Length of overnight fasting and six-year weight change in the Cancer Prevention Study-3.

BACKGROUND: Longer overnight fasting (ONF) is a potential strategy for weight control. While promising, the evidence from large population-based studies is limited. OBJECTIVE: To examine the association of self-reported ONF duration with three- and six-year weight change in the American Cancer Society's Cancer Prevention Study-3 (CPS-3) prospective cohort. METHODS: U.S. adult CPS-3 participants completed a 24-hour validated meal and snack timing and frequency grid (weekday and weekend) in 2015, from which weighted ONF hours were calculated. Participants reported body weight in 2015, 2018 and 2021. Three- and six-year weight change (kg, and % body weight) were assessed. RESULTS: Among 104,420 mostly female (78.5%) participants aged 52.7 +/- 9.5 (SD) years followed for six years, a one hour increase in ONF length was associated with a small but statistically significant reduction in weight gain over three- and six-year periods (multivariable-adjusted mean difference in % body weight= -0.02, 95% CI -0.05-0.00, p=0.03 and -0.04, 95 % CI, -0.07 to -0.01, p=0.007, respectively). The mean difference of 6-year % reduction in weight gain was slightly greater among individuals with overweight (-0.05, 95% CI, -0.10 to 0.00, p=0.05) and obesity (-0.06, 95% CI, -0.12 to 0.01, p=0.08) compared to those with healthy BMI (-0.03, 95% CI -0.07 to 0.01, p=0.13) or underweight (0.16, 95% CI, -0.04 to 0.36, p=0.13, pinteraction<0.0001). Stronger associations were observed among those ≤55 y than 56+ (pinteraction=0.01), and those with higher waist circumference (pinteraction<0.0001) but not by sex or earlier/later fasting period. CONCLUSIONS: Longer ONF was associated with slightly lower body weight in adult men and women over six years that was stronger among those with overweight or obesity, higher waist circumference, and those ≤age 55. The magnitude of weight change, though in the hypothesized direction, suggests that prolonged ONF may have modest impact on weight control over time.

Somatic cancer driver mutations are enriched and associated with inflammatory states in Alzheimer's disease microglia.

Alzheimer's disease (AD) is an age-associated neurodegenerative disorder characterized by progressive neuronal loss and pathological accumulation of the misfolded proteins amyloid-ß and tau1,2. Neuroinflammation mediated by microglia and brain-resident macrophages plays a crucial role in AD pathogenesis1-5, though the mechanisms by which age, genes, and other risk factors interact remain largely unknown. Somatic mutations accumulate with age and lead to clonal expansion of many cell types, contributing to cancer and many non-cancer diseases6,7. Here we studied somatic mutation in normal aged and AD brains by three orthogonal methods and in three independent AD cohorts. Analysis of bulk RNA sequencing data from 866 samples from different brain regions revealed significantly higher (~two-fold) overall burdens of somatic single-nucleotide variants (sSNVs) in AD brains compared to age-matched controls. Molecular-barcoded deep (>1000X) gene panel sequencing of 311 prefrontal cortex samples showed enrichment of sSNVs and somatic insertions and deletions (sIndels) in cancer driver genes in AD brain compared to control, with recurrent, and often multiple, mutations in genes implicated in clonal hematopoiesis (CH)8,9. Pathogenic sSNVs were enriched in CSF1R+ microglia of AD brains, and the high proportion of microglia (up to 40%) carrying some sSNVs in cancer driver genes suggests mutation-driven microglial clonal expansion (MiCE). Analysis of single-nucleus RNA sequencing (snRNAseq) from temporal neocortex of 62 additional AD cases and controls exhibited nominally increased mosaic chromosomal alterations (mCAs) associated with CH10,11. Microglia carrying mCA showed upregulated pro-inflammatory genes, resembling the transcriptomic features of disease-associated microglia (DAM) in AD. Our results suggest that somatic driver mutations in microglia are common with normal aging but further enriched in AD brain, driving MiCE with inflammatory and DAM signatures. Our findings provide the first insights into microglial clonal dynamics in AD and identify potential new approaches to AD diagnosis and therapy.

Single-cell genomics reveals region-specific developmental trajectories underlying neuronal diversity in the human hypothalamus.

The development and diversity of neuronal subtypes in the human hypothalamus has been insufficiently characterized. To address this, we integrated transcriptomic data from 241,096 cells (126,840 newly generated) in the prenatal and adult human hypothalamus to reveal a temporal trajectory from proliferative stem cell populations to mature hypothalamic cell types. Iterative clustering of the adult neurons identified 108 robust transcriptionally distinct neuronal subtypes representing 10 hypothalamic nuclei. Pseudotime trajectories provided insights into the genes driving formation of these nuclei. Comparisons to single-cell transcriptomic data from the mouse hypothalamus suggested extensive conservation of neuronal subtypes despite certain differences in species-enriched gene expression. The uniqueness of hypothalamic neuronal lineages was examined developmentally by comparing excitatory lineages present in cortex and inhibitory lineages in ganglionic eminence, revealing both distinct and shared drivers of neuronal maturation across the human forebrain. These results provide a comprehensive transcriptomic view of human hypothalamus development through gestation and adulthood at cellular resolution.

Comparative transcriptomics reveals human-specific cortical features.

The cognitive abilities of humans are distinctive among primates, but their molecular and cellular substrates are poorly understood. We used comparative single-nucleus transcriptomics to analyze samples of the middle temporal gyrus (MTG) from adult humans, chimpanzees, gorillas, rhesus macaques, and common marmosets to understand human-specific features of the neocortex. Human, chimpanzee, and gorilla MTG showed highly similar cell-type composition and laminar organization as well as a large shift in proportions of deep-layer intratelencephalic-projecting neurons compared with macaque and marmoset MTG. Microglia, astrocytes, and oligodendrocytes had more-divergent expression across species compared with neurons or oligodendrocyte precursor cells, and neuronal expression diverged more rapidly on the human lineage. Only a few hundred genes showed human-specific patterning, suggesting that relatively few cellular and molecular changes distinctively define adult human cortical structure.

A comparative atlas of single-cell chromatin accessibility in the human brain.

Recent advances in single-cell transcriptomics have illuminated the diverse neuronal and glial cell types within the human brain. However, the regulatory programs governing cell identity and function remain unclear. Using a single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq), we explored open chromatin landscapes across 1.1 million cells in 42 brain regions from three adults. Integrating this data unveiled 107 distinct cell types and their specific utilization of 544,735 candidate cis-regulatory DNA elements (cCREs) in the human genome. Nearly a third of the cCREs demonstrated conservation and chromatin accessibility in the mouse brain cells. We reveal strong links between specific brain cell types and neuropsychiatric disorders including schizophrenia, bipolar disorder, Alzheimer's disease (AD), and major depression, and have developed deep learning models to predict the regulatory roles of noncoding risk variants in these disorders.

Interindividual variation in human cortical cell type abundance and expression.

Single-cell transcriptomic studies have identified a conserved set of neocortical cell types from small postmortem cohorts. We extended these efforts by assessing cell type variation across 75 adult individuals undergoing epilepsy and tumor surgeries. Nearly all nuclei map to one of 125 robust cell types identified in the middle temporal gyrus. However, we found interindividual variance in abundances and gene expression signatures, particularly in deep-layer glutamatergic neurons and microglia. A minority of donor variance is explainable by age, sex, ancestry, disease state, and cell state. Genomic variation was associated with expression of 150 to 250 genes for most cell types. This characterization of cellular variation provides a baseline for cell typing in health and disease.

Transcriptomic cytoarchitecture reveals principles of human neocortex organization.

Variation in cytoarchitecture is the basis for the histological definition of cortical areas. We used single cell transcriptomics and performed cellular characterization of the human cortex to better understand cortical areal specialization. Single-nucleus RNA-sequencing of 8 areas spanning cortical structural variation showed a highly consistent cellular makeup for 24 cell subclasses. However, proportions of excitatory neuron subclasses varied substantially, likely reflecting differences in connectivity across primary sensorimotor and association cortices. Laminar organization of astrocytes and oligodendrocytes also differed across areas. Primary visual cortex showed characteristic organization with major changes in the excitatory to inhibitory neuron ratio, expansion of layer 4 excitatory neurons, and specialized inhibitory neurons. These results lay the groundwork for a refined cellular and molecular characterization of human cortical cytoarchitecture and areal specialization.

Transcriptomic diversity of cell types across the adult human brain.

The human brain directs complex behaviors, ranging from fine motor skills to abstract intelligence, but the diversity of cell types that support these skills has not been fully described. In this work, we used single-nucleus RNA sequencing to systematically survey cells across the entire adult human brain. We sampled more than three million nuclei from approximately 100 dissections across the forebrain, midbrain, and hindbrain in three postmortem donors. Our analysis identified 461 clusters and 3313 subclusters organized largely according to developmental origins and revealing high diversity in midbrain and hindbrain neurons. Astrocytes and oligodendrocyte-lineage cells also exhibited regional diversity at multiple scales. The transcriptomic census of the entire human brain presented in this work provides a resource for understanding the molecular diversity of the human brain in health and disease.

Single-cell DNA methylation and 3D genome architecture in the human brain.

Delineating the gene-regulatory programs underlying complex cell types is fundamental for understanding brain function in health and disease. Here, we comprehensively examined human brain cell epigenomes by probing DNA methylation and chromatin conformation at single-cell resolution in 517 thousand cells (399 thousand neurons and 118 thousand non-neurons) from 46 regions of three adult male brains. We identified 188 cell types and characterized their molecular signatures. Integrative analyses revealed concordant changes in DNA methylation, chromatin accessibility, chromatin organization, and gene expression across cell types, cortical areas, and basal ganglia structures. We further developed single-cell methylation barcodes that reliably predict brain cell types using the methylation status of select genomic sites. This multimodal epigenomic brain cell atlas provides new insights into the complexity of cell-type-specific gene regulation in adult human brains.

Comparative single-cell transcriptomic analysis of primate brains highlights human-specific regulatory evolution.

Enhanced cognitive function in humans is hypothesized to result from cortical expansion and increased cellular diversity. However, the mechanisms that drive these phenotypic innovations remain poorly understood, in part because of the lack of high-quality cellular resolution data in human and non-human primates. Here, we take advantage of single-cell expression data from the middle temporal gyrus of five primates (human, chimp, gorilla, macaque and marmoset) to identify 57 homologous cell types and generate cell type-specific gene co-expression networks for comparative analysis. Although orthologue expression patterns are generally well conserved, we find 24% of genes with extensive differences between human and non-human primates (3,383 out of 14,131), which are also associated with multiple brain disorders. To assess the functional significance of gene expression differences in an evolutionary context, we evaluate changes in network connectivity across meta-analytic co-expression networks from 19 animals. We find that a subset of these genes has deeply conserved co-expression across all non-human animals, and strongly divergent co-expression relationships in humans (139 out of 3,383, <1% of primate orthologues). Genes with human-specific cellular expression and co-expression profiles (such as NHEJ1, GTF2H2, C2 and BBS5) typically evolve under relaxed selective constraints and may drive rapid evolutionary change in brain function.

Dietaryindex: A User-Friendly and Versatile R Package for Standardizing Dietary Pattern Analysis in Epidemiological and Clinical Studies.

Background: Few standardized and open-source tools exist for calculating dietary pattern indexes from dietary intake data in epidemiological and clinical studies. Miscalculations of dietary indexes, with suspected erroneous findings, are occasionally noted in the literature. Objective: The primary aim is to develop and validate dietaryindex, a user-friendly and versatile R package that standardizes the calculation of dietary indexes. Methods: Dietaryindex utilizes a two-step process: an initial calculation of serving size for each food and nutrient category, followed by the calculation of individual dietary indexes. It includes generic functions that accept any preprocessed serving sizes of food groups and nutrients, with the standard serving sizes defined according to the methodologies used in well-known prospective cohort studies. For ease of use, dietaryindex also offers one-step functions that directly reference common datasets and tools, including the National Health and Nutrition Examination Survey (NHANES) and Block Food Frequency Questionnaire, eliminating the need for data preprocessing. At least two independent researchers validated the serving size definitions and scoring algorithms of dietaryindex. Results: Dietaryindex can calculate multiple dietary indexes of high interest in research, including Healthy Eating Index (HEI) - 2020, Alternative Healthy Eating Index 2010, Dietary Approaches to Stop Hypertension Index, Alternate Mediterranean Diet Score, Dietary Inflammatory Index, American Cancer Society 2020 dietary index, and Planetary Health Diet Index from the EAT-Lancet Commission. In our validation process, dietaryindex demonstrated full accuracy (100%) in all generic functions with two-decimal rounding precision in comparison to hand-calculated results. Similarly, using NHANES 2017-2018 data and ASA24 and DHQ3 example data, the HEI2015 outputs from dietaryindex aligned (99.95%-100%) with results using the SAS codes from the National Cancer Institute. Conclusions: Dietaryindex is a user-friendly, versatile, and validated informatics tool for standardized dietary index calculations. We have open-sourced all the validation files and codes with detailed tutorials on GitHub (https://github.com/jamesjiadazhan/dietaryindex).

Survey of parents of children with intellectual disabilities and/or autism who experience chronic constipation.

BACKGROUND: Constipation is common in children with intellectual disabilities and/or autism, but poorly researched. This study looks to understand parental knowledge, attitudes and management practices towards constipation in children with intellectual disabilities and/or autism. METHODS: A cross-sectional online survey developed with patient facing organisations was circulated to parents of children with intellectual disabilities and/or autism using an exponential and non-discriminatory snowballing method for recruitment. A smaller sample were purposively sampled for their in-depth experiences. RESULTS: Of 68 responses, people were open to discussing constipation and knowledgeable about risk factors. In the qualitative interviews, of 15 parents, they wanted to be treated as an expert in their child's care. They desired a service that was more responsive when in difficulty. While wanting more information about medication options, parents want a more holistic approach. CONCLUSIONS: Services need more emphasis on holistic management. Listening to parents and treating them as experts is important.

Change in Diet Quality and Meal Sources during the COVID-19 Pandemic in a Diverse Subset of Men and Women in the Cancer Prevention Study-3.

The COVID-19 pandemic resulted in restrictive measures that caused disruptions in behaviors that may have long-term consequences on diet, health, and chronic disease risk. The aim of this study was to assess longitudinal changes in diet quality from before to during the pandemic among 2335 adult participants (816 males and 1519 females; aged 36-78) of the Cancer Prevention Study-3 cohort. We compared dietary screeners conducted in 2018 and 2020 and calculated a diet quality score, which assigned higher points for recommended foods. Overall diet quality slightly improved among all participants from before to during the pandemic, particularly among males (+0.45 points, p < 0.001), White participants (+0.24 points, p < 0.001), and participants reporting weight loss (+0.66 points, p < 0.001 for 2.25 -< 4.5 kg loss; +1.04 points, p < 0.001 for ≥4.5 kg loss); change in diet quality did not differ by other sociodemographic factors. Reported consumption of most food groups decreased, especially whole grains (-0.17 servings/day, p < 0.001) and vegetables (-0.21 servings/day, p < 0.001), primarily among females, Black participants, and participants who gained ≥2.25 kg. The frequency of meals from outside the home decreased, especially in full-service restaurants (-0.47 times/week, p < 0.001) and for ready-to-eat meals (-0.37 times/week, p < 0.001). Declines in whole grain and vegetable consumption raise concerns for weight gain in these populations and increased risk of poor metabolic health and chronic disease.

Consistent and changing consumption of fast-food and full-service meals and 3-year weight change in a large population cohort study.

BACKGROUND: An average American consumes 3 meals weekly from fast-food or full-service restaurants, which contain more calories, fat, sodium, and cholesterol than meals prepared at home. OBJECTIVES: This study examined whether consistent and changing fast-food or full-service consumption was associated with weight change over a 3-y period. METHODS: Among 98,589 US adults from the American Cancer Society's Cancer Prevention Study-3, self-reported weight and fast-food and full-service consumption from 2015 and 2018 were examined using a multivariable-adjusted linear regression analysis to assess the association of consistent and changing consumption on 3-y weight change. RESULTS: Individuals who made no changes to their fast-food or full-service intake over the study period gained weight regardless of consumption frequency, although low consumers gained less weight than high consumers (low fast-food: ß = -1.08; 95% CI: -1.22, -0.93; low full-service: ß = -0.35; 95% CI: -0.50, -0.21; P < 0.001). Decreased fast-food intake during the study period (e.g., from high [>1 meal/wk] to low [≤0.5 meal/wk], high to medium [>0.5 to ≤1 meal/wk], or medium to low) and decreased full-service intake from high (≥1 meal/wk) to low (<1 meal/mo) were significantly associated with weight loss (high-low: ß = -2.77; 95% CI: -3.23, -2.31; high-medium: ß = -1.53; 95% CI: -1.72, -1.33; medium-low: ß = -0.85; 95% CI: -1.06, -0.63; high-low full-service: ß = -0.92; 95% CI: -1.36, -0.49; P < 0.001). Decreased consumption of both fast-food and full-service restaurant meals was associated with greater weight loss than decreasing fast-food alone (both: ß = -1.65; 95% CI: -1.82, -1.37; fast-food only: ß = -0.95; 95% CI: -1.12, -0.79; P < 0.001). CONCLUSIONS: Decreased consumption of fast-food and full-service meals over 3 y, particularly among high consumers at baseline, was associated with weight loss and may be an effective approach to weight loss. Moreover, decreasing both fast-food and full-service meal consumption was associated with a greater weight loss than decreasing only fast-food meal consumption.

Scalable in situ single-cell profiling by electrophoretic capture of mRNA using EEL FISH.

Methods to spatially profile the transcriptome are dominated by a trade-off between resolution and throughput. Here we develop a method named Enhanced ELectric Fluorescence in situ Hybridization (EEL FISH) that can rapidly process large tissue samples without compromising spatial resolution. By electrophoretically transferring RNA from a tissue section onto a capture surface, EEL speeds up data acquisition by reducing the amount of imaging needed, while ensuring that RNA molecules move straight down toward the surface, preserving single-cell resolution. We apply EEL on eight entire sagittal sections of the mouse brain and measure the expression patterns of up to 440 genes to reveal complex tissue organization. Moreover, EEL can be used to study challenging human samples by removing autofluorescent lipofuscin, enabling the spatial transcriptome of the human visual cortex to be visualized. We provide full hardware specifications, all protocols and complete software for instrument control, image processing, data analysis and visualization.

Multi-modal characterization and simulation of human epileptic circuitry.

Temporal lobe epilepsy is the fourth most common neurological disorder, with about 40% of patients not responding to pharmacological treatment. Increased cellular loss is linked to disease severity and pathological phenotypes such as heightened seizure propensity. While the hippocampus is the target of therapeutic interventions, the impact of the disease at the cellular level remains unclear. Here, we show that hippocampal granule cells change with disease progression as measured in living, resected hippocampal tissue excised from patients with epilepsy. We show that granule cells increase excitability and shorten response latency while also enlarging in cellular volume and spine density. Single-nucleus RNA sequencing combined with simulations ascribes the changes to three conductances: BK, Cav2.2, and Kir2.1. In a network model, we show that these changes related to disease progression bring the circuit into a more excitable state, while reversing them produces a less excitable, "early-disease-like" state.

Oligodendrocyte precursor cells ingest axons in the mouse neocortex.

Neurons in the developing brain undergo extensive structural refinement as nascent circuits adopt their mature form. This physical transformation of neurons is facilitated by the engulfment and degradation of axonal branches and synapses by surrounding glial cells, including microglia and astrocytes. However, the small size of phagocytic organelles and the complex, highly ramified morphology of glia have made it difficult to define the contribution of these and other glial cell types to this crucial process. Here, we used large-scale, serial section transmission electron microscopy (TEM) with computational volume segmentation to reconstruct the complete 3D morphologies of distinct glial types in the mouse visual cortex, providing unprecedented resolution of their morphology and composition. Unexpectedly, we discovered that the fine processes of oligodendrocyte precursor cells (OPCs), a population of abundant, highly dynamic glial progenitors, frequently surrounded small branches of axons. Numerous phagosomes and phagolysosomes (PLs) containing fragments of axons and vesicular structures were present inside their processes, suggesting that OPCs engage in axon pruning. Single-nucleus RNA sequencing from the developing mouse cortex revealed that OPCs express key phagocytic genes at this stage, as well as neuronal transcripts, consistent with active axon engulfment. Although microglia are thought to be responsible for the majority of synaptic pruning and structural refinement, PLs were ten times more abundant in OPCs than in microglia at this stage, and these structures were markedly less abundant in newly generated oligodendrocytes, suggesting that OPCs contribute substantially to the refinement of neuronal circuits during cortical development.

Sugar- and Artificially-Sweetened Beverages and Cancer Mortality in a Large U.S. Prospective Cohort.

BACKGROUND: Sugar-sweetened beverage (SSB) consumption may be associated with cancer mortality independent of, or indirectly through, established influences on increased body adiposity. METHODS: We examined the associations of SSBs and artificially-sweetened beverages (ASB) with mortality from all-cancers combined, obesity-related cancers combined, and 20 cancer types, among men and women in the Cancer Prevention Study-II (CPS-II) prospective cohort. In 1982, 934,777 cancer-free participants provided information on usual SSB and ASB consumption. Deaths were identified through 2016. Multivariable Cox proportional hazards regression models examined associations of beverage types with cancer mortality, without and with BMI adjustment. RESULTS: During follow-up, 135,093 CPS-II participants died from cancer. Consumption of ≥2 SSB drinks/day vs. never was not associated with all-cancer mortality, but was associated with increased risk of obesity-related cancers [HR, 1.05; 95% confidence intervals (CI), 1.01-1.08; Ptrend = 0.057], which became null after adjustment for BMI. SSBs were associated with increased mortality from colorectal (HR, 1.09; 95% CI, 1.02-1.17; Ptrend = 0.011), and kidney (HR, 1.17; 95% CI, 1.03-1.34; Ptrend = 0.056) cancers, which remained after BMI adjustment. A positive association of ASB consumption with obesity-related cancers (HR, 1.05; 95% CI, 1.01-1.08; Ptrend = 0.001) was null after controlling for BMI; however, an increased risk of pancreatic cancer was robust to BMI adjustment (HR, 1.11; 95% CI, 1.02-1.20; Ptrend < 0.008). CONCLUSIONS: SSB consumption was associated with higher mortality from certain cancers, partially mediated through obesity. Associations of ASB consumption and increased pancreatic cancer risk merit further study. IMPACT: Future research should consider the role of BMI in studies of sweetened beverages and cancer risk. These results should inform policy regarding sweetened beverage consumption.

Single nucleus multi-omics identifies human cortical cell regulatory genome diversity.

Single-cell technologies measure unique cellular signatures but are typically limited to a single modality. Computational approaches allow the fusion of diverse single-cell data types, but their efficacy is difficult to validate in the absence of authentic multi-omic measurements. To comprehensively assess the molecular phenotypes of single cells, we devised single-nucleus methylcytosine, chromatin accessibility, and transcriptome sequencing (snmCAT-seq) and applied it to postmortem human frontal cortex tissue. We developed a cross-validation approach using multi-modal information to validate fine-grained cell types and assessed the effectiveness of computational data fusion methods. Correlation analysis in individual cells revealed distinct relations between methylation and gene expression. Our integrative approach enabled joint analyses of the methylome, transcriptome, chromatin accessibility, and conformation for 63 human cortical cell types. We reconstructed regulatory lineages for cortical cell populations and found specific enrichment of genetic risk for neuropsychiatric traits, enabling the prediction of cell types that are associated with diseases.