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In this community effort, we compare measurements between 34 laboratories from 19 countries, utilizing mixtures of labelled authentic synthetic standards, to quantify by mass spectrometry four clinically used ceramide species in the NIST (National Institute of Standards and Technology) human blood plasma Standard Reference Material (SRM) 1950, as well as a set of candidate plasma reference materials (RM 8231). Participants either utilized a provided validated method and/or their method of choice. Mean concentration values, and intra- and inter-laboratory coefficients of variation (CV) were calculated using single-point and multi-point calibrations, respectively. These results are the most precise (intra-laboratory CVs ≤ 4.2%) and concordant (inter-laboratory CVs < 14%) community-derived absolute concentration values reported to date for four clinically used ceramides in the commonly analyzed SRM 1950. We demonstrate that calibration using authentic labelled standards dramatically reduces data variability. Furthermore, we show how the use of shared RM can correct systematic quantitative biases and help in harmonizing lipidomics. Collectively, the results from the present study provide a significant knowledge base for translation of lipidomic technologies to future clinical applications that might require the determination of reference intervals (RIs) in various human populations or might need to estimate reference change values (RCV), when analytical variability is a key factor for recall during multiple testing of individuals., (© 2024. The Author(s).)

Common and rare alleles are now being annotated across millions of human genomes, and omics technologies are increasingly being used to develop health and treatment recommendations. However, these alleles have not yet been systematically characterized relative to aerospace medicine. Here, we review published alleles naturally found in human cohorts that have a likely protective effect, which is linked to decreased cancer risk and improved bone, muscular, and cardiovascular health. Although some technical and ethical challenges remain, research into these protective mechanisms could translate into improved nutrition, exercise, and health recommendations for crew members during deep space missions., (© 2024. The Author(s).)

Future multi-year crewed planetary missions will motivate advances in aerospace nutrition and telehealth. On Earth, the Human Cell Atlas project aims to spatially map all cell types in the human body. Here, we propose that a parallel Human Cell Space Atlas could serve as an openly available, global resource for space life science research. As humanity becomes increasingly spacefaring, high-resolution omics on orbit could permit an advent of precision spaceflight healthcare. Alongside the scientific potential, we consider the complex ethical, cultural, and legal challenges intrinsic to the human space omics discipline, and how philosophical frameworks may benefit from international perspectives., (© 2024. The Author(s).)

As spaceflight becomes more common with commercial crews, blood-based measures of crew health can guide both astronaut biomedicine and countermeasures. By profiling plasma proteins, metabolites, and extracellular vesicles/particles (EVPs) from the SpaceX Inspiration4 crew, we generated "spaceflight secretome profiles," which showed significant differences in coagulation, oxidative stress, and brain-enriched proteins. While >93% of differentially abundant proteins (DAPs) in vesicles and metabolites recovered within six months, the majority (73%) of plasma DAPs were still perturbed post-flight. Moreover, these proteomic alterations correlated better with peripheral blood mononuclear cells than whole blood, suggesting that immune cells contribute more DAPs than erythrocytes. Finally, to discern possible mechanisms leading to brain-enriched protein detection and blood-brain barrier (BBB) disruption, we examined protein changes in dissected brains of spaceflight mice, which showed increases in PECAM-1, a marker of BBB integrity. These data highlight how even short-duration spaceflight can disrupt human and murine physiology and identify spaceflight biomarkers that can guide countermeasure development., (© 2024. The Author(s).)

The SpaceX Inspiration4 mission provided a unique opportunity to study the impact of spaceflight on the human body. Biospecimen samples were collected from four crew members longitudinally before (Launch: L-92, L-44, L-3 days), during (Flight Day: FD1, FD2, FD3), and after (Return: R + 1, R + 45, R + 82, R + 194 days) spaceflight, spanning a total of 289 days across 2021-2022. The collection process included venous whole blood, capillary dried blood spot cards, saliva, urine, stool, body swabs, capsule swabs, SpaceX Dragon capsule HEPA filter, and skin biopsies. Venous whole blood was further processed to obtain aliquots of serum, plasma, extracellular vesicles and particles, and peripheral blood mononuclear cells. In total, 2,911 sample aliquots were shipped to our central lab at Weill Cornell Medicine for downstream assays and biobanking. This paper provides an overview of the extensive biospecimen collection and highlights their processing procedures and long-term biobanking techniques, facilitating future molecular tests and evaluations.As such, this study details a robust framework for obtaining and preserving high-quality human, microbial, and environmental samples for aerospace medicine in the Space Omics and Medical Atlas (SOMA) initiative, which can aid future human spaceflight and space biology experiments., (© 2024. The Author(s).)

Spaceflight can change metabolic, immunological, and biological homeostasis and cause skin rashes and irritation, yet the molecular basis remains unclear. To investigate the impact of short-duration spaceflight on the skin, we conducted skin biopsies on the Inspiration4 crew members before (L-44) and after (R + 1) flight. Leveraging multi-omics assays including GeoMx™ Digital Spatial Profiler, single-cell RNA/ATAC-seq, and metagenomics/metatranscriptomics, we assessed spatial gene expressions and associated microbial and immune changes across 95 skin regions in four compartments: outer epidermis, inner epidermis, outer dermis, and vasculature. Post-flight samples showed significant up-regulation of genes related to inflammation and KRAS signaling across all skin regions. These spaceflight-associated changes mapped to specific cellular responses, including altered interferon responses, DNA damage, epithelial barrier disruptions, T-cell migration, and hindered regeneration were located primarily in outer tissue compartments. We also linked epithelial disruption to microbial shifts in skin swab and immune cell activity to PBMC single-cell data from the same crew and timepoints. Our findings present the inaugural collection and examination of astronaut skin, offering insights for future space missions and response countermeasures., (© 2024. The Author(s).)

Missions into Deep Space are planned this decade. Yet the health consequences of exposure to microgravity and galactic cosmic radiation (GCR) over years-long missions on indispensable visceral organs such as the kidney are largely unexplored. We performed biomolecular (epigenomic, transcriptomic, proteomic, epiproteomic, metabolomic, metagenomic), clinical chemistry (electrolytes, endocrinology, biochemistry) and morphometry (histology, 3D imaging, miRNA-ISH, tissue weights) analyses using samples and datasets available from 11 spaceflight-exposed mouse and 5 human, 1 simulated microgravity rat and 4 simulated GCR-exposed mouse missions. We found that spaceflight induces: 1) renal transporter dephosphorylation which may indicate astronauts' increased risk of nephrolithiasis is in part a primary renal phenomenon rather than solely a secondary consequence of bone loss; 2) remodelling of the nephron that results in expansion of distal convoluted tubule size but loss of overall tubule density; 3) renal damage and dysfunction when exposed to a Mars roundtrip dose-equivalent of simulated GCR., (© 2024. The Author(s).)

Spaceflight induces an immune response in astronauts. To better characterize this effect, we generated single-cell, multi-ome, cell-free RNA (cfRNA), biochemical, and hematology data for the SpaceX Inspiration4 (I4) mission crew. We found that 18 cytokines/chemokines related to inflammation, aging, and muscle homeostasis changed after spaceflight. In I4 single-cell multi-omics data, we identified a "spaceflight signature" of gene expression characterized by enrichment in oxidative phosphorylation, UV response, immune function, and TCF21 pathways. We confirmed the presence of this signature in independent datasets, including the NASA Twins Study, the I4 skin spatial transcriptomics, and 817 NASA GeneLab mouse transcriptomes. Finally, we observed that (1) T cells showed an up-regulation of FOXP3, (2) MHC class I genes exhibited long-term suppression, and (3) infection-related immune pathways were associated with microbiome shifts. In summary, this study reveals conserved and distinct immune disruptions occurring and details a roadmap for potential countermeasures to preserve astronaut health., (© 2024. The Author(s).)

Microgravity is associated with immunological dysfunction, though the mechanisms are poorly understood. Here, using single-cell analysis of human peripheral blood mononuclear cells (PBMCs) exposed to short term (25 hours) simulated microgravity, we characterize altered genes and pathways at basal and stimulated states with a Toll-like Receptor-7/8 agonist. We validate single-cell analysis by RNA sequencing and super-resolution microscopy, and against data from the Inspiration-4 (I4) mission, JAXA (Cell-Free Epigenome) mission, Twins study, and spleens from mice on the International Space Station. Overall, microgravity alters specific pathways for optimal immunity, including the cytoskeleton, interferon signaling, pyroptosis, temperature-shock, innate inflammation (e.g., Coronavirus pathogenesis pathway and IL-6 signaling), nuclear receptors, and sirtuin signaling. Microgravity directs monocyte inflammatory parameters, and impairs T cell and NK cell functionality. Using machine learning, we identify numerous compounds linking microgravity to immune cell transcription, and demonstrate that the flavonol, quercetin, can reverse most abnormal pathways. These results define immune cell alterations in microgravity, and provide opportunities for countermeasures to maintain normal immunity in space., (© 2024. The Author(s).)

Despite regulating overlapping gene enhancers and pathways, CREBBP and KMT2D mutations recurrently co-occur in germinal center (GC) B cell-derived lymphomas, suggesting potential oncogenic cooperation. Herein, we report that combined haploinsufficiency of Crebbp and Kmt2d induces a more severe mouse lymphoma phenotype (vs either allele alone) and unexpectedly confers an immune evasive microenvironment manifesting as CD8 + T-cell exhaustion and reduced infiltration. This is linked to profound repression of immune synapse genes that mediate crosstalk with T-cells, resulting in aberrant GC B cell fate decisions. From the epigenetic perspective, we observe interaction and mutually dependent binding and function of CREBBP and KMT2D on chromatin. Their combined deficiency preferentially impairs activation of immune synapse-responsive super-enhancers, pointing to a particular dependency for both co-activators at these specialized regulatory elements. Together, our data provide an example where chromatin modifier mutations cooperatively shape and induce an immune-evasive microenvironment to facilitate lymphomagenesis., (© 2024. The Author(s).)

Primary familial brain calcification (PFBC) is characterized by calcium deposition in the brain, causing progressive movement disorders, psychiatric symptoms, and cognitive decline. PFBC is a heterogeneous disorder currently linked to variants in six different genes, but most patients remain genetically undiagnosed. Here, we identify biallelic NAA60 variants in ten individuals from seven families with autosomal recessive PFBC. The NAA60 variants lead to loss-of-function with lack of protein N-terminal (Nt)-acetylation activity. We show that the phosphate importer SLC20A2 is a substrate of NAA60 in vitro. In cells, loss of NAA60 caused reduced surface levels of SLC20A2 and a reduction in extracellular phosphate uptake. This study establishes NAA60 as a causal gene for PFBC, provides a possible biochemical explanation of its disease-causing mechanisms and underscores NAA60-mediated Nt-acetylation of transmembrane proteins as a fundamental process for healthy neurobiological functioning., (© 2024. The Author(s).)

Exploring the molecular basis of disease severity in rare disease scenarios is a challenging task provided the limitations on data availability. Causative genes have been described for Congenital Myasthenic Syndromes (CMS), a group of diverse minority neuromuscular junction (NMJ) disorders; yet a molecular explanation for the phenotypic severity differences remains unclear. Here, we present a workflow to explore the functional relationships between CMS causal genes and altered genes from each patient, based on multilayer network community detection analysis of complementary biomedical information provided by relevant data sources, namely protein-protein interactions, pathways and metabolomics. Our results show that CMS severity can be ascribed to the personalized impairment of extracellular matrix components and postsynaptic modulators of acetylcholine receptor (AChR) clustering. This work showcases how coupling multilayer network analysis with personalized -omics information provides molecular explanations to the varying severity of rare diseases; paving the way for sorting out similar cases in other rare diseases., (© 2024. The Author(s).)

Astrocytes are intimately linked with brain blood vessels, an essential relationship for neuronal function. However, astroglial factors driving these physical and functional associations during postnatal brain development have yet to be identified. By characterizing structural and transcriptional changes in mouse cortical astrocytes during the first two postnatal weeks, we find that high-mobility group box 1 (Hmgb1), normally upregulated with injury and involved in adult cerebrovascular repair, is highly expressed in astrocytes at birth and then decreases rapidly. Astrocyte-selective ablation of Hmgb1 at birth affects astrocyte morphology and endfoot placement, alters distribution of endfoot proteins connexin43 and aquaporin-4, induces transcriptional changes in astrocytes related to cytoskeleton remodeling, and profoundly disrupts endothelial ultrastructure. While lack of astroglial Hmgb1 does not affect the blood-brain barrier or angiogenesis postnatally, it impairs neurovascular coupling and behavior in adult mice. These findings identify astroglial Hmgb1 as an important player in postnatal gliovascular maturation., (© 2023. Springer Nature Limited.)

Representing the most attractive and successful druggable receptors of the proteome, GPCRs regulate a myriad of physiological and pathophysiological functions. Although over half of present pharmaceuticals target GPCRs, the advancement of drug discovery is hampered by a lack of adequate screening tools, the majority of which are limited to probing agonist-induced G-protein and β-arrestin-2-mediated events as a measure of receptor activation. Here, we develop Tango-Trio, a comprehensive cell-based high-throughput platform comprising cumate-inducible expression of transducers, capable of the parallelized profiling of both basal and agonist-dependent GPCR activities. We capture the functional diversity of GPCRs, reporting β-arrestin-1/2 couplings, selectivities, and receptor internalization signatures across the GPCRome. Moreover, we present the construction of cumate-induced basal activation curves at approximately 200 receptors, including over 50 orphans. Overall, Tango-Trio's robustness is well-suited for the functional characterization and screening of GPCRs, especially for parallel interrogation, and is a valuable addition to the pharmacological toolbox., (© 2023. The Author(s).)

Motor skill learning relies on the plasticity of the primary motor cortex as task acquisition drives cortical motor network remodeling. Large-scale cortical remodeling of evoked motor outputs occurs during the learning of corticospinal-dependent prehension behavior, but not simple, non-dexterous tasks. Here we determine the response of corticospinal neurons to two distinct motor training paradigms and assess the role of corticospinal neurons in the execution of a task requiring precise modulation of forelimb movement and one that does not. In vivo calcium imaging in mice revealed temporal coding of corticospinal activity coincident with the development of precise prehension movements, but not more simplistic movement patterns. Transection of the corticospinal tract and optogenetic regulation of corticospinal activity show the necessity for patterned corticospinal network activity in the execution of precise movements but not simplistic ones. Our findings reveal a critical role for corticospinal network modulation in the learning and execution of precise motor movements., (© 2023. The Author(s).)

Viruses in built environments (BEs) raise public health concerns, yet they are generally less studied than bacteria. To better understand viral dynamics in BEs, this study assesses viromes from 11 habitats across four types of BEs with low to high occupancy. The diversity, composition, metabolic functions, and lifestyles of the viromes are found to be habitat dependent. Caudoviricetes species are ubiquitous on surface habitats in the BEs, and some of them are distinct from those present in other environments. Antimicrobial resistance genes are identified in viruses inhabiting surfaces frequently touched by occupants and in viruses inhabiting occupants' skin. Diverse CRISPR/Cas immunity systems and anti-CRISPR proteins are found in bacterial hosts and viruses, respectively, consistent with the strongly coupled virus-host links. Evidence of viruses potentially aiding host adaptation in a specific-habitat manner is identified through a unique gene insertion. This work illustrates that virus-host interactions occur frequently in BEs and that viruses are integral members of BE microbiomes., (© 2023. The Author(s).)

Social hierarchies exert a powerful influence on behavior, but the neurobiological mechanisms that detect and regulate hierarchical interactions are not well understood, especially at the level of neural circuits. Here, we use fiber photometry and chemogenetic tools to record and manipulate the activity of nucleus accumbens-projecting cells in the ventromedial prefrontal cortex (vmPFC-NAcSh) during tube test social competitions. We show that vmPFC-NAcSh projections signal learned hierarchical relationships, and are selectively recruited by subordinate mice when they initiate effortful social dominance behavior during encounters with a dominant competitor from an established hierarchy. After repeated bouts of social defeat stress, this circuit is preferentially activated during social interactions initiated by stress resilient individuals, and plays a necessary role in supporting social approach behavior in subordinated mice. These results define a necessary role for vmPFC-NAcSh cells in the adaptive regulation of social interaction behavior based on prior hierarchical interactions., (© 2023. The Author(s).)

Autophagy is a critical process in the regulation of muscle mass, function and integrity. The molecular mechanisms regulating autophagy are complex and still partly understood. Here, we identify and characterize a novel FoxO-dependent gene, d230025d16rik which we named Mytho (Macroautophagy and YouTH Optimizer), as a regulator of autophagy and skeletal muscle integrity in vivo. Mytho is significantly up-regulated in various mouse models of skeletal muscle atrophy. Short term depletion of MYTHO in mice attenuates muscle atrophy caused by fasting, denervation, cancer cachexia and sepsis. While MYTHO overexpression is sufficient to trigger muscle atrophy, MYTHO knockdown results in a progressive increase in muscle mass associated with a sustained activation of the mTORC1 signaling pathway. Prolonged MYTHO knockdown is associated with severe myopathic features, including impaired autophagy, muscle weakness, myofiber degeneration, and extensive ultrastructural defects, such as accumulation of autophagic vacuoles and tubular aggregates. Inhibition of the mTORC1 signaling pathway in mice using rapamycin treatment attenuates the myopathic phenotype triggered by MYTHO knockdown. Skeletal muscles from human patients diagnosed with myotonic dystrophy type 1 (DM1) display reduced Mytho expression, activation of the mTORC1 signaling pathway and impaired autophagy, raising the possibility that low Mytho expression might contribute to the progression of the disease. We conclude that MYTHO is a key regulator of muscle autophagy and integrity., (© 2023. The Author(s).)

Adult stem cells are indispensable for tissue regeneration, but their function declines with age. The niche environment in which the stem cells reside plays a critical role in their function. However, quantification of the niche effect on stem cell function is lacking. Using muscle stem cells (MuSC) as a model, we show that aging leads to a significant transcriptomic shift in their subpopulations accompanied by locus-specific gain and loss of chromatin accessibility and DNA methylation. By combining in vivo MuSC transplantation and computational methods, we show that the expression of approximately half of all age-altered genes in MuSCs from aged male mice can be restored by exposure to a young niche environment. While there is a correlation between gene reversibility and epigenetic alterations, restoration of gene expression occurs primarily at the level of transcription. The stem cell niche environment therefore represents an important therapeutic target to enhance tissue regeneration in aging., (© 2023. The Author(s).)

It has long been debated how humans resolve fine details and perceive a stable visual world despite the incessant fixational motion of their eyes. Current theories assume these processes to rely solely on the visual input to the retina, without contributions from motor and/or proprioceptive sources. Here we show that contrary to this widespread assumption, the visual system has access to high-resolution extra-retinal knowledge of fixational eye motion and uses it to deduce spatial relations. Building on recent advances in gaze-contingent display control, we created a spatial discrimination task in which the stimulus configuration was entirely determined by oculomotor activity. Our results show that humans correctly infer geometrical relations in the absence of spatial information on the retina and accurately combine high-resolution extraretinal monitoring of gaze displacement with retinal signals. These findings reveal a sensory-motor strategy for encoding space, in which fine oculomotor knowledge is used to interpret the fixational input to the retina., (© 2023. The Author(s).)

The neural substrates of depression may differ in men and women, but the underlying mechanisms are incompletely understood. Here, we show that depression is associated with sex-specific patterns of abnormal functional connectivity in the default mode network and in five regions of interest with sexually dimorphic transcriptional effects. Regional differences in gene expression in two independent datasets explained the neuroanatomical distribution of abnormal connectivity. These gene sets varied by sex and were strongly enriched for genes implicated in depression, synapse function, immune signaling, and neurodevelopment. In an independent sample, we confirmed the prediction that individual differences in default mode network connectivity are explained by inferred brain expression levels for six depression-related genes, including PCDH8, a brain-specific protocadherin integral membrane protein implicated in activity-related synaptic reorganization. Together, our results delineate both shared and sex-specific changes in the organization of depression-related functional networks, with implications for biomarker development and fMRI-guided therapeutic neuromodulation., (© 2022. The Author(s).)

Activation of microglia is a prominent pathological feature in tauopathies, including Alzheimer's disease. How microglia activation contributes to tau toxicity remains largely unknown. Here we show that nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, activated by tau, drives microglial-mediated tau propagation and toxicity. Constitutive activation of microglial NF-κB exacerbated, while inactivation diminished, tau seeding and spreading in young PS19 mice. Inhibition of NF-κB activation enhanced the retention while reduced the release of internalized pathogenic tau fibrils from primary microglia and rescued microglial autophagy deficits. Inhibition of microglial NF-κB in aged PS19 mice rescued tau-mediated learning and memory deficits, restored overall transcriptomic changes while increasing neuronal tau inclusions. Single cell RNA-seq revealed that tau-associated disease states in microglia were diminished by NF-κB inactivation and further transformed by constitutive NF-κB activation. Our study establishes a role for microglial NF-κB signaling in mediating tau spreading and toxicity in tauopathy., (© 2022. The Author(s).)

Abnormalities in brain glucose metabolism and accumulation of abnormal protein deposits called plaques and tangles are neuropathological hallmarks of Alzheimer's disease (AD), but their relationship to disease pathogenesis and to each other remains unclear. Here we show that succinylation, a metabolism-associated post-translational protein modification (PTM), provides a potential link between abnormal metabolism and AD pathology. We quantified the lysine succinylomes and proteomes from brains of individuals with AD, and healthy controls. In AD, succinylation of multiple mitochondrial proteins declined, and succinylation of small number of cytosolic proteins increased. The largest increases occurred at critical sites of amyloid precursor protein (APP) and microtubule-associated tau. We show that in vitro, succinylation of APP disrupted its normal proteolytic processing thereby promoting Aβ accumulation and plaque formation and that succinylation of tau promoted its aggregation to tangles and impaired microtubule assembly. In transgenic mouse models of AD, elevated succinylation associated with soluble and insoluble APP derivatives and tau. These findings indicate that a metabolism-linked PTM may be associated with AD., (© 2022. The Author(s).)

Naked mole-rats are among the most hypoxia-tolerant mammals. During hypoxia, their body temperature (T b ) decreases via unknown mechanisms to conserve energy. In small mammals, non-shivering thermogenesis in brown adipose tissue (BAT) is critical to T b regulation; therefore, we hypothesize that hypoxia decreases naked mole-rat BAT thermogenesis. To test this, we measure changes in T b during normoxia and hypoxia (7% O 2 ; 1-3 h). We report that interscapular thermogenesis is high in normoxia but ceases during hypoxia, and T b decreases. Furthermore, in BAT from animals treated in hypoxia, UCP1 and mitochondrial complexes I-V protein expression rapidly decrease, while mitochondria undergo fission, and apoptosis and mitophagy are inhibited. Finally, UCP1 expression decreases in hypoxia in three other social African mole-rat species, but not a solitary species. These findings suggest that the ability to rapidly down-regulate thermogenesis to conserve oxygen in hypoxia may have evolved preferentially in social species., (© 2021. The Author(s).)

In less than nine months, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) killed over a million people, including >25,000 in New York City (NYC) alone. The COVID-19 pandemic caused by SARS-CoV-2 highlights clinical needs to detect infection, track strain evolution, and identify biomarkers of disease course. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs and a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, viral, and microbial profiling. We applied these methods to clinical specimens gathered from 669 patients in New York City during the first two months of the outbreak, yielding a broad molecular portrait of the emerging COVID-19 disease. We find significant enrichment of a NYC-distinctive clade of the virus (20C), as well as host responses in interferon, ACE, hematological, and olfaction pathways. In addition, we use 50,821 patient records to find that renin-angiotensin-aldosterone system inhibitors have a protective effect for severe COVID-19 outcomes, unlike similar drugs. Finally, spatial transcriptomic data from COVID-19 patient autopsy tissues reveal distinct ACE2 expression loci, with macrophage and neutrophil infiltration in the lungs. These findings can inform public health and may help develop and drive SARS-CoV-2 diagnostic, prevention, and treatment strategies.

Neural stem/progenitor cells (NSPCs) persist over the lifespan while encountering constant challenges from age or injury related brain environmental changes like elevated oxidative stress. But how oxidative stress regulates NSPC and its neurogenic differentiation is less clear. Here we report that acutely elevated cellular oxidative stress in NSPCs modulates neurogenic differentiation through induction of Forkhead box protein O3 (FOXO3)-mediated cGAS/STING and type I interferon (IFN-I) responses. We show that oxidative stress activates FOXO3 and its transcriptional target glycine-N-methyltransferase (GNMT) whose upregulation triggers depletion of s-adenosylmethionine (SAM), a key co-substrate involved in methyl group transfer reactions. Mechanistically, we demonstrate that reduced intracellular SAM availability disrupts carboxymethylation and maturation of nuclear lamin, which induce cytosolic release of chromatin fragments and subsequent activation of the cGAS/STING-IFN-I cascade to suppress neurogenic differentiation. Together, our findings suggest the FOXO3-GNMT/SAM-lamin-cGAS/STING-IFN-I signaling cascade as a critical stress response program that regulates long-term regenerative potential.

Splicing varies across brain regions, but the single-cell resolution of regional variation is unclear. We present a single-cell investigation of differential isoform expression (DIE) between brain regions using single-cell long-read sequencing in mouse hippocampus and prefrontal cortex in 45 cell types at postnatal day 7 ( www.isoformAtlas.com ). Isoform tests for DIE show better performance than exon tests. We detect hundreds of DIE events traceable to cell types, often corresponding to functionally distinct protein isoforms. Mostly, one cell type is responsible for brain-region specific DIE. However, for fewer genes, multiple cell types influence DIE. Thus, regional identity can, although rarely, override cell-type specificity. Cell types indigenous to one anatomic structure display distinctive DIE, e.g. the choroid plexus epithelium manifests distinct transcription-start-site usage. Spatial transcriptomics and long-read sequencing yield a spatially resolved splicing map. Our methods quantify isoform expression with cell-type and spatial resolution and it contributes to further our understanding of how the brain integrates molecular and cellular complexity.

Adenovirus is a nuclear replicating DNA virus reliant on host RNA processing machinery. Processing and metabolism of cellular RNAs can be regulated by METTL3, which catalyzes the addition of N6-methyladenosine (m 6 A) to mRNAs. While m 6 A-modified adenoviral RNAs have been previously detected, the location and function of this mark within the infectious cycle is unknown. Since the complex adenovirus transcriptome includes overlapping spliced units that would impede accurate m 6 A mapping using short-read sequencing, here we profile m 6 A within the adenovirus transcriptome using a combination of meRIP-seq and direct RNA long-read sequencing to yield both nucleotide and transcript-resolved m 6 A detection. Although both early and late viral transcripts contain m 6 A, depletion of m 6 A writer METTL3 specifically impacts viral late transcripts by reducing their splicing efficiency. These data showcase a new technique for m 6 A discovery within individual transcripts at nucleotide resolution, and highlight the role of m 6 A in regulating splicing of a viral pathogen.

Resting state functional connectivity magnetic resonance imaging (fMRI) is a tool for investigating human brain organization. Here we identify, visually and algorithmically, two prevalent influences on fMRI signals during 440 h of resting state scans in 440 healthy young adults, both caused by deviations from normal breathing which we term deep breaths and bursts. The two respiratory patterns have distinct influences on fMRI signals and signal covariance, distinct timescales, distinct cardiovascular correlates, and distinct tendencies to manifest by sex. Deep breaths are not sex-biased. Bursts, which are serial taperings of respiratory depth typically spanning minutes at a time, are more common in males. Bursts share features of chemoreflex-driven clinical breathing patterns that also occur primarily in males, with notable neurological, psychiatric, medical, and lifespan associations. These results identify common breathing patterns in healthy young adults with distinct influences on functional connectivity and an ability to differentially influence resting state fMRI studies.

Optimal functioning of neuronal networks is critical to the complex cognitive processes of memory and executive function that deteriorate in Alzheimer's disease (AD). Here we use cellular and animal models as well as human biospecimens to show that AD-related stressors mediate global disturbances in dynamic intra- and inter-neuronal networks through pathologic rewiring of the chaperome system into epichaperomes. These structures provide the backbone upon which proteome-wide connectivity, and in turn, protein networks become disturbed and ultimately dysfunctional. We introduce the term protein connectivity-based dysfunction (PCBD) to define this mechanism. Among most sensitive to PCBD are pathways with key roles in synaptic plasticity. We show at cellular and target organ levels that network connectivity and functional imbalances revert to normal levels upon epichaperome inhibition. In conclusion, we provide proof-of-principle to propose AD is a PCBDopathy, a disease of proteome-wide connectivity defects mediated by maladaptive epichaperomes.

lncRNAs make up a majority of the human transcriptome and have key regulatory functions. Here we perform unbiased de novo annotation of transcripts expressed during the human humoral immune response to find 30% of the human genome transcribed during this process, yet 58% of these transcripts manifest striking differential expression, indicating an lncRNA phylogenetic relationship among cell types that is more robust than that of coding genes. We provide an atlas of lncRNAs in naive and GC B-cells that indicates their partition into ten functionally categories based on chromatin features, DNase hypersensitivity and transcription factor localization, defining lncRNAs classes such as enhancer-RNAs (eRNA), bivalent-lncRNAs, and CTCF-associated, among others. Specifically, eRNAs are transcribed in 8.6% of regular enhancers and 36.5% of super enhancers, and are associated with coding genes that participate in critical immune regulatory pathways, while plasma cells have uniquely high levels of circular-RNAs accounted for by and reflecting the combinatorial clonal state of the Immunoglobulin loci.

The DNA base modification N6-methyladenine (m 6 A) is involved in many pathways related to the survival of bacteria and their interactions with hosts. Nanopore sequencing offers a new, portable method to detect base modifications. Here, we show that a neural network can improve m 6 A detection at trained sequence contexts compared to previously published methods using deviations between measured and expected current values as each adenine travels through a pore. The model, implemented as the mCaller software package, can be extended to detect known or confirm suspected methyltransferase target motifs based on predictions of methylation at untrained contexts. We use PacBio, Oxford Nanopore, methylated DNA immunoprecipitation sequencing (MeDIP-seq), and whole-genome bisulfite sequencing data to generate and orthogonally validate methylomes for eight microbial reference species. These well-characterized microbial references can serve as controls in the development and evaluation of future methods for the identification of base modifications from single-molecule sequencing data.

Heterozygous de novo mutations in the neuronal protein Munc18-1 are linked to epilepsies, intellectual disability, movement disorders, and neurodegeneration. These devastating diseases have a poor prognosis and no known cure, due to lack of understanding of the underlying disease mechanism. To determine how mutations in Munc18-1 cause disease, we use newly generated S. cerevisiae strains, C. elegans models, and conditional Munc18-1 knockout mouse neurons expressing wild-type or mutant Munc18-1, as well as in vitro studies. We find that at least five disease-linked missense mutations of Munc18-1 result in destabilization and aggregation of the mutant protein. Aggregates of mutant Munc18-1 incorporate wild-type Munc18-1, depleting functional Munc18-1 levels beyond hemizygous levels. We demonstrate that the three chemical chaperones 4-phenylbutyrate, sorbitol, and trehalose reverse the deficits caused by mutations in Munc18-1 in vitro and in vivo in multiple models, offering a novel strategy for the treatment of varied encephalopathies.

The ApoE4 allele is associated with increased risk of small vessel disease, which is a cause of vascular cognitive impairment. Here, we report that mice with targeted replacement (TR) of the ApoE gene with human ApoE4 have reduced neocortical cerebral blood flow compared to ApoE3-TR mice, an effect due to reduced vascular density rather than slowing of microvascular red blood cell flow. Furthermore, homeostatic mechanisms matching local delivery of blood flow to brain activity are impaired in ApoE4-TR mice. In a model of cerebral hypoperfusion, these cerebrovascular alterations exacerbate damage to the white matter of the corpus callosum and worsen cognitive dysfunction. Using 3-photon microscopy we found that the increased white matter damage is linked to an enhanced reduction of microvascular flow resulting in local hypoxia. Such alterations may be responsible for the increased susceptibility to hypoxic-ischemic lesions in the subcortical white matter of individuals carrying the ApoE4 allele.

Oncolytic virus is an attractive anticancer agent that selectively lyses cancer through targeting cancer cells rather than normal cells. Although M1 virus is effective against several cancer types, certain cancer cells present low sensitivity to it. Here we identified that most of the components in the cholesterol biosynthesis pathway are downregulated after M1 virus infection. Further functional studies illustrate that mevalonate/protein farnesylation/ras homolog family member Q (RHOQ) axis inhibits M1 virus replication. Further transcriptome analysis shows that RHOQ knockdown obviously suppresses Rab GTPase and ATP-mediated membrane transporter system, which may mediate the antiviral effect of RHOQ. Based on this, inhibition of the above pathway significantly enhances the anticancer potency of M1 virus in vitro, in vivo, and ex vivo. Our research provides an intriguing strategy for the rational combination of M1 virus with farnesyl transferase inhibitors to enhance therapeutic efficacy.

Mutations in PTEN-induced kinase 1 (PINK1) result in a recessive familial form of Parkinson's disease (PD). PINK1 loss is associated with mitochondrial Ca 2+ mishandling, mitochondrial dysfunction, as well as increased neuronal vulnerability. Here we demonstrate that PINK1 directly interacts with and phosphorylates LETM1 at Thr192 in vitro. Phosphorylated LETM1 or the phospho-mimetic LETM1-T192E increase calcium release in artificial liposomes and facilitates calcium transport in intact mitochondria. Expression of LETM1-T192E but not LETM1-wild type (WT) rescues mitochondrial calcium mishandling in PINK1-deficient neurons. Expression of both LETM1-WT and LETM1-T192E protects neurons against MPP + -MPTP-induced neuronal death in PINK1 WT neurons, whereas only LETM1-T192E protects neurons under conditions of PINK1 loss. Our findings delineate a mechanism by which PINK1 regulates mitochondrial Ca 2+ level through LETM1 and suggest a model by which PINK1 loss leads to deficient phosphorylation of LETM1 and impaired mitochondrial Ca 2+ transport..

Fear can be highly adaptive in promoting survival, yet it can also be detrimental when it persists long after a threat has passed. Flexibility of the fear response may be most advantageous during adolescence when animals are prone to explore novel, potentially threatening environments. Two opposing adolescent fear-related behaviours-diminished extinction of cued fear and suppressed expression of contextual fear-may serve this purpose, but the neural basis underlying these changes is unknown. Using microprisms to image prefrontal cortical spine maturation across development, we identify dynamic BLA-hippocampal-mPFC circuit reorganization associated with these behavioural shifts. Exploiting this sensitive period of neural development, we modified existing behavioural interventions in an age-specific manner to attenuate adolescent fear memories persistently into adulthood. These findings identify novel strategies that leverage dynamic neurodevelopmental changes during adolescence with the potential to extinguish pathological fears implicated in anxiety and stress-related disorders.

Muscle-resident PDGFRβ(+) cells, which include pericytes and PW1(+) interstitial cells (PICs), play a dual role in muscular dystrophy. They can either undergo myogenesis to promote muscle regeneration or differentiate into adipocytes and other cells to compromise regeneration. How the differentiation and fate determination of PDGFRβ(+) cells are regulated, however, remains unclear. Here, by utilizing a conditional knockout mouse line, we report that PDGFRβ(+) cell-derived laminin inhibits their proliferation and adipogenesis, but is indispensable for their myogenesis. In addition, we show that laminin alone is able to partially reverse the muscle dystrophic phenotype in these mice at the molecular, structural and functional levels. Further RNAseq analysis reveals that laminin regulates PDGFRβ(+) cell differentiation/fate determination via gpihbp1. These data support a critical role of laminin in the regulation of PDGFRβ(+) cell stemness, identify an innovative target for future drug development and may provide an effective treatment for muscular dystrophy.

The common bed bug (Cimex lectularius) has been a persistent pest of humans for thousands of years, yet the genetic basis of the bed bug's basic biology and adaptation to dense human environments is largely unknown. Here we report the assembly, annotation and phylogenetic mapping of the 697.9-Mb Cimex lectularius genome, with an N50 of 971 kb, using both long and short read technologies. A RNA-seq time course across all five developmental stages and male and female adults generated 36,985 coding and noncoding gene models. The most pronounced change in gene expression during the life cycle occurs after feeding on human blood and included genes from the Wolbachia endosymbiont, which shows a simultaneous and coordinated host/commensal response to haematophagous activity. These data provide a rich genetic resource for mapping activity and density of C. lectularius across human hosts and cities, which can help track, manage and control bed bug infestations.

Changes in cell fate and identity are essential for endothelial-to-haematopoietic transition (EHT), an embryonic process that generates the first adult populations of haematopoietic stem cells (HSCs) from hemogenic endothelial cells. Dissecting EHT regulation is a critical step towards the production of in vitro derived HSCs. Yet, we do not know how distinct endothelial and haematopoietic fates are parsed during the transition. Here we show that genes required for arterial identity function later to repress haematopoietic fate. Tissue-specific, temporally controlled, genetic loss of arterial genes (Sox17 and Notch1) during EHT results in increased production of haematopoietic cells due to loss of Sox17-mediated repression of haematopoietic transcription factors (Runx1 and Gata2). However, the increase in EHT can be abrogated by increased Notch signalling. These findings demonstrate that the endothelial haematopoietic fate switch is actively repressed in a population of endothelial cells, and that derepression of these programs augments haematopoietic output.

Although zebrafish is used to model human diseases through mutational and morpholino-based knockdown approaches, there are currently no robust transgenic knockdown tools. Here we investigate the knockdown efficiency of three synthetic miRNA-expressing backbones and show that these constructs can downregulate a sensor transgene with different degrees of potency. Using this approach, we reproduce spinal muscular atrophy (SMA) in zebrafish by targeting the smn1 gene. We also generate different transgenic lines, with severity and age of onset correlated to the level of smn1 inhibition, recapitulating for the first time the different forms of SMA in zebrafish. These lines are proof-of-concept that miRNA-based approaches can be used to generate potent heritable gene knockdown in zebrafish.

Enhancers can regulate the transcription of genes over long genomic distances. This is thought to lead to selection against genomic rearrangements within such regions that may disrupt this functional linkage. Here we test this concept experimentally using the human X chromosome. We describe a scoring method to identify evolutionary maintenance of linkage between conserved noncoding elements and neighbouring genes. Chromatin marks associated with enhancer function are strongly correlated with this linkage score. We test >1,000 putative enhancers by transgenesis assays in zebrafish to ascertain the identity of the target gene. The majority of active enhancers drive a transgenic expression in a pattern consistent with the known expression of a linked gene. These results show that evolutionary maintenance of linkage is a reliable predictor of an enhancer's function, and provide new information to discover the genetic basis of diseases caused by the mis-regulation of gene expression.

Regulation of tyrosine hydroxylase gene (Th) transcription is critical for specifying and maintaining the dopaminergic neuronal phenotype. Here we define a molecular regulatory mechanism for Th transcription conserved in tetrapod vertebrates. We show that heterogeneous nuclear ribonucleoprotein (hnRNP) K is a transactivator of Th transcription. It binds to previously unreported and evolutionarily conserved G:C-rich regions in the Th proximal promoter. hnRNP K directly binds to C-rich single-stranded DNA within these conserved regions and also associates with double-stranded sequences when proteins, such as CRE-binding protein, are bound to an adjacent cis-regulatory element. The single DNA strands within the conserved G:C-rich regions adopt either G-quadruplex or i-motif secondary structures. We also show that small molecule-mediated stabilization of these secondary structures represses Th promoter activity. These data suggest that these secondary structures are targets for pharmacological modulation of the dopaminergic phenotype.

Alzheimer's dementia is a devastating and incurable disease afflicting over 35 million people worldwide. Amyloid-β (Aβ), a key pathogenic factor in this disease, has potent cerebrovascular effects that contribute to brain dysfunction underlying dementia by limiting the delivery of oxygen and glucose to the working brain. However, the downstream pathways responsible for the vascular alterations remain unclear. Here we report that the cerebrovascular dysfunction induced by Aβ is mediated by DNA damage caused by vascular oxidative-nitrosative stress in cerebral endothelial cells, which, in turn, activates the DNA repair enzyme poly(ADP)-ribose polymerase. The resulting increase in ADP ribose opens transient receptor potential melastatin-2 (TRPM2) channels in endothelial cells leading to intracellular Ca(2+) overload and endothelial dysfunction. The findings provide evidence for a previously unrecognized mechanism by which Aβ impairs neurovascular regulation and suggest that TRPM2 channels are a potential therapeutic target to counteract cerebrovascular dysfunction in Alzheimer's dementia and related pathologies.