Biology subjects

Zhao, Y.

Publications and source records attributed to Zhao, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Integrative multi-omics re-analysis of public sequencing and metabolomics repositories defines the mucosal immune and metabolic landscape of Helicobacter pylori infection and supports CagW as a rational mRNA-LNP vaccine antigen

Background. Helicobacter pylori colonises 40-45% of the global population, is a WHO Group I carcinogen, and is increasingly hard to eradicate amid rising resistance; no vaccine is licensed. CagW, a VirB6-family cagPAI component, has been advanced as a DNA-vaccine antigen that protects mice; a codon-optimised CagW-Fc mRNA in lipid nanoparticles (mRNA-LNP) is a candidate antigen design intended to engage humoral and cellular immunity. To define the infected gastric mucosal niche in which such a vaccine must operate, we mined public sequencing, single-cell and metabolomics repositories. Methods. NCBI GEO, MetaboLights and NCBI Protein were interrogated. Bulk transcriptomes of H. pylori-infected vs uninfected gastric epithelial cells (GSE295523; 24 libraries: HGC27, AGS, MKN45, GES-1) were re-analysed with a cell-line-blocked design; DEGs (BH-adjusted) were annotated via g:Profiler (GO, KEGG, Reactome) and 13 functional signatures scored. Single-cell composition was quantified from a mouse gastric scRNA-seq atlas (GSE224840; 16,434 cells) and sorted gastric CD8+ tissue-resident memory (TRM) vs splenic memory T cells (GSE216894). Targeted gastric metabolomics was re-analysed for epithelial ornithine decarboxylase (Odc) deletion (MTBLS3235) and cystathionine-gamma-lyase (Cth) deletion (MTBLS2851). CagW conservation was computed across 400 NCBI records (286 variants) with Kyte-Doolittle topology and four B cell epitope scales. Results. Infection induced 326 up- and 115 down-regulated genes (FDR < 0.05, |log2FC| > 1), dominated by TNF/NF-kappaB (KEGG hsa04668, P=5.5x10-11), integrated stress response (ATF4-CHAC1-DDIT3-TRIB3-ASNS) and Th17 chemokines (CXCL8 +3.43, CCL20 +1.32, CXCL1 +1.30, IL23A +1.02). MHC-I presentation rose (Delta-z +0.76; MHC-II unchanged); the arginine/polyamine module (SLC7A1 +1.19, ARG2 +1.33, SAT1 +1.50, ODC1 +0.58) was co-induced. scRNA-seq showed immune expansion in infected mucosa (39.8% vs 21.2% cells; OR 2.46; cell-level frequencies reported descriptively because cells are not independent; direction confirmed at sample level, n=4 vs 4), driven by plasmacytoid DCs (OR 8.9), neutrophils (OR 3.0), macrophages (OR 2.7) and cDCs (OR 2.1), with epithelial de-differentiation. Gastric CD8+ TRM cells showed a residency-effector program (Itgae +6.2, Cd69 +2.3, Gzmb +4.8, Ifng +5.3) with egress-gene loss (Sell -11.4, S1pr1 -9.7, Klf2 -5.2). Metabolomics confirmed both nodes: Odc deletion raised arginine (+3.08) and Cth deletion raised spermidine (+0.40) while lowering leukotriene B4 (-0.33). CagW was conserved across 286 variants (median 98.3% identity; 96.4% residues >=95% conserved), carried four transmembrane helices, and showed a surface-biased conserved epitope envelope at residues 505-530 and 63-79. Conclusions. Public multi-omics define an NF-kappaB/ATF4-driven, Th17-polarised, metabolically stressed niche where antigen-presenting cells expand, epithelium de-differentiates, and CD8+ TRM cells act locally. Here CagW is exceptional among cagPAI antigens: near-universal conservation plus an accessible, conserved surface. These findings provide a systems-level rationale for prioritising CagW as a conserved, epitope-accessible vaccine antigen and inform next-generation multi-antigen designs.

microbiology↗

bioRxiv2026

Multimodal Connectome Markers of Disruptive Behavior Problems in Youth: Distinct Patterns of Concurrent Severity and Prospective Symptom Change

Disruptive behaviors problems, such as irritability/anger and maladaptive aggression, are associated with altered connectivity across networks supporting emotion regulation. However, multimodal connectome markers of concurrent severity and prospective symptom change, including sex-specific patterns, remain unclear. Using a large, sex-balanced sample from the Adolescent Brain Cognitive Development (ABCD) Study, we applied multimodal connectome-based predictive modelling (CPM) to test whether resting-state functional (n = 7,556; 49.3% female) and structural (N = 8,978; 48.4%) connectivity predicted baseline disruptive behavior severity (at ages 9-10) and 2-year symptom change (from ages 9-10 to 11-12). Models were adjusted for covariates, permutation-tested, and FDR corrected. Secondary analyses tested sex-stratified models separately in males and females. In models emerging as significant, follow-up analyses tested for distinct vs shared predictive edges. Functional and structural connectomes predicted baseline severity ({rho} = 0.087 and {rho} = 0.071, respectively). Functional connectivity also predicted prospective symptom change ({rho} = 0.038), whereas prospective structural prediction was predominately with negative edges ({rho} = 0.026). Functional connectivity models were significant in both sexes, while prospective structural connectivity models were significant in males, but not females. Frontoparietal, medial frontal, sensorimotor, salience, cerebellar, and visual networks contributed across models. Cross-sectional models showed a more distributed pattern while prospective models showed a more focal pattern. These findings identify complementary, largely distinct functional and structural connectome patterns associated with current disruptive behavior risk and subsequent symptom change, supporting the importance of developmental timing, sex, and imaging modality in developing brain-based biomarkers of disruptive behavior risk in youth.

neuroscience↗

bioRxiv2026

Integrative multi-omics decoding of the host response to Klebsiella pneumoniae infection reveals an itaconate-centred metabolic-immune axis and a spatially organised inflammatory programme

Background: Klebsiella pneumoniae (Kp) is a leading cause of hospital and community acquired pneumonia and bacteraemia, and hypervirulent and carbapenem resistant lineages continue to expand worldwide. Although individual omics layers of Kp infection have been profiled, the host-level molecular logic that connects transcriptional, cellular, metabolic and spatial programmes has not been systematically integrated. Methods: We curated 16 public datasets spanning four modalities-bulk and dual host pathogen RNA-seq (six datasets; 118 libraries), single-cell RNA-seq (four datasets; {approx}164,000 cells), untargeted/targeted metabolomics (four datasets; 1,842 molecular features) and Visium spatial transcriptomics (two datasets; {approx}11,400 spots) covering murine lung infection models, hypervirulent and classical Kp strains, carbapenem-resistant isolates and human clinical specimens. Datasets were harmonised through a standardised pipeline (Salmon/DESeq2; Scanpy/Harmony; MetaboAnalyst; RCTD/spacexr), and integrated by WGCNA, GSVA, CellChat, Monocle3, SCENIC, joint-pathway analysis, RCTD deconvolution, and machine-learning feature selection (LASSO, random forest, SVM-RFE, XGBoost) with 1,000x bootstrap internal validation and independent external validation. Results: Kp infection evoked a coherent 920 gene neutrophil inflammatory response ({approx}520 up-/{approx}400 down-regulated genes) dominated by IL-17/TNF/NF-{kappa}B signalling, neutrophil degranulation, HIF-1-linked glycolysis and itaconate biosynthesis. A turquoise co-expression module (r = 0.86 with infection status) yielded a ten-gene hub signature (IRG1, S100A9, IL1B, CXCL2, SLC7A11, HMOX1, PTGS2, NFKBIA, LST1, FCGR3B). Single-cell profiling resolved twelve major lung lineages and eleven myeloid substates, tracing a Monocle3 trajectory from Cxcr2hi inflammatory neutrophils through an Irg1/Slc7a11hi oxidative phase into Cxcr4hi/Trem2hi suppressive states; CellChat identified S100A8/A9-TLR4 and CXCL2-CXCR2 as the most amplified axes (8.5x and 4.2x). Metabolomics revealed 30-fold pulmonary itaconate accumulation together with succinate and lactate elevation, glucose/glutamine depletion and glutathione collapse, and joint-pathway analysis positioned the TCA-cycle itaconate and ferroptosis modules as the strongest convergent perturbations. Spatial transcriptomics mapped these programmes onto five anatomical niches, with IRG1 expression peaking in the infection core (58% neutrophil content) and declining radially. A ten-gene host-response classification model reached AUC=0.958 in discovery, 0.937 in internal validation and 0.884-0.912 in two independent external cohorts (Brier score 0.071). Conclusions: By vertically integrating public multi-omics data, we define an itaconate-centred metabolic-immune axis that couples inflammatory myeloid expansion to spatially segregated metabolic niches, and we deliver a validated host-side molecular signature that discriminates Kp infection from non-infected states.

microbiology↗

bioRxiv2026

Brain state locks silent memories in a trained fly connectome

Complete connectomes now exist for insect nervous systems, but whether a fixed wiring diagram can learn, and what its memories consist of, is unknown. Here, we trained a spiking model of the whole male Drosophila central nervous system, 167,106 neurons with fixed wiring, to plan moves and to remember a visual cue. Spikes write the cue into neuronal adaptation, which holds it silently until the next input reads it out. Cells that hold the trace are enriched for transcripts of a calcium-activated potassium channel in fly cell atlases. The learned readout recalls the trace only in the brain state of learning: the same trace evokes a different response at another delay, and a disease-like loss of potassium current rewrites the trace and changes how it is read. Learning in the new state is spared. Memory failure can arise from a change of state rather than a loss of storage.

neuroscience↗

bioRxiv2026

Susceptibility and cellular responses of COPD airway epithelium to cigarette smoke in stem cell-derived ALI models

Despite cigarette smoke being the chief driver of chronic obstructive pulmonary disease (COPD), numerous patients maintain their smoking habits by opting for low-yield or slim cigarettes under the misconception of diminished health risks. Differential effects of high-tar conventional versus low-tar slim cigarettes on COPD airway epithelium remain poorly defined. We established an air-liquid interface (ALI) model using patient-derived COPD airway epithelial stem cells and exposed cultures to high-tar conventional smoke (TC), low-tar slim smoke (SC), or filtered air (NC). Both regimens induced IL-1{beta} inflammation, ciliary structural changes, and oxidative stress, with TC eliciting stronger responses. Single-cell RNA sequencing resolved six epithelial populations. In ciliated cells, hdWGCNA showed NC modules associated with cilium organization and assembly, SC adaptive defense responses, and TC more extensive reprogramming of ciliary functional networks. Basal cells displayed stress-pathway activation, squamous metaplasia signatures, and developmental regression toward a stem-like state, with TC producing approximately 1.7-fold stronger regression than SC. Secretory cells exhibited identity erosion, antimicrobial program downregulation, and a shift toward keratinization and squamous-oriented programs, again more pronounced under TC. These findings indicate that high-tar conventional smoke exerts greater influence than low-tar slim smoke on COPD airway epithelium across inflammatory, structural, and lineage-specific molecular dimensions, providing high-resolution insight into cell-type-specific responses to continued smoke exposure.

cell biology↗

bioRxiv2026

Widespread Presence of Catalytic RNAs in All Domains of Life

Very few ribozymes have been found in complex eukaryotes. Here, we developed a novel method, AbRiDGed, for highly accurate and sensitive ab initio self-cleaving ribozyme discovery. Our results indicate the existence of millions of self-cleaving ribozymes in humans and other species. We characterized ~150 novel classes of self-cleaving ribozymes, including one class which exhibits noncanonical biochemical properties and widespread presence in all domains of life and appears to play specific functions in complex organisms. These results revealed that the true complexity of the "ribozyme-ome" is several orders of magnitude higher than previously estimated. We demonstrate that newly identified self-cleaving ribozymes influence organismal fitness by regulating gene expression and enhancing transcriptome complexity in vivo. Moreover, these ribozymes hold promise as foundational tools for biotechnological applications, such as direct RNA detection technologies. Overall, catalytic RNAs are prevalent and diverse, and may represent a hidden regulatory layer in various organisms.

molecular biology↗

bioRxiv2026

Synthetic Poly(2-Oxazoline) Chimera 'Lipo-Polymers' as Replacements for Key Lipids in Lipid Nanoparticles

Lipid nanoparticles (LNPs) rely on specialized lipid components, many of which are proprietary and difficult to access or modify. Here, we introduce a polymer-based alternative to key LNP constituents through the development of chimera poly(2-oxazoline) (POx) synthetic lipid polymers (SLPs) and synthetic lipid oligomers (SLOs). These chimeric lipid-mimetic materials are designed for seamless incorporation into existing LNP architectures, enabling direct replacement of PEG-lipid conjugates and ionizable lipids, respectively. Chimeric SLPs comprise hydrophilic shell-forming POx blocks linked to hydrophobic lipid-inserting domains, whereas SLOs consist of hydrophobic POx oligomers terminated with ionizable amine-containing groups. Using mRNA-loaded LNPs, we demonstrate that first-generation POx-Chimera SLPs and SLOs functionally replace industry-standard lipid components while maintaining robust transfection activity in vitro and in vivo. Notably, SLO-containing formulations achieved expression levels comparable to, and in some cases exceeding, those of conventional lipid formulations. Together, these results establish POx-Chimera SLPs and SLOs as a versatile platform for polymer-centric LNP engineering, creating new opportunities to tune nanoparticle composition, biodistribution, and transgene expression while reducing reliance on proprietary lipids.

bioengineering↗

bioRxiv2026

Physiological Oscillations of Lateral Ventricular Volume Revealed by Fast fMRI: Effects of Aging and Vigilance

Abstract Background: Cyclic ventricular deformation may contribute to CSF displacement. However, the physiological mechanisms underlying ventricular motion remain poorly understood. This study characterized physiological oscillations of lateral ventricular volume (LVV) and examined their associations with aging and vigilance. Methods: We analyzed two fast resting-state fMRI scans from 99 adults aged 35-82 years. The first scan was acquired during eyes-open wakefulness, followed by 30 min of eyes-closed rest and a second scan during eyes-closed drowsiness. LVV was estimated for each image volume using automated ventricular segmentation, yielding time-resolved volume measurements. Temporal variability was quantified using the standard deviation and coefficient of variation, and spectral power was quantified for low-frequency, respiratory, and cardiac oscillations. Age-related associations were assessed using multiple linear regression adjusted for sex. Awake-drowsy differences were assessed using paired t-tests. False discovery rate correction was applied for multiple comparisons in all analyses. Results: After quality-control exclusions, 97 participants were analyzed. LVV exhibited distinct low-frequency, respiratory, and cardiac oscillatory components. Respiratory and cardiac oscillations accounted for 37.7% and 38.4% of total physiological spectral power, respectively, compared with 23.9% for low-frequency oscillations. Mean lateral ventricular volume increased with age (FDR-adjusted p = 0.010), while respiratory and cardiac power also showed positive associations with age, although neither survived FDR correction (both FDR-adjusted p = 0.067). The transition to eyes-closed drowsiness was associated with greater LVV fluctuation magnitude, reflected by increases in standard deviation (FDR-adjusted p < 0.001) and coefficient of variation (FDR-adjusted p = 0.028) and accompanied by increased low-frequency (FDR-adjusted p < 0.001) and respiratory power (FDR-adjusted p = 0.015). Mean LVV and cardiac power did not change significantly between conditions. Conclusions: LVV exhibits prominent respiratory and cardiac oscillations in addition to low-frequency fluctuations, suggesting that ventricular dynamics reflect multiple physiological influences rather than a single dominant physiological driver. Aging was primarily associated with ventricular enlargement, whereas reduced vigilance was accompanied by increased LVV fluctuation magnitude, primarily reflected in increased low-frequency and respiratory power, without a change in mean LVV. Time-resolved LVV provides a complementary approach for investigating the physiological regulation of ventricular dynamics in health and disease. Keywords: Cerebrospinal fluid; Lateral ventricular volume; Fast fMRI; Physiological oscillations; Cardiac pulsation; Respiration; Low-frequency oscillations; Aging; Vigilance

bioengineering↗

bioRxiv2026

An OmpX mRNA-LNP vaccine induces protective humoral and cellular immunity against Klebsiella pneumoniae in a C57BL/6 mouse model

Klebsiella pneumoniae (KP) poses a significant threat to global public health, and effective preventive measures are urgently needed. The widespread antibiotic resistance of KP underscores the critical need for vaccine-based strategies. Although numerous vaccine candidates against KP have been investigated, none has yet been licensed. mRNA-LNP vaccines against KP have only recently begun to be explored, and their ability to deliver conserved outer membrane protein antigens remains largely untested. In this study, we constructed an mRNA vaccine encoding the highly conserved outer membrane protein X (OmpX) of KP-the first mRNA-LNP vaccine targeting this antigen-and evaluated its immunogenicity and protective efficacy in mice. Immunoinformatics analyses predicted concordance between the in silico and in vivo outcomes: bioinformatics tools were used to evaluate the antigenicity, immunogenicity, physicochemical parameters, and secondary and tertiary structures of OmpX, and the C-ImmSim server was used to simulate the vaccine-elicited immune responses. We then performed mouse immunization and challenge experiments to characterize the humoral and cellular immune responses induced by the OmpX vaccine. The mRNA vaccine was immunogenic in mice, eliciting high titers of OmpX-specific antibodies. OmpX-Fc mRNA-LNPs also induced robust cell-mediated immunity, with significantly elevated IFN-{gamma} and IL-4 levels indicative of combined Th1/Th2 cellular responses. Serum from OmpX-immunized mice promoted significantly greater complement-mediated KP cell lysis than serum from the alum or luciferase mRNA-LNP control groups. Notably, OmpX-Fc mRNA-LNP immunization conferred complete protection against lethal KP challenge, with 100% survival and a significant reduction in visceral bacterial burden. These data establish a promising mRNA vaccine strategy for inducing broad immune responses and promoting serotype-independent immunity against KP.

microbiology↗

bioRxiv2026

Integrative reanalysis of public intracellular and vaccine-challenge transcriptomes, informed by single-cell and metabolomic atlases, refines antigen prioritisation for next-generation veterinary Brucella vaccines

Brucellosis is the most widespread bacterial zoonosis and a leading cause of abortion and infertility in cattle, sheep, goats and pigs. The live vaccines licensed for livestock (B. abortus S19 and RB51, B. melitensis Rev.1) give incomplete protection, retain residual virulence and induce antibodies that are indistinguishable from those of field infection, and no human vaccine is licensed. Antigen discovery for safer subunit and mRNA alternatives has proceeded one candidate at a time, so it remains unclear which immunogenic proteins are abundant in the intracellular niche that protective immunity must recognise. We reanalysed four public data layers: dual RNA-sequencing of intracellular B. abortus and infected murine macrophages (GSE100914), splenic transcriptomes from vaccinated and challenged mice (GSE254248), and single-cell (PRJCA054635) and metabolomic (ST003696, ST003692) atlases. Intracellular residence induced 704 genes and repressed 249, enriching translation functions 2.9-fold. Omp28/BP26 was the second most abundant intracellular transcript (35,652 TPM; log2FC 4.25), whereas the lipoproteins omp16, omp10 and omp19 were 116-637-fold less abundant. Vaccination left a germinal-centre and TCF1+ memory imprint rather than an effector-cytokine signature. A composite score combining abundance, induction and surface exposure reorders the candidate panel, placing Omp28/BP26 and omp2b above current candidates and defining concrete priorities for veterinary vaccine development.

immunology↗

bioRxiv2026

OmniRE-seq: A Dual-Method Framework for Decoding RNA Editing Dynamics in Disease and Therapy

RNA editing is a widespread post-transcriptional modification involved in transcriptome diversity and disease regulation. However, its quantification remains technically challenging. We present OmniRE-seq, a dual-framework combining RE-seq for bulk and scRE-seq for single-cell RNA-seq data, enabling multi-level quantification, differential analysis, and functional annotation of RNA editing. Using RE-seq, we profile editing alterations across diverse contexts -- including murine sepsis, MDS, drug-treated melanoma, and colorectal cancer -- demonstrating the utility of locus-, gene-, and burden-level metrics in capturing biologically meaningful differences. scRE-seq further resolves cell-type-specific editing patterns by aggregating cells of shared identity into pseudo-bulk units (Spots), enhancing editing signal while preserving cellular resolution in colorectal cancer. Integrated bulk and single-cell analysis identified convergent editing signals and therapeutic targets, exemplified in a mouse model of thymic atrophy treated with Ganoderma lucidum spores. Together, OmniRE-seq offers a standardized and scalable toolkit for dissecting RNA editing landscapes across resolution levels, advancing both mechanistic discovery and translational application in precision medicine.

bioinformatics↗

bioRxiv2026

Pediatric brain charts reveal ADHD biotypes with distinct developmental, molecular, and therapeutic profiles

Attention-deficit/hyperactivity disorder (ADHD) exhibits profound clinical heterogeneity; however, its underlying neurobiological substrates remain unclear. Here, leveraging large - scale structural magnetic resonance imaging data from 12,663 typically developing children and 1,699 children with ADHD across Chinese and United States cohorts, we construct population specific pediatric brain charts via hierarchical Bayesian normative modeling to quantify individual deviations in cortical morphology. Data-driven clustering identifies two robust, cross - ethnically replicable ADHD biotypes: an infra - normal biotype characterized by widespread cortical thinning and a supra - normal biotype marked by cortical thickening. Multivariate analyses reveal distinct brain-behavior architectures: cortical deviations correlate preferentially with symptom severity in the infra-normal biotype but with both symptom severity and cognitive performance in the supranormal biotype. Clinically, the infra-normal biotype demonstrates a superior response to methylphenidate over atomoxetine, whereas the supra-normal biotype responds comparably to both. Transcriptomic analyses identify biotype-specific molecular signatures converging with ADHD genome-wide association study (GWAS) risk loci but exhibiting distinct expression patterns in the brain. These findings classify ADHD heterogeneity into neurobiologically distinct, clinically informed biotypes, providing a mechanistic framework for precision psychiatry in pediatric ADHD.

neuroscience↗

bioRxiv2026

Mechanism of the Sequential Trajectory of Allosteric Ring Closure in Human Chaperonin TRiC

Eukaryotic chaperonin TRiC assists in the folding of approximately 10% of the cytosolic proteome through an ATP-driven chamber closure cycle, yet the underlying closure mechanism has remained elusive. Here, using cryo-EM and deep learning analyses, we resolve, at near-atomic resolution, the temporal engagement sequence of the eight distinct subunits during human TRiC ring closure triggered by ATP binding. TRiC closes its chamber through the stepwise extension of an apical helical network among subunits around the ring, coordinated with sequential rotations of the equatorial domains. Each subunit responds differently to ATP, rendering the overall motion asymmetric and ordered. This work delineates the complete structural trajectory of allosteric propagation during ATP-binding-induced, rather than hydrolysis-induced, ring closure, and establishes a mechanistic framework linking ATP-driven conformational dynamics to substrate handling.

biophysics↗

bioRxiv2026

Super-resolution imaging flow cytometry

Organelle architecture is a key determinant of cellular state. However, its heterogeneity remains difficult to quantify at population scale because conventional imaging flow cytometry is diffraction-limited. Here we present super-resolution imaging flow cytometry (SR-IFC), a high-throughput structured-illumination platform combining stroboscopic excitation, microfluidic hydrodynamic focusing and AI-assisted reconstruction. From three phase-shifted frames acquired along the flow axis, SR-IFC reconstructs images with approximately 118-nm isotropic in-plane resolution, about two-fold beyond the diffraction limit, at up to approximately 720 cells per second. In microbiology, SR-IFC resolves individual lipid droplets in nitrogen-starved Euglena gracilis and reveals spatial reorganization during lipid biogenesis missed by diffraction-limited imaging. In immunology, it maps activation-associated mitochondrial remodeling across primary human CD8+ T-cell differentiation, including a transient networked state. SR-IFC thus provides a broadly applicable platform for population-scale super-resolution phenotyping across diverse areas of biology.

bioengineering↗

bioRxiv2026

Evolution-Guided Engineering of Non-conserved N-Glycosylation Sites Improves Heterologous Production of Sulfotransferases

Escherichia coli features low cost, mature fermentation technology and high recombinant protein output, but the expression of glycosylation-dependent eukaryotic proteins therein is severely limited. Many glycosylated proteins are key tool enzymes for the biosynthesis of various pharmaceutical molecules, such as glycosaminoglycan-related synthases, but there is a lack of universal protein engineering methods to enable the adaptive evolution of these enzymes in E. coli. We established an evolution-guided optimization strategy that targets non-conserved N-glycosylation sites (EGO-NGly). The core of this strategy lies in the phylogeny-guided selective reversion of evolutionarily acquired N-glycosylation sequons to naturally occurring non-glycosylated residues from early-divergent orthologs, thereby improving heterologous production and residual activity stability for selected sulfotransferases in E. coli. Using heparin 3-O-sulfotransferase 1 (3OST-1) as a model enzyme apparatus in eukaryotic organisms, EGO-NGly extended the residual activity half-life by 4.5-fold. Structural analysis and molecular dynamics simulations indicate that greater surface rigidity and remodeling of intramolecular interaction networks contribute to the improved conformational stability of M7 in an aglycosylated host. EGO-NGly was further applied to a panel of 3OST and 6OST homologs, demonstrating its applicability to a subset of the tested enzymes. Furthermore, EGO-NGly can be orthogonally integrated with other complementary protein engineering strategies, as exemplified by its combination with the machine learning-aided Protein Repair One-Stop Service, generating variants with 8-fold and 17-fold extensions in residual activity half-life. Collectively, these results establish EGO-NGly as a practical and potentially cost-effective engineering strategy for improving the prokaryotic expression and functional performance of selected glycosylation-dependent eukaryotic sulfotransferases.

biochemistry↗

bioRxiv2026

Microrobot and Cellbot Locomotion in Confined Hydrogels and Cardiac Tissue Surfaces

Biohybrid cellbots and microrobots are a promising platform for targeted drug delivery, minimally invasive surgery, and tissue engineering. Here, we present a comparative analysis of the locomotion dynamics across synthetic substrates and ex vivo cardiac tissue to help bridge the gap presented by translating locomotion dynamics from idealized environments to biological interfaces. By subjecting the microrobots and cellbots to different substrates (specifically, cell culture dish, hydrogel, and ex vivo tissue) and applying rotating magnetic fields at varying frequencies, we characterized how surface boundary interactions and driving frequency dictate translation speed. We reveal that particles moving on tissue exhibit restricted movement based on trajectory and tissue fiber orientation. Finally, we discuss that while cellular integration introduces significant adhesive drag that reduces translation speeds, it simultaneously offers a unique clinical advantage. This study highlights the potential of balancing magnetic driving parameters against biological environments to optimize for various treatment timelines and efficiency across diverse biomedical applications.

bioengineering↗

bioRxiv2026

SubcellPert: Benchmarking Subcellular Perturbation Prediction

Perturbations change both RNA abundance and intracellular organization, but gene-count predictors omit the positions needed to model spatial responses. To address this limitation, we introduce \textbf{\textit{SubcellPert}}, a benchmark for perturbation-response prediction at single-RNA-molecule resolution. It comprises 14 imaging datasets with 13.88 million cells and 3.65 billion RNA molecules. Given source observations and a directed condition change, models generate target RNA populations in normalized two-dimensional coordinates. Two independently trained tasks separate abundance from localization: \textit{Oracle} predicts coordinates given exact target counts; \textit{Joint} predicts both. Eight baselines span source resampling, classical transport, and frozen foundation-model adapters. Fixed perturbation and cellular-context holdouts evaluate abundance error, target-gene coverage, and conditional spatial error. Through within-dataset analyses, we find context-dependent gains from spatial features in condition decoding. Interpreting spatial responses requires distinguishing changes within spatial clusters from population composition, and RNA fractions from enrichment relative to compartment geometry. Through comprehensive experiments, we find that Gaussian transport achieves the lowest Oracle spatial error on eight of eleven cellular-context OOD datasets. Audits show that better centroid accuracy can accompany worse radial distributions, and that 19 of 190 Joint adapter fits retain source counts after validation. SubcellPert opens a spatial frontier for virtual cell research, laying the groundwork for predictive models that capture both the molecular composition and intracellular organization of cellular responses. The code is available at \url{https://github.com/king159/SubcellPert}.

bioinformatics↗

bioRxiv2026

SF3B1 mutation generates novel peptides from long non-coding RNAs

While mRNA alternative splicing caused by somatic mutation in SF3B1 is well-characterized, long non-coding RNAs (lncRNAs) impacted by this mutation are completely unknown. Here, we report long-read sequencing of full-length transcripts from cell lines with and without SF3B1 mutation. We present ALLR (Annotation of human lncRNAs using Long-Read sequencing), a computational workflow to identify and quantify lncRNAs, and to annotate their potential roles in impacting cellular functions. Harnessing matched short-read RNA sequencing and proteomic data, we demonstrate that SF3B1 mutation generates thousands of novel lncRNAs with diverse levels of alternative splicing. Moreover, SF3B1 mutation-associated lncRNAs produce translatable peptides with putative functions, immunogenicity, and potential to bind proteins with impact on cellular signalling networks. 47 lncRNAs could also be detected in primary chronic lymphocytic leukemia (CLL) samples and are associated with significant risks towards adverse disease prognosis, in conjunction with other known CLL risk factors. Our work demonstrates the utility of long-read sequencing for lncRNA research and provides a new territory of functional RNA molecules that SF3B1 mutation may use to impact cancer.

genomics↗