Deciphering the role of extrachromosomal circular DNA in adipose stem cells from old and young donors

Abstract Background The functional impairment of adipose stem cells (ASCs) during aging limits their clinical transformation. Studies have shown that extrachromosomal circular DNAs (eccDNAs) are associated with tumor progression and cell aging, but the roles of eccDNAs in ASCs remain unknown. Method We conducted Circle sequencing (Circle-seq) to identify eccDNAs in ASCs isolated from young and old donors. The differentially expressed eccDNAs were calculated, annotated and validated via polymerase chain reaction. Results Thousands of eccDNAs were identified and comprehensively characterized. Most of them were GC-rich, < 1000 base pairs in size, and were enriched on chromosome 19 and 17 with a high density of Alu elements and genes, 2 kb upstream/downstream of genes and satellites. In total, 3025 eccDNAs were differentially expressed among the two ASC groups. Conjoint analysis of the Circle-seq results and previous RNA-seq results revealed that 73 eccDNAs and 55 genes exhibited the same differential expression between the two groups. KEGG and GO analyses revealed that genes encoding differentially expressed eccDNAs were enriched for cell adhesion, cellular senescence and TGF-β receptor signaling pathway. We also found that aged ASCs exhibited loss of eccDNAs, including CAMK2G (chr10: 75577899-75578176), TRABD2B (chr1: 48305638-48307008) and TRABD2B (chr1: 48305425 -48307091). Conclusion In this study, we elucidated the first eccDNA profile relating to ASCs and demonstrated that three eccDNAs are lost in aged ASCs, which may be potential biomarkers of stem cell aging and valuable targets for stem cell rejuvenation

Cysteamine Supplementation In Vitro Remarkably Promoted Rumen Fermentation Efficiency towards Propionate Production via Prevotella Enrichment and Enhancing Antioxidant Capacity

Cysteamine (CS) is a vital antioxidant product and nutritional regulator that improves the productive performance of animals. A 2 &times; 4 factorial in vitro experiment was performed to determine the effect of the CS supplementation levels of 0, 20, 40, and 60 mg/g, based on substrate weight, on the ruminal fermentation, antioxidant capacity, and microorganisms of a high-forage substrate (HF, forage:corn meal = 7:3) in the Statistical Analysis System Institute. After 48 h of incubation, the in vitro dry matter disappearance and gas production in the LF group were higher when compared with a low-forage substrate (LF, forge hay:corn meal = 3:7), which was analyzed via the use of the MIXED procedure of the HF group, and these increased linearly with the increasing CS supplementation (p &lt; 0.01). With regard to rumen fermentation, the pH and acetate were lower in the LF group compared to the HF group (p &lt; 0.01). However, the ammonia N, microbial crude protein, total volatile fatty acids (VFA), and propionate in the LF group were greater than those in the HF group (p &lt; 0.05). With the CS supplementation increasing, the pH, ammonia N, acetate, and A:P decreased linearly, while the microbial crude protein, total VFA, and propionate increased linearly (p &lt; 0.01). Greater antioxidant capacity was observed in the LF group, and the increasing CS supplementation linearly increased the superoxide dismutase, catalase, glutathione peroxidase, total antioxidant capacity, glutathione, and glutathione reductase, while it decreased the malondialdehyde (p &lt; 0.05). No difference occurred in the ruminal bacteria alpha diversity with the increasing CS supplementation, but it was higher in the LF group than in the HF group (p &lt; 0.01). Based on the rumen bacterial community, a higher proportion of Bacteroidota, instead of Firmicutes, was in the LF group than in the HF group. Furthermore, increasing the CS supplementation linearly increased the relative abundance of Prevotella, norank_f_F082, and Prevotellaceae_UCG-001 under the two substrates (p &lt; 0.05). Prevotella, norank_f_F082, and Prevotellaceae_UCG-001 were positively correlated with gas production, rumen fermentation, and antioxidant capacity in a Spearman correlation analysis (r &gt; 0.31, p &lt; 0.05). Overall, a CS supplementation of not less than 20 mg/g based on substrate weight enhanced the rumen fermentation and rumen antioxidant capacity of the fermentation system, and it guided the rumen fermentation towards glucogenic propionate by enriching the Prevotella in Bacteroidetes

Estimation of Energy Value and Digestibility and Prediction Equations for Sheep Fed with Diets Containing <i>Leymus chinensis</i> Hay

The objective of this study was to investigate the feeding value of sheepgrass, including its chemical composition, dry matter intake, nutrient digestibility, and available energy, as well as the prediction equations of dry matter intake (DMI), neutral detergent fiber digestibility (NDFD), dry matter digestibility (DMD), digestible energy (DE), and metabolizable energy (ME). Two independent experiments based on a completely randomized experimental design were performed to evaluate the feeding value. The results showed that there were significant relationships between chemical composition and DMI, digestibility, and available energy. The best-fit equations were as follows: DMI (g/d·W0.75) = 121.75 + 0.06CP (%) − 0.24EE (%) − 0.10ADF (%) − 0.60NDF (%) − 0.15OM (%) (R2 = 0.85, p 2 = 0.83, p 2 = 0.67, p 2 = 0.91, p 2 = 0.98, p < 0.01). This study found the energy value of sheepgrass to be 11 MJ/kg, which is similar to that of millet grass silage. The NDF was the main component that affected DMI and digestibility. Using a hay replacement ratio of 28.5% to determine the forage value of sheepgrass allowed accurate prediction equations to be established. The NDF demonstrated the strongest correlation with DMI, NDFD, OMD, DE, and ME. DE was estimated to be the best single predictor of ME

Inhibitory Effect Mediated by Deoxynivalenol on Rumen Fermentation under High-Forage Substrate

Deoxynivalenol (DON) is a type B trichothecene mycotoxin produced by Fusarium fungi. To investigate its ruminal degradability and its effect on rumen fermentation, a 2 &times; 5 factorial experiment was conducted in vitro with two feed substrates with different forage levels (high forage (HF), forage-to-concentrate = 4:1; low forage (LF), forage-to-concentrate = 1:4) and five DON additions per substrate (0, 5, 10, 15, and 20 mg/kg of dry matter). After 48 h incubation, the DON degradability in the HF group was higher than in the LF group (p &lt; 0.01), and it decreased along with the increase in DON concentrations (p &lt; 0.01), which varied from 57.18% to 29.01% at 48 h. In addition, the gas production rate, total VFA production and microbial crude protein decreased linearly against the increase in DON additions (p &lt; 0.05). Meanwhile, the proportion of CH4 in the fermentation gas end-products increased linearly, especially in the HF group (p &lt; 0.01). In brief, rumen microorganisms presented 29&ndash;57% of the DON degradation ability and were particularly significant under a high-forage substrate. Along with the increasing DON addition, the toxin degradability decreased, showing a dose-dependent response. However, DON inhibited rumen fermentation and increased methane production when it exceeded 5 mg/kg of dry matter

Microvesicles from human adipose stem cells promote wound healing by optimizing cellular functions via AKT and ERK signaling pathways

Abstract Background Human adipose stem cells (ASCs) have emerged as a promising treatment paradigm for skin wounds. Recent works demonstrate that the therapeutic effect of stem cells is partially mediated by extracellular vesicles, which comprise exosomes and microvesicles. In this study, we investigate the regenerative effects of isolated microvesicles from ASCs and evaluate the mechanisms how ASC microvesicles promote wound healing. Methods Adipose stem cell-derived microvesicles (ASC-MVs) were isolated by differential ultracentrifugation, stained by PKH26, and characterized by electron microscopy and dynamic light scattering (DLS). We examined ASC-MV effects on proliferation, migration, and angiogenesis of keratinocytes, fibroblasts, and endothelial cells both in vitro and in vivo. Next, we explored the underlying mechanisms by gene expression analysis and the activation levels of AKT and ERK signaling pathways in all three kinds of cells after ASC-MV stimulation. We then assessed the effect of ASC-MVs on collagen deposition, neovascularization, and re-epithelialization in an in vivo skin injury model. Results ASC-MVs could be readily internalized by human umbilical vein endothelial cells (HUVECs), HaCAT, and fibroblasts and significantly promoted the proliferation, migration, and angiogenesis of these cells both in vitro and in vivo. The gene expression of proliferative markers (cyclin D1, cyclin D2, cyclin A1, cyclin A2) and growth factors (VEGFA, PDGFA, EGF, FGF2) was significantly upregulated after ASC-MV treatment. Importantly, ASC-MVs stimulated the activation of AKT and ERK signaling pathways in those cells. The local injection of ASC-MVs at wound sites significantly increased the re-epithelialization, collagen deposition, and neovascularization and led to accelerated wound closure. Conclusions Our data suggest that ASC-MVs can stimulate HUVEC, HaCAT, and fibroblast functions. ASC-MV therapy significantly accelerates wound healing, and the benefits of ASC-MVs may due to the involvement of AKT and ERK signaling pathways. This illustrates the therapeutic potential of ASC-MVs which may become a novel treatment paradigm for cutaneous wound healing

LncRNA FTO-IT1 promotes glycolysis and progression of hepatocellular carcinoma through modulating FTO-mediated N6-methyladenosine modification on GLUT1 and PKM2

Abstract Background Long non-coding RNAs (LncRNAs) have been extensively studied to play essential roles in tumor progression. However, more in-depth studies are waiting to be solved on how lncRNAs regulate the progression of hepatocellular carcinoma (HCC). Methods Different expression levels of lncRNAs in HCC cells were compared by analysis of Gene Expression Omnibus and The Cancer Genome Atlas databases. The effects of lncRNA FTO Intronic Transcript 1 (FTO-IT1) on HCC cells were assessed by gain- and loss-of-function experiments. Colony formation assay, Edu assay, glucose uptake and lactic acid production assay were performed to evaluate the regulation of proliferation and glycolysis of HCC cells by FTO-IT1. The binding between protein interleukin enhancer binding factor 2/3 (ILF2/ILF3) and FTO-IT1 was determined by RNA pull-down, mass spectroscopy and RNA immunoprecipitation experiments. RNA stability assay, quantitative reverse transcription PCR and Western blot were employed to determine the regulatory mechanisms of FTO-IT1 on fat mass and obesity-associated (FTO). Methylated RNA immunoprecipitation assay was used to assessed the regulation of key enzymes of glycolysis by FTO. The role of FTO-IT1/FTO in vivo was confirmed via xenograft tumor model. Results LncRNA FTO-IT1, an intronic region transcript of FTO gene, was highly expressed in HCC and associated with poor prognosis of patients with HCC. FTO-IT1 was related to proliferation and glycolysis of HCC cells, and contributed to the malignant progression of HCC by promoting glycolysis. Mechanistically, FTO-IT1 induced stabilization of FTO mRNA by recruiting ILF2/ILF3 protein complex to 3’UTR of FTO mRNA. As a demethylase for N 6-methyladenosine (m6A), FTO decreased m6A modification on mRNAs of glycolysis associated genes including GLUT1, PKM2, and c-Myc which alleviated the YTH N6-methyladenosine RNA binding protein 2 (YTHDF2)-mediated mRNA degradation. Therefore, the upregulated expression of FTO-IT1 leaded to overexpression of GLUT1, PKM2, and c-Myc by which enhanced glycolysis of HCC. Meanwhile, it was found that c-Myc transcriptional regulated expression of FTO-IT1 by binding to its promoter area under hypo-glucose condition, forming a reciprocal loop between c-Myc and FTO-IT1. Conclusions This study identified an important role of the FTO-IT1/FTO axis mediated m6A modification of glycolytic genes contributed to glycolysis and tumorigenesis of HCC, and FTO-IT1 might be served as a new therapeutic target for HCC

Multifunctional ADM hydrogel containing endothelial cell-exosomes for diabetic wound healing

Non-healing wound, with limited treatment options, remains a prevalent complication of diabetes mellitus. The underlying causes wherein include oxidative stress injury, bacterial infection, cellular dysfunction, and persistent inflammation. Acellular Dermal Matrix (ADM), a wound dressing composed of natural extracellular matrix and abundant bioactive factors, has been successfully developed to treat various wounds, including burns and diabetic ulcers. Protocatechualdehyde (PA) &amp; trivalent iron ion (Fe3+) complex (Fe3+@PA) exhibits potential antioxidant and antibacterial properties. In this study, we developed a dual hydrogel network by combining Fe3+@PA complex-modified ADM with light-cured gelatin (GelMA), supplemented with exosomes derived from human umbilical vein endothelial cells (HUVEC-Exos), to create an ADM composite hydrogel system (ADM-Fe3+@PA-Exos/GelMA) with antioxidant, antibacterial, and cell-promoting functions for diabetic wound treatment. Through in vitro experiments, we investigated the biosafety, antioxidant and antibacterial properties of ADM composite hydrogel. Furthermore, we examined the protective effects of ADM composite hydrogel on diabetic wound. The above experiments collectively demonstrate that our ADM-Fe3+@PA-Exos/GelMA hydrogel promotes diabetic wound healing by eliminating bacterial infection, reduced the reactive oxygen species (ROS) levels, protecting cells against oxidative stress damage, promotingcollagen deposition and angiogenesis, which provides a promising strategy to optimize ADM for diabetic wound treatment

Exosomes from Adipose Stem Cells Promote Diabetic Wound Healing through the eHSP90/LRP1/AKT Axis

Oxidative damage is a critical cause of diabetic wounds. Exosomes from various stem cells could promote wound repair. Here, we investigated the potential mechanism by which exosomes from adipose-derived stem cells (ADSC-EXOs) promote diabetic wound healing through the modulation of oxidative stress. We found that ADSC-EXOs could promote proliferation, migration, and angiogenesis in keratinocytes, fibroblasts, and endothelial cells. Furthermore, ADSC-EXOs reduced the reactive oxygen species (ROS) levels in these cells and protected them against hypoxic and oxidative stress damage. Finally, the local injection of ADSC-EXOs at wound sites significantly increased collagen deposition and neovascularization while reducing ROS levels and cell death; thus, it led to accelerated diabetic wound closure. The mechanism underlying ADSC-EXO functions involved heat-shock protein 90 (HSP90) expressed on the cell surface; these functions could be inhibited by an anti-HSP90 antibody. Exosomal HSP90 could bind to the low-density lipoprotein receptor-related protein 1 (LRP1) receptor on the recipient cell membrane, leading to activation of the downstream AKT signaling pathway. Knockdown of LRP1 and inhibition of the AKT signaling pathway by LY294002 in fibroblasts was sufficient to impair the beneficial effect of ADSC-EXOs. In summary, ADSC-EXOs significantly accelerated diabetic wound closure through an exosomal HSP90/LRP1/AKT signaling pathway