The ENCODE mouse postnatal developmental time course identifies regulatory programs of cell types and cell states.

Postnatal genomic regulation significantly influences tissue and organ maturation but is under-studied relative to existing genomic catalogs of adult tissues or prenatal development in mouse. The ENCODE4 consortium generated the first comprehensive single-nucleus resource of postnatal regulatory events across a diverse set of mouse tissues. The collection spans seven postnatal time points, mirroring human development from childhood to adulthood, and encompasses five core tissues. We identified 30 cell types, further subdivided into 69 subtypes and cell states across adrenal gland, left cerebral cortex, hippocampus, heart, and gastrocnemius muscle. Our annotations cover both known and novel cell differentiation dynamics ranging from early hippocampal neurogenesis to a new sex-specific adrenal gland population during puberty. We used an ensemble Latent Dirichlet Allocation strategy with a curated vocabulary of 2,701 regulatory genes to identify regulatory "topics," each of which is a gene vector, linked to cell type differentiation, subtype specialization, and transitions between cell states. We find recurrent regulatory topics in tissue-resident macrophages, neural cell types, endothelial cells across multiple tissues, and cycling cells of the adrenal gland and heart. Cell-type-specific topics are enriched in transcription factors and microRNA host genes, while chromatin regulators dominate mitosis topics. Corresponding chromatin accessibility data reveal dynamic and sex-specific regulatory elements, with enriched motifs matching transcription factors in regulatory topics. Together, these analyses identify both tissue-specific and common regulatory programs in postnatal development across multiple tissues through the lens of the factors regulating transcription.

Identification of robust cellular programs using reproducible LDA that impact sex-specific disease progression in different genotypes of a mouse model of AD.

The gene expression profiles of distinct cell types reflect complex genomic interactions among multiple simultaneous biological processes within each cell that can be altered by disease progression as well as genetic background. The identification of these active cellular programs is an open challenge in the analysis of single-cell RNA-seq data. Latent Dirichlet Allocation (LDA) is a generative method used to identify recurring patterns in counts data, commonly referred to as topics that can be used to interpret the state of each cell. However, LDA's interpretability is hindered by several key factors including the hyperparameter selection of the number of topics as well as the variability in topic definitions due to random initialization. We developed Topyfic, a Reproducible LDA (rLDA) package, to accurately infer the identity and activity of cellular programs in single-cell data, providing insights into the relative contributions of each program in individual cells. We apply Topyfic to brain single-cell and single-nucleus datasets of two 5xFAD mouse models of Alzheimer's disease crossed with C57BL6/J or CAST/EiJ mice to identify distinct cell types and states in different cell types such as microglia. We find that 8-month 5xFAD/Cast F1 males show higher level of microglial activation than matching 5xFAD/BL6 F1 males, whereas female mice show similar levels of microglial activation. We show that regulatory genes such as TFs, microRNA host genes, and chromatin regulatory genes alone capture cell types and cell states. Our study highlights how topic modeling with a limited vocabulary of regulatory genes can identify gene expression programs in single-cell data in order to quantify similar and divergent cell states in distinct genotypes.

Antigen-specific age-related memory CD8 T cells induce and track Alzheimer's-like neurodegeneration.

Cerebral (Aß) plaque and (pTau) tangle deposition are hallmarks of Alzheimer's disease (AD), yet are insufficient to confer complete AD-like neurodegeneration experimentally. Factors acting upstream of Aß/pTau in AD remain unknown, but their identification could enable earlier diagnosis and more effective treatments. T cell abnormalities are emerging AD hallmarks, and CD8 T cells were recently found to mediate neurodegeneration downstream of tangle deposition in hereditary neurodegeneration models. The precise impact of T cells downstream of Aß/fibrillar pTau, however, appears to vary depending on the animal model used. Our prior work suggested that antigen-specific memory CD8 T (" hi T") cells act upstream of Aß/pTau after brain injury. Here we examine whether hi T cells influence sporadic AD-like pathophysiology upstream of Aß/pTau. Examining neuropathology, gene expression, and behavior in our hi T mouse model we show that CD8 T cells induce plaque and tangle-like deposition, modulate AD-related genes, and ultimately result in progressive neurodegeneration with both gross and fine features of sporadic human AD. T cells required Perforin to initiate this pathophysiology, and IFNγ for most gene expression changes and progression to more widespread neurodegenerative disease. Analogous antigen-specific memory CD8 T cells were significantly elevated in the brains of human AD patients, and their loss from blood corresponded to sporadic AD and related cognitive decline better than plasma pTau-217, a promising AD biomarker candidate. Our work is the first to identify an age-related factor acting upstream of Aß/pTau to initiate AD-like pathophysiology, the mechanisms promoting its pathogenicity, and its relevance to human sporadic AD. Significance Statement: This study changes our view of Alzheimer's Disease (AD) initiation and progression. Mutations promoting cerebral beta-amyloid (Aß) deposition guarantee rare genetic forms of AD. Thus, the prevailing hypothesis has been that Aß is central to initiation and progression of all AD, despite contrary animal and patient evidence. We show that age-related T cells generate neurodegeneration with compelling features of AD in mice, with distinct T cell functions required for pathological initiation and neurodegenerative progression. Knowledge from these mice was applied to successfully predict previously unknown features of human AD and generate novel tools for its clinical management.

A human embryonic limb cell atlas resolved in space and time.

Human limbs emerge during the fourth post-conception week as mesenchymal buds, which develop into fully formed limbs over the subsequent months1. This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common2. Decades of work with model organisms have defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterization of this process in humans has yet to be performed. Here we detail human embryonic limb development across space and time using single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells from a few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterized by different cell states regulated by separate gene expression programmes, and identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity. Through assembly of multiple anatomically continuous spatial transcriptomic samples using VisiumStitcher, we map cells across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of the mesenchyme in the autopod. Finally, we perform single-cell RNA sequencing on mouse embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species.

Validity of the Modified Defense and Veterans Pain Rating Scale Supplemented With Items Germane to Total Joint Replacement: Secondary Analysis From a Randomized Trial.

INTRODUCTION: Valid measures of pain are helpful to adjust treatment plans in real time after total joint replacement (TJR). We enhanced the existing Defense and Veterans Pain Rating Scale (DVPRS) with items related to pain at rest and movement, in specific reference to operative and nonoperative joints, creating the TJR-DVPRS. This manuscript is presented to validate the modified survey instrument. The aims of this psychometric study were to evaluate (1) the latent structure of the TJR-DVPRS, (2) the relationships among the pain dimensions represented on the TJR-DVPRS and the reference-standard Short-Form McGill Pain Questionnaire (version 2, SF-MPQ-2), and (3) the responsiveness of these two measures before and after TJR. MATERIALS AND METHODS: This report involves a secondary analysis of pain surveys from 135 veterans undergoing TJR at one center who participated in a randomized trial. The study was approved by the institutional review boards from participating institutions. The TJR-DVPRS and SF-MPQ-2 were completed preoperatively, during postoperative day 1, and 6 weeks post-surgery. Standard psychometric evaluations included correlations, principal component analysis, and internal consistency of survey items and subscales, using preoperative baseline data as a frame of reference. Responsiveness analysis included assessing both effect size and thresholds of clinically important change for survey subscales using data from all three time points. RESULTS: Two reliable subscales were identified for the TJR-DVPRS, one including items about pain intensity and interference related to the operative joint (Cronbach's α = .809) and the other including two pain items on the nonoperative joint. Combining the subscales indicated a two-factor solution: The first valid factor entailed the four subscales of the SF-MPQ-2 and the TJR-DVPRS operative joint subscales, indicating that these subscales validly represent the same pain dimension. The second valid factor entailed the TJR-DVPRS subscale addressing the nonoperative joint. Responsiveness analysis following accepted psychometric methods demonstrated significant decreases in pain from the preoperative period to 6 weeks postoperatively for all subscales. The TJR-DVPRS and SF-MPQ-2 subscales were similarly responsive, except for the SF-MPQ-2 neuropathic and TJR-DVPRS nonoperative joint subscales, which were minimally responsive during the preoperative to 6-week period. CONCLUSIONS: The TJR-DVPRS is valid for use among veterans undergoing TJR and poses significantly less respondent burden than does the SF-MPQ-2. The brevity and ease of use of the TJR-DVPRS make it a practical tool for use during surgical recovery to monitor pain intensity at rest and with movement in the operative joint, and to assess pain interference with activity, sleep, and mood. The TJR-DVPRS is at least as responsive as the SF-MPQ-2, but the SF-MPQ-2 neuropathic and TJR-DVPRS nonoperative joint subscales were minimally responsive. Limitations of this study include the small sample size, under-representation of women (which would be expected in the veteran population), and using only veterans. Future validations studies should include civilians and active military TJR patients.

Assessing Common Content and Responsiveness of the QoR-15 and the SF-8 in the Context of Recovery from Regional Anesthesia for Joint Replacement.

PURPOSE: There is no consensus regarding how best to measure postoperative quality of recovery after anesthesia/surgery (e.g., using 24-hour survey instruments) in veterans or active military. Our goals were to (1) describe health-related quality-of-life (HRQoL) and recovery across time in veterans, (2) examine the commonality between the quality of recovery (QoR-15) and short-form (SF) surveys (including the SF-8, 24-hour version), and (3) examine the responsiveness of these surveys. MATERIALS AND METHODS: This study was approved by the Institutional Review Boards from the University of Pittsburgh, the VA Pittsburgh, and the Human Research Protection Office of the Department of Defense. Secondary analyses of data were executed based on a randomized trial dataset of knee/hip arthroplasty patients, the study having recruited 135 total veterans. QoR-15 and HRQoL SF surveys were completed and self-reported before surgery (pre-op), on postoperative day 2 (PO-D2), and 6 weeks postoperatively. Descriptive statistics were used to examine scores across time. Common content was examined using Pearson's r. Responsiveness was examined using distribution-based methods. RESULTS: Average veteran age was 67 year, 89% were male, 88% white, and average body mass index was 33 kg∙m-2. QoR-15 scores declined from pre-op to PO-D2 but were higher than pre-op at 6 weeks. SF physical component summary (PCS) scores were low both pre-op and PO-D2, but were elevated over baseline at 6 weeks. SF mental component summary (MCS) scores declined from baseline to PO-D2 but were higher than pre-op at 6 weeks. Associations of the QoR-15 total score and PCS/MCS were medium/large and statistically significant at P ≤ .01. Both instruments were responsive to changes. CONCLUSION: QoR-15 and SF-8 have high content commonality and performed similarly in veterans across time. SF-8 has added benefits of (1) brevity, (2) assessment of physical and mental health components, and (3) being normed to the general population. The SF-8, if used without the QoR-15 in tandem in future study of anesthesia-related outcomes, would need to be supplemented by separate questions addressing postoperative nausea/vomiting (a frequent outcome after anesthesia that is relevant to same-day and next-day mobilization after elective joint replacement surgery).

Day-One Pain Reductions After Hip and Knee Replacement When Buprenorphine-Clonidine-Dexamethasone Is Added to Bupivacaine Nerve/Plexus Blocks: A Randomized Clinical Trial.

OBJECTIVE: To compare pain outcome reports of patients undergoing hip or knee replacement who received single-injection nerve/plexus blocks with plain bupivacaine (BPV) with those of patients who received injections of buprenorphine-clonidine-dexamethasone (BCD) admixed with BPV. DESIGN: Prospective, parallel-arm, randomized, double-blind trial. SETTING: A single veterans' hospital. SUBJECTS: Ninety-eight veterans scheduled for total hip or knee replacement surgery with spinal as the primary anesthetic. METHODS: Participants were randomized to BPV-BCD or plain BPV groups. They underwent nerve/plexus blocks in the L2-L4 and L4-S3 distributions in advance of joint replacement surgery. The primary outcome was change in pain from baseline during the postoperative day, as assessed by the total pain score on the short-form McGill Pain Questionnaire-v2 (SF-MPQ-2). Secondary outcomes were pain during movement, pain interference, range of motion, mobility, and quality of recovery. RESULTS: On postoperative day one, the SF-MPQ-2 total score for the BPV-BCD group demonstrated greater pain reduction than that of the plain BPV group (mean difference 1.8 points, 95% confidence interval 0.6 to 3.0, P = 0.003). The BPV-BCD group also had larger reductions in pain during movement in the surgical joint and less pain interference, along with increased range of hip and knee flexion, compared with the plain BPV group. Outcomes of mobility and quality of recovery were not different between groups. CONCLUSIONS: Preoperative BPV-BCD blocks in the L2-L4 and L4-S3 nerve distributions for hip and knee replacements led to less pain on postoperative day one and increased knee and hip range of motion, compared with plain BPV blocks. TRIAL REGISTRATION: ClinicalTrials.gov ID NCT02891798.

Extended Perineural Analgesia After Hip and Knee Replacement When Buprenorphine-Clonidine-Dexamethasone Is Added to Bupivacaine: Preliminary Report from a Randomized Clinical Trial.

OBJECTIVE: We tested the hypothesis that buprenorphine-clonidine-dexamethasone (BCD) extends perineural analgesia compared with plain bupivacaine (BPV) nerve blocks used for hip and knee replacement surgery. DESIGN: Prospective, parallel-arms, randomized, double-blind trial. SETTING: A single veterans' hospital. SUBJECTS: Seventy-eight veterans scheduled for total hip or knee replacement with plans for spinal as the primary anesthetic. METHODS: Participants underwent nerve/plexus blocks at L2-L4 and L4-S3 in advance of hip or knee joint replacement surgery. Patients were randomized to receive BPV-BCD or plain BPV in a 4:1 allocation ratio. Patients answered four block duration questions (listed below). Time differences between treatments were analyzed using the t test. RESULTS: Significant (P < 0.001) prolongation of the time parameters was reported by patients after the BPV-BCD blocks (N = 62) vs plain BPV (N = 16). The time until start of postoperative pain was 26 vs 11 hours (mean difference = 15 hours, 95% CI = 8 to 21). The time until no pain relief from the blocks was 32 vs 15 hours (mean difference = 17 hours, 95% CI = 10 to 24). The time until the numbness wore off was 37 vs 21 hours (mean difference = 16 hours, 95% CI = 8 to 23). The time until the worst postoperative pain was 39 vs 20 hours (mean difference = 19 hours, 95% CI = 11 to 27). CONCLUSIONS: BPV-BCD provided 26-39 hours of perineural analgesia in the L2-L4 and L4-S3 nerve distributions after hip/knee replacement surgery, compared with 11-21 hours for plain BPV.

Author Correction: An atlas of dynamic chromatin landscapes in mouse fetal development.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An atlas of dynamic chromatin landscapes in mouse fetal development.

The Encyclopedia of DNA Elements (ENCODE) project has established a genomic resource for mammalian development, profiling a diverse panel of mouse tissues at 8 developmental stages from 10.5 days after conception until birth, including transcriptomes, methylomes and chromatin states. Here we systematically examined the state and accessibility of chromatin in the developing mouse fetus. In total we performed 1,128 chromatin immunoprecipitation with sequencing (ChIP-seq) assays for histone modifications and 132 assay for transposase-accessible chromatin using sequencing (ATAC-seq) assays for chromatin accessibility across 72 distinct tissue-stages. We used integrative analysis to develop a unified set of chromatin state annotations, infer the identities of dynamic enhancers and key transcriptional regulators, and characterize the relationship between chromatin state and accessibility during developmental gene regulation. We also leveraged these data to link enhancers to putative target genes and demonstrate tissue-specific enrichments of sequence variants associated with disease in humans. The mouse ENCODE data sets provide a compendium of resources for biomedical researchers and achieve, to our knowledge, the most comprehensive view of chromatin dynamics during mammalian fetal development to date.