Surgical outcomes and risk factors for surgical complications after en bloc resection following reconstruction with 3D-printed artificial vertebral body for thoracolumbar tumors.

BACKGROUND: The outcomes of patients with tumors of the thoracolumbar spine treated with en bloc resection (EBR) using three-dimensional (3D)-printed endoprostheses are underreported. METHODS: We retrospectively evaluated patients with thoracolumbar tumors who underwent surgery at our institution. Logistic regression analysis was performed to identify the potential risk factors for surgical complications. Nomograms to predict complications were constructed and validated. RESULTS: A total of 53 patients with spinal tumors underwent EBR at our hospital; of these, 2 were lost to follow-up, 45 underwent total en bloc spondylectomy, and 6 were treated with sagittal en bloc spondylectomy. The anterior reconstruction materials included a customized 3D-printed artificial vertebral body (AVB) in 10 cases and an off-the-shelf 3D-printed AVB in 41 cases, and prosthesis mismatch occurred in 2 patients reconstructed with the off-the-shelf 3D-printed AVB. The median follow-up period was 21 months (range, 7-57 months). Three patients experienced local recurrence, and 5 patients died at the final follow-up. A total of 50 perioperative complications were encountered in 29 patients, including 25 major and 25 minor complications. Instrumentation failure occurred in 1 patient, and no prosthesis subsidence was observed. Using a combined surgical approach was a dependent predictor of overall complications, while Karnofsky performance status score, lumbar spine lesion, and intraoperative blood loss ≥ 2000 mL were predictors of major complications. Nomograms for the overall and major complications were constructed using these factors, with C-indices of 0.850 and 0.891, respectively. CONCLUSIONS: EBR is essential for the management of thoracolumbar tumors; however, EBR has a steep learning curve and a high complication rate. A 3D-printed AVB is an effective and feasible reconstruction option for patients treated with EBR.

Dark Side of Ammonium Nitrogen in Paddy Soil with Low Organic Matter: Stimulation of Microbial As(V) Reduction and As(III) Transfer from Soil to Rice Grains.

The bioavailability of arsenic (As) is influenced by ammonium (NH4+-N) fertilization, but the underlying mechanisms controlling As transformation in soil-rice systems are still not fully understood. The effects of two NH4+-N fertilizers, urea and NH4HCO3, on the transformation of As in a paddy soil with low organic matter content and transfer in rice plants were investigated. Treatments with urea and NH4HCO3 significantly increased arsenite (As(III)) concentration in porewater, bioavailable As in rhizosphere soil, and the relative abundance of the As(V) respiratory reductase gene (arrA) and As(III) methyltransferase gene (arsM). Furthermore, the relative expression of As transporter genes in rice roots, such as OsLsi1, OsLsi2, and OsLsi3, was upregulated, and the translocation efficiency of As(III) from rice roots to brown rice was promoted. Subsequently, As(III) accumulation in brown rice significantly increased. Therefore, attention should be paid to As-contaminated paddy fields with NH4+-N fertilization.

Retention and transformation of exogenous Hg in acidic paddy soil under alternating anoxic and oxic conditions: Kinetic and mechanistic insights.

To estimate the risks and developing remediation strategies for the mercury (Hg)-contaminated soils, it is crucial to understand the mechanisms of Hg transformation and migration in the redox-changing paddy fields. In present study, a Hg-spiked acidic paddy soil (pH 4.52) was incubated under anoxic conditions for 40 d and then under oxic conditions for 20 d. During anoxic incubation, the water-soluble, exchangeable, specifically adsorbed, and fulvic acid-complexed Hg decreased sharply, whereas the humic acid-complexed Hg, organic, and sulfide-bound Hg gradually increased, which were mainly ascribed to the enhanced adsorption on the surface of soil minerals with an increase in soil pH, complexation by organic matters, precipitation as HgS, and absorption by soil colloids triggered by reductive dissolution of Fe(III) oxides. By contrast, after oxygen was introduced into the system, a gradual increase in available Hg occurred with decreasing soil pH, decomposition of organic matters and formation of Fe(III) oxides. A kinetic model was established based on the key elementary reactions to quantitatively estimate transformation processes of Hg fractions. The model matched well with the modified Tessier sequential extraction data, and suggested that large molecular organic matter and humic acid dominated Hg complexation and immobilization in acidic paddy soils. The content of methylmercury increased and reached its peak on anoxic 20 d. Sulfate-reducing bacteria Desulfovibrio and Desulfomicrobium were the major Hg methylating bacteria in the anoxic stage whereas demethylating microorganisms Clostridium_sensu_stricto_1 and Clostridium_sensu_stricto_12 began to grow after oxygen was introduced. These new dynamic results provided new insights into the exogenous Hg transformation processes and the model could be used to predict Hg availability in periodically flooded acidic paddy fields.

Distinct arsenic uptake feature in rice reveals the importance of N fertilization strategies.

The environmental behavior of arsenic (As) is commonly affected by the biogeochemical processes of iron (Fe) and nitrogen (N). In this study, field experiments were conducted to explore As uptake in rice and As translation and distribution in As-contaminated iron-rich paddy soils after applying different forms of N fertilizers, including urea (CO(NH2)2), ammonium bicarbonate (NH4HCO3), nitrate of potash (KNO3), and ammonium bicarbonate + nitrate of potash (NH4HCO3 + KNO3). The results indicated that applying nitrate N fertilizer inhibited the reduction and dissolution of As-bearing iron minerals and promoted microbial-mediated As(III) oxidation in flooded soil, thus reducing the soil As bioavailability. The concentrations of total As and inorganic As ratio (iAs/TAs) in rice grain decreased by 32.4 % and 15.4 %, respectively. However, the application of ammonium nitrogen promoted the reductive dissolution of As-bearing iron minerals and stimulated microbial As(V) reduction in flooded soil, leading to the release of As from soil to porewater. The total As concentration and inorganic As uptake ratio in rice grain increased by 20.1 % and 6.2 %, respectively, when urea was applied, and by 29.6 % and 10.5 %, respectively, when ammonium bicarbonate was applied. However, the simultaneous application of NH4+ and NO3- had no significant effect on As concentration in rice grain and its transformation in paddy soils. Ammonium nitrogen enhanced the organic As concentration in rice grain because the increased As(III) promoted As methylation in soil. In contrast, nitrate decreased the organic As uptake by rice grain because the decreased As(III) diminished As methylation in soil. The results provide reasonable N fertilization strategies for regulating the As biogeochemical process and reducing the risk of As contamination in rice.

Novel carboxymethyl chitosan/N-acetylneuraminic acid hydrogel for the protection of Pediococcus pentosaceus.

In this study, carboxymethyl chitosan (CMCS) and N-acetylneuraminic acid (NeuAc) were used to develop C-NeuAc hydrogels to encapsulate Pediococcus pentosaceus QK-1. The mechanical properties, thermal stability, in vitro degradation, and pH sensitivity of the hydrogel were evaluated. The C-NeuAc concentration required for optimal hydrogel performance was 3% (w/v). Hydrogel swelling behaviour was effectively assessed by Fickian diffusion and Schott's second-order kinetic models. The hydrogel demonstrated excellent biocompatibility and low in vitro cytotoxicity. An in vitro assay revealed that the viability of Pediococcus pentosaceus QK-1 in C-NeuAc had decreased by only 1.41 log (CFU/ mL) after exposure to simulated acidic gastric fluid. Moreover, the survival rate of the encapsulated and free Pediococcus pentosaceus QK-1 cells were 80.1% and virtually zero, respectively, after passage through the gastrointestinal tract. It was empirically determined that low temperature and freeze-drying were the ideal condition and method to ensure storage longevity of the hydrogel-encapsulated probiotic. Hence, C-NeuAc hydrogel is highly desirable as a food-grade probiotic delivery vehicle.

Unique feature of Fe-OM complexes for limiting Cd accumulation in grains by target-regulating gene expression in rice tissues.

The excessive accumulation of cadmium (Cd) in rice grains is highly determined by the expression of specific genes in different tissues. Targeted gene regulation in rice plants is a long-standing challenge. Herein, a new strategy for regulating target gene expression responsible for Cd absorption and translocation in roots and leaves was developed by complexing Fe(II) with organic matter (i.e., Fe-OM) with the optimal mass ratio of 1. Results showed that Fe-OM noticeably reduced the grain Cd content from 0.48 ± 0.04 mg kg-1 to 0.25 ± 0.03 mg kg-1, exhibiting a significantly higher capacity in mitigating Cd accumulation in grains than Fe(II) or OM alone. The translocation factor (TF) was reduced from 0.14 (control) to 0.08 by Fe-FA from root to grain, which could be due to the preferential Cd translocation to leaves (i.e., TFroot to leaves was enhanced four times by the complex of Fe(II) with fulvic acid (Fe-FA). Further gene analysis revealed that the cooperative effects of OsNramp1 and OsNramp5 downregulation in roots/stems and OsLCT1 upregulation in leaves contributed to the mitigation of Cd in grains. This work provides a new strategy to regulating target gene expression in specific tissues to alleviate Cd accumulation in grains.

Phase Change Energy Storage Elastic Fiber: A Simple Route to Personal Thermal Management.

A novel thermoplastic polyurethane (TPU) PCFs possessing a high loaded ratio and high elasticity was simply prepared by vacuum absorption following wet spinning, then coated by waterborne polyurethane (WPU). Octadecane (OCC), hexadecanol (HEO), and stearic acid (SA), which have different tendencies to form hydrogen bonds with TPU, were selected as PCMs, and their thermal behavior, thermal storge properties, and elasticity were systematically studied, respectively. The hierarchical pore structure though from the sheath to the core part of TPU filaments weakened the influence of the nonfreezing layer and hydrogen bond on the crystallization behavior of PCMs. The resulting HEO/TPU fiber has the highest enthalpy of 208.1 J/g compared with OCC and SA. Moreover, the HEO/TPU fiber has an elongation at break of 354.8% when the phase change enthalpy is as high as 177.8 J/g and the phase change enthalpy is still 174.5 J/g after fifty cycles. After ten tensile recovery cycles, the elastic recovery rate of HEO/TPU fiber was only 71.3%. When the HEO in the fiber was liquid state, the elastic recovery rate of HEO/TPU fiber promoted to 91.6%. This elastic PCFs have excellent thermal cycle stability, elastic recovery, and temperature sensitivity. It has great application potential in the fields of flexible wearable devices, intelligent fabrics, and temperature sensors.

Multiple effects of nitrate amendment on the transport, transformation and bioavailability of antimony in a paddy soil-rice plant system.

Nitrate (NO3-) is known to be actively involved in the processes of mineralization and heavy metal transformation; however, it is unclear whether and how it affects the bioavailability of antimony (Sb) in paddy soils and subsequent Sb accumulation in rice. Here, the effects of NO3- on Sb transformation in soil-rice system were investigated with pot experiments over the entire growth period. Results demonstrated that NO3- reduced Sb accumulation in brown rice by 15.6% compared to that in the control. After amendment with NO3-, the Sb content in rice plants increased initially and then gradually decreased (in roots by 46.1%). During the first 15 days, the soil pH increased, the oxidation of Sb(III) and sulfides was promoted, but the reduction of iron oxide minerals was inhibited, resulting in the release of adsorbed and organic-bound Sb from soil. The microbial arsenite-oxidizing marker gene aoxB played an important role in Sb(III) oxidation. From days 15 to 45, after NO3- was partially consumed, the soil pH decreased, and the reductive dissolution of Fe(III)-bearing minerals was enhanced; consequently, iron oxide-bound Sb was transformed into adsorbed and dissolved Sb species. After day 45, NO3- was completely reduced, Sb(V) was evidently reduced to Sb(III), and green rust was generated gradually. Thus, the available Sb decreased due to its enhanced affinity for iron oxides. Moreover, NO3- inhibited the reductive dissolution of iron minerals, which ultimately caused low Sb availability. Therefore, NO3- can chemically and biologically reduce the Sb availability in paddy soils and alleviate Sb accumulation in rice. This study provides a potential strategy for decreasing Sb accumulation in rice in the Sb-contaminated sites.

Enhanced immobilization of arsenic and cadmium in a paddy soil by combined applications of woody peat and Fe(NO3)3: Possible mechanisms and environmental implications.

Organic matter (OM) plays an important role in the mobility of heavy metal(loid)s. Peat containing abundant OM can be used as an organic fertilizer improving physical and chemical properties of soil. Previous studies indicated that the immobilization of heavy metal(loid)s by peat is affected by the presence of metal oxides and/or hydroxides and that Fe-enriched peat is very effective in immobilizing metal(loid)s. Accordingly, we hypothesize that simultaneous application of peat and Fe-containing compounds may pronouncedly immobilize heavy metal(loid)s. In this study, the effects of the combined applications of woody peat and Fe(NO3)3 on As and Cd mobilities and accumulations in rice during the whole growth period were investigated by a pot experiment. The combined applications of woody peat and Fe(NO3)3 significantly decreased As(III) and Cd in porewater due to pH increases induced by applications of Fe(NO3)3, and these decreases were enhanced with increasing Fe(NO3)3. In addition, simultaneous application of peat and Fe(NO3)3 significantly decreased mobile portions of As and Cd but significantly increased their immobile portions. Increasing Fe(NO3)3 increased the amount of As immobilized by poorly crystalline Fe oxides. The formation of Fe plaques and production of poorly crystalline Fe oxides were enhanced by Fe(NO3)3 addition, which also contributed to the immobilization of As and Cd in soil. Overall, the combined applications of woody peat and Fe(NO3)3 provided a strategy for simultaneously immobilizing As and Cd in soils and further alleviating their accumulations from soil to rice plants. In paddy soil, the frequent occurrence of iron redox activity due to the alternating wetting and drying cycles provided favorable conditions for interactions between Fe and OM, and this process and its associated metal(loid) immobilization may be more important than we thought and need further study.

The translocation of antimony in soil-rice system with comparisons to arsenic: Alleviation of their accumulation in rice by simultaneous use of Fe(II) and NO3.

Antimony (Sb) accumulation in rice grains is a potential risk to human health. This study aims to develop agronomic practices that can reduce the accumulation of Sb in rice grain in contaminated soil. A pot culture experiment was conducted to investigate the effects of co-application of ferrous iron and nitrate (Fe(II) + NO3-) in paddy soils on Sb uptake by rice. The co-application of Fe(II) and NO3- promoted abiotic/biotic Fe(II) oxidation and mineralization in the rhizosphere soil and formation of Fe plaques, consequently, Sb bioavailability was significantly reduced by enhancing Sb immobilization on the newly formed Fe(III) (hydr)oxides. The results were compared with those for arsenic (As) in the same trial and it was shown that the two metalloids have different translocation behavior in the soil-rice plant system. The adsorption of Sb, especially the Sb(V), on Fe(III) (hydr)oxides was more significantly enhanced by the decreased soil pH after the application of Fe(II) + NO3- than that of As. The uptake of Sb by the roots of rice was much more difficult but it was much easier to be transported from the rice straw to the grains compared to As. The differences might be mainly caused by the different uptake mechanisms of Sb and As by rice plants from paddies. The bioavailable As(III) would be much more efficient in entering into the rice roots than Sb(III) through the aquaporin channel due to its much smaller ionic radius; the bioavailable As(V), entering into the rice roots via phosphate transporters, would also be more efficient in taking up by roots than Sb(V), which pathway from soil to rice roots remains unclear. These findings provide new insights into Sb biogeochemical behavior in soil-rice plant systems and demonstrate that co-application of Fe(II) and NO3- could be a promising strategy for safely-utilizing Sb contaminated sites in the future.

Simultaneous alleviation of cadmium and arsenic accumulation in rice by applying zero-valent iron and biochar to contaminated paddy soils.

The fates of cadmium (Cd) and arsenic (As) in paddy fields are generally opposite; thus, the inconsistent transformation of Cd and As poses large challenges for their remediation. In this study, the impacts of zero valent iron (ZVI) and/or biochar amendments on Cd and As bioavailability were examined in pot trials with rice. Comparison with the untreated soil, both Cd and As accumulation in different rice tissues decreased significantly in the ZVI-biochar amendments and the Cd and As accumulation in rice decreased with increasing ZVI contents. In particular, the concentrations of Cd (0.15 ± 0.01 mg kg-1) and As (0.17 ± 0.01 mg kg-1) in rice grains were decreased by 93% and 61% relative to the untreated soil, respectively. A sequential extraction analysis indicated that with increasing Fe ratios in the ZVI-biochar mixtures, bioavailable Cd and As decreased, and the immobilized Cd and As increased. Furthermore, high levels of Fe, Cd, and As were detected in Fe plaque of the ZVI-biochar amendments in comparison with the single biochar or single ZVI amendments. The ZVI-biochar mixture may have a synergistic effect that simultaneously reduces Cd and As bioavailability by increasing the formation of amorphous Fe and Fe plaque for Cd and As immobilization. The single ZVI amendment significantly decreased As bioavailability, while the single biochar amendment significantly reduced the bioavailability of Cd compared with the combined amendments. Hence, using a ZVI-biochar mixture as a soil amendment could be a promising strategy for safely-utilizing Cd and As co-contaminated sites in the future.

Ctenopharyngodon idella PERK (EIF2AK3) decreases cell viability by phosphorylating eIF2α under ER stress.

As an upstream kinase of eIF2α, protein kinase RNA-like ER (endoplasmic reticulum) kinase (PERK) is a type I transmembrane protein located in ER in eukaryotic cells. PERK is mainly composed of two domains, the intracavitary domain for BIP protein combination and the dissociative C-terminal region containing a typical serine/threonine kinase domain which promotes the phosphorylation of eIF2α. In this study, we cloned a PERK (also known as EIF2AK3) gene from grass carp (Ctenopharyngodon idella). The full-length cDNA of grass carp PERK (CiPERK) is 5192 bp including a 176 bp of 5' untranslated region, a 1719 bp of 3' untranslated region and a 3297 bp of the longest open reading frame (ORF) encoding 1098 amino acids. Phylogenetic analysis exhibits that CiPERK shares a high degree of sequence homology to the counterparts in other teleosts. RT-PCR indicated that CiPERK expression was significantly up-regulated following the stimulation with TM (tunicamycin). To study the function of CiPERK, the N-terminal sequence of CiPERK and CiGRP78 sequence were separately subcloned into the expression vectors pCMV-HA and pCMV-Flag for co-immunoprecipitation and GST-Pulldown assays. The assays indicated that CiPERK and CiGRP78 can combine with each other in normal conditions. However, under ER stress (TM stimulation) CiPERK can improve the eIF2α phosphorylation level. In addition, CCK assay showed the overexpression of CiPERK in CIK cells decreases the cell viability.

A mechanism underlies fish GRP78 protection against Pb2+ toxicity.

Heavy metal exposure impacts basic cellular processes and results in serious toxicological effects. Pb2+ can activate the response to endoplasmic reticulum (ER) stress by protein denaturation, changing intracellular calcium homeostasis, and inducing cell death. As an ER retention protein, 78-kDa glucose-regulated protein (GRP78) can relieve the Pb2+-induced ER stress and enhance cell viability. We previously showed that heavy metal ions such as Pb2+ etc. are harmful to fish cell lines in a time- and dose-dependent manner. The phenomenon is accompanied by the increasing accumulation of grass carp GRP78 (CiGRP78), which can protect the cells from heavy metal ion cytotoxicity. Here, we investigated the mechanism in which CiGRP78 exerted its protective function. Using metal ions affinity elution method and fluorescent spectral analysis, we showed that CiGRP78 could respectively form a complex with Calcium, Lead and Cadmium ions, especially with Lead ion in vitro. However, another ER retention protein CiGRP94 could not bind to Pb2+, highlighting the functional differentiation might exist in CiGRP78 and CiGRP94 in regulating heavy metal cytotoxicity. Our results suggested that CiGRP78 might increase cellular tolerance to Pb2+ via the direct interaction with it.

Identification of grass carp (Ctenopharyngodon idella) XBP1S as a primary member in ER stress.

X-box binding protein 1 (XBP1), a vital basic leucine zipper transcription factor for the related gene transcription in endoplasmic reticulum (ER) stress, belongs to the CREB/ATF family. In mammals, XBP1S is the activated one of XBP1 isoform. In order to study the role of fish XBP1S, we cloned and identified the XBP1S (KU509247) from grass carp (Ctenopharyngodon idella) (named CiXBP1S) by homologous cloning and RACE technique. The full length of CiXBP1S is 1694 bp along with 124 bp of 5' UTR, 418 bp of 3' UTR and the longest open reading frame (1152 bp) encoding a polypeptide of 383 amino acids with a well conserved DNA binding domain (BRLZ domain). CiXBP1S shares significant homology to zebrafish XBP1S (∼90%) at amino acid level. RT-PCR showed that the expression of CiXBP1S was ubiquitous in all tested grass carp tissues and was significantly up-regulated under the stimulation with tunicamycin (Tm) in CIK (C. idellus kidney) cells. To study the molecular mechanism of transcriptional regulation for XBP1 signaling pathway in fish, we cloned grass carp XBP1 promoter sequence. Its promoter is 1036 bp in length and divided into two distinct regions in which an ER stress response element (ERSE) exists in the proximal region. Meanwhile, grass carp ATF6 (CiATF6N) and CiXBP1S were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind resin. Gel mobility shift assay showed that CiATF6N and CiXBP1S had the high affinity with CiXBP1 promoter sequence in vitro. Co-transfection of pcDNA3.1-CiATF6 (or pcDNA3.1-CiXBP1S respectively) with pGL3-CiXBP1P2 (or pGL3-CiXBP1P1 respectively) into epithelioma papulosum cyprini (EPC) cells showed that CiATF6 and CiXBP1S played a positive role in CiXBP1S transcription. CiXBP1S also had high affinity with CiGRP78 and CiGRP94 promoter sequences. In addition, recombinant plasmids of pGL3-CiGRP78P and pGL3-CiGRP94P were constructed and transiently co-transfected with pcDNA3.1-CiXBP1S (pcDN3.1-CiXBP1S-nBRLZ, respectively) into EPC cells. The result showed that CiXBP1S can activate CiGRP78 and CiGRP94 promoters.

Arsenic mobility and bioavailability in paddy soil under iron compound amendments at different growth stages of rice.

Iron (Fe)-based solids can reduce arsenic (As) mobility and bioavailability in soils, which has been well recognized. However, to our knowledge, there are few studies on As uptake at different growth stages of rice under Fe compound amendments. In addition, the formation of Fe plaques at different growth stages of rice has also been rarely reported. Therefore, the present study was undertaken to investigate As mobility and bioavailability in paddy soil under Fe compound amendments throughout the whole growth stage of rice plants. Amendments of poorly crystalline Fe oxides (PC-Fe), FeCl2+NaNO3 and FeCl2 reduced grain As by 54% ± 3.0%, 52% ± 3.0% and 46% ± 17%, respectively, compared with that of the non-amended control. The filling stage was suggested to be the key stage to take measures to reduce As uptake. At this stage, all soil amendments significantly reduced As accumulation in rice plants. At the maturation stage, PC-Fe amendment significantly reduced mobile pools and increased immobile pools of soil As. Besides, PC-Fe treatment promoted the transformation of Fe fractions from dissolved Fe to adsorbed, poorly crystalline and free Fe oxides. Moreover, significant positive correlations between soil Fe fractions and As fractions were found. Accordingly, we hypothesized that Fe compound amendments might affect the concentration distribution of Fe fractions first and then affect As fractionation in soil and its bioavailability to rice plants indirectly. The formation of Fe plaques varied with growth stages and different treatments. Significantly negative correlations between mobile pools of As and Fe or As in Fe plaques indicated that Fe plaques could immobilize mobile As in soils and thus affect As bioavailability. Overall, the effect of the soil amendments on reduction of As uptake varied with growth stages and different treatments, and further research on the key stage for reducing As uptake is still required.

Heavy metal contaminations in soil-rice system: source identification in relation to a sulfur-rich coal burning power plant in Northern Guangdong Province, China.

Heavy metal contents (As, Cd, Cr, Cu, Hg, Ni, Pb, and Zn) in 99 pairs of soil-rice plant samples were evaluated from the downwind directions of a large thermal power plant in Shaoguan City, Guangdong Province, China. Results indicate that there is a substantial buildup of As, Cd, Cu, Pb, and Zn in the predominant wind direction of the power plant. The significant correlations between S and heavy metals in paddy soil suggest that the power plant represents a source of topsoil heavy metals in Shaoguan City due to sulfur-rich coal burning emissions. Elevated Cd concentrations were also found in rice plant tissues. Average Cd (0.69 mg kg(-1)) and Pb (0.39 mg kg(-1)) contents in rice grain had exceeded their maximum permissible limits (both were 0.2 mg kg(-1)) in foods of China (GB2762-2005). The enrichment of Cd and Pb in rice grain might pose a potential health risk to the local residents.

The availabilities of arsenic and cadmium in rice paddy fields from a mining area: The role of soil extractable and plant silicon.

Adequate silicon (Si) can greatly boost rice yield and improve grain quality through alleviating stresses associated with heavy metals and metalloids such as arsenic (As) and cadmium (Cd). The soil plant-available Si is relatively low in South China due to severe desilicification and allitization of the soils in this region. Conversely, pollution of heavy metals and metalloids in the soils of this region occurs widely, especially As and Cd pollution in paddy soil. Therefore, evaluating the plant availability of Si in paddy soil of South China and examining its correlation with the availability of heavy metals and metalloids are of great significance. Accordingly, in our study, 107 pairs of soil and rice plant samples were collected from paddy fields contaminated by As and Cd in South China. Significantly positive correlations between Si in rice plants and Si fractions in soils extracted with citric acid, NaOAc-HOAc buffer, and oxalate-ammonium oxalate buffer suggest that these extractants are more suitable for use in extracting plant-available Si in the soils of our present study. Significantly negative correlations between different Si fractions and As or Cd in rice plant tissues and negative exponential correlations between the molar ratios of Si to As/Cd in rice roots, straws, husks or grains and As/Cd in rice grains indicate that Si can significantly alleviate the accumulation of As/Cd from soils to the rice plants. Finally, a contribution assessment of soil properties to As/Cd accumulation in rice grains based on random forest showed that in addition to Si concentrations in soil or rice plants, other factors such as Fe fractions and total phosphorus also contributed largely to As/Cd accumulation in rice grains. Overall, Si exhibited its unique role in mitigating As or Cd stress in rice, and our study results provide strong field evidence for this role.

Ctenopharyngodon idella NF-κB subunit p65 modulates the transcription of IκBα in CIK cells.

NF-κB is an important transcription factor for regulating the multiple inflammatory and immune related gene transcription. It can bind with the nuclear factor κB site within the promoter of target genes to regulate their transcriptions. p65, the all-important subunit of NF-κB, is ubiquitously expressed in cells. In the present study, we cloned and identified the p65 subunit from grass carp (Ctenopharyngodon idella) (named Cip65) by homologous cloning and RACE technique. The full length of Cip65 cDNA is 2481 bp along with 9 bp 5' UTR, 639 bp 3' UTR and the largest open reading frame (1833 bp) encoding a polypeptide of 610 amino acids with a well conserved Rel-homology domain (RHD) in N-terminal and a putative transcription activation domain (TAD) in C-terminal. Cip65 gathers with other teleost p65 proteins to form a fish-specific clade clearly distinct from those of mammalian and amphibian counterparts on the phylogenetic tree. In CIK (C. idellus kidney) cells, the expression of Cip65 was significantly up-regulated under the stimulation with Poly I:C. As one member of the NF-κB inhibitor protein (IκB) family, IκBα can dominate the activity of NF-κB by interacting with it. To study the molecular mechanisms of negative feedback loop of NF-κB signaling in fish, we cloned grass carp IκBα (CiIκBα) promoter sequence. CiIκBα promoter is 414 bp in length containing two RelA binding sites and a putative atypical TATA-box. Meanwhile, Cip65 and its mutant proteins including C-terminus deletion mutant of Cip65 (Cip65-ΔC) and N-terminus deletion mutant of Cip65 (Cip65-ΔN) were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind resin. In vitro, Cip65 rather than Cip65-ΔC and Cip65-ΔN showed high affinity with CiIκBα promoter sequence by gel mobility shift assays. In vivo, the cotransfection of pcDNA3.1-Cip65 (or pcDNA3.1-Cip65-ΔC, pcDNA3.1-Cip65-ΔN respectively) with pGL3-CiIκBα and pRL-TK renilla luciferase plasmid into CIK cells showed that pcDNA3.1-Cip65 rather than pcDNA3.1-Cip65-ΔC and pcDNA3.1-Cip65-ΔN, can increase the luciferase activity. Taken together, these results suggested that Cip65 can regulate the expression of CiIκBα and works as a negative feedback loop in NF-κB pathway.

Grass carp (Ctenopharyngodon idella) ATF6 (activating transcription factor 6) modulates the transcriptional level of GRP78 and GRP94 in CIK cells.

ATF transcription factors are stress proteins containing alkaline area-leucine zipper and play an important role in endoplasmic reticulum stress. ATF6 is a protective protein which regulates the adaptation of cells to ER stress by modulating the transcription of UPR (Unfolded Protein Response) target genes, including GRP78 and GRP94. In the present study, a grass carp (Ctenopharyngodon idella) ATF6 full-length cDNA (named CiATF6, KT279356) has been cloned and identified. CiATF6 is 4176 bp in length, comprising 159 nucleotides of 5'-untranslated sequence, a 1947 nucleotides open reading frame and 2170 nucleotides of 3'-untranslated sequences. The largest open reading frame of CiATF6 translates into 648 aa with a typical DNA binding domain (BRLZ domain) and shares significant homology to the known ATF6 counterparts. Phylogenetic reconstruction confirmed its closer evolutionary relationship with other fish counterparts, especially with Zebrafish ATF6. RT-PCR showed that CiATF6 was ubiquitously expressed and significantly up-regulated after stimulation with thermal stress in all tested grass carp tissues. In order to know more about the role of CiATF6 in ER stress, recombinant CiATF6N with His-tag was over-expressed in Rosetta Escherichia coli, and the expressed protein was purified by affinity chromatography with Ni-NTA His-Bind Resin. In vitro, gel mobility shift assays were employed to analyze the interaction of CiATF6 protein with the promoters of grass carp GRP78 and GRP94, respectively. The result has shown that CiATF6 could bind to these promoters with high affinity by means of its BRLZ mainly. To further study the transcriptional regulatory mechanism of CiATF6, Dual-luciferase reporter assays were applied. Recombinant plasmids of pGL3-GRP78P and pGL3-CiGRP94P were constructed and transiently co-transfected with pcDNA3.1-CiATF6 (pcDN3.1-CiATF6-nBRLZ, respectively) into C. idella kidney (CIK) cells. The result has shown that CiATF6 could activate CiGRP78 and CiGRP94 promoters.

The transcription regulation analysis of Ctenopharyngodon idellus PKR and PKZ genes.

Protein kinase R (PKR), the double-stranded RNA-activated protein kinase, exists in mammalian and fish. PKZ, a PKR-like protein kinase containing Z-DNA binding domains, just exists in fish. PKR and PKZ work synergistically in the antiviral defense by inhibiting intracellular protein translation. The transcriptional factor IRF3 (interferon regulatory factor 3) acts as a key regulator of type I IFN (Interferon) and ISG (interferon stimulated gene). On the basis of the cloned CiIRF3 previously, CiIRF3 with His-tag was over-expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatography with Ni-NTA His-Bind Resin. In this study, we have demonstrated that grass carp (Ctenopharyngodon idellus) PKR (CiPKR) and PKZ (CiPKZ) genes were inducible by Poly I:C in C. idella kidney (CIK) cells. So, they might be implicated in the intracellular antiviral activity. To understand the up regulatory mechanism of CiPKR and CiPKZ genes upon virus induction, we constructed wild type (pGL3-CiPKR-luc and pGL3-CiPKZ-luc) and the mutant (pGL3-CiPKR-nISRE-luc and pGL3-CiPKZ-nISRE-luc) reporter gene vectors according to the promoter sequences of CiPKR (KJ704845) and CiPKZ (KJ704844). In vitro, gel mobility shift assays demonstrated that CiIRF3 can combine CiPKR and CiPKZ promoters with high affinity. However, CiIRF3 bound to the mutants CiPKR-nISRE and CiPKZ-nISRE faintly. Whereafter, the recombinant plasmids of pGL3-CiPKR-luc, pGL3-CiPKZ-luc were transiently co-transfected with pcDNA3.1-CiIRF3, pcDNA3.1-CiIRF7 respectively into CIK cells. Cell transfection assays indicated that CiIRF3 and CiIRF7 up-regulated the transcriptional level of CiPKR and CiPKZ. The results also revealed that the consensus sequence of ISRE (interferon stimulated response element) is an important regulatory element for the transcriptional initiation of CiPKR and CiPKZ.