Nature Communications

Endurance exercise imposes extreme metabolic demands on the adult human brain, raising the question of how core brain function is preserved under physiological challenge. We previously showed that marathon running induces reversible reductions in myelin within specific white-matter tracts, suggesting adaptive structural change under metabolic stress. Here, we asked whether this process is functionally tolerated. Neurophysiological recordings revealed maintained conduction latencies across motor, somatosensory, visual, and auditory pathways within 48 hours after race completion, indicating intact axonal signal transmission despite reduced myelin content. Cognitive testing revealed selective and transient modulation of higher-order processing, including attenuated practice-related gains in processing speed and short-lived increases in interference, whereas visuomotor speed and executive flexibility were preserved. All cognitive measures normalized one month after the race, supporting an adaptive framework linking myelin change with preserved brain function under extreme metabolic stress.

Citrate is a central intermediate metabolite linking the tricarboxylic acid cycle and lipid biosynthesis. Tools for monitoring of spatiotemporal citrate dynamics are critical for getting a better understanding of cellular metabolism. Here, we develope genetically encoded excitation ratiometric biosensors for citrate, based on our previous intensiometric green fluorescence protein-based citrate biosensor, Citron1. We find that a single mutation in the Citron1 chromophore-forming tripeptide provided an excitation ratiometric response. Further rounds of directed evolution yield highly responsive variants, exhibiting citrate-dependent fluorescence changes between two excitation peaks. When expressed in mammalian cells, these biosensors enable citrate dynamics to be monitored in both the cytosol and mitochondria. Comparative analysis across multiple human breast cancer cell lines uncovers cell line-specific differences in citrate levels and their heterogeneity, which could be linked to their malignancy. Furthermore, flow cytometry-based measurements in mouse embryonic stem cells demonstrate the proteomics signatures underlying the population-level variability in citrate concentrations and citrate rewiring during stem cell differentiation. Together, these results show that these excitation ratiometric citrate biosensors enable quantitative, compartment-resolved, and population-scale analysis of cellular metabolism.

NLRP3 is an innate immune sensor of a broad range of stimuli, which upon activation forms a multiprotein inflammasome complex triggering caspase-1 activation, IL-1{beta} and IL-18 maturation, and inflammatory cell death. The canonical NLRP3 activation pathway has been well characterized from a structural perspective. It involves the association of NLRP3 with membranes in the form of inactive oligomeric "cage" complexes, which, upon activation, convert to an active oligomeric NLRP3 disc. NLRP3 structural rearrangements during non-classical NLRP3 activation pathways, however, remain unknown. Here, we report a novel mode of NLRP3 activation utilized by the NLRP3 homolog from zebrafish. The cryo-EM structure of zebrafish NLRP3 shows that, unlike human NLRP3, it forms disc-shaped heptamers that undergo further trimerization, resulting in a 21-mer oligomeric arrangement. Surprisingly, a single zebrafish NLRP3 heptamer cannot arrange its PYD domains into a PYD helix and therefore requires a trimer of heptamers to form a PYD filament that enables ASC oligomerization. Furthermore, zebrafish NLRP3 does not associate with the Golgi network, nor does it form inactive "cage" oligomers or interact with NEK7. Thus, our data demonstrate an ancestral non-canonical structural mechanism of NLRP3 activation, which may shed light on alternative NLRP3 activation pathways present in humans.

Endosymbiosis of phytoplankton in heterotrophic hosts is ecologically important and has led to key evolutionary innovations. However, the dynamic molecular processes underlying endosymbiosis establishment remain poorly understood. Here, using large-particle sorting and liquid chromatography-tandem mass spectrometry, we unravel heterogeneous changes in proteomes of the cosmopolitan ciliate Paramecium and algal endosymbiont Chlorella from engulfment to stable endosymbiosis. The initial digestion sees a sharp decline of intracellular Chlorella cells, along with host cellular reorganization involving a reduction of the cortex-localized defensive organelles, trichocysts, and proteins for intracellular transport and recycling. The remaining Chlorella cells enter a bottleneck stage characterized by energy production and cell cycle commitment before active proliferation. Comparison of Paramecium with successful and failed endosymbiosis further identifies a solute carrier transporter that potentially mediates metabolic homeostasis of the endosymbiotic system. Our study reveals inter-organismal coordination during the transition from predator-prey to host-endosymbiont relationships. The approach of time-course single-cell dual proteomics can be useful for investigating diverse interactions between microbial eukaryotes.

Adenylyl cyclases (ACs) convert ATP into the second messenger cAMP, thus directly influencing cellular signaling in response to a wide variety of stimuli. Despite their physiological importance, structural studies of isoform-specific AC regulation are compounded by difficulties in AC expression and purification. Here, we designed a chimeric construct AC95, combining human AC9 as a molecular scaffold and incorporating the catalytic-allosteric core of human AC5. Cryo-EM analysis of AC95 at 3.5 [A] resolution revealed a state of AC95 bound to both ATPS and forskolin, demonstrating that the chimera reproduces AC5-like allosteric regulation while retaining the structural features of the AC9 scaffold. Although AC95 chimera retained the ability to bind to and be activated by forskolin, it lost the ability to be autoinhibited by the C2b domain of AC9. Moreover, AC95 is insensitive to inhibition by specific AC5 inhibitors SQ22,536 and NKY80, suggesting that these molecules may target a site distinct from the catalytic-allosteric core of AC5 grafted into the AC95 chimeric construct. Our results establish a generalizable approach for investigating isoform-specific regulation of membrane ACs by small molecules, offering a potential path for structure-based drug discovery targeting distinct AC isoforms.

A large fraction of marine protists, particularly the smallest ones, belong to uncultured lineages that lack genomic data, limiting insights into their ecological roles and evolutionary strategies. Here, we generated 325 single-cell amplified genomes (SAGs) from 2-5 {micro}m planktonic protists, which resulted in 147 genomes from dominant marine species at varying levels of completeness (40 of them >50%). These genomes matched the in situ community, with Prymnesiophyceae, Mamiellophyceae and Chrysophyceae dominating pigmented cells and MAST, Choanoflagellata and Picozoa prevailing among heterotrophic colourless cells. This resource allowed us to describe the genomic architecture of marine protist species, and revealed a pronounced genome streamlining in ecologically successful lineages. Comparative analyses highlighted unique functions enriched in photosynthetic and heterotrophic taxa (including motility, signal transduction, digestion and secondary metabolism), and revealed a broad distribution of gene families with adaptive traits such as polyketide synthases and rhodopsins. This large-scale single-cell genomics dataset provides a mechanistic foundation for investigating functional diversity, ecological strategies and genome evolution in the ocean.

1. Diptera represent a diverse insect order, including vectors of human and animal pathogens. Their accurate species identification remains a major bottleneck in ecological and epidemiological studies. Morphological identification requires taxonomic expertise, while molecular methods are costly and not universally reliable. Wing geometric morphometrics offers an alternative, but manual landmark annotation is time-consuming and introduces observer bias. 2. We developed ITHILDIN, an automated pipeline for landmark and semilandmark annotation of Diptera wings, combining UNet++ segmentation and an Hourglass landmark prediction model. Using mosquitoes as the primary model system, we extended an existing repository with 5,793 additional images. Models were trained on 5991 annotations of landmarks and segmentations and then evaluated on 12,522 images across 34 taxa. We assessed landmark prediction accuracy against human observers and ML-morph, evaluated species identification using Linear Discriminant Analysis on 17 homologous landmarks and 52 semilandmarks, and tested out-of-distribution generalisation by reproducing an independent study. Transferability was demonstrated by adapting the pipeline to the Dipteran families Drosophilidae and Glossinidae. 3. The Hourglass model achieved a mean landmark error of 4.5 pixels (95% CI: 4.3-4.6), within human observer variability (4.7 pixels, 95% CI: 4.4-5.0) and substantially outperforming ML-Morph (12.7 pixels, 95% CI: 11.1-14.2). The semilandmark-based approach for species identification achieved 91% balanced accuracy across 34 taxa, comparable to CNN performance (94%). On out-of-distribution data, the landmark pipeline generalised substantially better than the CNN and a soft-voting ensemble of the landmark and CNN classifiers achieved 88% balanced accuracy on a replicated study. 4. Combining geometric morphometrics with deep learning provides a reproducible, interpretable, and generalisable alternative to black-box CNN classifiers for Diptera wing analysis. By acting as a consistent single observer comparable to human annotation, the system eliminates inter-observer bias, enabling large-scale and cross-study morphometric analyses of Dipteran wings. The system is publicly available at www.ithildin.bnitm.de and transferable to other Diptera families with moderate retraining effort. Data availabilityImages used in this study are accessible under CC BY 4.0 license at https://doi.org/10.6019/S-BIAD1478. Downloadable and installable docker application can be accessed on the applications git page: https://anonymous.4open.science/r/ITHILDIN-4313/

Nitrogen fixation plays a central role in primary productivity and nitrogen cycling in aquatic ecosystems, yet its distribution among cyanobacterial lineages remains incompletely understood. Biological nitrogen fixation is energetically costly and highly oxygen-sensitive, imposing constraints in oxygenic phototrophs. The unicellular cyanobacterial genus Synechocystis has long been regarded as strictly non-diazotrophic. Here, we report that Synechocystis sp. LKSZ1 possesses a functional nitrogen fixation system. Comparative genomics revealed that LKSZ1 is distinct from other Synechocystis strains and uniquely harbors a complete nif gene. Phylogenetic and structural analyses indicate acquisition via horizontal gene transfer from filamentous cyanobacteria. Physiological assays demonstrated photoautotrophic growth under nitrogen-depleted conditions and nitrogenase activity under microoxic to anaerobic conditions. Disruption of nifK abolished both growth and activity. These findings show that ecological nitrogen limitation and host compatibility can enable functional integration of horizontally acquired nitrogen fixation.

Propiconazole (PCZ) is widely misused growth regulator in leafy Brassica vegetables. Developing green strategies for managing plant architecture has become an urgent agricultural priority. Here, we identified from a membrane-protein-defective yeast library a P4-ATP phospholipid flippase, aminophospholipid ATPase 3 (ALA3), as a target sensitive to PCZ. ALA3 exhibits high binding affinity for PCZ, which inhibits its ATPase activity. Knockdown of ALA3 rendered yeast, Arabidopsis, and Brassica rapa less sensitive to PCZ and conferred a growth-inhibited phenotype. This dwarfing phenotype is mediated through the interaction between ALA3 and CYP51G1 that jointly acts within the brassinosteroid regulatory pathway. Furthermore, we identified lead compounds A01 and A15 as ALA3-targeting agents, and compared to PCZ, they display superior binding affinity and reduced toxicity. Our work establishes ALA3 as a key mediator of PCZ-induced dwarfism and provides dual strategies--creating promising varieties through gene editing and developing targeted green pesticides--to reduce PCZ use. TeaserTargeting ALA3 reduces PCZ use through gene-edited varieties and green pesticides.

AO_SCPLOWBSTRACTC_SCPLOWAphids threaten crop productivity through phloem feeding and the transmission of plant viruses. Aphis gossypii, in particular, is a widespread and damaging pest of worldwide cultivated melon. Resistance to its emerging CUC1 clone in Europe remains poorly characterized. Here, we dissected the genetic architecture of melon resistance to CUC1 using complementary traits that capture multiple stages of the aphid-melon interaction: plant attractiveness to aphids, acceptance, aphid colonization, and multiplication. Genome-wide association studies (GWAS) in a diversity panel of 174 accessions identified a quantitative trait locus (QTL) for attractiveness on chromosome 6, while analyses in a complementary panel of 212 accessions revealed QTLs for plant acceptance by aphids on chromosomes 3, 8, and 12. Colonization and multiplication traits further highlighted resistance QTLs on chromosomes 5 and 12, the latter supported by both SNP-based GWAS and bulk-segregant analysis. Pan-NLRome k-mer- and graph- based GWAS, together with Vat presence/absence association analyses, provided allele-level resolution of the QTL on chromosome 5, corresponding to the Vat region. Leveraging allelic diversity at this locus, we functionally characterized 20 Vat homologs with four R65aa motifs within their leucine-rich repeat (LRR) domain and demonstrated the capacity of R65aa-type Vat alleles to confer clone-specific resistance. Resistance-conferring alleles limited virus multiplication, such as Cucumber mosaic virus (CMV), when transmitted by five A. gossypii clones, including CUC1. Together, our results revealed multiple genetic determinants underlying quantitative resistance to the A. gossypii CUC1 clone in melon and highlighted the central role of Vat homologs in resistance to both A. gossypii and the viruses it vectors. These findings provide strategic targets for pyramiding resistance loci acting at different stages of the pest life cycle to enhance durable protection against these biotic threats.

We report the identification and functional validation of a 7SK RNA polymerase III promoter in the Mediterranean fruit fly, Ceratitis capitata. CRISPR/Cas9-based genetic control strategies for this global agricultural pest, including gene drives and precision guided sterile insect approaches, require efficient guide RNA expression, yet only a single U6 Pol III promoter had previously been validated for this purpose in C. capitata, and no 7SK promoter had been characterised in any Tephritid species. Using comparative genomics with Drosophila orthologues, we identified a previously unannotated 7SK gene in the C. capitata genome, confirmed its transcriptional activity by RT-PCR, and demonstrated that the cloned promoter drives functional guide RNA expression in CRISPR/Cas9-mediated knockouts of the white gene. Comparative analysis identified putative 7SK orthologues across the Tephritid fruit flies. The availability of this additional new Pol III promoter will enable multiplexed guide RNA strategies using distinct promoters, supporting more robust genetic control designs.

Plants produce the most diverse blends of specialized metabolites on earth. Natural products derived from plants are valuable resources for drug development, food chemistry, and crop resistance breeding. Phenotypes of specialized metabolite profiles can be captured by untargeted mass-spectrometry across species phylogeny, tissues, and genotypes. Here, we collected metabolic fingerprints of 17 Brassicaceae species across three tissues (paired leaf and root; flower) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive and negative ionization mode. Corresponding metadata has been refined for reuse according to ReDU guidelines, and for integration with public genomic and transcriptomic data. Standardization of in vitro growth conditions, and data processing workflows enables integration of acquired raw and processed data across platforms for single- and multi-omics analysis. Further, the inclusion of tissue-specific metabolic profiles across ploidy levels, as well as across crop species and wild relatives, makes this dataset a valuable resource for natural product discovery.

Persistent detection of high-risk human papillomavirus (HPV) is required for cervical carcinogenesis, yet the metabolic phenotype associated with distinct HPV transition states remains incompletely defined. We analyzed vaginal metabolomics data from 71 HIV-negative, non-smoking, premenopausal women without other sexually transmitted infections, grouped by three-visit HPV trajectories: persistent negative (NNN, n=20), late incident positivity (NNP, n=9), conversion with persistence (NPP, n=13), clearance after prior positivity (PPN, n=16), and persistent positive (PPP, n=13). After detection-based filtering, 186 putative and 64 quantitatively estimated metabolites were retained for integrated univariate, multivariate, network, pathway, and machine learning analyses. Global class separation was weak by PERMANOVA and by five-class classification, indicating that the vaginal metabolome does not reorganize broadly across all HPV states. In contrast, trajectory-specific signals were reproducible. The strongest pairwise contrast was NNP versus PPP (best cross-validated ROC AUC 0.778; permutation p=0.039). Glycolic acid was the dominant single metabolite, particularly for NNP versus PPP (Mann-Whitney p=6.96x10^-4, FDR=0.0446, AUROC=0.902; detection 88.9% versus 15.4%; combined abundance+detection FDR=0.0010). Persistent positivity was characterized by a focused uracil-high, methyl-donor/redox-low signature, including lower glycolic acid, S-adenosylmethionine, NAD+, and betaine, together with higher uracil. Ratio mining further sharpened discrimination, with uracil/S-adenosylmethionine and uracil/creatinine among the best PPP classifiers, and glucose 1-phosphate/isovaleric acid-valeric acid strongly separating NNP from NPP. These data support a model in which HPV trajectory is encoded by targeted metabolic states rather than a diffuse HPV-positive versus HPV-negative metabolomic shift.

How conserved stress responses are across the plant kingdom remains poorly understood. Here, we present a kingdom-wide stress transcriptome atlas of 36 Viridiplantae species, from chlorophytes to angiosperms, across nine abiotic and biotic stresses. The atlas integrates reanalyzed public RNA-seq data with new in-house stress experiments on three species representing basal lineages, yielding 13.6 million differential expression calls from over 3,200 manually curated control-treatment comparisons. We find that ancient gene families respond broadly but moderately, while lineage-specific families respond narrowly but intensely, revealing a division of labor in stress gene deployment. Stress response conservation decays with phylogenetic distance yet remains detectable across more than 700 million years of divergence, with upregulated genes diverging faster than downregulated genes. Functional co-occurrence analysis uncovers a deeply conserved growth-defence tradeoff alongside stress-specific transcriptional rewiring. Conserved stress co-expression modules undergo regulatory subfunctionalization through duplication, with whole-genome duplicate pairs preferentially retained within modules. Finally, DNA and RNA foundation models predict stress responsiveness from sequence alone (auROC 0.755), suggesting a partially conserved cis-regulatory code underlying stress responses across the kingdom.

Technological advances in biomedical sciences have accelerated multi-omics research, enabling high-resolution spatial mapping of diverse molecular compound classes. However, integrating spatial omics often requires serial tissue sections, limiting the alignment correlation across modalities. We present a single-section integrative multi-omics (SIMO) workflow that combines metabolite and lipid imaging with histopathology and region-specific proteomics. Using MALDI-MSI, tissue staining, and laser microdissection (LMD), SIMO delivers comprehensive metabolic, lipidomic, and proteomic insight from the same sample. Using mouse cardiac tissue we develop, control, and validate the methodology resulting in [~]60 imaged lipids and [~]60 imaged metabolites at 20 {micro}m pixel size and subsequently spatial proteomics by LMD, detecting over 5,000 proteins from the same tissue. To demonstrate the capabilities of the workflow in preclinical context, we apply SIMO to a metastasizing melanoma PDX model, identifying over 100 spatially localized lipids and metabolites, and over 5,000 proteins across metastases and non-tumor tissues in liver. SIMO enables precise ROI selection, statistical comparison of protein regulation, and alignment of metabolic and lipidomics pathways across spatial omics and region-specific proteomics, demonstrating its value as a spatial multi-omics platform.

Visual surface perception is a fundamental aspect of vision, yet its neural implementation remains poorly understood. Troxlers perceptual filling-in paradigm provides a tractable illusion for studying surface perception, in which a peripheral figure becomes perceptually assimilated into the surrounding background after a period of sustained fixation. Although neural correlates of this phenomenon have been reported in early visual cortex, the underlying mechanisms, particularly the contribution of feedback signaling, remain unresolved. Here we use ultra-high-field (7T) layer-fMRI to investigate perceptual filling-in in the human visual cortex. While experimentally controlling perceptual filling-in, we measured GE-BOLD responses in ten participants. Analyses across cortical depth in the independently localized figure representation in primary visual cortex (V1) revealed neural correlates of filling-in in deep cortical layers, which are associated with feedback input. These findings provide evidence that perceptual filling-in and visual surface perception in general are supported by feedback signals to early visual cortex.

Adenylyl cyclase 8 (AC8) is a Ca2+-sensitive membrane adenylyl cyclase highly expressed in the central nervous system. AC8-mediated intracellular cyclic adenosine monophosphate (cAMP) accumulation shapes synaptic function, plasticity, and memory formation and is tightly controlled by intracellular Ca2+ and G protein-coupled receptor signaling. Although it is well known that the effects of Ca2+ on AC8 activity are directly mediated by calmodulin, it has remained unclear until now whether other Ca2+-binding proteins also regulate AC8 function. Here, we identify the neuronal calcium sensor hippocalcin-like protein 1 (HPCAL1) as a direct interaction partner of AC8. Using biochemistry, structural proteomics, integrative modeling, cryo-EM and cell-based FRET approaches, we show that HPCAL1 associates with AC8 in a Ca2+-dependent manner. HPCAL1 shows weak positive modulation of AC8 activity in vitro, with sub micromolar affinity. HPCAL1 interacts with flexible regulatory regions of AC8, including the C1b domain and the N- and C-terminal regions. Interestingly, other members of the neuronal calcium sensor family can bind AC8 via the same sites. Together, our study reveals a previously unrecognized mode of Ca2+-dependent AC8 recognition by a group of neuronal calcium sensor proteins, which may be relevant to context-dependent regulation of cAMP signaling.

Structural complexity in the rice inflorescence (panicle) is determined by the activity of indeterminate meristems, which allow sequential branching events to occur but later acquire a determinate character that precludes the initiation of new growth axes. To better understand the underlying regulatory processes, we combined a detailed time-course of panicle development with tissue-specific sampling of meristems before and after the determinacy switch, monitoring global gene expression programs and hormone accumulation. This allowed the delineation of three dynamic transcriptional modules and the inference of gene regulatory networks highlighting hormone regulatory hubs associated with the indeterminate-to-determinate meristem transition. Our data confirm the importance of known regulators of inflorescence development and identify novel actors, notably genes encoding transcription factors, that were not previously described in the regulation of flowering. The combined biochemical and transcriptomic data support a model in which cytokinin signalling is particularly active during the proliferative branching phase, during which meristem maintenance is promoted while a feedback mechanism is also triggered, ultimately leading to the acquisition of determinate meristem fate later in development. HighlightInvestigating the molecular nature of rice inflorescence meristem determinacy, we studied temporal and spatial gene expression alongside hormone accumulation, identifying novel regulatory interactions and a key role for cytokinins.

Ramaswamy et al. recently reported in Nature Medicine that ChatGPT Health, a consumer-facing health AI tool, undertriaged 51.6% of true emergencies. It was also susceptible to social anchoring in a structured stress test of triage recommendations. We applied the same vignette-based benchmark to OpenEvidence, a widely used physician-facing AI platform for clinical decision support. The benchmark included 960 prompts across 21 clinical domains (Supplementary Table S3). OpenEvidence undertriaged 12.5% of emergencies, a four-fold reduction relative to ChatGPT Health. It also showed no anchoring effect. Its errors skewed in a safer direction, including 68.0% overtriage of Home presentations. In 65 of 960 responses (6.8%), it declined to assign a triage level. These refusals occurred only in symptom-only prompts and never in urgent or emergency cases. Performance improved when objective clinical data were provided. Under the same benchmark, a widely used physician-facing system showed a different safety profile from a consumer-facing one. This suggests that who a health AI is built for can shape how it fails.

EHMT1 and EHMT2 genes encode human euchromatin histone lysine methyltransferase 1 and 2 (EHMT1 alias GLP; EHMT2 alias G9a) that form heteromeric GLP/G9a complexes with essential roles in epigenetic regulation of gene expression. While EHMT1 haploinsufficiency has been established as the cause of Kleefstra syndrome 1, the pathogenesis of G9a dysfunction in human disease remains largely unknown. We identified seven de novo EHMT2 variants in patients with clinical presentation, episignatures, histone modifications and transcriptomic profiles similar to those of Kleefstra syndrome 1. In vitro studies revealed that these variants encode for structurally stable G9a proteins that are catalytically incompetent due to aberrant interactions either with histone H3 tail or with S-adenosylmethionine. Heterozygous mice carrying a patient-derived variant exhibited growth retardation, facial/skull dysmorphia and aberrant behavior. Here we report pathogenic EHMT2 variants that likely exert dominant-negative effect on GLP/G9a complexes and thus genocopy the EHMT1 haploinsufficiency via a distinct molecular mechanism, defining an autosomal dominant EHMT2-related Kleefstra syndrome.