Biology subjects

Liu, X.

Publications and source records attributed to Liu, X..

At least 73 records · Page 4Linked to original sources

bioRxiv2026

Reconstruction of the gastropod ancestral karyotype reveals lineage-specific chromosomal evolution and adaptations during habitat transitions

Gastropoda is the most diverse class of molluscs, with spectacular adaptive radiations across shallow to deep marine, freshwater, and terrestrial environments, yet the role of chromosomal restructuring behind these habitat transitions remain largely unexplored. This is in part due to most chromosomal genomes available are in the subclasses Caenogastropoda and Heterobranchia, with few representatives from the other four subclasses. Here, we assembled chromosome-level genomes for five representatives from those four subclasses: Vittina coromandeliana (Neritimorpha), Shinkailepas gigas (Neritimorpha), Turbo cornutus (Vetigastropoda), Neomphalus fretterae (Neomphaliones), and Cellana toreuma (Patellogastropoda), and reconstructed the ancestral gastropod karyotype consisting of 20 linkage groups (GLGs) as well as the ancestral karyotype of each subclass. Integrating macrosynteny and phylogenomic analyses across all six subclasses, we show that different lineages have adopted fundamentally different chromosomal strategies to colonize novel habitats: freshwater caenogastropods genomes are characterized by extensive end-to-end fusions, terrestrial heterobranchs by whole genome duplications, freshwater neritimorphs by lineage specific fissions plus fusion with mixing, and deep sea hydrothermal vent vetigastropods by widespread chromosomal restructuring, whereas hydrothermal vent neomphaliones and caenogastropods retain remarkably conserved ancestral karyotypes. Rearrangement breakpoints preferentially reside within topologically associating domains (TADs) rather than at boundaries, preserving higher order chromatin architecture. Functionally, convergent enrichment of the mucin type O glycan biosynthesis pathway in both freshwater and hydrothermal vent lineages is accompanied by lineage specific modifications, enhanced immune modulation in freshwater versus reinforced physicochemical shielding in the deep sea, reflecting divergent adaptive applications of this pathway. Our findings demonstrate that habitat transitions in gastropods are facilitated not by a uniform genomic mechanism, but by a diverse repertoire of lineage specific chromosomal strategies, highlighting the extraordinary genomic flexibility that underpins the remarkable gastropod diversity seen today.

evolutionary biology↗

bioRxiv2026

Structural basis of endogenous inverse agonism and subtype selectivity at melanocortin receptors

Endogenous inverse agonists suppress constitutive G protein-coupled receptor (GPCR) signaling, but their mechanisms remain poorly understood. Here we report cryo-EM structures of melanocortin-1 receptor (MC1R) bound to its endogenous inverse agonist, agouti signaling protein (ASIP) and melanocortin-4 receptor (MC4R) bound to agouti-related protein (AgRP). Together with previously reported structures and those we determined using extracellular nanobodies developed here, these data delineate a conformational continuum underlying receptor activation and silencing. Both inverse agonists occlude the orthosteric pocket as molecular corks and drive a shared TM3-centered extracellular remodeling. Against this common mechanism, subtype selectivity is encoded not by the conserved orthosteric pocket but by the divergent extracellular receptor surface, engaged through an ASIP C-terminal loop-dependent clasp. These findings establish a mechanistic framework for endogenous inverse agonism and identify the receptor periphery as a tractable target for subtype-selective modulation.

biophysics↗

bioRxiv2026

Cortical Hierarchy Dynamically Organizes Large-Scale Neural Propagation

Flexible behaviour depends on the continuous coordination of sensory-driven and internally guided processing, yet whether the cortical hierarchy spanning lower-order sensory to higher-order association systems dynamically organizes large-scale cortical propagation over time remains unclear. Here we combined source-resolved magnetoencephalography with Riemannian cortical-flow modelling to derive hierarchy consistency, a moment-to-moment measure of the alignment between cortical propagation and the principal sensory-to-association functional gradient. We found that large-scale cortical propagation was dynamically organized by the cortical hierarchy. Hierarchy consistency exhibited a reproducible low-frequency periodic component that defined a characteristic timescale for the continuous updating of propagation direction. This dynamic organization was coordinated by a distributed cortical switchboard spanning the default-mode, salience, control and limbic systems, and was constrained by structural connectivity and network-control architecture. It flexibly adapted to behavioural demands, with hierarchy consistency increasing across both sensorimotor and working-memory states, while its characteristic periodicity shifted in a task-dependent manner. Moreover, hierarchy-related propagation dynamics were systematically reorganized across ageing and associated with higher-order cognitive function. Together, these findings establish the cortical hierarchy as a dynamic organizing principle that continuously shapes the direction and temporal evolution of large-scale cortical propagation to support adaptive behaviour.

neuroscience↗

bioRxiv2026

Sobetirome, a thyroid hormone receptor beta agonist, is a potential therapeutic agent for pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease with limited treatment options. Our group previously identified the antifibrotic potential of thyroid hormone, triiodothyronine (T3); however, clinical translation of thyroid hormone therapy is limited by its systemic adverse effects. In this study, we investigate whether sobetirome, a selective and well tolerated thyroid hormone receptor beta (THRB) agonist, offers antifibrotic benefits of thyroid hormone while minimizing systemic toxicity. Our study reveals that sobetirome, administered via intraperitoneal or inhalational routes, effectively mitigates bleomycin-induced pulmonary fibrosis in mice, with no evidence of toxicity. We identified that sobetirome restores mitochondrial homeostasis via activating the THRB-PPARGC1a axis. This protects alveolar type II epithelial cells from injury-induced apoptosis while selectively inducing apoptosis and metabolic reprogramming in apoptosis resistant IPF fibroblasts. Cell-specific deletion of Ppargc1a in either alveolar epithelial cells or fibroblasts abolishes sobetirome-mediated protection, establishing PPARGC1a as an essential mediator of therapeutic response. Importantly, sobetirome reverses fibrosis-associated transcriptional programs in human IPF lung tissue, reducing expression of key fibrosis-associated genes, including collagen I alpha 1 (COL1A1), collagen III alpha 1 (COL3A1), periostin (POSTN), cathepsin K (CTSK), and Chitinase 3 Like 1 (CHI3L1), while promoting extracellular matrix remodeling, epithelial restoration, and tissue homeostasis. Collectively, our findings identify THRB activation as a novel metabolic strategy for reversing pulmonary fibrosis. Across complementary in vitro, in vivo, and human ex vivo models, sobetirome restores mitochondrial function, modulates apoptotic pathways in pathogenic cells, and promotes fibrosis resolution, highlighting its potential as a lung-targeted therapeutic approach for IPF and other fibrotic lung diseases.

systems biology↗

bioRxiv2026

Temozolomide Induces Aberrant RNA Alkylation and Widespread Translational Repression

Temozolomide (TMZ) is a frontline alkylating chemotherapy, yet its direct impact on RNA modification and global translation dynamics remains poorly understood. Here, we demonstrate that TMZ induces pervasive RNA alkylation causing severe translational impairment. TMZ directly deposits aberrant methyl groups onto single-stranded mRNA in vitro, creating physical lesions that lower translational efficiency. In glioblastoma cells, acute TMZ exposure triggers a rapid, widespread accumulation of m7G on cellular RNAs, leading to the significant attenuation of global protein synthesis. Nanopore direct RNA sequencing identified distinct guanine-specific error signatures and sequence context preferences associated with TMZ-induced damage. Using a quantitative yeast spike-in ribosome profiling strategy, we mapped this translational repression at transcript-level, revealing a global downregulation of translational efficiency. This widespread repression disproportionately targets highly interconnected networks essential for cellular proliferation, specifically chromosome organization. We show that the severity of this translational repression is driven by a transcript's coding guanine density, stability and translation initiation speed. Together, our findings suggest that TMZ-induced alkylation targets stable, highly translated, guanine-rich transcripts. This establishes aberrant RNA methylation and subsequent translational arrest as a potential mechanism of temozolomide cytotoxicity.

biochemistry↗

bioRxiv2026

A direct, MFRN-independent Fe(II) transfer pathway at mitochondria-lysosome contacts

Mitochondrial iron homeostasis is fundamental to respiration and redox balance, and its dysregulation is implicated in neurodegeneration, cardiomyopathy, and metabolic diseases. Although lysosomes harbor the major cellular iron reservoir, the prevailing model holds that mitochondria acquire Fe(II) directly from the cytosolic labile iron pool (LIP) via MFRN transporters. Here, we challenge the canonical view by identifying a direct, MFRN-independent Fe(II) transfer pathway at mitochondria-lysosome contacts (MLCs). This VPS39/TOMM22/SFXN1-coordinated pathway enables lysosome-to-mitochondria Fe(II) flux bypassing the cytosolic LIP. Using live-cell structured illumination microscopy (SIM), we visualize direct Fe(II) transfer specifically occurring at MLCs. Multiple lines of evidence confirm that VPS39 and TOMM22 stabilize MLCs, while SFXN1 serves as the core effector protein for this MLC-dependent Fe(II) transport. Notably, SFXN1 knockdown markedly reduces mitochondrial Fe(II) levels independent of its established serine transport function. This pathway reveals a major route for mitochondrial Fe(II) acquisition to support redox homeostasis.

cell biology↗

bioRxiv2026

β4-integrins safeguard nuclear mechanics to suppress prostate cancer progression

Prostate cancer (PCa) progression is accompanied by profound alterations in cell-extracellular matrix (ECM) adhesion, nuclear architecture and mechanical adaptability, yet the molecular mechanisms linking these processes remain poorly understood. Hemidesmosomes (HDs), formed by 6{beta}4-integrins, anchor epithelial cells to the basement membrane and couple extracellular forces to the intermediate filament (IF) cytoskeleton. Here, we identify a previously unrecognized tumor-suppressive function of {beta}4-integrins in preserving nuclear integrity in prostate epithelial cells. Loss of {beta}4-integrins disrupted the cytokeratin-5 network and its coupling to the nucleus, leading to nuclear softening, lamin remodeling, reduced heterochromatin content and enhanced confined migration. Unexpectedly, proximity-labeling proteomics revealed that {beta}4-integrins engage nuclear pore complex (NPC) components in an 6-independent manner, particularly upon HD disassembly. Selected interactions were validated using proximity ligation and co-immunoprecipitation assays. {beta}4-integrin loss was associated with enlarged nuclear pores and aberrant nucleocytoplasmic transport, including nuclear accumulation of YAP1. Consistent with these findings, reduced {beta}4-integrin expression in a large PCa tissue cohort correlated with altered nuclear morphology, adverse clinicopathological features, metastatic progression, and poor patient survival. Collectively, our study establishes {beta}4-integrins as a critical molecular link between cell-ECM adhesion, nuclear mechanics and genome integrity.

cancer biology↗

bioRxiv2026

Dissecting the TMEM132A-EGFR Dependency to Unlock Translational Therapeutic Opportunities for Pan-Solid Tumor

Solid tumors remain refractory to conventional treatments, yet cell surface proteins, by virtue of their extracellular accessibility and critical roles in tumor signaling, represent an attractive class of targets for precision-targeted therapy. Here, we report that TMEM132A is an essential and previously unrecognized pan-cancer target. TMEM132A interacts directly with EGFR and stabilizes its expression, thereby tethering EGFR at the plasma membrane and sustaining constitutive activation of lipid synthesis. Mechanistically, the TMEM132A-EGFR axis promotes lipogenesis by facilitating SREBP nuclear translocation, which in turn upregulates ACLY and ACSS2 expression to drive acetyl-CoA production and downstream lipid biosynthesis, ultimately disrupting lipid droplet homeostasis. To therapeutically target this axis, we developed a nanobody, LFNanoT132A#3, which effectively blocks the TMEM132A-EGFR interaction, abrogates downstream signaling activation, and potently inhibits proliferation across multiple solid tumor types. Notably, LFNanoT132A also exerts robust antitumor activity against H1975 xenografts, a model resistant to first- and second- generation EGFR inhibitors, underscoring its potential to overcome conventional drug resistance. Our findings establish TMEM132A#3 as a critical node in membrane-tethered oncogenic signaling and metabolic rewiring, and position LFNanoT132A#3 as a promising therapeutic candidate for precision cancer therapy.

cancer biology↗

bioRxiv2026

Decoding the Transcriptome Dark Matter: Construction of Single-Cell Whole-Transcriptome Regulatory Atlas by dropTotal

To profile unknown ncRNAs-"dark matter" in single cells, we developed dropTotal, a high-throughput droplet-based total RNA-seq method that uses dU-modified GAT primer with temperature-ramp hybridization and droplet merge barcoding to co-detect coding and non-coding transcripts with record sensitivity (>13,500 genes/cell, including >2,000 lncRNAs and >500 sncRNAs), compatible with fresh, frozen, fixed, and FFPE tissues. Applied to ~75,000 human glioma nuclei, it captured 60,313 genes (18,681 lncRNA, 19,859 mRNAs and 6,753 sncRNAs), enabling ncRNA-driven regulatory landscape construction. In oligodendroglioma, module analysis identified recurrence-associated ncRNA-centered modules linked to therapy resistance and invasion; in glioblastoma, six cellular states showed hundreds of state-specific unannotated ncRNAs with divergent functions, from MIR222HG-mediated immune modulation to SCIRT-driven hypoxia adaptation. Alternative splicing analysis identified 428 state-specific junction markers and mapped cell-state-specific alternative splicing regulation. dropTotal offers broad application for decoding the underlying ncRNA biology and single-cell whole transcriptome regulatory mechanisms in cellular identity and disease progression.

genomics↗

bioRxiv2026

The Genomic Landscape of Post-Black Death Epidemics in Northern Europe and the Caucasus

One of the most devastating events in human history, the Black Death (c.1347-1353), marked the beginning of the Second Plague Pandemic. After initially receding, plague returned in intermittent outbreaks throughout Europe, beginning with the pestis secunda. Despite its significance, the post-Black Death epidemiology of Yersinia pestis remains poorly understood. Here, we report 23 new Y. pestis genomes recovered from Second Plague Pandemic contexts across Scandinavia, the Netherlands, Iceland, and Armenia, spanning approximately 270 years. Applying a reproducible mutation-filtering pipeline to assess genetic diversity, we report both Black Death and post-Black Death lineages and document multiple waves of plague at individual cemetery sites. We identify four pestis secunda genomes, including three from Armenia, supporting the eastward dissemination of this lineage prior to its disappearance from Europe. We also resolve a previously under-characterised Branch 1A sub-lineage of Y. pestis and provide the first genomic evidence of plague in Iceland, resolving longstanding uncertainty over its presence on the island. Finally, by calling variants against a reconstructed ancestral reference, we identify clade-defining mutations, including nonsynonymous changes in metabolic and biofilm-related genes with predicted structural effects that may have contributed to shaping the epidemiological dynamics of the Second Plague Pandemic.

genetics↗

bioRxiv2026

Active control modulates memory-related activity during encoding and encoding-retrieval similarity

Self-directed learning allows learners to actively control their learning experience and has been shown to enhance memory compared with matched yoked learning. However, it remains unclear when and how active control modulates memory-related neural activity during learning and retrieval. We recorded electroencephalography (EEG) while participants encoded objects under active and yoked learning conditions and again during a delayed recognition test approximately 24 h later. We examined event-related potential (ERP) activity across earlier processing windows, including pre-stimulus slow potentials and early N2 activity, and later processing windows, including P300, late slow-wave, and post-stimulus slow-potential activity. We also examined encoding-retrieval similarity (ERS) between neural patterns during encoding and retrieval. Behaviorally, active control improved delayed recognition, with the advantage selectively expressed in detailed recognition. In the ERP analyses, earlier processing windows showed memory-related effects, with pre-stimulus slow-potential and N2 activity differentiating subsequently remembered from forgotten items, but were not modulated by active control. By contrast, active control modulated later memory-related ERP activity, with remembered-forgotten differences expressed during late stimulus-related and immediate post-stimulus processing only in the active condition. ERS showed a similar active-control modulation: memory-related encoding-retrieval pattern similarity was evident under active learning, but not under yoked learning, with this effect involving relatively late encoding and retrieval windows. Together, these findings support a constructive-processing account, suggesting that memory formation under active control depends more strongly on rich, detailed encoding representations that can be reinstated during retrieval.

neuroscience↗

bioRxiv2026

Multiple Particle Tracking via Velocity Filtering (MPT-vVF): a velocity filtering framework for robust tracking moving organelles in living cells

Living cells are highly dynamic and densely crowded environments in which organelles such as vesicles undergo continuous motion that is essential for cellular processes. Therefore, accurate tracking of individual organelles is crucial for understanding intercellular dynamics and functions. However, precise tracking of individual organelles in living cells remains challenging due to high organelle densities, frequent particle overlap, and the coexistence of stationary and motile organelles. In particular, stationary organelles can obscure the trajectories of moving organelles, leading to tracking errors and fragmented tracks. To overcome these challenges, we developed Multiple Particle Tracking via Velocity Filtering (MPT-vVF), an unbiased, semi-automated tracking framework that incorporates a mathematically derived velocity-filtering algorithm to selectively identify and track moving organelles with high accuracy in crowded intracellular environments. MPT-vVF integrates denoising, background subtraction, and a velocity-matching detection step that discriminates true particle motion from noise based on spatiotemporal continuity, followed by robust trajectory linking. We demonstrate that MPT-vVF can accurately resolve nanometer-scale displacements of immobilized beads, highlighting its high tracking precision. We also validate the robustness of MPT-vVF by quantifying the transport of brain-derived neurotrophic factor (BDNF)-mRFP-containing vesicles in living hippocampal neurons. Furthermore, MPT-vVF reveals that exposure to 50-nm nanoplastics impairs vesicular transport, reducing both travel length and speed of BDNF-containing vesicles in living neurons. These findings establish MPT-vVF as a powerful method for quantitative analysis of intracellular organelles in crowded living cells and suggest its broad application to biophysics, cell biology, and soft matter research.

biophysics↗

bioRxiv2026

Syndecan-4 exerts canonical heparan sulfate-dependent and noncanonical heparan sulfate-independent functions that regulate Aβ amyloid homeostasis

Background Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPGs) are components of the amyloid deposits in Alzheimers disease (AD) and other amyloidoses. HS and HSPGs are canonically thought to facilitate amyloid deposition by accelerating the aggregation of amyloidogenic proteins and impairing their clearance in a HS-dependent manner. Methods Leveraging insights from large-scale proteomic data, we focused on Syndecan-4 (Sdc4), the most increased transmembrane HSPG in the AD brain and in the brain of A{beta} amyloid depositing mice. We used proximity ligation assays (PLA) to evaluate the association of Sdc4 with A{beta} in situ and assessed the impacts of the Sdc4 ectodomain on A{beta} aggregation in vitro. Overexpression studies in cells, hiPSC-derived neurons, and mouse organotypic brain slice cultures (OBSCs) coupled with structure-function studies were used to investigate impacts on A{beta} production and APP processing. Finally, effects of overexpression of Sdc4 in vivo in the CRND8 amyloid deposition model were evaluated. Results Consistent with canonical roles, PLA demonstrated a spatial association of Sdc4 with amyloid deposits, and in vitro, the Sdc4 ectodomain accelerated A{beta} fibril formation in a HS-dependent manner. Unexpectedly, Sdc4 overexpression reduced A{beta} production in CHO cells, hiPSC-derived neurons, and OBSCs. These effects were accompanied by dramatic decreases in the levels of sAPP and C83 and increased immature APP in the cell. Sdc4 promoted altered APP localization into detergent resistant membrane domains and increased APP association with ATG5+/LC3+/Cathepsin D+ vesicles. Structure-function studies revealed that the transmembrane region mediates these effects in a glycosaminoglycan-independent manner. Sdc4 overexpression in the brain of APP mice significantly reduced amyloid deposition at an early age. Conclusions Sdc4 exerts paradoxical and mechanistically distinct effects that could impact AD pathogenesis differentially, potentially promoting A{beta} fibrillization extracellularly while suppressing APP processing and A{beta} production. Such data challenge the prevailing view that increased levels of HSPGs in AD are always pro-amyloidogenic and identify Sdc4 as a previously unrecognized regulator of amyloid homeostasis in AD.

neuroscience↗

bioRxiv2026

An inflammation-associated five-gene expression signature stratifies survival and immune states in lung adenocarcinoma: an integrative public-cohort analysis

Background: Inflammation and the tumor immune microenvironment contribute to lung adenocarcinoma (LUAD) progression, but the relationship among inflammation-linked transcriptional heterogeneity, patient survival, and immune-state variation remains incompletely defined. Objective: We aimed to identify inflammation-associated LUAD subtypes, derive a parsimonious survival-stratification signature, and characterize its immune and pathway context across public transcriptomic cohorts. Methods: Expression profiles and clinical data were obtained from TCGA-LUAD, GTEx normal lung, and GEO datasets GSE11969, GSE30219, GSE31210, and GSE40791. A curated set of 596 inflammation-related genes was used for consensus clustering. Differential-expression analysis, functional enrichment, univariate Cox regression, and LASSO-Cox modeling were integrated to construct a gene-expression risk score. The prognostic dataset comprised 730 cases and was randomly divided into training (n=502) and internal-validation (n=228) sets; 85 GSE30219 cases formed an external-validation cohort. Immune-cell enrichment, gene set enrichment analysis (GSEA), gene set variation analysis (GSVA), and pan-cancer analyses were used for biological contextualization. Results: The LUAD-versus-control comparison identified 1,305 differentially expressed genes, including 498 upregulated and 807 downregulated genes. Consensus clustering resolved two inflammation-associated subtypes and 67 subtype-associated genes, of which 64 were higher and 3 were lower in Cluster 1 relative to Cluster 2. Thirty-three genes overlapped between the tumor-control and subtype contrasts. LASSO-Cox regression selected CHRDL1, FDCSP, CXCL13, CYP4B1, and S100P. The 1-, 3-, and 5-year areas under the time-dependent receiver operating characteristic curve were 0.6625, 0.6581, and 0.6658 in the training set; 0.7422, 0.6537, and 0.6761 in internal validation; and 0.6560, 0.6387, and 0.6753 in external validation. Risk groups differed across multiple T-cell, B-cell, natural-killer-cell, myeloid, dendritic-cell, macrophage, and granulocyte signatures. Positive GSEA signals included cell cycle (normalized enrichment score [NES]=2.67; adjusted P=1.42 x 10-), DNA replication (NES=2.52; adjusted P=2.52 x 10-), and mismatch repair (NES=2.20; adjusted P=1.77 x 10-). Conclusions: The five-gene expression score separated LUAD survival groups and captured coordinated proliferative and immune transcriptional states. Its moderate discrimination supports further biological and clinical validation rather than immediate clinical application.

bioinformatics↗

bioRxiv2026

Human NLRC4 can act as a direct sensor for cytosolic flagellin

Innate immune cells sense pathogenic bacteria like Legionella pneumophila through patterns such as the protein flagellin, a critical component of the bacterial motility apparatus. Recognition of cytosolic flagellin in mouse immune cells is well understood and mediated by the receptors, neuronal apoptosis inhibitory protein (Naip) 5 or Naip6, which activate the Nlrc4 inflammasome multi-protein complex for initiating cell death or interleukin-1 family cytokine release. However, the role of human NAIP as a cytosolic flagellin sensor remains controversial. Using a multipronged approach, we demonstrate that in a reconstituted cell system human NLRC4 engaged Legionella FlaA flagellin directly (i.e. without the need for hNAIP), whereas human NAIP did not interact with FlaA. Ectopic cytosolic FlaA expression also induced NLRC4 oligomerization, a prerequisite for inflammasome activation, in the absence of NAIP. Unexpectedly, the presence of NAIP diminished the binding of NLRC4 to flagellins and subsequent interleukin-1{beta} release. Interestingly, in resting THP-1 cells, NAIP stably interacted with NLRC4, and during infection or stimulation with FlaA pro-inflammatory responses in THP-1 cells were predominantly NLRC4-dependent. Our data highlight NLRC4 as a putative direct sensor of cytosolic flagellins in the human system and NAIP as a potential negative regulator of flagellin sensing.

immunology↗

bioRxiv2026

Structural-functional calibration corrects single-neuron identity errors in volumetric calcium imaging

Volumetric calcium imaging is increasingly used to capture larger neuronal populations at higher throughput, but high-speed axial sampling can compromise single-neuron identity. Here we identify cross-plane identity duplication as a structured error in volumetric imaging: anisotropic axial blurring and plane-wise functional segmentation can repeatedly detect the same neuron across adjacent planes, creating duplicate functional nodes that inflate neuronal counts and distort network phenotypes. We developed Comprehensive Label-Guided (CLG) volumetric imaging, a structural-functional calibration framework that uses nuclear labels as stable three-dimensional identity anchors for calcium signals. CLG combines nuclear labeling, deep-learning-based 3D segmentation, anatomical registration and identity-guided trace reassignment. In larval zebrafish whole-brain recordings, CLG resolved ~30,000 redundant detections and reduced estimated neuronal counts by 37-46%. In mouse visual cortex, CLG consolidated ~40% of putative duplicates and recovered over 2,000 active neurons missed by calcium-only analysis. Across baseline and perturbed conditions, calibration stabilized graph-derived measurements of hub organization, long-range correlations and network resilience. CLG therefore defines an anatomy-constrained identity-calibration layer for reliable single-neuron-resolved volumetric imaging.

neuroscience↗

bioRxiv2026

Scarless SARS-CoV-2 Genome Engineering and Variant Analysis

In addition to causing cold and flu-like symptoms, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can also cause chronic longer-term diseases. Antiviral drugs, especially used combinatorially, have the potential to reduce the severity of individual infections and prevent the development of chronic disease. One of the safest and most versatile reverse genetics systems for SARS-CoV-2 studies is a bacterial artificial chromosome (BAC)-based system harboring the WA1 strain full-length genome and attenuating deletions in the accessory open reading frame 3a and 7b proteins (ORF3a and ORF7b, respectively). Here, a scarless genome engineering technique called En Passant mutagenesis was used to change one amino acid in the viral main protease (Mpro P132) into the residue present in contemporary Omicron strains (H132), in order to more accurately study protease inhibitors and resistance mechanisms. This recombinant, attenuated viral system yields antiviral EC50 values for the active component of approved drugs including nirmatrelvir (Paxlovid) and ensitrelvir (Xocova) and, importantly, also enables a parallel assessment of drug efflux. For instance, the antiviral potency of nirmatrelvir improves 50-fold by inhibiting the P-Glycoprotein (P-Gp) transporter with ritonavir or tariquidar, whereas the potency of ensitrelvir is unaffected. This system also enables the safe isolation and characterization of viral variants with reduced sensitivity to drugs, as evidenced by Mpro M49L compromising the efficacy of ensitrelvir. Together, these systems combine to provide safe, reliable, and quantitative approaches for Mpro variant analysis and drug testing without the biosafety concerns of conducting these experiments using wildtype isolates.

microbiology↗

bioRxiv2026

The C. elegans endonuclease NUC-1 acts in engulfing cells to degrade the apoptotic cell DNA

During C. elegans embryonic development, cells undergoing programmed cell death are engulfed by neighboring cells and degraded inside phagosomes. Here we characterize a DNase responsible for the degradation of the chromatin DNA of apoptotic cells. In the past, NUC-1, a homolog of mammalian DNase II, which is only active at acidic pH, was claimed to act in apoptotic cell nuclei for chromatin DNA degradation by some researchers, yet proposed to act in engulfing cells by others. We found that NUC-1 acts exclusively in engulfing cells to degrade apoptotic cell DNA. In nuc-1 mutant embryos, apoptotic cell chromatin DNA remains undegraded. We observed that being engulfed is necessary for the apoptotic chromatin DNA to be degraded. In addition, specific expression of nuc-1 in the engulfing but not dying cells rescues the nuc-1 mutant phenotype. Furthermore, blocking the fusion between lysosomes and a phagosome in engulfing cells blocks apoptotic chromatin DNA degradation. NUC-1 was reported to be a lysosome-located enzyme. We not only confirmed this localization pattern, but also further determined that NUC-1 does not reside in the nuclei of either apoptotic or live cells. This, together with our finding that the nucleus of an apoptotic cell is not acidic, indicates that NUC-1 does not act in the apoptotic cell nucleus; rather, it acts in the engulfing cell phagosomal lumen to degrade apoptotic chromatin DNA. Our work clarified a long-standing controversy regarding the action of NUC-1 and advanced our knowledge of the mechanisms that drive the degradation of specific components of dying cells.

genetics↗