Analysis of Differential Expression Proteins of Paclitaxel-Treated Lung Adenocarcinoma Cell A549 Using Tandem Mass Tag-Based Quantitative Proteomics.

BACKGROUND: Paclitaxel is widely used in the treatment of cancer and has a good effect in the treatment of non-small cell lung cancer. The combination of TMT proteomics and bioinformatics is used to systematically analyze the molecular mechanism of paclitaxel in the treatment of lung adenocarcinoma A549 cell, which is helpful to screen new therapeutic targets. METHODS: MTT assay was used to analyze the inhibitory effect of paclitaxel on the proliferation of A549 cells. The proteins were identified by TMT quantitative proteomics and the differential expression proteins (DEPs) database was constructed. The DEPs were enriched by Gene Ontology (GO) and KEGG pathway annotation. Based on the information in the STRING database, find the interaction between DEPs, and the protein-protein interaction (PPI) networks of DEPs were constructed and analyzed by using the Cytoscape software. According to the PPI network results, select the hub proteins from DEPs for WB verification. RESULTS: A total of 5449 proteins were identified in A549 by TMT proteomics. Compared with the control group, 281 DEPs were significantly up-regulated and 218 were significantly down-regulated after paclitaxel treatment. GO functional analysis, we found that the main functions of these DEPs are binding, catalytic activity, molecular function regulator and so on. They are mainly involved in cellular process, metabolic process, biological regulation and so on. KEGG analysis showed that the three most significant signal transduction pathways of DEPs enrichment were DNA replication, steroid biosynthesis, oxidative phosphorylation. In PPI network, there are 294 nodes among which CDK1, MCM2-5 and PCNA are located at the center of proteins interaction. WB analysis confirmed that the expression of CDK1 was significantly down-regulated, consistent with the TMT results. CONCLUSION: Paclitaxel significantly increased the expression of tubulin, binding tubulin to promote A549 cell death. In addition, paclitaxel significantly inhibited the expression of hub proteins, DNA replication and cell cycle pathways, thus killing lung adenocarcinoma cell A549. These findings will enhance the understanding of the mechanism of paclitaxel in the treatment of lung adenocarcinoma cell A549 and provide new valuable targets.

Trehalose-6-phosphate phosphatase E modulates ABA-controlled root growth and stomatal movement in Arabidopsis.

Trehalose plays important roles in plant growth and stress responses and is synthesized from trehalose-6-phosphate by trehalose-6-phosphate phosphatase (TPP). Here, we show that trehalose and abscisic acid (ABA) have synergistic effects on root growth and stomatal closure. The Arabidopsis thaliana genome contains ten genes encoding TPPs and the expression level of one, TPPE, and trehalose contents increased in response to ABA. In the presence of ABA, the ABA-responsive transcription factor ABA RESPONSE ELEMENT BINDING FACTOR2 (ABF2) directly binds to the TPPE promoter to activate its expression. Genetic analysis revealed that TPPE acts downstream of ABF2, which is supported by the findings that TPPE expression and trehalose content are reduced in the abf2 mutant and that a mutation in TPPE abolished the ABA-sensitive root elongation phenotype of 35S:ABF2 plants. Reactive oxygen species (ROS) accumulation in response to ABA failed to occur in tppe mutant plants, suggesting that TPPE is involved in ABA-controlled root elongation and stomatal movement by inducing ROS accumulation. This study uncovers a new branch of the ABA signaling pathway and provides a molecular basis for the role of trehalose in plant responses to abiotic stress.

Systematic Analysis of the DNA Methylase and Demethylase Gene Families in Rapeseed (Brassica napus L.) and Their Expression Variations After Salt and Heat stresses.

DNA methylation is a process through which methyl groups are added to the DNA molecule, thereby modifying the activity of a DNA segment without changing the sequence. Increasing evidence has shown that DNA methylation is involved in various aspects of plant growth and development via a number of key processes including genomic imprinting and repression of transposable elements. DNA methylase and demethylase are two crucial enzymes that play significant roles in dynamically maintaining genome DNA methylation status in plants. In this work, 22 DNA methylase genes and six DNA demethylase genes were identified in rapeseed (Brassica napus L.) genome. These DNA methylase and DNA demethylase genes can be classified into four (BnaCMTs, BnaMET1s, BnaDRMs and BnaDNMT2s) and three (BnaDMEs, BnaDML3s and BnaROS1s) subfamilies, respectively. Further analysis of gene structure and conserved domains showed that each sub-class is highly conserved between rapeseed and Arabidopsis. Expression analysis conducted by RNA-seq as well as qRT-PCR suggested that these DNA methylation/demethylation-related genes may be involved in the heat/salt stress responses in rapeseed. Taken together, our findings may provide valuable information for future functional characterization of these two types of epigenetic regulatory enzymes in polyploid species such as rapeseed, as well as for analyzing their evolutionary relationships within the plant kingdom.

Genome-Wide Identification and Characterization of FBA Gene Family in Polyploid Crop Brassica napus.

Fructose-1,6-bisphosphate aldolase (FBA) is a versatile metabolic enzyme involved in multiple important processes of glycolysis, gluconeogenesis, and Calvin cycle. Despite its significance in plant biology, the identity of this gene family in oil crops is lacking. Here, we performed genome-wide identification and characterization of FBAs in an allotetraploid species, oilseed rape Brassica napus. Twenty-two BnaFBA genes were identified and divided into two groups based on integrative analyses of functional domains, phylogenetic relationships, and gene structures. Twelve and ten B. napus FBAs (BnaFBAs) were predicted to be localized in the chloroplast and cytoplasm, respectively. Notably, synteny analysis revealed that Brassica-specific triplication contributed to the expansion of the BnaFBA gene family during the evolution of B. napus. Various cis-acting regulatory elements pertinent to abiotic and biotic stresses, as well as phytohormone responses, were detected. Intriguingly, each of the BnaFBA genes exhibited distinct sequence polymorphisms. Among them, six contained signatures of selection, likely having experienced breeding selection during adaptation and domestication. Importantly, BnaFBAs showed diverse expression patterns at different developmental stages and were preferentially highly expressed in photosynthetic tissues. Our data thus provided the foundation for further elucidating the functional roles of individual BnaFBA and also potential targets for engineering to improve photosynthetic productivity in B. napus.

Long noncoding RNA MEG3 inhibits breast cancer growth via upregulating endoplasmic reticulum stress and activating NF-κB and p53.

Long noncoding RNA (lncRNA) maternally expressed 3 (MEG3) has been implicated as a tumor suppressor gene in several human cancer types. However, little is known regarding its involvement and potential mechanism in human breast cancer. In this study, we explored the effect of MEG3 on the growth of human breast cancer cell line MDA-MB-231 in vitro and in vivo, and sought to elucidate the potential signaling mechanisms. Ectopic overexpression of MEG3 using a lentiviral vector Lv-MEG3 significantly inhibited breast cancer cell growth in vitro and a cancer xenograft growth in vivo. MEG3 overexpression led to marked increase of apoptosis in breast cancer cells as determined using flow cytometry and fragmented DNA labeling. Moreover, ectopic expression of MEG3 increased the expression of endoplasmic reticulum (ER) stress-related proteins required for unfolded protein response, including glucose-regulated protein 78 (GRP78), inositol-requiring enzyme 1 (IRE1), protein kinase RNA (PKR)-like ER kinase (PERK), and activated transcription factor 6 (ATF6), as well as proapoptotic proteins CCAAT/enhancer binding protein homologous protein (CHOP) and caspase-3. Finally, MEG3 overexpression markedly increased nuclear factor κB (NF-κB) expression, NF-κB translocation to the nucleus, and p53 expression, whereas pharmacological inhibition of NF-κB completely abolished MEG3-induced activation of p53. Together, these results suggest that MEG3 inhibits breast cancer growth and induces breast cancer apoptosis, partially via the activation of the ER stress, NF-κB and p53 pathways, and that NF-κB signaling is required for MEG3-induced p53 activation in breast cancer cells. Our results indicate targeting lncRNA MEG3 may represent a novel strategy for breast cancer therapy.

Clinicopathological significance of DAPK promoter methylation in non-small-cell lung cancer: a systematic review and meta-analysis.

BACKGROUND: Lung carcinogenesis is related to silencing of tumor suppressor genes and activation of oncogenes. The aim was to investigate the significance of death-associated protein kinase (DAPK) methylation in non-small-cell lung cancer (NSCLC) through a meta-analysis. METHODS: A detailed literature search was made in PubMed, Embase, and Web of Science databases. All analysis was performed with Review Manager 5.2. RESULTS: In total, 28 studies with a total of 2,148 patients were involved. The frequency of DAPK promoter hypermethylation was 40.50% in NSCLC, significantly higher than in nonmalignant lung tissue; the pooled OR was 5.69, P<0.00001. Additionally, DAPK promoter hypermethylation was significantly correlated with poor overall survival in patients with NSCLC. However, there was no significant difference found while comparing the rate of DAPK promoter hypermethylation in adenocarcinoma and squamous cell cancer. The rate of DAPK promoter hypermethylation was similar between stage III/IV and stage I/II. In addition, the data showed that DAPK promoter hypermethylation was not associated with smoking behavior in patients with NSCLC. CONCLUSION: DAPK promoter hypermethylation is correlated with risk of NSCLC and is a potential biomarker for prediction of poor prognosis in patients with NSCLC.

Multichannel electroanalytical devices for competitive ELISA of phenylethanolamine A.

Herein, we reported a disposable electroanalytical device for competitive enzyme-linked immunosorbent assay (ELISA) of phenylethanolamine A (PA). Conductive carbon tape coated with gold cluster served as the working electrode, while the counter and reference electrodes were fabricated on the filter paper by the screen-printing technique. Separating fabrication of the working electrode from other electrodes could make it possible for full utilization of the working electrode modification in bulk and no contamination of the immune-reagents on the counter and reference electrodes. The gold cluster played an important role in both immobilizing antigen and accelerating electron transfer. The eight-channel devices were utilized to detect PA on the strategy of competitive ELISA. The detection range and the limit of detection (LOD) using differential pulse voltammetry for PA were 0.005-60ngmL-1 and 2.6pgmL-1, respectively. And also, the linear range for PA performed by square wave voltammetry was 0.05-60ngmL-1 with the LOD value as 0.028ngmL-1. The results clearly demonstrated that the proposed electroanalytical devices could be successfully applied in immunoassay and might be further developed for determination of different analytes based on ELISA format.

Fluorescent nitrogen and sulfur co-doped carbon dots from casein and their applications for sensitive detection of Hg2+ and biothiols and cellular imaging.

Fluorescent nitrogen and sulfur co-doped carbon dots (NSCDs) were synthesized by a one-step pyrolysis strategy using casein as carbon, nitrogen and sulfur sources, and characterized by UV-vis spectrum, fluorescent spectrum, X-ray photoelectron spectroscopy (XPS) and FT-IR, etc. The synthesized NSCDs displayed a blue emission under ultraviolet illumination with a quantum yield of 31.8%, and a good aqueous solubility, photostability and biocompatibility. It was found that the fluorescence intensity of NSCDs could be selectively quenched by Hg2+, so NSCDs was used as an effective probe for the detection of Hg2+. The linear range and the limit of detection (LOD) of the fluorescent sensor based on NSCDs for the detection of Hg2+ were 0.01-0.25 µM and 6.5 nM, respectively. Spiked water samples were detected by the sensor with the recovery of 95.4-106.3% and relative standard deviation (RSD) of 3.6-8.6%. It was also observed that the quenched NSCDs-Hg2+ system could be restored by the addition of biothiols such as l-cysteine (Lcy), homocysteine (Hcy) and glutathione (GSH), thus NSCDs-Hg2+ system was employed as a fluorescent sensor for the detection of biothiols. The linear range and LOD of the NSCDs-Hg2+ system were 1-10 µM and 23.6 nM for Lcy, 0.2-2.5 µM and 12.3 nM for Hcy, and 0.1-2.0 µM and 16.8 nM for GSH, respectively. The NSCDs-Hg2+ system was applied for the detection of biothiols in serum samples with satisfied results. In addition, the study in vitro imaging HeLa cells revealed that the synthesized NSCDs could be used as effective fluorescent probes in cellular imaging without noticeable cytotoxicity.

Development of an immunoaffinity chromatography column for selective extraction of a new agonist phenylethylamine A from feed, meat and liver samples.

Phenylethanolamine A (PA) is a new emerged ß-adrenergic agonist that has been illegally used as an animal feed additive for growth promotion in China. In this study, an immunoaffinity chromatography (IAC) column for selective extraction of PA from swine feed, meat and liver samples was developed. The IAC column was constructed by covalently coupling specific polyclonal antibody (Ab) against PA to CNBr-activated Sepharose 4B and packed into a common solid phase extraction (SPE) cartridge. The extraction conditions including loading, washing and eluting solutions were carefully optimized. Under optimal conditions, the IAC column was characterized in terms of maximum capacity, selectivity, extraction recovery and stability. The maximum capacity of the ICA for PA extraction was found to be 239.4ng. For selectivity testing, 100ng of other three ß-adrenergic agonists (clenbuterol, ractopamine and salbutamol) was separately loaded onto the column, and it was observed that the tested compounds could not be captured on the column, e.g. the column could only selectively recognize PA. The recovery of the IAC for PA extraction was found within 96.47-101.98% when 10, 50 and 100ng PA were separately loaded onto IAC column. The IAC column was also applied to real sample extraction. Swine feed, meat and liver samples were collected and spiked with PA in range of 1.0-20ngg(-1). The spiked and unspiked samples were extracted by IAC column and measured by high performance liquid chromatography (HPLC). It was found that there was no detectable PA in the blank samples, and the extraction recoveries of the IAC for PA from the spiked samples were within 89.48-104.89%. The stability of the column was also tested. It was showed that after 35 times repeated usage, 60% of the maximum capacity was still remained. The proposed IAC was proven to be a feasible extraction method for PA from different matrices with the properties of high maximum capacity, selectivity, extraction efficiency and stability.

Supersandwich cytosensor for selective and ultrasensitive detection of cancer cells using aptamer-DNA concatamer-quantum dots probes.

In this work, a signal amplification supersandwich strategy was developed for highly selective and sensitive detection of cancer cells using aptamer-DNA concatamer-quantum dots (QDs) probes. First of all, electrode materials denoted as MWCNTs@PDA@AuNPs were fabricated by multiwall carbon nanotubes (MWCNTs), gold nanoparticles (AuNPs), and polydopamine (PDA) using a layer-by-layer technique. Then, the prepared bases as matrices were applied to bind concanavalin A (Con A), resulting in high stability, bioactivity, and capability for cell capture. Meanwhile, aptamer-DNA concatamer-QDs were designed via DNA hybridization followed by covalent assembling, which incorporated the specific recognition of the aptamer with the signal amplification of the DNA concatamer and QDs. With aptamer-DNA concatamer-QDs as recognizing probes, the model cancer cells (CCRF-CEM cells) were detected using a MWCNTs@PDA@AuNPs modified electrode with trapped Con A by means of fluorescence and electrochemical methods. The proposed supersandwich cytosensor showed high sensitivity with the detection limit of 50 cells mL(-1). More importantly, it could distinguish cancer cells from normal cells, which indicated the promising applications of our method in clinical diagnosis and treatment of cancers.

[Identify the original plant of Forsythia suspensa by nucleotide sequence].

OBJECTIVE: To investigate the means of distinguishing the original plant of Forsythia suspensa from confusion. METHODS: To amplify the chloroplast matK gene by PCR using a consensus primer set and determine their nucleotide sequence by PCR direct sequencing. The ITS sequences were gained from NCBI. The characteristic analysis of matK and ITS sequences were generated using Clustal aligned. RESULTS: There were 30 bp and 8 bp unique nucleotide in ITS and matK sequence in Forsythia suspensa. The matK gene and ITS sequences might be good molecular marker to be used to identify the original plant of Forsythia suspensa. CONCLUSION: The sequence analysis of matK gene ITS sequences might become the mean to identify the original plant of Forsythia suspensa.

Zinc finger protein 1 (ThZF1) from salt cress (Thellungiella halophila) is a Cys-2/His-2-type transcription factor involved in drought and salt stress.

Zinc finger proteins (ZFPs) play important roles in growth and development in both animals and plants. Recently, some Arabidopsis genes encoding distinct ZFPs have been identified. However, the physiological role of their homologues with putative zinc finger motif remains unclear. In the present study, a novel gene, ThZF1, was characterized from salt stressed cress (Thellungiella halophila, Shan Dong), encoding a functional transcription factor. ThZF1 contains two conserved C(2)H(2) regions and shares conserved domains, including DNA-binding motif, with Arabidopsis thaliana ZFP family members. The transcript of the ThZF1 gene was induced by salinity and drought. Transient expression analysis of ThZF1-GFP fusion protein revealed that ThZF1 was localized preferentially in nucleus. A gel-shift assay showed that ThZF1 specially bind to the wild-type (WT) EP2 element, a cis-element present in the promoter regions of several target genes regulated by ZFPs. Furthermore, a functional analysis demonstrated that ThZF1 was able to activate HIS marker gene in yeast. Finally, ectopic expression of ThZF1 in Arabidopsis mutant azf2 suggested that ThZF1 may have similar roles as Arabidopsis AZF2 in plant development as well as regulation of downstream gene.

AtHsfA2 modulates expression of stress responsive genes and enhances tolerance to heat and oxidative stress in Arabidopsis.

There is increasing evidence for considerable interlinking between the responses to heat stress and oxidative stress, and recent researches suggest heat shock transcription factors (Hsfs) play an important role in linking heat shock with oxidative stress signals. In this paper, we present evidence that AtHsfA2 modulated expression of stress responsive genes and enhanced tolerance to heat and oxidative stress in Arabidopsis. Using Northern blot and quantitative RT-PCR analysis, we demonstrated that the expression of AtHsfA2 was induced by not only HS but also oxidative stress. By functional analysis of AtHsfA2 knockout mutants and AtHsfA2 overexpressing transgenic plants, we also demonstrated that the mutants displayed reduced the basal and acquired thermotolerance as well as oxidative stress tolerance but the overexpression lines displayed increased tolerance to these stress. The phenotypes correlated with the expression of some Hsps and APX1, ion leakage, H202 level and degree of oxidative injuries. These results showed that, by modulated expression of stress responsive genes, AtHsfA2 enhanced tolerance to heat and oxidative stress in Arabidopsis. So we suggest that AtHsfA2 plays an important role in linking heat shock with oxidative stress signals.