Effect of Single Nucleotide Polymorphisms of Toll-Like Receptor 4 (TLR 4) on Reproductive Performance and Immune Function in Dairy Cows

In dairy cows, inflammatory diseases caused by infection with pathogenic bacteria post calving affect ovarian functions. This study examined the relationship between single-nucleotide polymorphisms (SNPs) of Toll-like receptor 4 (TLR4), reproductive performances [the number of artificial insemination (AI) application and days open], and immune cell functions (apoptosis and migration). Two hundred Holstein cows from the Obihiro University farm were included. The SNPs of TLR4 were genotyped by PCR-restriction fragment length polymorphism (PCR–RFLP) method. Polymorphonuclear leukocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) were isolated from whole blood. The number of AI application in the animals with T/C genotype in the TLR4 exon3 was lower than that in animals with C/C genotype (1.6 ± 0.2 and 2.2 ± 0.2, respectively). Among the animals with TLR4 exon3 polymorphisms, the days open was shorter for the T/C cows than that for C/C cows (100.7 ± 6.9 days and 136.6 ± 9.0 days, respectively). The SNPs in the TLR4 intron did not affect the number of AI and days open. The apoptosis percentage of PMNs treated with lipopolysaccharide (LPS; 0.001 and 1 μg/ml) tended to be lower in the T/C genotype compared to that in the C/C genotype. The transmigration rates of PMNs, and IL-1β production in PBMCs were tended to be higher for the animals with the T/C genotype compared to those for animals with the C/C genotype. Taken together, these results suggest that TLR4 polymorphisms offer a meaningful tool to judge the reproductive potential and immune activity in individual cows.     

Reevaluation of <Emphasis Type="Italic">Pholiota squarrosa</Emphasis> lectin-reactive haptoglobin as a pancreatic cancer biomarker using an improved ELISA system

An increase in Lewis- and core-type fucosylation of haptoglobin has been reported in patients with pancreatic cancer (PC), suggesting that fucosylated haptoglobin is a candidate PC biomarker. Previously, we developed a Pholiota squarrosa lectin antibody enzyme-linked immunosorbent assay (PhoSL-ELISA) system for the detection of core-fucosylated haptoglobin. However, with this methodology, positive results were only obtained for some patients with PC, demonstrating the need for a more sensitive detection system. In the current study, we developed an improved PhoSL-ELISA system with higher sensitivity to detect core-fucosylated haptoglobin using high-concentration urea as a denaturing agent with lectin to facilitate detection. We then reevaluated the performance of PhoSL reactive-core-fucosylated haptoglobin (PhoSL-HP) as a PC biomarker using the improved PhoSL-ELISA system. PhoSL-HP levels in the sera of patients with PC were significantly higher than those in healthy volunteers, with an area under the curve (AUC) value of 0.753. Furthermore, the AUC value of CA19–9 improved from 0.793 to 0.907 when combined with PhoSL-HP. Additionally, several CA19–9-negative cases among the patients with PC were diagnosed as positive for PhoSL-HP. In conclusion, PhoSL-HP detection using our improved ELISA system might allow PhoSL-HP to serve as a potential biomarker for PC and thus might be useful to complement the detection of CA19–9 in PC diagnosis.     

Identification of enzymes responsible for extracellular alginate depolymerization and alginate metabolism in Vibrio algivorus

Alginate is a marine non-food-competing polysaccharide that has potential applications in biorefinery. Owing to its large size (molecular weight >300,000 Da), alginate cannot pass through the bacterial cell membrane. Therefore, bacteria that utilize alginate are presumed to have an enzyme that degrades extracellular alginate. Recently, Vibrio algivorus sp. SA2^T was identified as a novel alginate-decomposing and alginate-utilizing species. However, little is known about the mechanism of alginate degradation and metabolism in this species. To address this issue, we screened the V. algivorus genomic DNA library for genes encoding polysaccharide-decomposing enzymes using a novel double-layer plate screening method and identified alyB as a candidate. Most identified alginate-decomposing enzymes (i.e., alginate lyases) must be concentrated and purified before extracellular alginate depolymerization. AlyB of V. algivorus heterologously expressed in Escherichia coli depolymerized extracellular alginate without requiring concentration or purification. We found seven homologues in the V. algivorus genome (alyB, alyD, oalA, oalB, oalC, dehR, and toaA) that are thought to encode enzymes responsible for alginate transport and metabolism. Introducing these genes into E. coli enabled the cells to assimilate soluble alginate depolymerized by V. algivorus AlyB as the sole carbon source. The alginate was bioconverted into l-lysine (43.3 mg/l) in E. coli strain AJIK01. These findings demonstrate a simple and novel screening method for identifying polysaccharide-degrading enzymes in bacteria and provide a simple alginate biocatalyst and fermentation system with potential applications in industrial biorefinery.

     

Biodegradation of toxic organic compounds using a newly isolated <Emphasis Type="Italic">Bacillus</Emphasis> sp. CYR2

The objective of this study was to isolate a new bacterium and investigate its ability for degradation of various toxic organic compounds. Based on 16S rRNA gene sequence and phylogenetic analysis, the isolated strain was identified as Bacillus sp. CYR2. Degradation of various toxic compounds and growth of CYR2 strain were evaluated with 2 and 4% inoculum sizes. All the experiments were conducted for 6 days, flasks were incubated at 30oC under 180 rpm. Among the 2 and 4% inoculum sizes, bacteria showed highest growth and toxic compounds degradation at 4% inoculum size. Especially, compared to 2% inoculum size, growth of the strain CYR2 at 4% inoculum size was increased by 15.1 folds with 4-secondarybutylphenol, 9.1 folds with phenol, and 5.4 folds with 4-tertiary-butylphenol. Strain CYR2 at 4% inoculum size showed highest removal of phenol (84 ± 5%), followed by 4-tertiary-butylphenol (66 ± 3%), 4-secondary-butylphenol (63 ± 5%) and 4-nonylphenol (57 ± 6%). Compared with 2% inoculum size, degradation ability of strain CYR2 with 4% inoculum size was enhanced by 3.45 times with 4-tertiary-octylphenol, and 2.53 times with 4-tertiarybutylphenol. Our results indicated that the newly isolated Bacillus sp. CYR2 can be used for in situ bioremediation of phenol and alkylphenols contaminated water.     

Environmental DNA analysis for estimating the abundance and biomass of stream fish

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Inhibitory effects of food additives derived from polyphenols on staphylococcal enterotoxin A production and biofilm formation by Staphylococcus aureus

In this study, we examined the inhibitory effects of 14 food additives derived from polyphenol samples on staphylococcal enterotoxin A (SEA) production and biofilm formation by Staphylococcus aureus. Tannic acid AL (TA), Purephenon 50 W (PP) and Polyphenon 70A (POP) at 0.25 mg/mL and Gravinol®-N (GN), Blackcurrant polyphenol AC10 (BP), and Resveratrol-P5 (RT) at 1.0 mg/mL significantly decreased SEA production by S. aureus C-29 (p < 0.05). TA, GN, BP, and RT significantly inhibited the expression of the sea gene in S. aureus C-29 (p < 0.05), while suppression attempts by PP and POP proved unsuccessful. After result analysis, it can be derived that TA, GN, BP, and RT inhibit the production of SEA. Of the six samples, each one significantly inhibited biofilm formation (p < 0.05). Food additives derived from polyphenols have viability to be used as a means to inhibit the enterotoxin production and control the biofilm formation of foodborne pathogens.     

Nuclear internal transcribed spacer‐1 as a sensitive genetic marker for environmental DNA studies in common carp Cyprinus carpio

The recently developed environmental DNA (eDNA) analysis has been used to estimate the distribution of aquatic vertebrates by using mitochondrial DNA (mtDNA) as a genetic marker. However, mtDNA markers have certain drawbacks such as variable copy number and maternal inheritance. In this study, we investigated the potential of using nuclear DNA (ncDNA) as a more reliable genetic marker for eDNA analysis by using common carp (Cyprinus carpio). We measured the copy numbers of cytochrome b (CytB) gene region of mtDNA and internal transcribed spacer 1 (ITS1) region of ribosomal DNA of ncDNA in various carp tissues and then compared the detectability of these markers in eDNA samples. In the DNA extracted from the brain and gill tissues and intestinal contents, CytB was detected at 95.1 ± 10.7 (mean ± 1 standard error), 29.7 ± 1.59 and 24.0 ± 4.33 copies per cell, respectively, and ITS1 was detected at 1760 ± 343, 2880 ± 503 and 1910 ± 352 copies per cell, respectively. In the eDNA samples from mesocosm, pond and lake water, the copy numbers of ITS1 were about 160, 300 and 150 times higher than those of CytB, respectively. The minimum volume of pond water required for quantification was 33 and 100 mL for ITS1 and CytB, respectively. These results suggested that ITS1 is a more sensitive genetic marker for eDNA studies of C. carpio.     

Manganese and cobalt activate zebrafish ovarian cancer G-protein-coupled receptor 1 but not GPR4

Mammalian ovarian G-protein-coupled receptor 1 (OGR1) is activated by some metals in addition to extracellular protons and coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebrafish OGR1, zebrafish GPR4, and human GPR4 (zOGR1, zGPR4, and hGPR4, respectively) could sense the metals and activate the intracellular signaling pathways. On one hand, we found that only manganese and cobalt of the tested metals stimulated SRE-promoter activities in zOGR1-overexpressed HEK293T cells. On the other hand, none of the metals tested stimulated the promoter activities in zGPR4- and hGPR4-overexpressed cells. The OGR1 mutant (H4F), which is lost to activation by extracellular protons, did not stimulate metal-induced SRE-promoter activities. These results suggest that zOGR1, but not GPR4, is also a metal-sensing G-protein-coupled receptor in addition to a proton-sensing G-protein-coupled receptor, although not all metals that activate hOGR1 activated zOGR1.     

Different population responses of three sympatric rodent species to acorn masting—the role of tannin tolerance

Rodent population dynamics are predicted to respond positively to the masting of acorns, but diverging results have been published. This study tested the hypothesis that population responses to acorn masting vary depending on differences in the tolerance to tannins of different rodent species. The effects of acorn abundance on the rodent population densities were analyzed using a dataset obtained in Hokkaido, Japan. Specifically, population fluctuations of three rodent species (Apodemus speciosus, A. argenteus, and Myodes rufocanus) and the abundance of Quercus crispula acorns have been monitored since 1992. Acorn production in previous years had a positive effect on the annual population growth rates of A. speciosus; however, this trend was not clear in the other two species. Tannin tolerance, assessed by body weight changes in an acorn feeding experiment, exhibited clear differences among the rodent species; namely, the body weight of A. speciosus increased, whereas that of the other two species decreased. The observed responses to acorn masting of populations of the three sympatric rodent species reflected tannin tolerance. It suggests that only populations of species with high tannin tolerance positively respond to acorn masting. Previous studies tend to overlook species-specific abilities of coping with tannins in acorns. Our results emphasize the necessity of evaluating the tannin tolerance of rodents and the tannin content of acorns to understand how rodent population dynamics and acorn production are related.