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Arsenic trioxide (As2O3) is one of the most effective drugs for the treatment of acute promyelocytic leukemia (APL), and induces the degradation of chimeric oncoprotein PML/RARα (P/R) and APL cell differentiation. Recent evidence has suggested that P/R fusion protein degradation by arsenic occurs through two steps, namely, rapid solubility change/shift of the P/R fusion protein following arsenic treatment (i.e., transfer of P/R protein from the soluble fraction to the insoluble pellet fraction), and subsequent degradation of these insoluble proteins. However, there is little information regarding the reversibility of arsenic induced P/R fusion protein solubility change as well as protein degradation in the insoluble fraction after removing arsenic. In this study, we used APL cell line NB4 or P/R and PML over-expressed 293T cells as well as HeLa cells to reveal the solubility change of P/R and PML by arsenic exposure, and further determined the fate of these insoluble proteins after the removal of arsenic. Here, for the first time, we found that arsenic induced P/R or PML protein solubility change is an irreversible process. Once arsenic induces a P/R or PML protein solubility change, these insoluble proteins could be degraded by the proteasomal pathway even without continuous arsenic treatment. However, PML and P/R proteins can be newly synthesized after the removal of arsenic, suggesting that great caution should be taken in the clinical therapy of APL patients before ending arsenic treatment.

This study identified the effect of salicylic acid on degradation of isoproturon in Arabidopsis. Three T-DNA insertion mutant lines pal1- 1, pal1- 2, and eps1- 1 defective in salicylic acid synthesis were tested, which showed higher isoproturon accumulation and a toxic symptom in the mutants. When treated with 5 mg/L salicylic acid, these lines displayed a lower level of isoproturon and showed an attenuated toxic symptom. An RNA-sequencing study identified 2651 (1421 up and 1230 down) differentially expressed genes (DEGs) in eps1- 1 and 2211 (1556 up and 655 down) in pal1- 2 mutant plants (2.0 fold change, p0.05). Some of the DEGs covered Phase I-III reaction components, like glycosyltransferases (GTs) and ATP-binding cassette transporters (ABCs). Using ultra performance liquid chromatography-time-of-flight-tandem-mass spectrometer/mass spectrometer (UPLC/Q-TOF-MS/MS), 13 Phase I and four Phase II metabolites were characterized. Of these, two metabolites 1-OH-isopropyl-benzene-O-glucoside and 4-isopropylphenol-S-2-methylbutanoyl-serine, have been identified and reported for the first time.

The PML/RARα fusion protein is the oncogenic driver in acute promyelocytic leukemia (APL). Although most APL cases are cured by PML/RARα-targeting therapy, relapse and resistance can occur due to drug-resistant mutations. Here we report that thermal stress destabilizes the PML/RARα protein, including clinically identified drug-resistant mutants. AML1/ETO and TEL/AML1 oncofusions show similar heat shock susceptibility. Mechanistically, mild hyperthermia stimulates aggregation of PML/RARα in complex with nuclear receptor corepressors leading to ubiquitin-mediated degradation via the SIAH2 E3 ligase. Hyperthermia and arsenic therapy destabilize PML/RARα via distinct mechanisms and are synergistic in primary patient samples and in vivo, including three refractory APL cases. Collectively, our results suggest that by taking advantage of a biophysical vulnerability of PML/RARα, thermal therapy may improve prognosis in drug-resistant or otherwise refractory APL. These findings serve as a paradigm for therapeutic targeting of fusion oncoprotein–associated cancers by hyperthermia. Significance: Hyperthermia destabilizes oncofusion proteins including PML/RARα and acts synergistically with standard arsenic therapy in relapsed and refractory APL. The results open up the possibility that heat shock sensitivity may be an easily targetable vulnerability of oncofusion-driven cancers. See related commentary by Wu et al., p. 300.

Developing a biotechnical system with rapid degradation of pesticide is critical for reducing environmental, food security and health risks. Here, we investigated a novel epigenetic mechanism responsible for the degradation of the pesticide atrazine (ATZ) in rice crops mediated by the key component CORONATINE INSENSITIVE 1a (OsCOI1a) in the jasmonate-signaling pathway. OsCOI1a protein was localized to the nucleus and strongly induced by ATZ exposure. Overexpression of OsCOI1a (OE) significantly conferred resistance to ATZ toxicity, leading to the improved growth and reduced ATZ accumulation (particularly in grains) in rice crops. HPLC/Q-TOF-MS/MS analysis revealed increased ATZ-degraded products in the OE plants, suggesting the occurrence of vigorous ATZ catabolism. Bisulfite-sequencing and chromatin immunoprecipitation assays showed that ATZ exposure drastically reduced DNA methylation at CpG context and histone H3K9me2 marks in the upstream of OsCOI1a. The causal relationships between the DNA demethylation (hypomethylatioin), OsCOI1a expression and subsequent detoxification and degradation of ATZ in rice and environment were well established by several lines of biological, genetic and chemical evidence. Our work uncovered a novel regulatory mechanism implicated in the defense linked to the epigenetic modification and jasmonate signaling pathway. It also provided a modus operandi that can be used for metabolic engineering of rice to minimize amounts of ATZ in the crop and environment.

Atrazine (ATZ) residue in farmland is one of the environmental contaminants seriously affecting crop production and food safety. Understanding the regulatory mechanism for ATZ metabolism and degradation in plants is important to help reduce ATZ potential toxicity to both plants and human health. Here, we report our newly developed engineered rice overexpressing a novel Phase II metabolic enzyme glycosyltransfearse1 (ARGT1) responsible for transformation of ATZ residues in rice. Our results showed that transformed lines, when exposed to environmentally realistic ATZ concentration (0.2-0.8 mg/L), displayed significantly high tolerance, with 8-27% biomass and 36-56% chlorophyll content higher, but 37-69% plasma membrane injury lower than untransformed lines. Such results were well confirmed by ARGT1 expression in Arabidopsis. ARGT1-transformed rice took up 1.6-2.7 fold ATZ from its growth medium compared to its wild type (WT) and accumulated ATZ 10%-43% less than that of WT. A long-term study also showed that ATZ in the grains of ARGT1-transformed rice was reduced by 30-40% compared to WT. The ATZ-degraded products were characterized by UPLC/Q-TOF-MS/MS. More ATZ metabolites and conjugates accumulated in ARGT1-transformed rice than in WT. Eight ATZ metabolites for Phase I reaction and 10 conjugates for Phase II reaction in rice were identified, with three ATZ-glycosylated conjugates that have never been reported before. These results indicate that ARGT1 expression can facilitate uptake of ATZ from environment and metabolism in rice plants.

Ametryn is a selective herbicide belonging to the triazine family and widely used for killing annual grasses or weeds in China and other parts of the world. However, reports on its environmental risk assessment with regard to soil and crop contamination are limited. In this study, accumulation of ametryn in wheat, maize, ryegrass and alfalfa crops along with ametryn residues in the soil planted with the plants were comparatively investigated. Soil enzyme activities and low molecular weight organic acids (LMWOAs), as well as antioxidant and degradation enzyme activities in plant tissues were measured. The maximum accumulation of ametryn was found in shoots and roots of wheat and alfalfa. Ryegrass had the maximum ametryn translocation factor (TF) from roots to shoots, with more than three times over the other crops. The ametryn residue in ryegrass-planted soil was much lower than that in soil planted with others. The residual content of ametryn in crop-planted soils was ordered as rhizosphere soil

There is a rising public concern on accumulation of harmful pesticides in environment and crops. Epigenetic alteration caused by environmental contaminants is one of the key factors in the etiology of environmentally-associated diseases. Growing evidence shows that harmful pesticide atrazine (ATZ) has a profound effect on DNA methylation in human genome, however, little is known about the epigenetic mechanism underlying ATZ accumulation and degradation in plants, particularly in edible plants growing in the ATZ-contaminated areas. This study investigated the atrazine elimination that was mediated by DNA methylation and histone modification in the food crop rice. Studies with two mutant Osmet1-1/2 defective in the genomic CG DNA methylation show significantly lower accumulation of atrazine than its wild-types. Profiling methylome and transcriptome of ATZ-exposed Osmet1 and wild-type identified many differentially methylated loci (≥2 fold change, p

Cadmium (Cd) is a toxic metal that contributes to human diseases such as pediatric cancer and cardiovascular dysfunction. Epigenetic modification caused by Cd exposure is the major factor in etiology of environmentally-relevant diseases. However, the underlying epigenetic mechanism for Cd uptake and accumulation in food crops, particularly those growing in Cd-contaminated environments, is largely unknown. This study investigated uncharacterized regulatory mechanisms and biological functions of global DNA hypomethylation at CG sites that are associated with gene expression for Cd detoxification and accumulation in the food crop rice. Mutation of the CG maintenance enzyme OsMET1 confers rice tolerance to Cd exposure. Genome-wide analysis of OsMET1 loss of function mutant Osmet1 and its wild type shows numerous loci differentially methylated and upregulated genes for Cd detoxification, transport and accumulation. We functionally identified a new locus for a putative cadmium tolerance factor (here termed as OsCTF) and demonstrated that Cd-induced DNA demethylation is the drive of OsCTF expression. The 3'-UTR of OsCTF is the primary site of DNA and histone (H3K9me2) demethylation, which is associated with higher levels of OsCTF transcripts detected in the Osmet1 and Ossdg714 mutant lines. Mutation of OsCTF in rice led to hypersensitivity to Cd and the Osctf line accumulated more Cd, whereas transfer of OsCTF back to the Osctf mutant completely restored the normal phenotype. Our work unveiled an important epigenetic mechanism and will help develop breeding crops that contribute to food security and better human health.

Antimony (Sb) belongs to the same group as arsenic (As) in the periodic table, and both share similar characteristics. However, Sb2O3 (SbIII) has no methylation capacity, unlike arsenic trioxide (As2O3). In the present study, we determined the effect of SbIII on NB4 cells and found that antimony could induce PML-RARα fusion protein degradation, reorganization of PML-NBs, and NB4 cell differentiation with low cytotoxicity. On the other hand, zinc finger motifs in PML protein are considered to be a key target binding site for arsenic-induced PML-RARα protein degradation. Interestingly, antimony and arsenic lost their ability to degrade PML-RARα fusion protein in NB4 cells following pretreatment with phenanthroline (i.e., chelator of zinc ions), indicating that the integrity of zinc finger motifs in PML-RARα fusion protein is a fundamental condition for inducing the protein's degradation by antimony and arsenic. Moreover, we found that SbIII could not induce mutant PML (e.g., A126V and L218P) solubility change and degradation, similar to As2O3. In contrast, we found that the organic antimony compound phenylstibine oxide (PSO) could induce mutant PML protein degradation. In conclusion, our results indicate that SbIII might also be a promising agent to treat acute promyelocytic leukemia, in the same manner as As2O3.

Accumulating pesticide (and herbicide) residues in soils have become a serious environmental problem. This study focused on identifying the removal of two widely used pesticides, isoproturon (IPU) and acetochlor (ACT), by a genetically developed paddy (or rice) plant overexpressing an uncharacterized glycosyltransferase (IRGT1). IRGT1 conferred plant resistance to isoproturon-acetochlor, which was manifested by attenuated cellular injury and alleviated toxicity of rice under isoproturon-acetochlor stress. A short-term study showed that IRGT1-transformed lines removed 33.3-48.3% of isoproturon and 39.8-53.5% of acetochlor from the growth medium, with only 59.5-72.1 and 58.9-70.4% of the isoproturon and acetochlor remaining in the plants compared with the levels in untransformed rice. This phenotype was confirmed by IRGT1-expression in yeast ( Pichia pastoris) which grew better and contained less isoproturon-acetochlor than the control cells. A long-term study showed that isoproturon-acetochlor concentrations at all developmental stages were significantly lower in the transformed rice, which contain only 59.3-69.2% (isoproturon) and 51.7-57.4% (acetochlor) of the levels in wild type. In contrast, UPLC-Q-TOF-MS/MS analysis revealed that more isoproturon-acetochlor metabolites were detected in the transformed rice. Sixteen metabolites of isoproturon and 19 metabolites of acetochlor were characterized in rice for Phase I reactions, and 9 isoproturon and 13 acetochlor conjugates were characterized for Phase II reactions in rice; of these, 7 isoproturon and 6 acetochlor metabolites and conjugates were reported in plants for the first time.

Acute promyelocytic leukemia (APL) is characterized by a reciprocal translocation between chromosomes 15 and 17, t(15;17), resulting in the expression of PML-RARα fusion protein, which disrupts the normal PML nuclear bodies (PML-NBs) to micro-speckled pattern, leading to loss of their original functions. Moreover, reformation of PML-NBs in APL by arsenic is considered as one of the important step for APL treatment. Leptomycin B (LMB), a nuclear export inhibitor, is commonly used to inhibit the proteins exporting from the nucleus to the cytoplasm. In the present study, we found that LMB could induce the reformation of PML-NBs in leukemia NB4 cells as well as in APL blast cells from the patients, implying that nuclear shuttle proteins might be involved in the reformation of PML-NBs. Herein, we further found that LMB totally lost the ability to induce PML-NBs reformation when the endogenous PML gene was knocked out, indicating that endogenous PML protein is probably involved in the reformation of PML-NBs. More interestingly, among all PML isoforms (i.e., seven isoforms), reformation of PML-NBs was only observed when co-transfection of PML-RARα with PML-I after LMB treatment. Similarly, deletion of nuclear export signal (NES) of PML-I could also reform PML-NBs, suggesting that the protein level of endogenous PML-I in nucleus is important for the reformation of PML-NBs that interfered by PML-RARα fusion protein. Additionally, LMB has synergistic effect with iAsIII on enhancing PML-RARα fusion protein degradation, and it might provide new insight into APL treatment at clinical level in the near future.

Propazine is a s-triazine herbicide widely used for controlling weeds for crop production. Its persistence and contamination in environment nagatively affect crop growth and food safety. Elimination of propazine residues in the environment is critical for safe crop production. This study identified a microbial community able to degrade propazine in a farmland soil. About 94% of the applied propazine was degraded within 11 days of incubation when soil was treated with 10 mg kg−1 propazine as the initial concentration. The process was accompanied by increased microbial biomass and activities of soil enzymes. Denaturing gradient gel electrophoresis (DGGE) revealed multiple bacterial strains in the community as well as dynamic change of the composition of microbial community with a reduced microbial diversity (H′ from 3.325 to 2.78). Tracking the transcript level of degradative genes AtzB, AtzC and TrzN showed that these genes were induced by propazine and played important roles in the degradation process. The activities of catalase, dehydrogenase and phenol oxidase were stimulated by propazine exposure. Five degradation products (hydroxyl-, methylated-, dimeric-propazine, ammeline and ammelide) were characterized by UPLC-MS2, revealing a biodegradation of propazine in soil. Several novel methylated and dimeric products of propazine were characterized in thepropazine-exposed soil. These data help understand the pathway, detailed mechanism and efficiency of propazine biodegradation in soil under realistic field condition.

Prometryn has been used in crop (e.g., corn and sorghum) field to prevent growth of annual grasses and broadleaf weeds for many years. As a moderately persistent herbicide in soil, prometryn may exert detrimental effects on environmental safety and crop production. The present study assessed the photodegradation of prometryn residues in soil by exploring a variety of factors such as soil moisture, temperature, and light exposure that potentially affect prometryn photodegradation. The dissipation rate of prometryn during a 14-day period of study was more than 90% under 15 (low pressure), 100, and 300 W (medium pressure) UV light exposure. The half-life of prometryn decay under UV light (53.5–116.4 h) was far less than that under xenon light (1131.6 h) and dark (3138.7 h) conditions. When the soil moisture (clay loam) was 60% of the field moisture capacity, it was most effective for prometryn photodegradation. The prometryn photodegradation on soil with 60% moisture level was increased with temperature and prometryn concentrations. The theoretical optimization scheme for eliminating prometryn in soil was recommended. The degraded products of prometryn under UV light and darkness were characterized using ultra high-performance liquid chromatography coupled to a linear ion trap-orbitrap hybrid mass spectrometer (UPLC-LTQ-orbitrap-MS/MS) and showed that prometryn decay in soil was through hydroxylation, dealkylation, and dethiomethylation pathways.

This study aimed to explore whether antenatal supplement of taurine can improve the brain development of fetal rats with intrauterine growth restriction (IUGR) via protein kinase A (PKA)-Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)/c-fos pathway. A total of 30 pregnant rats were randomly divided into the following three groups: control, IUGR, and IUGR with antenatal taurine supplementation groups. The expression of PKA, CaMKII, and c-fos mRNA and proteins in fetal rat brain tissues were detected by quantitative real time (qRT)-polymerase chain reaction (PCR) and Western blot, respectively; and the number of PKA-, CaMKII- and c-fos-positive cells was measured by immunohistochemistry. Western blot and qRT-PCR revealed that antenatal taurine supplementation significantly increased PKA, CaMKII, and c-fos protein and mRNA levels in fetal rats brain with IUGR (p < 0.05). Immunohistochemistry results showed that the numbers of PKA-, CaMKII-, and c-fos-positive cells were significantly decreased in the IUGR groups compared with the controls; while antenatal taurine could significantly increase the positive cell numbers (p < 0.05). So, we conclude that antenatal taurine improves IUGR fetal brain development which is associated with increasing the activity of PKA-CaMKII/c-fos signal pathway.

Mycoplasma bovis is a major pathogen causing arthritis, respiratory disease and mastitis in cattle. A better understanding of its genetic features and evolution might represent evidences of surviving host environments. In this study, multiple factors influencing synonymous codon usage patterns in M. bovis (three strains’ genomes) were analyzed. The overall nucleotide content of genes in the M. bovis genome is AT-rich. Although the G and C contents at the third codon position of genes in the leading strand differ from those in the lagging strand (p

Phytohormone salicylic acid (SA) has been documented to induce nitric oxide (NO) generation and stomatal closure in plants. However, the cellular components mediating these processes are limited. Here, we report that NO synthesis in guard cells and stomatal closure are markedly induced by SA in Arabidopsis wild type plants, whereas these effects caused by SA are suppressed significantly in noa1 T-DNA mutant plants. These results suggest that AtNOA1 regulates SA-triggered NO accumulation and stomatal closure in Arabidopsis.