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The naive T cell compartment undergoes multiple changes across age that associate with altered susceptibility to infection and autoimmunity. In addition to the acquisition of naive-like memory T cell subsets, mouse studies describe substantial molecular reprogramming of the naive compartment in adults compared with adolescents. However, these alterations are not well delineated in human aging. Using a new trimodal single cell technology (TEA-seq), we discovered that the composition and transcriptional and epigenetic programming of the naive T cell compartment in children (11-13 yrs) is distinct from that of older adults (55-65 yrs). Naive CD4 T cells, previously considered relatively resistant to aging, exhibited far more pronounced molecular reprogramming than naive CD8 T cells, in which alterations are preferentially driven by shifts in naive-like memory subsets. These data reveal the complex nature of the naive T cell compartment that may contribute to differential immune responses across the spectrum of human age.One Sentence Summary:The naive CD8 and CD4 T cell compartments in humans are heterogeneous and impacted differently with age, in which naive CD8 T cell subsets dramatically shift in composition and true naive CD4 T cells display significant molecular re-programming.

The human neonatal cerebellum is one-fourth of its adult size yet contains the blueprint required to integrate environmental cues with developing motor, cognitive and emotional skills into adulthood. Although mature cerebellar neuroanatomy is well studied, understanding of its developmental origins is limited. In this study, we systematically mapped the molecular, cellular and spatial composition of human fetal cerebellum by combining laser capture microscopy and SPLiT-seq single-nucleus transcriptomics. We profiled functionally distinct regions and gene expression dynamics within cell types and across development. The resulting cell atlas demonstrates that the molecular organization of the cerebellar anlage recapitulates cytoarchitecturally distinct regions and developmentally transient cell types that are distinct from the mouse cerebellum. By mapping genes dominant for pediatric and adult neurological disorders onto our dataset, we identify relevant cell types underlying disease mechanisms. These data provide a resource for probing the cellular basis of human cerebellar development and disease.

Long COVID or post-acute sequelae of SARS-CoV-2 (PASC) is a clinical syndrome featuring diverse symptoms that can persist for months after acute SARS-CoV-2 infection. The etiologies are unknown but may include persistent inflammation, unresolved tissue damage, or delayed clearance of viral protein or RNA. Attempts to classify subsets of PASC by symptoms alone have been unsuccessful. To molecularly define PASC, we evaluated the serum proteome in longitudinal samples from 55 PASC individuals with symptoms lasting ≥60 days after onset of acute infection and compared this to symptomatically recovered SARS-CoV-2 infected and uninfected individuals. We identified subsets of PASC with distinct signatures of persistent inflammation. Type II interferon signaling and canonical NF-κB signaling (particularly associated with TNF), were the most differentially enriched pathways. These findings help to resolve the heterogeneity of PASC, identify patients with molecular evidence of persistent inflammation, and highlight dominant pathways that may have diagnostic or therapeutic relevance.One Sentence SummarySerum proteome profiling identifies subsets of long COVID patients with evidence of persistent inflammation including key immune signaling pathways that may be amenable to therapeutic intervention.

Amorphous metal oxides expand the range of material parameters significantly compared to their crystalline counter parts. However, predictions of the exact nature of the amorphous phase and its effect on material properties are still elusive. Thorough structure-property investigations of well-known model systems are thus necessary before predictive control of useful material properties is obtained. In this work, we fabricate a series of photodeposited nickel (oxy)hydroxide (NiOx) thin films and anneal them at temperatures up to 1000 oC. EXAFS, XRD and XPS are used to determine the local structure, allowing us to correlate it to measured electrochemical properties. We find an amorphous Ni(OH)2-like local structure for annealing conducted below 250 oC, followed by an amorphous-to-amorphous phase transition to a NiO-like structure by 300 oC, thus supplying evidence for different amorphous polymorphs in this Ni-O system. Above 400 oC a cubic NiO XRD diffraction pattern is detected. Electrochemically, we find a stepwise increase of the onset overpotential at this transition, indicating a change in potential-determining step and possibly OER reaction mechanism. The Tafel slope decreases linearly with annealing temperature, which we attribute to a decrease in (Ni)OOH reaction intermediary coverage, supported by in-operando UV-Vis electrochromism. Furthermore, we find that the (Ni)OOH coordination is increasingly strained with annealing temperature, which manifests in higher electrochromic coloring rates and lower binding energies. We identify this as the root cause of the lowered intermediary coverage. Thus, nano-crystalline NiO should kinetically be a superior catalyst to amorphous Ni(OH)2. However, at our benchmarking value of 10 mA cm-2 the amorphous material exhibits lower overpotential, due to a combination of lower onset potential, large chemically active surface area and mass transport limitations under our conditions.

Summary Deep immune profiling is essential for understanding the human immune system in health and disease. Successful biological interpretation of this data requires consistent laboratory processing with minimal batch-to-batch variation. Here, we detail a robust pipeline for the profiling of human peripheral blood mononuclear cells by both high-dimensional flow cytometry and single-cell RNA-seq. These protocols reduce batch effects, generate reproducible data, and increase throughput. For complete details on the use and execution of this protocol, please refer to Savage et al. (2021)., Graphical abstract, Highlights • Details a robust pipeline for the profiling of human PBMC • Outline a cell thaw protocol compatible with flow cytometry and single cell RNA-seq • Focus on batch effect reduction with a bridging cell control and commercial buffers • Increased throughput with automated liquid handling and cell counting, Deep immune profiling is essential for understanding the human immune system in health and disease. Successful biological interpretation of this data requires consistent laboratory processing with minimal batch-to-batch variation. Here, we detail a robust pipeline for the profiling of human peripheral blood mononuclear cells by both high dimensional flow cytometry and single-cell RNA-seq. These protocols reduce batch effects, generate reproducible data, and increase throughput.

SARS-CoV-2 has infected over 160 million and caused more than 3 million deaths to date. Most individuals (>80%) have mild symptoms and recover in the outpatient setting, but detailed studies of immune responses have focused primarily on moderate to severe COVID-19. We deeply profiled the longitudinal immune response in individuals with mild COVID beginning with early time points post-infection (1-15 days) and proceeding through convalescence to >100 days after symptom onset. We correlated data from single cell analyses of peripheral blood cells, serum proteomics, virus-specific cellular and humoral immune responses, and clinical metadata. Acute infection was characterized by vigorous coordinated innate and adaptive activation, including an early cellular and proteomic signature that correlated with the amplitude of virus-specific humoral responses after day 30. We characterized signals associated with recovery and convalescence to define a new signature of inflammatory cytokines, gene expression, and chromatin accessibility that persists in individuals with post-acute sequelae of SARS-CoV-2 infection (PASC).

Cerebellar malformations are diverse congenital anomalies frequently associated with developmental disability. Although genetic and prenatal non-genetic causes have been described, no systematic analysis has been performed. Here, we present a large-exome sequencing study of Dandy-Walker malformation (DWM) and cerebellar hypoplasia (CBLH). We performed exome sequencing in 282 individuals from 100 families with DWM or CBLH, and we established a molecular diagnosis in 36 of 100 families, with a significantly higher yield for CBLH (51%) than for DWM (16%). The 41 variants impact 27 neurodevelopmental-disorder-associated genes, thus demonstrating that CBLH and DWM are often features of monogenic neurodevelopmental disorders. Though only seven monogenic causes (19%) were identified in more than one individual, neuroimaging review of 131 additional individuals confirmed cerebellar abnormalities in 23 of 27 genetic disorders (85%). Prenatal risk factors were frequently found among individuals without a genetic diagnosis (30 of 64 individuals [47%]). Single-cell RNA sequencing of prenatal human cerebellar tissue revealed gene enrichment in neuronal and vascular cell types; this suggests that defective vasculogenesis may disrupt cerebellar development. Further, de novo gain-of-function variants in PDGFRB, a tyrosine kinase receptor essential for vascular progenitor signaling, were associated with CBLH, and this discovery links genetic and non-genetic etiologies. Our results suggest that genetic defects impact specific cerebellar cell types and implicate abnormal vascular development as a mechanism for cerebellar malformations. We also confirmed a major contribution for non-genetic prenatal factors in individuals with cerebellar abnormalities, substantially influencing diagnostic evaluation and counseling regarding recurrence risk and prognosis.

The apolipoprotein E gene (APOE) is the strongest genetic risk factor for developing Alzheimer's disease (AD). Our recent identification of altered APOE DNA methylation in AD postmortem brain (PMB) prompted this follow-up study. Our goals were to (i) validate the AD-differential methylation of APOE in an independent PMB study cohort and (ii) determine the cellular populations (i.e., neuronal vs. non-neuronal) of AD PMB that contribute to this differential methylation. Here, we obtained an independent cohort of 57 PMB (42 AD and 15 controls) and quantified their APOE methylation levels from frontal lobe and cerebellar tissue. We also applied fluorescence-activated nuclei sorting (FANS) to separate neuronal nuclei from non-neuronal nuclei within the tissue of 15 AD and 14 control subjects. Bisulfite pyrosequencing was used to generate DNA methylation profiles of APOE from both bulk PMB and FANS nuclei. Our results provide independent validation that the APOE CGI holds lower DNA methylation levels in AD compared to control in frontal lobe but not cerebellar tissue. Our data also indicate that the non-neuronal cells of the AD brain, which are mainly composed of glia, are the main contributors to the lower APOE DNA methylation observed in AD PMB. Given that astrocytes are the primary producers of ApoE in the brain our results suggest that alteration of epigenetically regulated APOE expression in glia could be an important part of APOE's strong effect on AD risk.

Summary Multi-omic profiling of human peripheral blood is increasingly utilized to identify biomarkers and pathophysiologic mechanisms of disease. The importance of these platforms in clinical and translational studies led us to investigate the impact of delayed blood processing on the numbers and state of peripheral blood mononuclear cells (PBMC) and on the plasma proteome. Similar to previous studies, we show minimal effects of delayed processing on the numbers and general phenotype of PBMC up to 18 hours. In contrast, profound changes in the single-cell transcriptome and composition of the plasma proteome become evident as early as 6 hours after blood draw. These reflect patterns of cellular activation across diverse cell types that lead to progressive distancing of the gene expression state and plasma proteome from native in vivo biology. Differences accumulating during an overnight rest (18 hours) could confound relevant biologic variance related to many underlying disease states., Graphical abstract, Highlights • Studies of human blood cells and plasma are highly sensitive to process variability • Time variability distorts biology in cutting-edge single-cell and multiplex assays • Longitudinal, multi-modal, and aligned data enable data qualification and exploration • Dataset holds potential novel, multi-modal biological correlations and hypotheses, Molecular Physiology ; Immunology ; Proteomics ; Transcriptomics

Single-cell measurements of cellular characteristics have been instrumental in understanding the heterogeneous pathways that drive differentiation, cellular responses to signals, and human disease. Recent advances have allowed paired capture of protein abundance and transcriptomic state, but a lack of epigenetic information in these assays has left a missing link to gene regulation. Using the heterogeneous mixture of cells in human peripheral blood as a test case, we developed a novel scATAC-seq workflow that increases signal-to-noise and allows paired measurement of cell surface markers and chromatin accessibility: integrated cellular indexing of chromatin landscape and epitopes, called ICICLE-seq. We extended this approach using a droplet-based multiomics platform to develop a trimodal assay that simultaneously measures transcriptomics (scRNA-seq), epitopes, and chromatin accessibility (scATAC-seq) from thousands of single cells, which we term TEA-seq. Together, these multimodal single-cell assays provide a novel toolkit to identify type-specific gene regulation and expression grounded in phenotypically defined cell types.

The human apolipoprotein E (APOE) gene plays an important role in lipid metabolism. It has three common genetic variants, alleles ɛ2/ɛ3/ɛ4, which translate into three protein isoforms of apoE2, E3 and E4. These isoforms can differentially influence total serum cholesterol levels; therefore, APOE has been linked with cardiovascular disease. Additionally, its ɛ4 allele is strongly associated with the risk of Alzheimer's disease (AD), whereas the ɛ2 allele appears to have a modest protective effect for AD. Despite decades of research having illuminated multiple functional differences among the three apoE isoforms, the precise mechanisms through which different APOE alleles modify diseases risk remain incompletely understood. In this study, we examined the genomic structure of APOE in search for properties that may contribute novel biological consequences to the risk of disease. We identify one such element in the ɛ2/ɛ3/ɛ4 allele-carrying 3′-exon of APOE. We show that this exon is imbedded in a well-defined CpG island (CGI) that is highly methylated in the human postmortem brain. We demonstrate that this APOE CGI exhibits transcriptional enhancer/silencer activity. We provide evidence that this APOE CGI differentially modulates expression of genes at the APOE locus in a cell type-, DNA methylation- and ɛ2/ɛ3/ɛ4 allele-specific manner. These findings implicate a novel functional role for a 3′-exon CGI and support a modified mechanism of action for APOE in disease risk, involving not only the protein isoforms but also an epigenetically regulated transcriptional program at the APOE locus driven by the APOE CGI.

Marine picocyanobacteria, comprised of the genera Synechococcus and Prochlorococcus, are the most abundant and widespread primary producers in the ocean. More than 20 genetically distinct clades of marine Synechococcus have been identified, but their physiology and biogeography are not as thoroughly characterized as those of Prochlorococcus. Using clade-specific qPCR primers, we measured the abundance of 10 Synechococcus clades at 92 locations in surface waters of the Atlantic and Pacific Oceans. We found that Synechococcus partition the ocean into four distinct regimes distinguished by temperature, macronutrients and iron availability. Clades I and IV were prevalent in colder, mesotrophic waters; clades II, III and X dominated in the warm, oligotrophic open ocean; clades CRD1 and CRD2 were restricted to sites with low iron availability; and clades XV and XVI were only found in transitional waters at the edges of the other biomes. Overall, clade II was the most ubiquitous clade investigated and was the dominant clade in the largest biome, the oligotrophic open ocean. Co-occurring clades that occupy the same regime belong to distinct evolutionary lineages within Synechococcus, indicating that multiple ecotypes have evolved independently to occupy similar niches and represent examples of parallel evolution. We speculate that parallel evolution of ecotypes may be a common feature of diverse marine microbial communities that contributes to functional redundancy and the potential for resiliency.