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Improving revascularization is one of the major measures in fracture treatment. Moderate local inflammation triggers angiogenesis, whereas systemic inflammation hampers angiogenesis. Previous studies showed that Akkermansia muciniphila, a gut probiotic, ameliorates systemic inflammation by tightening the intestinal barrier. In this study, fractured mice intragastrically administrated with A. muciniphila were found to display better fracture healing than mice treated with vehicle. Notably, more preosteclasts positive for platelet-derived growth factor-BB (PDGF-BB) were induced by A. muciniphila at 2 weeks post fracture, coinciding with increased formation of type H vessels, a specific vessel subtype that couples angiogenesis and osteogenesis, and can be stimulated by PDGF-BB. Moreover, A. muciniphila treatment significantly reduced gut permeability and inflammation at the early stage. Dextran sulfate sodium (DSS) was used to disrupt the gut barrier to determine its role in fracture healing and whether A. muciniphila still can stimulate bone fracture healing. As expected, A. muciniphila evidently improved gut barrier, reduced inflammation and restored the impaired bone healing and angiogenesis in DSS-treated mice. Our results suggest that A. muciniphila reduces intestinal permeability and alleviates inflammation, which probably induces more PDGF-BB+ preosteoclasts and type H vessel formation in callus, thereby promoting fracture healing. This study provides the evidence for the involvement of type H vessels in fracture healing and suggests the potential of A. muciniphila as a promising strategy for bone healing. This article has an associated First Person interview with the first author of the paper.

Ginsenoside Compound K (GCK) is the main metabolite of natural protopanaxadiol ginsenosides with diverse pharmacological effects. Gut microbiota contributes to the biotransformation of GCK, while the effect of gut microbiota on the pharmacokinetics of GCK in vivo remains unclear. To illustrate the role of gut microbiota in GCK metabolism in vivo , a systematic investigation of the pharmacokinetics of GCK in specific pathogen free (SPF) and pseudo-germ-free (pseudo-GF) rats were conducted. Pseudo-GF rats were treated with non-absorbable antibiotics. Liquid chromatography tandem mass spectrometry (LC–MS/MS) was validated for the quantification of GCK in rat plasma. Compared with SPF rats, the plasma concentration of GCK significantly increased after the gut microbiota depleted. The results showed that GCK absorption slowed down, T max delayed by 3.5 h, AUC 0-11 increased by 1.3 times, CL z/F decreased by 0.6 times in pseudo-GF rats, and C max was 1.6 times higher than that of normal rats. The data indicated that gut microbiota played an important role in the pharmacokinetics of GCK in vivo .

Although the interaction between tumor cells and tumor‐associated macrophages (TAMs) has been widely studied; however, the mechanism of osteosarcoma cells in regulating the polarization of TAMs remains unclear. Exosomes from SAOS‐2 cells were isolated and validated by electron microscopy and Western blot. Transfection of indicated plasmids was applied to modify the expressions of miR‐181a‐5p and RAR‐related orphan receptor alpha (RORA). Flow cytometric analysis was carried out to analyze M1/M2 macrophage polarization. Quantitative real‐time PCR was performed to determine the levels of miR‐181a‐5p and RORA. Protein levels of CD63, CD81, RORA, CD163, CD206, IL‐10, CXCL10, and IL‐1β were evaluated by Western blot. The direct interaction of miR‐181a‐5p and RORA was validated by dual‐luciferase activity assay. The expression of miR‐181a‐5p was upregulated in osteosarcoma tissues and presented in SAOS‐2‐derived exosomes. SAOS‐2‐derived exosomes promoted the polarization of M2 macrophages by transferring miR‐181a‐5p. In addition, RORA was downregulated in osteosarcoma tissues and showed a negative correlation with miR‐181a‐5p. RORA was found to be the downstream target of miR‐181a‐5p in SAOS‐2 cells. Inhibition of RORA reversed the effects of miR‐181a‐5p knockdown on the polarization of M2 macrophages. The results showed that exosomal miR‐181a‐5p derived from osteosarcoma cells induced polarization of M2 macrophages via targeting RORA.

Osteoporosis is one of the most common metabolic bone diseases affecting millions of people. We previously found that harmine prevents bone loss in ovariectomized mice via increasing preosteoclast platelet‐derived growth factor‐BB (PDGF‐BB) production and type H vessel formation. However, the molecular mechanisms by which harmine promotes preosteoclast PDGF‐BB generation are still unclear. In this study, we revealed that inhibitor of DNA binding‐2 (Id2) and activator protein‐1 (AP‐1) were important factors implicated in harmine‐enhanced preosteoclast PDGF‐BB production. Exposure of RANKL‐induced Primary bone marrow macrophages (BMMs), isolated from tibiae and femora of mice, to harmine increased the protein levels of Id2 and AP‐1. Knockdown of Id2 by Id2‐siRNA reduced the number of preosteoclasts as well as secretion of PDGF‐BB in RANKL‐stimulated BMMs administrated with harmine. Inhibition of c‐Fos or c‐Jun (components of AP‐1) both reversed the stimulatory effect of harmine on preosteoclast PDGF‐BB production. Dual‐luciferase reporter assay analyses determined that PDGF‐BB was the direct target of AP‐1 which was up‐regulated by harmine treatment. In conclusion, our data demonstrated a novel mechanism involving in the production of PDGF‐BB increased by harmine, which may provide potential therapeutic targets for bone loss diseases.

Background Bone marrow mesenchymal stem cells (BMSCs) can undergo inadequate osteogenesis or excessive adipogenesis as they age due to changes in the bone microenvironment, ultimately resulting in decreased bone density and elevated risk of fractures in senile osteoporosis. This study aims to investigate the effects of osteocyte senescence on the bone microenvironment and its influence on BMSCs during aging. Results Primary osteocytes were isolated from 2-month-old and 16-month-old mice to obtain young osteocyte-derived extracellular vesicles (YO-EVs) and senescent osteocyte-derived EVs (SO-EVs), respectively. YO-EVs were found to significantly increase alkaline phosphatase activity, mineralization deposition, and the expression of osteogenesis-related genes in BMSCs, while SO-EVs promoted BMSC adipogenesis. Neither YO-EVs nor SO-EVs exerted an effect on the osteoclastogenesis of primary macrophages/monocytes. Our constructed transgenic mice, designed to trace osteocyte-derived EV distribution, revealed abundant osteocyte-derived EVs embedded in the bone matrix. Moreover, mature osteoclasts were found to release osteocyte-derived EVs from bone slices, playing a pivotal role in regulating the functions of the surrounding culture medium. Following intravenous injection into young and elderly mouse models, YO-EVs demonstrated a significant enhancement of bone mass and biomechanical strength compared to SO-EVs. Immunostaining of bone sections revealed that YO-EV treatment augmented the number of osteoblasts on the bone surface, while SO-EV treatment promoted adipocyte formation in the bone marrow. Proteomics analysis of YO-EVs and SO-EVs showed that tropomyosin-1 (TPM1) was enriched in YO-EVs, which increased the matrix stiffness of BMSCs, consequently promoting osteogenesis. Specifically, the siRNA-mediated depletion of Tpm1 eliminated pro-osteogenic activity of YO-EVs both in vitro and in vivo. Conclusions Our findings suggested that YO-EVs played a crucial role in maintaining the balance between bone resorption and formation, and their pro-osteogenic activity declining with aging. Therefore, YO-EVs and the delivered TPM1 hold potential as therapeutic targets for senile osteoporosis. Graphical abstract

Background: Small bowel adenocarcinoma (SBA) is a rare malignancy that accounts for 3% of all gastrointestinal tumors. We evaluated the clinical characteristics, outcomes, and prognostic factors of primary SBAs. Methods: We retrospectively analyzed the clinicopathological features and clinical outcomes of 300 patients with SBA from three institutions in China between January 2003 and July 2020. Overall survival (OS) was analyzed using the Kaplan–Meier method and it was statistically compared using the log-rank test. Single-variable and multivariate analyses were used to identify the significant correlates of OS. Results: The primary tumor was on the duodenal papilla in 156 patients (52%), in the duodenum in 60 patients (20%), and in the jejunum–ileum in 84 patients (28%). The median OS of the entire cohort was 32.5 months (range, 0-213 months), with a 1-year OS rate of 78.0%. For jejunoileal adenocarcinoma, advanced age, advanced T stage, advanced N stage, more positive lymph nodes, distant metastasis, high carcinoembryonic antigen (CEA), and lymphocyte-to-monocyte ratio < 2.32 predicted worse survival on single-variable analysis. Multivariate analysis showed that advanced age, advanced tumor node metastases (TNM) stage, high CEA level, high alpha fetoprotein (AFP) level, and low prealbumin level were independent prognostic factors for non-ampullary SBA. The independent prognostic factors for duodenal papilla adenocarcinoma included TNM Stage III, nerve invasion, low platelet/lymphocyte ratio, and high CA19-9. Conclusion: We found different independent prognostic factors for tumors at different locations. This finding warrants further investigation to ensure more effective management strategies for SBA.

The heavy metal lead has been shown to be associated with a genotoxic risk. Drosophila melanogaster is a model organism commonly utilized in genetic toxicology testing. The endosymbionts--Wolbachia are now very common in both wild populations and laboratory stocks of Drosophila. Wolbachia may induce resistance to pathogenic viruses, filarial nematodes and Plasmodium in fruit fly and mosquito hosts. However the effect of Wolbachia infection on the resistance of their hosts to heavy metal is unknown. Manipulating the lead content in the diet of Drosophila melanogaster, we found that lead consumption had no different effects on developmental time between Wolbachia-infected (Dmel wMel) and -uninfected (Dmel T) flies. While in Pb-contaminated medium, significantly reduced amount of pupae and adults of Dmel wMel were emerged, and Dmel wMel adults had significantly shorter longevity than that of Dmel T flies. Lead infusion in diet resulted in significantly decreased superoxide dismutase (SOD) activity in Dmel T flies (P<0.05), but not in Dmel wMel flies. Correspondingly, lead cultures induced a 10.8 fold increase in malonaldehyde (MDA) contents in Dmel T larvae (P<0.05). While in Dmel wMel larvae, it resulted in only a 1.3 fold increase. By quantitative RT-PCR, we showed that lead infused medium caused significantly increased expression level of relish and CecA2 genes in Dmel T flies (P<0.01). Lead cultures did not change dramatically the expression of these genes in Dmel wMel flies. These results suggest that Wolbachia infection decreased the resistance of Drosophila to lead likely by limiting the production of peroxides resulted from lead, thus being unable to activate the immunological pathway in the host to prevent them from lead damage. This represents a novel Wolbachia-host interaction and provides information that researchers working on Drosophila toxicology should take in consideration the presence of Wolbachia in the stocks they are analyzing.

Pectate lyases (Pels) have a vital function in degradation of the primary plant cell wall and the middle lamella and have been widely used in the industry. In this study, two pectate lyase genes, IDSPel16 and IDSPel17, were cloned from a sheep rumen microbiome. The recombinant enzymes were expressed in Escherichia coli and functionally characterized. Both IDSPel16 and IDSPel17 proteins had an optimal temperature of 60 ℃, and an optimal pH of 10.0. IDSPel16 was relatively stable below 60 °C, maintaining 77.51% residual activity after preincubation at 60 °C for 1 h, whereas IDSPel17 denatured rapidly at 60 °C. IDSPel16 was relatively stable between pH 6.0 and 12.0, after pretreatment for 1 h, retaining over 60% residual activity. IDSPel16 had high activity towards polygalacturonic acid, with a Vmax of 942.90 ± 68.11, whereas IDSPel17 had a Vmax of only 28.19 ± 2.23 μmol/min/mg. Reaction product analyses revealed that IDSPel17 liberated unsaturated digalacturonate (uG2) and unsaturated trigalacturonate (uG3) from the substrate, indicating a typical endo-acting pectate lyase (EC 4.2.2.2). In contrast, IDSPel16 initially generated unsaturated oligogalacturonic acids, then converted these intermediates into uG2 and unsaturated galacturonic acid (uG1) as end products, a unique depolymerization profile among Pels. To the best of our knowledge, the IDSPel16 discovered with both endo-Pel (EC 4.2.2.2) and exo-Pel (EC 4.2.2.9) activities. These two pectate lyases, particularly the relatively thermo- and pH-stable IDSPel16, will be of interest for potential application in the textile, food, and feed industries.Key points• Two novel pectate lyase genes, IDSPel16 and IDSPel17, were isolated and characterized from the sheep rumen microbiome.• Both IDSPel16 and IDSPel17 are alkaline pectate lyases, releasing unsaturated digalacturonate and unsaturated trigalacturonate from polygalacturonic acid.• IDSPel16, a bifunctional pectate lyase with endo-Pel (EC 4.2.2.2) and exo-Pel (EC 4.2.2.9) activities, could be a potential candidate for industrial application.

Although the interaction between tumor cells and tumor‐associated macrophages (TAMs) has been widely studied; however, the mechanism of osteosarcoma cells in regulating the polarization of TAMs remains unclear. Exosomes from SAOS‐2 cells were isolated and validated by electron microscopy and Western blot. Transfection of indicated plasmids was applied to modify the expressions of miR‐181a‐5p and RAR‐related orphan receptor alpha (RORA). Flow cytometric analysis was carried out to analyze M1/M2 macrophage polarization. Quantitative real‐time PCR was performed to determine the levels of miR‐181a‐5p and RORA. Protein levels of CD63, CD81, RORA, CD163, CD206, IL‐10, CXCL10, and IL‐1β were evaluated by Western blot. The direct interaction of miR‐181a‐5p and RORA was validated by dual‐luciferase activity assay. The expression of miR‐181a‐5p was upregulated in osteosarcoma tissues and presented in SAOS‐2‐derived exosomes. SAOS‐2‐derived exosomes promoted the polarization of M2 macrophages by transferring miR‐181a‐5p. In addition, RORA was downregulated in osteosarcoma tissues and showed a negative correlation with miR‐181a‐5p. RORA was found to be the downstream target of miR‐181a‐5p in SAOS‐2 cells. Inhibition of RORA reversed the effects of miR‐181a‐5p knockdown on the polarization of M2 macrophages. The results showed that exosomal miR‐181a‐5p derived from osteosarcoma cells induced polarization of M2 macrophages via targeting RORA.

Improving revascularization is one of the major measures in fracture treatment. Moderate local inflammation triggers angiogenesis, whereas systemic inflammation hampers angiogenesis. Previous studies showed that Akkermansia muciniphila (A. muc), a gut probiotic, ameliorates systemic inflammation by tightening intestinal barrier. In this study, fractured mice intragastrically administrated with A. muc were found to display better fracture healing than mice treated with vehicle. Notably, more preosteclasts positive for platelet-derived growth factor-BB (PDGF-BB) were induced by A. muc at 2 weeks post fracture, coinciding with increased formation of type H vessels, a specific vessel subtype that couples angiogenesis and osteogenesis and can be stimulated by PDGF-BB. Moreover, A. muc treatment significantly reduced gut permeability and inflammation at early stage. Dextran Sulfate Sodium (DSS) was used to disrupt the gut barrier to determine the role of gut barrier in fracture healing and whether A. muc still can stimulate bone fracture healing. As expected, A. muc evidently improved gut barrier, reduced inflammation, and restored the impaired bone healing and angiogenesis in DSS-treated mice. Our results suggest that A. muc reduces intestinal permeability and alleviates inflammation, which probably induces more PDGF-BB positive preosteoclasts and type H vessel formation in callus, thereby promoting fracture healing. This study provides the evidences about the involvement of type H vessels in fracture healing and suggests the potential of A. muc as a promising strategy for bone healing.