Biology subjects

Li, S.

Publications and source records attributed to Li, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

Behaviorally prioritized entity-relation structure captures human visual cortical representations of natural scenes

Understanding natural scenes requires identifying visible entities and representing how those entities are related. Recent studies have shown that artificial neural networks (ANNs), large language models (LLMs), and vision language models (VLMs) can predict visual cortical responses to natural images. However, the neural organization of relational scene meaning remains poorly understood, in part because these models typically encode scene content in global feature spaces that are difficult to decompose into separable entity and relation components. Here, we combined scene-graph annotations, behavioral measurements, and large-scale neural datasets to characterize structured relational representations during natural vision. We used RotatE, a knowledge-graph embedding model, to represent head-relation-tail triplets annotated for images from the 7T Natural Scenes Dataset. Triplet embeddings reliably captured cortical representational structure across the visual hierarchy. Behavioral judgments further revealed systematic differences in triplet accessibility associated with visual, relational, and graph properties. Prioritizing more behaviorally accessible triplets improved neural correspondence and explained unique variance beyond object co-occurrence, ANN image features, and LLM caption embeddings. Decomposing triplet representations into entity and relation components revealed partially dissociable cortical contributions, with lateral parietal cortex showing sensitivity to both. Triplet-based semantic information also remained spatially grounded: visual-field-specific triplet models preferentially predicted voxels with matching retinotopic preferences. Finally, cross-species comparison indicated that triplet-based semantic features were relatively more aligned with human high-level visual cortex than with macaque inferotemporal cortex. Together, these findings provide new insights into the representation of semantic relational information in the human visual cortex during natural scene perception.

neuroscience↗

bioRxiv2026

Stability of Antibody Repertoires in Astronauts During Long-Term Spaceflight

Long-duration spaceflight is known to disrupt several aspects of immune function yet its impact on the antiviral antibody repertoire remains unclear. We analyzed longitudinal plasma samples from 11 astronauts participating in a 6-month long spaceflight, including preflight, inflight, and postflight timepoints. Using a high-throughput serological method based on phage display of linear peptides from thousands of viruses we assessed the stability of antiviral antibodies over time. Across two phage libraries expressing different pathogen panels, we detected antibodies against Alphainfluenzavirus, Betainfluenzavirus, Enterovirus, and Orthopneumovirus consistently with the highest abundancy in all astronauts over all timepoints. Antibody levels against these infectious agents remained remarkably stable within individuals during and after spaceflight. In contrast, substantial heterogeneity was observed among astronauts, reflecting individualized immune histories and baseline antibody profiles. These results were confirmed by a different immunoassay using whole protein from the most targeted viruses. Together, these findings demonstrate that the antiviral antibody repertoire is largely resilient during prolonged spaceflight, highlighting preserved humoral memory despite potential dysregulation in cellular immune compartments.

immunology↗

bioRxiv2026

A bispecific antibody targeting a membrane-proximal ASGPR1 region promotes TNF-α recruitment and uptake

Abstract Recruiting an endocytic receptor through an antibody offers a route to redirect extracellular cytokines into cells. Here, we developed BiAb J31, an IgG like bispecific antibody linking TNF recognition to a membrane-proximal region of asialoglycoprotein receptor 1 (ASGPR1). J31 was isolated by immunization with an engineered ASGPR1 extracellular domain followed by screening against wild-type ASGPR1 and HepG2 cells. Its variable domains were combined with unmodified adalimumab variable regions using knobs into holes and CrossMab engineering, with L234A/L235A/P329G Fc substitutions. BiAb J31 retained HepG2 binding and engaged both antigens in bridging assays. Sequential binding experiments demonstrated recruitment of TNF to BiAb J31 treated cells. Live cell imaging showed uptake of fluorescent antibody-containing complexes and overlap with LysoTracker-positive compartments. In HepG2 cultures containing BiAb J31, TNF and a PE conjugated detection antibody, supernatant PE fluorescence fell by approximately 60% over 90 minutes. Truncation and synthetic-peptide assays localized J31 recognition to ASGPR1 residues 62~100, outside the carbohydrate-recognition domain, and AlphaFold 3 modeling proposed an Fv peptide interface. These findings identify a membrane-proximal ASGPR1 binding antibody that is compatible with bispecific engineering and supports cellular recruitment and uptake of TNF containing complexes. BiAb J31 provides a molecular starting point for antibody based cytokine redirecting approaches.

immunology↗

bioRxiv2026

Intermediate-Resolution Modeling of Dynamic DNAs and Their Phase Separation

DNA is a fundamental biomolecule in eukaryotic cells, playing central roles in processes ranging from genome organization and transcription to innate immune signaling. Recent studies have revealed that DNA can undergo protein-free phase separation in the presence of divalent cations, yet the underlying molecular mechanisms, including the interplay of base stacking, base pairing, electrostatics, and ion interactions, remain poorly understood. Here, we introduce an intermediate-resolution model for condensates of DNAs (iConDNA) that can capture key local and long-range structural features of dynamic DNAs and simulate their spontaneous phase transitions. By introducing explicit base stacking and pairing interactions, the iConDNA model not only reproduces major conformational properties of DNA homopolymers but also folds DNA hairpins and duplexes and captures their thermodynamic properties. With an effective model of explicit Mg2+, iConDNA successfully captures the temperature and magnesium concentration dependence of DNA properties. Together, these features enable iConDNA to qualitatively recapitulate homotypic DNA phase separation, providing a suitable tool to study DNA homotypic phase separation in biological and engineering applications.

biophysics↗

bioRxiv2026

Intrasegmental and propriospinal pre-phrenic interneuron circuitry in intact CNS and following cervical spinal cord injury

Cervical spinal cord injury (SCI) disrupts descending respiratory circuitry, resulting in debilitating and often persistent ventilatory deficits. Respiratory drive emerges within medulla from the rostral ventral respiratory group (rVRG), whose neurons project to C3-C6 phrenic motor neurons (PhMNs), which then innervate diaphragm, the primary muscle of inspiration. Though rVRG neurons make extensive monosynaptic connection with PhMNs, rVRG input to PhMNs can also be relayed through pre-phrenic interneurons (PP-INs) via polysynaptic pathways. However, the neuroanatomical connectivity between PP-INs and PhMNs remains incompletely understood. In both uninjured rats and the C2 hemisection (C2HS) model of cervical SCI, we performed tracing of PP-INs that were synaptically connected to PhMNs located in rostral (C3-C4) or caudal (C5-C6) portions of the phrenic nucleus by unilaterally injecting retrograde trans-synaptic tracer, pseudorabies (PRV), selectively into ventral or dorsal regions of hemi-diaphragm. We quantified numbers of PRV-labeled PP-INs individually at segments across cervical spinal cord, including separately in dorsal horn, intermediate gray, and ventral horn. We found that PP-INs are widespread throughout C1-C7 spinal cord in the uninjured condition. These PP-INs have a predominant intrasegmental connectivity pattern with PhMNs, though significant numbers of longer distance projecting propriospinal PP-INs also exist both rostral and caudal to the PhMN pool. Furthermore, while PP-INs connect both ipsilaterally and contralaterally with PhMNs, there is a strong bias to ipsilateral projection. C2HS induced major disconnection between PhMNs and ipsilesional propriospinal PP-INs located rostral to the SCI, but conversely induced limited plasticity in connectivity of intrasegmental PP-INs located within C3-C6 spinal cord. These findings greatly improve our knowledge about PP-IN circuitry and also provide important information to aid in developing approaches to target PP-IN plasticity for promoting spinal cord repair.

neuroscience↗

bioRxiv2026

Catalytic and inhibitory architecture of comammox ammonia monooxygenase

Complete ammonia oxidizers (comammox) are widespread nitrifiers that can dominate ammonia oxidation in diverse environments by efficiently converting ammonia to nitrate within a single cell, yet the molecular basis distinguishing their ammonia monooxygenase (AMO) from those of canonical bacterial AMO remains unresolved. Here we report cryo-electron microscopy (cryo-EM) structures of AMO from the comammox Nitrospira inopinata (NiAMO) in inhibitor-free and allylthiourea (ATU)-bound states at 2.47 and 2.68 angstrom, respectively. NiAMO displays distinctive auxiliary-subunit organization, copper-site configuration and hydrophobic-channel architecture. Integrative molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations support a methyl-plastoquinol (methyl-PQH2)-coupled, CuD-centric catalytic model, with CuC potentially facilitating quinone redox cycling. N. inopinata exhibited broad susceptibility to several known nitrification inhibitors, and ATU-bound NiAMO structure localized the inhibitor to the CuC-CuD region, accompanied by constriction of the hydrophobic channel, which is consistent with the competitive role of ATU demonstrated in recovery assays. Multi-omics analyses further revealed an energy-limited stress response to ATU, including induction of urea transport and utilization systems. Collectively, these findings define a methyl-PQ-linked catalytic and inhibitor-responsive architecture of comammox AMO and establish a mechanistic framework for lineage-aware management of nitrification in natural and engineered ecosystems.

biophysics↗

bioRxiv2026

Unifying physical and molecular coordinate systems across modalities in spatial biology

Establishing a unified physical and molecular coordinate system from fragmented multi-modal data is a longstanding challenge in biology. Here, we present MAPS, a modality-agnostic platform for spatial biology comprising (1) MAPS-alignment for ultrafast alignment of any modality, (2) MAPS-integration for both anchored and unanchored integration across orthogonal modalities for 3D multi-modal reconstruction, and (3) MAPS-Explorer for large-scale interactive 3D analysis. MAPS outperformed existing methods across extensive benchmarks on 34 datasets spanning 16 technology platforms and 6 modalities, while delineating fine-grained multi-modal tissue architectures across diverse biological systems in mouse and human. At cross-consortium scale, MAPS integrated 434 slices comprising 21 million cells from 18 atlases and 5 modalities to construct the most comprehensive 3D multi-modal mouse brain atlas. At individual laboratory scale, MAPS empowered routine 2D spatial assays to reconstruct continuous 3D multi-modal landscapes of human hepatocellular carcinoma, revealing the limitations of 2D spatial relationships and uncovering depth-dependent immune-state transitions.

bioinformatics↗

bioRxiv2026

Structural basis for catalytic and inhibitory divergence between archaeal and bacterial ammonia monooxygenases

Ammonia oxidation initiates nitrification and is closely linked to microbial N2O production. Ammonia monooxygenase (AMO) catalyzes the first and rate-limiting step of nitrification and is widespread across evolutionarily distinct ammonia-oxidizing archaea (AOA) and bacteria (AOB). The ocean is the largest biome for AOA and AOB, which have distinct ecological niches and markedly different sensitivities to nitrification inhibitors. However, the lack of archaeal AMO structures and inhibitor-bound AMO complexes has hindered mechanistic understanding of the architectural, catalytic, and inhibitory divergence between these two enzyme systems. Here, we report high-resolution cryo-electron microscopy (cryo-EM) structures of marine archaeal AMO captured in active and inactivated states within its native membrane environment, together with inhibitor-bound structures of estuarine bacterial AMO. Archaeal AMO forms an unexpected cup-shaped homotrimer composed of eight subunits per protomer and exhibits substantial architectural divergence from bacterial AMO. Integrated structural, biochemical, kinetic, and computational analyses reveal distinct periplasmic architectures, copper-center organization, and hydrophobic channels between archaeal and bacterial AMOs for ammonium acquisition, catalysis and inhibitor response. These findings provide a structural and mechanistic framework for understanding how archaeal and bacterial AMOs have diverged to distinct ammonia-oxidizing strategies and inhibitor susceptibilities across environmentally important ammonia oxidizers.

molecular biology↗

bioRxiv2026

A chromosome-scale reference genome and single-nucleus root atlas reveal the cellular and molecular basis of coastal adaptation in sand bean (Strophostyles helvola)

Plants adapted to saline coastal habitats provide valuable systems for understanding how natural selection reshapes stress-response programs, yet the cellular and molecular basis of this adaptation remains poorly resolved. Here, we generated a chromosome-scale reference genome and a single-nucleus transcriptomic atlas of roots from sand bean (Strophostyles helvola), a wild legume represented by salt-tolerant Beach and salt-sensitive Inland ecotypes. The atlas comprised 87,901 nuclei assigned to 25 transcriptional clusters representing 16 major root cell types. Salt exposure elicited cell-type-specific transcriptional responses that differed between Beach and Inland roots. Beach roots preferentially maintained respiration-, energy metabolism-, and protein-homeostasis-associated functions, whereas Inland roots showed stronger induction of canonical abiotic-stress, ABA, water-deficit, hypoxia, and oxidative-stress programs. Integrating baseline ecotype differences with salt-responsive expression revealed that many genes induced by salt in Inland roots were already expressed at higher levels in untreated Beach roots. This baseline-enriched configuration comprised a broadly distributed regulatory backbone, including ERF-family transcription factors and RING-type E3 ubiquitin ligases, together with cell-type-associated modules involving ion and water transport at the root-soil interface, redox and dehydration protection in outer-root tissues, endodermal barrier-associated genes, and vascular regulatory candidates. These findings support a model in which Beach salt tolerance is associated not with entirely distinct stress-response pathways, but with the pre-existing and spatially organized deployment of conserved protective programs that remain responsive to salt exposure. Our study establishes genomic and cellular resources for sand beans and provides a framework for investigating how natural variation in the regulation and cellular organization of conserved pathways contributes to environmental stress tolerance.

plant biology↗

bioRxiv2026

The MOS4-associated complex subunit MAC5A maintains meristem development by regulating transcription elongation in Arabidopsis

The precise coordination of co-transcriptional RNA processing with transcriptional elongation is essential for eukaryotic gene regulation, yet the machineries coupling these processes remain largely elusive. Here, we reveal a role for the spliceosome-associated MOS4-associated complex (MAC) in regulating plant development through the direct regulation of transcriptional elongation. We demonstrate that the MAC subunit MAC5A physically interacts with Elongator Protein 6 (ELP6), a core component of the transcriptional Elongator complex. Genetic analyses reveal that MAC5A and ELP6 function synergistically to regulate apical meristem activity. Importantly, the MAC5A-Elongator module is required for the efficient expression of the critical auxin efflux carrier PIN-FORMED1 (PIN1) by enhancing RNA Polymerase II (RNAPII) occupancy across the PIN1 locus. Furthermore, we show that this elongation-promoting function is not unique to MAC5A, as other core MAC components are similarly required for efficient transcription progression. Together, our findings uncover a splicing-elongation nexus where MAC5A likely acts as a molecular bridge between the spliceosome and the elongation polymerase. This functional coupling ensures the efficient transcription of key developmental regulators, providing a mechanistic framework for the coupling of RNA processing to transcription and suggesting a conserved principle of gene expression control across eukaryotes.

plant biology↗

bioRxiv2026

Tracking a major evolutionary transition to superorganismality

Major transitions in evolution are associated with the loss of independent reproduction by formerly autonomous units. Termites provide a powerful system for studying this process because they exhibit diverse social systems in which worker developmental and reproductive potential declines with increasing colony-level organismality. However, the evolutionary sequence and developmental genetic basis of these transitions remain unresolved. Here, using comparative developmental transcriptomics across seven termite species that differ in workers' reproductive potential, we reconstructed the evolutionary history of termite social systems. We found that linear caste development, in which workers retain full reproductive potential, represents the ancestral state of termites. Bifurcated caste development, in which workers partially lose reproductive potential early in development, evolved independently multiple times, with two origins subsequently giving rise to superorganisms with unipotent, sterile workers. Ancestral gene regulatory network (GRN) reconstruction revealed that linear caste development evolved through retention of a juvenile-like worker state and co-option of a conserved developmental GRN characterizing hemimetabolous insect nymphal development, in which juvenile hormone, ecdysone and TGF-{beta} signaling pathways play central roles. The convergent evolution of bifurcated caste development repeatedly co-opted the GRN underlying linear caste development, heterochronically shifting its activity to earlier developmental stages. Finally, we found that the evolution of termite superorganisms involved somatization of the worker caste and co-option of a conserved endocrine GRN for terminal differentiation. Together, these findings uncovered repeated routes to reduced workers' reproductive potential through GRN co-option and highlight striking parallels between superorganism evolution in social insects and organismal evolution in metazoans.

evolutionary biology↗

bioRxiv2026

The common evolution of fungicide persistence in cryptococcosis patients

Fungicide persistence, the ability of dormant fungal cells to survive lethal drug exposure, undermines treatment efficacy, yet its clinical evolution remains largely unexplored. We assembled a nationwide collection of clinical Cryptococcus neoformans isolates from China and revealed extensive inter strain variation in amphotericin B (AmB) persistence. This variation can arise from common evolutionary events in patients, generating previously undescribed high persistence cryptococcal variants refractory to AmB clearance. Genome wide fitness landscape analysis shows that persistence associated mutations generally impose minimal fitness costs, facilitating persistence evolution even in fungistatic resistant and hypervirulent genetic backgrounds. Machine learning identified deficient ACO2 expression as a key predictor of high persistence clinical isolates. ACO2 deficiency reproduces the high persistence phenotype by promoting POPC accumulation, which competes with AmB for its target. Moreover, the clinical stage antifungal T-2307 effectively eliminates high persistence strains across diverse genetic backgrounds. Collectively, our findings unveil common evolution of fungicide persistence in cryptococcosis patients, representing a previously overlooked clinical concern.

microbiology↗

bioRxiv2026

C1Q-associated adaptive myeloid remodelling accompanies early response to BCMA CAR-T therapy in multiple myeloma

BCMA-directed chimeric antigen receptor T cells induce deep responses in multiple myeloma, yet the immune ecology accompanying early response remains incompletely resolved. We reanalysed 171,971 single-cell transcriptomes from 25 peripheral-blood and bone-marrow specimens from ten patients. At day 30, responders showed concordant enrichment of C1Q and IFN-{gamma} programmes in blood and marrow myeloid pseudobulk profiles. Antigen-presentation genes were enriched in blood, whereas TGF-{beta} and hypoxia programmes were depleted in responding marrow. Cholesterol-efflux genes were not enriched in responders or after treatment. A composite C1Q-cholesterol score showed nominal associations with response and CD8 dysfunction in selected compartments, but none survived study-wide correction. The pathway results support an adaptive, antigen-presenting C1Q-associated programme rather than a uniformly suppressive C1Q macrophage model. This state-contingent interpretation of early myeloid remodelling requires prospective, patient-level validation before biomarker or causal claims are warranted.

immunology↗

bioRxiv2026

TxNova: recovery of recurrent unannotated intergenic splice loci from existing bulk RNA-seq alignments

Background. Reference catalogs such as GENCODE capture most stably expressed mammalian genes but may not include condition-restricted or low-abundance transcripts. Reads supporting unannotated intergenic splice junctions are generally absent from annotation-restricted gene-count matrices; transcript assemblers may reconstruct a subset as novel models, but those models are typically handled separately from the annotated gene-count universe. TxNova is a lightweight command-line tool that directly indexes these unannotated splices from existing BAM files - without an external assembler, gffcompare, or workflow manager - yielding candidate leads for bench validation rather than standalone discovery claims. Methods. TxNova retains CIGAR N junctions from STAR/HISAT2 BAM files that recur in >= 2 samples and are absent from a comprehensive annotation, clusters them into residual loci, and counts each locus alongside annotated genes in a unified matrix. Structure gates - canonical splice motif, same-strand distance, coverage valley, bridging-junction absence, minimum length - remove likely artifacts to yield structure-pass models. An optional contrast filter retains loci detected in treatment but nearly silent in control. Results. A residual splice is a recurrent, unannotated CIGAR N junction that does not overlap any annotated gene body; intronic and antisense channels are out of scope. Across four published mouse treatment arms (GSE221720, GSE166522, GSE157460, GSE193335), harvest catalogs yielded 464, 657, 789, and 594 loci. On GSE221720, 45% of loci (209/464) shared an exact intron with another series, versus 0.074% for excluded junctions; a coordinate-placement control yielded 0/5,000 matches for length-matched intergenic decoys. Masked-gene recovery reached 87.6% (176/201) overall and 99.4% (176/177) among genes with a leak junction - sensitivity rather than precision estimates. An optional contrast provides a presence/absence screen on interval TPM. Residual models are partial reconstructions requiring cloning, RACE, or targeted proteomics for confirmation. Conclusions. TxNova produces a reproducible intergenic residual-locus catalog from existing BAM files, with four downloadable mouse injury/infection catalogs. Cross-series recurrence and coordinate-based null controls support reproducibility above simple placement backgrounds but do not by themselves establish biological validity. The optional contrast step offers a practical screen for candidate leads warranting experimental validation.

bioinformatics↗

bioRxiv2026

SPARKLE: evidence-constrained correction of local RNA leakage in high-resolution spatial transcriptomics

High-resolution sequencing-based spatial transcriptomics, including Stereo-seq and Visium HD, aggregates dense capture units into cell-resolved expression matrices. During tissue processing and permeabilization, RNA released from source cells can spread to neighbouring capture locations, reducing cell-type specificity and biasing downstream analyses. Here we developed SPARKLE (Spatial Ambient RNA Kernel-based Leakage Estimator), a cell-level correction method that uses capture locations outside cell-segmentation masks as within-sample spatial evidence of leakage. SPARKLE fits sparse spatial kernels to out-of-mask observations to estimate a sample-level propagation scale and gene-specific leakage coefficients. It corrects only genes supported by out-of-mask goodness of fit and uses expression-dependent conservative shrinkage to protect highly expressing source cells. In ten simulated scenarios, SPARKLE achieved the highest cell-wise concordance in eight and the lowest RMSE in nine. In axolotl brain, mouse brain and human ovarian cancer, SPARKLE removed ectopic marker signal from neighbouring cells while retaining source-cell expression, improved agreement with independent single-cell and single-nucleus references, and recovered an inferred fibroblast-to-tumor COL1A2-SDC4 communication route that was obscured by ectopic COL1A2 expression. Conclusions remained stable across plausible spatial scales and background-bin sizes. Runtime scaled near-linearly with tissue-window area and was further accelerated on GPU. SPARKLE is therefore a reference-free, fast and scalable method for correcting local RNA leakage from evidence contained within each sample, improving the reliability of cell-type localization, tissue-compartment identification and cell-cell communication inference.

bioinformatics↗

bioRxiv2026

From distyly to stigma-height dimorphism: phylogenetic insights into the evolutionary breakdown of distyly in Jasminum

Background and AimsDistylous polymorphisms (distyly) are adaptations for plants to improve efficiency of cross-pollination and reduce pollen wastage. Theoretical models suggest that distyly evolves from stylar monomorphism via an intermediate stage of stigma-height dimorphism (SHD), however, this evolutionary scenario is only observed in few distylous lineages. The genus Jasminum have many species exhibit either distyly or SHD, providing an ideal opportunity to test models of the evolution of distyly. Focusing on the evolution of distyly, we investigated floral morphs and evaluated the occurrence of distyly and SHD in Jasminum using phylogenetic reconstruction and morphological analysis. MethodsWe investigated floral morphs and evaluated the occurrence of distyly and stigma-height dimorphism through morphology observation. To perform phylogenetic analysis, we sequenced plastomes of forty species from Jasminum and Chrysojasminum using Illumina next generation sequencing, and then constructed phylogenetic tree using maximum likelihood method and Bayesian inference. Based on the phylogenetic tree, we inferred the ancestral floral type through ancestral reconstruction. Key ResultsOur results suggest that the distyly originated in the common ancestor of Jasminum and Chrysojasminum. SHD occurs in several Jasminum species scattered in different clades of the phylogenetic tree, suggesting multiple independent reversions from distyly back to SHD. However, this transition is not associated with the loss of ancillary polymorphisms, as all the species examined in this study well retain dimorphic traits for other floral organs such as pollens and stigmas. Moreover, most species maintain strict heteromorphic self-incompatibility, while J. officinale has lost or at least partially lost self-incompatibility, suggesting that distyly is not always linked to self-incompatibility. ConclusionsIn Jasminum, breakdown of distyly resulted in evolutionary transitions to stigma-height dimorphism for multiple times, suggesting that distyly is not a stable floral polymorphism under certain selective forces. These findings advance our understanding on the evolution of distyly and plant reproductive systems.

plant biology↗

bioRxiv2026

scFair: Geometry-Aware Gene Budgets and Same-Rank Extension for Highly Variable Gene Selection

BackgroundHighly variable gene (HVG) selection begins almost every single-cell RNA-seq analysis. While ranking formulas have been compared extensively, the integer gene budget at which any ranking must be truncated is typically left to the user and habitually fixed near 2,000. Relying on such a convention carries hidden costs--lists that are too short erase subtle structure, whereas lists that are too long add noise and computational overhead. Moreover, because global rankings measure variance across all cells, markers for rare populations often lose the "variance vote count" to dominant bulk variation, leading to an unfair feature allocation at the hard cutoff. Whether this convention is defensible, and whether the budget and tail can be set from data without disturbing the ranking, has not been examined systematically. ResultsUnder a frozen seurat_v3 ranking, k-sweeps across 18 labeled datasets show that n = 2,000 is ARI-optimal on 1 of 18 datasets and that the best available budget is worth a mean ARI gain of +0.033 over it, establishing cardinality as a real and largely unexploited design axis. We present scFair, a Scanpy-compatible HVG layer that automates list length alone: geometry-aware auto_n sets a base size k from multi-seed density and stability features of an intermediate embedding (trading a modest, intentional compute increase for a safer data-driven default), and a same-rank append step acts as a conservative safeguard against cutoff unfairness by adding a short near-miss tail. The ranking is never recomputed or reweighted. On the 18-dataset panel, the default path improved Leiden-label agreement over HVG@2000 (median {Delta}ARI = +0.016; 13/5; Wilcoxon P = 0.0077) and outperformed the neighborhood-based selector triku at author defaults on 15/18 datasets (median +0.024; P = 0.004), while triku did not improve on HVG@2000. Controls locate the effect: cell-number-only rules do not beat HVG@2000, an FDR-chosen length imposed on the frozen ranking is flat, and a fixed HVG@2200 default is not a general substitute because it cannot produce the short lists that compact matrices call for. ConclusionsA fixed budget near 2,000 HVGs is frequently suboptimal, and list cardinality is a separable design axis that can be automated without changing the ranking formula. Effect sizes are modest, the short-list branch rests on four datasets, and rule thresholds were developed with partial overlap to the evaluation panel.

bioinformatics↗

bioRxiv2026

Antibiotic condensates activate magnesium signaling to drive drug tolerance

The spatial distribution of small molecules within cells shapes their biological activity, yet these distributions are generally assumed to be governed passively by reaction-driven electrochemical gradients. Here we show that aminoglycoside antibiotics actively control their own subcellular organization by undergoing phase separation with RNAs. Combining in vitro reconstitution, bacterial assays, and molecular dynamics simulations, we discovered that aminoglycosides coacervate with RNA through multivalent electrostatic interactions, displacing and releasing RNA-bound Mg2+. This condensate-dependent Mg2+ release remodels the cytosolic labile Mg2+ pool and activates magnesium signaling. This effect dampens the magnesium-starvation regulation, sustains ribosome activity, and shifts the cellular electrochemical state, promoting bacterial fitness. Because condensation occurs only above a defined concentration threshold, it generates a non-monotonic dose-response in which higher antibiotic concentrations paradoxically enhance bacterial survival. This antibiotic condensate-dependent Mg2+ signaling confers tolerance to multiple ribosome-targeting antibiotics simultaneously even in cells lacking resistance gene, while condensate dissolution restores antibiotic efficacy. Our findings establish antibiotic-driven phase separation as a previously unrecognized mechanism to encode cellular signaling and identify antibiotic condensates as a distinct functional unit underlying drug tolerance.

microbiology↗