Biology subjects

Jin, H.

Publications and source records attributed to Jin, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Expanding the therapeutic window of bacteria for cancer therapy

Engineered bacteria enable self-amplifying therapeutic delivery and immune activation, yet these same features drive systemic toxicity and off-target growth. Here, we create a "tumorobe" that overcomes these limitations by integrating reduced immunogenicity, tumour-restricted growth, and enhanced payload release. Specifically, we identify lipopolysaccharide modifications that reduce immunogenicity while preserving fitness, restrict growth to tumours using a hypoxia-lactate "AND" gate, and enhance extracellular payload release via lipoprotein deletion. This strain expands the maximum tolerated dose in a route-dependent manner by up to 1,000-fold. Across multiple murine models, including colorectal cancer and glioblastoma, the strain drives robust antitumour efficacy as both a standalone therapeutic and an immunotherapeutic delivery platform, achieving durable complete responses in up to 75% of colorectal tumours when armed with a cytokine payload. Together, these results establish a broadly applicable platform for safe and effective bacterial cancer therapy.

synthetic biology↗

bioRxiv2026

Engineered probiotics target granulomas to treat mycobacterial infection

Granulomas are a pathological niche for mycobacteria that remain difficult to access with conventional therapies. Here we show that probiotic Escherichia coli Nissle 1917 (EcN) infiltrates and colonizes granulomas and can be engineered as a delivery system that senses the local microenvironment and releases antimycobacterial payloads. Specifically, we leveraged the hypoxic microenvironment of necrotic granulomas to restrict bacterial growth, and developed a dual-mode lysis circuit combining intracellular and extracellular killing for sustained payload release with limited evolutionary escape. We further engineered EcN to express a mycobacteriophage-derived enzyme, generating a strain that suppressed drug-sensitive and drug-resistant mycobacteria in vitro and potentiated conventional antibiotic activity. Engineered probiotics also reduced mycobacterial burden in vivo, and in a murine model of rifampicin-resistant infection, combination treatment with pretomanid achieved greater bacterial reduction than either monotherapy. Together, these results establish a synthetic biology framework for targeting granulomatous infections with programmable living microbes.

synthetic biology↗

bioRxiv2026

Structural basis for differential inhibition of THIK1 by chemically related inhibitors

TWIK-related halothane-inhibited potassium channel 1 (THIK1) is a two-pore domain potassium (K2P) channel highly expressed in microglia that contributes to microglial homeostasis and neuroinflammatory responses. Pharmacological inhibition of THIK1 suppresses tonic K+ currents and attenuates NLRP3-dependent IL-1{beta} release, highlighting THIK1 as a potential therapeutic target for neuroinflammatory disorders. However, the structural basis of THIK1 inhibition remains unclear. Here we report cryo-electron microscopy (cryo-EM) structures of full-length human THIK1 in complex with C101248, a commercially available tool compound, and CVN293, a clinical-stage inhibitor that has completed Phase I evaluation. The two inhibitors occupy a similar site within the inner vestibule formed by TM2 and TM4 beneath the selectivity filter. Consistent with their closely related chemical scaffolds and shared binding site, both inhibitors remodel the Y273 inner gate but differ in their effects on the C-terminal gate. Electrophysiological analyses further demonstrate that the C-terminal gate contributes differently to inhibition by the two compounds. Together, our findings show how chemically related inhibitors exert different effects on THIK1 despite engaging a common binding site.

biophysics↗

bioRxiv2026

π-MNovo improves de novo peptide sequencing through microbial-domain adaptation and evidence-guided candidate selection

The high taxonomic and strain-level diversity of microbial communities makes it difficult for reference databases to fully represent the protein sequences present in metaproteomic samples, limiting database-dependent peptide identification. De novo peptide sequencing can recover peptide sequences directly from tandem mass spectra without relying on reference databases, providing complementary peptide evidence for metaproteomics. However, most existing de novo sequencing models were developed largely from non-microbial proteomic data and lack specific adaptation to microbial spectra. Here, we established a dedicated microbial spectral resource comprising more than 10 million annotated high-quality tandem mass spectra from 72 cultured microbial isolates, with peptide digests from each isolate separated into five high-pH reversed-phase fractions to increase the opportunity for deeper and more diverse peptide sampling. Using this resource, we developed {pi}-MNovo, which combines adaptation to microbial spectra with evidence-guided candidate selection to improve full-length peptide sequencing precision. On a seven-species external benchmark, {pi}-MNovo achieved 66.30% complete-peptide recall under the same residue-mass-based complete-peptide matching criterion applied to all models, exceeding four published models by 6.9-21.7% in relative recall, with consistent gains across species and peptide-length groups. In two independent metaproteomic datasets, {pi}-MNovo increased reference-supported taxonomic coverage, while synthetic-community analysis recapitulated the designed abundance ranking. From pFind-unidentified spectra, {pi}-MNovo recovered 1,587 pFind-unreported, reference-matched peptides that passed spectrum-evidence filtering. These results show that {pi}-MNovo expands recoverable peptide evidence and enhances the utility of de novo sequencing for metaproteomic analysis.

bioinformatics↗

bioRxiv2026

Correlation-aware discovery of co-occurring mutational signatures in cancer

Somatic mutations in cancer genomes record the activities of diverse mutational processes. Mutational signature analysis has advanced mechanistic understanding of mutagenesis and informed clinical decision-making, yet existing methods assume independence among signatures--an unrealistic assumption that can produce composite or contaminated signatures, reduce detection power, and yield inconsistent results. Here we present Cornet (CORrelated NMF ExTraction), a framework for mutational signature discovery that explicitly models co-occurring processes and jointly infers signatures and their correlation structure. Benchmarking on simulated data shows Cornet more accurately recovers distinct signatures under strong correlations. Applied to cancer genomes, Cornet enables unsupervised discovery of the colibactin-associated signature SBS88 in oral cancers and identifies the tobacco smoking signature SBS4 in bladder cancer, where it was previously thought absent. Cornet also uncovers a novel mutational process implicated in early-onset colorectal cancer and a signature arising from the interplay between tobacco smoking and ERCC2-mutation-driven nucleotide-excision repair deficiency. Together, these results demonstrate that modeling correlations among mutational processes is essential for high-resolution signature discovery and dissecting the mutational etiology of human cancer.

bioinformatics↗

bioRxiv2026

Deep reinforcement learning-driven discovery of a MsbA-targeted small-molecule antibiotic for the treatment of Acinetobacter baumannii infection

Antibiotics with new mechanisms are highly pursued to address the threat of infections caused by drug-resistant Gram-negative bacteria. Targeting MsbA, a key protein of the lipopolysaccharide biosynthesis pathway, represents a promising strategy to discover new classes of antibiotics. However, currently available MsbA-targeted molecules either lack sufficient potency or have unfavorable properties, necessitating expansion of chemical space. In this study, we chose the most promising cerastecin Cpd 4 as the template, and used two Artificial Intelligence (AI)-based tools, i.e. Link-INVENT and AutoMolDesigner for molecular design, performed chemical derivatization and antibacterial activity evaluation, which led to the discovery of Y-11 (MIC for A. baumannii: 0.5 g/mL). Encouragingly, Y-11 showed equivalent potency to Cpd4 for carbapenem-resistant A. baumannii, and less cytotoxicity and hemolysis as well as lower spontaneous resistance frequency. In vivo efficacy study demonstrated that Y-11 could effectively reduce bacterial loads in the mice infected by A. baumannii. The following mechanism study including molecular dynamics simulation, biochemical assay, and transmission electron microscope (TEM) analysis suggested that Y-11 inhibited the transport of lipooligosaccharide and impaired the formation of outer membrane, probably by competitively binding to the substrate binding site of MsbA and modulating ATPase activity. Taken together, we have discovered a MsbA-targeted small molecule Y-11 via AI-driven drug design, which provides a foundation for future antibiotic development.

biochemistry↗

bioRxiv2026

A shear lag model of the podocyte foot process network predicts a mechanical feedback loop driving progressive effacement

The podocyte foot process network forms the final barrier of the kidneys glomerular filtration system. Under mechanical stress this network is prone to injury in which podocytes lose connectivity to their neighbors and begin the progression toward effacement, but what governs its mechanical resilience is unknown. We show that the network is built like a lap joint: two major processes coupled through interdigitating foot processes, a configuration that behaves as a classical shear lag system, with force concentrating at the joint ends and decaying over a characteristic transfer length set by geometry and stiffness. A discrete network model reproduces the continuum shear lag solution and identifies a hierarchy among governing parameters, with cytoskeletal stiffening of the major process amplifying foot process force more potently than basement membrane stiffness. Applying the model to morphometric data from puromycin aminonucleoside nephrosis, a model of human minimal change disease and early focal segmental glomerulosclerosis, reveals a mechanical positive feedback loop: force concentration drives foot process loss, which raises force on surviving segments and accelerates further loss. This nonlinear amplification implies a threshold beyond which failure becomes self-sustaining, analogous to the critical crack length in fracture mechanics.

biophysics↗

bioRxiv2026

Cobamide-Dependent Dichloromethane Fermentation by Dehalobacter Reveals a Hidden Acetogenic Route for Organohalide Biotransformation

Halogenated one-carbon (C1) compounds, such as dichloromethane (DCM), drive critical fluxes in global carbon and halogen cycles. While the genus Dehalobacter is canonically defined by obligate organohalide respiration, its physiological and ecological roles in anaerobic C1 metabolism have remained fundamentally ambiguous. Here, we document a paradigm-shifting metabolic capacity within a sediment-derived microbial consortium: the autonomous fermentation of DCM by a novel population, Candidatus Dehalobacter formatiformans strain J1. Over successive transfers, strain J1 outcompeted co-existing Dehalobacterium formicoaceticum to become the overwhelmingly dominant population (>80% relative abundance), converting DCM stoichiometrically to acetate and formate (4:1) without auxiliary substrates. Genome-resolved metagenomics revealed that strain J1 couples a distinct mec gene cassette--mediating methyl-transfer reactions during DCM activation--to a complete Wood-Ljungdahl pathway for efficient C1 assimilation. Crucially, strain J1 lacks the complete genetic repertoire for de novo cobamide biosynthesis. Physiological validation confirmed that this fermentative pathway is strictly dependent on exogenous cobamides, exposing a profound reliance on community cross-feeding. These findings reveal an unexpected acetogenic lifestyle within Dehalobacter, a lineage historically viewed as comprising obligate organohalide-respiring bacteria. More broadly, this work identifies cobamide-dependent methyl-transfer metabolism as an ecological control on anaerobic DCM fermentation and expands the known roles of Dehalobacter in carbon-halogen cycling in anoxic environments.

microbiology↗

bioRxiv2026

Paving the way for plant-based bioproduction of (Z)-13-octadecenoic moth pheromone compounds

Sex pheromones are eco-friendly agents to control pest insects. Yet, the cost of synthetic pheromone production has restricted their use essentially to high value crops. Plant-based bioproduction of pheromone compounds offers a promising and sustainable alternative, opening up for the deployment of pheromone-mediated pest control also in row crops. The rice leaffolder Cnaphalocrocis medinalis uses C18 pheromone compounds and is a major rice pest in South China. We demonstrate that the fatty acid precursor of the main C18 pheromone component of C. medinalis, (Z)-13-octadecenal, can be biotechnologically produced using the oilseed crop Camelina sativa. First, we used deuterium-labelling to establish an elongation-based pathway for pheromone precursor formation from hexadecanoic (palmitic) acid via desaturation to (Z)-11-hexadecenoic acid and elongation to (Z)-13-octadecenoic acid. Using transient expression in Nicotiana benthamiana, we identified a minimal pathway consisting of a {Delta}11 desaturase (CmedDES1), an elongase (CmedELO1), and a fatty acyl reductase (CmedFAR2) for the heterologous biosynthesis of long-chain pheromone monounsaturated C18 precursors in planta. Co-expression of the insect desaturases CmedDES1 or CsupYPAQ with the Camelina elongase CsaKCS1 in Nicotiana benthamiana resulted in higher production of Z13-18 products compared to co-expression with insect elongases. We finally engineered Camelina sativa seeds through seed-specific co-expression of the Z11-16-forming insect desaturase CsupYPAQ and CsaKCS1 elongase, enabling the accumulation of C18 fatty acid pheromone precursors. Our study paves the way for sustainable production of the sex pheromone of C. medinalis and other C18 monounsaturated pheromone precursors for crop protection.

synthetic biology↗

bioRxiv2026

A Conserved Obg-type GTPase sustains protein synthesis and its physiological roles in eukaryotic cells

Translation is regulated both temporally and spatially to ensure that proteins are synthesized at the correct time and delivered to the proper subcellular location. Temporary slowing or halting of ribosomal movement along an mRNA, a physiological pausing, allows cells to coordinate protein production with internal cues and external signals. However, how cells distinguish such constructive pauses from harmful pathological stalls remains a fundamental unresolved question in molecular biology. Here, we report that a ribosome-binding GTPase (Rbg in yeast, and so-called Developmentally Regulated GTP-binding (Drg) in human) promotes protein biosynthesis in halted ribosomes in an evolutionarily conserved manner. We show that the essential bacterial Obg GTPase rescues cell growth and translation in Saccharomyces cerevisiae and human cells lacking their endogenous Rbg/Drg proteins in a GTPase-dependent manner. Furthermore, Obg enhances puromycin incorporation into stalled ribosomes in a manner requiring GTP hydrolysis. We reason that Rbg/Drg proteins act to prevent premature engagement of ribosome-associated quality-control or stress-response pathways. Under physiological conditions, proteomic analyses of Rbg1-bound ribosomes in yeast reveal that Rbg GTPases associate with ribosomes translating proteins essential for diverse functions, including those of the nucleus, ER, Golgi, mitochondria, and protein transport pathways. Finally, loss of even one of the two eukaryotic Rbg/Drg paralogs impedes cell-cycle progression, disrupts DNA replication, and compromises mitochondrial function. Together, these findings suggest that Rbg/Drg proteins enable cells across different domains of life to protect physiological ribosomal pauses, revealing a previously unrecognized layer of regulation in gene expression. Significance StatementHow cells distinguish productive ribosomal pauses from harmful stalls remains unclear. We identify a ribosome-binding GTPase--Rbg in yeast and Drg in humans--as a conserved regulator that protects physiological pauses and sustains protein synthesis. Remarkably, bacterial Obg functionally substitutes for eukaryotic Rbg/Drg in both yeast and human cells, revealing deep evolutionary conservation of this mechanism. Our data show that Obg promotes continued peptide-bond formation in a GTPase-dependent manner. Positioned near the ribosomal GTPase-associated center, these GTPases are also poised to compete with stringent-response and stress-activation factors that trigger translation shutdown. Together, the findings suggest that translation is controlled not only by GTPases that drive protein synthesis forward but also by Rbg/Drg GTPases that protect the catalytic cycle against premature arrest.

biochemistry↗

bioRxiv2026

Topographical archetypes of somatic mutagenesis in cancer

The genome of every cancer cell carries a record of the mutational processes that have acted throughout its history. Mutational signature analysis, which infers the activity of mutagenic processes from their characteristic base-change patterns, has become an indispensable tool for interpreting somatic mutations. However, this framework captures only which types of mutations a process generates and not where in the genome they occur -- a distribution influenced by replication timing, chromatin organization, transcription, DNA secondary structure, and other genomic features. Here, we present a generative probabilistic framework (MuTopia) that jointly infers mutational spectra and their genome-wide topography as nonlinear functions of genomic and epigenomic state. Applied to whole-genome sequencing data from 15 tumor types, MuTopia reveals that mutational processes fall into eight conserved topographic archetypes, or topotypes, shaped primarily by replication timing and chromatin state. Diverse mutational processes converge upon this limited repertoire, indicating that the genomic distribution of mutagenesis is constrained less by the source of damage than by how that damage is processed. Individual mutational processes exhibit state-dependent variation in their genomic distributions: the same signature can adopt distinct topotypes depending on repair proficiency and replication stress. For instance, SBS8 shifts from a canonical late-replicating profile in homologous recombination-proficient tumors to an early-replicating, stress-associated topotype in HR-deficient tumors, and replication stress similarly reshapes the genomic distribution of APOBEC editing. Topotypes, therefore, provide a classification of mutagenesis distinct from spectral signatures, capturing aspects of tumor biology that spectra alone cannot resolve.

bioinformatics↗

bioRxiv2026

B-Lactamase-Responsive Probe for Rapid and Non-invasive Detection of Tuberculosis through Induced Breath Analysis

Pulmonary tuberculosis (PTB) remains a major global public health challenge. Existing diagnostic approaches are generally limited by suboptimal sensitivity, prolonged turnaround times, high costs, and reliance on sputum samples. These constraints hinder large-scale implementation in resource-limited settings and substantially impede early screening and timely intervention. Targeting the characteristic expression of {beta}-lactamase in Mycobacterium tuberculosis, this study employed systematic screening of a molecular compound library and identified cefapirin sodium as a specific molecular probe. Upon selective hydrolysis by {beta}-lactamase, the probe releases hydrogen sulfide (H2S), which can be detected using a highly sensitive H2S sensing system, thereby enabling rapid and noninvasive identification of the pathogen. Gas chromatography-mass spectrometry (GC-MS) and colorimetric assays were used to validate the specificity of the enzymatic reaction and to confirm the accuracy of the generated product, collectively demonstrating the feasibility of the proposed detection platform. This method offers several advantages, including noninvasiveness, rapid response, and low cost, making it particularly suitable for application in resource-constrained regions. It provides a promising new strategy for early, point-of-care, and large-scale screening of pulmonary tuberculosis, with important implications for improving TB control efforts in high-burden settings.

bioengineering↗

bioRxiv2026

Homologous and Heterologous Immunization with a PIV5-Based Modified OspA Vaccine Confers Equivalent Protection Against Tick-Transmitted Borrelia burgdorferi

Vaccines targeting outer surface protein A (OspA) of Borrelia burgdorferi protect against Lyme disease by eliciting antibodies that neutralize spirochetes in the Ixodes scapularis tick midgut during engorgement before transmission occurs. We evaluated whether different delivery vehicles and administration routes for a modified OspA construct (OspABPBPk) differed in protective efficacy. Four groups of nine C3H-HeN mice were immunized with OspABPBPk delivered intranasally by live parainfluenza virus 5 (PIV5-OspABPBPk) or by subcutaneous administration of recombinant protein adjuvanted with aluminum hydroxide (rOspABPBPk) using three prime-boost regimens and a vector/adjuvant control: homologous intranasal PIV5-OspABPBPk (IN/IN), homologous subcutaneous rOspABPBPk (SC/SC), and heterologous intranasal PIV5-OspABPBPk prime/subcutaneous rOspABPBPk boost (IN/SC). Mice were challenged on day 90, approximately 2 months after the boost, with nymphal ticks infected with 19 strains of B. burgdorferi. All OspABPBPk-containing vaccines elicited high antigen-specific IgG antibody titers (geometric mean titers of 10^4-10^6, approximately 4 log10 higher than the control group), reduced B. burgdorferi loads in engorged nymphal ticks by 1.7-2 log10, neutralized B. burgdorferi motility in multi-strain cultures, and prevented recovery of viable spirochetes from tissues after tick challenge. While one mouse (1/9, 11%) in the heterologous IN/SC vaccinated group had detectable flaB DNA in tissues and seroreactivity to B. burgdorferi PepVF, culture of spirochetes from heart and bladder showed no evidence of active infection. Although homologous immunization produced the most consistent results in mice, different delivery vehicles and routes of immunization with OspABPBPk vaccines did not affect overall efficacy as assessed by recovery of viable B. burgdorferi from tissues.

microbiology↗

bioRxiv2026

Gene conversion is a key driver of diversity hotspots in M. tuberculosis antigens and virulence-associated loci

Despite the long-held view of Mycobacterium tuberculosis (Mtb) as a genetically conserved pathogen, many genomic regions remain poorly resolved due to high sequence homology and repetitive content. Using complete genome assemblies generated from long-read sequencing of 151 globally representative clinical isolates, we comprehensively analyzed genome-wide patterns of genetic diversity and evolution across the Mtb genome. Our analysis uncovers pronounced diversity hotspots within paralogous regions generated by recurrent gene conversion between homologous genes. In many cases, these hotspots exhibit more than an order of magnitude greater genetic diversity than the rest of the Mtb genome, which is otherwise characterized by remarkably low variation. Mutations within these regions display clustered substitution patterns, excess paralog-matching variants, and distinct mutational spectra consistent with ongoing gene conversion. Our analysis identifies over 300 individual gene conversion events distributed throughout the Mtb phylogeny. These gene conversion events occur predominantly within gene families associated with virulence and host-pathogen interactions, including the PE, PPE, and ESX families. Several of the most pronounced diversity hotspots occur in antigens encoded within paralogous regions. Among these, the vaccine candidate PPE18 harbors mutations in validated epitope sequences and predicted alterations in HLA-II binding. Together, these findings demonstrate that gene conversion actively shapes antigenic and virulence-associated diversity in Mtb.

genomics↗

bioRxiv2026

Developmentally Regulated GTP-binding Protein Drg1 defines a translational decision point that protects mitochondrial integrity

Coordination of the functionalities among key organelles is essential for maintaining the complexity and adaptability of the eukaryotic cell. Functionally versatile proteins, such as GTPases, often assume important roles in this process. A ribosome-binding GTPase called Developmentally-regulated GTP-binding (Drg) protein is a conserved family of GTPases recently implicated in promoting protein synthesis and regulating cytoskeletal dynamics, suggesting their importance for cell growth and proliferation. Here, we show that Drg1, one of the two Drg paralogues in eukaryotic cells, is critical for maintaining mitochondria morphology, dynamics, and function. Results from APEX2-proximity labeling show that Drg1 is in proximity to proteins associated with the nucleus, mitochondria, endoplasmic reticulum membrane, cytoplasmic ribosomal subunits, and cytoskeleton components. We demonstrate that Drg1 associates with the outer mitochondrial membrane and that loss of Drg1 leads to reduced mitochondrial membrane potential, decreased protein import, and ATP production. Data from our mammalian in vitro translation assay show that the presence of Drg1/Dfrp1 increases puromycin incorporation in the stalled ribosome, indicating a molecular mechanism of enhanced translation. Data from candidate approaches further reveal that loss of Drg1 leads to changes in mRNA abundance and translation of proteins critical for mitochondrial ATP production, fusion, and fission. Thus, at the molecular level, loss of cellular homeostasis that is caused by compromised translation of cytoplasmically synthesized proteins functionally related to mitochondria underpins the observed phenomenon. Given the evolutionary conservation of Drg proteins, our findings suggest that this mechanism is broadly shared across other living organisms. Scientific SignificanceMitochondria are essential organelles that not only generate ATP but also coordinate numerous cellular processes vital to cell survival and adaptation. Most mitochondrial proteins are synthesized in the cytoplasm and imported into the organelle. Our study reveals that the conserved ribosome-binding GTPase Drg1 facilitates translation of mitochondrial proteins at the outer mitochondrial membrane. By coupling cytoplasmic protein synthesis with mitochondrial function, Drg1 plays a critical role in maintaining mitochondrial morphology, dynamics, and functions. These findings provide new insights into the coordination between cytoplasmic translation, protein homeostasis, and organelle function, which informs future studies on diseases involving mitochondrial dysfunction and defective proteostasis.

molecular biology↗

bioRxiv2026

Small molecule agonists of 8-oxoguanine DNA glycosylase, OGG1

Base excision repair (BER) is the primary pathway that removes oxidatively-induced DNA base damage from the nuclear and mitochondrial genomes, with 8-oxoguanine DNA glycosylase (OGG1) initiating repair at the two most frequently-formed base lesions: 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoGua) and 2,6-diamino-4-oxo-5-formamidopyrimidine (FapyGua). Humans expressing a catalytically-compromised variant of OGG1 (S326C) are at increased risk for type 2 diabetes, Alzheimers disease, and Parkinsons disease. To potentially enhance the overall catalytic efficiency of this variant, a prior medicinal chemistry screen discovered seven chemically distinct agonists of OGG1 that stimulated activity in vitro and attenuated a paraquat (PQ) challenge in cultured cells. Herein, we developed structure-activity relationships around one specific core structure, F01. Using fluorescence-based DNA cleavage assays, we assessed the abilities of these compounds to stimulate the overall rate of OGG1 catalysis. Multiple compounds were identified that increased OGG1 activity on DNAs containing a site-specific 8-oxoGua by 2-fold or greater, with 9 compounds showing EC50 concentrations lower than F01 and were specific for OGG1. Selected agonists were shown to enhance OGG1-catalyzed release of 8-oxoGua and FapyGua from {gamma}-irradiated high-molecular-weight DNA using gas chromatography tandem mass spectrometry analyses. Since these assays did not reveal which step in the overall reaction was stimulated, we used a separation-of-function OGG1 mutant that possessed glycosylase, but not abasic-site (AP) lyase activity to demonstrate that the glycosylase step was not enhanced. In contrast, all agonists stimulated the AP lyase activity to levels equal to or greater than the magnitude of stimulation observed for overall chemistry, implicating agonist-mediated turnover as a potential contributor to the overall rate stimulation. The biological activities of selected agonists were evaluated in OGG1-deficient Kasumi-1 cells under conditions of paraquat (PQ)-induced oxidative stress, with several compounds mitigating PQ challenge.

biochemistry↗

bioRxiv2025

Differential Cerebral White Matter Tract Alterations in Generalized Anxiety Disorder Revealed by Ultra-High Field 7T Diffusion-Weighted Imaging

BackgroundGeneralized anxiety disorder (GAD) is characterized by chronic worry and emotional dysregulation, yet its underlying white matter (WM) architecture remains inconsistent in previous neuroimaging studies. This study aimed to delineate microstructural WM alterations in GAD using ultra-high field (7T) diffusion tensor imaging (DTI) and advanced correlational tractography, evaluating their associations with symptom severity and diagnostic-aided value. MethodsEighty-eight participants (27 GAD, 61 healthy controls, HCs) underwent 7T DTI scanning with 1.5-mm isotropic resolution. Whole-brain correlational tractography was applied to identify tracts exhibiting significant group-related differences in diffusion indices while controlling for demographic covariates. Associations with Hamilton Anxiety Rating Scale (HAM-A) and Penn State Worry Questionnaire (PSWQ) scores were examined, and machine learning-based models were used to assess the diagnostic-aided utility of identified WM features. ResultsTwo tract bundles showed significant fractional anisotropy (FA) alterations in GAD: decreased FA in the right prefrontal pathway (PFDR = 0.039) and increased FA in the right cingulum (PFDR < 0.001). The anterior portions of both tracts exhibited stronger effects of GAD. FA in the right cingulum positively correlated with HAM-A and PSWQ scores, indicating that greater microstructural coherence was associated with higher anxiety and worry severity (both PFDRs < 0.001). Inclusion of WM features could improve classification performance beyond neuropsychological measures alone. ConclusionsUltra-high field 7T tractometry revealed a differential pattern of WM abnormalities in GAD, suggesting weakened prefrontal control and hyper-integrated cingulate connectivity as structural correlates of emotional dysregulation. These findings indicate the potential of 7T DTI markers for advancing mechanistic and diagnostic understanding of GAD.

neuroscience↗

bioRxiv2025

Automated, modular assembly of reconstituted cell-free systems from in vitro-produced components

High-performance cell-free protein synthesis has transformative potential for synthetic biology, yet the prohibitive costs of PURE kits and the labor intensity of in-house preparation have restricted accessibility and scalability. We developed i-POPFLEX (Purified components OPtimized for FLEXible protein expression), a modular cell-free protein synthesis system in which 34 translation factors, individually synthesized in vitro, are assembled using automated liquid handling. This workflow minimizes manual input and supports parallelized production, generating complete, ready-to-use systems within two days. i-POPFLEX achieves up to 5-fold higher protein yields and a 95 % cost reduction (20-fold lower cost) compared with commercial kits. Its flexible architecture also enables selective component inclusion for genetic code reprogramming and site-specific incorporation of non-canonical amino acids. By coupling modular design with automation, i-POPFLEX provides an accessible, customizable, and economically viable platform for next-generation biomanufacturing workflows. Technology readinessi-POPFLEX is a reconstituted cell-free system (CFS) composed of 34 translational components that are individually synthesized in vitro and assembled by automation. This system achieves up to 5-fold higher protein yields with a 95 % cost reduction (20-fold lower; <USD 0.06 {micro}L-1) compared with commercial PURE kits (USD 1.36 {micro}L-1) and reduces preparation time from four days to two. The workflow has been validated across diverse protein and peptide synthesis applications, including genetic code reprogramming and site-specific incorporation of non-canonical amino acids, and integrates seamlessly with benchtop automated liquid-handling platforms. These capabilities place i-POPFLEX at Technology Readiness Level (TRL) 4-5: integrated components validated in a laboratory setting and advancing toward pilot-scale readiness. For full-scale deployment, several key challenges remain. Matching crude lysate costs (USD 0.03 {micro}L-1) will require bulk reagent production, optimized procurement strategies, and specialized equipment for high-throughput component synthesis. Enabling milliliter- and liter-scale production will demand improvements in upstream synthesis, downstream purification, and quality control to ensure consistent performance at industrial throughput. Overcoming these barriers would enable production of thousands to millions of fully assembled i-POPFLEX systems for biofoundry-scale enzyme engineering, metabolic pathway prototyping, and large-scale variant screening, democratizing reconstituted CFS technology, and accelerating adoption in next-generation biomanufacturing pipelines.

synthetic biology↗