PROJECT SUMMARY Osteoporosis is the most prevalent metabolic bone aging disease, mainly influenced by cells in the lineages of osteoblast (for bone formation) and osteoclast (for bone resorption). It is commonly characterized by low bone mineral density (BMD) with osteoporotic fractures (OFs) as the clinical end outcome. Both BMD and OFs have strong, but only share ~50% of their, (epi-)genetic determinations (some being sex-/ethnic-specific). The gut microbiome has a profound impact on human health, yet specific human genome and gut microbiome (epi-)genetic factors, and particularly their functional mechanisms for BMD and OF risk, are largely unknown. Our Overall Objective is to comprehensively identify/characterize in vivo, in humans (epi-)genes/gut bacterial species/environmental factors and their functional interactions/mechanisms/mediators for sex-/ethnic-general/specific osteoporosis risk. To do so, we will investigate the human genome and gut microbiome, for their impacts via cells and cell-cell interactions (CCIs) in both osteoclastogenic and osteoblastogenic lineages. Based on the solid progress made on our current U19 (AG055373) and leveraging the resources accumulated through other support (e.g., Louisiana Osteoporosis Study; LOS), we propose to fulfill the following Specific Aims: 1) Pioneer a comprehensive and highly innovative project to identify osteoporosis risk (epi-)genes in vivo simultaneously with human genome/gut microbiome (Proj 1), epigenome and single-cell transcriptome of osteoblast/osteoclast lineage cells (Proj 2), and metabolome (Proj 3), while assessing age/sex/ethnicity- specificity/generality of the identified molecular features (Proj 1-3). We will study BMD (both areal & volumetric–aBMD & vBMD), bone strength/quality (by finite element analyses – FEA and trabecular bone score - TBS), and OF. 2) Leverage the omics profiles to be generated in this U19 renewal and those accumulated in the ongoing U19 and LOS to impute (in Proj 2 & 3) to the full dataset all the omics features studied (in >5,600 subjects). 3) Efficiently/innovatively generate/integrate trans-omics interaction networks (Proj 1-3 & Core C), making causal inference to re-construct functional gene regulatory networks (GRN) within and across cells (for CCIs) and across our genome and gut microbiome. We will test these re-constructed GRNs to illuminate their functional significance and mechanisms for osteoporosis from the aspects of osteoblasto- and osteoclasto-genic lineages in vivo in humans. 4) Pursue in-depth follow-up functional studies (Proj 1 & 3) for specific molecular mechanisms and confirmation of the identified novel molecules, gut bacterial species, functions, and pathways. This U19 will include three Cores (Administrative, Clinical, Biostatistics/Bioinformatics) to efficiently support the necessary synergy among projects for the above analyses that all address age/sex/ethnicity-specific/general effects. This project promises to break new ground n...