Biofortification of iron content by regulating a NAC transcription factor in maize.

Iron (Fe) deficiency remains widespread among people in developing countries. To help solve this problem, breeders have been attempting to develop maize cultivars with high yields and high Fe concentrations in the kernels. We conducted a genome-wide association study and identified a gene, ZmNAC78 (NAM/ATAF/CUC DOMAIN TRANSCRIPTION FACTOR 78), that regulates Fe concentrations in maize kernels. We cultivated maize varieties with both high yield and high Fe concentrations in their kernels by using a molecular marker developed from a 42-base pair insertion or deletion (indel) in the promoter of ZmNAC78. ZmNAC78 expression is enriched in the basal endosperm transfer layer of kernels, and the ZmNAC78 protein directly regulates messenger RNA abundance of Fe transporters. Our results thus provide an approach to develop maize varieties with Fe-enriched kernels.

MIR164b represses iron uptake by regulating the NAC domain transcription factor5-Nuclear Factor Y, Subunit A8 module in Arabidopsis.

Recent findings have revealed the important roles of microRNAs (miRNAs) in the secondary responses to oxidative damage caused by iron (Fe) excess. However, the functional importance of miRNAs in plant responses to Fe deficiency remains to be explored. Here, we show that the expression level of miR164 in Arabidopsis (Arabidopsis thaliana) roots was repressed by Fe deficiency. Primary root length, lateral root number, ferric reductase activity, and mRNA abundance of IRON-REGULATED TRANSPORTER1 (IRT1) and FERRIC REDUCTION OXIDASE2 (FRO2) were higher in the mir164b mutant than in the wild-type (WT) under Fe-deficient conditions. Analysis of the Fe concentrations and ferric reductase activities in the roots of miR164 knockdown transgenic plants showed that members of the miR164 family had different functions in Fe-deficiency responses. Promoter::GUS analysis showed that NAM/ATAF/CUC (NAC) domain transcription factor5 (NAC5) is regulated at both transcriptional and posttranscriptional levels under Fe-deficient conditions. Transgenic Arabidopsis plants overexpressing NAC5 were more tolerant of Fe deficiency than the WT. NAC5 has transactivation activity and directly transactivates the expression of Nuclear Factor Y, Subunit A8 (NFYA8), as demonstrated by chromatin immunoprecipitation followed by quantitative polymerase chain reaction, electrophoretic mobility shift assay (EMSA), and dual-luciferase reporter assay. Like overexpression of NAC5, overexpression of NFYA8 increases primary root length, lateral root number, ferric reductase activity, and mRNA abundance of IRT1 and FRO2 under Fe-deficient conditions. Thus, MIR164b is important for Fe-deficiency responses by its regulation of the NAC5-NFYA8 module.

Changes of serum inflammatory factors and miR-145 expression in patients with osteoarthritis before and after treatment and their clinical value.

BACKGROUND: Osteoarthritis is a chronic degenerative disease with an incidence of 50% in people over 65 years old and 80% in people over 80 years old worldwide. It is the second leading reason of loss of working capacity after cardiovascular diseases and severely affects the society and families. Therefore, finding biological markers related to the diagnosis and treatment of osteoarthritis is of great significance in clinical practice. AIM: To observe the changes and clinical value of serum inflammatory factors and miR-145 expression in patients with osteoarthritis before and after treatment. METHODS: Eighty-three patients with knee osteoarthritis (observation group) who were admitted to our hospital from April 2013 to June 2015, and 60 healthy people (control group) during the same period were selected. After 4 wk of treatment, the levels of miR-145, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 were compared between the control group and the observation group before treatment. The correlation of miR-145, TNF-α, IL-6, and IL-10 levels with visual analogue scale (VAS), Lysholm, and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores was assessed by Pearson correlation analysis. The correlation of the expression of miR-145, TNF-α, IL-6, and IL-10 with Kellgren-Lawrence (K-L) grades was assessed by Spearman correlation analysis. The critical levels of miR-145, TNF-α, IL-6, and IL-10 in distinguishing different K-L grades were determined by receiver operating characteristic (ROC) curve analysis. RESULTS: The expression level of miR-145 in the observation group was significantly higher than that in the control group before treatment (P < 0.05). After treatment, the expression level of miR-145 in the observation group was significantly lower than that before treatment (P < 0.05). The levels of TNF-α and IL-6 in the observation group were significantly higher than those in the control group (P < 0.05), and the level of IL-10 was significantly lower than that in the control group (P < 0.05). After treatment, the levels of TNF-α and IL-6 in the observation group were significantly lower than those before treatment (P < 0.05), and IL-I0 level was significantly higher than that before treatment (P < 0.05). VAS and WOMAC scores were both positively correlated with miR-145, TNF-α, and IL-6 (P < 0.05), and negatively correlated with IL-10 (P < 0.05), while Lysholm scores were negatively correlated with miR-145, TNF-α, and IL-6 (P < 0.05), and positively correlated with IL-10 (P < 0.05). K-L grades were positively correlated with miR-145, TNF-α, and IL-6 (P < 0.05), and negatively correlated with IL-10 (P < 0.05). The area under the ROC curve (AUC) and specificity of TNF-α in differentiating K-L grades I-II were the highest, which were 0.785 and 97.45%, respectively, and miR145 had the highest sensitivity of 94.59%; the AUC and sensitivity of IL-6 in differentiating K-L grades II-III were the highest, which were 0.766 and 97.30%, respectively, and TNF-α had the highest specificity of 86.68%. CONCLUSION: MiR-145 and inflammatory factors have certain diagnostic value in osteoarthritis, and they are expected to become potential indicators for the diagnosis and evaluation of osteoarthritis in the future.

The PILNCR1-miR399 Regulatory Module Is Important for Low Phosphate Tolerance in Maize.

The regulation of adaptive responses to phosphorus (P) deficiency by the microRNA399 (miR399)/PHOSPHATE2 (PHO2) pathway has been well studied in Arabidopsis (Arabidopsis thaliana) but not in maize (Zea mays). Here, we show that miR399 transcripts are strongly induced in maize by phosphate (Pi) deficiency. Transgenic maize plants that overexpressed MIR399b accumulated excessive amounts of P in their shoots and displayed typical Pi-toxicity phenotypes. We reannotated ZmPHO2 with an additional 1,165 bp of the 5' untranslated region. miR399-guided posttranscriptional repression of ZmPHO2 was mainly observed in the P-efficient lines. We identified Pi-deficiency-induced long-noncoding RNA1 (PILNCR1) from our strand-specific RNA libraries. Transient expression assays in Nicotiana benthamiana and maize leaf protoplasts demonstrated that PILNCR1 inhibits ZmmiR399-guided cleavage of ZmPHO2 The abundance of PILNCR1 was significantly higher in P-inefficient lines than in P-efficient lines, which is consistent with the abundance of ZmmiR399 transcripts. These results indicate that the interaction between PILNCR1 and miR399 is important for tolerance to low Pi in maize.

RepSox improves viability and regulates gene expression in rhesus monkey-pig interspecies cloned embryos.

OBJECTIVE: To investigate the effect of the small molecule, RepSox, on the expression of developmentally important genes and the pre-implantation development of rhesus monkey-pig interspecies somatic cell nuclear transfer (iSCNT) embryos. RESULTS: Rhesus monkey cells expressing the monomeric red fluorescent protein 1 which have a normal (42) chromosome complement, were used as donor cells to generate iSCNT embryos. RepSox increased the expression levels of the pluripotency-related genes, Oct4 and Nanog (p < 0.05), but not of Sox2 compared with untreated embryos at the 2-4-cell stage. Expression of the anti-apoptotic gene, Bcl2, and the pro-apoptotic gene Bax was also affected at the 2-4-cell stage. RepSox treatment also increased the immunostaining intensity of Oct4 at the blastocyst stage (p < 0.05). Although the blastocyst developmental rate was higher in the group treated with 25 µM RepSox for 24 h than in the untreated control group (2.4 vs. 1.2%, p > 0.05), this was not significant. CONCLUSION: RepSox can improve the developmental potential of rhesus monkey-pig iSCNT embryos by regulating the expression of pluripotency-related genes.

Asclepiasterol, a novel C21 steroidal glycoside derived from Asclepias curassavica, reverses tumor multidrug resistance by down-regulating P-glycoprotein expression.

Multidrug resistance (MDR) mediated by P-glycoprotein (P-gp) is a major cause of cancer therapy failure. In this study, we identified a novel C21 steroidal glycoside, asclepiasterol, capable of reversing P-gp-mediated MDR. Asclepiasterol (2.5 and 5.0µM) enhanced the cytotoxity of P-gp substrate anticancer drugs in MCF-7/ADR and HepG-2/ADM cells. MDR cells were more responsive to paclitaxel in the presence of asclepiasterol, and colony formation of MDR cells was only reduced upon treatment with a combination of asclepiasterol and doxorubicin. Consistent with these findings, asclepiasterol treatment increased the intracellular accumulation of doxorubicin and rhodamine 123 (Rh123) in MDR cells. Asclepiasterol decreased expression of P-gp protein without stimulating or suppressing MDR1 mRNA levels. Asclepiasterol-mediated P-gp suppression caused inhibition of ERK1/2 phosphorylation in two MDR cell types, and EGF, an activator of the MAPK/ERK pathway, reversed the P-gp down-regulation, implicating the MAPK/ERK pathway in asclepiasterol-mediated P-gp down-regulation. These results suggest that asclepiasterol could be developed as a modulator for reversing P-gp-mediated MDR in P-gp-overexpressing cancer variants.

3'3-Diindolylmethane inhibits migration, invasion and metastasis of hepatocellular carcinoma by suppressing FAK signaling.

Late stage hepatocellular carcinoma (HCC) usually has a low survival rate because it has high potential of metastases and there is no effective cure. 3'3-Diindolylmethane (DIM) is the major product of the acid-catalyzed oligomerization of indole-3-carbinol present in cruciferous vegetables. DIM has been proved to exhibit anticancer properties. In this study, we explored the effects and molecular mechanisms of anti-metastasis of DIM on HCC cells both in vitro and in vivo. We chose two HCC cell lines SMMC-7721 and MHCC-97H that have high potential of invasion. The results showed that DIM inhibited the proliferation, migration and invasion of these two cell lines in vitro. In addition, in vivo study demonstrated that DIM significantly decreased the volumes of SMMC-7721 orthotopic liver tumor and suppressed lung metastasis in nude mice. Focal Adhesion Kinase (FAK) is found over activated in HCC cells. We found that DIM decreased the level of phospho-FAK (Tyr397) both in vitro and in vivo. DIM inhibition of phospho-FAK (Tyr397) led to down-regulation of MMP2/9 and decreased potential of metastasis. DIM also repressed the migration and invasion induced by vitronectin through inactivation of FAK pathway and down-regulation of MMP2/9 in vitro. We also found that pTEN plays a role in down-regulation of FAK by DIM. These results demonstrated that DIM blocks HCC cell metastasis by suppressing tumor cell migration and invasion. The anti-metastasis effect of DIM could be explained to be its down-regulated expression and activation of MMP2/9 partly induced by up-regulation of pTEN and inhibition of phospho-FAK (Tyr397).

Cadmium Induced Apoptosis in MG63 Cells by Increasing ROS, Activation of p38 MAPK and Inhibition of ERK 1/2 Pathways.

BACKGROUND/AIMS: Cadmium (Cd) induces apoptosis in different kinds of cells, including osteoblasts, both in vivo and in vitro. However, little is known about the mechanisms by which Cd induces apoptosis. METHODS: In the present study, we used the human osteosarcoma cell line MG63, which has characteristics similar to human osteoblasts, as an in vitro model to determine the cellular mechanisms by which Cd induces apoptosis. RESULTS: We found that short-term exposure to CdCl2 induced apoptosis in MG63 cells. Furthermore, the incubation of cells with CdCl2 significantly increased the level of phosphorylated p38MAPK and significantly decreased the phosphorylation of ERK1/2 in a concentration-dependent manner. Additionally, the inhibition of the phosphorylation of p38 MAPK by SB202190 protected MG63 cells from Cd-induced apoptosis. The incubation of MG63 cells with the ERK1/2 inhibitor PD98059 significantly increased apoptosis in MG63 cells. CdCl2 also significantly increased the intracellular levels of ROS. N-acetylcysteine (NAC) significantly reduced ROS levels and reversed the effects of CdCl2 on MAPK signaling. CONCLUSION: Our results suggested that Cd induced apoptosis in MG63 cells by increasing ROS, activation of p38 MAPK and inhibition of ERK1/2 pathways.

Thimerosal-induced apoptosis in mouse C2C12 myoblast cells occurs through suppression of the PI3K/Akt/survivin pathway.

BACKGROUND: Thimerosal, a mercury-containing preservative, is one of the most widely used preservatives and found in a variety of biological products. Concerns over its possible toxicity have reemerged recently due to its use in vaccines. Thimerosal has also been reported to be markedly cytotoxic to neural tissue. However, little is known regarding thimerosal-induced toxicity in muscle tissue. Therefore, we investigated the cytotoxic effect of thimerosal and its possible mechanisms on mouse C2C12 myoblast cells. METHODOLOGY/PRINCIPAL FINDINGS: The study showed that C2C12 myoblast cells underwent inhibition of proliferation and apoptosis after exposure to thimerosal (125-500 nM) for 24, 48 and 72 h. Thimerosal caused S phase arrest and induced apoptosis as assessed by flow cytometric analysis, Hoechst staining and immunoblotting. The data revealed that thimerosal could trigger the leakage of cytochrome c from mitochondria, followed by cleavage of caspase-9 and caspase-3, and that an inhibitor of caspase could suppress thimerosal-induced apoptosis. Thimerosal inhibited the phosphorylation of Akt(ser473) and survivin expression. Wortmannin, a PI3K inhibitor, inhibited Akt activity and decreased survivin expression, resulting in increased thimerosal-induced apoptosis in C2C12 cells, while the activation of PI3K/Akt pathway by mIGF-I (50 ng/ml) increased the expression of survivin and attenuated apoptosis. Furthermore, the inhibition of survivin expression by siRNA enhanced thimerosal-induced cell apoptosis, while overexpression of survivin prevented thimerosal-induced apoptosis. Taken together, the data show that the PI3K/Akt/survivin pathway plays an important role in the thimerosal-induced apoptosis in C2C12 cells. CONCLUSIONS/SIGNIFICANCE: Our results suggest that in C2C12 myoblast cells, thimerosal induces S phase arrest and finally causes apoptosis via inhibition of PI3K/Akt/survivin signaling followed by activation of the mitochondrial apoptotic pathway.

Melamine induces sperm DNA damage and abnormality, but not genetic toxicity.

Melamine is a category III carcinogen. However, its illegal addition to milk and other protein products has led to the pet melamine poisoning in USA and infant renal calculus cases in China. Its long-term toxicity is not clear and needs to be investigated urgently to appease the public panic. In this study, the effects of melamine on mutagenesis in prokaryotes and eukaryotes were tested by Ames test, in vitro mammalian chromosome aberration test, mouse micronucleus test and sperm abnormality test; the effects of melamine on DNA damage in sperm cells were observed by single cell gel electrophoresis, and the effects on malignant transformation were examined by malignant transformation experiment of normal human liver cell line L02. The results show that melamine has no mutagenic function in prokaryotes and eukaryotes in vitro and in vivo, and does not induce malignant cell transformation after long-term exposure. However, it has ability to increase sperm abnormality rate and DNA damage.

Cloning and functional analysis of the peanut iron transporter AhIRT1 during iron deficiency stress and intercropping with maize.

In previous research, iron-deficiency symptoms in peanut (Arachis hypgaea) were alleviated during anthesis by intercropping with maize. This benefit was associated with increased phytosiderophore secretion by maize and increased Fe(III)-chelate reductase activity by peanut. In the present study, we isolated the full-length cDNA of AhIRT1 (iron-regulated transporter 1) from peanut and characterized how iron deficiency and intercropping affected its iron-transporting ability. Functional complementation with AhIRT1 restored normal growth of the yeast mutant fet3fet4 (defective in both high- and low-affinity iron-uptake systems) under iron-deficiency conditions. Based on transient expression analysis, AhIRT1 was determined to be a membrane protein, which was consistent with a function in iron uptake. In peanut, transcript levels of AhIRT1 increased in both root and shoot under iron-deficiency conditions. In a pot experiment, AhIRT1 transcript levels in intercropped peanut were 10 times greater during anthesis than pre-anthesis, and transcript levels during anthesis were 40% greater in intercropped than in monocropped peanut.

Regulation of AhFRO1, an Fe(III)-chelate reductase of peanut, during iron deficiency stress and intercropping with maize.

Iron deficiency-induced chlorosis in peanut during anthesis was alleviated when peanut was intercropped with maize in field and pot experiments. Iron acquisition of graminaceous plants is characterized by the synthesis and secretion of the iron-chelating phytosiderophores. Compared to the roots of monocropped maize, the roots of maize intercropped with peanut always secreted higher amounts of phytosiderophores during peanut anthesis. For non-graminaceous plants, reduction of ferric to ferrous iron on the root surface is the rate-limiting step for mobilizing iron from soil. The full-length cDNA, AhFRO1, which is encoding an Fe(III)-chelate reductase, was isolated from peanut. AhFRO1 expression in yeast conferred Fe(III)-chelate reductase activity to the cells. Consistent with its function in iron uptake, AhFRO1 was determined to be a membrane protein by transient expression analysis. AhFRO1 mRNA accumulated under iron deficiency conditions. During pre-anthesis, the Fe(III)-chelate reductase activity and the transcript levels of AhFRO1 were similar in monocropped and intercropped peanut. When the iron deficiency-induced chlorosis developed in the monocropped peanuts, both the Fe(III)-chelate reductase activity of peanut and the transcript levels of AhFRO1 were higher in intercropped than in monocropped peanuts, which is consistent with the secretion of phytosiderophores by maize roots. We conclude that AhFRO1 in peanut and phytosiderophores from maize co-operate to improve the iron nutrition of peanut when intercropped with maize.

[Effects of indole-3-carbinol on the outcome of tumor and the changes of anti-oxidative system in null mice grafted with nasopharyngeal carcinoma].

OBJECTIVE: To observe the effects of indole-3-carbinol (I3C) on the outcome of the tumor as well as the changes of the anti-oxidative system in null mice grafted with nasopharyngeal carcinoma. METHODS: 48 BALB/c null mice were divided by means of random number table into control group (0.5% sodium carboxyl methyl cellulose), low dosage (0.02 g/kg), middle dosage (0.1 g/kg) and high dosage (0.5 g/kg) of I3C. The mice were administered with different solutions by gavage for 10 days before CNE1 cells were inoculated subcutaneously into the back (near the armpit) of the nude mice, then the solutions were continually administered by gavage. The tumor volume was measured and the tumor inhibitory rate was calculated. The level of malondialdehyde (MDA), the activity of superoxide dismutases (SOD), the activity of glutathione peroxidase (GSHPx) and the expression of cleaved caspase-3 were determined on the 31th day of the study. RESULTS: I3C could reduce the tumor volume [the tumor volumes of the control group, the middle dosage group and the high dosage group were (4.13 +/- 0.53) x 10(-6) m(3), (3.14 +/- 0.71) x 10(-6) m(3), (2.72 +/- 0.29) x 10(-6) m(3)], as compared with the control, the shrinkage of tumor volume of the middle dosage group and the high dosage group were significant (the t valued at 0.990 and 1.510, P < 0.01). The tumor inhibitory rates of 3 groups were 3.8%, 20.5% and 34.9%, respectively. The contents of MDA in the tumor tissue tended to decrease [the values of control group, the low dosage group, the middle dosage group and the high dosage group were (31.29 +/- 2.51) x 10(-6) mol/L, (30.12 +/- 2.37) x 10(-6) mol/L, (23.32 +/- 1.93) x 10(-6) mol/L, (16.45 +/- 1.43) x 10(-6) mol/L] (F = 98.752, P < 0.01), and that of the high and the middle dosage group could obviously be reduced (t = 8.970, 14.840, P < 0.01) as compared with the control. The activity of SOD seemed to be elevated according to the increase of I3C dosage [the values were (387.24 +/- 23.16) x 10(3) U/L, (399.37 +/- 34.45) x 10(3) U/L, (431.63 +/- 31.24) x 10(3) U/L, (476.45 +/- 44.67) x 10(3) U/L] (F = 53.444, P < 0.01). When compared with the control, the SOD activity of the middle and the high dosage group be obviously increased (t = 44.390, 89.210, P < 0.01). I3C could also elevate the GSHPx activity [the GSHPx values of the four groups were (226.98 +/- 18.35) x 10(3) U/L, (234.65 +/- 15.59) x 10(3) U/L, (247.72 +/- 22.73) x 10(3) U/L, (300.37 +/- 26.02) x 10(3) U/L] (F = 25.916, P < 0.01). The GSHPx of the high dosage group was enhanced remarkable (t = 73.390, P < 0.01) as compared with the control. The expression of cleaved caspase-3 (relative molecular weight = 19 000 000) seemed to be elevated according to the increase of I3C dosage and the relative expression levels of which were 0.87 +/- 0.01, 0.97 +/- 0.01, 1.02 +/- 0.06 and 1.14 +/- 0.02 (F = 39.864, P < 0.01). When compared with the control, the elevation of this kind of cleaved caspase-3 was considered statistical significant (the t values were 0.100, 0.086 and 0.303, respectively, P < 0.05). When I3C dosage increased, the expression of cleaved caspase-3 (relative molecular weight = 17 000 000) seemed to increased too [the relative expression levels of which were 0.00 +/- 0.00, 0.05 +/- 0.02, 0.11 +/- 0.02, 0.20 +/- 0.02 (F = 56.629, P < 0.01)], and the increase of this kind of cleaved caspase-3 was esteemed significantly as compared with those of the control (the t valued at 0.046, 0.103 and 0.193, respectively, P < 0.05). Linear correlate analysis showed that the correlation coefficients between the shrinkage of tumor volume and the expression of the two kinds of cleaved caspase-3 protein was -0.732 (t = 3.404, P < 0.01) and -0.901 (t = 6.642, P < 0.01). CONCLUSION: I3C could reduce the growth of tumor, the mechanism underlie it could be related to the decrease of the content of MDA as well as the elevated levels of SOD, GSHPx, and perhaps could be related to the apoptosis transduced by cleaved caspase-3.

[Establishment and application of oncogene over expressed human epithelial cell transformation model].

OBJECTIVE: To establish human bronchial epithelial cell lines over expressing oncogene and to investigate its application in detection of carcinogen-induced cell transformation. METHODS: Mediated by retrovirus infection, human telomerase catalytic subunit, hTERT was introduced into immortal human bronchial epithelial cells (16HBE) and followed by introduction of the oncogenic allele H-Ras(V12), or c-Myc or empty vector, creating cell lines 16HBETR, 16HBETM and 16HBETV, respectively. Biological characteristics of these cell lines including morphology, proliferation, and chromosomal aberration were examined to access whether they were transformed. Soft agar experiment and nude mice subcutaneous injection were performed using pre-transformed 16HBE cells induced by known carcinogens, nickel sulfate (NiSO4) and 7, 8, -dihydrodiol-9, 10-epoxide benzo[a] pyrene (BPDE). RESULTS: With detection of telomerase activity and Western blotting, the expression of target proteins was verified. Thus, the transgenic 16HBE cell lines were successfully established. Cells expressing oncogene H-Ras or c-Myc grew 30.3% or 10.4% faster than control cells. However, these cells failed to form colonies in soft agar or form tumor in nude mice. 16HBETR, 16HBETM cells obtained transformed phenotype at 5 wks, 11 wks, respectively after treatment with BPDE, which are 15 wks and 9 wks earlier than control cells 16HBETV (20 wks). Meanwhile, 16HBETR, 16HBETM cells obtained transformed phenotype at 11 wks, 14 wks, respectively after treatment with nickel sulfate, which are 21 wks and 18 wks earlier than control cells (32 wks). CONCLUSION: With the advantage of shorter latency, transgenic human cell transformation models could be used in potent carcinogen screening and applied to chemical-carcinogenesis mechanism study.