Multiomics analysis reveals the molecular mechanisms underlying virulence in Rhizoctonia and jasmonic acid-mediated resistance in Tartary buckwheat (Fagopyrum tataricum).

Rhizoctonia solani is a devastating soil-borne pathogen that seriously threatens the cultivation of economically important crops. Multiple strains with a very broad host range have been identified, but only 1 (AG1-IA, which causes rice sheath blight disease) has been examined in detail. Here, we analyzed AG4-HGI 3 originally isolated from Tartary buckwheat (Fagopyrum tataricum), but with a host range comparable to AG1-IA. Genome comparison reveals abundant pathogenicity genes in this strain. We used multiomic approaches to improve the efficiency of screening for disease resistance genes. Transcriptomes of the plant-fungi interaction identified differentially expressed genes associated with virulence in Rhizoctonia and resistance in Tartary buckwheat. Integration with jasmonate-mediated transcriptome and metabolome changes revealed a negative regulator of jasmonate signaling, cytochrome P450 (FtCYP94C1), as increasing disease resistance probably via accumulation of resistance-related flavonoids. The integration of resistance data for 320 Tartary buckwheat accessions identified a gene homolog to aspartic proteinase (FtASP), with peak expression following R. solani inoculation. FtASP exhibits no proteinase activity but functions as an antibacterial peptide that slows fungal growth. This work reveals a potential mechanism behind pathogen virulence and host resistance, which should accelerate the molecular breeding of resistant varieties in economically essential crops.

[Current quality standards of cattle bile and sheep bile quality standards and discussion on related problems].

To analyze quality standards of cattle bile and sheep bile, and to discuss the related problems in the standards. The results showed that physical forms of the related medicinal materials of cattle bile and sheep bile were chaotic, and the technical methods adopted in the quality standards were generally backward. In addition, there were still problems that some medicinal material standards lacked necessary test items, which were especially obvious in the relevant medicinal material standards of sheep bile and brought difficulties to quality evaluation and control. We suggest that physical forms of cattle bile and sheep bile in quality standards should be determined, and inspection items should be completed. Based on mainstream analytical technology, some technical methods of these standards should be improved.

Mechanisms of Intracellular Calcium Homeostasis in MC3T3-E1 Cells and Bone Tissues of Sprague-Dawley Rats Exposed to Fluoride.

Calcium homeostasis of osteoblasts (OBs) has an important role in the physiology and pathology of bone tissue. In order to study the mechanisms of intracellular calcium homeostasis, MC3T3-E1 cells and Sprague-Dawley rats were treated with different concentrations of fluoride. Then, we examined intracellular-free calcium ion ([Ca(2+)]i) in MC3T3-E1 cells as well as mRNA and protein levels of Cav1.2, the main subunit of L-type voltage-dependent calcium channels (VDCCs), Na(+)/Ca(2+) exchange carriers (NCS), and plasma membrane Ca(2+)-ATPase (PMCA), inositol 1,4,5-trisphosphate receptor (IP3R) channels, sarco/endoplasmic reticulum calcium ATPase 2b (SERCA2b)/ATP2A2 in vitro, and rat bone tissues in vivo. Our results showed that [Ca(2+)]i of fluoride-treated OBs increased in a concentration-dependent manner with an increase in the concentration of fluoride. We also found that the low dose of fluoride led to high expression levels of Cav1.2, NCS-1, and PMCA and low expression levels of IP3R and SERCA2b/ATP2A2, while the high dose of fluoride induced an increase in SERCA2b/ATP2A2 levels and decrease in Cav1.2, PMCA, NCS-1, and IP3R levels. These results demonstrate that calcium channels and calcium pumps of plasma and endoplasmic reticulum (ER) membranes keep intracellular calcium homeostasis by regulating Cav1.2, NCS-1, PMCA, IP3R, and SERCA2b/ATP2A2 expression.

Fluoride Affects Calcium Homeostasis by Regulating Parathyroid Hormone, PTH-Related Peptide, and Calcium-Sensing Receptor Expression.

Parathyroid hormone (PTH), PTH-related peptide (PTHrP), and calcium-sensing receptor (CaSR) play important roles in maintaining calcium homeostasis. Here, we study the effect of fluoride on expression of PTH, PTHrP, and CaSR both in vitro and in vivo. MC3T3-E1 cells and Sprague-Dawley rats were treated with different concentrations of fluoride. Then, the free calcium ion concentration in cell culture supernatant and serum were measured by biochemical analyzer. The expression of PTH, PTHrP, and CaSR was analyzed by qRT-PCR and Western blot. We found that the low dose of fluoride increased ionized calcium (i[Ca(2+)]) and the high dose of fluoride decreased i[Ca(2+)] in cell culture supernatant. The low dose of fluoride inhibited the PTH and PTHrP expression in MC3T3-E1 cells. The high dose of fluoride improved the PTHrP expression in MC3T3-E1 cells. Interestingly, we found that NaF decreased serum i[Ca(2+)] in rats. Fluoride increased CaSR expression at both messenger RNA (mRNA) and protein levels in MC3T3-E1 cells and rats. The expression of PTHrP protein was inhibited by fluoride in rats fed regular diet and was increased by fluoride in rats fed low-calcium diet. Fluoride also increased the expression of PTH, NF-kappaB ligand (RANKL), and osteoprotegerin (OPG) in rats. The ratio of RANKL/OPG in rats fed low-calcium food in presence or absence of fluoride was significantly increased. These results indicated that fluoride might be able to affect calcium homeostasis by regulating PTH, PTHrP, and CaSR.

Insulin sensitizing effects of oligomannuronate-chromium (III) complexes in C2C12 skeletal muscle cells.

BACKGROUND: It was known that the insulin resistance in skeletal muscle is a major pathogenic factor in diabetes mellitus. Therefore prevention of metabolic disorder caused by insulin resistance and improvement of insulin sensitivity are very important for the therapy of type 2 diabetes. In the present study, we investigated the ability of marine oligosaccharides oligomannuronate and its chromium (III) complexes from brown alga to enhance insulin sensitivity in C2C12 skeletal muscle cells. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that oligomannuronate, especially its chromium (III) complexes, enhanced insulin-stimulated glucose uptake and increased the mRNA expression of glucose transporter 4 (GLUT4) and insulin receptor (IR) after their internalization into C2C12 skeletal muscle cells. Additionally, oligosaccharides treatment also significantly enhanced the phosphorylation of proteins involved in both AMP activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathways in C2C12 cells, indicating that the oligosaccharides activated both the insulin signal pathway and AMPK pathways as their mode of action. Moreover, oligosaccharides distributed to the mitochondria after internalization into C2C12 cells and increased the expression of transcriptional regulator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), carnitine palmitoyl transferase-1 (CPT-1), and phosphorylated acetyl-CoA carboxylase (p-ACC), which suggested that the actions of these oligosaccharides might be associated with mitochondria through increasing energy expenditure. All of these effects of marine oligosaccharides were comparable to that of the established anti-diabetic drug, metformin. In addition, the treatment with oligosaccharides showed less toxicity than that of metformin. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that oligomannuonate and its chromium (III) complexes improved insulin sensitivity in C2C12 skeletal muscle cells, and acted as a novel glucose uptake stimulator with low toxicity, and could be used as dietary supplementary or potential drug for type 2 diabetes mellitus.

Effects of fluoride on the intracellular free Ca2+ and Ca2+-ATPase of kidney.

In the present study, the effect of fluoride on intracellular free calcium ([Ca2+]i) and Ca2+-ATPase of renal cells were examined. Some paradoxical experimental results about the mechanism of fluoride toxicity were observed. In vivo, 48 Wistar rats were divided into 4 groups, and half of rats were treated with sodium fluoride (NaF) by drinking water (per liter of tap water containing 100 mg F-). Compared with the respective control, the level of [Ca2+]i of the kidney in two fluoride-treated rats obviously increased (p < 0.05); and the activity of Ca2+-ATPase in 100 mg F-/L groups with a standard diet did not significantly increase, and the enzyme activity in 100-mg F-/L group with a low-calcium diet decreased significantly compared to the 100 mg F-/L group with a standard diet (p < 0.05). In vitro, renal tubular cells were cultured and respectively exposed to 1.0, 5.0, 7.5, and 12.5 mg/L fluoride in the culture medium. Results showed the significantly elevated activity of Ca2+-ATPase in the cells exposed to 1.0 and 5.0 mg/L fluoride (p < 0.05), and this enzyme activity indicated inhibitory trend in cells of the 7.5- and 12.5-mg/L fluoride-treated group. To sum up, the effect of fluoride on Ca2+-ATPase is a similar to a dose-effect relationship phenomenon characterized by low-dose stimulation and high-dose inhibition, and the increase of [Ca2+]i probably plays a key role on the mechanism of renal injury in fluorosis.