HSP90AB1 as the Druggable Target of Maggot Extract Reverses Cisplatin Resistance in Ovarian Cancer.

Cisplatin resistance is a crucial factor affecting ovarian cancer patient's survival rate, but the primary mechanism underlying cisplatin resistance in ovarian cancer remains unclear, and this prevents the optimal use of cisplatin therapy. Maggot extract (ME) is used in traditional Chinese medicine for patients with comas and patients with gastric cancer when combined with other drug treatments. In this study, we investigated whether ME enhances the sensitivity of ovarian cancer cells to cisplatin. Two ovarian cancer cells-A2780/CDDP and SKOV3/CDDP-were treated with cisplatin and ME in vitro. SKOV3/CDDP cells that stably expressed luciferase were subcutaneously or intraperitoneally injected into BALB/c nude mice to establish a xenograft model, and this was followed by ME/cisplatin treatment. In the presence of cisplatin, ME treatment effectively suppressed the growth and metastasis of cisplatin-resistant ovarian cancer in vivo and in vitro. RNA-sequencing data showed that HSP90AB1 and IGF1R were markedly increased in A2780/CDDP cells. ME treatment markedly decreased the expression of HSP90AB1 and IGF1R, thereby increasing the expression of the proapoptotic proteins p-p53, BAX, and p-H2AX, while the opposite effects were observed for the antiapoptotic protein BCL2. Inhibition of HSP90 ATPase was more beneficial against ovarian cancer in the presence of ME treatment. In turn, HSP90AB1 overexpression effectively inhibited the effect of ME in promoting the increased expression of apoptotic proteins and DNA damage response proteins in SKOV3/CDDP cells. Inhibition of cisplatin-induced apoptosis and DNA damage by HSP90AB1 overexpression confers chemoresistance in ovarian cancer. ME can enhance the sensitivity of ovarian cancer cells to cisplatin toxicity by inhibiting HSP90AB1/IGF1R interactions, and this might represent a novel target for overcoming cisplatin resistance in ovarian cancer chemotherapy.

Maggot extracts chemo-prevent inflammation and tumorigenesis accompanied by changes in the intestinal microbiome and metabolome in AOM/DSS-induced mice.

Inflammatory responses and intestinal microbiome play a crucial role in the progression of colitis-associated carcinoma (CAC). The traditional Chinese medicine maggot has been widely known owing to its clinical application and anti-inflammatory function. In this study, we investigated the preventive effects of maggot extract (ME) by intragastric administration prior to azoxymethane (AOM) and dextran sulfate sodium (DSS)-induced CAC in mice. The results showed that ME had superior advantages in ameliorating disease activity index score and inflammatory phenotype, in comparison with the AOM/DSS group. The number and size of polypoid colonic tumors were decreased after pre-administration of ME. In addition, ME was found to reverse the downregulation of tight junction proteins (zonula occluden-1 and occluding) while suppressing the levels of inflammatory factors (IL-1ß and IL-6) in models. Moreover, Toll-like receptor 4 (TLR4) mediated intracellular nuclear factor-κB (NF-κB)-containing signaling cascades, including inducible nitric oxide synthase and cyclooxygenase-2, and exhibited decreasing expression in the mice model after ME pre-administration. 16s rRNA analysis and untargeted-metabolomics profiling of fecal samples inferred that ME revealed ideal prevention of intestinal dysbiosis in CAC mice, accompanied by and correlated with alterations in the composition of metabolites. Overall, ME pre-administration might be a chemo-preventive candidate in the initiation and development of CAC.

Functional characterization and transcriptional activity analysis of Dryopteris fragrans farnesyl diphosphate synthase genes.

Farnesyl diphosphate synthase (FPS), a key enzyme of the terpene metabolic pathway, catalyzes the precursor of sesquiterpene compounds farnesyl diphosphate (FPP) synthesis, and plays an important role in regulating plant growth and development. Dryopteris fragrans is a medicinal plant rich terpenoids. In this study, the function of the gene was verified in vitro and in vivo, the promoter of the gene was amplified and its transcriptional activity was analyzed. In the present study, we report the molecular cloning and functional characterization of DfFPS1 and DfFPS2, two FPS genes from D. fragrans. We found that the two genes were evolutionarily conserved. Both DfFPS genes were highly expressed in the gametophyte and mature sporophyte leaves, and their expression levels increased in response to methyl jasmonate (MeJA) and high temperature. Both DfFPS proteins were localized in the cytoplasm and could catalyze FPP synthesis in vitro. We also found that the overexpression of DfFPS genes in tobacco plants promoted secondary metabolite accumulation but exhibited negligible effect on plant growth and development. However, the transgenic plants exhibited tolerance to high temperature and drought. The promoters of the two genes were amplified using fusion primer and nested integrated polymerase chain reaction (FPNI-PCR). The promoter sequences were truncated and their activity was examined using the ß-glucuronidase (GUS) gene reporter system in tobacco leaves, and we found that both genes were expressed in the stomata. The transcriptional activity of the promoters was found to be similar to the expression pattern of the genes, and the transcriptional core regions of the two genes were mainly between -943 bp and -740 bp of proDfFPS1. Therefore, we present a preliminary study on the function and transcriptional activity of the FPS genes of D. fragrans and provide a basis for the regulation of terpene metabolism in D. fragrans. The results also provide a novel basis for the elucidation of terpene metabolic pathways in ferns.

Functional Characterization of Potato UBC13-UEV1s Genes Required for Ubiquitin Lys63 Chain to Polyubiquitination.

Ubiquitin-conjugating enzymes (E2s/UBC) are components of the ubiquitin proteasome system (UPS), and the ubiquitin-conjugating enzyme variant (UEV) is one of E2s (ubiquitin-conjugating enzymes, UBC) subfamily. The UEVs and UBC13 play an auxiliary role in mediating Lys63-linked polyUb chain assembly, which is correlated with target protein non-proteolytic functions, such as DNA repair or response to stress. However, the collaborative mechanism of StUBC13 (homologue of AtUBC13) and StUEVs (the UEVs in potato) involved in potato are not fully understood understood. Here, we identified two StUBC13 and seven StUEVs from potato genome. We analyzed protein motif and conserved domain, gene structure, phylogenetic features, cis-acting elements of StUBC13 and StUEVs. Subsequently, we screened StUBC13 partners protein and verified interaction between StUBC13 and StUEVs using yeast two-hybrid, split luciferase complementation (SLC) and bimolecular fluorescence complementation (BiFC) approach. The expression profile and qRT-PCR analysis suggested that StUBC13 and StUEVs gene exhibited a tissue-specific expression and were induced by different stress. Overall, this investigative study provides a comprehensive reference and view for further functional research on StUBC13 and StUEV1s in potato.

Stu-miR827-Targeted StWRKY48 Transcription Factor Negatively Regulates Drought Tolerance of Potato by Increasing Leaf Stomatal Density.

Stomata are specialized portals in plant leaves to modulate water loss from plants to the atmosphere by control of the transpiration, thereby determining the water-use efficiency and drought resistance of plants. Despite that the stomata developmental progression is well-understood at the molecular level, the experimental evidence that miRNA regulates stomata development is still lacking, and the underlying mechanism remains elusive. This study demonstrates the involvement of stu-miR827 in regulating the drought tolerance of potato due to its control over the leaf stomatal density. The expression analysis showed that stu-miR827 was obviously repressed by drought stresses and then rapidly increased after rewatering. Suppressing the expression of stu-miR827 transgenic potato lines showed an increase in stomatal density, correlating with a weaker drought resistance compared with wildtype potato lines. In addition, StWRKY48 was identified as the target gene of stu-miR827, and the expression of StWRKY48 was obviously induced by drought stresses and was greatly upregulated in stu-miR827 knockdown transgenic potato lines, suggesting its involvement in the drought stress response. Importantly, the expression of genes associated with stomata development, such as SDD (stomatal density and distribution) and TMM (too many mouths), was seriously suppressed in transgenic lines. Altogether, these observations demonstrated that suppression of stu-miR827 might lead to overexpression of StWRKY48, which may contribute to negatively regulating the drought adaptation of potato by increasing the stomatal density. The results may facilitate functional studies of miRNAs in the process of drought tolerance in plants.

Transcriptome Analysis Provides Insights into Potentilla bifurca Adaptation to High Altitude.

Potentilla bifurca is widely distributed in Eurasia, including the Tibetan Plateau. It is a valuable medicinal plant in the Tibetan traditional medicine system, especially for the treatment of diabetes. This study investigated the functional gene profile of Potentilla bifurca at different altitudes by RNA-sequencing technology, including de novo assembly of 222,619 unigenes from 405 million clean reads, 57.64% of which were annotated in Nr, GO, KEGG, Pfam, and Swiss-Prot databases. The most significantly differentially expressed top 50 genes in the high-altitude samples were derived from plants that responded to abiotic stress, such as peroxidase, superoxide dismutase protein, and the ubiquitin-conjugating enzyme. Pathway analysis revealed that a large number of DEGs encode key enzymes involved in secondary metabolites, including phenylpropane and flavonoids. In addition, a total of 298 potential genomic SSRs were identified in this study, which provides information on the development of functional molecular markers for genetic diversity assessment. In conclusion, this study provides the first comprehensive assessment of the Potentilla bifurca transcriptome. This provides new insights into coping mechanisms for non-model organisms surviving in harsh environments at high altitudes, as well as molecular evidence for the selection of superior medicinal plants.

Integrated mRNA and miRNA Transcriptome Analysis Suggests a Regulatory Network for UV-B-Controlled Terpenoid Synthesis in Fragrant Woodfern (Dryopteris fragrans).

Fragrant woodfern (Dryopteris fragrans) is a medicinal plant rich in terpenoids. Ultraviolet-B (UV-B) light could increase concentration of terpenoids. The aim of this study was to analyze how UV-B regulates the terpenoid synthesis of the molecular regulatory mechanism in fragrant woodfern. In this study, compared with the control group, the content of the terpenes was significantly higher in fragrant woodfern leaves under UV-B treatment for 4 days (d). In order to identify how UV-B regulates the terpenoid metabolic mechanism in fragrant woodfern, we examined the mRNAs and small RNAs in fragrant woodfern leaves under UV-B treatment. mRNA and miRNA-seq identified 4533 DEGs and 17 DEMs in the control group compared with fragrant woodfern leaves under UV-B treatment for 4 d. mRNA-miRNA analysis identified miRNA target gene pairs consisting of 8 DEMs and 115 miRNAs. The target genes were subjected to GO and KEGG analyses. The results showed that the target genes were mainly enriched in diterpene biosynthesis, terpenoid backbone biosynthesis, plant hormone signal transduction, MEP pathway and MVA pathway, in which miR156 and miR160 regulate these pathways by targeting DfSPL and DfARF, respectively. The mRNA and miRNA datasets identified a subset of candidate genes. It provides the theoretical basis that UV-B regulates the terpenoid synthesis of the molecular regulatory mechanism in fragrant woodfern.

Genome-wide identification of U-box genes and protein ubiquitination under PEG-induced drought stress in potato.

Protein ubiquitination is one of the most important posttranslational modifications in eukaryotic cells, and it is involved in a variety of biological processes, including abiotic stress response. The ubiquitination modification is highly specific, which depends on the accurate recognition of substrate proteins by ubiquitin ligase. Plant U-box (PUB) proteins are a class of ubiquitin ligases, multiple members of which have shown to participate in water-deficit stress in Arabidopsis and rice. U-box gene family and large-scale profiling of the ubiquitome in potato has not been reported to date, although it is one of the most important food crops. The identified 66 U-box genes from the potato genome database were unevenly distributed on 10 chromosomes. These StPUBs have a large number of tandem repeat sequences. Analysis of gene expression characteristics revealed that many StPUBs responded to abiotic stress. Three hundred and fourteen lys modification sites were identified under PEG-induced drought stress, which were distributed on 200 proteins, with 25 differential ubiquitination modification sites, most of which were up-regulated. The ubiquitination modification in potato protein was enhanced under PEG-induced drought stress, and U-box ubiquitin ligase was involved. This study provides an overall strategy and rich data set to clarify the effects of ubiquitination on potatoes under PEG-induced drought stress and the ubiquitination modification involved in potato U-box genes in response to PEG-induced drought stress.

Albicanol Alleviates D-Galactose-Induced Aging and Improves Behavioral Ability Via by Alleviating Oxidative Stress-Induced Damage.

Albicanol is a natural terpenoid derived from Dryopteris fragrans. Herein, we assessed the ability of Albicanol to protect against oxidative stress-induced senescence. Using a murine model of D-galactose (D-gal)-induced aging, we determined that Albicanol treatment can reverse D-gal-mediated learning impairments and behavioral changes, while also remediating brain tissue damage in treated mice. We found that serum SOD, CAT, GSH-Px, and T-AOC levels were significantly decreased in aging mice, and that Albicanol treatment significantly increased the serum levels of these antioxidant enzymes. We additionally evaluated the impact of Albicanol treatment on the Keap1/Nrf2/ARE signaling pathway, and found that it was able to decrease Keap1 expression while increasing the expression of Nrf2, thereby activating this signaling pathway, suppressing oxidative damage, and enhancing the expression of downstream target genes including SOD, GSH, GST, HO-1, and NQO1 in this murine aging model system. Albicanol treatment also inhibited the secretion of inflammatory TNF-a and IL-1b. Together, these data indicated that Albicanol can activate Nrf2 pathway-related genes, thereby inhibition of delayed aging by alleviating oxidative stress-induced damage.

Potato E3 ubiquitin ligase PUB27 negatively regulates drought tolerance by mediating stomatal movement.

The ubiquitin-proteasome system (UPS) is one of the main ways of eukaryotic protein degradation and post-translational modification. It has proven as an essential process for plants to respond to abiotic stresses. Plant U-box (PUB) protein acts as a ubiquitin ligase, which recognizes and ubiquitinates the target proteins. Many PUBs have been involved in water stress in Arabidopsis and rice, but similar comprehensive studies in potato remained limited. In this study, the overexpressed and interfered transgenic potato plants of StPUB27 were obtained and their performances were evaluated under osmotic stress. The result showed that overexpression of StPUB27 accelerated the dehydration of detached leaves companied with greater stomatal conductance, while the down-regulated StPUB27 expression by RNA interference (RNAi) showed a smaller stomatal conductance and a lower rate of water loss in detached leaves, thus showing higher tolerance to osmotic stress. In addition, no significant changes in the proline content were observed between StPUB27 overexpressed and RNAi potato plants. The result demonstrated that potato E3 ubiquitin ligase PUB27 may negatively regulate drought tolerance by mediating stomatal conductance.

A potato RING-finger protein gene StRFP2 is involved in drought tolerance.

The ubiquitin/26S proteasome pathway is widely related to plant growth and metabolism and response to treatment by specifically degrading ubiquitin-modified proteins, including RING-finger-type E3 ubiquitin ligase (RING). The RING finger protein (RFP) gene family, determining the specificity of the ubiquitination process, is numerous and complex in function. In this study, we constructed a pCEGFP-StRFP2 fusion protein expression vector and transformed it into tobacco to achieve transient expression, thereby confirming that StRFP2 is localized in the cell membrane and cytoplasm. The result of qRT-PCR analysis showed that StRFP2 gene was significantly expressed in potato leaves, and the expression level of StRFP2 was significantly up-regulated under drought treatment. The transgenic plants of overexpressing StRFP2 gene were obtained with Agrobacterium tumefaciens-mediated transformation. Plant height, stem diameter, root length, fresh weight and root-shoot ratio of transgenic plants were significantly higher than those of non-transgenic plants (WT), indicating that the growth of plants was significantly promoted after overexpression of StRFP2 gene. Under PEG osmotic stress, the expressional level of StRFP2 in transgenic potato plants was significantly higher than that of WT. Furthermore, the free proline content and CAT activity in transgenic plants were higher than WT, on the contrary, MDA was lower than WT, and transgenic plants have stronger water retention capacity under simulated drought stress treatment, which indicated that StRFP2 could strengthen the tolerance of plants responding to drought stress. The above evidence strongly suggested that the StRFP2 gene is obviously up-regulated expression by drought stress, thereby enhancing the drought tolerance of the potato.

Genome-wide identification and expression analysis of the E2 gene family in potato.

E2 (ubiquitin conjugating enzymes) is an important part of the ubiquitin-proteasome pathway. These enzymes have a significant role to play during plant growth and development, which can response to various stresses. To date, the E2 family has been reported in some high plants, but the genome-wide characterization of this gene family in potato remains unknown. In the present study, 57 putative StUBCs were identified, which were clustered into eight subgroups based on phylogeny. The introns varied in numbers 0 to 9. The highest numbers of introns were 5, which accounted for 31.57%. The analysis of gene duplication showed that 22 StUBC genes were involved in 13 segmental duplication events, while no tandem duplication was found in StUBC genes. According to gene ontology analysis (GO), StUBC family major function is protein binding and ion binding. The RNA sequencing data revealed that 15 StUBC genes were highly expressed in different organs and tubers. 27 StUBC genes were up-regulated under 50 µM ABA treatments. Moreover, the RNA-seq data and qRT-PCR analysis indicated that 17 StUBC genes responded to heat stress. 8 StUBC genes responded to salt stress according to qRT-PCR analysis, and StUBC2, StUBC12, StUBC30 and StUBC13 were predominant expression. The result of this research could provide valuable information to insight into potato E2 family and establish a foundation for further to elucidate function of E2 genes.

Functional analysis of StDWF4 gene in response to salt stress in potato.

The DWARF4 (DWF4) gene encodes a C-22 hydroxylase which is pivotal for brassinosteroids (BRs) biosynthesis. In this research, aimed to understand the molecular mechanism of DWF4 on regulation of potatoes tolerance to salt stress, DWF4 was cloned from potato, named as StDWF4. Its 1476 bp open reading frame encodes a protein of 491 amino acids. The StDWF4-overexpressing (OE) and interference-expressing (RNAi) transgenic potato plants were acquired using Agrobacterium-mediated transformation, respectively. Tissue specific analysis using Quantitative real-time polymerase chain reaction (qRT-PCR) demonstrated that the StDWF4 gene expressed in the leaves, stems and roots of the transgenic and un-transgenic (NT) plants, with specially increased (StDWF4-OE)/reduced (StDWF4-RNAi) expression in the roots. The content of malondialdehyde (MDA) in StDWF4-OE potato plants was lower than that of NT, and proline content was higher than that of NT. MDA and proline content in StDWF4-OE and NT under salt-stress was significantly higher than that of the control and was increased at different sampling times. The content of soluble protein, soluble sugar and the activities of superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX) was higher in the StDWF4-OE plantlets at varied salt treatment time than in the NT potatoes. Reduction of H2O2 content in the StDWF4-OE plants was observed. All above plant physiology indicators in the StDWF4-RNAi potatoes showed opposite variation trends. The results proved that the overexpressing of StDWF4 in potato plantlets can enhance the salt resistance by alleviating the negative effects of salt-stress. However, its interference expression in potato plants depresses the salt resistance. The results lay the groundwork for intensive study of BRs regulation in potato growth and development, and will help us to reveal the molecular mechanisms of how the BRs signaling regulate potato salt tolerance.

Selenium level in the environment and the population of Zhoukoudian area, Beijing, China.

The objective of this study was to detect the selenium level in the environment and the population of Zhoukoudian area, Beijing, and to discuss the influence of various factors on serum selenium level. The soil, drinking water, and foodstuff samples and venous blood samples of 401 individuals were obtained to determine the selenium level by gas chromatograph equipped with a (63)Ni electron capture detector (ECD). The selenium level was 0.210+/-0.013 microg/g in soil, 0.017 microg/L+/-0.002 in drinking water, 0.034+/-0.002 microg/g in rice, and 0.034+/-0.012 microg/g in wheat flour. This index showed that the Zhoukoudian area of Beijing was a moderate or marginal level selenium ecological landscape. The mean serum selenium level of the population was 75.01+/-28.35 microg/L, ranging between 35.2 and 160.4 microg/L. A total of 279 (69.6%) individuals exhibited serum selenium level below 80 microg/L, which is the lowest threshold for the activity of glutathione peroxidases (GPx) in vivo. A total of 35 (8.5%) individuals exhibited serum selenium level below 45 microg/L. It is widely recommended that below this value (45 microg/L) there is an increased risk of cardiovascular disease and cancer. Multiple linear regression analysis showed that serum selenium level was positively associated with body mass index (beta=0.137; P=0.011), serum total cholesterol TC (beta=0.785; P=0.000), however, negatively associated with systolic blood pressure (beta=-0.172; P=0.023), serum triglyceride (beta=-0.170; P=0.007), high density lipoprotein-cholesterol (beta=-0.121; P=0.027), and low high density lipoprotein-cholesterol (beta=-0.568; P=0.027).