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A neural signature of serial dependence has been found, which mirrors the attractive bias of visual information seen in behavioral experiments.

Papua New Guinea (PNG) hosts distinct environments mainly represented by the ecoregions of the Highlands and Lowlands that display increased altitude and a predominance of pathogens, respectively. Since its initial peopling approximately 50,000 years ago, inhabitants of these ecoregions might have differentially adapted to the environmental pressures exerted by each of them. However, the genetic basis of adaptation in populations from these areas remains understudied. Here, we investigated signals of positive selection in 62 highlanders and 43 lowlanders across 14 locations in the main island of PNG using whole-genome genotype data from the Oceanian Genome Variation Project (OGVP) and searched for signals of positive selection through population differentiation and haplotype-based selection scans. Additionally, we performed archaic ancestry estimation to detect selection signals in highlanders within introgressed regions of the genome. Among highland populations we identified candidate genes representing known biomarkers for mountain sickness (SAA4, SAA1, PRDX1, LDHA) as well as candidate genes of the Notch signaling pathway (PSEN1, NUMB, RBPJ, MAML3), a novel proposed pathway for high altitude adaptation in multiple organisms. We also identified candidate genes involved in oxidative stress, inflammation, and angiogenesis, processes inducible by hypoxia, as well as in components of the eye lens and the immune response. In contrast, candidate genes in the lowlands are mainly related to the immune response (HLA-DQB1, HLA-DQA2, TAAR6, TAAR9, TAAR8, RNASE4, RNASE6, ANG). Moreover, we find two candidate regions to be also enriched with archaic introgressed segments, suggesting that archaic admixture has played a role in the local adaptation of PNG populations.

MOTIVATION: Somatic mosaicism has been implicated in several developmental disorders, cancers, and other diseases. Short tandem repeats (STRs) consist of repeated sequences of 1-6 bp and comprise >1 million loci in the human genome. Somatic mosaicism at STRs is known to play a key role in the pathogenicity of loci implicated in repeat expansion disorders and is highly prevalent in cancers exhibiting microsatellite instability. While a variety of tools have been developed to genotype germline variation at STRs, a method for systematically identifying mosaic STRs is lacking. RESULTS: We introduce prancSTR, a novel method for detecting mosaic STRs from individual high-throughput sequencing datasets. prancSTR is designed to detect loci characterized by a single high-frequency mosaic allele, but can also detect loci with multiple mosaic alleles. Unlike many existing mosaicism detection methods for other variant types, prancSTR does not require a matched control sample as input. We show that prancSTR accurately identifies mosaic STRs in simulated data, demonstrate its feasibility by identifying candidate mosaic STRs in Illumina whole genome sequencing data derived from lymphoblastoid cell lines for individuals sequenced by the 1000 Genomes Project, and evaluate the use of prancSTR on Element and PacBio data. In addition to prancSTR, we present simTR, a novel simulation framework which simulates raw sequencing reads with realistic error profiles at STRs. AVAILABILITY AND IMPLEMENTATION: prancSTR and simTR are freely available at https://github.com/gymrek-lab/trtools. Detailed documentation is available at https://trtools.readthedocs.io/.

The impact of variations in the three-dimensional structure of the genome has been recognized, but solid cancer tissue studies are limited. Here, we performed integrated deep Hi-C sequencing with matched whole-genome sequencing, whole-genome bisulfite sequencing, 5-hydroxymethylcytosine (5hmC) sequencing and RNA sequencing across a cohort of 80 biopsy samples from patients with metastatic castration-resistant prostate cancer. Dramatic differences were present in gene expression, 5-methylcytosine/5hmC methylation and in structural variation versus mutation rate between A and B (open and closed) chromatin compartments. A subset of tumors exhibited depleted regional chromatin contacts at the AR locus, linked to extrachromosomal circular DNA (ecDNA) and worse response to AR signaling inhibitors. We also identified topological subtypes associated with stark differences in methylation structure, gene expression and prognosis. Our data suggested that DNA interactions may predispose to structural variant formation, exemplified by the recurrent TMPRSS2-ERG fusion. This comprehensive integrated sequencing effort represents a unique clinical tumor resource.

Patterns of transcriptional activity are encoded in our genome through regulatory elements such as promoters or enhancers that, paradoxically, contain similar assortments of sequence-specific transcription factor (TF) binding sites1-3. Knowledge of how these sequence motifs encode multiple, often overlapping, gene expression programs is central to understanding gene regulation and how mutations in non-coding DNA manifest in disease4,5. Here, by studying gene regulation from the perspective of individual transcription start sites (TSSs), using natural genetic variation, perturbation of endogenous TF protein levels and massively parallel analysis of natural and synthetic regulatory elements, we show that the effect of TF binding on transcription initiation is position dependent. Analysing TF-binding-site occurrences relative to the TSS, we identified several motifs with highly preferential positioning. We show that these patterns are a combination of a TF's distinct functional profiles-many TFs, including canonical activators such as NRF1, NFY and Sp1, activate or repress transcription initiation depending on their precise position relative to the TSS. As such, TFs and their spacing collectively guide the site and frequency of transcription initiation. More broadly, these findings reveal how similar assortments of TF binding sites can generate distinct gene regulatory outcomes depending on their spatial configuration and how DNA sequence polymorphisms may contribute to transcription variation and disease and underscore a critical role for TSS data in decoding the regulatory information of our genome.

Amyloid β (Aβ) peptides accumulating in the brain are proposed to trigger Alzheimers disease (AD). However, molecular cascades underlying their toxicity are poorly defined. Here, we explored a novel hypothesis for Aβ42 toxicity that arises from its proven affinity for γ-secretases. We hypothesized that the reported increases in Aβ42, particularly in the endolysosomal compartment, promote the establishment of a product feedback inhibitory mechanism on γ-secretases, and thereby impair downstream signaling events. We conducted kinetic analyses of γ-secretase activity in cell-free systems in the presence of Aβ, as well as cell-based and ex vivo assays in neuronal cell lines, neurons, and brain synaptosomes to assess the impact of Aβ on γ-secretases. We show that human Aβ42 peptides, but neither murine Aβ42 nor human Aβ17-42 (p3), inhibit γ-secretases and trigger accumulation of unprocessed substrates in neurons, including C-terminal fragments (CTFs) of APP, p75, and pan-cadherin. Moreover, Aβ42 treatment dysregulated cellular homeostasis, as shown by the induction of p75-dependent neuronal death in two distinct cellular systems. Our findings raise the possibility that pathological elevations in Aβ42 contribute to cellular toxicity via the γ-secretase inhibition, and provide a novel conceptual framework to address Aβ toxicity in the context of γ-secretase-dependent homeostatic signaling.

Brain microvessels possess the unique properties of a blood-brain barrier (BBB), tightly regulating the passage of molecules from the blood to the brain neuropil and vice versa. In models of brain injury, BBB dysfunction and the associated leakage of serum albumin to the neuropil have been shown to induce pathological plasticity, neuronal hyper-excitability, and seizures. The effect of neuronal activity on BBB function and whether it plays a role in plasticity in the healthy brain remain unclear. Here we show that neuronal activity induces modulation of microvascular permeability in the healthy brain and that it has a role in local network reorganization. Combining simultaneous electrophysiological recording and vascular imaging with transcriptomic analysis in rats, and functional and BBB-mapping MRI in human subjects, we show that prolonged stimulation of the limb induces a focal increase in BBB permeability in the corresponding somatosensory cortex that is associated with long-term synaptic plasticity. We further show that the increased microvascular permeability depends on neuronal activity and involves caveolae-mediated transcytosis and transforming growth factor β signaling. Our results reveal a role of BBB modulation in cortical plasticity in the healthy brain, highlighting the importance of neurovascular interactions for sensory experience and learning.

How is the information-processing architecture of the human brain organised, and how does its organisation support consciousness? Here, we combine network science and a rigorous information-theoretic notion of synergy to delineate a synergistic global workspace, comprising gateway regions that gather synergistic information from specialised modules across the human brain. This information is then integrated within the workspace and widely distributed via broadcaster regions. Through functional MRI analysis, we show that gateway regions of the synergistic workspace correspond to the human brains default mode network, whereas broadcasters coincide with the executive control network. We find that loss of consciousness due to general anaesthesia or disorders of consciousness corresponds to diminished ability of the synergistic workspace to integrate information, which is restored upon recovery. Thus, loss of consciousness coincides with a breakdown of information integration within the synergistic workspace of the human brain. This work contributes to conceptual and empirical reconciliation between two prominent scientific theories of consciousness, the Global Neuronal Workspace and Integrated Information Theory, while also advancing our understanding of how the human brain supports consciousness through the synergistic integration of information.

Tandem repeats are frequent across the human genome, and variation in repeat length has been linked to a variety of traits. Recent improvements in long read sequencing technologies have the potential to greatly improve tandem repeat analysis, especially for long or complex repeats. Here, we introduce LongTR, which accurately genotypes tandem repeats from high-fidelity long reads available from both PacBio and Oxford Nanopore Technologies. LongTR is freely available at https://github.com/gymrek-lab/longtr and https://zenodo.org/doi/10.5281/zenodo.11403979 .

Findings from this study provide further reassuring evidence that infant exposure through human milk received from lactating individuals who require treatment with remdesivir is negligible.

Extrachromosomal DNA is a common cause of oncogene amplification in cancer. The non-chromosomal inheritance of ecDNA enables tumors to rapidly evolve, contributing to treatment resistance and poor outcome for patients. The transcriptional context in which ecDNAs arise and progress, including chromosomally-driven transcription, is incompletely understood. We examined gene expression patterns of 870 tumors of varied histological types, to identify transcriptional correlates of ecDNA. Here, we show that ecDNA-containing tumors impact four major biological processes. Specifically, ecDNA-containing tumors up-regulate DNA damage and repair, cell cycle control, and mitotic processes, but down-regulate global immune regulation pathways. Taken together, these results suggest profound alterations in gene regulation in ecDNA-containing tumors, shedding light on molecular processes that give rise to their development and progression.

Human milk oligosaccharides (HMOs) are a diverse class of carbohydrates which support the health and development of infants. The vast health benefits of HMOs have made them a commercial target for microbial production; however, producing the approximately 200 structurally diverse HMOs at scale has proved difficult. Here we produce a diversity of HMOs by leveraging the robust carbohydrate anabolism of plants. This diversity includes high-value and complex HMOs, such as lacto-N-fucopentaose I. HMOs produced in transgenic plants provided strong bifidogenic properties, indicating their ability to serve as a prebiotic supplement with potential applications in adult and infant health. Technoeconomic analyses demonstrate that producing HMOs in plants provides a path to the large-scale production of specific HMOs at lower prices than microbial production platforms. Our work demonstrates the promise in leveraging plants for the low-cost and sustainable production of HMOs.

The aim of this project was to increase willingness to receive the influenza vaccine to the optimal rate of ≥ 70%. Low acuity adult patients who visited an Emergency Department (ED) were assessed regarding their willingness to receive the influenza vaccine before and after an educational intervention that included a provider recommendation and an educational handout. A total of seventy-six patients (n = 76) were assessed. Patients willingness to receive the influenza vaccine rose from 29% pre-intervention to 72% post-intervention without disrupting the clinical flow in a busy ED. Similar vaccine educational strategies can be applied to influenza and other vaccines in EDs  to increase vaccination willingness in patients, including those who use the ED as a primary point of contact for healthcare, decreasing the burden of influenza illness in the community.

Variants in cis-regulatory elements link the noncoding genome to human pathology; however, detailed analytic tools for understanding the association between cell-level brain pathology and noncoding variants are lacking. CWAS-Plus, adapted from a Python package for category-wide association testing (CWAS), enhances noncoding variant analysis by integrating both whole-genome sequencing (WGS) and user-provided functional data. With simplified parameter settings and an efficient multiple testing correction method, CWAS-Plus conducts the CWAS workflow 50 times faster than CWAS, making it more accessible and user-friendly for researchers. Here, we used a single-nuclei assay for transposase-accessible chromatin with sequencing to facilitate CWAS-guided noncoding variant analysis at cell-type-specific enhancers and promoters. Examining autism spectrum disorder WGS data (n = 7280), CWAS-Plus identified noncoding de novo variant associations in transcription factor binding sites within conserved loci. Independently, in Alzheimers disease WGS data (n = 1087), CWAS-Plus detected rare noncoding variant associations in microglia-specific regulatory elements. These findings highlight CWAS-Pluss utility in genomic disorders and scalability for processing large-scale WGS data and in multiple-testing corrections. CWAS-Plus and its user manual are available at https://github.com/joonan-lab/cwas/ and https://cwas-plus.readthedocs.io/en/latest/, respectively.

Tissue-clearing and labeling techniques have revolutionized brain-wide imaging and analysis, yet their application to clinical formalin-fixed paraffin-embedded (FFPE) blocks remains challenging. We introduce HIF-Clear, a novel method for efficiently clearing and labeling centimeter-thick FFPE specimens using elevated temperature and concentrated detergents. HIF-Clear with multi-round immunolabeling reveals neuron circuitry regulating multiple neurotransmitter systems in a whole FFPE mouse brain and is able to be used as the evaluation of disease treatment efficiency. HIF-Clear also supports expansion microscopy and can be performed on a non-sectioned 15-year-old FFPE specimen, as well as a 3-month formalin-fixed mouse brain. Thus, HIF-Clear represents a feasible approach for researching archived FFPE specimens for future neuroscientific and 3D neuropathological analyses.

Obesity, and the associated metabolic syndrome, is a risk factor for increased disease severity with a variety of infectious agents, including influenza virus. Yet, the mechanisms are only partially understood. As the number of people, particularly children, living with obesity continues to rise, it is critical to understand the role of host status on disease pathogenesis. In these studies, we use a diet-induced obese ferret model and tools to demonstrate that, like humans, obesity resulted in notable changes to the lung microenvironment, leading to increased clinical disease and viral spread to the lower respiratory tract. The decreased antiviral responses also resulted in obese animals shedding higher infectious virus for a longer period, making them more likely to transmit to contacts. These data suggest that the obese ferret model may be crucial to understanding obesitys impact on influenza disease severity and community transmission and a key tool for therapeutic and intervention development for this high-risk population.

Health equity is the state in which everyone has fair and just opportunities to attain their highest level of health. The field of human genomics has fallen short in increasing health equity, largely because the diversity of the human population has been inadequately reflected among participants of genomics research. This lack of diversity leads to disparities that can have scientific and clinical consequences. Achieving health equity related to genomics will require greater effort in addressing inequities within the field. As part of the commitment of the National Human Genome Research Institute (NHGRI) to advancing health equity, it convened experts in genomics and health equity research to make recommendations and performed a review of current literature to identify the landscape of gaps and opportunities at the interface between human genomics and health equity research. This Perspective describes these findings and examines health equity within the context of human genomics and genomic medicine.

Monosomy 7 and del(7q) are among the most common and poorly understood genetic alterations in myelodysplastic neoplasms and acute myeloid leukemia. Chromosome band 7q22 is a minimally deleted segment in myeloid malignancies with a del(7q). However, the rarity of second hit mutations supports the idea that del(7q22) represents a contiguous gene syndrome. We generated mice harboring a 1.5 Mb germline deletion of chromosome band 5G2 syntenic to human 7q22 that removes Cux1 and 27 additional genes. Hematopoiesis is perturbed in 5G2+/del mice but they do not spontaneously develop hematologic disease. Whereas alkylator exposure modestly accelerated tumor development, the 5G2 deletion did not cooperate with KrasG12D, NrasG12D, or the MOL4070LTR retrovirus in leukemogenesis. 5G2+/del mice are a novel platform for interrogating the role of hemopoietic stem cell attrition/stress, cooperating mutations, genotoxins, and inflammation in myeloid malignancies characterized by monosomy 7/del(7q).

ImportanceIncreasing influenza vaccination rates is a public health priority. One method recommended by the US Centers for Disease Control and Prevention and others is for health systems to send reminders nudging patients to be vaccinated.ObjectiveTo evaluate and compare the effect of electronic health record (EHR)-based patient portal reminders vs text message reminders on influenza vaccination rates across a health system.Design, setting, and participantsThis 3-arm randomized clinical trial was conducted from September 7, 2022, to April 30, 2023, among primary care patients within the University of California, Los Angeles (UCLA) health system.InterventionsArm 1 received standard of care. The health system sent monthly reminder messages to patients due for an influenza vaccine by portal (arm 2) or text (arm 3). Arm 2 had a 2 × 2 nested design, with fixed vs responsive monthly reminders and preappointment vs no preappointment reminders. Arm 3 had 1 × 2 design, with preappointment vs no preappointment reminders. Preappointment reminders for eligible patients were sent 24 and 48 hours before scheduled primary care visits. Fixed reminders (in October, November, and December) involved identical messages via portal or text. Responsive portal reminders involved a September message asking patients about their plans for vaccination, with a follow-up reminder if the response was affirmative but the patient was not yet vaccinated.Main outcomes and measuresThe primary outcome was influenza vaccination by April 30, 2023, obtained from the UCLA EHR, including vaccination from pharmacies and other sources.ResultsA total of 262 085 patients (mean [SD] age, 45.1 [20.7] years; 237 404 [90.6%] adults; 24 681 [9.4%] children; 149 349 [57.0%] women) in 79 primary care practices were included (87 257 in arm 1, 87 478 in arm 2, and 87 350 in arm 3). At the entire primary care population level, none of the interventions improved influenza vaccination rates. All groups had rates of approximately 47%. There was no statistical or clinically significant improvement following portal vs text, preappointment reminders vs no preappointment reminders (portal and text reminders combined), or responsive vs fixed monthly portal reminders.Conclusions and relevanceAt the population level, neither portal nor text reminders for influenza vaccination were effective. Given that vaccine hesitancy may be a major reason for the lack of impact of portal or text reminders, more intensive interventions by health systems are needed to raise influenza vaccination coverage levels.Trial registrationClinicalTrials.gov Identifier: NCT05525494.

Viral infections remain a major risk in immunocompromised pediatric patients, and virus-specific T cell (VST) therapy has been successful for treatment of refractory viral infections in prior studies. We performed a phase II multicenter study (NCT03475212) for the treatment of pediatric patients with inborn errors of immunity and/or post allogeneic hematopoietic stem cell transplant with refractory viral infections using partially-HLA matched VSTs targeting cytomegalovirus, Epstein-Barr virus, or adenovirus. Primary endpoints were feasibility, safety, and clinical responses (>1 log reduction in viremia at 28 days). Secondary endpoints were reconstitution of antiviral immunity and persistence of the infused VSTs. Suitable VST products were identified for 75 of 77 clinical queries. Clinical responses were achieved in 29 of 47 (62%) of patients post-HSCT including 73% of patients evaluable at 1-month post-infusion, meeting the primary efficacy endpoint (>52%). Secondary graft rejection occurred in one child following VST infusion as described in a companion article. Corticosteroids, graft-versus-host disease, transplant-associated thrombotic microangiopathy, and eculizumab treatment correlated with poor response, while uptrending absolute lymphocyte and CD8 T cell counts correlated with good response. This study highlights key clinical factors that impact response to VSTs and demonstrates the feasibility and efficacy of this therapy in pediatric HSCT.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infection in young children and the second leading cause of infant death worldwide. While global circulation has been extensively studied for respiratory viruses such as seasonal influenza, and more recently also in great detail for SARS-CoV-2, a lack of global multi-annual sampling of complete RSV genomes limits our understanding of RSV molecular epidemiology. Here, we capitalise on the genomic surveillance by the INFORM-RSV study and apply phylodynamic approaches to uncover how selection and neutral epidemiological processes shape RSV diversity. Using complete viral genome sequences, we show similar patterns of site-specific diversifying selection among RSVA and RSVB and recover the imprint of non-neutral epidemic processes on their genealogies. Using a phylogeographic approach, we provide evidence for air travel governing the global patterns of RSVA and RSVB spread, which results in a considerable degree of phylogenetic mixing across countries. Our findings highlight the potential of systematic global RSV genomic surveillance for transforming our understanding of global RSV spread.

Donor human milk (DHM) from a milk bank is the recommended feeding method for preterm infants when the mothers own milk (MOM) is not available. Despite this recommendation, information on the possible contamination of donor human milk and its impact on infant health outcomes is poorly characterised. The aim of this systematic review is to assess contaminants present in DHM samples that preterm and critically ill infants consume. The data sources used include PubMed, EMBASE, CINAHL and Web of Science. A search of the data sources targeting DHM and its potential contaminants yielded 426 publications. Two reviewers (S. T. and D. L.) conducted title/abstract screening through Covidence software, and predetermined inclusion/exclusion criteria yielded 26 manuscripts. Contaminant types (bacterial, chemical, fungal, viral) and study details (e.g., type of bacteria identified, study setting) were extracted from each included study during full-text review. Primary contaminants in donor human milk included bacterial species and environmental pollutants. We found that bacterial contaminants were identified in 100% of the papers in which bacterial contamination was sought (16 papers) and 61.5% of the full data set (26 papers), with the most frequently identified genera being Staphylococcus (e.g., Staphylococcus aureus and coagulase-negative Staphylococcus) and Bacillus (e.g., Bacillus cereus). Chemical pollutants were discovered in 100% of the papers in which chemical contamination was sought (eight papers) and 30.8% of the full data set (26 papers). The most frequently identified chemical pollutants included perfluoroalkyl substances (six papers), toxic metal (one paper) and caffeine (one paper). Viral and fungal contamination were identified in one paper each. Our results highlight the importance of establishing standardisation in assessing DHM contamination and future studies are needed to clarify the impact of DHM contaminants on health outcomes.

The Genome Aggregation Database (gnomAD), widely recognized as the gold-standard reference map of human genetic variation, has largely overlooked tandem repeat (TR) expansions, despite the fact that TRs constitute ∼6% of our genome and are linked to over 50 human diseases. Here, we introduce the TR-gnomAD (https://wlcb.oit.uci.edu/TRgnomAD), a biobank-scale reference of 0.86 million TRs derived from 338,963 whole-genome sequencing (WGS) samples of diverse ancestries (39.5% non-European samples). TR-gnomAD offers critical insights into ancestry-specific disease prevalence using disparities in TR unit number frequencies among ancestries. Moreover, TR-gnomAD is able to differentiate between common, presumably benign TR expansions, which are prevalent in TR-gnomAD, from those potentially pathogenic TR expansions, which are found more frequently in disease groups than within TR-gnomAD. Together, TR-gnomAD is an invaluable resource for researchers and physicians to interpret TR expansions in individuals with genetic diseases.

In the human genome, heterozygous sites refer to genomic positions with a different allele or nucleotide variant on the maternal and paternal chromosomes. Resolving these allelic differences by chromosomal copy, also known as phasing, is achievable on a short-read sequencer when using a library preparation method that captures long-range genomic information. TELL-Seq is a library preparation that captures long-range genomic information with the aid of molecular identifiers (barcodes). The same barcode is used to tag the reads derived from the same long DNA fragment within a range of up to 200 kilobases (kb), generating linked-reads. This strategy can be used to phase an entire genome. Here, we introduce a TELL-Seq protocol developed for targeted applications, enabling the phasing of enriched loci of varying sizes, purity levels, and heterozygosity. To validate this protocol, we phased 2-200 kb loci enriched with different methods: CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis for the longest fragments, CRISPR/Cas9-mediated protection from exonuclease digestion for mid-size fragments, and long PCR for the shortest fragments. All selected loci have known clinical relevance: BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, and PKI3CA. Collectively, the analyses show that TELL-Seq can accurately phase 2-200 kb targets using a short-read sequencer.

BACKGROUND: The interpretation of relative vaccine effectiveness (rVE) of improved influenza vaccines is complex. Estimation of burden averted is useful to contextualise their potential impact across different seasons. For the population aged under 65 years in Australia, this study estimated the additional morbidity and mortality that could be averted using improved influenza vaccines. METHODS: We used observed, season-specific (2015-2019) influenza notification and influenza-coded hospitalisation frequencies and published modelled estimates of influenza-associated hospitalisations and deaths that occurred under the prevailing influenza vaccination coverage scenario. After back-calculating to the estimated burden in the population without vaccination, we applied published standard influenza vaccine effectiveness and coverage estimates to calculate the burden potentially averted by standard and improved influenza vaccines. A plausible range of rVE values were used, assuming 50% coverage. RESULTS: The percentage point difference in absolute vaccine effectiveness (VE) of an improved vaccine compared to a standard vaccine is directly proportional to its rVE and inversely proportional to the effectiveness of the standard vaccine. The incremental burden averted by an improved vaccine is a function of both its difference in absolute VE and the severity of the influenza season. Assuming an rVE of 15% with 50% coverage, the improved vaccine was estimated to additionally avert 1517 to 12,641 influenza notifications, 287 to 1311 influenza-coded hospitalisations and 9 to 33 modelled all-cause influenza deaths per year compared to the standard vaccine. CONCLUSIONS: Improved vaccines can have substantial clinical and population impact, particularly when the effectiveness of standard vaccines is low, and burden is high.

Dynamically organized chromatin complexes often involve multiplex chromatin interactions and sometimes chromatin-associated RNA1-3. Chromatin complex compositions change during cellular differentiation and ageing, and are expected to be highly heterogeneous among terminally differentiated single cells4-7. Here we introduce the multinucleic acid interaction mapping in single cells (MUSIC) technique for concurrent profiling of multiplex chromatin interactions, gene expression and RNA-chromatin associations within individual nuclei. When applied to 14 human frontal cortex samples from older donors, MUSIC delineated diverse cortical cell types and states. We observed that nuclei exhibiting fewer short-range chromatin interactions were correlated with both an older transcriptomic signature and Alzheimers disease pathology. Furthermore, the cell type exhibiting chromatin contacts between cis expression quantitative trait loci and a promoter tends to be that in which these cis expression quantitative trait loci specifically affect the expression of their target gene. In addition, female cortical cells exhibit highly heterogeneous interactions between XIST non-coding RNA and chromosome X, along with diverse spatial organizations of the X chromosomes. MUSIC presents a potent tool for exploration of chromatin architecture and transcription at cellular resolution in complex tissues.

Pangenome references address biases of reference genomes by storing a representative set of diverse haplotypes and their alignment, usually as a graph. Alternate alleles determined by variant callers can be used to construct pangenome graphs, but advances in long-read sequencing are leading to widely available, high-quality phased assemblies. Constructing a pangenome graph directly from assemblies, as opposed to variant calls, leverages the graph's ability to represent variation at different scales. Here we present the Minigraph-Cactus pangenome pipeline, which creates pangenomes directly from whole-genome alignments, and demonstrate its ability to scale to 90 human haplotypes from the Human Pangenome Reference Consortium. The method builds graphs containing all forms of genetic variation while allowing use of current mapping and genotyping tools. We measure the effect of the quality and completeness of reference genomes used for analysis within the pangenomes and show that using the CHM13 reference from the Telomere-to-Telomere Consortium improves the accuracy of our methods. We also demonstrate construction of a Drosophila melanogaster pangenome.

The harsh climate of Arabia has posed challenges in generating ancient DNA from the region, hindering the direct examination of ancient genomes for understanding the demographic processes that shaped Arabian populations. In this study, we report whole-genome sequence data obtained from four Tylos-period individuals from Bahrain. Their genetic ancestry can be modeled as a mixture of sources from ancient Anatolia, Levant, and Iran/Caucasus, with variation between individuals suggesting population heterogeneity in Bahrain before the onset of Islam. We identify the G6PD Mediterranean mutation associated with malaria resistance in three out of four ancient Bahraini samples and estimate that it rose in frequency in Eastern Arabia from 5 to 6 kya onward, around the time agriculture appeared in the region. Our study characterizes the genetic composition of ancient Arabians, shedding light on the population history of Bahrain and demonstrating the feasibility of studies of ancient DNA in the region.

We assessed the causal relation of four glycemic traits and type 2 diabetes liability with 167 metabolites using Mendelian randomization with various sensitivity analyses and a reverse Mendelian randomization analysis. We extracted instruments for fasting glucose, 2-h glucose, fasting insulin, and glycated hemoglobin from the Meta-Analyses of Glucose and Insulin-related traits Consortium (n = 200,622), and those for type 2 diabetes liability from a meta-analysis of multiple cohorts (148,726 cases, 965,732 controls) in Europeans. Outcome data were from summary statistics of 167 metabolites from the UK Biobank (n = 115,078). Fasting glucose and 2-h glucose were not associated with any metabolite. Higher glycated hemoglobin was associated with higher free cholesterol in small low-density lipoprotein. Type 2 diabetes liability and fasting insulin were inversely associated with apolipoprotein A1, total cholines, lipoprotein subfractions in high-density-lipoprotein and intermediate-density lipoproteins, and positively associated with aromatic amino acids. These findings indicate hyperglycemia-independent patterns and highlight the role of insulin in type 2 diabetes development. Further studies should evaluate these glycemic traits in type 2 diabetes diagnosis and clinical management.

AIM: Psychedelics have recently gained attention as potential therapeutic agents for various psychiatric disorders. Previous research has highlighted that a diminished sense of self, commonly termed ego-dissolution is a pivotal feature of the psychedelic-induced state. While the Ego-Dissolution Inventory (EDI) is a widely acknowledged instrument for measuring this phenomenon, no Japanese version has been available. This study aimed to develop a Japanese version of the EDI. METHODS: We adhered to the Guidelines for Best Practices in the Translation and Cultural Modification Process for Patient-Reported Outcomes Instruments: Document from the ISPOR Committee on Translation and Cultural Modification during our translation approach. Two Japanese psychiatrists independently conducted initial translations, and a consolidated version was achieved via mutual agreement. This version was then back-translated to English and assessed by the original authors for consistency. The repetitive modification process was conducted in continuous dialogues with the original authors until they accepted the concluding back-translated version. RESULTS: The finalized, approved back-translated version of the EDI is presented in the accompanying figure. In addition, the authorized Japanese version of the EDI is included in the Appendix. CONCLUSIONS: In this study, we successfully developed the Japanese version of the EDI. This instrument will assist in assessing ego-dissolution experiences associated with psychedelic-assisted therapy among Japanese speakers. Additional studies are necessary to evaluate the reliability and validity of this newly translated instrument.

BACKGROUND AND OBJECTIVE: Prior Epstein-Barr virus (EBV) infection is associated with an increased risk of pediatric-onset multiple sclerosis (POMS) and adult-onset multiple sclerosis (MS). It has been challenging to elucidate the biological mechanisms underlying this association. We examined the interactions between candidate human leukocyte antigen (HLA) and non-HLA variants and childhood EBV infection as it may provide mechanistic insights into EBV-associated MS. METHODS: Cases and controls were enrolled in the Environmental and Genetic Risk Factors for Pediatric MS study of the US Network of Pediatric MS Centers. Participants were categorized as seropositive and seronegative for EBV-viral capsid antigen (VCA). The association between prior EBV infection and having POMS was estimated with logistic regression. Interactions between EBV serostatus, major HLA MS risk factors, and non-HLA POMS risk variants associated with response to EBV infection were also evaluated with logistic regression. Models were adjusted for sex, age, genetic ancestry, and the mothers education. Additive interactions were calculated using relative risk due to interaction (RERI) and attributable proportions (APs). RESULTS: A total of 473 POMS cases and 702 controls contributed to the analyses. Anti-VCA seropositivity was significantly higher in POMS cases compared to controls (94.6% vs 60.7%, p

Birth-death models play a key role in phylodynamic analysis for their interpretation in terms of key epidemiological parameters. In particular, models with piecewise-constant rates varying at different epochs in time, to which we refer as episodic birth-death-sampling (EBDS) models, are valuable for their reflection of changing transmission dynamics over time. A challenge, however, that persists with current time-varying model inference procedures is their lack of computational efficiency. This limitation hinders the full utilization of these models in large-scale phylodynamic analyses, especially when dealing with high-dimensional parameter vectors that exhibit strong correlations. We present here a linear-time algorithm to compute the gradient of the birth-death model sampling density with respect to all time-varying parameters, and we implement this algorithm within a gradient-based Hamiltonian Monte Carlo (HMC) sampler to alleviate the computational burden of conducting inference under a wide variety of structures of, as well as priors for, EBDS processes. We assess this approach using three different real world data examples, including the HIV epidemic in Odesa, Ukraine, seasonal influenza A/H3N2 virus dynamics in New York state, America, and Ebola outbreak in West Africa. HMC sampling exhibits a substantial efficiency boost, delivering a 10- to 200-fold increase in minimum effective sample size per unit-time, in comparison to a Metropolis-Hastings-based approach. Additionally, we show the robustness of our implementation in both allowing for flexible prior choices and in modeling the transmission dynamics of various pathogens by accurately capturing the changing trend of viral effective reproductive number.

Protein knockdown with an inducible degradation system is a powerful tool for studying proteins of interest in living cells. Here, we adopted the auxin-inducible degron (AID) approach to detail Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) function in latency maintenance and inducible viral lytic gene expression. We fused the mini-auxin-inducible degron (mAID) tag at the LANA N-terminus with KSHV bacterial artificial chromosome 16 recombination, and iSLK cells were stably infected with the recombinant KSHV encoding mAID-LANA. Incubation with 5-phenyl-indole-3-acetic acid, a derivative of natural auxin, rapidly degraded LANA within 1.5 h. In contrast to our hypothesis, depletion of LANA alone did not trigger lytic reactivation but rather decreased inducible lytic gene expression when we stimulated reactivation with a combination of ORF50 protein expression and sodium butyrate. Decreased overall lytic gene induction seemed to be associated with a rapid loss of KSHV genomes in the absence of LANA. The rapid loss of viral genomic DNA was blocked by a lysosomal inhibitor, chloroquine. Furthermore, siRNA-mediated knockdown of cellular innate immune proteins, cyclic AMP-GMP synthase (cGAS) and simulator of interferon genes (STING), and other autophagy-related genes rescued the degradation of viral genomic DNA upon LANA depletion. Reduction of the viral genome was not observed in 293FT cells that lack the expression of cGAS. These results suggest that LANA actively prevents viral genomic DNA from sensing by cGAS-STING signaling axis, adding novel insights into the role of LANA in latent genome maintenance.IMPORTANCESensing of pathogens' components is a fundamental cellular immune response. Pathogens have therefore evolved strategies to evade such cellular immune responses. KSHV LANA is a multifunctional protein and plays an essential role in maintaining the latent infection by tethering viral genomic DNA to the host chromosome. We adopted the inducible protein knockdown approach and found that depletion of LANA induced rapid degradation of viral genomic DNA, which is mediated by innate immune DNA sensors and autophagy pathway. These observations suggest that LANA may play a role in hiding KSHV episome from innate immune DNA sensors. Our study thus provides new insights into the role of LANA in latency maintenance.

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140-kb unique coding region flanked by 30-40 copies of a 0.8-kb terminal repeat (TR) sequence. A gene enhancer recruits transcription-related enzymes by having arrays of transcription factor binding sites. Here, we show that KSHV TR possesses transcription regulatory function with latency-associated nuclear antigen (LANA). Cleavage under targets and release using nuclease demonstrated that TR fragments were occupied by LANA-interacting histone-modifying enzymes in naturally infected cells. The TR was enriched with histone H3K27 acetylation (H3K27Ac) and H3K4 tri-methylation (H3K4me3) modifications and also expressed nascent RNAs. The sites of H3K27Ac and H3K4me3 modifications were also conserved in the KSHV unique region among naturally infected primary effusion lymphoma cells. KSHV origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with that of TR. In the Ori-Lyt region, the LANA and LANA-interacting proteins colocalized with an H3K27Ac-modified nucleosome along with paused RNA polymerase II. The KSHV transactivator KSHV replication and transcription activator (K-Rta) recruitment sites franked the LANA-bound nucleosome, and reactivation evicted the LANA-bound nucleosome. Including TR fragments in reporter plasmid enhanced inducible viral gene promoter activities independent of the orientations. In the presence of TR in reporter plasmids, K-Rta transactivation was drastically increased, while LANA acquired the promoter repression function. KSHV TR, therefore, functions as an enhancer for KSHV inducible genes. However, in contrast to cellular enhancers bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA nuclear body.IMPORTANCEEnhancers are a crucial regulator of differential gene expression programs. Enhancers are the cis-regulatory sequences determining target genes' spatiotemporal and quantitative expression. Here, we show that Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeats fulfill the enhancer definition for KSHV inducible gene promoters. The KSHV enhancer is occupied by latency-associated nuclear antigen (LANA) and its interacting proteins, such as CHD4. Neighboring terminal repeat (TR) fragments to lytic gene promoters drastically enhanced KSHV replication and transcription activator and LANA transcription regulatory functions. This study, thus, proposes a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates viral inducible gene expression.

Vital to the treatment of influenza is the use of antivirals such as Oseltamivir (Tamiflu) and Zanamivir (Relenza); however, antiviral resistance is becoming an increasing problem for these therapeutics. The RNA-dependent RNA polymerase acidic N-terminal (PAN) endonuclease, a critical component of influenza viral replication machinery, is an antiviral target that was recently validated with the approval of Baloxavir Marboxil (BXM). Despite its clinical success, BXM has demonstrated susceptibility to resistance mutations, specifically the I38T, E23K, and A36 V mutants of PAN. To better understand the effects of these mutations on BXM resistance and improve the design of more robust therapeutics, this study examines key differences in protein-inhibitor interactions with two inhibitors and the I38T, E23K, and A36 V mutants. Differences in inhibitor binding were evaluated by measuring changes in binding to PAN using two biophysical methods. The binding mode of two distinct inhibitors was determined crystallographically with both wild-type and mutant forms of PAN. Collectively, these studies give some insight into the mechanism of antiviral resistance of these mutants.

BACKGROUND: Our previous study established a 2-dose regimen of high-dose trivalent influenza vaccine (HD-TIV) to be immunogenically superior compared to a 2-dose regimen of standard-dose quadrivalent influenza vaccine (SD-QIV) in pediatric allogeneic hematopoietic cell transplant (HCT) recipients. However, the durability of immunogenicity and the role of time post-HCT at immunization as an effect modifier are unknown. METHODS: This phase II, multi-center, double-blinded, randomized controlled trial compared HD-TIV to SD-QIV in children 3-17 years old who were 3-35 months post-allogeneic HCT, with each formulation administered twice, 28-42 days apart. Hemagglutination inhibition (HAI) titers were measured at baseline, 28-42 days following each dose, and 138-222 days after the second dose. Using linear mixed effects models, we estimated adjusted geometric mean HAI titer ratios (aGMR: HD-TIV/SD-QIV) to influenza antigens. Early and late periods were defined as 3-5 and 6-35 months post-HCT, respectively. RESULTS: During 3 influenza seasons (2016-2019), 170 participants were randomized to receive HD-TIV (n = 85) or SD-QIV (n = 85). HAI titers maintained significant elevations above baseline for both vaccine formulations, although the relative immunogenic benefit of HD-TIV to SD-QIV waned during the study. A 2-dose series of HD-TIV administered late post-HCT was associated with higher GMTs compared to the early post-HCT period (late group: A/H1N1 aGMR = 2.16, 95% confidence interval [CI] = [1.14-4.08]; A/H3N2 aGMR = 3.20, 95% CI = [1.60-6.39]; B/Victoria aGMR = 1.91, 95% CI = [1.01-3.60]; early group: A/H1N1 aGMR = 1.03, 95% CI = [0.59-1.80]; A/H3N2 aGMR = 1.23, 95% CI = [0.68-2.25]; B/Victoria aGMR = 1.06, 95% CI = [0.56-2.03]). CONCLUSIONS: Two doses of HD-TIV were more immunogenic than SD-QIV, especially when administered ≥6 months post-HCT. Both groups maintained higher titers compared to baseline throughout the season. CLINICAL TRIALS REGISTRATION: NCT02860039.

Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in Drosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria.

X-chromosomal genetic variants are understudied but can yield valuable insights into sexually dimorphic human traits and diseases. We performed a sex-stratified cross-ancestry X-chromosome-wide association meta-analysis of seven kidney-related traits (n = 908,697), identifying 23 loci genome-wide significantly associated with two of the traits: 7 for uric acid and 16 for estimated glomerular filtration rate (eGFR), including four novel eGFR loci containing the functionally plausible prioritized genes ACSL4, CLDN2, TSPAN6 and the female-specific DRP2. Further, we identified five novel sex-interactions, comprising male-specific effects at FAM9B and AR/EDA2R, and three sex-differential findings with larger genetic effect sizes in males at DCAF12L1 and MST4 and larger effect sizes in females at HPRT1. All prioritized genes in loci showing significant sex-interactions were located next to androgen response elements (ARE). Five ARE genes showed sex-differential expressions. This study contributes new insights into sex-dimorphisms of kidney traits along with new prioritized gene targets for further molecular research.

Influenza virus infection alters the promoter DNA methylation of key immune response-related genes, including type-1 interferons and proinflammatory cytokines. However, less is known about the effect of the influenza vaccine on the epigenome. We utilized a targeted DNA methylation approach to study the longitudinal effects (day 0 pre-vaccination and day 28 post-vaccination) on influenza vaccination responses in peripheral blood mononuclear cells. We found that baseline, pre-vaccination methylation profiles are associated with pre-existing, protective serological immunity. Additionally, we identified 481 sites that were differentially methylated between baseline and day 28 post-vaccination. These were enriched for genes involved in the regulation of the RIG-I signaling pathway, an important regulator of viral responses. Our results suggest that DNA methylation changes to components of the RIG-I pathway are associated with vaccine effectiveness. Therefore, immunization strategies that target this pathway may improve serological responses to influenza vaccination.

BACKGROUND: Carriers of the 1q21.1 distal and 15q11.2 BP1-BP2 copy number variants exhibit regional and global brain differences compared with noncarriers. However, interpreting regional differences is challenging if a global difference drives the regional brain differences. Intraindividual variability measures can be used to test for regional differences beyond global differences in brain structure. METHODS: Magnetic resonance imaging data were used to obtain regional brain values for 1q21.1 distal deletion (n = 30) and duplication (n = 27) and 15q11.2 BP1-BP2 deletion (n = 170) and duplication (n = 243) carriers and matched noncarriers (n = 2350). Regional intra-deviation scores, i.e., the standardized difference between an individuals regional difference and global difference, were used to test for regional differences that diverge from the global difference. RESULTS: For the 1q21.1 distal deletion carriers, cortical surface area for regions in the medial visual cortex, posterior cingulate, and temporal pole differed less and regions in the prefrontal and superior temporal cortex differed more than the global difference in cortical surface area. For the 15q11.2 BP1-BP2 deletion carriers, cortical thickness in regions in the medial visual cortex, auditory cortex, and temporal pole differed less and the prefrontal and somatosensory cortex differed more than the global difference in cortical thickness. CONCLUSIONS: We find evidence for regional effects beyond differences in global brain measures in 1q21.1 distal and 15q11.2 BP1-BP2 copy number variants. The results provide new insight into brain profiling of the 1q21.1 distal and 15q11.2 BP1-BP2 copy number variants, with the potential to increase understanding of the mechanisms involved in altered neurodevelopment.

The UCSC Genome Browser (https://genome.ucsc.edu) is a web-based genomic visualization and analysis tool that serves data to over 7,000 distinct users per day worldwide. It provides annotation data on thousands of genome assemblies, ranging from human to SARS-CoV2. This year, we have introduced new data from the Human Pangenome Reference Consortium and on viral genomes including SARS-CoV2. We have added 1,200 new genomes to our GenArk genome system, increasing the overall diversity of our genomic representation. We have added support for nine new user-contributed track hubs to our public hub system. Additionally, we have released 29 new tracks on the human genome and 11 new tracks on the mouse genome. Collectively, these new features expand both the breadth and depth of the genomic knowledge that we share publicly with users worldwide.

Craniofacial phenotyping is critical for both syndrome delineation and diagnosis because craniofacial abnormalities occur in 30% of characterized genetic syndromes. Clinical reports, textbooks, and available software tools typically provide two-dimensional, static images and illustrations of the characteristic phenotypes of genetic syndromes. In this work, we provide an interactive web application that provides three-dimensional, dynamic visualizations for the characteristic craniofacial effects of 95 syndromes. Users can visualize syndrome facial appearance estimates quantified from data and easily compare craniofacial phenotypes of different syndromes. Our application also provides a map of morphological similarity between a target syndrome and other syndromes. Finally, users can upload 3D facial scans of individuals and compare them to our syndrome atlas estimates. In summary, we provide an interactive reference for the craniofacial phenotypes of syndromes that allows for precise, individual-specific comparisons of dysmorphology.

This scientific commentary refers to ‘Human herpesvirus 6A and axonal injury before the clinical onset of multiple sclerosis’ by Grut et al. (https://doi.org/10.1093/brain/awad374).

BackgroundSleep disturbances are a prevalent and complex comorbidity in neurodevelopmental disorders (NDDs). Dup15q syndrome (duplications of 15q11.2-13.1) is a genetic disorder highly penetrant for NDDs such as autism and intellectual disability and it is frequently accompanied by significant disruptions in sleep patterns. The 15q critical region harbors genes crucial for brain development, notably UBE3A and a cluster of gamma-aminobutyric acid type A receptor (GABAAR) genes. We previously described an electrophysiological biomarker of the syndrome, marked by heightened beta oscillations (12-30 Hz) in individuals with Dup15q syndrome, akin to electroencephalogram (EEG) alterations induced by allosteric modulation of GABAARs. Those with Dup15q syndrome exhibited increased beta oscillations during the awake resting state and during sleep, and they showed profoundly abnormal NREM sleep. This study aims to assess the translational validity of these EEG signatures and to delve into their neurobiological underpinnings by quantifying sleep physiology in chromosome-engineered mice with maternal (matDp/ + mice) or paternal (patDp/ + mice) inheritance of the full 15q11.2-13.1-equivalent duplication, and mice with duplication of just the UBE3A gene (Ube3a overexpression mice; Ube3a OE mice) and comparing the sleep metrics with their respective wildtype (WT) littermate controls.MethodsWe collected 48-h EEG/EMG recordings from 35 (23 male, 12 female) 12-24-week-old matDp/ + , patDp/ + , Ube3a OE mice, and their WT littermate controls. We quantified baseline sleep, sleep fragmentation, spectral power dynamics during sleep states, and recovery following sleep deprivation. Within each group, distinctions between Dup15q mutant mice and WT littermate controls were evaluated using analysis of variance (ANOVA) and student's t-test. The impact of genotype and time was discerned through repeated measures ANOVA, and significance was established at p

In vitro evolution and whole genome analysis has proven to be a powerful method for studying the mechanism of action of small molecules in many haploid microbes but has generally not been applied to human cell lines in part because their diploid state complicates the identification of variants that confer drug resistance. To determine if haploid human cells could be used in MOA studies, we evolved resistance to five different anticancer drugs (doxorubicin, gemcitabine, etoposide, topotecan, and paclitaxel) using a near-haploid cell line (HAP1) and then analyzed the genomes of the drug resistant clones, developing a bioinformatic pipeline that involved filtering for high frequency alleles predicted to change protein sequence, or alleles which appeared in the same gene for multiple independent selections with the same compound. Applying the filter to sequences from 28 drug resistant clones identified a set of 21 genes which was strongly enriched for known resistance genes or known drug targets (TOP1, TOP2A, DCK, WDR33, SLCO3A1). In addition, some lines carried structural variants that encompassed additional known resistance genes (ABCB1, WWOX and RRM1). Gene expression knockdown and knockout experiments of 10 validation targets showed a high degree of specificity and accuracy in our calls and demonstrates that the same drug resistance mechanisms found in diverse clinical samples can be evolved, discovered and studied in an isogenic background.

Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.

While the involvement of actin polymerization in cell migration is well-established, much less is known about the role of transmembrane water flow in cell motility. Here, we investigate the role of water influx in a prototypical migrating cell, the neutrophil, which undergoes rapid, directed movement to sites of injury, and infection. Chemoattractant exposure both increases cell volume and potentiates migration, but the causal link between these processes are not known. We combine single-cell volume measurements and a genome-wide CRISPR screen to identify the regulators of chemoattractant-induced neutrophil swelling, including NHE1, AE2, PI3K-gamma, and CA2. Through NHE1 inhibition in primary human neutrophils, we show that cell swelling is both necessary and sufficient for the potentiation of migration following chemoattractant stimulation. Our data demonstrate that chemoattractant-driven cell swelling complements cytoskeletal rearrangements to enhance migration speed.

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.