Ebola virus disease outbreaks can often be controlled, but require rapid response efforts frequently with profound operational complexities. Mathematical models can be used to support response planning, but it is unclear if models improve the prior understanding of experts.

In Escherichia coli, RNase E, a central enzyme in RNA processing and mRNA degradation, contains a catalytic N-terminal domain, a membrane-targeting sequence (MTS), and a C-terminal domain (CTD). We investigated how MTS and CTD influence RNase E localization, diffusion, and function. Super-resolution microscopy revealed that ~93% of RNase E localizes to the inner membrane and exhibits slow diffusion similar to polysomes. Comparing the native amphipathic MTS with a transmembrane motif showed that the MTS confers slower diffusion and stronger membrane binding. The CTD further slows diffusion by increasing mass but unexpectedly weakens membrane association. RNase E mutants with partial cytoplasmic localization displayed enhanced co-transcriptional degradation of lacZ mRNA. These findings indicate that variations in the MTS and the presence of the CTD shape the spatiotemporal organization of RNA processing in bacterial cells, providing mechanistic insight into how RNase E domain architecture influences its cellular function.

Inferring the causes of illness is a culturally universal example of causal thinking. We tested the hypothesis that making causal inferences about biological processes (e.g. illness) depends on the animacy semantic network. Participants (n=20) undergoing fMRI read two-sentence vignettes that elicited implicit causal inferences across sentences, either about the emergence of illness or about the mechanical breakdown of inanimate objects, in addition to noncausal control vignettes. All vignettes were about people and were linguistically matched. The same participants performed localizer tasks: language, logical reasoning, and mentalizing. Inferring illness causes, relative to all control conditions, selectively engaged a portion of the precuneus (PC) previously implicated in the semantic representation of animates (e.g. people, animals). Neural responses to causal inferences about illness were adjacent to but distinct from responses to mental state inferences, suggesting a neural mind/body distinction. We failed to find evidence for domain-general responses to causal inference. Causal inference is supported by content-specific semantic networks that encode causal knowledge.

The intrinsic ability of injured neurons to degenerate and regenerate their axons facilitates nervous system repair; however, this ability is not engaged in all neurons and injury locations. Here, we investigate the regulation of a conserved axonal injury response pathway with respect to the location of damage in branched motoneuron (MN) axons in Drosophila larvae. The dileucine zipper kinase (DLK; also known as MAP3K12 in mammals and Wallenda (Wnd) in Drosophila) is a key regulator of diverse responses to axonal injury. In three different populations of MNs, we observed the same striking result that Wnd/DLK signaling becomes activated only in response to injuries that remove all synaptic terminals. Injuries that spared even a small part of a synaptic terminal were insufficient to activate Wnd/DLK signaling, despite the presence of extensive axonal degeneration. The regulation of injury-induced Wnd/DLK signaling occurs independently of its previously known regulator, the Hiw/PHR ubiquitin ligase. We propose that Wnd/DLK signaling regulation is linked to the trafficking of a synapse-to-nucleus axonal cargo and that this mechanism enables neurons to respond to impairments in synaptic connectivity.

Signaling receptors often encounter multiple ligands and have been shown to respond selectively to generate appropriate, context-specific outcomes. At thermal equilibrium, ligand specificity is limited by the relative affinities of ligands for their receptors. Here, we present a non-equilibrium model in which receptors overcome thermodynamic constraints to preferentially signal from specific ligands while suppressing others. In our model, multi-site phosphorylation and active receptor degradation act in concert to regulate ligand specificity, with receptor degradation, a common motif in eukaryotes, providing a previously under-appreciated layer of control. Here, ligand-bound receptors undergo sequential phosphorylation, with progression restarted by ligand unbinding or receptor turnover. High-affinity complexes are kinetically sorted toward degradation-prone states, while low-affinity complexes are sorted toward inactivated states, both limiting signaling. As a result, network activity is maximized for ligands with intermediate affinities. This mechanism explains paradoxical experimental observations in receptor tyrosine kinase signaling, including non-monotonic dependence of signaling output on ligand affinity and kinase activity. Given the ubiquity of multi-site phosphorylation and ligand-induced degradation across signaling receptors, we propose that kinetic sorting may be a general non-equilibrium ligand-discrimination strategy used by multiple signaling receptors.

The centromeric protein-A (CENP-A) is an evolutionarily conserved histone H3 variant that marks the identity of the centromeres. Several mechanisms regulate the centromeric deposition of CENP-A as its mislocalization causes erroneous chromosome segregation, leading to aneuploidy-based diseases, including cancers. The most crucial deposition factor is a CENP-A specific chaperone, HJURP (Scm3 in budding yeast), which specifically binds to CENP-A. However, the discovery of HJURP as a DDR (DNA damage repair) protein and evidence of its binding to Holliday junctions in vitro indicate a CENP-A-deposition-independent role of these chaperones. In this study, using budding yeast, we demonstrate that Scm3 is crucial for the DDR pathway as Scm3-depleted cells are sensitive to DNA damage. We further observe that Scm3 depletion genetically interacts with the rad52 DDR mutant and is compromised in activating DDR-mediated arrest. We demonstrate that Scm3 associates with DNA damage sites and undergoes posttranslational modifications upon DNA damage. Overall, from this report and earlier studies on HJURP, we conclude that DDR functions of CENP-A chaperones are conserved across eukaryotes. The revelation that these chaperones promote genome stability in more than one pathway has clinical significance.

Gastric cancer remains a diagnostic and therapeutic challenge worldwide. Improved prognostic biomarkers could aid treatment planning across surgical, neoadjuvant, and adjuvant settings. We evaluated a novel liquid-biopsy approach integrated with esophagogastroduodenoscopy (EGD) by analyzing gastric fluid DNA (gfDNA) from a large cohort (n=1056) to assess its diagnostic utility and prognostic value in gastric cancer. In this exploratory study, gfDNA concentration was measured in patients with normal gastric mucosa, peptic diseases, preneoplastic conditions, or cancer. Variables included sex, gastric fluid pH, proton-pump inhibitor use, tumor subtype, stage, and outcomes. gfDNA levels were significantly higher in gastric cancer than in all comparison groups (mean 26.86 ng/µL; 95% CI 20.05-33.79; p=3.61 × 10e-12) and as compared to non-malignant controls (mean 10.77 ng/µL; 95% CI 9.23-12.33; p=9.55 × 10e-13) and preneoplastic states (mean 10.10 ng/µL; 95% CI 7.59-12.60; p=1.10 × 10e-5). Advanced tumors (T3) exhibited higher gfDNA than earlier stages (T2 or below; mean 25.66 vs 15.12 ng/µL; p=5.97 × 10e-4). In a subset of gastric cancer patients, gfDNA >1.28 ng/µL associated with longer progression-free survival (p=0.009) and correlated with increased tumor-infiltrating immune cells (p=0.001); this association remained after adjusting for stage (p=0.014). Elevated gfDNA supports gastric cancer presence in the general human population and may inform disease management when combined with tissue biopsies. Importantly, gfDNA shows prognostic potential in established gastric cancer, where higher gfDNA content may paradoxically relate to better outcomes, potentially linked to immune-cell infiltration. These findings warrant further validation and integration with complementary diagnostic modalities to enhance accuracy and clinical utility.

Ammonia is a morphogen in Dictyostelium and is known to arise from the catabolism of proteins and RNA. However, we show that extracellular adenosine deamination catalysed by 'adenosine deaminase-related growth factor' (ADGF) is a major source of ammonia and demonstrate a direct role of ammonia in tip organizer development. The tip formed during early development in Dictyostelium functions analogously to the embryonic organizer of higher vertebrates. Dictyostelium strains carrying mutations in the gene adgf fail to establish an organizer, and this could be reversed by exposing the mutants to volatile ammonia. Interestingly, Klebsiella pneumoniae physically separated from the Dictyostelium adgf mutants in a partitioned dish also rescues the mound arrest phenotype. Both the substrate, adenosine and the product, ammonia, regulate adgf expression, and ADGF acts downstream of the histidine kinase DhkD in regulating tip formation. Thus, the consecutive transformation of extracellular cAMP to adenosine, and adenosine to ammonia is integral steps during Dictyostelium development. Remarkably, in higher vertebrates, adgf expression is elevated during gastrulation and thus adenosine deamination may be a conserved process driving organizer development in different organisms.

Sharp-wave ripples (SWRs) are hippocampal network oscillations associated with memory consolidation. They are characterized by the co-occurrence of fast and slow field potentials across CA1 layers: the fast-frequency oscillations, known as ripples, are prominent in the pyramidal cell layer, where they coincide with increased neuronal spiking, while slower negative transients, referred to as sharp waves, occur simultaneously in the stratum radiatum. SWRs have traditionally been considered globally synchronous across the hippocampus; however, recent evidence suggests that ripples may be less synchronous than previously thought, particularly between the two hemispheres (Villalobos et al., 2017). In this study, we revisited this question using a unique dataset from probes spanning the septo-temporal axis of CA1 in rats. Our results demonstrate that ripples are phase-locked within but not between hemispheres, although their occurrence remains time-locked across both the septo-temporal axis and hemispheres. We also observed a similar synchronicity pattern for spiking activity: neurons are locally phase-coupled and globally time-coupled to ripple events. Interneurons exhibit a much stronger phase coupling to both ipsilateral and contralateral ripples than pyramidal neurons. These findings suggest that ripples are locally phase-coupled through pyramidal-interneuron interactions, with global time-locking likely driven by a common bilateral CA3 input and potentially modulated by interneuron circuits.

Axon guidance cues direct nerves in the heart during development, disease, and regeneration. These cues determine cardiac innervation patterning by regulating the balance between chemo-attraction and chemo-repulsion. However, the role of one of the most crucial ligand-receptor combinations among axon guidance molecules, the Slit chemo-active ligands and their Roundabout (Robo) transmembrane receptors, remains unknown during cardiac innervation patterning. To test if Slit-Robo signalling is important for cardiac innervation guidance, we analysed Slit and Robo mouse knock-outs. Constitutive Slit2-/- ventricles showed significantly reduced innervation, while Slit3-/- hearts showed temporary increased levels of innervation compared to wild-type littermate controls. Whereas innervation was not affected in Robo2-/-mice, the phenotype seen in Slit2-/- ventricles was recapitulated in Robo1-/- mice. Detailed expression analysis identified expression of Slit2 ligand in the endothelium of the coronary vessels, while Slit3 was highly present in the coronary smooth muscle wall and in the innervation. Both the Robo1 and Robo2 receptors were present in the nerves and at low levels in the vessels. Knocking out Slit2 specifically in the endothelium recapitulated the defects seen in the constitutive Slit2-/- hearts. Ex vivo axon guidance cultures showed that attraction of axons extending from the ganglia was strongly reduced in ventricles with absence of endothelial Slit2 compared to wild-type controls. In the absence of endothelial Slit2, adult mice showed reduced response to challenging the sympathetic innervation. In conclusion, we have identified an important new chemo-active Slit2-Robo1 pathway required for correct cardiac innervation development.

Hypoplastic left heart syndrome (HLHS) is the most lethal congenital heart disease (CHD) whose genetic basis remains elusive, likely due to oligogenic complexity. To identify regulators of cardiomyocyte (CM) proliferation relevant to HLHS, we performed a genome-wide siRNA screen in human iPSC-derived CMs, revealing ribosomal protein (RP) genes as the most prominent effectors of CM proliferation. Whole-genome sequencing of 25 HLHS proband-parent trios similarly showed enrichment of rare RP gene variants, including a damaging RPS15A promoter variant shared in a familial CHD case. Cross-species functional analyses demonstrated that perturbation of RP genes impairs cardiac growth: knockdown of RPS15A, RPS17, RPL26L1, RPL39, or RPS15 reduced CM proliferation, caused cardiac malformations in Drosophila, and produced hypoplastic or dysfunctional hearts in zebrafish. Genetic interactions between RP genes and key cardiac transcription factors (TBX5 and NKX2-7) further support their developmental role. Importantly, p53 suppression or Hippo activation partially rescued RP deficiency phenotypes. Together, these findings implicate RP genes as critical regulators of cardiogenesis and candidate contributors to HLHS.

Neuronal function is intimately tied to axodendritic polarity. Neurotransmitter release, for example, is usually the role of the axon. There are widespread exceptions to this rule, however, including many mammalian neuronal types that can release neurotransmitter from their dendrites. In the mouse olfactory bulb, closely related subclasses of dopaminergic interneuron differ markedly in their polarity, with one subtype lacking an axon entirely. These axon-bearing and anaxonic dopaminergic subclasses have distinct developmental profiles and sensory responses, but how their fundamental polarity differences translate to functional outputs remains entirely unknown. Here, we provide anatomical evidence for distinct neurotransmitter release strategies among these closely related dopaminergic subtypes: anaxonic cells release from their dendrites, while axon-bearing neurons release exclusively from their intermittently myelinated axon. These structural differences are linked to a clear functional distinction: anaxonic, but not axon-bearing, dopaminergic neurons are capable of self-inhibition. Our findings suggest that variations in polarity can produce striking distinctions in neuronal outputs, and that even closely related neuronal subclasses may play entirely separate roles in sensory information processing.

The regulation of gene expression is an integral cellular process orchestrated by epigenetic marks like histone modifications. Perturbations in the activity or abundance of epigenetic factors can lead to tumorigenesis. Remarkably, several metabolites influence the epigenetic landscape in cells. Here, we investigated the interplay between a highly expressed epigenetic factor, YEATS2, and a metabolic enzyme, GCDH, in regulating epithelial-to-mesenchymal transition in head and neck cancer. We report that the histone reader YEATS2 is responsible for increased invasive potential in head and neck cancer in an SP1-dependent manner. YEATS2 functions by maintaining histone crotonylation, and its abrogation leads to a global decrease in the H3K27cr mark. Mechanistically, we report that YEATS2 maintains high promoter H3K27cr levels by assisting in the recruitment of crotonyltransferase p300 at the promoter of the EMT-promoting gene SPARC. Furthermore, we found that the addition of the H3K27cr mark is also dependent on the crotonyl-CoA-producing enzyme GCDH. Overall, we describe a novel mechanism of interplay between epigenetics and metabolism in head and neck tumorigenesis, which results in the enhanced expression of EMT-related genes in a histone crotonylation-dependent manner.

While genetically encoded Ca2+ indicators are valuable for visualizing neural activity, their speed and sensitivity have had limited performance when compared to chemical dyes and electrophysiology, particularly at synaptic compartments. We addressed these limitations by engineering a suite of next-generation GCaMP8-based indicators, targeted to presynaptic boutons, active zones, and postsynaptic compartments at the Drosophila neuromuscular junction. We first validated these sensors to be superior to previous versions and synthetic dyes. Next, we developed a Python-based analysis program, CaFire, which enables the automated quantification of evoked and spontaneous Ca²+ signals. Using CaFire, we show a ratiometric presynaptic GCaMP8m sensor accurately captures physiologically relevant presynaptic Ca2+ changes with superior sensitivity and similar kinetics compared to chemical dyes. Moreover, we test the ability of an active zone-targeted, ratiometric GCaMP8m sensor to report differences in Ca²+ between release sites. Finally, a newly engineered postsynaptic GCaMP8m, positioned near glutamate receptors, detects quantal events with temporal and signal resolution comparable to electrophysiological recordings. These next-generation indicators and analytical methods demonstrate that GCaMP8 sensors, targeted to synaptic compartments, can now achieve the speed and sensitivity necessary to resolve Ca2+ dynamics at levels previously only attainable with chemical dyes or electrophysiology.

The macaque genus includes 25 species with diverse social systems, ranging from low to high social tolerance grades. Such interspecific behavioral variability provides a unique model to tackle the evolutionary foundation of primate social brain. Yet, the neuroanatomical correlates of these social tolerance grades remain unknown. To address this question, we expressed social tolerance grades within a novel cognitive framework and analyzed post-mortem structural scans from 12 macaque species. Our results show that amygdala volume is a subcortical predictor of macaques' social tolerance, with high tolerance species exhibiting larger amygdala than low tolerance ones. We further investigated the developmental trajectory of amygdala across social grades and found that intolerant species showed a gradual increase in relative amygdala volume across the lifespan. Unexpectedly, tolerant species exhibited a decrease in relative amygdala volume across the lifespan, contrasting with the age-related increase observed in intolerant species-a developmental pattern previously undescribed in primates. Taken together, these findings provide valuable insights into the cognitive, neuroanatomical, and evolutionary basis of primates' social behaviors.

Oxytocin (OXT), a primitive nonapeptide known to regulate reproduction and social behaviors, is synthesized primarily in the hypothalamus and is secreted via the hypophyseal-portal system of the posterior pituitary gland. In line with the premise that pituitary hormones, traditionally thought of as regulators of single targets, display an array of central and peripheral actions, we found that OXT directly affects bone and body composition. The effect of OXT on bone remodeling is physiologically relevant, as elevated OXT levels during pregnancy and lactation cause calcium mobilization from the maternal skeleton for intergenerational calcium transfer towards fetal bone mineralization. There is an equally large body of evidence that has established the presence of OXT receptors (OXTRs) in the brain through which central functions, such as social bonding, and peripheral functions, such as the regulation of body composition, are exerted. To purposefully address effects of OXT on the brain, we used RNAscope to map OXT and OXTR expression, at the single transcript level, in the whole female and male mouse brains. Identification of brain nuclei with the highest OXT and OXTR transcript density sheds further light on functional OXT nodes that could be further interrogated experimentally to define new physiologic circuitry.

Mitochondrial membranes harbor the electron transport chain (ETC) that powers oxidative phosphorylation (OXPHOS) and drives the synthesis of ATP. Yet, under physiological conditions, the OXPHOS proteins operate as higher-order supercomplex (SC) assemblies, although their functional role remains poorly understood and much debated. By combining large-scale atomistic and coarse-grained molecular simulations with analysis of cryo-electron microscopic data and statistical as well as kinetic models, we show here that the formation of the mammalian I/III2 supercomplex reduces the molecular strain of inner mitochondrial membranes by altering the local membrane thickness and leading to an accumulation of both cardiolipin and quinone around specific regions of the SC. We find that the SC assembly also affects the global motion of the individual ETC proteins with possible functional consequences. On a general level, our findings suggest that molecular crowding and strain effects provide a thermodynamic driving force for the SC formation, with a possible flux enhancement in crowded biological membranes under constrained respiratory conditions.

Sliding clamps like PCNA are crucial processivity factors for replicative polymerases, requiring specific clamp loaders for loading onto DNA. The human alternative clamp loader CTF18-RFC interacts with the leading strand polymerase Pol ε and loads PCNA onto primer/template DNA using its RFC pentameric module. Here, we provide a structural characterization of the human CTF18-RFC complex and its interaction with PCNA. Our cryo-EM data support that the Ctf8 and Dcc1 subunits of CTF18-RFC, which form the regulatory module interacting with Pol ε, are flexibly tethered to the RFC module. A 2.9 Å cryo-EM structure shows the RFC module bound to PCNA in an autoinhibited conformation similar to the canonical RFC loader, marking the initial step of the clamp-loading reaction. The unique RFC1 (Ctf18) large subunit of CTF18-RFC, which based on the cryo-EM map shows high relative flexibility, is anchored to PCNA through an atypical low-affinity PIP box in the AAA+ domain and engages the RFC5 subunit using a novel β-hairpin at the disordered N-terminus. We show that deletion of this β-hairpin impairs the CTF18-RFC-PCNA complex stability, slows down clamp loading, and decreases the rate of primer synthesis by Pol ε. Our research identifies distinctive structural characteristics of the human CTF18-RFC complex, providing insights into its role in PCNA loading and the stimulation of leading strand synthesis by Pol ε.

DNA aptamers are short, single-stranded DNA molecules that bind specifically to a range of targets such as proteins, cells, and small molecules. Typically, they are utilized in the development of therapeutic agents, diagnostics, drug delivery systems, and biosensors. Although aptamers perform well in controlled extracellular environments, their intracellular use has been less explored due to challenges of expressing them in vivo. In this study, we employed the bacterial retron system Eco2 to express a DNA light-up aptamer in Escherichia coli. Our data confirms that structure-guided insertion of the aptamer domain into the non-coding region of the retron enables reverse transcription and biosynthesis of functional aptamer constructs in bacteria. The purified DNA aptamer synthesized under intracellular conditions shows comparable activity to a chemically synthesized control. Our findings demonstrate that retrons can be used to express short DNA aptamers within living cells, potentially broadening and optimizing their application in intracellular settings.

Distal renal tubular acidosis (dRTA) is a disorder characterized by the inability of the collecting duct system to secrete acids during metabolic acidosis. The pathophysiology of dominant or recessive SLC4A1 variant-related dRTA has been linked with the mis-trafficking defect of mutant kAE1 protein. However, in vivo studies in kAE1 R607H dRTA mice and humans have revealed a complex pathophysiology implicating a loss of kAE1-expressing intercalated cells and intracellular relocation of the H+-ATPase in the remaining type-A intercalated cells. These cells also displayed accumulation of ubiquitin and p62 autophagy markers. The highly active transport properties of collecting duct cells require the maintenance of cellular energy and homeostasis, a process dependent on intracellular pH. Therefore, we hypothesized that the expression of dRTA variants affects intracellular pH and autophagy pathways. In this study, we report the characterization of newly identified dRTA variants and provide evidence of abnormal autophagy and degradative pathways in mouse inner medullary collecting duct cells and kidneys from mice expressing kAE1 R607H dRTA mutant protein. We show that reduced transport activity of the kAE1 variants correlated with increased cytosolic pH, reduced ATP synthesis, attenuated downstream autophagic pathways pertaining to the fusion of autophagosomes and lysosomes and/or lysosomal degradative activity. Our study elucidated a close relationship between the expression of defective kAE1 proteins, reduced mitochondrial activity, and decreased autophagy and protein degradative flux.