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bioRxiv2025

Light and chloroplast redox state modulate the progression of tobacco leaf infection by Pseudomonas syringae pv tabaci

Light significantly influences plant stress responses, with chloroplasts playing a pivotal role as both energy providers and light sensors. They communicate with the nucleus through retrograde signals, including secondary metabolites and reactive oxygen species (ROS). To investigate the contribution of chloroplast redox biochemistry to biotic responses, we studied the interactions of tobacco leaves expressing the alternative electron shuttle flavodoxin with virulent and nonhost Pseudomonas syringae pathovars under light and dark conditions. Flavodoxin is reported to limit light-dependent ROS propagation and over-reduction of the photosynthetic electron transport system under stress. Light intensified the hypersensitive response against the nonhost pathovar tomato (Pto), but slowed disease progression caused by the virulent pathovar tabaci (Pta). Flavodoxin mitigated light responses during both interactions, including decreased ROS levels, reduced stromule occurrence, and lower phytoalexin production, with different signatures depending on the pathovar. Similar leaf metabolic profiles were observed in the dark for both strains, with a general up-regulation of sugars, metabolic intermediates, and amino acids. In the light, instead, Pta increased sugars and intermediates, while Pto decreased them. Our results suggest that HR-like responses are elicited in the light even during virulent interactions, and that light effects are related to signals originating at the photosynthetic machinery. Highlights- Light inhibits disease progression during a tobacco-Pseudomonas virulent interaction. - Light exacerbates the hypersensitive response (HR) during a nonhost interaction. - HR-like responses are elicited in the light even during virulent interactions. - Plastid-targeted flavodoxin decreases plant damage only in photoperiod. - The chloroplast redox state modulates plant biotic response.

plant biology↗

bioRxiv2025

Context-dependent translation inhibition as a novel oncology therapeutic modality

Inhibitors of protein synthesis, including anisomycin, homoharringtonine, and other natural products bind in the peptidyl-transferase center (PTC) of the eukaryotic ribosome to inhibit translation. Recent work has demonstrated that some PTC-binding antibiotics act in a sequence-selective manner, inhibiting translation elongation at specific amino acids while the polypeptide is engaged in the PTC. However, this phenomenon has yet to be documented for compounds that inhibit translation by the human ribosome. Here we use structure-based design to guide synthesis of molecules called interdictors that bind to the human ribosome PTC and act in a context-selective manner to inhibit translation elongation. Using ribosome profiling, in combination with in vitro biochemistry and cryo-electron microscopy, we characterize the context selectivity of unique analogues and observe their preferred interactions with nascent chain residues with complementary properties. Furthermore, we present a structure for an interdictor bound to a portion of the MYC protein at [~] 1.9 [A] resolution and identify resulting structural rearrangements in both the nascent chain and ribosomal RNA. In cells, we document how these compounds differentially impact the ribotoxic stress response pathway which monitors ribosome collisions and can trigger apoptosis. Finally, we confirm their tumor growth inhibition activity after oral dosing in cell line derived xenografts in mice using the MDA-MB-231 model for triple-negative breast cancer. Together, our data establish sequence-selective inhibition of translation as a novel small-molecule therapeutic modality for historically difficult to address cancers by targeting translation of oncogenic dependency factors in the human ribosome PTC.

cancer biology↗

bioRxiv2025

The Lipocone Superfamily: A Unifying Theme In Metabolism Of Lipids, Peptidoglycan And Exopolysaccharides, Inter-Organismal Conflicts And Immunity

Wnt proteins are critical signaling molecules in developmental processes across animals. Despite intense study, their evolutionary roots have remained enigmatic. Using sensitive sequence analysis and structure modeling, we establish that the Wnts are part of a vast assemblage of domains, the Lipocone superfamily, defined here for the first time. It includes previously studied enzymatic domains like the phosphatidylserine synthases (PTDSS1/2) and the TelC toxin domain from Streptococcus intermedius, the enigmatic VanZ proteins, the animal Serum Amyloid A (SAA) and a further host of uncharacterized proteins in a total of 30 families. Although the metazoan Wnts are catalytically inactive, we present evidence for a conserved active site across this superfamily, versions of which are consistently predicted to operate on head groups of either phospholipids or polyisoprenoid lipids, catalyzing transesterification and phosphate-containing head group cleavage reactions. We argue that this superfamily originated as membrane proteins, with one branch (including Wnt and SAA) evolving into diffusible versions. By comprehensively analyzing contextual information networks derived from comparative genomics, we establish that they act in varied functional contexts, including regulation of membrane lipid composition, extracellular polysaccharide biosynthesis, and biogenesis of bacterial outer-membrane components, like lipopolysaccharides. On multiple occasions, members of this superfamily, including the bacterial progenitors of Wnt and SAA, have been recruited as effectors in biological conflicts spanning inter-organismal interactions and anti-viral immunity in both prokaryotes and eukaryotes. These findings establish a unifying theme in lipid biochemistry, explain the origins of Wnt signaling and provide new leads regarding immunity across the tree of life. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/632903v3_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@d3bf0dorg.highwire.dtl.DTLVardef@1a3f929org.highwire.dtl.DTLVardef@3b20b9org.highwire.dtl.DTLVardef@1e16029_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

A Paper-Droplet Hybrid System for Efficient Blood Plasma Separation in Multiplex Diagnostics

Single PAD-based lateral flow blood plasma separation techniques suffer from low sensitivity, inconsistent performance, and extended turnaround times, hindering point- of-care whole blood multiplexing. Integrating multifunctional microfluidic approach could offer a paradigm shift, providing rapid, high-throughput blood testing with reduced sample alongside streamlined downstream automation. Herein we report a novel low-cost sessile-drop and paper hybrid blood plasma separation technique that achieves efficient ([≥] 98 %) separation within seconds ([~]70 s). Evaporation-driven natural convection within a sessile blood droplet facilitates effective mixing, while capillary forces prevalent in the paper structure selectively transport plasma to the detection zones, all without any externally applied motive force. The separated plasma is successfully analysed for simultaneous glucose and albumin detection using colorimetric assay. This method demonstrates high accuracy in predicting glucose and albumin levels in healthy and diseased samples when benchmarked against an Automated Biochemistry Analyzer. A 3D foldable all-in-one paper microfluidic device, complemented by a MATLAB based application (GPS App.) has been developed for quantitative assessment during on-field diagnostics. This study presents a rapid, efficient, and user-friendly alternative to traditional flow-based separation, providing reliable detection in resource-limited settings.

bioengineering↗

bioRxiv2025

Genetically encoded mechano-sensors with versatile readouts and compact size

Mechanical forces are critical for virtually all fundamental biological processes, yet quantification of mechanical forces at the molecular scale in vivo remains challenging. Here, we present a new strategy using calibrated coiled coils as genetically encoded, compact, tunable, and modular mechano-sensors to substantially simplify force measurement in vivo, via diverse readouts (luminescence, fluorescence and analytical biochemistry) and instrumentation readily available in biology labs. We demonstrate the broad applicability and ease-of-use of these coiled coil mechano-sensors by measuring forces during cytokinesis (formin Cdc12) and endocytosis (epsin Ent1) in yeast, force distributions in nematode axons ({beta}-spectrin UNC-70), and forces transmitted to the nucleus (mini-nesprin-2G) and within focal adhesions (vinculin) in mammalian cells. We report discoveries in intracellular force transmission that have been elusive to existing tools.

cell biology↗

bioRxiv2025

Automatically Defining Protein Words for Diverse Functional Predictions Based on Attention Analysis of a Protein Language Model

Understanding the relationship between protein sequence and function remains a longstanding challenge in bioinformatics, and to date the lions share of related tools parse proteins at the domain or motif levels. Here, we define "protein words" as an alternative to "motif" for studying proteins and functional prediction applications. We first developed an unsupervised tool we term Protein Wordwise, which parses analyte protein sequences into protein words by analyzing attention matrices from a protein language model (PLM) through a community detection algorithm. We then developed a supervised sequence-function prediction model called Word2Function, for mapping protein words to GO terms through feature importance analysis. We compared the prediction performance of our protein word-based toolkit with a motif-based method (PROSITE) for multiple protein function datasets. We also assembled a functionally diverse data resource we term PWNet to support evaluation of protein words for predicting functional residues across 10 tasks (e.g., diverse biomolecular binding, catalysis, and ion-channel activity). Our toolkit outperforms PROSITE in all the examined datasets and tasks. By abandoning domains and instead using attention matrices from a PLM for automatic, systematic, and annotation-agnostic parsing of proteins, our toolkit both outperforms currently available tools for functional annotations at the residue and whole-protein levels and suggests innovative forms of protein analysis well-suited to the post-AlphaFold era of biochemistry.

bioinformatics↗

bioRxiv2025

Hyperglycemia worsens smooth muscle foam cell formation through PI3Kgamma-dependent defective autophagy

BackgroundDiabetes significantly increases the risk of cardiovascular complications, particularly through the mechanisms of atherosclerosis. In this context, uncontrolled hyperglycaemia is a key contributor to arterial dysfunctions. However, the specific effects of elevated glucose levels on smooth muscle cells (SMC)-derived foam cells formation remain poorly defined. MethodsUsing a wire injury-based accelerated atherosclerosis mouse model combined to lipid biochemistry and cellular lipid imaging approaches, we analysed molecular mechanisms involved in SMC derived foam cell formation. ResultsOur findings demonstrated that hyperglycaemia induced a specific lipid loading in SMC in an accelerated atherosclerosis mice model. In vitro, high glucose concentration negatively affected autophagic process independently of lipid uptake and efflux in human and mouse primary SMC. Consistently, treatment with autophagy activators successfully reduced lipid droplet formation. Mechanistically, we identified that PI3K{psi} activity impaired autophagy through TFEB phosphorylation promoting SMC foam cells formation in response to high glucose. ConclusionThese findings shed light on the mechanisms underlying SMC homeostasis disruption under hyperglycemic conditions and underscore the potential therapeutic window for PI3K{psi} inhibitor in managing cardiovascular diseases in diabetic patients.

cell biology↗

bioRxiv2025

Genome-wide identification of genes involved in beetle odoriferous defensive stink gland function recognizes Laccase2 as the phenoloxidase responsible for toxic para-benzoquinone synthesis

Exocrine glands have evolved several times independently in Coleoptera to produce defensive chemical compounds with repellent, antimicrobial, or toxic effects. Research on such glands had focused on morphological or chemical ecology methods. However, modern genetic approaches were missing to better understand this biological process. With the rise of the red flour beetle, Tribolium castaneum, as a model for studies of development and pest biology, molecular genetic tools are now available to also study the safe generation of toxic compounds in defensive stink glands. Using the RNA-interference-based, genome-wide, phenotypic screen "iBeetle" and the re-analysis of gland-specific transcriptomics based on a significantly improved genome annotation, we could identify 490 genes being involved in odoriferous stink gland function. In the iBeetle screen, 247 genes were identified, of which we present here 178 genes identified during iBeetles 3rd phase, while the transcriptomics analyses identified 249 genes, with six genes being identified in both functional genomics approaches. Of these 490 genes, only about 40% of these genes have molecularly characterized homologs in the vinegar fly, while for 213 genes no fly homologs were recognized and for 13 genes no gene ontology at all was identified. This highlights the importance of genome-wide gene identification in tissues that have not been previously analyzed to recognize potentially new gene functions. Gene ontology analysis revealed "SNARE interactions in vesicular transport", "Lysosome", "Pancreatic secretion", and "MAPK signaling pathway - fly" as key pathways. Additionally, many of the genes are encoding enzymes, transcription factors, transporters, or are involved in membrane trafficking. As the phenoloxidase responsible for generating the toxic para-benzoquinones in the stink glands of the beetle, we could identify laccase2, which is expressed in the last secretory cell in contact with the cuticule-lined organelle, where the toxic compounds are safely produced before being released into the gland reservoir. Author summaryCertain beetles produce toxic substances to defend themselves against microorganisms or predators. For the generation of such chemicals, the beetles have specialized glands, which are organized in a way that they can produce and store the toxic compounds without harming themselves. The morphology and biochemistry of such glands had been intensively studied. However, the genes that are involved in this process had not been recognized. Here we present the identification of about 500 genes that are involved in the function of beetle defensive glands, which presents a starting point for a detailed molecular understanding of the process of safe production of toxic substances. A key feature for the production is the use of non-harmful precursors that are only finally processed by a specifically secreted enzyme called Laccase2 to become toxic in a cuticle-shielded organelle.

genetics↗

bioRxiv2025

The expression of IL-35 in the prophase of liver failure and its preliminary exploration for the mechanism of immunoregulation by IL-35 and glucocorticoids

Background and AimsTo determine appropriate dosages for mice models in liver failure prophase and explore IL-35 and cytokine levels. Also, investigate changes in Treg/ Th17 ratio, IL-35, and IL-17 in patients with HBV-induced liver failure before and after treatment. MethodsWe induced liver failure in mice with different doses of concanavalin and assessed severity through serum biochemistry and pathology. Mice were divided into control, model, IL-35 plasmid, anti-IL-35, and dexamethasone groups. IL-35 and IL-17 expressions in liver tissue were evaluated via immunohistochemistry, fluorescence staining, and ELISA. Treg/Th17 ratio and IL-35 and IL-17 levels in peripheral blood of patients were measured using flow cytometry and ELISA, compared to 22 healthy individuals. ResultsThe optimal dose for the liver failure model was 20 g/ kg concanavalin. The IL-35 plasmid and dexamethasone groups showed significantly lower serum TBil, ALT, AST, IL-4, IL-17, TNF- , and liver histopathology scores compared to the model group, while the anti-IL-35 group showed higher levels (P<0. 05). In HBV-PLF patients, TBil, ALT, and AST significantly decreased, and PTA increased after glucocorticoid treatment (P<0. 05). Treg/ Th17 ratio was lower in HBV-PLF patients compared to the healthy group (P<0. 05), with higher IL-35 and IL-17 levels (P<0. 05). Post-treatment, Treg increased and Th17 decreased significantly; IL-17 and Treg/ Th17 ratios increased, while IL-35 decreased (P<0.05). IL-35 positively correlated with the Treg/Th17 ratio. Conclusions 20 g/ kg concanavalin proper dose for mice model. IL-35 may protect in liver failure. Glucocorticoids help maintain immune balance, prevent failure, increase ratios and IL-35 in HBV-PLF patients, and IL-35 related to Treg/Th17 balance.

immunology↗

bioRxiv2025

Endogenous RALF peptide function is required for powdery mildew host colonization

The receptor kinase FERONIA (FER) is a susceptibility factor for biotrophic powdery mildew fungal pathogens in Arabidopsis thaliana, but the underlying molecular mechanisms remain largely unknown. FER is required for the perception of endogenous RAPID ALKALINIZATION FACTOR (RALF) peptides to control various aspects of plant growth, development and immunity. RALFs are perceived by FER-LORELEI-LIKE GPI-ANCHORED PROTEIN (LLG) heterocomplexes to induce cellular responses and bind to LEUCINE-RICH REPEAT EXTENSIN (LRX) proteins as structural components of the cell wall. Combining genetics, cell biology and biochemistry, we found that FERs endogenous RALF ligands are necessary for full colonization success of the powdery mildew species Erysiphe cruciferarum. We reveal that LLGs and LRXs are also powdery mildew susceptibility factors. We show that cell wall remodeling and apoplastic pH homeostasis, hallmark features of RALF function, support powdery mildew reproductive success. Moreover, we provide data that RALF-dependent powdery mildew pathogenesis is partially independent of FER. Powdery mildew fungi likely do not produce RALF peptide mimics, suggesting their reliance on endogenous RALFs for successful host colonization. We propose that powdery mildew fungi require RALF-mediated modulation of apoplastic pH and pectin re-modelling for successful host colonization, highlighting a new susceptibility mechanism by obligate biotrophic fungi.

plant biology↗

bioRxiv2025

A novel expression system for imaging single-molecule fluorescence in Haloferax volcanii WR806 enables visualization of Cas1 involvement in UV-light associated DNA repair

Fluorescence microscopy has become an indispensable tool in biological research, offering powerful approaches to study protein dynamics and molecular biochemistry in vivo. Among archaea, Haloferax volcanii has emerged as a particularly well-suited model organism for imaging studies, with a growing toolkit of established fluorescent markers, plasmids, and promoter systems. Recent advances in single-molecule imaging techniques have created new opportunities through WR806, a carotenoid-free strain providing reduced autofluorescence background. However, existing plasmid-based expression systems in WR806 show critical limitations in protein expression control and challenges with protein aggregation. To address these limitations, we developed pUE001, a novel expression system specifically designed for WR806. This system achieves precise expression control by decoupling selection and induction through strategic implementation of the trpA selection marker. Through comprehensive characterization, we demonstrate that pUE001 provides superior control over protein expression compared to the previously established pTA962 system. It enables linear, titratable expression of diverse proteins -- from the highly regulated CRISPR-Cas component Cas1 to the abundant structural protein FtsZ1 -- while preventing protein aggregation that could compromise native cellular functions. Additionally, we performed a comprehensive analysis of WR806 to show that carotenoid depletion does not affect native cellular physiology. Finally, to demonstrate the systems utility, we investigated the role of Cas1 in UV-induced DNA repair using single-particle tracking photoactivated localization microscopy (sptPALM). Our findings reveal significant, dose-dependent changes in Cas1 mobility following UV-light induced damage, providing evidence for its involvement in DNA repair processes and offering new insights into the expanding roles of CRISPR-Cas systems beyond adaptive immunity.

microbiology↗

bioRxiv2025

Genetically encoded protein oscillators for FM streaming of single-cell data

Wireless devices use frequency modulation (FM) to reliably transmit information. Here, we establish a biochemical analogue of this paradigm using genetically encoded protein oscillators (GEOs) as carrier signals for real-time streaming of single-cell data. The GEO platform leverages evolutionarily diverse MinDE-family ATPase and Activator modules to generate fast synthetic protein oscillations in cells, where the waveform is controlled by both circuit biochemistry and intrinsic cellular physiology. This allows for diverse data encoding strategies, including noise-resistant FM reporters that broadcast proteasomal degradation or transcriptional dynamics; and autoencoder circuits that directly couple shifts in cell physiology to GEO waveform. Deploying GEOs in human embryonic stem cells allowed us to track single-cell developmental states in living populations during differentiation, revealing distinct patterns of spatiotemporal heterogeneity along endoderm, mesoderm, and ectoderm trajectories. The GEO platform establishes a dynamically controllable biochemical carrier signal, unlocking new high-fidelity FM data-encoding paradigms for continuous single-cell analysis.

synthetic biology↗

bioRxiv2025

The renal response to FGF23 shifts from phosphaturia towards inflammation in murine kidney disease models

BackgroundFGF23 excess is associated with morbidity and mortality, but the role of excessive circulating FGF23 concentrations as a mere biomarker or causative factor of pathology is controversial. Here, we investigated the consequences of FGF23 excess in kidney disease. MethodsThis study used three independent disease models: i) anti-glomerular basement membrane (Anti-GBM) disease in male C57BL/6 mice, ii) Adriamycin (doxorubicin)-induced nephropathy in female BALB/c mice, and iii) male DBA/2J mice fed an adenine-containing diet. Anti-GBM and Adriamycin mice and matched control mice received intravenous injections of recombinant FGF23 1{micro}g or vehicle for six consecutive days (Anti-GBM) or once (Adriamycin model), with dissection 24h after the last injection. Adenine mice underwent organ harvesting after 15 weeks to establish ex vivo precision-cut kidney slices (PCKS) and 24h treatment with recombinant FGF23 or vehicle. In addition to histological and biochemical profiling, we assessed serum cytokines, biochemistry and renal transcriptomes and histology of mice and patients with IgA nephropathy. RNAseq data and published transcriptomes underwent gene set enrichment, bulk ligand-receptor interaction analysis and cell-type decomposition. ResultsMice with Anti-GBM disease showed decreased glomerular filtration rate, albuminuria and renal tubular casts. FGF23 treatment increased phosphaturia, but also circulating soluble TNF receptor-1. Renal transcriptomes revealed FGF23-driven proinflammatory transcriptional signatures in murine Anti-GBM and also adverse Vcam1, Pdgfrb and chemokine ligand-receptor signaling in Anti-GBM but not in healthy mice. FGF23 increased transcriptome-inferred renal macrophage content in Anti-GBM mice. Findings were confirmed by immunofluorescence. In Adriamycin-induced nephropathy and in PCKS from the adenine nephropathy model, a short-term FGF23 excess caused expression of proinflammatory transcripts. Finally, human data revealed associations between histopathological or transcriptome-inferred renal immune cell infiltration and circulating FGF23 concentrations. ConclusionFGF23-driven patterns of proinflammatory gene and protein expression or leukocyte overabundance in the kidney were observed in several different models or states of FGF23 excess. The present data provide evidence that excess FGF23 directly drives inflammation in kidney disease and may serve as a therapeutic target.

molecular biology↗

bioRxiv2025

Correlative In Situ Cryo-ET Reveals Cellular and Viral Remodeling Associated with Selective HIV-1 Core Nuclear Import

Lentiviruses like HIV-1 infect non-dividing cells by traversing the nuclear pore, but studying this process has been challenging due to its scarcity and dynamic nature in infected cells. Here, we developed a robust cell-permeabilization system that recapitulates HIV-1 nuclear import and established an integrated cryo-correlative workflow combining cryo-CLEM, cryo-FIB, and cryo-ET for targeted imaging of this process. These advancements enabled the successful capture of 1,899 HIV-1 cores at various stages of nuclear import. Statistical and structural analyses of native wild-type and mutant cores revealed that HIV-1 nuclear import depends on both capsid elasticity and nuclear pore adaptability, as well as nuclear factors such as CPSF6. Brittle cores fail to enter the nuclear pore complex (NPC), while CPSF6-binding-deficient cores stall inside the NPC, resulting in impaired nuclear import. Intriguingly, nuclear pores function as selective filters favoring the import of smaller, tube-shaped cores. Our study opens new avenues for dissecting the biochemistry and structural biology of HIV-1 nuclear import as well as downstream events including core uncoating and potentially integration, with unprecedented detail.

microbiology↗

bioRxiv2025

H2A.Z facilitates Sox2-nucleosome interaction by promoting DNA and histone H3 tail mobility

Epigenetic regulation of chromatin structure is strongly influenced by histone variants and post-translational modifications. The conserved histone variant H2A.Z has been functionally linked to the pioneer factors Sox2 and Oct4, which open chromatin and activate cell fate-specific transcriptional programs. However, the molecular basis of this interaction is not well understood. Here, we combine biochemistry, NMR spectroscopy, and molecular dynamics (MD) simulations to investigate how H2A.Z nucleosome dynamics influence pioneer factor binding. We find that H2A.Z enhances Sox2 and Oct4 association at distinct positions within 601 nucleosomes, correlating with increased DNA accessibility and altered H3 N-terminal tail dynamics. We show that the H3 tail competes with Sox2 for DNA binding and is more efficiently displaced with H2A.Z, while also allowing for unique Sox2-H3 tail interactions. MD simulations reveal that H2A.Z promotes DNA unwrapping, increases inter-gyre spacing, and enhances H3 tail flexibility, while simultaneously reducing contacts with DNA and with the H2A.Z C-terminal tail. This destabilizing effect is DNA-sequence dependent and prominent in the less stable Lin28B nucleosome, which Sox2 appears to substantially reshape. Together, our results suggest that H2A.Z promotes pioneer factor binding by increasing DNA accessibility and reducing histone tail competition, with broad implications for epigenetic regulation and chromatin recognition.

biophysics↗

bioRxiv2025

Widely Used Variants of the Farquhar-von-Caemmerer-Berry Model Can Cause Errors in Parameter Estimation

AO_SCPLOWBSTRACTC_SCPLOWThe Farquhar-von-Caemmerer-Berry (FvCB) model is the most widely-used mechanistic model of C3 net CO2 assimilation, and it plays a significant role in plant physiology, ecology, climate science, and Earth system modeling. As use of the model has grown, multiple variants have appeared across publications. Although many of these are commonly used, there has not been a detailed investigation of existing variants and their impacts on results and interpretations. Here we summarize the types of variants and their prevalence in the literature, and we present a comprehensive comparison of differences between them. A key finding is that a common variant that uses the minimum of assimilation rates rather than the minimum of carboxylation rates, which we call the "min-A variant," makes different predictions than the original "min-W variant," yet appears in approximately half of highly-cited publications and software tools that use the FvCB model. Another concern is that although leaf biochemistry restricts the range of CO2 partial pressures where limitations due to triose phosphate utilization (TPU) can occur, this restriction is commonly omitted from the models equations. Among other potential issues, these variations can introduce errors exceeding 20% when estimating photosynthetic parameter values from CO2 response curves. It is therefore important to be aware of this source of error when fitting the model, to avoid using the min-A variant, and to include the biochemically-derived CO2 threshold for TPU limitations. CO_SCPLOWENTRALC_SCPLOW TO_SCPLOWHEMEC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWOFC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWTHEC_SCPLOW MO_SCPLOWANUSCRIPTC_SCPLOWThe Farquhar-von-Caemmerer-Berry model of CO2 assimilation plays a key role in plant research, but many publications use variants of the model that differ from the original and can potentially introduce errors in photosynthetic parameter estimates. NO_SCPLOWOVELC_SCPLOW RO_SCPLOWESULTSC_SCPLOW, IO_SCPLOWDEASC_SCPLOWO_SCPCAP, C_SCPCAPO_SCPLOWORC_SCPLOW MO_SCPLOWETHODSC_SCPLOWUsing a literature survey, FvCB model variants are categorized, and some are found to make contradictory predictions. Comparisons against A-Ci curves show that the "min-W variant" exhibits the best performance, especially at low CO2 concentrations.

plant biology↗

bioRxiv2025

Tissue-specific mutagenesis from endogenous guanine damageis suppressed by Polκ and DNA repair

Knowledge of mutational patterns has expanded significantly, but a persistent challenge is to link these complex patterns to the underlying molecular mechanism or source of DNA damage, especially when that damage is endogenous and not driven by environmental mutagens. Technological advances now allow us to catalogue mutation across tissues or even closely related cell types, but the results are largely descriptive until we identify the endogenous sources of mutation and how they differ across tissues. Here, we combine mouse genetics, advanced sequencing, biochemistry and mass spectrometry to provide a detailed mechanistic understanding of endogenous mutagenesis. We reveal that endogenous guanine adducts are significant drivers of tissue-specific mutagenesis, while the interwoven actions of DNA polymerase Pol{kappa} and DNA repair mechanisms are pivotal in mitigating mutagenesis. For the first time, we use untargeted DNA adductomics to characterize new sources of endogenous DNA damage. Our novel approach to understanding endogenous mutational landscapes reveals previously unseen mutational processes and points to vast potential for new discoveries.

molecular biology↗

bioRxiv2025

Immiscible proteins compete for RNA binding to order condensate layers

Biomolecular condensates mediate diverse and essential cellular functions by compartmentalizing biochemical pathways. Many condensates have internal subdomains with distinct compositional identities. A major challenge lies in dissecting the multicomponent logic that relates biomolecular features to emergent condensate organization. Nuclear paraspeckles are paradigmatic examples of multi-domain condensates, comprising core and shell layers with distinct compositions that are scaffolded by the lncRNA NEAT1, which spans both layers. A prevailing model of paraspeckle assembly proposes that core proteins bind directly and specifically to core-associated NEAT1 domains. Combining informatics and biochemistry, we unexpectedly find that the essential core proteins FUS and NONO bind and condense preferentially with shell-associated NEAT1 domains. The shell protein TDP-43 exhibits similar NEAT1 domain preferences on its own but forms surfactant-like shell layers around core protein-driven condensates when both are present. Together, experiments and physics-based simulations suggest that competitive RNA binding and immiscibility between core and shell proteins orders paraspeckle layers. More generally, we propose that sub-condensate organization can spontaneously arise from a balance of collaborative and competitive protein binding to the same domains of a lncRNA. SignificanceThe cellular milieu is spatially organized into compartments called biomolecular condensates that exhibit rich internal organization which shapes their functions. Often comprising multiple proteins and RNAs, a major question concerns how molecular-scale features relate to emergent condensate forms. Here we study nuclear paraspeckles, archetypal multi-domain condensates comprising distinct core and shell layers assembled around a layer-spanning RNA scaffold. We find that different proteins associated with each layer all bind preferentially to the same shell-associated domains of the RNA scaffold. Core and shell proteins are inherently immiscible, establishing a competition that redirects core proteins to a suboptimal, core-associated domain of the scaffold. Our work reveals how a combination of competitive RNA binding and protein immiscibility can spatially organize multicomponent condensate subdomains.

biophysics↗