Effects of castration and eucalyptus oil supplementation on growth performance, nutrient digestibility, and blood-immunity indicators of male Holstein calves.

The purpose of this study was to investigate the effects of early castration and eucalyptus oil (EUC) supplementation on dry matter intake (DMI), growth performance, and immune response of Holstein calves. Fifty-six male Holstein calves 52 d old and with an initial body weight (BW) of 63.5 ± 5.27 kg were used. The animals were blocked by BW and randomly assigned into 1 of the 4 treatment groups in a randomized complete block design with a 2 (no castration vs. castration) × 2 (without vs. with EUC) factorial arrangement of treatments. The treatments were (1) uncastrated calves fed without EUC, (2) uncastrated calves fed 0.5 g/d EUC (EUC group), (3) castrated calves (steers) fed without EUC (castrated group), and (4) steers fed with 0.5 g/d EUC (castrated + EUC). The experiment was 8 wk long, including pre- and postweaning (weaned at 72 d). The EUC × castrated interactions were not significant for DMI, growth performance, nutrient digestibility, and immune response. Castration did not affect the DMI, final BW, average daily gain (ADG), or feed efficiency, except that the ADG was greater for bull calves than for steers at postweaning. Supplementation with EUC increased DMI pre- and postweaning and increased the ADG of weaned calves. Digestibility in the total digestive tract was not affected by castration (except for organic matter digestibility), whereas adding EUC improved the digestibility of dry matter, acid detergent fiber, and crude protein. Blood concentration of IL-6 at d 94 was decreased by feeding EUC. These results indicate that the EUC could be fed to either intact or castrated dairy calves to promote growth and health postweaning; castration before weaning may reduce ADG and cause inflammatory stress without affecting feed intake or feed efficiency.

Mesenchymal stem cells-derived extracellular vesicles protect against oxidative stress-induced xenogeneic biological root injury via adaptive regulation of the PI3K/Akt/NRF2 pathway.

Xenogeneic extracellular matrices (xECM) for cell support have emerged as a potential strategy for addressing the scarcity of donor matrices for allotransplantation. However, the poor survival rate or failure of xECM-based organ transplantation is due to the negative impacts of high-level oxidative stress and inflammation on seed cell viability and stemness. Herein, we constructed xenogeneic bioengineered tooth roots (bio-roots) and used extracellular vesicles from human adipose-derived mesenchymal stem cells (hASC-EVs) to shield bio-roots from oxidative damage. Pretreatment with hASC-EVs reduced cell apoptosis, reactive oxygen species generation, mitochondrial changes, and DNA damage. Furthermore, hASC-EV treatment improved cell proliferation, antioxidant capacity, and odontogenic and osteogenic differentiation, while significantly suppressing oxidative damage by activating the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and nuclear factor erythroid 2 (NFE2)-related factor 2 (NRF2) nuclear translocation via p62-associated Kelch-like ECH-associated protein 1 (KEAP1) degradation. Inhibition of PI3K/Akt and Nrf2 knockdown reduced antioxidant capacity, indicating that the PI3K/Akt/NRF2 pathway partly mediates these effects. In subcutaneous grafting experiments using Sprague-Dawley rats, hASC-EV administration significantly enhanced the antioxidant effect of the bio-root, improved the regeneration efficiency of periodontal ligament-like tissue, and maximized xenograft function. Conclusively, therefore, hASC-EVs have the potential to be used as an immune modulator and antioxidant for treating oxidative stress-induced bio-root resorption and degradation, which may be utilized for the generation and restoration of other intricate tissues and organs.

Biomimetic, biodegradable and osteoinductive treated dentin matrix/α-calcium sulphate hemihydrate composite material for bone tissue engineering.

It is still a huge challenge for bone regenerative biomaterial to balance its mechanical, biological and biodegradable properties. In the present study, a new composite material including treated dentin matrix (TDM) and α-calcium sulphate hemihydrate (α-CSH) was prepared. The optimal composition ratio between TDM and α-CSH was explored. The results indicate that both components were physically mixed and structurally stable. Its compressive strength reaches up to 5.027 ± 0.035 MPa for 50%TDM/α-CSH group, similar to human cancellous bone tissues. Biological experiments results show that TDM/α-CSH composite exhibits excellent biocompatibility and the expression of osteogenic related genes and proteins (ALP, RUNX2, OPN) is significantly increased. In vivo experiments suggest that the addition of TDM for each group (10%, 30%, 50%) effectively promotes cell proliferation and osteomalacia. In addition, 50% of the TDM/α-CSH combination displays optimal osteoconductivity. The novel TDM/α-CSH composite is a good candidate for certain applications in bone tissue engineering.

TSF/FHA induces osteogenic differentiation of Mc3t3 cells via Pygo2 dependent Wnt/ß-catenin signaling pathway.

OBJECTIVES: This study aimed to investigate whether tussah silk fibroin (TSF)/fluoridated hydroxyapatite (FHA) can promote osteogenic differentiation of Mc3t3 cells and explore the role of Wnt/ß-catenin signaling in this process. METHODS: TSF/FHA was gained via freeze drying technique and cyclic phosphate immersion method. The relative expression levels of bone-related genes and proteins of Mc3t3 cells seeded on different materials were examined by RT-qPCR and Western blotting. Knockdown or overexpression of Pygo2 in Mc3t3 cells was achieved using lentiviral transfection. Cell proliferation, the expression of bone-related genes and proteins were subsequently examined. Animal experiment was also performed to observe the osteogenesis effect. RESULTS: Different ratios of fluorine of TSF/FHA accelerated the osteogenic differentiation of Mc3t3 cells and increased the Pygo2 expression. The Wnt/ß-catenin signaling pathway was activated after TSF/FHA induction, accompanied by the increased expression of related genes. In SD rats with skull defect, the newly formed bone increased significantly and the Pygo2 overexpressing Mc3t3 cells promoted osteogenesis. However, Pygo2 knockdown markedly compromised the osteogenesis of Mc3t3 cells after TSF/FHA induction. CONCLUSION: TSF/FHA facilitates osteogenic differentiation of Mc3t3 cells via upregulating Pygo2 and activating Wnt/ß-catenin signaling pathway.

Subtype-based analysis of cell-in-cell structures in non-small cell lung cancer.

Lung cancer is ranked as the leading cause of cancer-related death worldwide, and the development of novel biomarkers is helpful to improve the prognosis of non-small cell lung cancer (NSCLC). Cell-in-cell structures (CICs), a novel functional surrogate of complicated cell behaviors, have shown promise in predicting the prognosis of cancer patients. However, the CIC profiling and its prognostic value remain unclear in NSCLC. In this study, we retrospectively explored the CIC profiling in a cohort of NSCLC tissues by using the "Epithelium-Macrophage-Leukocyte" (EML) method. The distribution of CICs was examined by the Chi-square test, and univariate and multivariate analyses were performed for survival analysis. Four types of CICs were identified in lung cancer tissues, namely, tumor-in-tumor (TiT), tumor-in-macrophage (TiM), lymphocyte-in-tumor (LiT), and macrophage-in-tumor (MiT). Among them, the latter three constituted the heterotypic CICs (heCICs). Overall, CICs were more frequently present in adenocarcinoma than in squamous cell carcinoma (P = 0.009), and LiT was more common in the upper lobe of the lung compared with other lobes (P = 0.020). In univariate analysis, the presence of TiM, heCIC density, TNM stage, T stage, and N stage showed association with the overall survival (OS) of NSCLC patients. Multivariate analysis revealed that heCICs (HR = 2.6, 95% CI 1.25-5.6) and lymph node invasion (HR = 2.6, 95% CI 1.33-5.1) were independent factors associated with the OS of NSCLC. Taken together, we profiled the CIC subtypes in NSCLC for the first time and demonstrated the prognostic value of heCICs, which may serve as a type of novel functional markers along with classical pathological factors in improving prognosis prediction for patients with NSCLC.

α-Hemihydrate calcium sulfate/n-hydroxyapatite combined with metformin promotes osteogenesis in vitro and in vivo.

This study aimed to examine the effects of loading different concentrations of metformin onto an α-hemihydrate calcium sulfate/nano-hydroxyapatite (α-CSH/nHA) composite. The material characteristics, biocompatibility, and bone formation were compared as functions of the metformin concentration. X-ray diffraction results indicated that the metformin loading had little influence on the phase composition of the composite. The hemolytic potential of the composite was found to be low, and a CCK-8 assay revealed only weak cytotoxicity. However, the metformin-loaded composite was found to enhance the osteogenic ability of MC3T3-E1 cells, as revealed by alkaline phosphate and alizarin red staining, real-time PCR, and western blotting, and the optimal amount was 500 µM. RNA sequencing results also showed that the composite material increased the expression of osteogenic-related genes. Cranial bone lacks muscle tissue, and the low blood supply leads to poor bone regeneration. As most mammalian cranial and maxillofacial bones are membranous and of similar embryonic origin, the rat cranial defect model has become an ideal animal model for in vivo experiments in bone tissue engineering. Thus, we introduced a rat cranial defect with a diameter of 5 mm as an experimental defect model. Micro-computed tomography, hematoxylin and eosin staining, Masson staining, and immunohistochemical staining were used to determine the effectiveness of the composite as a scaffold in a rat skull defect model. The composite material loaded with 500 µM of metformin had the strongest osteoinduction ability under these conditions. These results are promising for the development of new methods for repairing craniofacial bone defects.

Enhancement of recombinant human IL-24 (rhIL-24) protein production from site-specific integrated engineered CHO cells by sodium butyrate treatment.

Interleukin-24 (IL-24) has specific inhibitory effects on the proliferation of various tumor cells with almost no toxicity to normal cells. The antitumor activity of recombinant human IL-24 protein produced in mammalian cells is much higher than that of bacteria, but its expression level is extremely low. Sodium butyrate (NaBu) was utilized as a media additive to increase protein expression in Chinese hamster ovary cells. The site-specific integrated engineered cells FCHO/IL-24 were treated with NaBu under different culture conditions (10% and 0.5% serum adherent culture, 0.5% serum suspension culture). First, 3 days of 1 mmol/L NaBu treatment significantly increased rhIL-24 expression level in FCHO/IL-24 cells by 119.94 ± 1.5% (**p < 0.01), 57.49 ± 2.4% (**p < 0.01), and 20.17 ± 3.03% (*p < 0.05) under the above culture conditions. Second, NaBu has a time- and dose-dependent inhibitory effect on FCHO/IL-24 proliferation and induces G0/G1 phase arrest. Under 10% and 0.5% serum adherent culture, G0/G1 phase cells were increased by 11.3 ± 0.5% (**p < 0.01) and 15.0 ± 2.6% (**p < 0.01), respectively. No induction of apoptosis was observed under a high dosage of NaBu treatment. These results suggest that NaBu increases rhIL-24 secretion via inhibiting cell cycle progression, thereby trapping cells in the highly productive G0/G1 phase. Finally, with increasing NaBu dose, glucose concentration increased (**p < 0.01) while lactic acid and ammonia concentrations reduced significantly (**p < 0.01) in 10% and 0.5% serum adherent culture supernatant. RNA-seq showed that NaBu treatment affected multiple tumor and immune-related pathways. In conclusion, NaBu treatment dramatically promoted rhIL-24 production in engineered FCHO/IL-24 cells by altering downstream pathways and inducing G0/G1 cell arrest with little effect on apoptosis.

Adaptation process of engineered cell line FCHO/IL-24 stably secreted rhIL-24 in serum-free suspension culture.

Interleukin-24 (IL-24) displays tumor cell-specific proliferation inhibition in vitro and in vivo. Recombinant human IL-24 (rhIL-24) has significantly higher activity, yet significantly lower expression level in mammalian cells than in bacteria. To further realize therapeutic potential of IL-24, we enhanced rhIL-24 expression in mammalian cell systems by adapting engineered Flp-InTMCHO/IL-24 (FCHO/IL-24) cells (adherent cultured in Ham's F12 medium with 10% serum) to serum-free suspension culture. First, MTT assay showed that among four different media (F12, DMEM/F12, 1640 and DMEM), DMEM/F12 medium was the most suitable media for lower-serum adherent culture. Then, cells were adherently cultured in DMEM/F12 with serum concentration reduced from 10% to 0.5% in a gradient manner. Compared to cells in 10% serum, cells in 0.5% serum displayed significantly lower relative cell viability by 40%, increased G0/G1 phase arrest (8.5 ± 2.4%, p < 0.05), decreased supernatant rhIL-24 concentration by 73%, and altered metabolite profiles, such as glucose, lactate and ammonia concentration. Next, the cells were directly adapted to 0.5% serum suspension culture in 125 mL shake flask at 119 rpm with the optimal cell seeding density of 5 × 105 cells/mL (3.3 times higher than that of adherent culture), under which the concentration of rhIL-24 in culture medium was stable at 3.5 ng/mL. Finally, cells adapted to 0.5% serum proliferated better in serum-free medium Eden™-B300S with higher rhIL-24 expression level compared to CDM4CHO. The successful adaptation of engineered cells FCHO/IL-24 laid foundation for adapting cells from adherent culture to suspension serum-free culture to mass produce rhIL-24 protein for therapeutic purposes.

Treated dentin matrix induces odontogenic differentiation of dental pulp stem cells via regulation of Wnt/ß-catenin signaling.

Treated dentin matrix (TDM) is an ideal scaffold material containing multiple extracellular matrix factors. The canonical Wnt signaling pathway is necessary for tooth regeneration. Thus, this study investigated whether the TDM can promote the odontogenic differentiation of human dental pulp stem cells (hDPSCs) and determined the potential role of Wnt/ß-catenin signaling in this process. Different concentrations of TDM promoted the dental differentiation of the hDPSCs and meanwhile, the expression of GSK3ß was decreased. Of note, the expression of the Wnt/ß-catenin pathway-related genes changed significantly in the context of TDM induction, as per RNA sequencing (RNA seq) data. In addition, the experiment showed that new dentin was visible in rat mandible cultured with TDM, and the thickness was significantly thicker than that of the control group. In addition, immunohistochemical staining showed lower GSK3ß expression in new dentin. Consistently, the GSK3ß knockdown hDPSCs performed enhanced odotogenesis compared with the control groups. However, GSK3ß overexpressing could decrease odotogenesis of TDM-induced hDPSCs. These results were confirmed in immunodeficient mice and Wistar rats. These suggest that TDM promotes odontogenic differentiation of hDPSCs by directly targeting GSK3ß and activating the canonical Wnt/ß-catenin signaling pathway and provide a theoretical basis for tooth regeneration engineering.

Subtype-Based Analysis of Cell-in-Cell Structures in Esophageal Squamous Cell Carcinoma.

Cell-in-cell (CIC) structures are defined as the special structures with one or more cells enclosed inside another one. Increasing data indicated that CIC structures were functional surrogates of complicated cell behaviors and prognosis predictor in heterogeneous cancers. However, the CIC structure profiling and its prognostic value have not been reported in human esophageal squamous cell Carcinoma (ESCC). We conducted the analysis of subtyped CIC-based profiling in ESCC using "epithelium-macrophage-leukocyte" (EML) multiplex staining and examined the prognostic value of CIC structure profiling through Kaplan-Meier plotting and Cox regression model. Totally, five CIC structure subtypes were identified in ESCC tissue and the majority of them was homotypic CIC (hoCIC) with tumor cells inside tumor cells (TiT). By univariate and multivariate analyses, TiT was shown to be an independent prognostic factor for resectable ESCC, and patients with higher density of TiT tended to have longer post-operational survival time. Furthermore, in subpopulation analysis stratified by TNM stage, high TiT density was associated with longer overall survival (OS) in patients of TNM stages III and IV as compared with patients with low TiT density (mean OS: 51 vs 15 months, P = 0.04) and T3 stage (mean OS: 57 vs 17 months, P=0.024). Together, we reported the first CIC structure profiling in ESCC and explored the prognostic value of subtyped CIC structures, which supported the notion that functional pathology with CIC structure profiling is an emerging prognostic factor for human cancers, such as ESCC.

Expression, Purification, and Polyethylene Glycol Site-Specific Modification of Recombinant Human Interleukin 24 in Escherichia coli.

Interleukin 24 (IL-24) has a broad spectrum of specific antitumor activities without affecting normal cells. The recombinant human IL-24 (rhIL-24) expressed in E. coli has low biological activity due to lack of necessary glycosylation modification. In this study, based on the modification of the non-glycosylated IL-24 with polyethylene glycol (PEG), we aimed to improve the stability and prolong its half-life in vivo. Firstly, the recombinant plasmid containing the hIL-24 cDNA was prepared by the prokaryotic-expression plasmid pET-28a and transformed into E. coli BL21. After induced by isopropyl ß-D-thiogalactoside (IPTG), the target protein rhIL-24 was expressed as insoluble inclusion body, which was solubilized and denatured by 6 M guanidine hydrochloride. The denatured rhIL-24 was diluted to refold in the optimized buffer overnight at the protein concentration of 0.1 mg/mL. The refolded rhIL-24 was mainly in the form of soluble aggregate, but high-purity monomer rhIL-24 was obtained through size exchange chromatography with the addition of SDS in elution buffer. The tertiary structure of rhIL-24 was confirmed by fluorescence spectroscopy. Western blot analysis showed that rhIL-24 could be site-specifically modified by mPEG5000-ALD. Methyl thiazolyl tetrazolium (MTT) assay showed no significant difference between mPEG5000-ALD-rhIL-24 and rhIL-24 in inhibiting the growth of melanoma cell line A375 in vitro. Pharmacokinetic studies showed that PEG modification could significantly improve the stability and prolong the half-life of rhIL-24 from 8.41 to 13.2 h. The data strongly suggested that mPEG-ALD 5000 could site-specifically modify rhIL-24 expressed in E. coli. The PEG modification significantly prolonged the half-life of rhIL-24 without reducing its antitumor activity in vitro.