Characterization of spheres derived from canine mammary gland adenocarcinoma cell lines

There is increasing evidence for the presence of cancer stem cells in several solid tumors, and these cancer stem cells have a potential role in tumor initiation, aggression, and recurrence. The stem cell-like properties of spheres derived from canine mammary tumors remain largely elusive. We attempted to induce sphere formation using four cell lines of canine mammary adenocarcinoma, and characterized the spheres derived from a CHMp line in vitro and in vivo. The CHMp-derived spheres showed predominantly CD44⁺CD24⁻ population, higher expression of stem cell-related genes, such as CD133, Notch3 and MDR, and higher resistance to doxorubicin compared with the CHMp-derived adherent cells. Xenograft transplantations in nude mice demonstrated that only 1 × 10⁴ sphere cells were sufficient for tumor formation. Use of the sphere assay on these sphere-derived tumors showed that sphere-forming cells were present in the tumors, and were maintained in serial transplantation. We propose that spheres derived from canine mammary adenocarcinoma cell lines possess a potential characteristic of cancer stem cells. Spheres derived from canine mammary tumors could be a powerful tool with which to investigate novel therapeutic drugs and to elucidate the molecular and cellular mechanisms that underlie tumorigenesis.     

Formation of carcinogenic 4(5)-methylimidazole in Maillard reaction systems

4(5)-Methylimidazole has received the attention of federal and state regulatory agencies because of its carcinogenicity and common presence in foods and beverages. In the present study, the formation of 4(5)-methylimidazole in Maillard reaction model systems consisting of D-glucose/NH(3), L-rhamnose/NH(3), methylglyoxal/NH(3), and methylglyoxal/formaldehyde/NH(3) was investigated. 4(5)-Methylimidazole was formed at levels ranging from 0.49 to 0.71 mg/mL in the d-glucose/NH(3) model system. The formation of 4(5)-methylimidazole was slightly higher in the L-rhamnose/NH(3) system (0.91 mg/mL) than in the d-glucose/NH(3) system (0.71 mg/mL) under the conditions used in the present study. A methylglyoxal/NH(3) system produced significantly higher levels of 4(5)-methylimidazole (5.70 mg/mL), suggesting that methylglyoxal is an important precursor of 4(5)-methylimidazole. Ammonolysis of methylglyoxal, which is one of the glucose degradation products, was proposed to form formamide, which subsequently reacted with 2-aminopropanal (α-aminocarbonyl intermediate) formed from methylglyoxal to give 4- or 5-methylimidazole. The levels of 4(5)-methylimidazole found in commercial cola soft drinks range from 0.30 μg/mL (brand 3) to 0.36 μg/mL (brands 1 and 5).     

High levels of sugars and fructan in mature seed of sweet wheat lacking GBSSI and SSIIa enzymes

Sweet wheat (SW), which lacks functional granule-bound starch synthase I (GBSSI) and starch synthase IIa (SSIIa), accumulates high levels of free sugars in immature seeds. Here, we examined the effects of the lack of these two enzymes on mature kernel composition. Whole grain flour of SW had higher levels of sugars, particularly maltose, slightly higher ash and protein content, approximately two to three times higher lipid levels, and about twice as much total dietary fiber as parental or wild-type lines. Considerably higher levels of low-molecular-weight soluble dietary fiber (LMW-SDF), largely consisting of fructan, were also detected in SW. Although there were no differences in total amino acid levels, the free amino acid content of SW was approximately 4-fold higher than that of wild type, and the levels of certain free amino acids such as proline were particularly high. Thus, we were able to clearly demonstrate that the lack of GBSSI and SSIIa caused dramatic changes in mature seed composition in SW. These compositional changes suggest that SW flour may provide health benefits when used as a food ingredient.     

Palatability and physicochemical properties of sausages prepared via lactic acid fermentation and drying at low temperature

The present study aimed to assess the palatability of sausages undergoing low‐temperature fermentation and drying process. Lactobacillus sakei D‐1001 or Lactobacillus salivarius A001 were used as starter cultures for fermentation, and the following properties of the sausages were investigated: colony‐forming units of lactic acid bacteria; concentrations of lactic acid, protein, peptides, and free amino acids; distribution of protein; composition of free amino acids; and physical properties and taste. Alterations in the composition of proteins, peptides, and free amino acids as well as in various physical properties were caused by fermentation by lactic acid bacteria. A sensory test indicated that the palatability of the fermented sausages was greater than that of the non‐fermented sausages, particularly in terms of hardness and juiciness. This was considered to be due to protein degeneration and changes in the physical properties of the sausages as a result of fermentation by lactic acid bacteria. However, the taste of the fermented sausages was sourer than that of the non‐fermented sausages, and therefore, inferior. Our study revealed that the palatability of the sausages in terms of hardness and juiciness were increased by low‐temperature fermentation by lactic acid bacteria and the drying process.     

The relationship of readily decomposable organic matter of the paddy soil to its colloidal complexes

The soil nitrogen of paddy soil is found to exist in the form of organic and inorganic nitrogen. In general, the content of inorganic nitrogen is so low that it is mainly occupied by organic nitrogen. Such soil organic nitrogen is characterized by large resistance to decomposition by soil microorganisms, and so its mineralization occurs to a small extent in most cases. However, as revealed by M. Shioiri¹⁾ and T. Harada et al. ^2,3), by some appropriate treatment of paddy soil, a part of soil organic nitrogen becomes decomposed by microorganisms and mineralized under flooded condition. Among various treatment, for example, are Presented the followings: (1) air-drying of soil Prior to flooding; (2) raising the soil temperature during the flooded condition; (3) raising the soil reaction (pH 9.0) by addition of weak alkali solution; (4) the addition of neutral salts such as Nafiuoride, Na-phosphate, Na-oxalate etc., which make soil humus peptizable.     

Model-based forecasting of twig canker incidence of bacterial spot of peach in Fukushima Prefecture

Bacterial spot caused by Xanthomonas arboricola pv. pruni (Xap) is the most important disease that affects peach production. A disease-forecasting model was developed to help growers decide when to apply bactericides and remove diseased, last-year twigs. To predict the incidence of “spring cankers”, peach twigs damaged by Xap, we used 12 years (2009–2020) of data from Fukushima Prefecture to develop a forecasting system using a hierarchical Bayesian model (HBM). The model included the number of fields with a bacterial spot incidence (BSI) on leaves?≥?10% in late September of the previous season and the number of days with rain (≥?10 mm/day) and maximum wind speed (≥?5 m/s) during the previous October as predictors. Using a best-fit cutoff value based on a receiver operating characteristic (ROC) curve, the model achieved a 0.836 accuracy, 0.804 sensitivity, 0.847 specificity, 0.847 precision, and 0.712 F-measure. The model was validated using a fourfold cross-validation (CV) procedure and achieved an average accuracy of 0.847. Thus, the model explained 65.7% of the variability compared to observed frequencies with predicted probabilities of twig canker incidence (TCI)?≥?2% from April to May 2009 to 2020 in Fukushima Prefecture. These results suggest that this disease-forecasting model using HBM based on 12 years of historical data can be used to predict the risk of twig cankers of bacterial spot of peach.

     

Effect of nucleotides supplementation to low‐fish meal feed on long‐chain polyunsaturated fatty acid composition of juvenile rainbow trout Oncorhynchus mykiss

A feeding trial was conducted to evaluate the effect of nucleotides supplementation to low‐fish meal feed on growth and fatty acid composition of rainbow trout. Six isonitrogenous (42% crude protein) and isolipidic (18% crude lipid) diets were formulated containing fish meal and plant ingredients as main protein sources. The control diet was a basal diet without supplementation of nucleotides, and five experimental diets were prepared by supplementing one of the five different nucleotides in the form of 5′‐monophosphate (0.15%), that is inosine (IMP), adenosine (AMP), guanosine (GMP), uridine (UMP) and cytidine (CMP) onto basal diet. Two hundred forty juvenile rainbow trout with an initial average body weight 9.8 g were randomly distributed into twelve aquaria. After 15 weeks of feeding period, growth performance and feed utilization of rainbow trout were not significantly different among dietary treatments. Dietary GMP, UMP and CMP tended to accumulate crude lipid in the muscle and whole fish body. Moreover, dietary GMP, UMP and CMP significantly increased hepatic 18:3n‐3 and long‐chain homologue 18:4n‐3 and 20:4n‐3 contents. Hepatic 18:2n‐6 content showed also increase in fish fed GMP, UMP and CMP diets, but decreased in long‐chain homologue 20:3n‐6 and 20:4n‐6 contents. Decrease in 20:4n‐6, 20:5n‐3 and 22:6n‐3 contents was also found in the muscle of fish fed IMP, GMP and CMP diets. The present study clearly showed that there was no positive effect of dietary nucleotides on growth of fish, but dietary nucleotides particularly GMP, UMP and CMP altered polyunsaturated fatty acid composition of rainbow trout.     

The profile of urinary lipid metabolites in healthy dogs

Polyunsaturated fatty acids, including arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), are converted to hundreds of lipid mediators by cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 (CYP), or through non-enzymatic processes, and they reflect inflammatory states of the body. We comprehensively analyzed lipid metabolites in dog urine using a liquid chromatograph-mass spectrometry (LC-MS/MS) to describe their metabolic characteristics. We detected 31 AA-derived metabolites, four EPA-derived metabolites, and a DHA-derived metabolite in all urine samples. Among AA-derived metabolites, 15, 5, 3, and 8 were generated by COX, LOX, CYP, and non-enzymatic oxidation respectively. This study will be the first step to use profiles of urinary lipid metabolites for better understanding and diagnosis of canine diseases.     

Seasonality of serum levels of vitellogenin in male Japanese whiting, Sillago japonica, reared under natural temperature and photoperiod

ABSTRACT:   Because blood vitellogenin (Vg) has been considered a biomarker for environmental estrogens, the basal levels of Vg and 17β-estradiol (E₂) were determined in male Japanese whiting reared under natural conditions. Serum levels of Vg and E₂ were measured and gonadal development was assessed by gonadosomatic index (GSI) and histological observation in 8–10 male fish at monthly intervals throughout the annual reproductive cycle. Serum E₂ was <60 pg/mL throughout the study period. In contrast, serum Vg exhibited seasonal changes: serum levels of Vg gradually increased from April to May (mean 63 ± 13 ng/mL and 124 ± 48 ng/mL in April and May, respectively), and then reached a peak value (mean 352 ± 68 ng/mL) in June. Thereafter, serum Vg gradually decreased, reaching undetectable levels (<50 ng/mL) in October. Serum levels of Vg tended to increase in the male fish in which the GSI was >1%. Histological observation revealed that testes in such male fish were in active spermatogenesis and then all of the testes of male fish in which serum Vg decreased to ND levels were regressed. These results suggest that Vg productive potency (sensitivity to estrogens) may increase in the spermatogenic stage, resulting in production of Vg in response to very low levels of natural or xenobiotic estrogens.     

Generation of PDX‐1 mutant porcine blastocysts by introducing CRISPR/Cas9‐system into porcine zygotes via electroporation

Recently, we established the GEEP (“gene editing by electroporation of Cas9 protein”) method, in which the CRISPR/Cas9 system, consisting of a Cas9 protein and single guide RNA (sgRNA), is introduced into pig zygotes by electroporation and thus induces highly efficient targeted gene disruption. In this study, we examined the effects of sgRNA on the blastocyst formation of porcine embryos and evaluated their genome‐editing efficiency. To produce an animal model for diabetes, we targeted PDX‐1 (pancreas duodenum homeobox 1), a gene that is crucial for pancreas development during the fetal period and whose monoallelic disruption impairs insulin secretion. First, Cas9 protein with different sgRNAs that targeted distinct sites in the PDX‐1 exon 1 was introduced into in vitro‐fertilized zygotes by the GEEP method. Of the six sgRNAs tested, three sgRNAs (sgRNA1, 2, and 3) successfully modified PDX‐1 gene. The blastocyst formation rate of zygotes edited with sgRNA3 was significantly (p < 0.05) lower than that of control zygotes without the electroporation treatment. Our study indicates that the GEEP method can be successfully used to generate PDX‐1 mutant blastocysts, but the development and the efficiency of editing the genome of zygotes may be affected by the sgRNA used for CRISPR/Cas9 system.