Biomechanical cues play an essential role in sculpting organ formation. Comprehending how cardiac cells perceive and respond to biomechanical forces is a biological process with significant medical implications that remains poorly understood. Here, we show that biomechanical forces activate endocardial id2b (inhibitor of DNA-binding 2b) expression, thereby promoting cardiac contractility and valve formation in zebrafish. Taking advantage of the unique strengths of zebrafish, particularly the viability of embryos lacking heartbeats, we systematically compared the transcriptomes of hearts with impaired contractility to those of control hearts. This comparison identified id2b as a gene sensitive to blood flow. By generating a knock-in reporter line, our results unveiled the presence of id2b in the endocardium, and its expression is sensitive to both pharmacological and genetic perturbations of contraction. Furthermore, id2b loss-of-function resulted in progressive heart malformation and early lethality. Combining RNA-seq analysis, electrophysiology, calcium imaging, and echocardiography, we discovered profound impairment in atrioventricular (AV) valve formation and defective excitation-contraction coupling in id2b mutants. Mechanistically, deletion of id2b reduced AV endocardial cell proliferation and led to a progressive increase in retrograde blood flow. In the myocardium, id2b directly interacted with the bHLH component tcf3b (transcription factor 3b) to restrict its activity. Inactivating id2b unleashed its inhibition on tcf3b, resulting in enhanced repressor activity of tcf3b, which subsequently suppressed the expression of nrg1 (neuregulin 1), an essential mitogen for heart development. Overall, our findings identify id2b as an endocardial cell-specific, biomechanical signaling-sensitive gene, which mediates intercellular communications between endocardium and myocardium to sculpt heart morphogenesis and function.

The cardiac conduction system (CCS) orchestrates the electrical impulses that enable coordinated contraction of the cardiac chambers. The T-box transcription factors TBX3 and TBX5 are required for CCS development and associated with overlapping and distinct human CCS diseases. We evaluated the coordinated role of Tbx3 and Tbx5 in the murine ventricular conduction system (VCS). We engineered a compound Tbx3:Tbx5 conditional knockout allele for both genes located in cis on mouse chromosome 5. Conditional deletion of both T-box transcriptional factors in the VCS, using the VCS-specific MinKCreERT2, caused loss of VCS function and molecular identity. Combined Tbx3 and Tbx5 deficiency in the adult VCS led to conduction defects, including prolonged PR and QRS intervals and elevated susceptibility to ventricular tachycardia. These electrophysiological defects occurred prior to detectable alterations in cardiac contractility or histologic morphology, indicative of a primary conduction system defect. Tbx3:Tbx5 double-knockout VCS cardiomyocytes revealed a transcriptional shift toward non-CCS-specialized working myocardium, indicating a change to their cellular identity. Furthermore, optical mapping revealed a loss of VCS-specific conduction system propagation. Collectively, these findings indicate that Tbx3 and Tbx5 coordinate to control VCS molecular fate and function, with implications for understanding cardiac conduction disorders in humans.

Education is related to a wide variety of beneficial health, behavioral, and societal outcomes. However, whether education causes long-term structural changes in the brain remains unclear. A pressing challenge is that individuals self-select into continued education, thereby introducing a wide variety of environmental and genetic confounders. Fortunately, natural experiments allow us to isolate the causal impact of increased education from individual (and societal) characteristics. Here, we exploit a policy change in the UK (the 1972 ROSLA act) that increased the amount of mandatory schooling from 15 to 16 years of age to study the impact of education on long-term structural brain outcomes in the UK Biobank. Using regression discontinuity (RD) - a causal inference method - we find no evidence of an effect from an additional year of education on any structural neuroimaging outcomes. This null result is robust across modalities, regions, and analysis strategies. An additional year of education is a substantial cognitive intervention, yet we find no evidence for sustained experience-dependent plasticity. Our results provide a challenge for prominent accounts of cognitive or 'brain reserve' theories which identify education as a major protective factor to lessen adverse aging effects. Our preregistered findings are one of the first implementations of RD on neural data - opening the door for causal inference in population-based neuroimaging.

Dendritic morphology is a defining characteristic of neuronal subtypes. In Drosophila, heterotypic olfactory receptor neurons (ORNs) expressing different receptors display diverse dendritic morphologies, but whether such diversity exists among homotypic ORNs remains unclear. Using serial block-face scanning electron microscopy on cryofixed tissues, we analyzed the majority of CO2-sensing neurons (ab1C) and their odor-sensing neighbors (ab1D) in the Drosophila melanogaster antenna. Surprisingly, ab1C neurons featured flattened, sheet-like dendrites-distinct from the cylindrical branches typical of odor-sensing neurons-and displayed remarkable diversity, ranging from plain sheets to tube-like structures that enclose several neighboring dendrites, forming 'dendrite-within-dendrite' structures. Similarly, ab1D dendrites varied from simple, unbranched forms to numerously branched morphologies. These findings suggest that morphological heterogeneity is common even among homotypic ORNs, potentially expanding their functional adaptability and ranges of sensory physiological properties.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by two major diagnostic criteria - persistent deficits in social communication and interaction, and the presence of restricted, repetitive patterns of behavior (RRBs). Evidence from both human and animal model studies of ASD suggests that alteration of striatal circuits, which mediate motor learning, action selection, and habit formation, may contribute to the manifestation of RRBs. CNTNAP2 is a syndromic ASD risk gene, and loss of function of Cntnap2 in mice is associated with RRBs. How the loss of Cntnap2 impacts striatal neuron function is largely unknown. In this study, we utilized Cntnap2-/- mice to test whether altered striatal neuron activity contributes to aberrant motor behaviors relevant to ASD. We find that Cntnap2-/- mice exhibit enhanced cortical drive of direct pathway striatal projection neurons (dSPNs). This enhanced drive is due to increased intrinsic excitability of dSPNs, which make them more responsive to cortical inputs. We find that Cntnap2-/- mice exhibit spontaneous repetitive behaviors, increased motor routine learning, perseveration, and cognitive inflexibility. Increased corticostriatal drive may therefore contribute to the acquisition of repetitive, inflexible behaviors in Cntnap2 mice.

The ability to calibrate learning according to new information is a fundamental component of an organism's ability to adapt to changing conditions. Yet, the exact neural mechanisms guiding dynamic learning rate adjustments remain unclear. Catecholamines appear to play a critical role in adjusting the degree to which we use new information over time, but individuals vary widely in the manner in which they adjust to changes. Here, we studied the effects of a low dose of methamphetamine (MA), and individual differences in these effects, on probabilistic reversal learning dynamics in a within-subject, double-blind, randomized design. Participants first completed a reversal learning task during a drug-free baseline session to provide a measure of baseline performance. Then they completed the task during two sessions, one with MA (20 mg oral) and one with placebo (PL). First, we showed that, relative to PL, MA modulates the ability to dynamically adjust learning from prediction errors. Second, this effect was more pronounced in participants who performed moderately low at baseline. These results present novel evidence for the involvement of catecholaminergic transmission on learning flexibility and highlights that baseline performance modulates the effect of the drug.

Regulatory T cells (Tregs) are essential for maintaining immune tolerance in both lymphoid and non-lymphoid tissues. We discovered that layilin, a C-type lectin receptor, is predominantly expressed on Tregs in skin. Layilin was highly expressed on a subset of clonally expanded 'effector' Tregs in both healthy and psoriatic human skin. Layilin-expressing Tregs exhibited a transcriptional profile indicative of enhanced adhesion. Deletion of layilin in Tregs in mice in vivo resulted in significantly attenuated skin inflammation. Mechanistically, layilin enhanced in vitro human Treg adhesion via modulation of lymphocyte function-associated antigen-1, resulting in distinct cytoskeletal alterations consistent with enhanced focal adhesion and lamellipodia formation. Taken together, we define layilin as a critical regulator of Treg-suppressive capacity by modulating motility and adhesion in a non-lymphoid tissue.

Affiliative social connections facilitate well-being and survival in numerous species. Engaging in social interactions requires positive or negative motivational drive, elicited through coordinated activity across neural circuits. However, the identity, interconnectivity, and functional encoding of social information within these circuits remains poorly understood. Here, we focus on downstream projections of dorsal raphe nucleus (DRN) dopamine neurons (DRNDAT) in mice, which we previously implicated in social motivation alongside an aversive affective state. We show that three prominent DRNDAT projections - to the bed nucleus of the stria terminalis (BNST), central amygdala (CeA), and posterior basolateral amygdala (BLP) - play separable roles in behavior, despite substantial collateralization. Photoactivation of the DRNDAT-CeA projection promoted social behavior and photostimulation of the DRNDAT-BNST projection promoted exploratory behavior, while the DRNDAT-BLP projection supported place avoidance, suggesting a negative affective state. Downstream regions showed diverse receptor expression, poising DRNDAT neurons to act through dopamine, neuropeptide, and glutamate transmission. Furthermore, we show ex vivo that the effect of DRNDAT photostimulation on downstream neuron excitability depended on region and baseline cell properties, resulting in excitatory responses in BNST cells and diverse responses in CeA and BLP. Finally, in vivo microendoscopic cellular-resolution recordings in the CeA with DRNDAT photostimulation revealed a correlation between social behavior and neurons excited by social stimuli, suggesting that increased dopamine tone may recruit different CeA neurons to social ensembles. Collectively, these circuit features may facilitate a coordinated, but flexible, response in the presence of social stimuli that can be flexibly guided based on the internal social homeostatic need state of the individual.

Thalamocortical activity is known to orchestrate sensory gating and consciousness switching. The precise thalamic regions involved, or the firing patterns related to the unconsciousness, remain unclear. Interestingly, the highly -expressed thalamic T-type calcium currents have been considered as a candidate for the ionic mechanism for the generation of thalamocortically driven change in conscious state. Here, we tested the hypothesis that Cav3.1 T-type channels in the mediodorsal thalamic nucleus (MD) might control neuronal firing during unconsciousness using Cav3.1 T-type channel knockout (KO) and knockdown (KD) mice under natural sleep and ethanol-induced unconsciousness. During natural sleep, the MD neurons in KO mice showed general characteristics of sustained firing across sleep stages. We found that KO and MD-specific KD mice showed enhanced resistance to ethanol. During the ethanol-induced unconscious state, wild-type (WT) MD neurons showed a significant reduction in neuronal firing from baseline with increased burst firing, whereas Cav3.1 KO neurons showed well-sustained neural firing, within the level of wakefulness, and no burst firing. Further, 20 Hz optogenetic and electrical activation of MD neurons mimicked the ethanol resistance behavior in WT mice. These results suggest that maintaining MD neural firing at a wakeful level is sufficient to induce resistance to ethanol-induced hypnosis in WT mice. This work has important implications for the design of treatments for consciousness disorders using thalamic stimulation of deeper nuclei, including the targeting of the mediodorsal thalamic nucleus.

Synaptic plasticity underlies the brain's ability to learn and adapt. While experiments in brain slices have revealed mechanisms and protocols for the induction of plasticity between pairs of neurons, how these synaptic changes are coordinated in biological neuronal networks to ensure the emergence of learning remains poorly understood. Simulation and modeling have emerged as important tools to study learning in plastic networks, but have yet to achieve a scale that incorporates realistic network structure, active dendrites, and multi-synapse interactions, key determinants of synaptic plasticity. To rise to this challenge, we endowed an existing large-scale cortical network model, incorporating data-constrained dendritic processing and multi-synaptic connections, with a calcium-based model of functional plasticity that captures the diversity of excitatory connections extrapolated to in vivo-like conditions. This allowed us to study how dendrites and network structure interact with plasticity to shape stimulus representations at the microcircuit level. In our exploratory simulations, plasticity acted sparsely and specifically, firing rates and weight distributions remained stable without additional homeostatic mechanisms. At the circuit level, we found plasticity was driven by co-firing stimulus-evoked functional assemblies, spatial clustering of synapses on dendrites, and the topology of the network connectivity. As a result of the plastic changes, the network became more reliable with more stimulus-specific responses. We confirmed our testable predictions in the MICrONS datasets, an openly available electron microscopic reconstruction of a large volume of cortical tissue. Our results quantify at a large scale how the dendritic architecture and higher-order structure of cortical microcircuits play a central role in functional plasticity and provide a foundation for elucidating their role in learning.

Humans and animals exhibit a remarkable ability to selectively filter out irrelevant information based on context. However, the neural mechanisms underlying this context-dependent selection process remain elusive. Recently, the issue of discriminating between two prevalent selection mechanisms-input modulation versus selection vector modulation-with neural activity data has been highlighted as one of the major challenges in the study of individual variability underlying context-dependent decision-making (CDM). Here, we investigated these selection mechanisms through low-rank neural network modeling of the CDM task. We first showed that only input modulation was allowed in rank-one neural networks and additional dimensions of network connectivity were required to endow neural networks with selection vector modulation. Through rigorous information flow analysis, we gained a mechanistic understanding of why additional dimensions are required for selection vector modulation and how additional dimensions specifically contribute to selection vector modulation. This new understanding then led to the identification of novel neural dynamical signatures for selection vector modulation at both single neuron and population levels. Together, our results provide a rigorous theoretical framework linking network connectivity, neural dynamics, and selection mechanisms, paving the way towards elucidating the circuit mechanisms when studying individual variability in context-dependent computation.

Translation initiation in eukaryotes requires a 40 S ribosome loaded with initiator tRNA which scans for an initiation codon. The initiator tRNA is usually recruited to the ribosome as part of a ternary complex composed of initiator tRNA, eIF2, and GTP. Although initiator tRNA recruitment was originally ascribed to another factor, eIF2A, it was later disproven and shown to occur via eIF2. Nonetheless, eIF2A is still considered a translation initiation factor because it binds the ribosome and shows genetic interactions with other initiation factors such as eIF4E. The exact function of eIF2A during translation initiation, however, remains unclear. Here, we use ribosome profiling and luciferase reporter assays to systematically test in HeLa cells the role of eIF2A in translation initiation, including translation of upstream ORFs. Since eIF2A is thought to take over the function of eIF2 when eIF2 is inhibited, we also test conditions where the integrated stress response is activated. In none of our assays, however, could we detect a role of eIF2A in translation initiation. It is possible that eIF2A plays a role in translation regulation in specific conditions that we have not tested here, or that it plays a role in a different aspect of RNA biology.

Menopausal hormone therapy (MHT) is generally thought to be neuroprotective, yet results have been inconsistent. Here, we present a comprehensive study of MHT use and brain characteristics in females from the UK Biobank.

In Escherichia coli, replisome and replication fork assembly is initiated by DnaB helicase loading at the chromosomal origin oriC via its interactions with the DnaA initiator and the DnaC helicase loader. Upon replication fork arrest, the replisome including DnaB dissociates from the stalled fork. Replication fork progression is rescued by primosomal protein PriA- or PriC-dependent pathway in which PriA and PriC promote reloading of DnaB in different mechanisms. However, the mechanism responsible for rescue of blocked replication initiation at oriC remains unclear. Here, we found that PriC rescued blocked replication initiation in cells expressing an initiation-specific DnaC mutant, in mutant cells defective in DnaA-DnaB interactions, and in cells containing truncated oriC sequence variants. PriC rescued DnaB loading at oriC even in the absence of Rep helicase, a stimulator of the PriC-dependent replication fork restart pathway. These results of in vitro reconstituted assays concordantly suggest that this initiation-specific rescue mechanism provides a bypass of the DnaA-DnaB interaction for DnaB loading by PriC-promoted loading of DnaB to the unwound oriC region. These findings expand understanding of mechanisms sustaining the robustness of replication initiation and specific roles for PriC in the genome maintenance.

The role of beta band activity in cortico-basal ganglia interactions during motor control has been studied extensively in resting-state and for simple movements, such as button pressing. However, little is known about how beta oscillations change and interact in more complex situations involving rapid changes of movement in various contexts. To close this knowledge gap, we combined magnetoencephalography (MEG) and local field potential recordings from the subthalamic nucleus (STN) in Parkinson's disease patients to study beta dynamics during initiation, stopping, and rapid reversal of rotational movements. The action prompts were manipulated to be predictable vs. unpredictable. We observed movement-related beta suppression at motor sequence start, and a beta rebound after motor sequence stop in STN power, motor cortical power, and STN-cortex coherence. Despite involving a brief stop of movement, no clear rebound was observed during reversals of turning direction. At the cortical level, beta power decreased bilaterally following reversals, but more so in the hemisphere ipsilateral to movement, due to a floor effect on the contralateral side. In the STN, power modulations varied across patients, with patients displaying brief increases or decreases of high-beta power. Importantly, cue predictability affected these modulations. Event-related increases of STN-cortex beta coherence were generally stronger in the unpredictable than in the predictable condition. In summary, this study reveals the influence of movement context on beta oscillations in basal ganglia-cortex loops when humans change ongoing movements according to external cues. We find that movement scenarios requiring higher levels of caution involve enhanced modulations of subthalamo-cortical beta synchronization. Furthermore, our results confirm that beta oscillations reflect the start and end of motor sequences better than movement changes within a sequence.

Oxidative phosphorylation has emerged as a critical therapeutic vulnerability of M. tuberculosis (Mtb). However, it is unknown how intracellular bacterial pathogens such as Mtb maintain respiration during infection despite the chemical effectors of host immunity. Mtb synthesizes diisonitrile lipopeptides that tightly chelate copper, but the role of these chalkophores in host-pathogen interactions is also unknown. We demonstrate that M. tuberculosis chalkophores maintain the function of the heme-copper bcc:aa3 respiratory supercomplex under copper limitation. Chalkophore deficiency impairs Mtb survival, respiration to oxygen, and ATP production under copper deprivation in culture, effects that are exacerbated by loss of the heme-dependent Cytochrome BD respiratory oxidase. Our genetic analyses indicate that the maintenance of respiration is the major cellular target of chalkophore-mediated copper acquisition. M. tuberculosis lacking chalkophore biosynthesis is attenuated in mice, a phenotype that is also severely exacerbated by loss of the CytBD respiratory oxidase. We find that the host immune pressure that attenuates chalkophore-deficient Mtb is independent of adaptive immunity and neutrophils. These data demonstrate that chalkophores counter host-inflicted copper deprivation and highlight a multilayered system by which M. tuberculosis maintains respiration during infection.

We present a method for spatially resolving the electric field potential throughout the entire volume of the human brain from electroencephalography (EEG) data. The method is not a variation of the well-known 'source reconstruction' methods, but rather a direct solution to the EEG inverse problem based on our recently developed model for brain waves that demonstrates the inadequacy of the standard 'quasi-static approximation' that has fostered the belief that such a reconstruction is not physically possible. The method retains the high temporal/frequency resolution of EEG, yet has spatial resolution comparable to (or better than) functional MRI (fMRI), without its significant inherent limitations. The method is validated using simultaneous EEG/fMRI data in healthy subjects, intracranial EEG data in epilepsy patients, comparison with numerical simulations, and a direct comparison with standard state-of-the-art EEG analysis in a well-established attention paradigm. The method is then demonstrated on a very large cohort of subjects performing a standard gambling task designed to activate the brain's 'reward circuit'. The technique uses the output from standard extant EEG systems and thus has potential for immediate benefit to a broad range of important basic scientific and clinical questions concerning brain electrical activity. By offering an inexpensive and portable alternative to fMRI, it provides a realistic methodology to efficiently promote the democratization of medicine.

Spiking activity along synaptic circuits linking primary somatosensory (S1) and motor (M1) areas is fundamental for sensorimotor integration in cortex. Circuits along the ascending somatosensory pathway through mouse hand/forelimb S1 and M1 were recently described in detail (Yamawaki et al., 2021). Here, we characterize the peripherally evoked spiking dynamics in these two cortical areas. Brief (5 ms) optogenetic photostimulation of the hand generated short (~25 ms) barrages of activity first in S1 (onset latency 15 ms) then M1 (10 ms later). The estimated propagation speed was 20-fold faster from hand to S1 than from S1 to M1. Amplitudes in M1 were strongly attenuated. Responses were typically triphasic, with suppression and rebound following the initial peak. Evoked activity in S1 was biased to middle layers, consistent with thalamocortical connectivity, while that in M1 was biased to upper layers, consistent with corticocortical connectivity. Parvalbumin (PV) inhibitory interneurons were involved in each phase, accounting for three quarters of the initial spikes generated in S1, and their selective photostimulation sufficed to evoke suppression and rebound in both S1 and M1. Partial silencing of S1 by PV activation during hand stimulation reduced the M1 sensory responses. Overall, these results characterize how evoked spiking activity propagates along the hand/forelimb transcortical loop, and illuminate how in vivo cortical dynamics relate to the underlying synaptic circuit organization in this system.

The management of Leishmania donovani (LD), responsible for fatal visceral leishmaniasis (VL), faces increasing challenges due to rising drug unresponsiveness, leading to increasing treatment failures. While hypolipidemia characterizes VL, LD, a cholesterol auxotroph, relies on host lipid scavenging for its intracellular survival. The aggressive pathology, in terms of increased organ parasite load, observed in hosts infected with antimony-unresponsive-LD (LD-R) as compared to their sensitive counterparts (LD-S), highlights LD-R's heightened reliance on host lipids. Here, we report that LD-R-infection in mice promotes fluid-phase endocytosis in the host macrophages, selectively accumulating neutral lipids while excluding oxidized-low-density lipoprotein (LDL). LD-R enhances the fusion of endocytosed LDL-vesicles with its phagolysosomal membrane and inhibits cholesterol mobilization from these vesicles by suppressing NPC-1. This provides LD-R amastigotes with excess lipids, supporting their rapid proliferation and membrane synthesis. This excess LDL-influx leads to an eventual accumulation of neutral lipid droplets around LD-R amastigotes, thereby increasing their unresponsiveness toward Amphotericin-B, a second-line amphiphilic antileishmanial. Notably, VL patients showing relapse with Amphotericin-B treatment exhibited significantly lower serum LDL and cholesterol than cured cases. Treatment with Aspirin, a lipid droplet blocker, reduced lipid droplets around LD-R amastigotes, restoring Amphotericin-B responsiveness.

The structural basis for the pharmacology of human G protein-coupled receptors (GPCRs), the most abundant membrane proteins and the target of about 35% of approved drugs, is still a matter of intense study. What makes GPCRs challenging to study is the inherent flexibility and the metastable nature of interaction with extra- and intracellular partners that drive their effects. Here, we present a molecular dynamics (MD) adaptive sampling algorithm, namely multiple walker supervised molecular dynamics (mwSuMD), to address complex structural transitions involving GPCRs without energy input. We first report the binding and unbinding of the vasopressin peptide from its receptor V2. Successively, we present the complete transition of the glucagon-like peptide-1 receptor (GLP-1R) from inactive to active, agonist and Gs-bound state, and the guanosine diphosphate (GDP) release from Gs. To our knowledge, this is the first time the whole sequence of events leading from an inactive GPCR to the GDP release is simulated without any energy bias. We demonstrate that mwSuMD can address complex binding processes intrinsically linked to protein dynamics out of reach of classic MD.