Nature

Task fMRI1 and electrophysiology2 have revealed distributed, linked cortical patches with shared category preferences (e.g., faces, objects, places)1,3-5, smaller than cytoarchitectonic areas. Resting-state functional connectivity (RSFC) similarly showed that somato-cognitive action network (SCAN) nodes interleave with effectors (foot, hand, mouth), subdividing the precentral gyrus6. Here, using multiple precision functional mapping (PFM) modalities (RSFC, task, lags), we discovered that most of association cortex is organized like face processing and SCAN, with small, discrete patches interconnected into chains. Such patch-chains densely tile prefrontal cortex but are largely absent from primary cortex. Cortico-striatal connectivity is organized such that patches of the same chain connect to the same striatal location. Within chains, infra-slow fMRI signals are ordered in time. RSFC-defined chains align with task fMRI localizers (e.g., visual, motor, pain). Chains are absent at birth and emerge in the first year of life, suggesting their formation is at least partially experience-driven. Cytoarchitectonic areas are subdivided by patches, and patches in the same chain are distributed across different cytoarchitectures. Chains represent parallel ordered processing streams that are separated by information domain and behavioral goals, not cytoarchitectonics. Functional subdivision of architectonics into smaller patches, interlinked to form cross-architecture chains, enable greater parallelization and flexible specialization of processing.

Large language models embedded in autonomous agents process trusted instructions and untrusted data in one context window, leaving them open to direct and indirect prompt injection. In healthcare this is not hypothetical: a 2025 JAMA Network Open study found commercial medical LLMs followed injected instructions in 94.4% of simulated patient encounters, including life threatening recommendations . Yet the clinically decisive problem we quantify here is different. Most real clinical threats protected health information PHI exfiltration, cross patient access, bulk export, out of scope advice are fluent, legitimate looking requests that carry no attack signal, so even a state of the art injection detector passes them. Existing runtime guardrails trade safety against latency: model based auditors are accurate but add hundreds of milliseconds of Python inference, while lexical filters are fast but blind to obfuscated or semantically disguised payloads. We present QFIRE, an inline, provider agnostic prompt firewall implemented as a single self contained Rust toolchain proxy, CLI, and benchmark harness. QFIRE combines three mechanisms: (i) positive security scope constraints, which restrict a model call to a declared natural language purpose and block out of scope drift even when no overt attack token is present; (ii) an asynchronous detector graph that runs N rules and their detector nodes concurrently, cheapest checks first; and (iii) a de obfuscation pass that decodes Base64 hex ROT13, folds homoglyphs and leetspeak, and strips zero width characters before detection. QFIRE ships 106 versioned firewall rules and a dedicated HIPAA Safe Harbor 18 identifier PHI panel, and runs a local DeBERTa v3 injection classifier via embedded ONNX Runtime. On 1968 public prompt injection and jailbreak prompts QFIREs deterministic hybrid attains F1 0.86, statistically tied with Metas state of the art PromptGuard 2 0.86 and above protectai DeBERTa v3 0.83; lexical baselines lag 0.16 to 0.50. Our central result is on QFIRE HealthBench, a new 2000 prompt healthcare benchmark we build and release with real garak and Microsoft PyRIT payloads. There the same PromptGuard-2 recovers only 0.40 recall DeBERTa v3 0.57, because most clinical threats carry no injection signal; QFIREs combined scope plus PHI chain reaches 0.83 recall F1 0.87 at a calibrated 0.08 false positive rate. Generic injection detection, even state of the art, is therefore necessary but not sufficient for healthcare agents. A bare LLM judge also closes most of this static corpus gap F1 0.90; QFIREs contribution beyond static accuracy is auditable determinism, bounded latency, and adaptive robustness, where the bare judge falls to 34 to 59% recall section 5.5. End to end, placing QFIRE in front of a tool using agent over a mock EHR sandbox cuts the agents harmful action rate from 0.38 to 0.00 at a 0.13 benign utility cost. All code, rules, corpora snapshots, and scripts are released, and every table regenerates from a single make paper target against local models with no paid API keys.

Bacterial, plant, and animal cells synthesize nucleotide immune signals as a conserved strategy to defend against viral infection1-4. In bacteria, Thoeris anti-phage defense systems convert nicotinamide adenine dinucleotide (NAD+) into the cyclic ADP-ribose signals 2'cADPR and 3'cADPR to activate downstream effectors and restrict viral replication5-8. Phage proteins can bind and sequester Thoeris signals6,9-13, but no mechanisms are known to degrade the exceptionally stable 2'cADPR and 3'cADPR molecules and terminate immune activation. Here we use a forward biochemical screen to discover the mycobacteriophage protein RyDEP as the founding member of an enzyme family that cleaves 2'cADPR and 3'cADPR to inactivate Thoeris defense. We show that RyDEP is a glycosidase that cleaves the ribose-ribose linkage in 2' and 3' cADPR immune signals to both inactivate host defense and enable direct restoration of NAD+. A crystal structure of the RyDEP-3'cADPR complex in the post-cleavage state explains the molecular basis of immune signal degradation and reveals surprising homology with the Repeat12 domain of animal ryanodine receptors (RyRs) that control calcium flux and muscle contraction14,15. We demonstrate that diverse phage RyDEP proteins tune RyR-domain activity to either degrade or sequester immune signals. Our results define RyR-domain proteins as regulators of nucleotide immune signaling and explain how viruses subvert host antiviral defense.

The endosymbiotic evolution of plastids and mitochondria was central to the origin and success of eukaryotes. One of the most prominent molecular machineries thought to have disappeared early in eukaryote evolution is the multi-subunit bacterial DNA polymerase III (DNApol-III), which is the principal enzyme complex supporting DNA replication in bacteria. Here, we combined worldwide metagenomics and cultivation to characterise the mosaic genomic landscape of abundant phytoplankton lineages of Teleaulax (Cryptophyceae), which contain an endosymbiotically-derived nucleomorph genome. Unexpectedly, the nuclear, plastid and nucleomorph genomes of Teleaulax contain ubiquitously expressed genes for plastid-targeted DNApol-III subunits. These genes shed light on the functioning of Teleaulax genomes when sequestered by the ciliate Mesodinium during its kleptoplastidic photosynthetic activity1-3. In particular, the alpha subunit gene (encoding the polymerase activity), which resides in the nucleomorph genome, is continuously expressed in Mesodinium in controlled laboratory experiments. This provides a mechanistic explanation for the replication of Teleaulax plastid genomes weeks after the nuclear genome is lost4. Beyond Teleaulax and close relatives, we also identified genes encoding plastid-targeted DNApol-III subunits (including alpha) in nuclear genomes of unicellular and multicellular lineages of Archaeplastida that form, along with those of Cryptophyceae, monophyletic clades firmly positioned within Cyanobacteria. Together, our results reveal a previously overlooked retention of bacterial DNA replication machinery from plastid primary endosymbiosis in Archaeplastida, its acquisition by Cryptophyceae during secondary endosymbiosis, and its direct role in contemporary plankton as a facilitator of kleptoplastidic photosynthetic activity by heterotrophic ciliates.

Iron mineralization has profoundly influenced Earths biogeochemical history1,2, yet the specific mechanisms underlying banded iron formation (BIF) remain unresolved3-5. Here we profile the microbiomes of Holocene sediments beneath the Larsen C Ice Shelf (LCIS), Antarctica6-8, through stratigraphic analysis of sedimentary ancient DNA combined with metagenomics. Distinct microbial phases aligned with glacial facies boundaries, with sub-ice shelf communities dominated by chemolithoautotrophs including an uncultured Thermodesulfovibrionia. This bacterium, visualized by fluorescence in situ hybridization and designated Candidatus Mariimomonas ferrooxydans (phylum Nitrospirota), emerged as a keystone taxon with high network centrality. Its genome encodes Cyc2, a fused porin-cytochrome outer membrane protein implicated in Fe(II) oxidation. Heterologous expression of Cyc2 in Escherichia coli confirmed its ability to catalyze iron oxidation, supporting iron precipitation under dark, anoxic conditions. These pristine LCIS sediments, unaltered since the last glacial maximum, provide a modern analogue for synglacial BIFs deposited during Neoproterozoic Snowball Earth events. Our findings deliver direct genomic and functional evidence for chemolithotrophic iron oxidation, challenge phototroph-centric models of BIF genesis, and highlight microbial iron cycling as a recurring force in Earths geochemical evolution. Beyond Earth, these insights inform interpretations of iron deposits on other planetary bodies.

Innate immune systems detect molecular signatures of infection to initiate antiviral defence1-3, yet the identity of pathogen-associated signals that distinguish phage from host nucleic acids remains incompletely understood. While recent work has shown that nucleic acid structures can act as triggers for bacterial defense systems4-7, how these structural signals are coupled with immune activation remain unclear. Here we show that forked DNA structures activate a helicase-nuclease immune complex in type III Druantia through a processing-dependent mechanism. Using cryo-electron microscopy and biochemical reconstitution, we find that the exonuclease DruH processes 3' DNA termini to generate 5' overhangs that recruit and activate the helicase-nuclease DruE at duplex-single-stranded DNA junctions. Structural analysis of the DruE-DruH complex reveals how substrate-dependent assembly remodels an autoinhibited helicase dimer into an active DNA degradation complex. Functional assays demonstrate that coordinated nuclease and helicase activities enable efficient degradation of forked DNA substrates and mediate phage defense without detectable host toxicity. Together, our findings define a mechanism in which enzymatic processing of replication-associated DNA structures licenses immune activation, providing a framework for how nucleic acid architecture is coupled to effector activation in bacterial immunity.

Panarthropod vision exhibits extraordinary morphological and functional diversity, yet the sensory biology of tardigrades--microscopic extremophiles renowned for their resilience--remains poorly understood. In the model tardigrade Hypsibius exemplaris, we uncover an unprecedented expansion of opsin genes, with over 100 paralogs constituting the largest known opsin repertoire in any animal. Paradoxically, the visual system is structurally minimalist: a paired, inverse pigment-cup ocellus embedded within the brain lobes, forming a single-pixel, dual-receptor organ. Integrating genomic, phylogenetic, molecular expression, and ultrastructural analyses, we show that directional vision relies on a single rhabdomeric opsin (He-R-Opsin-V), localized to microvilli of the rhabdomeric cell. A ciliary photoreceptor with a lamellated cilium co-expresses two ciliary opsins (He-C-Opsin-1 and -2), suggesting non-visual light detection. These and other non-visual opsins are differentially expressed in the brain, gut, storage cells, and peripheral tissues, implicating them in circadian regulation, neuromodulation, ecdysis, digestion, and environmental sensing. Crucially, the eye is an internalized epidermal vesicle, not a cerebral derivative, challenging long-standing assumptions about its evolutionary origin. These findings reveal how extreme miniaturization drives sensory system simplification in visual organs while enabling parallel evolutionary innovation in non-visual photoreception. This study establishes a new paradigm for sensory evolution in microscale animals.

Animals use the location of visual stimuli to select appropriate actions1-5, and the upper and lower visual field often carry different ecological and behavioral meaning6-9. In mice, the superior colliculus is a key central hub that transforms visual input into orienting, defensive, and approach behaviors3,10-13. Its superficial layers receive retinotopically organized input from the retina and contain genetically defined cell types with distinct downstream projections, including wide-field neurons that project to the lateral posterior thalamus and narrow-field neurons that target the parabigeminal nucleus and deeper collicular layers14-18. These features raise the question of whether circuits of the superior colliculus are repeated across visual space or exhibit visual-field-dependent specializations. Here, we show that the mouse superficial superior colliculus contains visual-field-dependent circuit modules. Dual-color rabies tracing revealed that wide-field and narrow-field neurons receive input from a largely shared set of brain regions, whereas upper- and lower-field domains differ in how they sample those inputs. Some source regions preferentially innervate one visual-field domain, producing biased regional input strength, while others contain topographically segregated projecting neurons that target upper- or lower-field domains. MAPseq showed that most superficial collicular neurons project to single downstream targets, with upper- and lower-field populations differing in target probability. Two-photon calcium imaging further showed that wide-field neurons in upper- and lower-field domains differ in stimulus selectivity. Together, these findings reveal a visual-field-dependent wiring logic that biases how the superior colliculus samples inputs and routes signals to downstream pathways. HighlightsO_LIWide- and narrow-field neurons receive broadly overlapping inputs C_LIO_LIUpper- and lower-field domains differ in input strength and topographic organization C_LIO_LIMost superficial collicular neurons project to a single target C_LIO_LIVisual field position biases downstream target probability C_LI

Pathogens hijack macrophages by triggering pathological cAMP surges that block phagolysosomal killing--a defect mirrored in phagocytes from refractory colitis. We identify a host-encoded, pathogen-specific surge-protector comprised of a three-protein toggle: The innate sensor NOD2 binds and masks an evolutionarily conserved motif in GIV that activates trimeric-GTPase Gi, enforcing a biphasic surge-to-plunge cAMP-program: early, NOD2*GIV assembly permits a brief, tolerogenic cAMP rise, whereas subsequent GIV*Gi engagement collapses cAMP to drive phagolysosomal fusion and microbial clearance. Structural, biochemical, and ultrastructural analyses reveal how molecular toggling imposes precise spatial and temporal control. Pharmacogenomic perturbations pinpoint cAMP-PKA hyperactivation as the defining lesion in GIV-deficient macrophages. Functional studies in primary macrophages and human gut organoid co-cultures show that toggling the NOD2*GIV*Gi-axis is necessary and sufficient to convert tolerant macrophages into microbicidal machines that preserve mucosal barrier integrity. These findings uncover a druggable cAMP-control pathway with therapeutic promise in colitis. GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/715116v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@6f7c6corg.highwire.dtl.DTLVardef@151d439org.highwire.dtl.DTLVardef@1441b12org.highwire.dtl.DTLVardef@4d76e3_HPS_FORMAT_FIGEXP M_FIG C_FIG eTOC BlurbPathogens hijack macrophages by inducing cAMP surges that help them evade clearance. Anandachar et al. identify a host "toggle switch" in which NOD2 and G proteins compete for GIV, driving a rapid and robust surge-to-plunge transition in cAMP. This temporal switch limits tolerogenic signaling, restores microbial clearance and barrier integrity, and unveils a targetable host pathway in infection and IBD. HighlightsO_LIPathogens exploit cAMP surges in macrophages to block phagolysosomal killing of microbes C_LIO_LIGIV acts as a molecular "toggle" linking NOD2 sensing to Gi-mediated cAMP control C_LIO_LIStructural and mutagenesis studies reveal mutually exclusive binding of NOD2 and Gi to GIV C_LIO_LIPharmacogenomic perturbations pinpoint PKA, not EPAC, as the critical downstream effector C_LIO_LIOrganoid co-cultures show NOD2-GIV-PKA crosstalk safeguards microbial clearance and gut barrier integrity C_LI

Walking involves coordinate rhythmic movements at every joint of every leg. Central pattern generator (CPG) circuits in the spinal or ventral nerve cord provide such a rhythmic drive to all the legs1-3. In turn, each leg provides rhythmic sensory feedback through peripheral proprioceptive neurons2,4-6. Disentangling contributions from these two rhythmic drives, has been a long-standing hurdle in uncovering both the structure and function of neural-circuits governing the generation of a coordinated walking output2,3,7. Here, using the highly tractable Drosophila model and a novel sensory-deprivation paradigm, we uncovered central and peripheral neural pathways underlying walking pattern generation. We provide evidence that each leg is governed by its own CPG module with an inherent cycle period that is unmasked when proprioceptive feedback is reduced. We find that contact driven load inputs and descending brain inputs are critical for coordinating the intra-leg movements and shaping the microstructure of a single legs step-cycle. We show that central coupling pathways underlie inter-leg coordination and that proprioceptive inputs and descending brain commands can flexibly modulate this coupling in a task-specific manner. We identify co-stimulation of specific descending neurons as a mechanism for speeding-up this walking rhythm. Finally, by constraining connectome search8 based on the empirical results, we identify putative neural-circuit motifs underlying generation of a coordinated six-legged walking pattern.

Leading biomedical resources rely on genome variation in Britain1-3, but the historical processes that shaped present-day fine-scale diversity remain debated4-13. Here we sequenced 1039 ancient shotgun genomes from Britain (median 1.4-fold coverage), primarily dating to the first millennium CE. We imputed [~]660 million variants in the UK Biobank14-16 and employed genealogy-based ancestry reconstruction. We found an association between Iron Age consanguinity and matrilineal burial practices17, later disrupted following the Roman Conquest. Despite this societal impact, only 20% of Roman-period individuals carried detectable ancestry from outside Britain. In contrast, from the 6th century CE we detect widespread influx of ancestry in over 70% of individuals in southernAnglo-Saxon Britain, with limited local admixture. We find previously underappreciated heterogeneity, with ancestries associated with Central and Southern Europe rising in prevalence from the 7th century CE. We demonstrate distinct Scandinavian-related ancestry in many Viking-associated contexts, but show that the population-level impact of the Viking Age in Britain was limited. Finally, we detect pre-medieval selection on variants linked with key immunity genes TLR10-TLR1 and IRF8. These results identify population-level and selective processes that shape variation and disease risk in Britain today.

Maternal inheritance of mitochondrial DNA (mtDNA) is a near-universal feature of eukaryotes1, yet the mechanisms that ensure this by preventing paternal mtDNA inheritance have remained unclear. In both Drosophila and humans, mtDNA is actively eliminated from sperm during spermatogenesis, producing mature sperm whose mitochondria lack their genomes2-5. Here we identify Hotaru, a previously uncharacterized, testis-specific GIY-YIG endonuclease, as a central player in this process. We find that Hotaru is expressed in elongated spermatids, localizes to the mitochondrial matrix, and is required for paternal mtDNA elimination. In hotaru mutants, sperm retain mtDNA at levels comparable to those present before the elimination process. Genetic and biochemical analyses show that Hotaru selectively recognizes and cleaves cruciform DNA structures within the mtDNA control region. Together, these findings identify a dedicated nuclease that enforces mitochondrial genome elimination in the animal male germline and reveal that an unexpected structural feature of mtDNA serves as the molecular determinant of its destruction. By recognizing DNA structure rather than specific sequence motifs, this mechanism is inherently robust to the high mutation rate of mitochondrial genomes.

For the brain to compute, electrical signals must propagate over the membranes of individual neurons, connecting synaptic inputs to synaptic outputs1. Complex neuronal morphologies coupled with the spatial organization of synaptic inputs and outputs enable diverse voltage transformations that underlie cell-type specific computations2,3. However, measuring these transformations in vivo has remained challenging, leaving a crucial gap in our mechanistic understanding of single neuron computation. Here, we develop ASAP7y, a genetically encoded voltage indicator with unprecedented subthreshold sensitivity and expanded excitation compatibility in both mice and flies. We leveraged ASAP7y combined with two-photon random-access microscopy to record sensory stimulus-evoked voltage dynamics with millisecond, subcellular, and subthreshold resolution along the neurites of individual neurons in Drosophila. We found remarkable heterogeneity in voltage propagation across cell-types, delineating a fundamental axis of electrical diversity. Leveraging a nanoscale EM reconstruction of the visual system4, we modeled the electrotonic properties of single neurons spanning 717 cell types, revealing how morphology shapes voltage transformations. Finally, we demonstrate that confined voltage propagation creates substrates for local computation, producing subcellular domains with distinct feature selectivity across multiple cell types. These results provide mechanistic insight into how critical single neuron computations arise and reveal parallel processing in single neurons.

Across brain regions and behaviors, neural population activity unfolds as temporally structured sequences that underlie perception, memory, and precisely timed actions1-10. However, how neural circuits transform continuous information streams into transient patterns of activity over time remains poorly understood. A long-standing hypothesis for cerebellar learning posits that the granule cell (GC) layer segments sensory and motor information arriving via mossy fibers (MFs) into temporal basis sets that enable precisely timed motor and cognitive commands11-15. Measurements of such basis sets have been elusive. Using high-speed multiphoton calcium imaging of MF and GC responses to whisker air puff stimulation, we show that prolonged MF activity is transformed into temporally sharpened GC responses that form a sparse population sequence tiling the sensory event in time. Temporal sparsity of GC sequences varied between cerebellar regions. By combining in vivo glutamate imaging with ex vivo synaptic recordings, we identify heterogeneous MF-GC synaptic strength and short-term plasticity as the mechanisms underlying region-specific temporal sparsification. Mathematical modeling predicted that region-specific MF-GC synaptic dynamics generate temporally sparse GC sequences with distinct statistics specifically suited for learning across different timescales. Thus, heterogeneous synaptic dynamics provide a biological substrate for shaping population activity in time, setting the temporal precision of sensorimotor associations underlying adaptive behavior. One-sentence summaryDiverse short-term synaptic dynamics transform input activity patterns into temporally sparse neural sequences in the cerebellar cortex, providing a mechanistic basis for precise temporal learning.

Mounting evidence from surgical type II focal cortical dysplasia (FCD) tissues and mouse models have recently shown that dysmorphic neurons carrying MTOR mutations (DNs) in FCD exhibit hallmarks of cellular senescence. Building on pioneering work from the Baulac group identifying cellular senescence as a feature of mTOR-pathway FCD, a recent study by Ribierre et al. (2024) [1] proposed oral dasatinib and quercetin (DQ) as a therapy that partially decreases the load of mutant, senescent neurons and thus reduces seizure occurrence in FCD mice. Using a different mouse strain and a different gain-of-function mutation in MTOR, our data confirm the presence of senescence hallmarks in FCD mice, but do not support one of the conclusions of Ribierre et al.--that DQ acts as a senolytic in an FCD mouse model--and we propose an alternative interpretation. We longitudinally tracked individual cell fate using two-photon microscopy and complemented these data with EEG monitoring and immunohistochemistry. Immunohistochemical analyses were performed within the same sections using multiple markers, allowing direct identification of mutant neurons and assessment of senescence-associated labeling. While we observed a detectable reduction in a senescence-associated marker, consistent with a senomorphic effect, it did not translate into a change in seizure phenotype, despite treatment timing and dosing matching those in the original study. For detailed materials and methods, see Extended Methods.

Fossilized traces of neuropils, nerves and ganglia have demonstrated that cerebral organization in Cambrian arthropods conforms to a ground pattern defining one of todays two existing euarthropod clades, Mandibulata and Chelicerata1-8. Artiopoda - a third clade including trilobites and soft-bodied relatives - persisted until the late Carboniferous9,10, but its cerebral organization has remained unknown. Here we identify and reconstruct fossilized neural traces of the artiopodan Xandarella spectaculum10, which reveal an expanded prosocerebrum associated with paired ocelli, a truncated protocerebrum supplied by substantial lateral eyes, and salient deutocerebral antennular lobes. This arrangement predicts reliance on chemosensory-guided foraging, with visual processing largely limited to dorsal orientational cues and simple local motion signals. The artiopodan brain thus reveals clade-specific modifications of homologous domains of the euarthropod cerebral ground pattern4,6-8 established in the early Cambrian.

Organisms must regulate metabolic resources such as oxygen (O2) and nutrients despite environmental variability and the energetic costs of their own actions1-3. Such regulation can occur reactively, through homeostatic corrections of recent imbalances, or predictively, through allostatic adjustments that anticipate future demand4,5. Predictive regulation is particularly important because metabolic resources often continue to be consumed for seconds to minutes after motor actions cease as tissues repay incurred costs, making it advantageous to prevent depletion before it occurs6. However, the cellular and circuit mechanisms for allostatic control remain largely unknown5,7,8. Using whole-brain neuronal and astroglial imaging and O2 measurements in behaving zebrafish, we identified a noradrenergic-astroglial circuit that detects, anticipates, and prevents internal O2 depletion. We found that swimming exacerbated internal hypoxia with a multi-second delay, but behavioral adaptations occurred before such self-generated hypoxia manifested, suggesting predictive control, confirmed using computational modeling. Noradrenergic neurons in the nucleus of the solitary tract directly detected brain hypoxia and received efference copies of swimming actions; these inputs summed at the level of membrane voltage to increase spiking and norepinephrine release when actions and resource scarcity co-occurred. Astroglia integrated noradrenergic input into prolonged Ca2+ elevation that tracked the O2 cost of recent actions and thereby predicted O2 debt relative to O2 availability, rising ~8 s before O2 fell. This astroglial prediction reorganized brain-wide activity to suppress locomotion and promote respiration, preempting O2 depletion. Silencing noradrenergic neurons or astroglial signaling abolished these hypoxia coping behaviors, whereas selective activation evoked them. This neuronal-astroglial mechanism constitutes a predictive control system that integrates physiological state with behavioral intent to avert metabolic crisis, revealing a cellular substrate for proactive energy management.

Summary paragraphWhen cells enter S phase, bidirectional DNA replication is initiated through the kinase-regulated recruitment of three activators (Cdc45, GINS and Pol epsilon) to a duplex DNA-loaded double hexamer of MCM ATPases. Together these proteins form two CMGE helicases that establish divergent replication forks as they become separated1. To understand CMGE biogenesis, we reconstituted the pre-Initiation Complex with purified yeast proteins. The cryo-EM structure shows a set of firing factors caught in the act of assembling two symmetric CMGEs. We show how stepwise complex formation reshapes MCM in preparation for DNA opening and we explain how ATP promotes firing-factor ejection and CMGE maturation. While we find that Sld2 promotes GINS recruitment to MCM as expected, it also aids efficient separation of the CMGE dimer, and it is essential for lagging strand ejection from MCM. These findings have direct implications for our understanding of the metazoan Sld2 ortholog, RECQL4, pointing to a replication-fork establishment mechanism conserved across eukaryotes.

The virulence of emerging Gram-negative pathogens frequently arises from toxins delivered by the type II secretion system1. Cryo-EM single particle analysis and cryo-electron tomography and have defined the outer membrane secretin pore in detail, but the organisation of proteins within the periplasm and inner membrane that form the pilus assembly platform is not well resolved2,3. Here we combine AlphaFold4 models with single particle cryo-EM to define the organisation of the pilus assembly platform. We show that CLM heterotrimers form a continuous link from the cytoplasmic ATPase, across the inner membrane and periplasm, to the base of the secretin channel. AlphaFold models of the inner membrane spanning rotor and cytoplasmic ATPase fit readily within the cryo-EM density. The resolved secretion system exhibits an offset between the inner membrane assembly platform and the outer membrane secretin pore, together with profound asymmetry and an unexpectedly open periplasmic architecture. This architecture provides a route by which large, folded proteins access the secretion channel from the periplasm and suggests that substrate engagement may trigger the final steps in secretion system assembly leading to secretion.

How can humans remember and imagine without vision? Mental time travel, the ability to re-experience past events and envision future ones, is widely assumed to rely on visual imagery and the construction of mental scenes. Blindness provides a critical test of this assumption. Across behavioral interviews, language analyses, and multimodal neuroimaging in congenitally blind, late-blind, and sighted individuals, we show that blind individuals, even those blind from birth, mentally time travel as vividly as sighted people, but construct their inner worlds differently. Sighted participants relied on perceptual detail and activated classic scene-processing regions, whereas blind participants emphasized thoughts and emotions and recruited reorganized occipital cortex. Connectivity analyses revealed strengthened coupling between occipital and medial temporal regions, indicating adaptive reconfiguration of the episodic system. The brain does not require images to imagine: it flexibly builds internal experiences using the representational resources available. Summary ParagraphHow humans reconstruct events that are no longer available to the senses is a fundamental but unresolved question. Remembering the past and imagining the future, known as mental time travel, is a defining feature of human cognition, shaping identity and guiding decisions{superscript 1},{superscript 2}. Prevailing theories assume that such constructions depend on visual imagery, with the minds eye reconstructing events on a visuospatial stage3,. Blindness provides a critical test of this assumption. Across extensive behavioral interviews and multimodal neuroimaging, we find that mental time travel remains phenomenologically intact in blindness, but its scaffolding changes fundamentally. Sighted individuals rely on perceptual detail and activate regions specialized for visual scenes, whereas blind individuals, whether blind from birth or later in life, emphasize thoughts and emotions and reorganize occipital cortex for conceptual strategies,. These contrasting strategies map onto distinct neural signatures, revealing a dissociation between perceptual and conceptual routes to episodic simulation. Together, these findings reveal that the brains capacity to construct internal experience rests on conceptual scaffolding, not perceptual re-creation.