SMC (structural maintenance of chromosomes) protein complexes are ring-shaped molecular motors essential for genome folding. Despite recent progress, the detailed molecular mechanism of DNA translocation in concert with the ATP-driven conformational changes of the complex remains to be clarified. In this study, we elucidated the mechanisms of SMC action on DNA using all-atom and coarse-grained molecular dynamics simulations. We first created a near-atomic full-length model of a prokaryotic SMC–kleisin complex based on experimental structures and implemented ATP-dependent conformational changes using a structure-based coarse-grained model. We further incorporated key protein–DNA hydrogen-bond interactions derived from fully atomistic simulations. Extensive simulations of the SMC complex with 800 base pairs of duplex DNA over the ATP cycle observed unidirectional DNA translocation by the SMC complex. The process exhibited a step size of ~200 base pairs, wherein the SMC complex captured a DNA segment of about the same size within the SMC ring in the engaged state, followed by its pumping into the kleisin ring as ATP was hydrolyzed. Analysis of trajectories identified the asymmetric path of the kleisin as a critical factor for the observed unidirectionality.
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The ant nest serves as the skeleton of the ant superorganism. Similar to a skeleton, the nest expands as the colony grows and requires repair after catastrophic events. We experimentally compared nest excavation in colonies seeded from a single mated queen and allowed to grow for 6 months to excavation triggered by a catastrophic event in colonies with fixed demographics, where the age of each worker, including the queen, is known. The areas excavated by equal group sizes differed significantly between these conditions: heterogeneous populations in naturally growing colonies as well as cohorts of young ants dig larger areas than old ant cohorts. Moreover, we find that younger ants tend to dig slanted tunnels while older ants dig straight down. This is a novel form of age polyethism, where an ant’s age dictates not only her likelihood to engage in a task but also the way she performs the task. We further present a quantitative model that predicts that under normal growth, digging is predominantly performed by the younger ants, while after a catastrophe, all ants dig to restore lost nest volume. The fact that the nests of naturally growing colonies exhibit slanted tunnels strengthens this prediction. Finally, our results indicate how a colony’s demographic and physical history are sketched into the current structure of its nest.
Endophilin A3-mediated clathrin-independent endocytosis (EndoA3-mediated CIE) contributes to the internalization of immunoglobulin-like proteins, including key immune synapse components. Here, we identify ICAM1 as a novel EndoA3-dependent cargo, alongside ALCAM. We demonstrate that both proteins subsequently follow retromer-dependent retrograde transport to the <i>trans</i>-Golgi network (TGN) in cancer cells. From there, we propose that they undergo polarized redistribution to the plasma membrane, where they contribute to immune synapse formation between cancer cells and cytotoxic CD8 T cells. Disruption of EndoA3 or retromer components significantly affects the response of autologous cytotoxic CD8 T cells, as evidenced by reduced cytokine production and secretion, but increased lytic activity, while proliferation and later activation marker expression remain intact. This is accompanied by diminished ICAM1 density at the immune synapse, where we observe it arriving via polarized vesicular transport, indicating altered synapse organization. Indeed, cancer cells lacking EndoA3-mediated CIE or retromer form enlarged immune synapses that fail to sustain full T cell cytokine secretion, suggesting a compensatory attempt by T cells to overcome the defective synapse, while likely promoting more transient contacts that potentially favor serial killing. Together, these findings reveal that EndoA3-mediated CIE and retrograde transport act in concert in cancer cells to relocate immune synapse components via the Golgi, thereby fine-tuning the balance between cytotoxic T cell cytokine secretion and lytic activity. These insights contribute to a better understanding of the mechanisms governing immune synapse formation and organization, providing a necessary foundation for the long-term identification of new strategies to enhance T cell–mediated anti-tumor immunity.
Classical psychedelics induce complex visual hallucinations in humans, generating percepts that are coherent at a low level, but which have surreal, dream-like qualities at a high level. While there are many hypotheses as to how classical psychedelics could induce these effects, there are no concrete mechanistic models that capture the variety of observed effects in humans, while remaining consistent with the known pharmacological effects of classical psychedelics on neural circuits. In this work, we propose the ‘oneirogen hypothesis,’ which posits that the perceptual effects of classical psychedelics are a result of their pharmacological actions inducing neural activity states that truly are more similar to dream-like states. We simulate classical psychedelics’ effects via manipulating neural network models trained on perceptual tasks with the Wake-Sleep algorithm. This established machine learning algorithm leverages two activity phases: a perceptual phase (wake) where sensory inputs are encoded, and a generative phase (dream) where the network internally generates activity consistent with stimulus-evoked responses. We simulate the action of psychedelics by partially shifting the model to the ‘Sleep’ state, which entails a greater influence of top-down connections, in line with the impact of psychedelics on apical dendrites. The effects resulting from this manipulation capture a number of experimentally observed phenomena, including the emergence of hallucinations, increases in stimulus-conditioned variability, and large increases in synaptic plasticity. We further provide a number of testable predictions which could be used to validate or invalidate our oneirogen hypothesis.
Genomes assume a complex 3D architecture in the interphase cell nucleus. Yet the molecular mechanisms that determine global genome architecture are only poorly understood. To identify mechanisms of higher-order genome organization, we performed high-throughput imaging-based CRISPR knockout screens targeting 1064 genes encoding nuclear proteins in multiple human cell lines. We assessed changes in the distribution of centromeres at single-cell resolution as surrogate markers for global genome organization. The screens revealed multiple major regulators of spatial distribution of centromeres, including components of the nucleolus, kinetochore, cohesins, condensins, and the nuclear pore complex. Alterations in centromere distribution required progression through the cell cycle and acute depletion of mitotic factors with distinct functions altered centromere distribution in the subsequent interphase. These results identify molecular determinants of spatial centromere organization, and they show that orderly progression through mitosis shapes interphase genome architecture.
Establishing a cell type-specific chromatin landscape is crucial for the maintenance of cell identity during embryonic development. However, our knowledge of how this landscape is set during vertebrate embryogenesis has been limited, due to the lack of methods to jointly detect chromatin modifications and gene expression in the same cell. Here we present a multimodal measurement of full-length transcriptome and histone modifications in individual cells during early embryonic development in zebrafish. We show that before the formation of germ layers, the chromatin and transcription states of cells are uncoupled and become progressively connected during gastrulation and somitogenesis. Silencing of developmental genes is achieved by local spreading of repressive chromatin together with cell type-specific demethylation. Combining transcription factor (TF) expression and chromatin states within an interpretable machine learning model, we classify TFs as lineage-specific activators and repressors and identify a subset of TFs that are epigenetically regulated. Altogether, our data resolves the dynamic relationship between chromatin and transcription during early vertebrate development and clarifies how these two layers interact to establish cell identity.
Protein-protein interactions underlie nearly all cellular processes. With the advent of protein structure prediction methods such as AlphaFold2 (AF2), models of specific protein pairs can be built extremely accurately in most cases. However, determining the relevance of a given protein pair remains an open question. It is presently unclear how to use best structure-based tools to infer whether a pair of candidate proteins indeed interacts with one another: ideally, one might even use such information to screen among candidate pairings to build up protein interaction networks. Whereas methods for evaluating quality of modeled protein complexes have been co-opted for determining which pairings interact (e.g. pDockQ and iPTM), there have been no rigorously benchmarked methods for this task. Here, we introduce PPIscreenML, a classification model trained to distinguish AF2 models of interacting protein pairs from AF2 models of compelling decoy pairings. We find that PPIscreenML outperforms methods such as pDockQ and iPTM for this task, and further that PPIscreenML exhibits impressive performance when identifying which ligand/receptor pairings engage one another across the structurally conserved tumor necrosis factor superfamily (TNFSF). Analysis of benchmark results using complexes not seen in PPIscreenML development strongly suggests that the model generalizes beyond training data, making it broadly applicable for identifying new protein complexes based on structural models built with AF2.
Layer-dependent functional magnetic resonance imaging (fMRI) is a promising yet challenging approach for investigating layer-specific functional connectivity (FC). Achieving a brain-wide mapping of layer-specific FC requires several technical advancements, including sub-millimeter spatial resolution, sufficient temporal resolution, functional sensitivity, global brain coverage, and high spatial specificity. Although gradient echo (GE)-based echo planar imaging (EPI) is commonly used for rapid fMRI acquisition, it faces significant challenges due to the draining-vein contamination. In this study, we addressed these limitations by integrating velocity-nulling (VN) gradients into a GE-BOLD fMRI sequence to suppress vascular signals from the vessels with fast-flowing velocity. The extravascular contamination from pial veins was mitigated using a GE-EPI sequence at 3T rather than 7T, combined with phase regression methods. Additionally, we incorporated advanced techniques, including simultaneous multi-slice (SMS) acceleration and NOise Reduction with DIstribution Corrected principal component analysis (NORDIC PCA) denoising, to improve temporal resolution, spatial coverage, and signal sensitivity. This resulted in a VN fMRI sequence with 0.9 mm isotropic spatial resolution, a repetition time (TR) of 4 s, and brain-wide coverage. The VN gradient strength was determined based on results from a button-pressing task. Using resting-state data, we validated layer-specific FC through seed-based analyses, identifying distinct connectivity patterns in the superficial and deep layers of the primary motor cortex (M1), with significant inter-layer differences. Further analyses with a seed in the primary sensory cortex (S1) demonstrated the reliability of the method. Brain-wide layer-dependent FC analyses yielded results consistent with prior literature, reinforcing the efficacy of VN fMRI in resolving layer-specific functional connectivity. Given the widespread availability of 3T…
The orbitofrontal cortex (OFC) is critical to identifying task structure and to generalizing appropriately across task states with similar underlying or hidden causes. This capability is at the heart of OFCs proposed role in a network responsible for cognitive mapping, and its loss can explain many deficits associated with OFC damage or inactivation. Substance use disorder is defined by behaviors that share much in common with these deficits, such as an inability to modify learned behaviors in the face of new information about undesired consequences. One explanation for this similarity would be if addictive drugs impacted the ability of OFC to recognize underlying similarities, hidden states, that allow information learned in one setting to be used in another. To explore this possibility, we trained rats to self-administer cocaine and then recorded single-unit activity in lateral OFC as these rats performed in an odor sequence task consisting of unique and shared positions. In well-trained controls, we observed chance decoding of sequence at shared positions and near chance decoding even at unique positions, reflecting the irrelevance of distinguishing these positions in the task. By contrast, in cocaine-experienced rats, decoding remained significantly elevated, particularly at the positions that had superficial sensory differences that were collapsed in controls across learning. These neural differences were accompanied by increases in behavioral variability at these positions. A tensor component analysis showed that this effect of reduced generalization after cocaine use also extended across positions in the sequences. These results show that prior cocaine use disrupts the normal identification of hidden states by OFC.
Critical Gram-negative pathogens, like <i>Pseudomonas</i>, <i>Stenotrophomonas,</i> and <i>Burkholderia</i>, are now resistant to most antibiotics. Complex resistance profiles, together with synergistic interactions between these organisms, increase the likelihood of treatment failure in distinct infection settings, for example in the lungs of cystic fibrosis (CF) patients. Here, we discover that cell envelope protein homeostasis pathways underpin both antibiotic resistance and cross-protection in CF-associated bacteria. We find that inhibition of oxidative protein folding inactivates multiple species-specific resistance proteins. Using this strategy, we sensitize multidrug-resistant <i>Pseudomonas aeruginosa</i> to β-lactam antibiotics and demonstrate promise of new treatment avenues for the recalcitrant emerging pathogen <i>Stenotrophomonas maltophilia</i>. The same approach also inhibits cross-protection between resistant <i>S. maltophilia</i> and susceptible <i>P. aeruginosa</i>, allowing eradication of both commonly co-occurring CF-associated organisms. Our results provide the basis for the development of next-generation strategies that target antibiotic resistance, while also impairing specific interbacterial interactions that enhance the severity of polymicrobial infections.
The speed of voluntary movements varies systematically, with some individuals moving consistently faster than others across different actions. These variations, conceptualized as vigor, reflect a time–effort–accuracy tradeoff in motor planning. How do two mechanically coupled partners with different individual vigors collaborate, e.g. to move a table together? Here, we show that such dyads coordinate goal-directed movements with minimal interaction force, exhibiting a <i>dyadic vigor</i> with similar characteristics as individual vigor. The emerging dyadic motor plan is strongly influenced by the slower partner, whose vigor predicts dyadic vigor, with effects lasting beyond practice. Computational modeling with stochastic optimal control reveals the critical role of partners’ movement timing uncertainty and vigor in shaping coordination, allowing us to predict dyadic movements from individual behavior across diverse conditions. These findings shed light on the mechanisms underlying human collaboration and may be used in applications ranging from physical training and rehabilitation to collaborative robotics for manufacturing.
Alpha-band neural oscillations (8–13 Hz) are theorized to phasically inhibit visual processing based, in part, on results showing that pre-stimulus alpha phase predicts detection (i.e., hit rates). However, recent failures to replicate and a lack of a mechanistic understanding regarding how alpha impacts detection have called this theory into question. We recorded EEG while six observers (6020 trials each) detected near-threshold Gabor targets embedded in noise. Using signal detection theory (SDT) and reverse correlation, we observed an effect of occipital and frontal pre-stimulus alpha phase on sensitivity (d'), not criterion. Hit and false alarm rates were counterphased, consistent with a reduction in internal noise during optimal alpha phases. Perceptual reports were also more consistent when two identical stimuli were presented during the optimal phase, suggesting a decrease in internal noise rather than signal amplification. Classification images revealed sharper spatial frequency and orientation tuning during the optimal alpha phase, implying that alpha phase shapes sensitivity by modulating sensory tuning towards relevant stimulus features.
A new biologically-informed training paradigm enables protein language models to predict affinity maturation trajectories for antibodies.
Neural network architecture determines its functional output. However, the detailed mechanisms are not well characterized. In this study, we focused on the neural network architectures of male and hermaphrodite <i>Caenorhabditis elegans</i> and the association with sexually dimorphic behaviors. We applied graph theory and computational neuroscience methods to systematically discern the features of these two neural networks. Our findings revealed that a small percentage of sexual-specific neurons exerted dominance throughout the entire male neural network, suggesting males prioritized sexual-related behavior outputs. Based on the structural and dynamical characteristics of two complete neural networks, sub-networks containing sex-specific neurons and their immediate neighbors, or sub-networks exclusively comprising sex-shared neurons, we predicted dimorphic behavioral outcomes for males and hermaphrodites. To verify the prediction, we performed behavioral and calcium imaging experiments and dissected a circuit that is specific for the increased spontaneous local search in males for mate-searching. Our research sheds light on the neural circuits that underlie sexually dimorphic behaviors in <i>C. elegans</i> and provides significant insights into the interconnected relationship between network architecture and functional outcomes at the whole-brain level.
Cre-loxP technology, a cornerstone in fate mapping and in vivo gene function studies, faces challenges in achieving precise and efficient conditional mutagenesis through inducible systems. This study introduces two innovative genetic tools designed to overcome these limitations. The first, roxCre, enables DreER-mediated Cre release, paving the way for intersectional genetic manipulation that permits increased precision and efficiency. The second, loxCre, facilitates conditional gene targeting by allowing CreER lines to induce Cre expression with significantly enhanced efficiency. These tools incorporate a fluorescent reporter for genetic lineage tracing, simultaneously revealing efficient gene knockout in cells marked by the reporter. These strategies hold great potential for precise and efficient exploration of lineage-specific gene functions, marking a significant advancement in genetic research methodologies.
Tripartite multidrug efflux pumps that span the cell envelope are essential for antibiotic resistance in Gram-negative bacteria. Here, we report cryo-EM structures of two endogenous efflux complexes from <i>Escherichia coli</i>: a TolC–YbjP subcomplex at 3.56 Å resolution and the complete TolC–YbjP–AcrABZ pump at 3.39 Å. Structural analysis reveals that YbjP, a previously uncharacterized lipoprotein, binds TolC in a 3:3 stoichiometry, bridging the TolC protomers at their equatorial domain. Clear density of the mature YbjP’s N-terminal Cys19 indicates that YbjP is anchored to the outer membrane by an N-terminal lipid moiety. Notably, YbjP remains bound as TolC undergoes AcrA-induced opening, suggesting that this accessory protein accommodates the conformational change. The AcrB trimer simultaneously presents three distinct conformational states (L, T, and O), capturing a complete transport cycle. These high-resolution structures provide insights into the architecture and mechanism of clinically relevant efflux machinery, identifying YbjP as a previously unrecognized structural component that contributes to TolC positioning, and may assist in its membrane localization.
mRNA translation involves multiple regulatory steps, but how translation elongation influences protein output remains unclear. Using SunTag live-cell imaging and mathematical modeling, we quantified translation dynamics in single mRNAs across diverse coding sequences. Our Totally Asymmetric Exclusion Process (TASEP)-based Hidden Markov Model revealed a strong coordination between initiation and elongation rates, resulting in consistently low ribosome density (≤12% occupancy) across all reporters. This coupling persisted under pharmacological inhibition of the elongation factor eIF5A, where proportional decreases in both initiation and elongation rates maintained homeostatic ribosome density. In contrast, eIF5A knockout cells exhibited a significant decrease in ribosome density, suggesting altered coordination. Together, these results highlight a dynamical coupling of initiation and elongation rates at the single-mRNA level, preventing ribosome crowding and maintaining translational homeostasis in mammalian cells.
Humans perform tasks involving the manipulation of inputs regardless of how these signals are perceived by the brain, thanks to representations that are invariant to the stimulus modality. In this paper, we present modality-agnostic decoders that leverage such modality-invariant representations to predict which stimulus a subject is seeing, irrespective of the modality in which the stimulus is presented. Training these modality-agnostic decoders is made possible thanks to our new large-scale fMRI dataset SemReps-8K, released publicly along with this paper. It comprises six subjects watching both images and short text descriptions of such images, as well as the conditions during which the subjects were imagining visual scenes. We find that modality-agnostic decoders can perform as well as modality-specific decoders and even outperform them when decoding captions and mental imagery. Furthermore, a searchlight analysis revealed that large areas of the brain contain modality-invariant representations. Such areas are also particularly suitable for decoding visual scenes from the mental imagery condition.
Fluid flow has a major effect on the aggregation and fragmentation of bacterial colonies. Yet, a generic framework to understand and predict how hydrodynamics affects colony size remains elusive. This study investigates how fluid flow affects the formation and maintenance of large colonial structures in cyanobacteria, using an experimental technique that precisely controls hydrodynamic conditions. We performed experiments on laboratory cultures and lake samples of the cyanobacterium <i>Microcystis</i>, while their colony size distribution was measured simultaneously by direct microscopic imaging. We demonstrate that extracellular polymeric substances (EPS)-embedded cells formed by cell division exhibit significant mechanical resistance to shear forces. However, at elevated hydrodynamic stress levels (exceeding those typically generated by surface wind mixing), these colonies experience fragmentation through an erosion process. We also show that single cells can aggregate into small colonies due to fluid flow. However, the structural integrity of these flow-induced colonies is weaker than that of colonies formed by cell division. We provide a mathematical analysis to support the experiments and demonstrate that a population model with two categories of colonies describes the measured size distributions. Our results shed light on the specific conditions wherein flow-induced fragmentation and aggregation of cyanobacteria are decisive and indicate that colony formation under natural conditions is mainly driven by cell division, although flow-induced aggregation could play a role in dense bloom events. These findings can be used to improve prediction models and mitigation strategies for toxic cyanobacterial blooms and also offer potential applications in other areas, such as algal biotechnology or medical settings where the dynamics of biological aggregates play a significant role.
Cone-rod dystrophy (CRD) is a macular degeneration disorder characterized by initial cone cell degeneration. Mutations in CDHR1, a photoreceptor-specific cadherin, have been found to be associated with the incidence of CRD. While studying the function of CDHR1, we observed that the localization of the zebrafish homologue, cdhr1a, resembles that of calyceal process (CPs). When co-labeling CPs using pcdh15b, we observed that cdhr1a, in the outer segment (OS), juxtaposes with pcdh15b, found in the CP. Similar localization patterns were detected in human, macaque, xenopus, ducks, gerbil, and mouse. Using immunoprecipitation and K652 cell aggregation assays, we demonstrate that pcdh15b and cdhr1a can interact and thus potentially link the OS and CP. To analyze the consequences of OS-CP interactions in CRD, we established a <i>cdhr1a</i> mutant line (<i>cdhr1a<sup>fs*146</sup></i>). Homozygous <i>cdhr1a<sup>fs*146</sup></i> mutants exhibit minor cone OS defects starting at 15 dpf and severe OS disruption and cell loss by 3 months. Shortening of CPs coincided with cone OS defects which were significantly exacerbated when combined with the loss of pcdh15b. Rod OS defects were mild and delayed until 3–6 months. In conclusion, we propose that cdhr1a and pcdh15b function to link cone OSs with CPs and maintain OS integrity.
Intermittent fasting (IF) has emerged as a powerful dietary intervention with profound metabolic benefits, yet the tissue-specific molecular mechanisms underlying these effects remain poorly understood. In this study, we employed comprehensive proteomics and transcriptomics analysis to investigate the systemic and organ-specific adaptations to IF in male C57BL/6 mice. Following a 16 hr daily fasting regimen (IF16) over 4 months, IF reduced blood glucose, HbA1c, and cholesterol levels while increasing ketone bodies, indicative of enhanced metabolic flexibility. Proteomic profiling of the liver, skeletal muscle, and cerebral cortex revealed tissue-specific responses, with the liver exhibiting the most pronounced changes, including upregulation of pathways involved in fatty acid oxidation, ketogenesis, and glycan degradation, and downregulation of steroid hormone and cholesterol metabolism. In muscle, IF enhanced pyruvate metabolism, fatty acid biosynthesis, and AMPK signaling, while suppressing oxidative phosphorylation and thermogenesis. The cerebral cortex displayed unique adaptations, with upregulation of autophagy, PPAR signaling, and metabolic pathways, and downregulation of TGF-beta and p53 signaling, suggesting a shift toward energy conservation and stress resilience. Notably, Serpin A1c emerged as the only protein commonly upregulated across all three tissues, highlighting its potential role in systemic adaptation to IF. Integrative transcriptomic and proteomic analyses revealed partial concordance between mRNA and protein expression, underscoring the complexity of post-transcriptional regulation. Shared biological signaling processes were identified across tissues, suggesting unifying mechanisms linking metabolic changes to cellular communication. Our findings reveal both conserved and tissue-specific responses by which IF may optimize energy utilization, enhance metabolic flexibility, and promote cellular resilience.
A third of patients with the pediatric cerebellar tumor Medulloblastoma (MB) have mutations that activate Sonic hedgehog (SHH) signaling (SHH-MB subgroup). The contribution of secondary mutations to tumor severity, however, is not clear. <i>PTEN</i> mutations are enriched in the SHH-1 subtype that has the lowest survival rate. Widespread heterozygous loss of <i>Pten</i> in two SHH-MB mouse models increases penetrance and accelerates onset of differentiated tumors. We delineated cellular and transcriptional changes that accelerate tumor growth and cause differentiation using a sporadic SHH-MB mouse model expressing oncogenic SmoM2 in rare cerebellar granule cell precursors (GCPs) and scRNA-seq analysis. Homozygous but not heterozygous sporadic loss of <i>Pten</i> resulted in rapid acceleration of tumor growth and end-stage disease by 40 days, compared to ~25% survival in control SmoM2 mice at 100 days. Heterozygous <i>PTEN</i> mutations, therefore, should negatively impact disease outcome primarily with germline mutations. Loss of <i>Pten</i> in normal or SmoM2-expressing GCPs increased proliferation and enhanced progenitor state initially, but by 12 days <i>Pten</i> mutant SmoM2 tumors were highly differentiated due to increased survival of non-proliferating GCPs. Furthermore, macrophage infiltration and cytotoxicity appeared reduced in differentiated regions of tumors lacking <i>Pten</i>, indicating cell nonautonomous changes could also contribute to accelerated tumor growth.
The mechanisms guiding nocturnal insect migration remain poorly understood. Although many species are thought to use the geomagnetic field, the sensory basis of magnetic orientation in insects has yet to be clarified. We developed an indoor experimental system to investigate the integration of geomagnetic and visual cues in the seasonal orientation of a globally distributed pest moth, the fall armyworm (<i>Spodoptera frugiperda</i>), a highly invasive species which in the past decade has colonized almost all potentially habitable regions of the globe. Our results demonstrate that fall armyworms require both geomagnetic and visual cues for accurate migratory orientation, with visual cues being indispensable for magnetic orientation. When visual and geomagnetic cues are placed in conflict, moths become disoriented, although not immediately, indicating that sensory recognition of the conflict requires time to process. We also show that the absence of visual cues leads to a significant loss of flight stability, which likely explains the disruption in orientation. Our findings highlight that visual cues are critical for stable magnetic orientation in the fall armyworm, offering a basis for future investigations of visual-magnetic integration in noctuid migrants.
The transcription factor (TF) regulatory network in <i>Pseudomonas aeruginosa</i> is complex and involves multiple regulators that respond to various environmental signals and physiological cues by regulating gene expression. However, the biological functions of at least half of its 373 putative TFs remain uncharacterised. Herein, chromatin immunoprecipitation sequencing (ChIP-seq) was used to investigate the binding sites of 172 TFs in the <i>P. aeruginosa</i> PAO1 strain. The results revealed 81,009 significant binding peaks in the genome, more than half of which were located in the promoter regions. To further decode the diverse regulatory relationships among TFs, a hierarchical network was assembled into three levels: top, middle, and bottom. Thirteen ternary regulatory motifs revealed flexible relationships among TFs in small hubs, and a comprehensive co-association atlas was established, showing the enrichment of seven core associated clusters. Twenty-four TFs were identified as the master regulators of virulence-related pathways. The pan-genome analysis revealed the conservation and evolution of TFs in <i>P. aeruginosa</i> complex and other species. A web-based database combining existing and new data from ChIP-seq and the high-throughput systematic evolution of ligands by exponential enrichment was established for searching TF-binding sites. This study provides important insights into the pathogenic mechanisms of <i>P. aeruginosa</i> and related bacteria and is expected to contribute to the development of effective therapies for infectious diseases caused by this pathogen.