[Effects of the Application of Irrigation Water Containing Zn at the Key Growth Period on the Uptake and Transport of Cd in Rice].

In this study， a field experiment was conducted to examine the effects of the application of irrigation water containing Zn at the key growth period （booting stage and filling stage） on exchangeable Cd content in the soil， Cd concentration in pore water， and Cd uptake and transport in rice in a Cd-contaminated paddy field in Liuyang City， Hunan Province. The results indicated that： ① the application of irrigation water containing Zn during the key growth period could inhibit the releasing process of exchangeable Cd from the soil into pore water. Compared with that in the control， the content of exchangeable Cd in soil was slightly changed， but the concentration of Cd in soil pore water at the mature stage was significantly reduced by 16.7%-57.6%. ② The application of irrigation water containing Zn at the key growth period could significantly reduce the Cd content in various parts of rice. Cd contents in root， stem， and brown rice with the application of irrigation water containing 20 mg·L-1 Zn before the booting and the filling stage （BF1） were significantly decreased by 56.0%， 83.8%， and 85.2%， respectively. ③ Compared with the application of 100 mg·L-1 irrigation water containing Zn， the application of 20 mg·L-1 irrigation water containing Zn significantly reduced the uptake and transport of Cd in rice， and the translocation factor （TF） of Cd from rice roots to stems was also significantly reduced by 12.5%-56.3%， with the B1 and BF1 treatments reaching significant levels. These results suggested that the application of irrigation water containing Zn could significantly reduce the uptake and accumulation of Cd in rice， and the application of 20 mg·L-1 irrigation water containing Zn before the booting and filling stage could effectively realize the safe production of Cd-contaminated paddy fields.

[Effects of Straw Removal Measure on Soil Cd Bioavailability and Rice Cd Accumulation].

A field experiment was conducted in a lightly Cd-contaminated rice field in Ningxiang City, Hunan Province, to study the effects of straw removal measures on the soil Cd bioavailability and rice Cd accumulation. The results showed that:① two consecutive seasons of straw removal measures (T1-T4 treatments) effectively increased soil pH by 0.04-0.58 units, reduced soil organic matter by 0.68%-25.87%, and reduced the Cd content of rhizosphere soil by 3.76%-12.78%. ② The proportions of Cd in the acid-extractable fraction and oxidizable fraction decreased, and the proportion of Cd in the residual fraction increased. Furthermore, straw removal measures significantly reduced the bioavailability of Cd in rhizosphere soil, and the Cd contents in TCLP, DTPA, and CaCl2 extracts all significantly decreased compared with those in CK. ③ The straw removal measure could significantly reduce the content of DOC and Cd in soil pore water; and the contents of Cd in soil pore water decreased by 4.54%-40.00% and 2.75%-67.34% under the straw removal measure (T1-T4) for two consecutive seasons, respectively, indicating that DOC was one of the key factors affecting the content of Cd in soil pore water. ④ Two consecutive straw removal measures (T1-T4) reduced the accumulation of Cd in different rice tissues, among which, under the treatment of all straw and root removal (T4), the Cd contents of brown rice in late rice planting in 2020 and early rice planting in 2021 decreased by 18.52% and 39.69%, respectively. Therefore, full or partial removal of straw in Cd-contaminated rice fields is a powerful measure to reduce the risk of exceeding Cd levels in brown rice.

Simultaneous alleviation of Cd availability in contaminated soil and accumulation in rice (Oryza sativa L.) by Fe-Mn oxide-modified biochar.

Fe-Mn oxide-modified biochar (BC-FM) was used to remediate Cd-contaminated soil and mitigate Cd accumulation in rice. The roles of Fe and Mn in soil Cd immobilization and in controlling Cd uptake by rice were investigated via X-ray photoelectron spectroscopy (XPS) characterization and chemical analysis. Fe and Mn loaded on BC-FM increased the removal efficiencies of CaCl2 extractable Cd in soil and Cd in pore water compared to those in only biochar (BC)-treated soil, with maximum removal rates at 67.9 % and 77.8 %, respectively. The XPS results indicated that the redox reactions of the Fe-Mn oxides on BC-FM surface affected Cd immobilization in the soil. The Fe (II/III) components on BC-FM were primarily converted to Fe3O4 in the soil system, which may form stable complexes with Cd2+ (Fe-O-Cd) during the entire rice growth period, and Cd may be bound to MnO or Mn2O3 in the form of CdMn2O4. The excellent adsorption performance of BC-FM enhanced by Fe-Mn oxides reduced the available Cd in the soil and stimulated Fe and Mn transport in rice, thereby inhibiting Cd accumulation in the aerial parts of rice. Cd concentrations in brown rice under BC-FM treatments reached the national safety standard (0.2 mg/kg, GB2762-2017). And BC-FM significantly increased the biomass of brown rice with a maximum rate of 26.8 %. These findings suggest that BC-FM could be used as an efficient material for Cd-contaminated soil remediation, and Fe-Mn plays important role in immobilizing Cd in soil and reducing Cd transport in rice.

Adsorption Characteristics and Mechanisms of Fe-Mn Oxide Modified Biochar for Pb(II) in Wastewater.

This study prepared iron-manganese oxide-modified biochar (FM-BC) by impregnating rice straw biochar (BC) with a mixed solution of ferric nitrate and potassium permanganate. The effects of pH, FM-BC dosage, interference of coexisting ions, adsorption time, incipient Pb(II) concentration, and temperature on the adsorption of Pb(II) by FM-BC were investigated. Moreover, the Pb(II) adsorption mechanism of FM-BC was analyzed using a series of characterization techniques. The results showed that the Fe-Mn oxide composite modification significantly promoted the physical and chemical functions of the biochar surface and the adsorption capacity of Pb(II). The specific surface area of FM-BC was 18.20 times larger than that of BC, and the maximum Pb(II) adsorption capacity reached 165.88 mg/g. Adsorption kinetic tests showed that the adsorption of Pb(II) by FM-BC was based on the pseudo-second-order kinetic model, which indicated that the adsorption process was mainly governed by chemical adsorption. The isothermal adsorption of Pb(II) by FM-BC conformed to the Langmuir model, indicating that the adsorption process was spontaneous and endothermic. Characterization analyses (Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy) showed that the adsorption mechanism of Pb(II) by FM-BC was mainly via electrostatic adsorption, chemical precipitation, complexation, ion exchange, and the transformation of Mn2O3 into MnO2. Therefore, FM-BC is a promising adsorbent for Pb(II) removal from wastewater.

[Effects of Phosphorus Sufficiency and Deficiency on Cadmium Uptake and Transportation by Rice].

Phosphorus (P) is an essential nutrient element for crop growth. The effects of P surplus or deficit on Cd absorption and transport in rice in Cd-polluted farmland is not clear. The effects of P deficiency and P sufficiency on Cd uptake, transport, and accumulation in rice under Cd stress were investigated by applying different levels of phosphorus (NaH2PO4) in a hydroponic experiment. The results showed that:① with the increase in ρ(P) (1.5-48.0 mg·L-1), the biomass in all parts of the rice plants had no obvious change, and the contents of photosynthetic pigment (chlorophyll a, chlorophyll b, and carotenoid) firstly ascended and then descended; high concentrations of P inhibited the synthesis of photosynthetic pigments. ② Under Cd stress, when the P was deficient (1.5-6.0 mg·L-1) or sufficient (12.0-48.0 mg·L-1), the Cd content in different parts of the rice increased with the increase in P addition level, and the maximum increase in Cd content in brown rice was 132.1% and 191.2%, respectively. ③ The P/Cd of brown rice showed a piecewise decreasing rule under P deficiency and P sufficiency, and the Cd content in brown rice was significantly negatively correlated with P/Cd (P<0.01). These results indicated that elevating phosphorus concentration when rice was under both the conditions of P deficiency and P sufficiency could promote the uptake and transport of Cd by rice roots under Cd stress, thus increasing the accumulation of Cd in aboveground parts and the risk of excessive Cd in rice.

Enhancing Cd(II) adsorption on rice straw biochar by modification of iron and manganese oxides.

Metal oxide-modified biochar showed excellent adsorption performance in wastewater treatment. Iron nitrate and potassium permanganate were oxidative modifiers through which oxygen-containing groups and iron-manganese oxides could be introduced into biochar. In this study, iron-manganese (Fe-Mn) oxide-modified biochar (BC-FM) was synthesized using rice straw biochar, and the adsorption process, removal effect, and the mechanism of cadmium (Cd) adsorption on BC-FM in wastewater treatment were explored through batch adsorption experiments and characterization (SEM, BET, FTIR, XRD, and XPS). Adsorption kinetics showed that the maximum adsorption capacity of BC-FM for Cd(II) was 120.77 mg/g at 298 K, which was approximately 1.5-10 times the amount of adsorption capacity for Cd(II) by potassium-modified or manganese-modified biochar as mentioned in the literature. The Cd(II) adsorption of BC-FM was well fit by the pseudo-second-order adsorption and Langmuir models, and it was a spontaneous and endothermic process. Adsorption was mainly controlled via a chemical adsorption mechanism. Moreover, BC-FM could maintain a Cd removal rate of approximately 50% even when reused three times. Cd(II) capture by BC-FM was facilitated by coprecipitation, surface complexation, electrostatic attraction, and cation-π interaction. Additionally, the loaded Fe-Mn oxides also played an important role in the removal of Cd(II) by redox reaction and ion exchange in BC-FM. The results suggested that BC-FM could be used as an efficient adsorbent for treating Cd-contaminated wastewater.

Differential expression of Wilms' tumour 1 gene in porcine urogenital organs during development.

Wilms' tumour 1 gene (WT1) is essential for the development of mammalian urogenital system. However, the expression pattern of WT1 in the development of porcine urogenital organs is still unclear. Here, we examined the expression of WT1 mRNA and protein in porcine kidneys, ovaries and testes from embryonic days 35 and 60 (E35d, E60d, n = 3) to the newborn (0d, n = 4) and adult (210d, n = 3) stages, using real-time PCR and immunofluorescent staining. Real-time PCR analysis showed that porcine kidneys, ovaries and testes all expressed high level of WT1 mRNAs, especially in adult testes (p < 0.05 or 0.01 vs. kidney and ovary, respectively). Morphologically, characteristic microstructures of the kidneys, ovaries and testes were observed and discerned at all four stages. Immunofluorescently, WT1 expression was detected in a dynamic and context-specific pattern during the development of these organs. Taken together, porcine urogenital organs express relatively high levels of WT1 mRNA. Dynamical and context-specific expression profile of WT1 in these organs occurs during their development, implying its close association with the development and function of porcine kidney, ovary and testis.

Sodium Fluoride Affects DNA Methylation of Imprinted Genes in Mouse Early Embryos.

Fluorine is reported to affect embryonic development, but the underlining mechanism is unclear. The modification of DNA methylation of the H19 and Peg3 genes is important in embryonic development. Therefore, the effect of fluorine on methylation of H19 and Peg3 during early mouse embryos was studied. It was shown that the H19 gene was significantly downmethylated in E2.5, E3.5, and E4.5 embryos from pregnant mice treated with 120 mg/l NaF in drinking water for 48 h. But methylation of both H19 and Peg3 genes was disrupted when the parent male mice were treated with NaF for 35 days. H19 DNA methylation decreased significantly, while Peg3 was almost completely methylated. However, when pregnant mice, mated with NaF-treated male mice, were again treated with NaF for 48 h, either H19 or Peg3 methylation in the embryos decreased significantly. In addition, the mRNA level of H19 considerably increased in E3.5 and E4.5 embryos from NaF-treated pregnant mice. Further, the expression of DNMT1 decreased significantly after NaF treatment. Conclusively, we demonstrated that fluorine may adversely affect early embryonic development by disrupting the methylation of H19 and Peg3 through downregulation of DNMT1.

Sodium butyrate-induced histone hyperacetylation up-regulating WT1 expression in porcine kidney fibroblasts.

OBJECTIVES: Wilms' tumor 1 gene (WT1) is essential for the development of kidney and histone acetylation and is involved in its expression regulation in mice. However, whether WT1 expression is associated with histone acetylation in porcine kidney cells is unclear. Here, the effect of histone deacetylase inhibitor sodium butyrate (NaBu)-induced hyperacetylation on WT1 expression in porcine kidney fibroblasts (PKF) was examined. RESULTS: Treatments of NaBu (1, 3, 6 mM) for 24 h increased PKF viability, and 24, 48 h-treatments of 1 mM NaBu enhanced PKF proliferation. WT1 mRNA levels were significantly elevated in NaBu-treated (1, 3 mM for 24, 48 h, respectively) PKF samples. Consistently, strengthened expression of WT1 protein and histone acetylation level were detected in NaBu-treated PKF cells. CONCLUSION: Together, NaBu-induced hyperacetylation up-regulates WT1 expression in PKF, suggesting the involvement of histone acetylation in the transcriptional modulation of WT1 in porcine kidney cells.

Effects of WT1 gene downregulation on apoptosis in porcine fetal fibroblasts.

Wilms' tumor gene 1 (WT1) is located on chromosome 11p13. Besides a role in the development of Wilms' tumor, specific mutations in the Zn finger region are found in Denys-Drash syndrome and Frasier syndrome, both characterized by urogenital abnormalities, sometimes in combination with Wilms' tumor. Our past study shows that WT1 is expressed in porcine kidney fibroblasts (PKFs) and swine testis cells (ST cells) and is essential for the maintenance of the development and survival of PKFs and ST cells. But we do not know whether WT1 gene was expressed in porcine fetal fibroblasts or not. To further explore whether WT1 was expressed in porcine fetal fibroblasts (PFFs) and its contribution to cell apoptosis, RT-PCR, immunocytochemical staining, and Western blot were used to detect the expression of WT1, the recombinant plasmids of pLV3-WT1 short hairpin ribonucleic acid (shRNA) were used to downregulate the WT1 gene in porcine fetal fibroblasts, and the role of WT1 in cell proliferation was examined by apoptosis analysis also. Our results indicated that WT1 was expressed in PFFs, the pLV3-WT1 shRNA dramatically reduced the expression of WT1, and downregulation of WT1 directly led to early cell apoptosis by downregulating the expression of antiapoptotic gene Bcl-2 and upregulating the expression of proapoptotic gene Bax in PFFs. Our results demonstrate that WT1 is also essential for the maintenance of the survival of PFFs.

miRNAs promote generation of porcine-induced pluripotent stem cells.

The pigs have similarities of organ size, immunology and physiology with humans. Porcine-induced pluripotent stem cells (piPSCs) have great potential application in regenerative medicine. Here, we established piPSCs induced from porcine fetal fibroblasts by the retroviral overexpression of Oct4, Sox2, Klf4, and c-Myc. The piPSCs not only express pluripotent markers but also have the capacity for differentiation in vivo and in vitro, including EB and teratoma formation. We supplemented microRNAs during the induction process because miR-302a, miR-302b, and miR-200c have been reported to be highly expressed in human and mouse embryonic stem cells and in iPSCs. In this study, we found that the overexpression of miR-302a, miR-302b, and miR-200c effectively improved the reprogramming efficiency and reduced the induction time for piPSCs in the OSKM and OSK induction systems. Due to the similar induction efficiency of 4F-induced piPSCs or of three factors combined with miR-302a, miR-302b, and miR-200c (3F-miRNA-induced piPSCs), we recommend the addition of miRNAs instead of c-Myc to reduce the tumorigenicity of piPSCs.

Effects of TET1 knockdown on gene expression and DNA methylation in porcine induced pluripotent stem cells.

TET1 is implicated in maintaining the pluripotency of embryonic stem cells. However, its precise effects on induced pluripotent stem cells (iPSCs), and particularly on porcine iPSCs (piPSCs), are not well defined. To investigate the role of TET1 in the pluripotency and differentiation of piPSCs, piPSCs were induced from porcine embryonic fibroblasts by overexpression of POU5F1 (OCT4), SOX2, KLF4, and MYC (C-MYC). siRNAs targeting to TET1 were used to transiently knockdown the expression of TET1 in piPSCs. Morphological abnormalities and loss of the undifferentiated state of piPSCs were observed in the piPSCs after the downregulation of TET1. The effects of TET1 knockdown on the expression of key stem cell factors and differentiation markers were analyzed to gain insights into the molecular mechanisms underlying the phenomenon. The results revealed that knockdown of TET1 resulted in the downregulated expression of pluripotency-related genes, such as LEFTY2, KLF2, and SOX2, and the upregulated expression of differentiation-related genes including PITX2, HAND1, GATA6, and LEF1. However, POU5F1, MYC, KLF4, and NANOG were actually not downregulated. Further analysis showed that the methylation levels of the promoters for POU5F1 and MYC increased significantly after TET1 downregulation, whereas there were no obvious changes in the promoters of SOX2, KLF4, and NANOG. The methylation of the whole genome increased, while hydroxymethylation slightly declined. Taken together, these results suggest that TET1 may play important roles in the self-renewal of piPSCs and the maintenance of their characteristics by regulating the expression of genes and the DNA methylation.

The effects of the WT1 gene on apoptosis and development-related gene expression in porcine kidney fibroblasts and swine testis cells.

The Wilm's tumor 1 gene (WT1) encodes zinc finger proteins that function as tumor suppressors, and play important roles in the development of the genito-urinary system and other organs. Its precise function in the development of porcine tissues and organs, which is an attractive transplantation resource for certain human diseases, is still unclear. Here, we sought to define the role of WT1 in porcine kidney and testis tissues using porcine kidney fibroblasts (PKFs) and swine testis (ST) cells as in vitro models, both of which express WT1. The recombinant plasmids pLV3-WT1 shRNA and pIRES2 -WT1-EGFP were constructed to respectively down- and up-regulate the WT1 gene in porcine cells. The role of WT1 in cell proliferation was investigated by RT-PCR, immunocytochemical staining, apoptosis analysis, and Western blot. The pLV3-WT1 shRNA dramatically reduced WT1 expression at both the transcription and protein levels. The down-regulation of WT1 directly led to early cell apoptosis, and changes in Sf1, Sox9, and Gdnf gene expression in PKFs and ST cells. In contrast, up-regulation of WT1 gave no obvious phenotype in ST cells. Our results demonstrate that WT1 is essential for the survival of PKFs and ST cells because it regulates apoptosis- and development-related genes in the cells; however, no obvious effect was observed when WT1 was over-expressed.