Science Journals

With the continuous rise in antibiotic resistance, novel methods that can reveal currently unknown antibiotic resistance mechanisms are essential to prepare and inform health responses and novel antibiotic discovery campaigns. Here, we built a library of species representative of the genus <i>Mycobacterium</i> and determined their antibiotic resistance profiles, allowing for the first time systematic multispecies comparisons. Analyzing antibiotic resistance in the context of other closely related yet diverse organisms revealed species with truly exceptional traits as well as general principles underpinning antibiotic resistance. Among these, we reveal that intrabacterial accumulation of antibiotics does not correlate with their potency at the species level. Our data also reveals that rifamycin resistance in mycobacteria is dominantly caused by antibiotic modification, contrary to what has been observed in <i>Mycobacterium tuberculosis</i>. Our data provides a solid starting point for the exploration of novel determinants of antibiotic resistance. We illustrate the utility of this species-level approach to discovery of novel traits by characterizing a previously unrecognized rifamycin-inactivating enzyme group that is present in a wide range of bacterial genera.

Insulin degrading enzyme (IDE) is a dimeric M16A zinc metalloprotease that degrades amyloidogenic peptides diverse in shape and sequence, including insulin and amyloid-β, to prevent toxic amyloid fibril formation. IDE has a hollow catalytic chamber formed by two ~55 kDa N- and C- domains (IDE-N and IDE-C, respectively), in which peptides bind, unfold, and are repositioned for proteolysis. IDE is known to transition between a closed state, poised for catalysis, and an open state, able to release cleavage products and bind a new substrate. Here, we present six cryo-EM structures of the IDE dimer at 3.0–5.1 Å resolution, obtained in the presence of a sub-saturating concentration of insulin. Combining cryo-EM heterogeneity analysis with all-atom molecular dynamics (MD) simulations, we identified the structural basis and key residues for IDE conformational dynamics that were not previously revealed by IDE static structures. Notably, R668 serves as a molecular latch mediating the open-close transition and facilitates key protein motions through charge-swapping interactions at the IDE-N/C interface. Our small-angle X-ray scattering analysis and enzymatic assays of an R668A mutant indicate a profound alteration of conformational dynamics and catalytic activity. By integrating coarse-grained MD simulations, our analysis reveals that IDE unfolds its substrates through the coordinated motion between IDE-N and IDE-C, as well as β-sheet formation between IDE and insulin. Additionally, our time-resolved cryo-EM analysis uncovers IDE allostery within the IDE dimer. Collectively, our findings demonstrate the strength of combining experimental and computational approaches to probe protein dynamics and pave the way for developing substrate-specific modulators of IDE activity.

TFIIH is an essential transcription initiation factor for RNA polymerase II (RNApII). This multi-subunit complex comprises two modules that are physically linked in <i>Saccharomyces cerevisiae</i> by the subunit Tfb3 (MAT1 in metazoans). The Core Module, with two DNA-dependent ATPases and several additional subunits, promotes DNA unwinding. The Kinase Module phosphorylates the C-terminal domain (CTD) of RNApII subunit Rpb1, initiating a cycle of CTD modifications that coordinate the exchange of initiation and elongation factors. Why these two disparate activities are bundled into one factor is not obvious, but the connection may provide temporal coordination during early initiation. When Tfb3 is split into two parts to uncouple the TFIIH modules, the resulting cells are viable but grow very slowly. Chromatin immunoprecipitation of the split TFIIH shows that the Core Module, but not the Kinase, is properly recruited to promoters. Instead of the normal promoter-proximal peak, high CTD Serine 5 phosphorylation is seen throughout transcribed regions. Therefore, coupling the TFIIH modules is necessary to localize and limit CTD kinase activity to early stages of transcription. These results are consistent with the idea that the two TFIIH modules began as independent functional entities that later became connected by Tfb3 during early eukaryotic evolution.

The superior colliculus (SC) is traditionally considered a brain region that functions as an interface between processing visual inputs and generating eye movement outputs. Although its role as a primary reflex center is thought to be conserved across vertebrate species, evidence suggests that the SC has evolved to support higher-order cognitive functions, including spatial attention. When it comes to oculomotor areas, such as the SC, it is critical that high precision fixation and eye movements are maintained even in the presence of signals related to ongoing changes in cognition and brain state, both of which have the potential to interfere with eye position encoding and movement generation. In this study, we recorded spiking responses of neuronal populations in the SC while two rhesus macaque monkeys performed a memory-guided saccade task and found that the activity of some of the neurons fluctuated over tens of minutes. By leveraging the statistical power afforded by high-dimensional neuronal recordings, we were able to identify a low-dimensional pattern of activity that was correlated with pupil size and simultaneously recorded data in the prefrontal cortex (PFC), consistent with slow changes in the monkeys’ arousal levels while they were performing the task. Importantly, we found that the spiking responses of deep-layer SC neurons were less correlated with this brain-wide arousal signal, and that neural activity associated with changes in pupil size and saccade tuning did not overlap in population activity space with movement initiation signals. Taken together, these findings provide a framework for understanding how signals related to cognition and arousal can be embedded in the population activity of oculomotor structures without compromising the fidelity of the motor output.

Animals respond to tactile stimulations of the body with location-appropriate behavior, such as aimed grooming. These responses are mediated by mechanosensory neurons distributed across the body, whose axons project into somatotopically organized brain regions corresponding to body location. How mechanosensory neurons interface with brain circuits to transform mechanical stimulations into location-appropriate behavior is unclear. We previously described the somatotopic organization of bristle mechanosensory neurons (BMNs) around the <i>Drosophila</i> head that elicit a sequence of location-aimed grooming movements (Eichler et al., 2024). Here, we use a serial section electron microscopy reconstruction of a full adult fly brain to identify nearly all of BMN pre- and postsynaptic partners uncovering circuit pathways that control head grooming. Postsynaptic partners dominate the connectome and are both excitatory and inhibitory. We identified an excitatory cholinergic hemilineage (hemilineage 23b), a developmentally related group of neurons that elicits aimed head grooming and exhibits differential connectivity with BMNs from distinct head locations, revealing a lineage-based somatotopically organized parallel circuit architecture. Presynaptic partners provide extensive BMN presynaptic inhibition, consistent with models of sensory gain control as a mechanism of suppressing grooming movements and controlling the sequence. This work provides the first comprehensive map of a somatotopically organized connectome, and reveals how this organization could shape grooming. It also reveals the mechanosensory interface with the brain, illuminating fundamental features of mechanosensory processing, including feedforward excitation and inhibition, feedback inhibition, somatotopic circuit organization, and developmental origins.

Organisms have evolved protective strategies that are geared toward limiting cellular damage and enhancing organismal survival in the face of environmental stresses, but how these protective mechanisms are coordinated remains unclear. Here, we define a requirement for neural activity in mobilizing the antioxidant defenses of the nematode <i>Caenorhabditis elegans</i> both during chronic oxidative stress and prior to its onset. We show that acetylcholine-deficient mutants are particularly vulnerable to chronic oxidative stress. We find that extended oxidative stress mobilizes a broad transcriptional response which is strongly dependent on both cholinergic signaling and activation of the muscarinic G-protein acetylcholine-coupled receptor (mAChR) GAR-3. Gene enrichment analysis revealed a lack of upregulation of proteasomal proteolysis machinery in both cholinergic-deficient and <i>gar-3</i> mAChR mutants, suggesting that muscarinic activation is critical for stress-responsive upregulation of protein degradation pathways. Further, we find that GAR-3 overexpression in cholinergic motor neurons prolongs survival during chronic oxidative stress. Our studies demonstrate neuronal modulation of antioxidant defenses through cholinergic activation of G protein-coupled receptor signaling pathways, defining new potential links between cholinergic signaling, oxidative damage, and neurodegenerative disease.

Bacteria precisely regulate the place and timing of their cell division. One of the best-understood systems for division site selection is the Min system in <i>Escherichia coli</i>. In <i>E. coli</i>, the Min system displays remarkable pole-to-pole oscillation, creating a time-averaged minimum at the cell’s geometric center, which marks the future division site. Interestingly, the Gram-positive model species <i>Bacillus subtilis</i> also encodes homologous proteins: the cell division inhibitor MinC and the Walker-ATPase MinD. However, <i>B. subtilis</i> lacks the activating protein MinE, which is essential for Min dynamics in <i>E. coli</i>. We have shown before that the <i>B. subtilis</i> Min system is highly dynamic and quickly relocalizes to active sites of division. This raised questions about how Min protein dynamics are regulated on a molecular level in <i>B. subtilis</i>. Here, we show with a combination of in vitro experiments and in vivo single-molecule imaging that the ATPase activity of <i>B. subtilis</i> MinD is activated by membrane binding. Additionally, both monomeric and dimeric MinD bind to the membrane, and binding of ATP to MinD is a prerequisite for fast membrane detachment. Single-molecule localization microscopy data confirm membrane binding of monomeric MinD variants. However, only wild-type MinD enriches at cell poles and sites of ongoing division, likely due to interaction with MinJ. Monomeric MinD variants and locked dimers remain distributed along the membrane and lack the characteristic pattern formation. Single-molecule tracking data further support that MinD has a freely diffusive population, which is increased in the monomeric variants and a membrane-binding defective mutant. Thus, MinD dynamics in <i>B. subtilis</i> under the tested conditions do not require any unknown protein component and can be fully explained by MinD’s binding and unbinding kinetics with the membrane. The spatial organization of MinD relies on the short-lived …

Here, we review the history, advancements, and broad utility of the NTR/prodrug system, and suggest future strategies for developing versatile ablation models. As a chemogenetic tool, the nitroreductase (NTR)/prodrug system enables precise spatiotemporal control over cell ablation. The technology leverages bacterial NTR enzymes (e.g. <i>nfsB</i>) to convert inert prodrugs into cytotoxic agents, thereby allowing researchers to induce targeted cell death. Although the NTR/prodrug approach was first implemented in transgenic mice, it was subsequently adapted to zebrafish, where it has been extensively optimized and applied. Consequently, zebrafish remain the primary focus of this review. Nevertheless, the utility of the NTR/prodrug system has expanded to other important model organisms, including <i>Drosophila</i>, <i>Nematostella</i>, <i>Xenopus</i>, medaka, and rats, enabling detailed studies of tissue damage and regeneration. This review highlights how the NTR system has been deployed to model a spectrum of human diseases, including Parkinson’s disease, retinal degeneration, demyelinating disorders, and kidney disease. These models provide valuable platforms to study pathogenesis in vivo. Furthermore, the precise and controllable nature of NTR ablation makes it an ideal tool for high-throughput chemical and genetic screens aimed at discovering pro-regenerative and protective compounds. The development of NTR2.0, an enzyme variant with over 100-fold greater activity, along with more potent prodrugs such as ronidazole (RNZ), has dramatically broadened experimental possibilities. These improvements permit chronic ablation and long-term disease modeling at well-tolerated drug concentrations. Here, we present some key considerations, including transgenic design for optimal cell-type specificity, calibrating expression levels for desired ablation kinetics, and suitable controls to allow interpretation. These best practices will allow the researcher to develop a precise…

Functional brain network organisation, measured by functional connectivity (FC), reflects key neurodevelopmental processes for healthy development. Early exposure to adversity, for example undernutrition, affects neurodevelopment, observable via disrupted FC, and leads to poorer outcomes from preschool age onwards. We assessed longitudinally the impact of early growth trajectories on developmental FC in a rural Gambian population from age 5–24 months. To investigate how these early trajectories relate to later childhood outcomes, we assessed cognitive flexibility at 3–5 years. We observed that early physical growth before the fifth month of life drove optimal developmental trajectories of FC that in turn predicted cognitive flexibility at pre-school age. In contrast to previously studied developmental populations, this Gambian sample exhibited long-range interhemispheric FC that decreased with age. Our results highlight the measurable effects that poor growth in early infancy has on brain development and the possible subsequent impact on pre-school age cognitive development, underscoring the need for early life interventions throughout global settings of adversity.

Collagen is the most abundant protein in the human body and has an important role in healthy tissue as well as in a range of prevalent diseases. Medical research and diagnostics, hence, call for means of mapping collagen in vivo. Magnetic resonance imaging (MRI) is a natural candidate for this task, offering full 3D capability and versatile contrast non-invasively. However, collagen has so far been invisible to MRI due to extremely short lifetime of its resonances. Here, we report the direct imaging of collagen in vivo by magnetic resonance on the microsecond scale. The dynamics of resonance signals from collagen were first assessed in samples of bovine tendon and cortical bone. On this basis, imaging was performed at echo times down to 10 microseconds, yielding collagen-specific depiction by echo subtraction. The same approach was then extended for use in vivo, enabling direct collagen imaging of a human forearm. This capability suggests significant promise for biomedical science and clinical use.

Pseudouridine (Ψ) modifications are the most abundant RNA modifications; however, their distribution and functional significance in bacteria remain largely unexplored compared to eukaryotic systems. In this study, we present the first transcriptome-wide and quantitative mapping of Ψ modifications across five diverse bacterial species (<i>Bacillus cereus</i>, <i>Escherichia coli</i>, <i>Klebsiella pneumoniae</i>, <i>Pseudomonas aeruginosa</i>, and <i>Pseudomonas syringae</i>) at single-base resolution, utilizing the optimized baBID-seq method for bacterial RNA. Our analysis revealed growth phase-dependent dynamics of pseudouridylation in bacterial tRNA and mRNA, particularly in genes enriched in core metabolic pathways. Comparative analysis demonstrated evolutionarily conserved features of Ψ modifications, such as dominant motif contexts, Ψ clustering within operons, etc. Functional analysis indicated Ψ modifications affect bacterial mRNA stability, translation, and interactions with specific RNA-binding proteins in response to changing cellular demands during growth phase transitions. The integrated computational analysis on local RNA architecture was conducted to elucidate the structure-dependent Ψ modifications in bacterial RNA. Furthermore, we developed an integrated deep learning framework, combining LSTM-transformer-GNN-based neural networks (pseU_NN) to capture both RNA sequence and local structure features for effective prediction of Ψ-modified sites. Overall, our study provides valuable insights into the landscapes of bacterial RNA Ψ modifications and establishes a foundation for future mechanistic investigations on bacterial Ψ functions.

Sequence and structure comparisons between alpha and beta connexins, Cx43 and Cx26, revealed that Cx43 has a motif, the carbamylation motif, that confers CO₂-sensitivity on a subset of beta connexins. By using a fluorescent dye loading assay, whole cell patch clamp recordings and real-time measurement of ATP release via GRAB_ATP, we have demonstrated that Cx43 hemichannels open in a highly CO₂-sensitive manner over the range 20–70 mmHg. Mutational analysis confirms that the equivalent residues to those in Cx26, known to be involved in mediating the effects of CO₂ on gating of hemichannels and gap junction channels, also mediate Cx43 hemichannel gating. These data predict that Cx43 will be partially open at resting physiological levels of PCO₂. In acute mouse hippocampal slices, we have demonstrated a CO₂-dependent enhancement of synaptic transmission that was blocked by the Cx43-selective mimetic peptide Gap26. Our data resolves an inconsistency in the literature between in vivo studies suggesting that Cx43 hemichannels are at least partially open at rest and in vitro studies performed in the absence of HCO₃^-/CO₂ buffering that show Cx43 hemichannels are shut.

Cholesterol homeostasis is fundamental to cellular function, and its disruption underlies a wide range of human diseases. However, the contribution of cholesterol biosynthesis to auditory physiology remains poorly understood. HSD17B7 (17β-Hydroxysteroid dehydrogenase type 7) catalyzes the conversion of zymosterone to zymosterol, a key step in the post-lanosterol cholesterol biosynthetic pathway. Here, we found that Hsd17b7 is highly enriched in sensory hair cells of zebrafish and mice. The deficiency of Hsd17b7 reduced intracellular cholesterol levels in HEI-OC1 cells and zebrafish hair cells, thereby compromising MET and acoustic startle responses. A heterozygous nonsense variant (c.544G&gt;T; p.E182*) in <i>HSD17B7</i> was identified in an individual with bilateral profound hearing loss. mRNA of c.544G&gt;T HSD17B7 failed to rescue the impaired MET and acoustic startle response of hsd17b7 mutants. Mechanistically, the mutation decreases mRNA abundance and significantly reduces protein. Moreover, expression of the p.E182* mutation disrupted the interaction between HSD17B7 and the ER retention receptor RER1, leading to aberrant subcellular localization and altered cholesterol distribution, thereby exacerbating HC dysfunction. Together, our findings suggest a conserved and essential role for HSD17B7-mediated cholesterol biosynthesis in sensory hair cell function and identify HSD17B7 as a candidate gene for sensorineural hearing loss.

The glutamine-binding protein GlnBP is part of an ATP-binding cassette transporter system in <i>Escherichia coli</i> and uses two well-characterized conformational states, an open ligand-free and a closed-liganded state, to facilitate active amino-acid uptake. Existing literature on its ligand-binding mechanism lacked sufficient evidence to univocally assign the kinetic type of binding mechanism for GlnBP: ligand binding prior to conformational change, that is an induced fit, or the conformational selection, in which the ligand binds the matching conformation from a pre-existing ensemble. Since such mechanistic questions are relevant for our fundamental understanding of how this and other biomacromolecules regulate cellular processes, we here revisit the question for GlnBP. We present a biochemical and biophysical analysis using a combination of calorimetry, single-molecule and surface-plasmon resonance spectroscopy, and molecular dynamics simulations. We found that both apo- and holo-GlnBP show no detectable exchange between open and (semi-)closed conformations on timescales between 100 ns and 10 ms and that ligand binding and conformational changes in GlnBP are correlated. A global analysis of our experimental results suggests that the conformational selection model is only compatible with GlnBP for the extreme scenario of very fast conformational exchange between the open and closed states on timescales &lt;100 ns. In contrast, all data remains compatible with an induced-fit mechanism, where the ligand binds GlnBP prior to conformational rearrangements. Importantly, our work demonstrates that it is an intricate task to identify the type of kinetic binding mechanism and that this requires not only a sufficient set of data, but also an integrative experimental and theoretical framework to address the question. Based on this concept, we propose that various protein systems, for which so far only insufficient kinetic data are available, should be revisited.

Liu Q, Xiao Q, Sun Z, Wang B, Wang L, Wang N, Wang K, Song C, Yang Q. 2021. Exosome component 1 cleaves single-stranded DNA and sensitizes human kidney renal clear cell carcinoma cells to poly(ADP-ribose) polymerase inhibitor. eLife 10:e69454. doi: 10.7554/eLife.69454. Published 23 June 2021 Following post-publication review of our manuscript, we identified errors in the manuscript. In Figure 1—figure supplement 1A, the top-ranked genes of each substitution type (Supplementary file 1) were used for Gene Ontology (GO) enrichment analysis with Metascape (metascape.org). During data processing, the data corresponding to the C>T/G>A substitution were misapplied to the C>G/G>C group. The C>T/G>A mutation should be shown to be enriched in GO:0140053 mitochondrial gene expression, GO:0000959, GO:0007005, GO:1902775 and GO:0034620. We have updated Figure 1—figure supplement 1A and source data accordingly. Because the findings and conclusions were based on the original data, these corrections do not affect the conclusion that substitution mutations were enriched in ‘mitochondrial gene expression’. In Figure 2J and Figure 2—figure supplement 1D, the labels for the A>G/T>C and A>C/T>G mutations were inadvertently reversed. In addition, the mutation frequency of A>G/T>C should be 3.13% but not 31.3%. The figures and source data have been corrected accordingly. These corrections do not affect the conclusion for C>A/G>T mutation. In Figure 2B-2D and Figure 5E, illustrative images based on the incomplete experiments were mistaken as final experimental images. During the handover of research work, illustrative images based on incomplete experiments were applied to show the study information and figure layout. Due to a miscommunication between the authors, the illustrative images (EXOSC1 and several groups in Figure 2B-2D, and TUHR14TKB cells in Figure 5E) were mistaken as final images for publication. To rectify these issues, the authors reviewed all original data and experiment…

Trained immunity involves the reprogramming of innate immune cells after an initial exposure, resulting in heightened inflammatory responses to subsequent stimuli and enhanced bactericidal capacity during infection. However, this pro-inflammatory state could also exacerbate chronic conditions like inflammatory bowel disease (IBD), which is characterized by persistent inflammation and microbial imbalance. It remains unclear how trained immunity influences IBD pathogenesis and whether it can be harnessed therapeutically. In our study, pretreatment with β-glucan reprogrammed bone marrow hematopoietic progenitors and peripheral monocytes, inducing a profound shift in monocyte plasticity and significantly reducing the severity of dextran sulfate sodium (DSS)-induced colitis. Adoptive transfer of bone marrow or peripheral monocytes from β-glucan-trained mice into naive mice conferred robust protection against colitis, demonstrating that this protective effect is transferable. Trained mice also displayed improved clearance of intestinal bacterial infections. Single-cell RNA sequencing revealed an expansion of reparative Cx3cr1⁺ macrophages derived from Ly6C^hi monocytes, correlating with accelerated colonic epithelial regeneration. Collectively, these findings reveal how β-glucan-induced trained immunity modulates monocyte differentiation to ameliorate experimental colitis, highlighting the potential of harnessing trained immunity as a therapeutic strategy to recalibrate innate immune responses and restore gut homeostasis in IBD, shedding light for future clinical applications.

Cohesin is a DNA tethering complex essential for chromosome structure and function. In fission yeast, defects in the cohesin loader Mis4 result in chromosome segregation defects and dysregulated expression of genes near chromosome ends. A genetic screen for suppressors of the thermosensitive growth defect of <i>mis4-G1487D</i> identified several hypomorphic mutants of the Target of Rapamycin Complex 1 (TORC1), a conserved kinase that integrates cellular signals to regulate growth and metabolism through substrate-specific phosphorylation. Here, we demonstrate that the TORC1 pathway modulates cohesin functions in chromosome segregation and gene expression. In the context of compromised cohesin loading, the incidence of chromosome segregation defects was modulated by the growth medium in a TORC1-dependent manner. Pharmacological or genetic downregulation of TORC1 activity restored cohesin binding to its chromosomal sites and improved mitotic chromosome segregation. Notably, reduced TORC1 activity also increased cohesin binding and chromosome transmission fidelity in wild-type cells. These results suggest that environmental cues influence chromosome stability via TORC1. Biochemically, TORC1 co-purified with cohesin and reduced TORC1 activity correlated with decreased phosphorylation of specific residues on Mis4 and cohesin. Mutations in cohesin that mimic the non-phosphorylated state mirrored the effects of TORC1 downregulation, showing that TORC1 is part of the network that controls cohesin phosphorylation to modulate its functions. Finally, we show that the functional interaction between TORC1 and Mis4 extends to the regulation of stress-responsive genes. Our findings reveal a TORC1–cohesin link that may facilitate cellular adaptation to environmental changes. Given that TORC1 inhibitors and calorie restriction extend lifespan in diverse species, this connection raises the intriguing possibility that cohesin-mediated changes in chromosome structure contribute to th…

The blood–brain barrier (BBB) protects the brain from circulating metabolites and plays central roles in neurological diseases. Endothelial cells (ECs) of the BBB are enwrapped by mural cells including pericytes and vascular smooth muscle cells (vSMCs) that regulate angiogenesis, vessel stability and barrier function. To explore mural cell control of the BBB, we investigated neurovascular phenotypes in zebrafish <i>pdgfrb</i> mutants that lack brain pericytes and vSMCs. As expected, mutants showed an altered cerebrovascular network with mispatterned capillaries. Unexpectedly, mutants displayed no BBB leakage at larval stages of development. This suggests that pericytes and vSMCs are not essential for normal BBB function in developing zebrafish. Instead, we observed juvenile and adult BBB disruption occurring at ‘hotspot’ focal hemorrhages at large vessel aneurysms. ECs at leakage hotspots showed induction of caveolae on abluminal surfaces and structural defects including basement membrane thickening and disruption. Our work suggests that capillary pericytes primarily regulate cerebrovascular patterning in development and vSMCs of major arteries protect from hemorrhage and BBB breakdown in older zebrafish. The fact that young zebrafish have a functional BBB in the absence of mural cells calls for renewed interrogation of mural cell control of the BBB throughout vertebrate evolution.

Neural circuits evolved to produce variable cognitive processes through adaptive mechanisms operating within a background of developmental and functional constraints. Understanding how this conflict is resolved requires a comparative framework encapsulating clear behavioural variation. We leverage Heliconiini butterflies to examine how selection shaped the evolution of the central complex and mushroom bodies, two insect integration centres involved in navigation. The evolution of systematic spatial foraging in <i>Heliconius</i> has led to changes in brain morphology and learning and memory profiles over a short evolutionary timescale. Here, we show that in contrast to massively expanded mushroom bodies, the central complex is strongly conserved in size and general architecture. However, we identify divergences in the expression of a neuropeptide, Allatostatin A, in the noduli, and in the numbers of GABA-ergic ring neurons and their branching in the fan-shaped body, which are essential members of the anterior compass pathway. These differences are rare examples of divergence inside the central complex network matching expectations of where evolutionary adaptability might occur. We conclude that due to the contrasting volumetric conservation of the central complex, and the massive differences in the mushroom bodies, their circuit logics must determine distinct responses to selection associated with divergent foraging behaviours.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection poses a major threat to public health, and understanding the mechanism of viral replication and virion release would help identify therapeutic targets and effective drugs for combating the virus. Herein, we identified E3 ubiquitin protein ligase Itchy homolog (ITCH) as a central regulator of SARS-CoV-2 at multiple steps and processes. ITCH enhances the ubiquitination of viral envelope and membrane proteins and mutual interactions of structural proteins, thereby aiding in virion assembly. ITCH-mediated ubiquitination also enhances the interaction of viral proteins to the autophagosome receptor p62, promoting their autophagosome-dependent secretion. Additionally, ITCH disrupts the trafficking of the protease furin and the maturation of cathepsin L, thereby suppressing their activities in cleaving and destabilizing the viral spike protein. Furthermore, ITCH exhibits robust activation during the SARS-CoV-2 replication stage, and SARS-CoV-2 replication is significantly decreased by genetic or pharmacological inhibition of ITCH. These findings provide new insights into the mechanisms of the SARS-CoV-2 life cycle and identify a potential target for developing treatments for the virus-related diseases.

Alternative splicing (AS) of pre-mRNA plays a crucial role in tissue-specific gene regulation, with disease implications due to splicing defects. Predicting and manipulating AS can therefore uncover new regulatory mechanisms and aid in therapeutic design. We introduce TrASPr+BOS, a generative AI model with Bayesian Optimization for predicting and designing RNA for tissue-specific splicing outcomes. Transformer for Alternative Splicing Prediction (TrASPr) is a multi-transformer model that can handle different types of AS events and generalize to unseen cellular conditions. It then serves as an oracle, generating labeled data to train a Bayesian Optimization for Splicing (BOS) algorithm to design RNA for condition-specific splicing outcomes. We show TrASPr+BOS outperforms existing methods, enhancing tissue-specific AUPRC by up to 1.8-fold and capturing tissue-specific regulatory elements. We validate hundreds of predicted novel tissue-specific splicing variations and confirm new regulatory elements using dCas13. We envision TrASPr+BOS as a light yet accurate method researchers can probe or adopt for specific tasks.

The ability of newborns to distinguish between different voices helps them to establish verbal memories from a very early age.

RAB5-GTP activation of the multiprotein VPS34 complex II (VPS34-CII) is critical for endosomal sorting and maturation, phagocytosis, and receptor downregulation. RAB5-GTP activates VPS34-CII by binding to a helical insertion in the C2 domain of VPS34 on the BECLIN1/UVRAG-containing adaptor arm of the complex. The autophagy complex, VPS34 complex I (VPS34-CI), features a unique ATG14L subunit in place of the VPS34-CII UVRAG subunit, and we found that this distorts the adaptor arm to alter the VPS34 RAB-GTPase binding pocket so that it preferentially binds RAB1-GTP. Surprisingly, our higher-resolution single-particle cryo-EM structure of VPS34-CII showed a second RAB5-GTP binding site on the VPS15 solenoid region. This site (VPS15-RAB5-site) appears to be the primordial RAB5-binding region. A mutant in the helical insertion of the C2 domain of human VPS34 that mimics the <i>Saccharomyces cerevisiae</i> sequence abolishes RAB5 binding to VPS34. Mutation of the VPS15-RAB5-site ortholog in <i>S. cerevisiae</i> VPS15 resulted in defective CPY sorting, loss of colocalisation with the RAB5 ortholog Vps21, and loss of binding to Vps21 in vitro. Evolutionary expansion from one to two RAB5-orthologue binding sites may have increased membrane binding and VPS34-CII activity to adapt to more complex endocytic systems.

Multiplexed assays of variant effects (MAVEs) make it possible to measure the functional impact of all possible single amino acid residue substitutions in a protein in a single experiment. Combination of variant effect data from several such experiments provides the opportunity to conduct large-scale analyses of variant effect scores measured across proteins, but can be complicated by variations in the phenotypes that are probed across experiments. Thus, using variant effect datasets obtained with similar MAVE techniques can help reveal general rules governing the effects of amino acid variation for a single molecular phenotype. In this work, we accordingly combined data from six individual variant abundance by massively parallel sequencing (VAMP-seq) experiments and analysed a total of 31,614 variant effect scores reporting solely on the impact of single amino acid residue substitutions on the cellular abundance of proteins. Using our combined variant effect dataset, we derived and analysed a collection of amino acid substitution matrices describing the average impact on cellular abundance of all residue substitution types in different structural environments. We found that the substitution matrices predict the cellular abundance of protein variants with surprisingly high accuracy when given structural information only in the form of whether a residue is buried or exposed. We thus propose our substitution matrix-based predictions as strong baselines for future abundance model development.

Coleoid cephalopods, a subclass of mollusks that includes octopuses, cuttlefish, and squid, exhibit sophisticated biological features, such as dynamic and neurally driven camouflage behavior, inter-individual communication, single-lens camera-like eyes, the largest brains among invertebrates, and a distinctive embryonic development. The common cuttlefish <i>Sepia officinalis</i> has served as a model organism in various research fields, spanning biophysics, neurobiology, behavior, evolution, ecology, and biomechanics. More recently, it has become a model to investigate the neural mechanisms underlying cephalopod camouflage, using quantitative behavioral approaches alongside molecular techniques to characterize the identity, evolution, and development of neuronal cell types. Despite significant interest in this animal, a high-quality, annotated genome of this species is still lacking. To address this, we sequenced and assembled a chromosome-scale genome for <i>S. officinalis</i>. Our assembly spans 5.68 billion base pairs and comprises 1n=47 repeat-rich chromosome scaffolds. This was unexpected because the haploid karyotypes of other decapods indicate 46 chromosomes. Detailed comparisons of our data to those from published decapod genome assemblies and to another recent genome assembly of <i>S. officinalis</i> (itself suggesting 1n=49 chromosomes) in fact revealed clear homologies between 46 scaffolds across all the datasets. In-depth comparison of datasets reveals highly repetitive regions at discordant scaffold boundaries and suggests that the true karyotype of <i>S. officinalis</i> is probably 1n=46 chromosomes, a likely ancestral and if true, conserved decapod karyotype. Our results include a comprehensive gene annotation and full-length transcript prediction, which we used to characterize orthologous gene families across mollusks. We identified several large-scale expansions specific to cephalopods, with many genes specific to neural or non-neural tissues of …