International regulatory considerations pertaining to the development of stem cell-based veterinary medicinal products

Stem cell-based products (SCPs) are an emerging field of veterinary medicine that focuses on the regeneration, repair, or replacement of damaged tissues or organs. However, there are some issues in applying the traditional regulatory guideline for the approval of SCPs as veterinary medicinal products. This article describes the positions of Korea, US, and EU regarding SCPs, and compares the regulatory guidelines of each country for their safety evaluation. Although there are some differences in the regulatory guidelines, similar considerations in identifying the quality of SCPs and their safety has adopted. Overall, these guidelines need to be harmonized among countries.     

Temperature sensitivity: a cell character determined by obligate endosymbionts in amoebas

A strain of Amoeba proteus has lost its ability to survive at temperatures above 26 degrees C as a result of becoming dependent on endosymbiotic bacteria that are psychrophile-like. The observed temperature sensitivity develops in fewer than 200 host cell generations (18 months of culture) after the host cells are experimentally infected with the symbionts.     

Land use types with different fertilization management affected isotope ratios of bulk and water-extractable C and N of soils in an intensive agricultural area

Journal of Soils and Sediments - This study was conducted to investigate variations in the stable isotope ratios of carbon (δ13C) and nitrogen (δ15N) of agricultural soils under different...     

Development of a model to predict dietary metabolizable energy from digestible energy in beef cattle

Understanding the utilization of feed energy is essential for precision feeding in beef cattle production. We aimed to assess whether predicting the metabolizable energy (ME) to digestible energy (DE) ratio (MDR), rather than a prediction of ME with DE, is feasible and to develop a model equation to predict MDR in beef cattle. We constructed a literature database based on published data. A meta-analysis was conducted with 306 means from 69 studies containing both dietary DE and ME concentrations measured by calorimetry to test whether exclusion of the y-intercept is adequate in the linear relationship between DE and ME. A random coefficient model with study as the random variable was used to develop equations to predict MDR in growing and finishing beef cattle. Routinely measured or calculated variables in the field (body weight, age, daily gain, intake, and dietary nutrient components) were chosen as explanatory variables. The developed equations were evaluated with other published equations. The no-intercept linear equation was found to represent the relationship between DE and ME more appropriately than the equation with a y-intercept. The y-intercept (−0.025 ± 0.0525) was not different from 0 (P = 0.638), and Akaike and Bayesian information criteria of the no-intercept model were smaller than those with the y-intercept. Within our growing and finishing cattle data, the animal’s physiological stage was not a significant variable affecting MDR after accounting for the study effect (P = 0.213). The mean (±SE) of MDR was 0.849 (±0.0063). The best equation for predicting MDR (n = 106 from 28 studies) was 0.9410 ( ± 0.02160) +0.0042 ( ± 0.00186) × DMI (kg) – 0.0017 ( ± 0.00024) × NDF(% DM) – 0.0022 ( ± 0.00084) × CP(% DM). We also presented a model with a positive coefficient for the ether extract (n = 80 from 22 studies). When using these equations, the observed ME was predicted with high precision (R² = 0.92). The model accuracy was also high, as shown by the high concordance correlation coefficient (>0.95) and small root mean square error of prediction (RMSEP), <5% of the observed mean. Moreover, a significant portion of the RMSEP was due to random bias (> 93%), without mean or slope bias (P > 0.05). We concluded that dietary ME in beef cattle could be accurately estimated from dietary DE and its conversion factor, MDR, predicted by the dry matter intake and concentration of several dietary nutrients, using the 2 equations developed in this study.     

3,4-Dihydroxyflavone acts as an antioxidant and antiapoptotic agent to support bovine embryo development in vitro

The effects of two antioxidants, superoxide dismutase (SOD) and the flavonoid 3,4-dihydroxyflavone (DHF), on bovine embryo development in vitro were examined. Blastocyst development, total cell and inner cell mass (ICM) numbers, intracellular levels of reactive oxygen species (ROS), apoptotic indices and gene expression levels were examined before and after treatment of day 2 bovine embryos (≥2-4 cells) with various concentrations of 3,4-DHF or SOD for 6 days. Statistical analysis was performed using analysis of variance, with significance defined at the P<0.05 level. SOD had no significant effect on bovine embryo development at any tested concentration (control, 32.8%; 300 U/ml, 33.9%; 600 U/ml, 24.2%). In contrast, 10 μM 3,4-DHF promoted higher blastocyst development (39.3%) than any other concentration (control, 26.7%; 1 μM, 30.3%; 50 μM, 29.5%; 100 μM, 20.5%). Compared with 300 U/ml SOD, 10 μM 3,4-DHF resulted in significantly higher blastocyst development (44.2%) (control, 31.5%; SOD 300 U/ml, 33.6%). Treatment with 3,4-DHF increased the ICM cell number and reduced intracellular ROS production and apoptotic cell numbers. When O(2) tension was decreased from 20% (high tension) to 5% (low tension), embryo development rates were doubled regardless of 3,4-DHF treatment. Under high O(2) tension, 10 μM 3,4-DHF treatment may render bovine embryo development similar to a low O(2) tension environment. The best blastocyst development was obtained under low O(2) tension plus 10 μM 3,4-DHF treatment. The relative expression levels of antioxidant (MnSOD), antiapoptotic (Survivin, Bax inhibitor) and growth-related genes (IFN-τ, Glut-5) were significantly increased after 3,4-DHF treatment, while the expression levels of oxidant (Sox) and apoptotic genes (Caspase-3 and Bax) were reduced. These results suggest that 3,4-DHF may promote the in vitro development of bovine embryos through its antioxidant and antiapoptotic effects.     

Production of transgenic bovine cloned embryos using piggybac transposition

Transgenic research on cattle embryos has been developed to date using viral or plasmid DNA delivery systems. In this study, a different gene delivery system, piggybac transposition, was employed to investigate if it can be applied for producing transgenic cattle embryos. Green or red fluorescent proteins (GFP or RFP) were transfected into donor fibroblasts, and then transfected donor cells were reprogrammed in enucleated oocytes through SCNT and developed into pre-implantation stage embryos. GFP was expressed in donor cells and in cloned embryos without any mosaicism. Induction of RFP expression was regulated by doxycycline treatment in donor fibroblasts and pre-implantational stage embryos. In conclusion, this study demonstrated that piggybac transposition could be a mean to deliver genes into bovine somatic cells or embryos for transgenic research.     

A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle

A high proportion of purebred Hampshire pigs carries the dominant RN- mutation, which causes high glycogen content in skeletal muscle. The mutation has beneficial effects on meat content but detrimental effects on processing yield. Here, it is shown that the mutation is a nonconservative substitution (R200Q) in the PRKAG3 gene, which encodes a muscle-specific isoform of the regulatory gamma subunit of adenosine monophosphate-activated protein kinase (AMPK). Loss-of-function mutations in the homologous gene in yeast (SNF4) cause defects in glucose metabolism, including glycogen storage. Further analysis of the PRKAG3 signaling pathway may provide insights into muscle physiology as well as the pathogenesis of noninsulin-dependent diabetes mellitus in humans, a metabolic disorder associated with impaired glycogen synthesis.     

Tangeretin reduces ultraviolet B (UVB)-induced cyclooxygenase-2 expression in mouse epidermal cells by blocking mitogen-activated protein kinase (MAPK) activation and reactive oxygen species (ROS) generation

The present study examined the effects of tangeretin, a polymethoxylated flavonone present in citrus fruits, on ultraviolet B (UVB)-induced cyclooxygenase-2 (COX-2) expression in JB6 P+ mouse skin epidermal cells. Tangeretin suppressed UVB-induced COX-2 expression and transactivation of nuclear factor-κB and activator protein-1 in JB6 P+ cells. Moreover, tangeretin blocked UVB-induced phosphorylation of Akt and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and p38, and attenuated the phosphorylation of MAPK kinases 1/2, 3/6, and 4. Tangeretin also limited the endogenous generation of reactive oxygen species (ROS), thereby protecting the cells against oxidative stress. However, tangeretin did not scavenge the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and influence the nicotinamide adenine dinucleotide phosphate oxidase activity. These results suggest that the anti-inflammatory effects of tangeretin stem from its modulation of cell signaling and suppression of intracellular ROS generation. Tangeretin may have a potent chemopreventive effect in skin cancer.     

Natural Gradient Drift Tests for Assessing the Feasibility of In Situ Aerobic Cometabolism of Trichloroethylene and Evaluating the Microbial Community Change

The objective of this study is to develop a method for using the single-well natural gradient drift test (SWNGDT) in the field to assess in situ aerobic cometabolism of trichloroethylene (TCE) and to analyze microbial community changes. The SWNGDT was performed in a monitoring well installed in a TCE-contaminated aquifer in Wonju, South Korea. The natural gradient drift biostimulation test (NGDBT) and surrogate test (NGDST) were performed by injecting dissolved solutes (bromide (a tracer), toluene (a growth substrate), ethylene (a nontoxic surrogate substrate to probe for TCE transformation activity), dissolved oxygen (DO, an electron acceptor), and nitrate (nutrient)) into the aquifer. Push?Cpull blocking tests (PPBT) were also performed to examine whether the monooxygenase of toluene oxidizers is involved in the degradation of toluene and the transformation of ethylene. Through the NGDBT, NGDST, and PPBT, we confirmed that the addition of toluene and oxygen in these field tests stimulated indigenous toluene utilizers to cometabolize aerobically TCE, with the following results: (1) the observed simultaneous utilization of toluene and DO; (2) the transformation of ethylene to ethylene oxide and propylene to propylene oxide; and (3) the transformation of TCE. Furthermore, the results of restriction fragment length polymorphism suggested that the microbial community shifts and the microbes capable of transforming TCE are stimulated by injecting the growth substrate, toluene.