Toll-Like Receptor 4 Reduces Oxidative Injury via Glutathione Activity in Sheep

Toll-like receptor 4 (TLR4) is an important sensor of Gram-negative bacteria and can trigger activation of the innate immune system. Increased activation of TLR4 can lead to the induction of oxidative stress. Herein, the pathway whereby TLR4 affects antioxidant activity was studied. In TLR4-overexpressing sheep, TLR4 expression was found to be related to the integration copy number when monocytes were challenged with lipopolysaccharide (LPS). Consequently, production of malondialdehyde (MDA) was increased, which could increase the activation of prooxidative stress enzymes. Meanwhile, activation of an antioxidative enzyme, glutathione peroxidase (GSH-Px), was increased. Real-time PCR showed that expression of activating protein-1 (AP-1) and the antioxidative-related genes was increased. By contrast, the expression levels of superoxide dismutase 1 (SOD1) and catalase (CAT) were reduced. In transgenic sheep, glutathione (GSH) levels were dramatically reduced. Furthermore, transgenic sheep were intradermally injected with LPS in each ear. The amounts of inflammatory infiltrates were correlated with the number of TLR4 copies that were integrated in the genome. Additionally, the translation of γ-glutamylcysteine synthetase (γ-GCS) was increased. Our findings indicated that overexpression of TLR4 in sheep could ameliorate oxidative injury through GSH secretion that was induced by LPS stimulation. Furthermore, TLR4 promoted γ-GCS translation through the AP-1 pathway, which was essential for GSH synthesis

Overexpression of Toll-Like Receptor 4 Contributes to Phagocytosis of Salmonella Enterica Serovar Typhimurium via Phosphoinositide 3-Kinase Signaling in Sheep

Background/Aims: Phagocytosis of bacteria by monocytes/macrophages can trigger the immune response and the clearance of bacteria. This innate immune response involves Toll-like receptor 4 (TLR4). However, much remains unknown about the mechanism of TLR4-regulated phagocytosis of Salmonella enterica serovar Typhimurium (S. typhimurium) within sheep monocytes/macrophages. Here, we aimed to address these knowledge gaps by infecting transgenic sheep overexpressing TLR4 with S. typhimurium and examining the phagocytic mechanisms involved. Methods: Transgenic sheep were generated by microinjection of the constructed plasmids into fertilized eggs. Monocytes/macrophages isolated from sheep blood were stimulated with LPS and S. typhimurium. Phagocytosis-related factor expression, phagocytic ability, and adhesion were then determined. TLR4/phosphatidylinositide 3-kinase (PI3K) signaling was inhibited to investigate if this pathway is involved in changes in bacterial internalization in sheep. Results: We found that TLR4 overexpression effectively activated the PI3K signaling pathway and upregulated the expression of scavenger receptors. Additionally, actin polymerization and adhesive capacity were both enhanced in TLR4-overexpressing sheep monocytes/macrophages. TLR4 inhibition decreased S. typhimurium phagocytosis by reducing the actin polymerization and adhesive capacity of cells. Furthermore, inhibition of PI3K markedly impaired TLR4-dependent phagocytosis by restraining actin polymerization and scavenger receptor expression and reduced the adhesive capacity of the monocytes/macrophages. Conclusion: Our findings indicate that overexpression of TLR4 enhances phagocytosis through PI3K signaling and the subsequent activation of actin polymerization and scavenger receptors in sheep monocytes/macrophages infected with S. typhimurium

AANAT transgenic sheep generated via OPS vitrified-microinjected pronuclear embryos and reproduction efficiency of the transgenic offspring

Background The open pulled straw (OPS) vitrification method has been successfully applied in mouse, pig, and goat embryos as well as in buffalo oocytes, but it has not yet been applied to the microinjected embryos. This study examined the effects of OPS vitrification on embryo development and the reproductive capacity of the transgenic offspring in order to establish a method for preservation of microinjected embryos. Methods Ovine pronuclear embryos were microinjected with the exogenous aralkylamine N-acetyltransferase gene (AANAT), frozen by the OPS method, and subsequently thawed for embryo transplantation. Pregnancy rate, lambing rate, survival rate, average birth weight and transgenic positive rate as well as reproduction efficiency and hormone level of the transgenic offspring were investigated to analyze the effect of OPS vitrification on microinjectd pronuclear embryos. Results No significant differences were observed in the birth rate, lamb survival rate and transgenic positive rate between the frozen and non-frozen AANAT-microinjected pronuclear embryos. The average birth weight of the frozen embryos offspring was greater than that of the non-frozen embryos. Importantly, the transgenic offspring that overexpressed the AANAT gene showed improved ovulation efficiency and lambing rate by regulating their hormone levels. Conclusions The OPS vitrification approach may be a valuable method in microinjected- embryo transfer technology, which could reserve embryos and result in fewer unnecessary animal sacrifices. In addition, the AANAT+ transgenic offspring exhibited improved reproductive capacity on account of regulation effect of melatonin on reproductive hormone. These data may provide available references for human-assisted reproduction

Irreversible electroporation ablation area enhanced by synergistic high- and low-voltage pulses.

Irreversible electroporation (IRE) produced by a pulsed electric field can ablate tissue. In this study, we achieved an enhancement in ablation area by using a combination of short high-voltage pulses (HVPs) to create a large electroporated area and long low-voltage pulses (LVPs) to ablate the electroporated area. The experiments were conducted in potato tuber slices. Slices were ablated with an array of four pairs of parallel steel electrodes using one of the following four electric pulse protocols: HVP, LVP, synergistic HVP+LVP (SHLVP) or LVP+HVP. Our results showed that the SHLVPs more effectively necrotized tissue than either the HVPs or LVPs, even when the SHLVP dose was the same as or lower than the HVP or LVP doses. The HVP and LVP order mattered and only HVPs+LVPs (SHLVPs) treatments increased the size of the ablation zone because the HVPs created a large electroporated area that was more susceptible to the subsequent LVPs. Real-time temperature change monitoring confirmed that the tissue was non-thermally ablated by the electric pulses. Theoretical calculations of the synergistic effects of the SHLVPs on tissue ablation were performed. Our proposed SHLVP protocol provides options for tissue ablation and may be applied to optimize the current clinical IRE protocols

Comparative Evaluation of the Physiochemical Properties, and Antioxidant and Hypoglycemic Activities of <i>Dendrobium officinale</i> Leaves Processed Using Different Drying Techniques

Dendrobium officinale leaves have the potential to be processed into natural antioxidants, functional foods, and food additives. To maximally maintain their quality, fresh D. officinale leaves were dehydrated using different drying methods, i.e., hot air drying (HD), microwave drying (MD), infrared drying (IRD), and freeze drying (FD), and then the physicochemical properties, microstructure, and biological activities of the dried samples were compared. The results showed that, with the FD method, the samples had a porous microstructure, maintained the highest phenolic content, and demonstrated the highest antioxidant and hypoglycemic activities. Among the three thermal drying methods, with the IRD method, the samples retained higher phenolic contents, showed stronger DPPH free-radical scavenging, ferric ion reducing, ferrous ion chelating, and α-glucosidase inhibitory abilities, and more strongly promoted glucose metabolism in insulin-resistant HL-7702 cells than the samples with the MD and HD methods. These results suggested that FD was the most suitable method. However, IRD might be a promising alternative, owing to the high cost and long time needed for FD for the large-scale drying of D. officinale leaves

Melatonin-Mediated Development of Ovine Cumulus Cells, Perhaps by Regulation of DNA Methylation

Cumulus cells of pre-pubertal domestic animals are dysfunctional, perhaps due to age-specific epigenetic events. This study was designed to determine effects of melatonin treatment of donors on methylation modification of pre-pubertal cumulus cells. Cumulus cells from germinal vesicle stage cumulus oocyte complexes (COCs) were collected from eighteen lambs which were randomly divided into control group (C) and melatonin group given an 18 mg melatonin implant subcutaneous (M). Compared to the C group, the M group had higher concentrations of melatonin in plasma and follicular fluid (p &lt; 0.05), greater superovulation, a higher proportion of fully expanded COCs, and a lower proportion of apoptotic cumulus cells (p &lt; 0.05). Real-time PCR results showed that melatonin up-regulated expression of genes MT1, Bcl2, DNMT1, DNMT3a and DNMT3b, but down-regulated expression of genes p53, Caspase 3 and Bax (p &lt; 0.05). Furthermore, melatonin increased FI of FITC (global methylation level) on cumulus cells (p &lt; 0.05). To understand the regulation mechanism, the DNMTs promoter methylation sequence were analyzed. Compared to the C group, although there was less methylation at two CpG sites of DNMT1 (p &lt; 0.05) and higher methylation at two CpG sites of DNMT3a (p &lt; 0.05), there were no significant differences in methylation of the detected DNMT1 and DNMT3a promoter regions. However, there were lower methylation levels at five CpG sites of DNMT3b, which decreased methylation of detected DNMT3b promoter region on M group (p &lt; 0.05). In conclusion, alterations of methylation regulated by melatonin may mediate development of cumulus cells in lambs

Integrated aggregate turnover and soil organic carbon sequestration using rare earth oxides and 13C isotope as dual tracers

The formation, stabilization and breakdown processes of soil aggregates determine soil organic carbon (SOC) sequestration, in turn, soil aggregate dynamics are mediated by SOC changes. However, the interactions between them remain elusive. Herein, three types of ¹³C-labelled residues were added to two textured soils. Rare earth oxides (REOs) and ¹³C isotope were used as dual tracers to simultaneously track aggregate transfer pathways and SOC sequestration during a 56-day incubation period. Residue-derived CO₂ followed the sequence of Vetch > Maize > Decomposed maize during the first two weeks. Residue-derived CO₂ was significantly negatively correlated with the aggregate turnover time in both investigated soils (P < 0.01), indicating that aggregate turnover was a controlling factor of residue decomposition in addition to its inherent features. Generally, residue addition decreased the aggregate turnover time in the sequence of Vetch < Maize < Decomposed maize. In Red clay soil, macroaggregates attained a higher turnover rate than that of microaggregates, while a similar change pattern was not observed in Sandstone soil with residue application. Aggregates turnover occurred faster in Sandstone soil than in Red clay soil under a given residue application. The aggregate turnover time was significantly reciprocally correlated with the residue-derived C sequestration rate (P < 0.01), suggesting that aggregate turnover was the key factor in C sequestration. A C flow conceptual model was proposed, residue-derived C firstly accumulated in macroaggregates in the formation process, and then relocated from macroaggregates to microaggregates with the breakdown processes at the mid-to-late stage. This study highlights the importance of aggregate turnover in SOC sequestration and demonstrates that these interactions are further affected by residue features and soil texture.ISSN:0016-7061ISSN:1872-625

Fibrin Facilitates Mesenchymal Stem Cells to Ameliorate Rats with Polycystic Ovary Syndrome

Polycystic ovarian syndrome (PCOS) is a ubiquitous hormonal disorder and induces female infertility and heterogeneous syndromes, for which there is still no effective treatment. Thanks to the properties of immunomodulatory and endocrine regulation, mesenchymal stem cells (MSCs) have been widely used in various disease types. There were few reports for MSCs injected to ovaries due to the size limitation and complicated vascular network. Here, we develop one simple and efficient approach to deliver and stabilize MSCs in the outside of the ovary without blood leaking through the fibrin gelation, which also possesses excellent biocompatibility to support MSC survival. Notably, the transplantation of MSCs, encapsulated in fibrin hydrogel, could rescue ovarian function more efficiently compared to only MSC control in terms of elevated estradiol (E2) and progesterone (P) levels, diminished gonadotropins (LH/FSH), testosterone (T), and transforming growth factor-&beta;1 (TGF-&beta;1) levels, regular estrous cycles, enhanced number of granulosa cells, and reduced number of immature cystic follicles. The size and weight of the ovary increased for MSCs both within and without fibrin in PCOS rat models in two weeks. Moreover, we have shown the versatility of fibrin hydrogel as a cell-compatible platform for advanced stem cell translation, including identifying novel mechanisms of cell survival support, tissue development, and regenerative medicine

Study on the Atmospheric Diffusion of Airborne Radionuclide under LOCA of Offshore Floating Nuclear Power Plants Based on CALPUFF

Studying the migration and diffusion of radionuclides plays an important role in emergency decision making and accident mitigation of floating nuclear power plants. Based on the CALPUFF model, this paper simulates the spatial distribution and concentration distribution of airborne radionuclides 131I diffusion under the conditions of sailing and power supply under LOCA (Loss-of-Coolant Accident) of the floating nuclear power plant, and the influence of four meteorological parameters, namely wind speed, cloudiness, temperature and air pressure, on the migration was analyzed using sensitivity analysis. The results show that the wind direction affects the diffusion direction of 131I, and the concentration of 131I decreases with the increase in the diffusion distance; under the same conditions, the radionuclides diffuses farther and the affected area is larger under the sailing condition. Wind speed is the dominant factor affecting the diffusion of radionuclides, followed by the cloud amount parameter, temperature parameter, and air pressure parameter. The research results can provide theoretical support for emergency responses to nuclear accidents in offshore floating nuclear power plants