Effect of Different Edible Trichosanthes Germplasm on Its Seed Oil to Enhance Antioxidant and Anti-Aging Activity in Caenorhabditis elegans.

The seeds of various Trichosanthes L. plants have been frequently used as snacks instead of for traditional medicinal purposes in China. However, there is still a need to identify the species based on seeds from Trichosanthes germplasm for the potential biological activities of their seed oil. In this study, 18 edible Trichosanthes germplasm from three species were identified and distinguished at a species level using a combination of seed morphological and microscopic characteristics and nrDNA-ITS sequences. Seed oil from the edible Trichosanthes germplasm significantly enhanced oxidative stress tolerance, extended lifespan, delayed aging, and improved healthspan in Caenorhabditis elegans. The antioxidant activity of the seed oil exhibits a significant positive correlation with its total unsaturated fatty acid content among the 18 edible Trichosanthes germplasm, suggesting a genetic basis for this trait. The biological activities of seed oil varied among species, with T. kirilowii Maxim. and T. rosthornii Harms showing stronger effects than T. laceribractea Hayata.

Extracellular vesicle-derived TP53BP1, CD34, and PBX1 from human peripheral blood serve as potential biomarkers for the assessment and prediction of vascular aging.

BACKGROUND: Vascular aging is an important pathophysiological basis for the senescence of various organs and systems in the human body, and it is a common pathogenetic trigger for many chronic diseases in the elderly. METHODS: The extracellular vesicles (EVs) from young and aged umbilical vein endothelial cells were isolated and identified by qPCR the differential expression levels of 47 mRNAs of genes closely related to aging in the two groups. RESULTS: There were significant differences in the expression levels of 18 genes (we noted upregulation in PLA2G12A, TP53BP1, CD144, PDE11A, FPGT, SERPINB4, POLD1, and PPFIBP2 and downregulation in ATP2C2, ROBO2, RRM2, GUCY1B1, NAT1-14, VEGFR2, WTAPP1, CD146, DMC1, and GRIK2). Subsequent qPCR identification of the above-mentioned genes in PBMCs and plasma-EVs from the various age groups revealed that the trend in expression levels in peripheral blood plasma-EVs of the different age groups was approximately the same as that in PBMCs. Of these mRNAs, the expression of four genes-PLA2G12A, TP53BP1, OPRL1, and KIAA0895-was commensurate with increasing age. In contradistinction, the expression trend of four genes (CREG1, PBX1, CD34, and SLIT2) was inversely proportional to the increase in age. Finally, by taking their intersection, we determined that the expression of TP53BP1 was upregulated with increasing human age and that CD34 and PBX1 were downregulated with increasing age. CONCLUSION: Our study indicates that human peripheral blood plasma-EV-derived TP53BP1, CD34, and PBX1 potentially comprise a noninvasive biomarker for assessing and predicting vascular aging.

Atranorin driven by nano materials SPION lead to ferroptosis of gastric cancer stem cells by weakening the mRNA 5-hydroxymethylcytidine modification of the Xc-/GPX4 axis and its expression.

Gastric cancer is a highly malignant tumor. Gastric cancer stem cells (GCSCs) are the main causes of drug resistance, metastasis, recurrence, and poor prognosis. As a secondary metabolite of lichen, Atranorin has a variety of biological effects, such as antibacterial, anti-inflammatory, analgesic, and wound healing; however, its killing effect on GCSCs has not been reported. In this study, we constructed Atranorin complexes comprising superparamagnetic iron oxide nanoparticles (SPION) (Atranorin@SPION). In vitro and in vivo experiments confirmed that Atranorin@SPION could significantly inhibit the proliferation, invasion, angiogenesis, and tumorigenicity of CD44+/ CD24+ GCSCs, and induce oxidative stress injury, Fe2+ accumulation, and ferroptosis. Quantitative real-time reverse transcription PCR and western blotting results showed that Atranorin@SPION not only reduced the expression levels of GCSC stem cell markers and cell proliferation and division markers, but also significantly inhibited the expression levels of key molecules in the cystine/glutamate transporter (Xc-)/glutathione peroxidase 4 (GPX4) and Tet methylcytosine dioxygenase (TET) family proteins. The results of high performance liquid chromatography-mass spectrometry and Dot blotting showed that Atranorin@SPION significantly inhibited the mRNA 5­hydroxymethylcytidine modification of GCSCs. Meanwhile, the results of RNA immunoprecipitation-PCR also indicated that Atranorin@SPIONs significantly reduced the 5-hydroxymethylcytidine modification level of GPX4 and SLC7A11 mRNA 3' untranslated region in GCSCs, resulting in a decrease in their stability, shortening their half-lives and reducing translation activity. Therefore, this study revealed that Atranorin@SPIONs induced ferroptosis of GCSCs by weakening the expression of the Xc-/GPX4 axis and the 5-hydroxymethylcytidine modification of mRNAs in the pathway, thereby achieving their therapeutic effect on gastric cancer.

siRNA@superparamagnetic iron oxide nanoparticles attenuate physiological toxicity of DEHP by suppressing autophagy pathway activities in Caenorhabditis elegans.

Bis(2-ethylhexyl)ortho-phthalate (DEHP) is a widely used plasticizer in polyvinyl chloride materials. Considering its widespread application, it has become a major environmental pollutant and can cause endocrine, reproductive system, and gastrointestinal disorders. Herein we aimed to elucidate the mechanisms via which DEHP causes cytotoxicity in Caenorhabditis elegans and assess whether siRNA@superparamagnetic iron oxide nanoparticles (SPIONs) can attenuate this effect. On exposing C. elegans to 10 µM DEHP, its physiological functions and gene expression levels were markedly affected. RNA-seq and Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that DEHP exposure significantly activated the autophagy-animal signal transduction pathway in the somatic cells of C. elegans. Subsequently, the surface of SPIONs was loaded with siRNAs and transfected into C. elegans. Transmission electron microscopy showed that SPIONs could smoothly enter the somatic cells of C. elegans. Further, qPCR showed that the expression levels of autophagy pathway-related genes, namely Atg-2, Epg-9, Atg-18, Bec-1, and Atg-16.2, in the siRNA@SPION intervention group were significantly lower than those in the control group. Biochemical and physiological test results suggested that siRNA@SPION complexes attenuated DEHP-induced physiological toxicity and oxidative stress damage in C. elegans. Collectively, our findings indicated that DEHP markedly affects the physiological activity of C. elegans, induces changes in gene expression levels, and activates the autophagy signal transduction pathway and that siRNA@SPION complexes suppress such toxic effects by silencing the expression of genes involved in the autophagy signal transduction pathway.

Superparamagnetic iron oxide nanoparticles drive miR-485-5p inhibition in glioma stem cells by silencing Tie1 expression.

Gliomas are highly malignant nervous system tumours. Studies shown that cancer stem cells are one of the main reasons underlying recurrence, metastasis, and poor prognosis in glioma cases. Our previous studies have found that superparamagnetic iron oxide nanoparticles (SPIONs) can act as nucleic acid carriers to drive intracellular overexpression of these nucleic acids. In this study, CD44+/CD133+ glioma stem cells (HuGSCs) were first isolated from surgically resected tissues from patients. qPCR and western blot results showed that Tie1 expression in HuGSCs was significantly higher thanexpression in CD44-/CD133- glioma cells. Bioinformatic analysis and luciferase reporter assays showed that miR-485-5p binds to specific loci on the 3'-UTR of Tie1 mRNA to inhibit Tie1 expression. Subsequently, miR-485-5p/miR-mut and SPION complexes were transfected into HuGSCs. Transmission electron microscopy showed that a highly dense metallic electron cloud is present in HuGSCs. At the same time, in vivo and in vitro studies showed that miR-485-5p@SPIONs can significantly inhibit HuGSC proliferation, invasion, tumourigenicity, and angiogenesis. In-depth analysis showed that Tie1 interacts with neuronal growth factors such as FGF2, BDNF, GDNF, and GFAP. qPCR and western blot results showed that in miR-485-5p@SPIONs-HuGSCs, the expression levels of Tie1 and stem cell markers (Oct4, Sox2, Nanog, CD44, and CD133), and even FGF2, BDNF, GDNF, and GFAP were significantly lower than thelevels in the control group (miR-mut@SPIONs-HuGSCs). Therefore, this study showedthat Tie1 is an important factor that maintains glioma stem cell activity. SPIONs drive miR-485-5p overexpression in cells and inhibit endogenous Tie1 expression to downregulate the protein expression levels of Fgf2/GDNF/GFAP/BDNF and significantly weaken the in vivo and in vitro viability of gliomas.

Fabrication of superwetting, antimicrobial and conductive fibrous membranes for removing/collecting oil contaminants.

In order to remove/collect organic contaminants from polluted water, polypyrrole/silver nanoparticles (PPy/Ag NPs) have been loaded onto spandex fabric using the method of in situ redox-oxidation polymerization to achieve a specific membrane. Observations showed that the original hydrophobic fabric became superhydrophilic and superoleophobic underwater (with an underwater oil contact angle (OCA) of 160°). The as-prepared specimen could effectively remove the oil from an oil-in-water emulsion. After further hydrophobic modification, the specimen was transformed into a fabric that possessed durable superhydrophobicity and superlipophilicity (with a water contact angle (WCA) of 159°), which could collect the oil from a water-in-oil emulsion. Apparently, the two types of fibrous membranes completely satisfied the conditions for removing/collecting organic contaminants from opposite types of water/oil mixtures. The durable evaluation results exhibited the outstanding resistance of both fibrous membranes to friction and acidic and basic scouring agents. Additionally, the multifunctional fabric membrane also possessed excellent electrical conductivity and antibacterial activities towards S. aureus, B. subtilis, and E. coli, which will greatly promote developments in the textile industry and provide a bright future for fabric-based materials.

Superparamagnetic Iron Oxide Nanoparticles Induce Ferroptosis of Human Ovarian Cancer Stem Cells by Weakening Cellular Autophagy.

Human ovarian cancer stem cells (HuOCSCs) are the main source of ovarian cancer recurrence, metastasis, and drug resistance. Superparamagnetic iron oxide nanoparticles (SPIONs) are well-known nucleic acid or drug carriers owing to their controllable properties, superior stability, and easy modification. However, whether SPIONs can inhibit the activity of HuOCSCs by inducing ferroptosis remains unclear. In the present study, we isolated CD44+ /CD133+ HuOCSCs from tumours of four patients with clear cell ovarian cancer and added 0.2 mM SPIONs for mixed culture. Transmission electron microscopy showed that SPION-treated HuOCSCs contained multiple high-density electron clouds. Prussian blue staining showed high concentrations of iron ions in the cells. In vitro , SPIONs treatment of HuOCSCs inhibited cell proliferation, migration, and soft agar clone formation, weakened their resistance to multiple chemotherapeutics, and induced cell death. In vivo , SPIONs pretreatment of HuOCSCs significantly reduced their tumour-forming ability and induced angiogenesis in nude mice. Further, SPIONs induced the accumulation of reactive oxygen species in HuOCSCs and induced oxidative stress. qPCR analysis indicated that SPIONs-treated HuOCSCs had reduced expression of tumour stem cell markers (CD117, NANOG, CD133, and SOX2), cell proliferation factors (KI67, CCND), autophagy-related factors (ATG3, ATG5, MAP1ALC3a, MAP1ALC3b, and MAP1ALC3c), and certain negative regulators of ferroptosis, while the mRNA expression levels of cell death-related proteins (BAK1 and BID), and certain positive regulators of ferroptosis were significantly increased. Overall, our findings suggest that SPIONs induce oxidative stress and decrease autophagy activity in ovarian cancer stem cells, activate ferroptosis, and inhibit their proliferation, invasion, drug resistance, and tumorigenic ability.

Superparamagnetic iron oxide nanoparticle-mediated expression of miR-326 inhibits human endometrial carcinoma stem cell growth.

Background: Previously, our group confirmed the presence of a subset of cancer stem cells in the tissues of endometrial carcinoma (ie, human endometrial carcinoma stem cells [HuECSCs]). However, the mechanisms by which microRNAs regulate the growth of HuECSCs remain elusive. Methods: We loaded miR-326 onto superparamagnetic iron oxide nanoparticles (miR-326@SPION) and transfected them into HuECSCs. Results: In the present study, we found that the expression levels of members of the G-protein coupled receptor 91 (GPR91)/signal transducer and activator of transcription 3 (STAT3)/vascular endothelial growth factor (VEGF) pathway were significantly elevated in CD44+/CD133+ HuECSCs. Luciferase reporter assays indicated that the succinate receptor 1 (SUCNR1) gene, also known as the G-protein coupled receptor 91 (GPR91) gene, was one of the potential targets of miR-326. Transmission electron microscopy revealed that the SPIONs could cross the cell membrane and accumulate in the cytoplasm. The overexpression of miR-326 significantly inhibited the proliferation and cell cycle progression of HuECSCs in vitro. MiR-326 overexpression also effectively inhibited the invasion and angiogenic capacities of HuECSCs in the extracellular matrix. Meanwhile, miR-326 overexpression significantly inhibited the tumorigenicity and tumour neovascularization capacity of HuECSCs in nude mice. Both quantitative real-time PCR and Western blotting confirmed that overexpression of miR-326 significantly reduced the expression of members of the GPR91/STAT3/VEGF pathway in HuECSCs, and the activity (level of phosphorylation) of key molecules in this pathway was also reduced. Conclusion: Collectively, we confirmed that SPIONs are highly efficient nanocarriers for nucleic acids, on which the loading of miR-326 inhibited the activation of the GPR91/STAT3/VEGF signaling pathway and significantly attenuated the activity of stem cells in endometrial carcinoma, both in vitro and in vivo.

Enterovirus 71 induces apoptosis of SH­SY5Y human neuroblastoma cells through stimulation of endogenous microRNA let-7b expression.

Enterovirus 71 (EV71) is a pathogenic microorganism that causes hand, foot and mouth disease. However, the epigenetic mechanisms behind how EV71 regulates host cell proliferation and apoptosis are unclear. In the present study, the ability of EV71 to induce apoptosis was analyzed in the SH-SY5Y human neuroblastoma cell line and the effect of this virus on the mRNA expression levels of various apoptotic markers, miRNA let-7b and cyclin D1 (CCND1), was also investigated. The results demonstrated that EV71 induced SH-SY5Y cell apoptosis. An MTT assay revealed a significant inhibitory effect of EV71 on cell proliferation between 12-72 h post injection, compared with the control group. Furthermore, quantitative polymerase chain reaction and western blot analyses demonstrated that expression level of the apoptosis inhibitor Bcl-2 was markedly reduced, but the expression levels of the apoptosis-promoting factors Bax, caspase-7, caspase­3 and active caspase-3 were markedly higher in the SH-SY5Y cells 12-48 h after EV71 infection, compared with the non-infected cells. In addition, flow cytometric assays revealed that EV71 arrested the cell cycle of host SH-SY5Y cells. Northern blot analysis revealed a marked miRNA let-7b hybridization signal in the EV71 virus-infected group compared with the non-infected group. Furthermore, western blotting confirmed that the CCND1 protein expression levels were significantly reduced in EV71-infected SH-SY5Y cells. EV71-inhibited SH-SY5Y proliferation was abrogated using let-7b specific 2'-O-Methyl-RNA, which inhibited endogenous miRNA let-7b expression. Thus, EV71 regulated the host SH­SY5Y cell cycle and cell proliferation via stimulating endo-genous miRNA let-7b and directly targeting CCND1, therefore EV71 is a potential candidate for antiviral therapy.

The induction of maturation on dendritic cells by TiO2 and Fe(3)O(4)@TiO(2) nanoparticles via NF-κB signaling pathway.

Nanomaterials are increasingly used in many fields, including drug vectors and vaccine formulation. In this study, nano-TiO(2) and magnetic Fe(3)O(4)@TiO(2) were synthesized and their abilities to activate dendritic cells were investigated. The signaling pathway involved in their effects on the cellular functions was also explored. First, nano-TiO(2) and Fe(3)O(4)@TiO(2) were prepared with diameters of 82nm and 63nm, and zeta potentials of 41.5mV and 30.2mV, respectively. The magnetic property of Fe(3)O(4)@TiO(2) was detected to be 12.9emu/g. Both kinds of nanoparticles were proved to have good biocompatibility in vitro. Second, the exposure of nano-TiO2 and Fe(3)O(4)@TiO(2)caused an increased expression of TNF-α, CD86 and CD80, and besides, Fe(3)O(4)@TiO(2)showed a certain up-regulation on MHC-II. The cellular uptake of Ovalbumin on BMDCs could be strongly improved by nano-TiO2 and Fe(3)O(4)@TiO(2)as detected via flow cytometer and confocal observation. Further investigation revealed that nano-TiO(2) and Fe(3)O(4)@TiO(2)significantly increased the NF-κB expression in the nucleus, indicating that the NF-κB signaling pathway was involved in the dendritic cell maturation. Our results suggested that nano-TiO(2) and Fe(3)O(4)@TiO(2)may function as a useful vector to promote vaccine delivery in immune cells, and Fe(3)O(4)@TiO(2)provided a possibility to deliver and track vaccines via its magnetofection.

Application of a NotI subtraction and methylation­specific genome subtractive hybridization technique in the detection of genomic DNA methylation differences between hydatidiform moles and villi.

Previous studies indicate that epigenetic modifications play an important role in transcriptional regulation and contribute to the pathogenesis of gestational trophoblastic disease, including complete hydatidiform moles (CHMs). However, the underlying mechanisms and the critical genes have not been clearly identified. In the present study, we developed a novel technique, NotI subtraction and methylation-specific genome subtractive hybridization (MS-G-SH), as a method of screening for methylation changes between hydatidiform moles and villi. Following NotI subtraction and hybridization, three different positive DNA clones were found in 110 random clones of DNA samples. Most importantly, two DNA clones having long CpG islands and high homology with exons of insulin-like growth factor 2 (IGF2) and transforming growth factor-ß (TGF-ß) were identified using bioinformatic tools. After bisulfite treatment and methylation-specific PCR, the specific methylation of certain exons of IGF2 and TGF-ß was identified. In addition, the mRNA expression levels of these two genes were markedly different. In conclusion, this novel MS-G-SH technique is an alternative and effective approach for the detection of specific DNA methylation.

pH-sensitive strontium carbonate nanoparticles as new anticancer vehicles for controlled etoposide release.

Strontium carbonate nanoparticles (SCNs), a novel biodegradable nanosystem for the pH-sensitive release of anticancer drugs, were developed via a facile mixed solvent method aimed at creating smart drug delivery in acidic conditions, particularly in tumor environments. Structural characterization of SCNs revealed that the engineered nanocarriers were uniform in size and presented a dumbbell-shaped morphology with a dense mass of a scale-like spine coating, which could serve as the storage structure for hydrophobic drugs. Chosen as a model anticancer agent, etoposide was effectively loaded into SCNs based on a simultaneous process that allowed for the formation of the nanocarriers and for drug storage to be accomplished in a single step. The etoposide-loaded SCNs (ESCNs) possess both a high loading capacity and efficient encapsulation. It was found that the cumulative release of etoposide from ESCNs is acid-dependent, and that the release rate is slow at a pH of 7.4; this rate increases significantly at low pH levels (5.8, 3.0). Meanwhile, it was also found that the blank SCNs were almost nontoxic to normal cells, and ESCN systems were evidently more potent in antitumor activity compared with free etoposide, as confirmed by a cytotoxicity test using an MTT assay and an apoptosis test with fluorescence-activated cell sorter (FACS) analysis. These findings suggest that SCNs hold tremendous promise in the areas of controlled drug delivery and targeted cancer therapy.

Establishment and characterization of multi-drug resistant, prostate carcinoma-initiating stem-like cells from human prostate cancer cell lines 22RV1.

Multi-drug resistance is an important element which leads to ineffectiveness of chemotherapeutics. To identify subpopulations of cancerous prostate cells with multi-drug resistance and cancer stem-cell properties has recently become a major research interest. We identified a subpopulation from the prostate cancer cell line 22RV1, which have high surface expression of both CD117 and ABCG2. We found this subpopulation of cells termed CD117(+)/ABCG2(+) also overexpress stem cells markers such as Nanog, Oct4, Sox2, Nestin, and CD133. These cells are highly prolific and are also resistant to treatment with a variety of chemotherapeutics such as casplatin, paclitaxel, adriamycin, and methotrexate. In addition, CD117(+)/ABCG2(+) cells can readily establish tumors in vivo in a relatively short time. To investigate the mechanism of aggressive tumor growth and drug resistance, we examined the CpG islands on the ABCG2 promoter of CD117(+)/ABCG2(+) cells and found they were remarkably hypomethylated. Furthermore, chromatin immunoprecipitation assays revealed high levels of both histone 3 acetylation and H3K4 trimethylation at the CpG islands on the ABCG2 promoter. Our these data suggest that CD117(+)/ABCG2(+) cells could be reliably sorted from the human prostate cancer cell line 22RV1, and represent a valuable model for studying cancer cell physiology and multi-drug resistance. Furthermore, identification and study of these cells could have a profound impact on selection of individual treatment strategies, clinical outcome, and the design or selection of the next generation of chemotherapeutic agents.

Human amniotic epithelial cell feeder layers maintain mouse embryonic stem cell pluripotency via epigenetic regulation of the c-Myc promoter.

Mouse embryonic stem cells (ESCs) are typically cultured on a feeder layer of mouse embryonic fibroblasts (MEFs), with leukemia inhibitory factor (LIF) added to maintain them in an undifferentiated state. We have previously shown that human amniotic epithelial cells (hAECs) can be used as feeder cells to maintain mouse ESC pluripotency, but the mechanism for this is unknown. In the present study, we found that CpG islands 5' of the c-Myc gene remain hypomethylated in mouse ESCs cultured on hAECs. In addition, levels of acetylation of histone H3 and trimethylation of histone H3K4 in the c-Myc gene promoter were higher in ES cells cultured on hAECs than those in ES cells cultured on MEFs. These data suggested that hAECs can alter mouse ESC gene expression via epigenetic modification of c-Myc, providing a possible mechanism for the hAEC-induced maintenance of ESCs in an undifferentiated state.

Cloning and characterization of a novel Hsp100/Clp gene (osClpD) from Oryza sativa.

A novel osClpD gene, encoding a highly conservative ClpD subfamily member, was first isolated and characterized from Oryza sativa. The full-length cDNA of osClpD gene was 3140 bp and contained a 2884 bp open reading frame encoding a 938 amino acid protein. The phylogenetic tree and blast search showed that OSClpD belonged to the ClpD subfamily of the Hsp100/Clp family, and contained all protein motifs characteristic for the ClpD subfamily of Hsp100/Clp proteins. The real-time quantitative PCR analysis proved that it was inducible by water deficit and temperature stress in vegetative tissues.

cDNA cloning and expression analysis of the rice (Oryza sativa L.) RNase L inhibitor.

A subtractive cDNA library was constructed using rice (Oryza sativa L.) callus cDNA as driver and differentiating callus cDNA as tester. A novel cDNA fragment encoding RNase L inhibitor (RLI) was isolated by screening the subtractive library, which had a higher expression level in differentiating callus than in callus. The full-length cDNA of rice-RLI was obtained by the method of rapid amplification of cDNA ends, which contained a 1812-bp open reading frame encoding a 604 amino acid polypeptide. Homologous analysis showed that rice-RLI contained the conserved motifs (two repeated P-loops, two ATP-binding boxes and an iron-sulfur binding motif). The fluorescence quantitative PCR analysis showed that it was a constitutive expressed gene but up-regulated in abiotic stress (low temperature and NaCl treatment) and down-regulated under the treatments of NAA and IAA.

[Cloning and expression of maize fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase (mF2KP)].

A 2469 bp cDNA encoding entire fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase (F2KP) was cloned from maize (Zea mays L.cv. Ziyunuo 1) by the methods of RT-PCR and rapid amplification of cDNA ends(RACE), on the basis of AF007582, which was isolated from "Yedan 4". The cDNA was designated as mF2KP and the GenBank accession number is AF334143, which contains a 2226 bp open reading from (ORF), encoding a 741 residue polypeptide. There are some differences between the F2KP genes of two maize varieties. The length of 3' non-coding region of mF2KP is 38 bp shorter than that of AF007582. On the 1592th, 1593th and 1605th positions of mF2KP, there is an additional nucleotide respectively compared with AF007582, which cause a shifted reading frame in a small region. Northern blot showed that the expression of mF2KP were significantly different among maize tissues. The Transcription of mF2KP in stem was lower than those in leaves, kernel leaves and male inflorescence, but much higher than that in immature seeds.