Wei Xu

Living cells regulate biochemical reactions through compartmentalization, achieving high efficiency by precisely controlling spatial and temporal factors. Inspired by this principle, we developed multicompartmentalized artificial cells using liquid–liquid phase separation (LLPS) to regulate cascade enzymatic reactions. Glucose oxidase (GOx) and horseradish peroxidase (HRP) were separately encapsulated into PEGylated and non‐PEGylated liposomes, forming enzyme‐loaded nanoreactors. These nanoreactors were assembled within a PEG/dextran aqueous two‐phase system, allowing controlled spatial arrangement of nanoreactors. Depending on PEGylation, liposomes localized either at the microdroplet interface or inside the lumen, creating four distinct artificial cell models in one step. Among them, the organization with GOx‐loaded liposomes at the interface (Models 1 and 2) exhibited the highest catalytic efficiency. This enhancement arose from improved substrate accessibility, reduced diffusion barriers, and optimized nanoreactor separation. Other models with less favorable spatial arrangements showed slower reaction rates. Our results highlight how spatial organization within artificial cells can critically influence cascade reaction performance. This modular and biomimetic strategy offers a versatile platform for designing synthetic cells, programmable biocatalysts, and functional microreactors for applications in biosensing, metabolic engineering, and therapeutics.

Purpose: Chemoresistance remains a key hurdle in osteosarcoma therapy. This study aims to delineate the role and underlying mechanisms of spermidine (SPD) in osteosarcoma chemoresistance. Experimental Design: Using osteosarcoma cell lines and xenografts, we combined flow cytometry, Western blotting, proteomic mass spectrometry, and RNA sequencing to characterize SPD-driven changes in cellular pathways and resistance signatures. We tested whether pharmacologic inhibition of SPD biosynthesis, alone or in combination with standard chemotherapy, improves therapeutic response in vivo. Results: Following chemotherapy, either cisplatin (CDP) or doxorubicin (DOX), apoptotic osteosarcoma cells exhibit an upregulation of ornithine decarboxylase 1 and SPD synthase, key enzymes involved in SPD synthesis, resulting in heightened levels of this polyamine. SPD diminishes the therapeutic efficacy of CDP and DOX in osteosarcoma cells, both in vitro and in vivo. Mechanistically, SPD enhances β-catenin activity, which subsequently upregulates genes associated with cancer stemness and ATP-binding cassette transporters, both of which are implicated in drug resistance. Furthermore, pharmacologic inhibition of SPD synthesis using α-difluoromethylornithine markedly increases the chemosensitivity of osteosarcoma cells to CDP and DOX. Conclusions: These findings illuminate the critical role of apoptotic cell metabolites in mediating treatment resistance and suggest that targeting SPD may offer a promising therapeutic strategy to augment the effectiveness of chemotherapy in osteosarcoma.

Background: MicroRNAs are known to regulate carcinogenesis of osteosarcoma. Although, miR-16-5p is known to exert inhibitory effects on several forms of cancers, its effects on the growth and invasion of osteosarcoma have not been studied. Methods: We collected human osteosarcoma specimens and adjacent tissues to detect the expression of miR-16-5p by real-time polymerase chain reaction, immunoblotting, and immunohistochemistry. The proliferation, migration, and invasion of MG63 and HOS cells following miR-16-5p overexpression and inhibition were detected with cell counting kit-8, wound healing assay, and Transwell assay, respectively. An expression vector carrying a mutated 3′-untranslated region of mothers against decapentaplegic homolog 3 (Smad3) was constructed. Results: The results showed that miR-16-5p expression was downregulated in osteosarcoma tissues and cells as compared with adjacent counterparts, while Smad3 was overexpressed in osteosarcoma cells. The overexpression of miR-16-5p resulted in the inhibition of the proliferation, migration, and invasion of osteosarcoma cells and enhanced the therapeutic effect of cisplatin. These effects were attenuated with miR-16-5p expression inhibition. In cells transfected with miR-16-5p mimic, Smad3 expression decreased, while this effect was absent in the cells carrying mutated Smad3. Conclusions: Therefore, miR-16-5p inhibits the growth and invasion of osteosarcoma by targeting Smad3.

The hypoxic state of the brain tissue surrounding craniocerebral injury induces an increase in the secretion of HIF‑1α during the healing process. HIF‑1α can promote mesenchymal stem cell (MSC) migration to ischemic and hypoxic sites by regulating the expression levels of molecules such as stromal cell‑derived factor‑1 (SDF‑1) in the microenvironment. Stem cells express the SDF‑1 receptor C‑X‑C chemokine receptor type 4 (CXCR4) and serve a key role in tissue repair, as well as a number of physiological and pathological processes. The present study aimed to determine the role of HIF‑1α/SDF‑1/CXCR4 signaling in the process of accelerated fracture healing during craniocerebral injury. Cultured MSCs underwent HIF‑1α knockdown to elucidate its effect on the proliferative ability of MSCs, and the effect of SDF‑1 in MSCs was investigated. It was also determined whether HIF‑1α could promote osteogenesis via SDF‑1/CXCR4 signaling and recruit MSCs. The results indicated that HIF‑1α knockdown suppressed MSC proliferation in vitro, and SDF‑1 promoted cell migration via binding to CXCR4. Furthermore, HIF‑1α knockdown inhibited MSC migration via SDF‑1/CXCR4 signaling. Considering the wide distribution and diversity of roles of SDF‑1 and CXCR4, the present results may form a basis for the development of novel strategies for the treatment of craniocerebral injury.

Inflammation can promote the maturity of osteoclasts and bone resorption in many bone disease such as osteoporosis and arthritis. Here, we aimed to investigate the inhibitory effects of deacylcynaropicrin (DAC) on osteoclastogenesis and bone resorption induced by RANKL. Bone-marrow-derived macrophages were used for assessing the influence of DAC on polarization of macrophages and osteoclastogenesis in vitro. Inducible nitric oxide synthase (iNOS) and CD206, as well as osteoclastogenesis markers, nuclear factor of activated T-cells 1 (NFATc1), and c-Fos, were qualitatively analyzed by immunofluorescence, flow cytometry, reverse transcription polymerase chain reaction, and Western blotting. The results showed that DAC significantly inhibited osteoclastogenesis by suppressing the expression levels of c-Fos and NFATc1 through nuclear factor-κB, c-Jun N-terminal kinase (JNK), and Akt pathway. Moreover, immunohistochemistry and enzyme-linked immunosorbent assays showed that DAC reduced the release of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 in vivo. Finally, DAC also promoted macrophage polarization from M1 to M2 types. In conclusion, these results demonstrated that DAC suppressed RANKL-induced inflammation and osteoclastogenesis and therefore it can be used as a potential treatment for osteoporosis, arthritis, osteolysis, and aseptic loosening of artificial prostheses.

Mesenchymal stem cells (MSCs) have drawn great attention because of their therapeutic potential. It has been suggested that intra‐venous infused MSCs could migrate the site of injury to help repair the damaged tissue. However, the mechanism for MSC migration is still not clear so far. In this study, we reported that hypoxia increased chemotaxis migration of MSCs. At 4 and 6 hours after culturing in hypoxic (1% oxygen) conditions, the number of migrated MSCs was significantly increased. Meanwhile, hypoxia also increased the expression of HIF‐1α and SDF‐1. Using small interference RNA, we knocked down the expression of HIF‐1α in MSCs to study the role of HIF‐1α in hypoxia induced migration. Our data indicated that knocking down the expression of HIF‐1α not only abolished the migration of MSCs, but also reduced the expression of SDF‐1. Combining the results of migration assay and expression at RNA and protein level, we demonstrated a novel mechanism that controls the increase of MSCs migration. This mechanism involved HIF‐1α mediated SDF‐1 expression. These findings provide new insight into the role of HIF‐1α in the hypoxia induced MSC migration and can be a benefit for the development of MSC‐based therapeutics for wound healing.

Objectives: This study aims to reveal the roles and related mechanisms of Lnc RNA B4 GALT 1‐ AS 1 in osteosarcoma ( OS ) cells stemness and migration. Materials and methods: Real‐time quantitative PCR ( RT ‐ qPCR ) was used to detect the expression of several Lnc RNA s in OS tissues and normal adjacent tissues and in OS mammospheres and cells. Cell viability, transwell migration, tumour spheres formation and in vivo tumour formation assays were used to examine the effects of Lnc RNA B4 GALT 1‐ AS 1 on OS progression. In addition, RNA immunoprecipitation ( RIP ) and Luciferase reporter assays were performed to determine the binding site of RNA ‐binding protein HuR on B4 GALT 1‐ AS 1 and transcriptional factor YAP . Immunofluorescence analysis was used to examine YAP nuclear‐cytoplasm translocation. Results: Lnc RNA B4 GALT 1‐ AS 1 expression was significantly increased in OS tissues and cells spheres. Knockdown of B4 GALT 1‐ AS 1 inhibited OS cells proliferation, migration, stemness and chemotherapeutic sensitivity. Mechanistically, B4 GALT 1‐ AS 1 recruited HuR to enhance YAP mRNA stability and thus its transcriptional activity. Conclusions: We indicate that lnc RNA B4 GALT 1‐ AS 1 promotes OS cells stemness and migration via recruiting HuR to enhance YAP activity.

Objectives: This work aims to reveal the roles and related mechanisms of RNA binding protein PUM 2 in osteosarcoma progression. Materials and methods: Transcriptome analysis based on RNA sequencing data, real‐time quantitative PCR ( RT ‐ qPCR ), and western blot analysis were used to detect the expression of RBP s and mi RNA s in OS and normal adjacent tissues, and the correlation between them in OS tissues. RT ‐ qPCR , western blot, cell viability, transwell migration, tumour spheres formation and in vivo tumour formation assays were used to examine the effects of RBP PUM 2 on OS progression. Additionally, RNA immunoprecipitation ( RIP ) assay combined with RNA sequencing was performed to determine the binding site of RBP PUM 2 on STARD 13 3′ UTR . Luciferase reporter and RIP assays were used to confirm the binding of mi RNA s or PUM 2 on STARD 13 3′UTR. Results: PUM 2 and STARD 13 expression was significantly decreased in OS tissues, and positively correlated. Overexpression of PUM 2 or STARD 13 3′ UTR inhibited OS cells proliferation, migration, and stemness. Mechanistically, PUM 2 competitively bound to STARD 13 3′ UTR with miR‐590‐3p and miR‐9. The inhibition of PUM 2 on OS cells progression was attenuated by STARD 13 knockdown or related mi RNA s overexpression. Conclusion: PUM 2 suppresses OS progression via partly and competitively binding to STARD 13 3′ UTR with mi RNA s.

The treatment for osteoporosis involves inhibiting bone resorption and osteoclastogenesis. Glycyrrhizin (GLY) is a triterpenoid saponin glycoside known to be as the most medically efficacious component of the licorice plant. It has strong anti-inflammatory, antioxidant, and antitumor properties. We investigated the effect of GLY on osteoclastogenesis, bone resorption, and intracellular oxidative stress and its molecular mechanisms. In vitro osteoclastogenesis assays were performed using bone marrow monocytes with and without glycyrrhizin. We also evaluated the effects of glycyrrhizin on the secretion of TNF-α, IL-1β, and IL-6 in LPS-stimulated RAW 264.7 cells using ELISA. The effects of glycyrrhizin on the expression of osteoclast-related genes, such as Nfatc1, c-fos, Trap, and cathepsin K (CK), were investigated by RT-PCR. Intracellular reactive oxygen species (ROS) were detected in receptor activator of nuclear factor kappa-Β ligand (RANKL)-stimulated osteoclasts in the presence and absence of glycyrrhizin. During the inhibition of osteoclastogenesis by glycyrrhizin, phosphorylation of AMPK, Nrf2, NF-κB, and MAPK was analyzed using western blotting. Our results showed that glycyrrhizin significantly inhibited RANKL-induced osteoclastogenesis, downregulated the expression of NFATc1, c-fos, TRAP, CK, DC-STAMP, and OSCAR, and inhibited p65, p38, and JNK. Glycyrrhizin was found to significantly decrease the secretion of inflammatory cytokines (TNF-α, IL-1β, and IL-6). Additionally, glycyrrhizin reduced the formation of ROS in osteoclasts by inducing AMPK phosphorylation and nuclear transfer of NRF2, resulting in an upregulation of antioxidant enzymes, such as HO-1, NQO-1, and GCLC. In summary, we found that glycyrrhizin inhibited RANKL-induced osteoclastogenesis. It was also indicated that glycyrrhizin could reduce oxidative stress by inhibiting the MAPK and NF-κB pathways and activating the AMPK/NRF2 signaling. Therefore, glycyrrhizin may be used as an effective therapeutic agent against osteoporosis and bone resorption.