Biology subjects

Liu, P.

Publications and source records attributed to Liu, P..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Photometallobiocatalytic Asymmetric Radical-Mediated Cross-Coupling of Organotrifluoroborate Salts and Pyridotriazoles

The cooperative integration of photoredox catalysis and metalloenzyme catalysis has emerged as a powerful strategy for enabling stereoselective radical transformations beyond the capabilities of either catalytic mode alone. Herein, we report a photometallobiocatalytic enantioselective intermolecular C-C cross-coupling of pyridotriazoles and secondary alkyltrifluoroborate salts through cooperative catalysis between an organic photosensitizer and an engineered protoglobin. By combining visible-light-mediated radical generation with enzymatic activation of pyridotriazoles to form reactive Fe carbenoid intermediates, this transformation enabled highly enantioselective radical C-C bond formation through a proposed outer-sphere coupling mechanism. Through biocatalyst mining and directed evolution, engineered Aeropyrum pernix protoglobin catalysts were developed that catalyzed this radical C-C coupling with excellent efficiency and stereocontrol. The photobiocatalytic platform exhibited a broad substrate scope with respect to both secondary alkyltrifluoroborate salts and pyridotriazoles, affording a range of valuable N-heterocyclic products in excellent yields and enantioselectivities. Mechanistic studies supported the involvement of radical intermediates and revealed spontaneous binding between the photocatalyst eosin B and the engineered metalloenzyme. By leveraging cooperative photometallobiocatalysis, this work established an underexplored strategy for asymmetric intermolecular radical cross-coupling via an outer-sphere mechanism, further expanding the catalytic repertoire of transition-metal carbenoid chemistry. Entry for the Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/744224v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@39f55borg.highwire.dtl.DTLVardef@11b3dfeorg.highwire.dtl.DTLVardef@1832ac2org.highwire.dtl.DTLVardef@6c4f56_HPS_FORMAT_FIGEXP M_FIG An enantioselective photometallobiocatalytic cross-coupling of pyridotriazoles and secondary alkyltrifluoroborate salts is developed. Cooperative catalysis using eosin B and an engineered protoglobin combines visible-light-mediated radical generation with enzymatic metal carbenoid activation, affording valuable N-heterocyclic products in excellent yield and enantioselectivity through an outer-sphere radical coupling pathway. C_FIG

biochemistry↗

bioRxiv2026

Humanized Anti-PD-1 Antibodies Generated Using The Conditional Kernel-Elastic Autoencoder

The human immune system excels at generating highly effective antibodies through natural selection and somatic hypermutation, but adapting these antibodies for therapeutic use, referred to as "antibody medicine-likeness", requires careful consideration of biochemical and physiological properties. Traditional redesign methods are often slow and limited in scope. In this study, we introduce a machine learning-based approach to evolve new anti-PD-1 antibodies within a chemically informed latent space using a conditional kernel-elastic autoencoder (CKEA) between nivolumab and pembrolizumab, both of which bind the FG-loop "hotspot" of PD-1 in the most distantly related orientations, differing by 174{degrees}. This generative framework is designed to preserve favorable therapeutic features while exploring variants with different potency, ultimately for improved potency. To evaluate structural and functional viability, we performed molecular dynamics (MD) simulations of the generated antibody - PD-1 complexes and described their MD properties. These simulations reveal detailed free-energy landscapes and identify stable binding conformations, providing a strong basis for experimental validation. To validate our designs, we expressed and experimentally tested the antibodies for binding affinity to PD-1. Upon expression and purification, three out of six designed antibodies exhibited some binding to PD-1, whose properties could likely be improved using other computational saturation mutagenesis or laboratory evolution. Our results demonstrate the potential of artificial intelligence (AI)-guided interpolation methods to generate novel, high-affinity antibodies with therapeutic promise, offering a powerful strategy for next-generation antibody development. SYNOPSIS TOCCombination of MD simulations with machine learning algorithms could revolutionize the antibody-breeding sciences to lead to new antibody discovery that is compatible with or better than naturally occurring antibodies. Topics of Content (TOC)Fingerprints of R86 finger of PD-1 are recognized by our designed P2N_2 anti-PD-1 antibody according to our MD simulations O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/742579v1_ufig1.gif" ALT="Figure 1000"> View larger version (49K): org.highwire.dtl.DTLVardef@130fd5aorg.highwire.dtl.DTLVardef@1495cd8org.highwire.dtl.DTLVardef@16e822forg.highwire.dtl.DTLVardef@25004c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

A conserved vesicular circuit enables metabolic adaptation and maintains systemic heme homeostasis

Metabolic homeostasis depends on adaptive control of intracellular metabolite flux, yet how such control is reconfigured when canonical transport pathways fail is unknown. Here we define a conserved vesicular circuit that preserves systemic heme balance by rerouting intracellular heme flux. We show that loss of the intestinal heme exporter MRP-5 in Caenorhabditis elegans causes lethal heme sequestration within endolysosomal compartments. This defect is bypassed by disabling the vesicular adaptor AP-3, which stabilizes and reroutes the heme importer HRG-1, restoring heme export without increasing cytosolic heme. Unbiased genetics and transcriptomics identify two previously uncharacterized SLC49A family members, HRG-13 and HRG-14, as heme exporters with distinct affinities that engage in a vesicular importer-exporter handoff. Live imaging reveals heme-enhanced contacts between HRG-1 and SLC49A-containing vesicles, consistent with direct vesicular transfer. This circuitry extends to vertebrates as disruption of the SLC49A3 homolog impairs erythropoiesis in zebrafish and causes intracellular heme overload, premature hemoglobinization and apoptosis in differentiating human erythroid cells. Together, these findings establish SLC49A3 proteins as conserved heme exporters and uncover a general principle of metabolic adaptation in which reprogramming intracellular compartmentalization, rather than increasing nutrient supply, restores systemic homeostasis.

cell biology↗

bioRxiv2026

Conserved and species-specific STIM autoinhibition revealed by AlphaFold3-guided dissection

Store-operated Calcium (Ca2+) entry (SOCE), mediated by dynamic STIM-Orai coupling, is crucial for cellular signaling, yet the molecular architecture of STIM autoinhibition remains poorly defined. Using an AlphaFold3-guided functional validation strategy combining FRET-based biosensors, mutagenesis, and Ca2+ imaging, we systematically dissected the CC1-SOAR autoinhibitory interface in both human STIM1 and C. elegans STIM (cSTIM). Our results reveal a conserved hydrophobic core essential for maintaining the resting state in both species. Interestingly, cSTIM possesses an extended polar interaction network that strengthens autoinhibition and correlates with slower activation kinetics. Species-specific polar networks differentially modulate interface stability, providing an additional layer of regulatory tuning. This integrated approach not only validates AlphaFold3 as a powerful tool for studying dynamic regulatory interfaces but also establishes a structural framework for understanding STIM activation and disease-associated mutations, offering mechanistic insights with therapeutic implications for SOCE-related disorders.

developmental biology↗

bioRxiv2026

A Novel Syngeneic Mouse Model to Study Immune Evasion in Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and patient outcomes have remained largely unchanged despite advances in multimodal therapy. Immune checkpoint inhibitors (ICIs), which block the PD-1/PD-L1 axis to restore T cell-mediated anti-tumor immunity, have emerged as a promising treatment strategy. However, response rates remain below 20% in HNSCC, underscoring the need to better understand mechanisms of immune evasion within the tumor microenvironment. Syngeneic mouse models are essential for studying tumor-immune interactions, yet currently available HNSCC models are limited. Here, we report the development of a novel FVB/NJ-derived syngeneic HNSCC model generated from 7,12-dimethylbenz(a)anthracene (DMBA)-induced primary on floor of mouth/buccal tumors, designated FMOC1, FMOC2, and FMOC3 (FVB/NJ Mouse Oral Cancer). In vitro, all FMOC cell lines exhibited robust proliferative capacity with distinct proliferation kinetics. In vivo, all FMOC cell lines exhibited characteristic HNSCC histopathology, including cytokeratin 5 positivity, and were tumorigenic in immunodeficient NCG mice; however, in syngeneic immunocompetent mice, only FMOC1 demonstrated sustained tumor growth at orthotopic and flank sites, whereas FMOC2 and FMOC3 tumors underwent spontaneous regression within 2 weeks, indicating differential immune-dependent tumorigenicity among the lines. Consistent with this, depletion of CD4+ and/or CD8+ T cells restored tumor growth in FMOC2 and FMOC3 models, indicating a critical role for T cell-mediated immunity in tumor suppression. Notably, FMOC1 tumors were responsive to anti-PD-L1 and anti-CTLA-4 therapy, supporting their utility for evaluating immunotherapeutic strategies. Collectively, these findings establish the FMOC model as a novel and versatile platform to study tumor-immune interactions and immune evasion mechanisms in HNSCC, with potential applications in preclinical immunotherapy development.

cancer biology↗

bioRxiv2026

Tailoring Precise Genomic Integration Toward Isolate-to-Industry Strain Development for Scalable High-Titer Production of Polyhydroxyalkanoate

Halophilic chassis has emerged as a promising biomanufacturing platform for industrial polyhydroxyalkanoate (PHA) production. However, challenges still remain in improving the production capacity, scalability and robustness, thereby lowering cost to meet market demands. Here, a high-performing halophilic strain Halomonas LY03 was isolated with over 38% glucose- to-PHA conversion rate and broad non-grain substrate utilization capability. Multidimensional tools, including algorithm-guided high-expression neutral integration site (HENIS) screening toolkit designated SiteSeek, stop codon (TAA)-dependent enhancement of gene expression and recombinase-mediated large-fragment (> 9 kb) genomic integration, were then developed to enable precise, efficient and interference-free genomic integrative expression. Using these tools, various chromosomally engineered strains were rapidly constructed to achieve high-level production of poly-3-hydroxybutyrate (PHB, 151 g L-{superscript 1}) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB, 139 g L-{superscript 1}) under high cell-density fermentation (up to 186 g L-1 cell dry weight) in a 5-L bioreactor. Scalability was demonstrated at 2-m3 and 20-m3 industry-scale fermentations, yielding up to 134 g L-{superscript 1} PHB and 127 g L-{superscript 1} P34HB (6.1 mol% 4HB). Building on the proven robustness, a two-stage continuous fermentation (TCF) process was developed using a twin-bioreactor system at 5-L and 20-m3 scales, where stable and sustained PHA production lasted over 260 h and 160 h, respectively. Techno-economic analysis revealed a substantial cost-reduction space of 48% compared with conventional fed-batch process. This study demonstrates a successful paradigm for engineering a newly isolated strain toward robust, high-titer and cost-competitive PHA production across lab-to-industry scales.

synthetic biology↗

bioRxiv2026

Influence of Primary Coordination Sphere on Anion Rebound Selectivity in Nonheme Fe Enzyme-Catalyzed C(sp3)-H Functionalization: A Comparative Experimental and Computational Study of EgtB and ACCO

Developing enzymatic mechanisms for C-F bond formation remains a long-standing challenge. Here, we repurposed the biosynthetic nonheme Fe enzyme EgtB, which features a three-histidine facial triad, to catalyze C(sp3)-H fluorination reactions. Directed evolution of EgtB afforded two new-to-nature fluorine atom transferases with opposite enantiopreference, EgtBCHF1 and EgtBCHF2, with up to 28-fold improved total activity. In contrast to our previously evolved nonheme Fe fluorine atom transfer biocatalyst ACCOCHF, which contains a two-histidine-one-carboxylate facial triad, the evolved EgtBCHF variants displayed unexpected hydroxylation activity. 18O-labeling experiments showed that the hydroxy group originated from water rather than residual O2. Computational studies suggested that the three-histidine-supported Fe(III) center exhibits enhanced Lewis acidity compared to the two-histidine-one-carboxylate system, allowing deprotonation of Fe(III)-bound water to form a Fe(III)-OH species to catalyze radical hydroxylation. Primary coordination-sphere mutagenesis in EgtB and ACCO further supported the critical role of Fe coordination chemistry in controlling radical rebound reactivity and selectivity. Computational studies revealed that Fe coordination chemistry strongly influences both fluorine atom abstraction and radical rebound, with the intrinsic C-X (X = F, OH, and N3) bond forming radical rebound preference following the order N3 > OH > F. Furthermore, multivariate linear regression analysis revealed that fluorine atom abstraction is primarily governed by the intrinsic Fe-F bond strength, whereas fluorine rebound is predominantly controlled by the electronic structure of the Fe(III) intermediate. Together, these findings provide mechanistic insights into nonheme Fe enzymology and reprogramming toward selective radical rebound reactions, including challenging C-H fluorination. Table of Contents (TOC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/737789v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@143b503org.highwire.dtl.DTLVardef@2102aaorg.highwire.dtl.DTLVardef@135e7fdorg.highwire.dtl.DTLVardef@11b68a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Strict OOD Antigen-to-Antibody Retrieval with CDR-Aware Slot Late Interaction

Antigen-specific antibody retrieval aims to rank candidate antibodies for a target antigen, providing an early virtual-screening step before structural modeling or experimental validation. Existing sequence-based antibody-antigen interaction studies often formulate the problem as pairwise binding prediction, and random or non-clustered evaluations can over-estimate generalization when related antigens appear across training and test data. We study a strict antigen-cluster out-of-distribution (OOD) retrieval setting in which test antigens come from sequence clusters unseen during training. This setting is difficult because binding is driven by local epitope-CDR complementarity, while available databases mainly contain observed positive complexes and lack reliable negative labels for unlabeled candidates. We propose Ab-CASLR, an antibody CDR-aware slot late-interaction retriever that encodes antigens with ESM-2, encodes antibodies with IgBert, constrains antibody-side latent slots to complementarity-determining regions (CDRs), and scores local slot compatibility instead of single-vector global similarity. On a strict OOD benchmark with 849 antigen queries and 869 candidate antibodies, the model achieves 7.42% Hits@10, outperforming k-mer homology transfer at 5.53% Hits@10 and yielding 6.28-fold enrichment over exact random screening at K = 10. Ablations and diagnostics show that CDR-constrained antibody slots remain diverse, whereas antigen-side latent slots collapse into similar summaries. These results support CDR-aware local antibody representation as a useful inductive bias for early binder recovery under strict OOD evaluation, while antigen-side epitope grounding remains unresolved.

bioinformatics↗

bioRxiv2026

Distinct RNA engagements define genome import and replication elongation in alphaviruses

Membrane-associated replication complexes (RCs) are a hallmark of alphaviruses, yet the mechanisms by which viral RNA is delivered into these compartments and coordinated with RNA synthesis remain incompletely understood. Here, we present a cryo-electron microscopy (cryo-EM) structure of the Chikungunya virus (CHIKV) RC core, consisting of nsP1, nsP2, and nsP4, in complex with the replicative RNA substrates, revealing two spatially distinct RNA-binding sites. A single-stranded RNA (ssRNA) engages the nsP2 helicase and extends toward a pore formed at the nsP1-nsP4 interface, whereas a double-stranded RNA (dsRNA) is accommodated within the central catalytic pocket of the nsP4 polymerase in an elongation configuration. Structural analysis suggests a helicase-assisted RNA threading model in which the 5' end of the viral genome is guided toward the spherule lumen through the RC pore. Concurrently, the nsP4 polymerase engages dsRNA in a manner consistent with RNA synthesis. This work provides a structural framework for understanding RNA trafficking and enzymatic coordination in viral replication organelles. Significance StatementLike many positive-sense RNA viruses, alphavirus replication occurs within membrane-bound spherules, but how viral RNA is delivered into these compartments and coupled to RNA synthesis has remained unclear. Here, we show that the CHIKV RC core contains two functionally distinct RNA-engagement modules: an nsP2-associated ssRNA path directed toward the nsP1-nsP4 pore, and an nsP4-bound duplex RNA positioned in an elongation-like polymerase channel. These observations support a model in which alphavirus RCs integrate genome import and RNA synthesis within a single membrane-associated molecular machine. This work provides a structural framework for understanding RNA trafficking, replication-organelle function, and potential antiviral targeting of alphavirus replication.

microbiology↗

bioRxiv2026

Changes in the translational landscape during red clover necrotic mosaic virus infection

Viruses alter host gene expression to create a proviral environment while the host simultaneously regulates gene expression to restrict the virus spread. Owing to RNA virus complete reliance on the host translational machinery, it is important to assess the translational control during virus infection. Therefore, we used ribosome profiling (ribo-seq) paired with RNAseq to observe how red clover necrotic mosaic virus (RCNMV) infection of Arabidopsis plants alters cellular gene expression at the levels of mRNA abundance and translation efficiency. We determined that at 5 days post-inoculation (dpi), the translational response to RCNMV infection is enriched in genes of the innate immune system. Expression of a tumor necrosis factor receptor-associated factor (TRAF)-like protein, a regulator of development and immune response, was translationally but not transcriptionally upregulated early in systemic infection. By 8 dpi, many pathways were regulated/dysregulated, and unfolded protein response (UPR) genes were transcriptionally upregulated but with reduced translation efficiency. Ribosome profiling of RCNMV RNAs revealed (i) -1 programmed ribosomal frameshifting at 7.5-8.0%, the first direct measurement of frameshift efficiency in infected cells for any plant virus; (ii) that coat protein is translated at extremely high efficiency, while the RNA-dependent RNA polymerase is translated least efficiently, and (iii) an unexpected extremely strong ribosomal pause site in the open reading frame that encodes the movement protein. To our knowledge, this is the first genome-wide study that assesses the translational control of gene expression in plants infected with a virus from the large and diverse Tombusviridae family. ImportancePositive strand RNA viruses usurp the hosts translation machinery to synthesize viral proteins. Moreover, translation of host mRNAs is altered by virus infection, both as part of the host immune response and by the virus to inhibit host defenses. To assess all these changes globally, we used ribosome profiling of plants infected with a member of the large and ubiquitous Tombusviridae family. We identified key host genes and pathways that were differentially altered in translation efficiency, giving us an understanding of host responses not detectable by conventional RNA sequencing. Moreover, ribosome profiling revealed (i) the most accurate calculation of efficiency of ribosomal frameshifting during infection for any plant virus, (ii) the extremely high level of translation of viral coat protein, and (iii) an unexpected strong ribosomal pause site in the movement protein gene. This work provides understanding of a new dimension of gene expression control in plant-virus interactions.

microbiology↗

bioRxiv2026

OmicOS: A Comprehensive Omics Ecosystem Infrastructure and Agent System for the AI Era

Biology has accumulated a vast ecosystem of omics methods, but much of this ecosystem remains built for expert humans rather than scientific agents. Methods are scattered across Python packages, R/Bioconductor and CRAN workflows, command-line tools, incompatible data containers and implicit object states, making even routine analyses difficult for an AI system to choose, execute and verify reliably. Here we introduce OmicOS, a comprehensive omics ecosystem infrastructure and agent system that turns OmicVerse V2, an open-source omics community, into an executable foundation for agentic biology. OmicVerse V2 provides the community substrate: scalable AnnDataOOM-compatible rust backends, agent-friendly Python algorithms for single-cell, spatial, bulk and multi-omics analysis, interfaces to single-cell foundation models, and Python-native reconstructions of historically R-centred Bioconductor/CRAN-style workflows. OmicOS makes this substrate actionable by registering analytical functions as state-aware capability contracts, allowing agents to inspect live data objects, select valid methods, execute controlled workflows and record provenance. The result is not a fixed pipeline, but a programmable omics environment in which agents compose real analyses from verified community methods rather than inventing tools. Across external and purpose-built benchmarks, OmicOS ranked first among the evaluated systems, reaching 81.2% on BiomniBench. Adding OmicVerse to a minimal agent improved task completion by up to 34.2 percentage points with qwen-3.6-35b, and controlled ablations showed that the gains came from registry-grounded execution rather than from larger models, documentation retrieval or unrestricted tool exposure. The same infrastructure scaled to atlas-sized data, reproduced R-centred workflows in Python and converted external pathology software into agent-usable skills. In a discovery task starting from a whole-body spatial map and the term "Alzheimers disease", OmicOS composed a non-canonical workflow that integrated spatial expression, genetic association, eQTL and colocalization evidence to nominate a colon epithelial risk axis centred on PICALM, CD2AP and CR1. Together, OmicVerse and OmicOS define an open foundation for AI-era omics, showing how a community of biological methods can be transformed into a reliable, extensible and agent-operable system for discovery. HighlightO_LIOmicVerse 2.0 consolidates 694 methods spanning 11 omics domains into agent-callable high-level APIs. C_LIO_LIRebuildR automatically reconstructs and evolves R/Bioconductor methods as Python-native implementations under output-equivalence gates. C_LIO_LIOmicOS establishes a state-of-the-art omics agent harness, ranking first on general omics benchmarks across models and substantially improving the analytical capability of local open-source models. C_LIO_LICompositional use of ecosystem modules nominates a colon epithelial axis associated with Alzheimers disease risk. C_LIO_LIExternal algorithm packages supporting automatic iterative evolution can be integrated into the OmicOS ecosystem. C_LI

bioinformatics↗

bioRxiv2026

Double-strand break end configuration and 3D genome architecture are crucial for chromosomal translocation

Chromosomal translocations originate from erroneous rejoining of DNA double-strand break (DSB) ends and mechanisms of their formation remain largely unknown. Here we reveal DSB end configuration and three-dimensional (3D) genome architecture cooperatively shape both translocation frequency and junctional indel pattern. By integrating multiplex CRISPR/Cas9 editing with an optimized translocation assay, we find that translocation frequency correlates with spatial proximity. In addition, templated insertions emerge as a defining structural feature of translocation junctions induced by Cas9-mediated staggered cleavage and confirmed by DISCOVER-seq. Interestingly, translocation frequency is correlated with cleavage geometry. Finally, engineered Cas9 variants with altered DSB end configurations enable programmable modulation of junctional indel patterns. These findings shed significant insights into mechanisms of recurrent chromosomal rearrangements in human diseases and have interesting implications in safe applications of genome editing.

cell biology↗

bioRxiv2026

Nature-inspired Nanoparticle Adiposomes Enable Targeted Delivery of Hydrophobic Drug for Anti-Cancer Treatment

The predominance of hydrophobic molecules in drug pipelines poses a fundamental delivery challenge in the aqueous physiological environment. Inspired by the endogenous lipid-transport machinery of lipid droplets and lipoproteins, adiposome was developed as a biocompatible platform for hydrophobic drug delivery. Here, docetaxel (DTX) was selected as a model hydrophobic chemotherapeutic agent to evaluate the therapeutic versatility of adiposome. Non-targeted DTX-loaded adiposomes (DTX-Ad) reduced systemic toxicity by avoiding the allergenic excipients Tween 80 and ethanol used in commercial DTX formulations. The cytotoxic mechanism and intracellular responses of DTX-Ad were comprehensively characterized. To boost antitumor efficacy, three tumor-targeting DTX-Ad formulations were subsequently engineered. Lung-targeting DTX-adiposomes (Lung@DTX-Ad), mediated by AAM-B1-28-CRGDK fusion peptide/NRP1 axis, enhanced antitumor efficacy in a lung metastasis model. BCMA-targeting DTX-adiposomes (BCMA@DTX-Ad), conferred by biotin-avidin-mediated antibody conjugation, achieved sustained suppression in a multiple myeloma model. Liver-targeting DTX-adiposomes (Liver@DTX-Ad), enabled by the ApoE/LDLR axis, outperformed both commercial DTX injection and sorafenib in hepatocellular carcinoma models. Taken together, these results demonstrate adiposome as a promising and broadly applicable drug delivery platform, with considerable potential for clinical translation across diverse malignancies.

cancer biology↗

bioRxiv2026

Engineering xylose metabolism for diverse polyhydroxyalkanoates synthesis in Halomonas TD

Engineering the biosynthesis of fully biodegradable polyhydroxyalkanoates (PHAs) from non-food and renewable feedstocks like lignocellulose is becoming an attractive strategy for sustainable biomanufacturing. However, the efficiency and diversity of PHA synthesis from lignocellulosic hydrolysate (LH), mainly containing glucose and xylose, still remains challenge. Here, Halomonas TD, a cost-effective PHA-producing chassis, was developed to utilize glucose and xylose (or LH) for effective production of poly-3-hydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] by engineering the phosphoketolase pathway-dependent xylose metabolism in the genome, yielding 50.1 g L-1 PHB and 42.6 g L-1 P(3HB-co- 11.4 mol% 4HB) under fed-batch condition. Subsequently, the introduction of Weimberg pathway was found be able to synthesize terpolymer consisting of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate (3HV), namely [P(3HB-co-4HB-co-3HV)], from glucose and xylose only due to the isoenzyme activity of keto-acid decarboxylase encoded by kivD, which converts xylose-derived intermediate 2,5-dioxopentanoate and -ketoglutarate into butanedial (4HB synthesis) and 2-ketobutyrate (3HV synthesis), respectively. Finally, a tailored-made xylose-induced system was constructed to achieve exquisite xylose transmembrane transportation control for improved synthesis of terpolymer P(3HB-co-4.5 mol% 4HB-co- 3.0 mol% 3HV), reaching to 6.3 g L-1 under shake-flask condition, together with the co-expression of fine-tuned phosphoketolase and Weimberg pathways. This study provides a feasible and sustainable alternative for lignocellulosic resources valorization powered by the engineered Halomonas TD capable of efficient xylose utilization and diverse PHAs synthesis.

bioengineering↗

bioRxiv2026

Interplay of stability and dynamics in the optimization of a highly proficient de novo enzyme

The development of highly active de novo enzymes that catalyze new-to-nature reactions is becoming increasingly possible. Here, we identify the features underlying the catalytic efficiency of a highly proficient de novo enzyme, from the original computational design to an optimized catalyst obtained through two rounds of directed evolution. Computational, spectroscopic, and biochemical studies reveal successfully designed features, including precise alignment of catalytic residues, transition state stabilization, and environmental tuning. In the most evolved enzyme, binding of a transition state analog also led to widespread increases in backbone conformational stability throughout the protein, except within a helix near the active site entrance, where the introduction of Gly and Pro increased dynamics and catalytic activity. Thus, the entire protein contributes to catalysis in the most optimized enzyme. Also, the initial design considered only the transition state but not substrate binding, leading to a dynamic Michaelis complex prior to optimization. These studies show the multiple features that need to be optimized to achieve high activity in a designed enzyme.

biochemistry↗

bioRxiv2026

Fully Modified SpyCas9 Guide RNAs Enable Robust Genome Editing In Cells and In Vivo

Precision engineering of CRISPR/Cas9 components has advanced genome editing toward therapeutic applications. Completely chemically stabilized guide RNAs (gRNAs) have the potential to improve in vivo editing efficacy while enabling greater flexibility in delivery strategies. However, previous generations of fully modified guides have been associated with reduced Cas9 activity. Here, we employed an iterative, structure-guided optimization strategy to systematically introduce chemical modifications at each position of SpyCas9 gRNAs. Extending beyond commonly used nucleotide modifications, we incorporated 2-amino-RNA, 4-thio-RNA, and extended nucleic acid (exNA) to generate gRNA designs in which 90-100% of the nucleotides are sugar- or backbone-modified. Although certain modification patterns exhibit sequence-dependent variability, we have established a growing repertoire of guides that consistently maintain or enhance editing efficacy when applied both in vitro and in vivo. Collectively, our heavily and fully modified gRNAs hold potential for applications in nuclease editing, base editing, and other genome editing tools. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/725424v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1118f08org.highwire.dtl.DTLVardef@1c57069org.highwire.dtl.DTLVardef@1572148org.highwire.dtl.DTLVardef@14a111e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

cGAS bends unpaired DNA to form an unconventional structure that hyperactivates the innate immune response

cGAS is a pattern-recognition receptor for dsDNA and forms 2cGAS:2DNA dimers followed by oligomerization into phase-separated condensates when fully-complementary DNA is studied. However, many DNAs are not fully complementary. We report that DNA with unpaired regions such as those found during transcription, recombination or replication (designated as bubble-DNA, Bu-DNA) causes cGAS hyper-activation. Hyperactivation is observed by Bu-DNA embedded in linear DNA, circular DNA, plasmid DNA and mitochondria DNA. Bu-DNA binds significantly more tightly to the cGAS catalytic domain than paired-DNA but suppresses condensation. Cryo-EM and single-molecule FRET reveal that cGAS forms 2cGAS:1DNA complexes by bending Bu-DNA into a V-shape using the unpaired region as a hinge, limiting its oligomeric state. This uncovers a novel mode of cGAS activation attributed to pattern diversity within pattern ligands.

immunology↗

bioRxiv2026

Simultaneous brain-wide single-cell recording resolves spatiotemporal memory architecture

Many fundamental mammalian brain functions emerge from the coordinated activity of cells distributed across large, brain-wide networks. To understand these processes in healthy and diseased states, ideally one would simultaneously measure and analyze single-cell activity at the brain-wide scale, an enduring challenge for live-measurement approaches that often face an inherent tradeoff between spatial resolution and scale. Here, we present GLOBE (sinGle-cell spatiotemporaL recOrding Brain-widE), a technology for brain-wide single-cell recording of cellular activity in vivo with spatiotemporal resolution, physiological sensitivity, and parallelization-accelerated readout. GLOBE leverages genetically encoded intracellular protein tape recorders and a high-throughput computational platform for integrated image and signal analysis. GLOBE records analog signal amplitudes across a continuous time axis, requires only standard light microscopy for in situ readout, and is compatible with expansion microscopy and RNA readouts. We applied GLOBE to simultaneously record transcriptional activity of the immediate early gene Fos in up to 219,703 neurons simultaneously across a single mouse brain over 5.5 continuous days, with a timestamp precision of 3.1-6.7 hours (median absolute error), a local recording density of 69-90% of neurons per imaging field of view, and a post-mortem imaging readout speed of 2.9 seconds per neuron on average. GLOBE resolves the brain-wide spatiotemporal structure of single-cell activity, revealing that Fos transcriptional dynamics associated with fear learning and memory retrieval are distributed across the brain with region-specific temporal heterogeneity, and that the variance of this structure scales down as the number of sampled cells increases. We envision GLOBE to have broad applications for dissecting and decoding physiological and pathological processes at the brain-wide scale.

neuroscience↗