Background:IGF2 is an imprinted growth factor essential for fetal development. A single nucleotide variation at a conserved ZBED6 binding site within Igf2 intron 3 induces post-natal IGF2 expression and resulted in increased lean mass in multiple species. While the role of IGF2 in muscle growth is established, its impact on the adult skeleton remains incompletely defined. Methods:We studied 13-week-old male and female Igf2 G/A knock-in mice carrying the pig-derived G ➔ A substitution that prevents ZBED6 binding. We quantified Igf2 expression in bone and visceral tissues, measured body and organ size, assessed femoral geometry and microarchitecture by micro-CT, examined growth plate morphology, evaluated bone turnover markers (P1NP, CTX1), and tested whole-bone mechanical properties. Results:Igf2 G/A mice exhibited increased size, body weight, length, and kidney mass, while liver mass trended higher. Igf2 mRNA levels were elevated in kidney, liver, and bone tissues. Femurs demonstrated greater length and larger periosteal perimeter, with increased cortical area in both sexes but no changes in cortical thickness or bone mineral density. Trabecular parameters remained unchanged in males but improved in females, characterized by higher BV/TV, increased trabecular thickness and number, and reduced spacing. Growth plate metrics were predominantly unaffected, except for a modest increase in mean thickness observed in Igf2 G/A females. Serum P1NP and CTX1 levels showed no genotype-dependent differences. Mechanical testing revealed reduced elastic modulus in both sexes of the Igf2 G/A compared to wildtype and lower ultimate stress in females, while other mechanical properties remained unchanged. Circulating IGF1 and IGFBP3 levels as well as bone expression of Igf1/Igf1r/Igfbp3 were unchanged, suggesting small if any impact of the greater GH-IGF axis on the phenotype. Conclusions:Post-natal IGF2 expression alters the adult murine skeleton by augmenting longitudinal growth and cortical accrual and inducing female-specific trabecular gains, with selective decrements in material properties. These data establish IGF2 as a regulator of postnatal bone architecture and mechanics and extend the functional scope of the conserved ZBED6-IGF2 regulatory axis to the skeleton.

Denosumab is a monoclonal anti-RANKL antibody that inhibits bone resorption, increases bone mass, and reduces fracture risk. Denosumab discontinuation causes an extensive wave of rebound resorption, but the cellular mechanisms remain poorly characterized. We utilized in situ hybridization (ISH) as a direct approach to identify the cells that activate osteoclastogenesis through the RANKL/OPG pathway. ISH was performed across species, skeletal sites, and following recombinant OPG (OPG:Fc) and parathyroid hormone 1-34 (PTH) treatment of mice. OPG:Fc treatment in mice induced an increased expression of RANKL mRNA mainly in trabecular, but not endocortical bone surface cells. Additionally, a decreased expression of OPG mRNA was detected in bone surface cells and osteocytes of both compartments. A similar but more pronounced effect on RANKL and OPG expression was seen one hour after PTH treatment. These findings suggest that bone surface cells and osteocytes conjointly regulate the activation of osteoclastogenesis, and that OPG:Fc treatment induces a local accumulation of osteoclastogenic activation sites, ready to recruit and activate osteoclasts upon treatment discontinuation. Analysis of publicly available single-cell RNA sequencing (scRNAseq) data from murine bone marrow stromal cells revealed that Tnfsf11+ cells expressed high levels of Mmp13, Limch1, and Wif1, confirming their osteoprogenitor status. ISH confirmed co-expression of Mmp13 and Tnfsf11 in bone surface cells of both vehicle- and OPG:Fc-treated mice. Under physiological conditions of human/mouse bone, RANKL is expressed mainly by osteoprogenitors proximate to the osteoclasts, while OPG is expressed mainly by osteocytes and bone-forming osteoblasts.

Emerging evidence suggests a significant role of gut microbiome in bone health. Aging is well recognized as a crucial factor influencing the gut microbiome. In this study, we investigated whether age-dependent microbial change contributes to age-related bone loss in CB6F1 mice. The bone phenotype of 24-month-old germ-free (GF) mice was indistinguishable compared to their littermates colonized by fecal transplant at 1-month-old. Moreover, bone loss from 3 to 24-month-old was comparable between GF and specific pathogen-free (SPF) mice. Thus, GF mice were not protected from age-related bone loss. 16S rRNA gene sequencing of fecal samples from 3-month and 24-month-old SPF males indicated an age-dependent microbial shift with an alteration in energy and nutrient metabolism potential. An integrative analysis of 16S predicted metagenome function and LC-MS fecal metabolome revealed an enrichment of protein and amino acid biosynthesis pathways in aged mice. Microbial S-adenosyl methionine metabolism was increased in the aged mice, which has previously been associated with the host aging process. Collectively, aging caused microbial taxonomic and functional alteration in mice. To demonstrate the functional importance of young and old microbiome to bone, we colonized GF mice with fecal microbiome from 3-month or 24-month-old SPF donor mice for 1 and 8 months. The effect of microbial colonization on bone phenotypes was independent of the microbiome donors’ age. In conclusion, our study indicates age-related bone loss occurs independent of gut microbiome.

This article has been corrected: The authors found an error in Figure 5G, which depicts transwell migration assays assessing the effects of miR-15a-3p inhibition on HUVEC cells. The central panel, illustrating migration of HUVECs treated with exosomes derived from DFU individuals (Dia-Exos), was replaced with the appropriate image from the original set of experiments. This inadvertent error does not in any way compromise the validity or integrity of the study's findings. The authors take full responsibility for this oversight and sincerely apologize for any inconvenience caused.

Human milk guides the structure and function of microbial commensal communities that colonize the nursing infant gut. Indigestible molecules dissolved in human milk establish a microbiome often dominated by bifidobacteria capable of utilizing these substrates. Interestingly, urea accounts for ~15% of total human milk nitrogen, representing a potential reservoir for microbiota that may be salvaged for critical metabolic operations during lactation and neonatal development. Accordingly, B. infantis strains are competent for urea nitrogen utilization, constituting a previously hypothetical phenotype in commensal bacteria hosted by humans. Urease gene expression, downstream nitrogen metabolic pathways, and enzymatic activity are induced during urea utilization to yield elevated ammonia concentrations. Moreover, biosynthetic networks relevant to infant nutrition and development are transcriptionally responsive to urea utilization including branched chain and other essential amino acids. Importantly, isotopically labeled urea nitrogen is broadly distributed throughout the expressed B. infantis proteome. This incisively demonstrates that the previously inaccessible urea nitrogen is incorporated into microbial products available for infant host utilization. In aggregate, B. infantis possesses the requisite phenotypic foundation to participate in human milk urea nitrogen recycling within its infant host and thus may be a key contributor to nitrogen homeostasis early in life.

Bifidobacterium longum subsp. infantis (B. infantis) utilizes oligosaccharides secreted in human milk as a carbohydrate source. These human milk oligosaccharides (HMOs) integrate the nitrogenous residue N-acetylglucosamine (NAG), although HMO nitrogen utilization has not been described to date. Herein, we characterize the B. infantis nitrogen utilization phenotype on two NAG-containing HMO species, LNT and LNnT. This was characterized through in vitro growth kinetics, incorporation of isotopically labeled NAG nitrogen into the proteome, as well as modulation of intracellular 2-oxoglutarate levels while utilizing HMO nitrogen. Further support is provided by comparative transcriptomics and proteomics that identified global regulatory networks deployed during HMO nitrogen utilization. The aggregate data demonstrate that B. infantis strains utilize HMO nitrogen with the potential to significantly impact fundamental and clinical studies, as well as enable applications.

Compositional and functional alterations to the gut microbiota during aging are hypothesized to potentially impact our health. Thus, determining aging-specific gut microbiome alterations is critical for developing microbiome-based strategies to improve health and promote longevity in the elderly. In this study, we performed a meta-analysis of publicly available 16S rRNA gene sequencing data from studies investigating the effect of aging on the gut microbiome in mice. Aging reproducibly increased gut microbial alpha diversity and shifted the microbial community structure in mice. We applied the bioinformatic tool PICRUSt2 to predict microbial metagenome function and established a random forest classifier to differentiate between microbial communities from young and old hosts and to identify aging-specific metabolic features. In independent validation data sets, this classifier achieved an area under the receiver operating characteristic curve (AUC) of 0.75 to 0.97 in differentiating microbiomes from young and old hosts. We found that 50% of the most important predicted aging-specific metabolic features were involved in carbohydrate metabolism. Furthermore, fecal short-chain fatty acid (SCFA) concentrations were significantly decreased in old mice, and the expression of the SCFA receptor Gpr41 in the colon was significantly correlated with the relative abundances of gut microbes and microbial carbohydrate metabolic pathways. In conclusion, this study identified aging-specific alterations in the composition and function of the gut microbiome and revealed a potential relationship between aging, microbial carbohydrate metabolism, fecal SCFA, and colonic Gpr41 expression. IMPORTANCE Aging-associated microbial alteration is hypothesized to play an important role in host health and longevity. However, investigations regarding specific gut microbes or microbial functional alterations associated with aging have had inconsistent results. We performed a meta-analysis across 5 independent studies to investigate the effect of aging on the gut microbiome in mice. Our analysis revealed that aging increased gut microbial alpha diversity and shifted the microbial community structure. To determine if we could reliably differentiate the gut microbiomes from young and old hosts, we established a random forest classifier based on predicted metagenome function and validated its performance against independent data sets. Alterations in microbial carbohydrate metabolism and decreased fecal short-chain fatty acid (SCFA) concentrations were key features of aging and correlated with host colonic expression of the SCFA receptor Gpr41. This study advances our understanding of the impact of aging on the gut microbiome and proposes a hypothesis that alterations in gut microbiota-derived SCFA-host GPR41 signaling are a feature of aging.

Still's disease is a severe inflammatory syndrome characterized by fever, skin rash and arthritis affecting children and adults. Patients with Still's disease may also develop macrophage activation syndrome, a potentially fatal complication of immune dysregulation resulting in cytokine storm. Here we show that mTORC1 (mechanistic target of rapamycin complex 1) underpins the pathology of Still's disease and macrophage activation syndrome. Single-cell RNA sequencing in a murine model of Still's disease shows preferential activation of mTORC1 in monocytes; both mTOR inhibition and monocyte depletion attenuate disease severity. Transcriptomic data from patients with Still's disease suggest decreased expression of the mTORC1 inhibitors TSC1/TSC2 and an mTORC1 gene signature that strongly correlates with disease activity and treatment response. Unrestricted activation of mTORC1 by Tsc2 deletion in mice is sufficient to trigger a Still's disease-like syndrome, including both inflammatory arthritis and macrophage activation syndrome with hemophagocytosis, a cellular manifestation that is reproduced in human monocytes by CRISPR/Cas-mediated deletion of TSC2. Consistent with this observation, hemophagocytic histiocytes from patients with macrophage activation syndrome display prominent mTORC1 activity. Our study suggests a mechanistic link of mTORC1 to inflammation that connects the pathogenesis of Still's disease and macrophage activation syndrome.

Biopolymer microgels were designed to encapsulate and protect probiotics during storage and passage through the upper human gastrointestinal tract (GIT), but then release them in the colon. An anaerobic probiotic strain (Bifidobacterium pseudocatenulatum G7; BPG7) was encapsulated within calcium alginate microgels. In some cases, either colloidal antacid (CaCO3) or colloidal antacid and nanoemulsion (nE) lipid droplets were co-encapsulated with the probiotics. These additives were used to control the porosity and/or internal pH of the microgels. Initially, the viability of free and encapsulated probiotics was evaluated when they were stored at 4 degrees C for 28 days. The free probiotics rapidly degraded during storage. Encapsulation greatly improved the stability of the probiotics, with the effectiveness depending on the formulation: antacid-microgels > control microgels > antacid-nE-microgels. These effects were mainly attributed to the ability of the colloidal CaCO3 particles to inhibit molecular diffusion processes. The ability of the microgels to protect the probiotics under simulated gastrointestinal tract (GIT) conditions was then assessed. The free probiotics were completely inactivated when exposed to gastric and small intestine conditions. In contrast, the antacid- and antacid-nE-microgels were highly effective at improving the viability of the probiotics during passage through the upper GIT. Moreover, these microgels were fermented under simulated colonic conditions, thereby releasing the probiotics. These results suggest that antacid-loaded calcium alginate microgels can be used for the encapsulation, protection, and colonic delivery of probiotics. Nevertheless, further studies are required using in vivo models.

Beyond proteins, milk contains an array of smaller nitrogen (N)-containing components generally referred to as nonprotein nitrogen (NPN). These include milk protein–derived peptides, peptide hormones, amino acids (AAs), N-containing oligosaccharides, N-containing monosaccharides, nucleic acids (DNA and RNA), free nucleotides, and small molecules, including urea, uric acid, ammonia, creatine, creatinine, carnitine, and polyamines. Human milk (HM) is unique in that NPN comprises up to 25% of the total N, compared to 5% in cow milk. Many of these milk components have functions in infants; for example, urea N can be used to synthesize nonessential AAs or converted by the gut microbiota as an N source. In addition, some milk protein–derived peptides have antimicrobial, antihypertensive, and other activities. This chapter reviews components of the NPN fraction of HM, their physiological relevance, typical amounts in milk, factors that affect their concentrations, the analytical techniques used to determine their concentrations, and areas for future research.

In this study, we used bioinformatics tools, and experiments with patient tissues and human mesenchymal stem cells (hMSCs) to identify differentially regulated genes (DEGs) and microRNAs (miRNAs) that promote postmenopausal osteoporosis. By analyzing the GSE56815 dataset from the NCBI GEO database, we identified 638 DEGs, including 371 upregulated and 267 downregulated genes, in postmenopausal women with low bone density. Enrichment and protein-protein interaction network analyses showed that TP53, RPS27A, and VEGFA were the top three hub genes with the highest degree of betweenness and closeness centrality. TargetScanHuman and DIANA software analyses and dual luciferase reporter assays confirmed that miR-16a-5p directly targets the 3'UTR of VEGFA. Postmenopausal patients with osteoporosis showed higher miR-16-5p and lower VEGFA levels than those without osteoporosis (n=10 each). VEGFA levels were higher in miR-16-5p knockdown hMSCs and were reduced in miR-16-5p-overexpressing hMSCs. mRNA expression of osteogenic markers, ALP, OCN, and RUNX2, as well as calcium deposition based on Alizarin red staining, correlated inversely with miR-16-5p levels and correlated positively with VEGFA levels. These findings suggest that miR-16-5p suppresses osteogenesis by inhibiting VEGFA expression and is a promising target for postmenopausal osteoporosis therapy.

Osteoporosis is a metabolic disease affecting 40% of postmenopausal women. It is characterized by decreased bone mass per unit volume and increased risk of fracture. We investigated the molecular mechanism underlying osteoporosis by identifying the genes involved in its development. Osteoporosis-related genes were identified by analyzing RNA microarray data in the GEO database to detect genes differentially expressed in osteoporotic and healthy individuals. Enrichment and protein interaction analyses carried out to identify the hub genes among the deferentially expressed genes revealed TP53, MAPK1, CASP3, CTNNB1, CCND1, NOTCH1, CDK1, IGF1, ERBB2, CYCS to be the top 10 hub genes. In addition, p53 had the highest degree score in the protein-protein interaction network. In vivo and in vitro experiments showed that TP53 gene expression and serum p53 levels were upregulated in osteoporotic patients and a mouse osteoporosis model. The elevated p53 levels were associated with decreases in bone mass, which could be partially reversed by knocking down p53. These findings suggest p53 may play a central role in the development of osteoporosis.

Although osteoporosis is one of the most common chronic age-related diseases, there is currently no gold standard for treatment. Evidence suggests resveratrol, a natural polyphenolic compound, may be helpful in the treatment of osteoporosis and other diseases. However, the molecular mechanisms underlying the anti-osteoporotic effects of resveratrol remain largely unknown. In the present study, KEGG pathway enrichment analysis of resveratrol-targeted genes identified 33 associated pathways, 12 of which were also involved in osteoporosis. In particular, the MDM2/p53 signaling pathway was identified as a potential key pathway among the shared pathways. In vitro experiments indicated that MDM2-mediated p53 degradation induced osteoblast differentiation, and resveratrol could partially reverse p53-dependent inhibition of osteogenic differentiation. These findings suggest resveratrol may alleviate osteoporosis at least in part by modulating the MDM2/p53 signaling pathway.

BACKGROUND Osteoarthritis (OA) is a common disorder in the elderly. OA influences the daily life of patients and has become a worldwide health problem. It is still unclear whether the pathogenesis mechanism is different between males and females. This study investigated the differentially expressed genes (DEGs) and explored the different signaling pathways of OA between males and females. MATERIAL AND METHODS Data sets of GSE55457, GSE55584, and GSE12021 were retrieved from Gene Expression Omnibus to conduct DEGs analysis. Enrichment analysis of Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology term was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics tool. The protein interaction network was constructed in Cytoscape 3.7.2. qRT-PCR was then performed to validate the expression of hub genes in OA patients and healthy people. RESULTS In total, 4 co-upregulated and 10 co-downregulated genes were identified. We found that enriched pathways were different between males and females. BCL2L1, EEF1A1, EEF2, HNRNPD, and PABPN1 were considered as hub genes in OA pathogenesis in males, while EEF2, EEF1A1, RPL37A, FN1 were considered as hub genes in OA pathogenesis in females. Consistent with the bioinformatics analysis, the qRT-PCR analysis also showed that the gene expression of BCL2L1, HNRNPD, and PABPN1 was significantly lower in male OA patients. In contrast, EEF2, EEF1A1, and RPL37A were significantly lower in female OA patients. CONCLUSIONS The DEGs identified may be involved in different OA disease progression mechanisms between males and females, and they are considered as treatment targets or prognosis markers for males and females. The pathogenesis mechanism is sex-dependent.

Background: As a common metabolic disorder, osteoporosis is characterized by decreasing bone mass density and increased possibility of fragility fracture. The incidence of senile osteoporosis increases year by year. There is no gold standard of treatment for osteoporosis. Tomatidine is the aglycone derivative of tomatine, having the ability to treat various diseases, including osteoporosis. However, the mechanism by which tomatidine improves osteoporosis has not been fully elucidated. Tomatidine is a potential and promising drug for osteoporosis. Material/Methods: In this study, the KEGG pathways that tomatidine-targeted genes enriched in were obtained using bioinformatics methods. The KEGG pathways involved in osteoporosis that were also associated with tomatidine-targeted genes were selected. After analysis of these pathways, essential genes that may be involved in this biological process were identified and validated experimentally. Results: We found 110 osteoporosis related KEGG pathways and 76 tomatidine-targeted genes-related KEGG pathways were obtained. 39 shared KEGG pathways were identified. The top 5 pathways were: pathway of chronic myeloid leukemia, pathway of B cell receptor signaling, pathway in cancer, bladder cancer pathway, and progesterone-mediated oocyte maturation pathway. MAPK1, MAP2K1, MAPK3, RAF1 were involved in all the 5 pathways. The p53 signaling pathway and the MAPK signaling pathway were involved in the 5 KEGG pathways. In vitro experiments showed that downregulating p53 expression could be potentially protective for osteoporosis. Conclusions: Tomatidine can improve osteoporosis, and one of the mechanisms of its action is achieved by modulating p53. Tomatidine may be a promising drug for osteoporosis.

Osteoporosis is a common metabolic bone disease that affects about 40% of postmenopausal women. Treatment options for osteoporosis are limited, however. Icariin is an herbal substance that has been shown to improve bone mass, but the mechanisms are largely unknown. Using bioinformatics analysis, we have identified the hub genes and KEGG pathways shared between icariin-targeted genes and osteoporosis. The top five shared KEGG pathways were the Toll-like receptor signaling pathway, adipocytokine pathway, neurotrophin signaling pathway, NOD-like receptor signaling, and B cell receptor signaling pathway; the hub genes were RELA, NFKBIA, and IKBKB, belonging to the NF-κB family. The identified icariin-targeted genes are involved in inflammation, insulin resistance, apoptosis, and immune responses, and regulate the PI3K-Akt, NF-κB, MAPK, and JNK signaling pathways. Our in vitro data show that icariin inhibits apoptosis in human mesenchymal stem cells by suppressing JNK/c-Jun signaling pathway. Together, these findings indicate that icariin exerts its anti-osteoporotic function by inhibiting JNK/c-Jun signaling pathway, and suggest that icariin may be a promising treatment option for osteoporosis.

Diabetic foot ulcers are a common complication of diabetes, and are usually incurable in the clinic. Exosomes (carriers that transfer endogenous molecules) from diabetic patients' blood have been demonstrated to suppress diabetic wound repair. In this study, we investigated the effects of circulating exosomal microRNA-15a-3p (miR-15a-3p) on diabetic wound repair. Exosomes were extracted from diabetic patients' blood, and were found to inhibit diabetic wound repair in vitro and in vivo. miR-15a-3p was upregulated in diabetic exosomes, and impaired wound healing. When miR-15a-3p was knocked down in diabetic exosomes, their negative effects were partially reversed both in vitro and in vivo. NADPH oxidase 5 (NOX5) was identified as a potential target of miR-15a-3p, and the inhibition of NOX5 reduced the release of reactive oxygen species, thereby impairing the functionality of human umbilical vein endothelial cells. In summary, inhibition of circulating exosomal miR-15a-3p accelerated diabetic wound repair by activating NOX5, providing a novel therapeutic target for diabetic foot ulcer therapy.