs were grouped into cosmopolitan and geographically restricted lineages. We identified 27 cosmopolitan GV species spanning multiple viral lineages, including families of Imitervirales, Pimascovirales, and mirusviruses order Styxvirales. The cosmopolitan species were characterized by their larger genomes and expanded gene repertoires of host-interaction functions, which may facilitate interactions with diverse hosts and contribute to their global distributions. The presence of geographically restricted species and the stronger distance-decay in community similarity observed in freshwater than marine ecosystems suggest that physical connectivity between ecosystems is an important factor influencing GV dispersal. We identified 312 and 177 GV MAGs almost exclusively associated with the epilimnion and hypolimnion, respectively. This water-layer preference of individual MAGs was highly consistent across lakes, suggesting conserved vertical partitioning in association with the thermal stratification of the water column. Overall, our findings reveal that GV biogeography in deep freshwater lakes is structured by the combined influence of horizontal dispersal limitation, vertical partitioning, and lineage-specific evolutionary histories.

microbiology↗

bioRxiv2026

Inferring disruption of directed graphs using LIKA reveals altered protein phosphorylation networks in schizophrenia

MotivationKinases regulate a multitude of protein functions, and their dysregulation is pivotal for many human diseases. Direct measurement of kinase activity, however, is often challenging; therefore, inferring activity from the behavior of their substrates is a widely adopted strategy. Nonetheless, traditional methods typically oversimplify the underlying network, ignoring that any particular substrate can be phosphorylated by multiple kinases. ResultsWe present LIKA, a likelihood-based framework for inferring kinase activity from phosphoproteomic data. By modeling the many-to-many structure of kinase-substrate interactions, LIKA achieves high efficiency, even with limited data, while capturing network complexity. Simulation and cell line analyses confirm the robustness and accuracy of LIKA. Importantly, analysis of a phosphoproteomic dataset from schizophrenia and control subjects reveals novel dysregulated kinases. Availability and ImplementationThe implementation code and publicly available data are provided at: https://github.com/lujingz/LIKA.

bioinformatics↗

bioRxiv2026

IGF2BP3 amplifies antiviral innate immunity with implications for autoimmune diseases

The insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is a known N6-methyladenosine (m6A) reader, but its role in antiviral innate immunity is unknown. Here, we identify IGF2BP3 as a critical positive regulator of antiviral responses. Viral infection and interferon (IFN) stimulation upregulate IGF2BP3, establishing a feedforward loop that potentiates virus-induced activation of the TBK1-IRF3 and NF-{kappa}B pathways, thereby amplifying type I interferon (IFN-I) production, and restricting viral replication in human and murine cells and in vivo. Mechanistically, IGF2BP3 directly binds and stabilizes MAVS and TBK1 mRNAs and promotes their translation by facilitating recruitment to the eIF4F/PABP-associated initiation complex. Upon infection, IGF2BP3 relocalizes to antiviral stress granules (avSGs), where it scaffolds the RIG-I-G3BP1 complex to enhance viral RNA sensing. Notably, IGF2BP3 is aberrantly upregulated in patients with systemic lupus erythematosus (SLE) and in Trex1 knockout (KO) mice, and pharmacological inhibition by curcumol suppresses IFN-I-driven pathology and improves survival. Collectively, our findings establish IGF2BP3 as a central feedforward circuit that couples viral RNA sensing to the control of RNA stability and translation of key signaling molecules, and reveals its potential as a therapeutic target in interferon-associated autoimmune diseases. Significance StatementAntiviral immunity demands rapid and coordinated gene expression, yet how RNA-binding proteins link viral recognition to downstream signaling is poorly understood. We reveal that the m6A reader IGF2BP3 acts as a central amplifier of antiviral innate immunity. Induced by both viruses and interferons, IGF2BP3 enhances viral RNA sensing, stabilizes key signaling transcripts, and boosts their translation, creating a self-reinforcing feedforward circuit that strengthens interferon responses. Beyond host defense, IGF2BP3 is aberrantly elevated in interferon-driven autoimmunity, and its pharmacological inhibition reduce disease pathology in vivo. Our findings uncover a previously unrecognized mechanism that integrates RNA metabolism, stress-granule signaling, and translational control to regulate innate immunity, offering new therapeutic perspectives for both infectious and autoimmune disease.

immunology↗

bioRxiv2026

A Conserved Flexible N-Terminal Domain Tunes the Calcium Sensitivity of Sorcin by Stabilizing Its Active Conformation

Sorcin is a penta-EF-hand Ca2+-binding protein that acts as a Ca2+ sensor and regulator of Ca2+ homeostasis. Although the structure and Ca2+-dependent activation of Sorcin is well characterized, the function of its flexible N-terminal domain (NTD) remains unclear. We combined sequence analysis, Ca2+-induced aggregation assays, multidimensional NMR spectroscopy, and long-timescale molecular dynamics (MD) simulations to define the NTDs role in Sorcin activation. Sequence comparisons showed that the NTD is conserved across vertebrates despite its intrinsic disorder, indicating functional importance. NTD deletion markedly reduced Ca2+ responsiveness. Relative to full-length Sorcin, the construct with NTD truncation required more than twofold higher Ca2+ concentrations and over tenfold higher protein concentrations to initiate aggregation, while aggregation kinetics slowed by nearly three orders of magnitude. Temperature-dependent measurements yielded an apparent activation energy of 36.7 kJ{middle dot}mol-1, consistent with aggregation driven by Ca2+-induced conformational activation rather than denaturation. NMR chemical shift perturbations localized the effects of NTD removal to the EF-hand Ca2+-binding loops and adjacent D-helix. Apo-state MD simulations revealed transient intra- and intermolecular NTD-SCBD contacts that explain some perturbations and support direct and allosteric regulation. Ca2+-bound simulations further showed that SCBD departs more readily from the crystallographic active conformation than full-length Sorcin, indicating that the NTD stabilizes the active state through dynamic contacts. Together, these findings establish the NTD as a critical regulator that enhances Ca2+ responsiveness by shifting Sorcins conformational equilibrium toward the active state and suggest that flexible N-terminal extensions regulate signaling within the penta-EF-hand protein family.

biophysics↗

bioRxiv2026

IKKβ as a putative non-covalent and quinone-mediated covalent target of 4-methylcatechol in RANKL/NF-κB signaling: a combined computational and experimental analysis

Excessive osteoclast activity contributes to pathological bone loss in osteoporosis, rheumatoid arthritis, and osteolytic malignancies. The effects of small catechol derivatives on receptor activator of nuclear factor-{kappa}B ligand (RANKL)-induced osteoclastogenesis remain poorly understood. This study investigated the effects of 4-methylcatechol (4-MC) on RANKL-induced NF-{kappa}B activation and osteoclast differentiation. 4-MC reduced RANKL-induced NF-{kappa}B luciferase activity in HEK-293T/RANK cells. 4-MC also suppressed RANKL-induced TRAP activity in RAW264.7 cells in a concentration-dependent manner and reduced the number of TRAP-positive multinucleated osteoclasts, without affecting cell viability. Molecular docking predicted non-covalent binding of 4-MC within the ATP-binding hinge region of IKK{beta} (PDB: 4KIK), forming a close polar contact with Glu97, predicted hydrogen bonds with Cys99, and a hydrophobic contact with Ile165, within the pocket occupied by the co-crystallized inhibitor K252a. Covalent docking predicted that the oxidized quinone form of 4-MC engages Cys179 in the IKK{beta} activation loop. Quantum chemical calculations confirmed a markedly higher electrophilicity index for the oxidized quinone than for the parent catechol, supporting this mechanism. In silico ADMET profiling indicated favorable drug-likeness and safety. These findings identify IKK{beta} as a plausible molecular target of 4-MC through both non-covalent and covalent mechanisms. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/741661v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1d9f82eorg.highwire.dtl.DTLVardef@134a24borg.highwire.dtl.DTLVardef@8aeb91org.highwire.dtl.DTLVardef@6fb85d_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2026

Genetic background diversifies mutational paths but converges on membrane depolarization for bleomycin resistance in yeast

Drug resistance evolution is shaped by both selective agent and genetic background in which resistant mutations arise, but most experimental evolution studies rely on a single genetic background. Here we performed parallel experimental evolution across six genetically diverged Saccharomyces cerevisiae strains, propagating 48 replicate populations for [~]225 generations under the selection of DNA-damaging chemotherapeutic bleomycin. Over 70% of the evolved populations acquired resistance with the magnitude and repeatability of adaptation varying markedly across genetic backgrounds. Whole-genome sequencing showed that bleomycin elevated single-nucleotide and insertion-deletion mutation rates, and induced background-specific aneuploidies and structural variants, including Ty-element-mediated translocations and tandem duplications. Notably, mutation rates varied up to 10-fold among backgrounds, with the most commonly-used lab strain exhibiting the highest mutagenic susceptibility, underscoring that evolutionary insights derived from single-background models may not universally generalize across a species. Despite this background-dependent genomic heterogeneity, we identified recurrent loss-of-function mutations in PMA1 and PTK2 that arose independently across nearly all genetic backgrounds and conferred strong resistance. Functional and biophysical assays revealed that these mutations impair Pma1p H+-ATPase activity and depolarize the plasma membrane, thereby limiting the cellular uptake of the positively charged drug. Our findings demonstrate that while the mutational routes to drug resistance are heavily shaped by genetic background, a convergent, background-independent mechanism centered on membrane potential regulation can dominate phenotypic adaptation. This work underscores the necessity of integrating genetic diversity into experimental evolution paradigm and nominates plasma membrane potential as an important lever for modulating chemotherapeutic efficacy.

genetics↗

bioRxiv2026

Catch-bond engineering surpasses high-affinity maturation for T cell receptor therapies against solid tumors

The sensitivity of T cell receptors (TCRs) has traditionally been attributed to their affinity, a principle that has long underpinned both T cell biology and TCR engineering. Currently, high-affinity maturation remains the predominant strategy employed to enhance TCR sensitivity; however, this approach has been associated with severe off-target toxicities in clinical settings. In this study, using the only FDA-approved TCR-T therapy as a clinical reference, we demonstrate that force-induced catch bonds, rather than static affinity, primarily determine TCR sensitivity and facilitate the development of more effective TCR engineering strategies. Among polar and charged amino acids, histidine was determined to be the most effective residue for pinpointing engineering hotspots. TCRs engineered to form catch bonds exhibited superior performance compared to the FDA-approved TCR subjected to high-affinity maturation, improving the efficacy of TCR-T cell therapies against solid tumors without eliciting off-target toxicity or alloreactivity. Mechanistically, a correlation was observed between TCR sensitivity and the strength of catch bonds, whereas no such relationship was found with affinity. The duration of T cell-tumor cell interactions, immunological synapse formation, and the intensity of subsequent signaling are governed by catch bonds rather than by affinity. Structural and computational investigations have demonstrated that force-induced reconfiguration of the ligand-receptor interface, along with the formation of a novel hydrogen bond network, augments the specificity of interactions between the TCR and antigenic peptides. Furthermore, bispecific T cell engagers utilizing the TCR scaffold also formed catch bonds with tumor antigens, indicating intrinsic mechanosensory properties of the extracellular domains of TCRs and suggesting a novel therapeutic modality based on catch-bond-engineered TCRs.

immunology↗

bioRxiv2026

Uncovering key genes related to potato tuber starch concentration from the tuber bulking, maturation stage to storage

Potato is the worlds fourth major staple crop, and tuber starch is a critical dietary energy source and a food processing raw material. In this study, hybrid progenies of the high-starch cultivar Huasheng No.7 and China main commercial varieties were used as materials, high-throughput sequencing was performed on tuber samples at tuber bulking, maturation and storage stages. Combined with bioinformatics analysis, parent-progeny resequencing, SNP screening, the regulatory mechanism of tuber starch metabolism was explored. The results showed significant stage-specific transcriptional reprogramming in potato tubers: few differentially expressed genes (DEGs) were identified at tuber bulking and maturation stages, but massive transcriptional changes occurred during storage. XET family genes regulated cell wall remodeling and carbon translocation across all stages, and WRKY transcription factors specifically controlled starch homeostasis in stored tubers. The DEGs at different stage were induced by the SNPs from their parent, and the allele from the high-starch parent, Huasheng 7 may contribute the high-starch allele to the offsprings. One key gene, Soltu.DM.02G019910, which encoded {beta}-glucosidase, have a negatively relationship with tuber starch concentration. The low-starch potato breeding tubers shown higher significantly {beta}-glucosidase activity than high-starch breeding population at maturation stage. This study clarifies the molecular regulatory network of potato tuber starch metabolism and screens core regulatory genes from the tuber bulking stage to tuber storage, thereby providing theoretical support and genetic resources for molecular breeding of high-starch and storage-resistant potato varieties.

plant biology↗

bioRxiv2026

Effects of different shading levels on growth, physiology and leaf surface micromorphology of Sorbus sibirica "Dong Hong" seedlings

BackgroundSorbus sibirica Dong Hong is a promising ornamental cultivar, but its optimal light conditions remain unclear. AimsThis study evaluated the effects of shading on seedling growth, physiology, root morphology, and leaf surface micromorphology. MethodsOne-year-old seedlings were grown under full sunlight (CK) and 30%, 50%, or 70% shade for 100 days. Growth, biomass, root traits, chlorophyll, invertase, soluble protein, stomatal characteristics, and epicuticular wax morphology were determined. ResultsShading significantly affected all measured traits. The 30% shade treatment produced the greatest seedling height, which increased by 118.18% compared with CK, and the highest chlorophyll content, which increased by 120.21%. Soluble protein content was slightly increased, whereas invertase activity decreased under moderate shading. Although total biomass decreased by 18.63%, root development remained relatively stable under 30% shade, with slight increases in total root length and average root diameter. Stomatal density was highest under this treatment, and the epicuticular wax structure remained relatively regular. In contrast, 70% shade markedly inhibited biomass accumulation and root development. ConclusionsModerate shading, particularly 30%, provided the most favorable light environment for Sorbus sibirica Dong Hong seedlings and is recommended for summer nursery cultivation in Northeast China.

plant biology↗

bioRxiv2026

CNVeil resolves haplotype-specific copy number and uncovers subclonal architecture hidden from total copy number profiling in single-cell cancergenomes

Single-cell DNA sequencing (scDNA-seq) resolves copy number variation (CNV) at single-cell resolution, revealing tumor heterogeneity and subclonal structure. Most existing methods, however, infer only total copy number. Haplotype-resolved copy number, which captures allelic imbalance and clonal evolution, remains far less developed, largely because low coverage, allelic dropout, and technical noise in scDNA-seq make phased allelic inference substantially harder than total copy number estimation. We present CNVeil, a haplotype-aware framework that infers total, allele-specific, and chromosome-scale haplotype-resolved copy number from scDNA-seq data. CNVeil first builds robust total copy number profiles through highly variable bin selection, hierarchical clustering, subclone-aware ploidy estimation, and cross-cell consensus segmentation. Using this profile as a stable scaffold, it infers allele-specific copy number with an expectation-maximization algorithm applied to heterozygous SNP allele counts, then reconstructs haplotype-specific copy number by enforcing coherent haplotype orientation across adjacent segments via dynamic programming. We benchmarked CNVeil against 12 state-of-the-art methods, including eight total copy number callers, two allele-specific callers, and two haplotype-resolved callers, across 20 simulated and real datasets spanning six experimental settings, including high-multiplexed single-nucleus sequencing, Acoustic Cell Tagmentation (ACT), and 10x Chromium. This constitutes the largest comparative evaluation of single-cell copy number inference methods to date. CNVeil consistently outperformed existing tools in segmentation accuracy, ploidy inference, subclone identification, and allele-specific copy number estimation. In a breast cancer multi-omics (wellDR-seq) cohort, CNVeil uncovered haplotype-specific subclonal diversification invisible to total copy number analysis alone and linked allele-specific copy number states to transcriptional variation. By transforming sparse single-cell allelic signals into chromosome-scale haplotype-resolved profiles, CNVeil closes a major methodological gap and provides a scalable framework for studying tumor evolution and functional genomic heterogeneity at single-cell resolution.

bioinformatics↗

bioRxiv2026

Diagnostic Performance of Fluorescence-Based Rapid On-Site Specimen Evaluation for Helicobacter pylori Antimicrobial Susceptibility Testing Before and After Algorithm Optimization: A Two-Round Comparative Study

ObjectiveThis study was designed to evaluate the diagnostic performance of fluorescence-based rapid on-site specimen evaluation (F-ROSE) for antimicrobial susceptibility testing of Helicobacter pylori (H. pylori) and to compare test performance before and after iterative optimization of fluorescence image acquisition and of the artificial intelligence (AI) recognition algorithm, using two consecutive rounds of paired testing, so as to provide trial data supporting clinical implementation of rapid susceptibility testing. MethodsForty patients with a urea breath test (UBT) or rapid urease test (RUT) positive for H. pylori within the preceding 2 weeks, together with strongly positive endoscopic findings, were prospectively enrolled. In order of enrollment they were allocated to two rounds in which F-ROSE was compared with culture-based susceptibility testing. Twenty patients were tested in round 1 on an automated fluorescence immunoassay scanner running the original algorithm; a further 20 were tested in round 2 with the optimized algorithm. With in vitro culture and E-test as the reference standard, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of F-ROSE were calculated for each round for amoxicillin, levofloxacin hydrochloride and clarithromycin, and agreement between methods was assessed with Cohens kappa. Receiver operating characteristic (ROC) analysis was used to identify the optimal cutoff of the fluorescence residual rate (FRR) and to determine how far threshold adjustment improved performance. ResultsCulture succeeded in 15 patients in round 1 and in 14 patients in round 2, leaving 29 evaluable samples. Overall diagnostic performance after algorithm optimization was clearly better than before. Pooled across the three antibiotics, sensitivity was 92.9%, specificity 71.0% and accuracy 77.8% before optimization; after optimization sensitivity rose to 100.0%, specificity increased slightly to 74.2% and accuracy to 81.0%. NPV reached 100.0% for all three agents after optimization, and no resistant isolate was missed. The gain was largest for clarithromycin, for which sensitivity rose from 85.7% to 100.0%, accuracy from 66.7% to 78.6% and kappa from 0.348 to 0.571, indicating a clear improvement in agreement between the two methods. ROC analysis showed that drug-specific optimal cutoffs derived from the Youden index improved specificity appreciably compared with the uniform 50% FRR threshold currently applied, the gain being most evident for levofloxacin hydrochloride. ConclusionsWith sharper fluorescence images and an improved AI recognition algorithm, F-ROSE performs better in H. pylori susceptibility testing, and the reduction in missed resistant strains is particularly noteworthy; the assay is therefore a plausible option for rapid susceptibility screening in clinical practice. Multicenter studies with larger samples are still required to confirm the stability of the technique.

microbiology↗