21 results on '"Xu, Kun"'
Search Results
2. Genome-wide analysis and expression profiles of glyoxalase gene families in Chinese cabbage (Brassica rapa L).
- Author
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Yan, Guixin, Xiao, Xin, Wang, Nian, Zhang, Fugui, Gao, Guizhen, Xu, Kun, Chen, Biyun, Qiao, Jiangwei, and Wu, Xiaoming
- Subjects
CHINESE cabbage ,GENE expression in plants ,GLYOXALASE ,EFFECT of heavy metals on plants ,CHROMOSOME duplication - Abstract
The glyoxalase pathway is composed of glyoxalase I (GLYI) and glyoxalase II (GLYII) and is responsible for the detoxification of a cytotoxic metabolite methylglyoxal (MG) into the nontoxic S-D-lactoylglutathione. The two glyoxalase enzymes play a crucial role in stress tolerance in various plant species. Recently, the GLY gene families have well been analyzed in Arabidopsis, rice and soybean, however, little is known about them in Chinese cabbage (Brassica rapa). Here, 16 BrGLYI and 15 BrGLYII genes were identified in the B. rapa genome, and the BrGLYI and BrGLYII proteins were both clustered into five subfamilies. The classifications, chromosomal distributions, gene duplications, exon–intron structures, localizations, conserved motifs and promoter cis-elements were also predicted and analyzed. In addition, the expression pattern of these genes in different tissues and their response to biotic and abiotic stresses were analyzed using publicly available data and a quantitative real-time PCR analysis (RT-qPCR). The results indicated that the expression profiles of BrGLY genes varied among different tissues. Notably, a number of BrGLY genes showed responses to biotic and abiotic stress treatments, including Plasmodiophora brassicae infection and various heavy metal stresses. Taken together, this study identifies BrGLYI and BrGLYII gene families in B. rapa and offers insight into their roles in plant development and stress resistance, especially in heavy metal stress tolerance and pathogen resistance. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
3. Trait-Based Community Assembly along an Elevational Gradient in Subalpine Forests: Quantifying the Roles of Environmental Factors in Inter- and Intraspecific Variability.
- Author
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Luo, Ya-Huang, Liu, Jie, Tan, Shao-Lin, Cadotte, Marc William, Wang, Yue-Hua, Xu, Kun, Li, De-Zhu, and Gao, Lian-Ming
- Subjects
SUBALPINE zone ,FORESTS & forestry ,PLANT communities ,PLANT anatomy ,SOIL moisture ,ENVIRONMENTAL sciences - Abstract
Understanding how communities respond to environmental variation is a central goal in ecology. Plant communities respond to environmental gradients via intraspecific and/or interspecific variation in plant functional traits. However, the relative contribution of these two responses to environmental factors remains poorly tested. We measured six functional traits (height, leaf thickness, specific leaf area (SLA), leaf carbon concentration (LCC), leaf nitrogen concentration (LNC) and leaf phosphorus concentration (LPC)) for 55 tree species occurring at five elevations across a 1200 m elevational gradient of subalpine forests in Yulong Mountain, Southwest China. We examined the relative contribution of interspecific and intraspecific traits variability based on community weighted mean trait values and functional diversity, and tested how different components of trait variation respond to different environmental axes (climate and soil variables). Species turnover explained the largest amount of variation in leaf morphological traits (leaf thickness and SLA) across the elevational gradient. However, intraspecific variability explained a large amount of variation (49.3%–76.3%) in three other traits (height, LNC and LPC) despite high levels of species turnover. The detection of limiting similarity in community assembly was improved when accounting for both intraspecific and interspecific variability. Different components of trait variation respond to different environmental axes, especially soil water content and climatic variables. Our results indicate that intraspecific variation is critical for understanding community assembly and evaluating community response to environmental change. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
4. Prevalence, Awareness, Treatment and Control of Diabetes Mellitus in a Chinese Population.
- Author
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Yue, Jiqiang, Mao, Xuhua, Xu, Kun, Lü, Lingshuang, Liu, Sijun, Chen, Feng, and Wang, Jianming
- Subjects
TREATMENT of diabetes ,DIABETES prevention ,DISEASE prevalence ,CHINESE people ,REGRESSION analysis ,DISEASES - Abstract
Objective: The purpose of this study is to evaluate the prevalence, awareness, treatment and glycemic control of diabetes mellitus (DM) in a Chinese population. The findings from this study are expected to offer scientific evidence to better prevent and control the growing number of reported and untreated cases. Methods: A cross-sectional survey was conducted in Jiangsu, China. We recruited permanent residents over 18 years of age from eight towns in Jintan (JT) and six towns in Yangzhong (YZ) using a three-stage stratified cluster sampling method. The rates of DM prevalence, awareness, treatment and control as well as their related factors were analyzed. Results: A total number of 15404 people were entered into the analysis. The DM prevalence, awareness, treatment and control rates were 7.31%, 58.35%, 51.87% and 14.12%, respectively. Multivariable logistic regression analysis showed that being female was positively related to prevalence (OR = 1.21, 95% CI: 1.07–1.37), awareness (OR = 1.52, 95% CI: 1.19–1.93), treatment (OR = 1.48, 95% CI: 1.17–1.88) and control (OR = 1.87, 95% CI: 1.30–2.67) of DM. Having a family history of diabetes was significantly correlated with DM risk (OR = 1.86, 95% CI: 1.37–2.54) and increased awareness (OR = 3.12, 95% CI: 2.19–4.47), treatment (OR = 3.47, 95% CI: 2.45–4.90) and control (OR = 1.81, 95% CI: 1.22–2.68) of DM. Former smoking status (OR = 1.82, 95% CI: 1.23–2.71), overweight (OR = 2.11, 95% CI: 1.72–2.60) and obesity (OR = 3.46, 95% CI: 2.67–4.50) were related to the risk of DM. Additionally, we found current drinking status to be positively correlated with DM risk (OR = 1.30, 95% CI: 1.01–1.66) and negatively correlated with DM awareness (OR = 0.41, 95% CI: 0.29–0.59) and treatment (OR = 0.41, 95% CI: 0.29–0.59). Our study highlights the high prevalence and inadequate awareness, treatment and control of DM in the Chinese population. Conclusions: Management and prevention of DM-related complications should be considered an essential strategy by governments and society. This study assessed the reasons why DM has been increasing and established the first step in determining where to start regarding preventative methods. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
5. Massive Endoscopic Screening for Esophageal and Gastric Cancers in a High-Risk Area of China.
- Author
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Zheng, Xianzhi, Mao, Xuhua, Xu, Kun, Lü, Lingshuang, Peng, Xianzhen, Wang, Min, Xu, Guisheng, Hua, Zhaolai, Wang, Jianping, Xue, Hengchuan, Wang, Jianming, and Lu, Cheng
- Subjects
DIAGNOSIS of esophageal cancer ,CANCER diagnosis ,STOMACH cancer ,ENDOSCOPY ,MEDICAL screening ,ESOPHAGEAL cancer patients ,STOMACH cancer patients ,TREATMENT of esophageal cancer - Abstract
Objective: This study aims to describe the findings from a massive endoscopic screening program in a high-risk area of China and to evaluate the prognosis of patients diagnosed through endoscopic screening compared with those diagnosed at usual hospital visits because of illness. Methods: In 2006, an early detection and treatment program was initiated in Yangzhong county, China. Local residents aged 40–69 years were eligible for free endoscopic screening. Endoscopic examination was performed with Lugol’s iodine staining, followed by biopsies. Patients diagnosed with esophageal or gastric cancer were referred for treatment and followed to assess their long-term survival status. Results: From 2006 through 2012, we screened 12453 participants, including 5334 (42.8%) men and 7119 (57.2%) women. The average age was 52.8±8.0 years. We detected 166 patients with upper digestive tract cancers, including 106 cancers in the esophagus (detection rate: 0.85%) and 60 cancers in the stomach (detection rate: 0.48%). Of these patients, 98.11% with esophageal cancer and 100% with gastric cancer were defined as at the early stage. In the process of follow-up, 17 patients died from cancer-related causes, and the median survival time was greater than 85 months. The overall survival rates for 1, 3 and 5 years were 98.0%, 90.0% and 89.0%, respectively. A significant positive effect was observed for the long-term survival of patients diagnosed through massive endoscopic screening. Conclusions: In a high-risk population, massive endoscopic screening can identify early stage carcinoma of esophageal and gastric cancers and improve patients’ prognosis through early detection and treatment. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
6. Extensive Analysis of GmFTL and GmCOL Expression in Northern Soybean Cultivars in Field Conditions.
- Author
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Guo, Guangyu, Xu, Kun, Zhang, Xiaomei, Zhu, Jinlong, Lu, Mingyang, Chen, Fulu, Liu, Linpo, Xi, Zhang-Ying, Bachmair, Andreas, Chen, Qingshan, and Fu, Yong-Fu
- Subjects
- *
SOYBEAN farming , *EXPERIMENTAL agriculture , *GENETIC overexpression , *PLANTS , *PHOTOPERIODISM , *ARABIDOPSIS thaliana - Abstract
The FLOWERING LOCUS T (FT) gene is a highly conserved florigen gene among flowering plants. Soybean genome encodes six homologs of FT, which display flowering activity in Arabidopsis thaliana. However, their contributions to flowering time in different soybean cultivars, especially in field conditions, are unclear. We employed six soybean cultivars with different maturities to extensively investigate expression patterns of GmFTLs (Glycine max FT-like) and GmCOLs (Glycine max CO-like) in the field conditions. The results show that GmFTL3 is an FT homolog with the highest transcript abundance in soybean, but other GmFTLs may also contribute to flower induction with different extents, because they have more or less similar expression patterns in developmental-, leaf-, and circadian-specific modes. And four GmCOL genes (GmCOL1/2/5/13) may confer to the expression of GmFTL genes. Artificial manipulation of GmFTL expression by transgenic strategy (overexpression and RNAi) results in a distinct change in soybean flowering time, indicating that GmFTLs not only impact on the control of flowering time, but have potential applications in the manipulation of photoperiodic adaptation in soybean. Additionally, transgenic plants show that GmFTLs play a role in formation of the first flowers and in vegetative growth. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
7. Structural and Biochemical Analysis of Tyrosine Phosphatase Related to Biofilm Formation A (TpbA) from the Opportunistic Pathogen Pseudomonas aeruginosa PAO1.
- Author
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Xu, Kun, Li, Shanshan, Yang, Wen, Li, Kan, Bai, Yuwei, Xu, Yueyang, Jin, Jin, Wang, Yingying, and Bartlam, Mark
- Subjects
- *
PSEUDOMONAS aeruginosa infections , *PROTEIN-tyrosine phosphatase , *BIOCHEMISTRY , *MOLECULAR structure , *BIOFILMS , *OPPORTUNISTIC infections , *CELL communication - Abstract
Biofilms are important for cell communication and growth in most bacteria, and are responsible for a number of human clinical infections and diseases. TpbA (PA3885) is a dual specific tyrosine phosphatase (DUSP) that negatively regulates biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa PAO1 by converting extracellular quorum sensing signals into internal gene cascade reactions that result in reduced biofilm formation. We have determined the three-dimensional crystal structure of wild-type TpbA from P. aeruginosa PAO1 in the phosphate-bound state and a TpbA (C132S) mutant with phosphotyrosine. Comparison between the phosphate-bound structure and the previously reported ligand-free TpbA structure reveals the extent of conformational changes that occur upon substrate binding. The largest changes occur in the functional loops that define the substrate binding site, including the PTP, general acid and α4-α5 loops. We further show that TpbA efficiently catalyzes the hydrolysis of two phosphotyrosine peptides derived from the periplasmic domain of TpbB (YfiN, PA1120), with a strong preference for dephosphorylating Tyr48 over Tyr62. This work adds to the small repertoire of DUSP structures in both the ligand-free and ligand-bound states, and provides a starting point for further study of the role of TpbA in biofilm formation. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
8. The Effects of Harvesting Media on Biological Characteristics and Repair Potential of Neural Stem Cells after Traumatic Brain Injury.
- Author
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Liu, Shengliang, Li, Zhuying, Fu, Jin, Sun, Liang, Xu, Fengyan, Harada, Toshihide, Lou, Yu, Chu, Ming, Sun, Qi, Xu, Kun, Zhang, Rui, Jin, Lianhong, Xiao, Hui, and Wu, Shuliang
- Subjects
BRAIN injury treatment ,NEURAL stem cell transplantation ,STEM cell culture ,CELL proliferation ,CEREBROSPINAL fluid ,LABORATORY mice - Abstract
Various solutions are utilized widely for the isolation, harvesting, sorting, testing and transplantation of neural stem cells (NSCs), whereas the effects of harvesting media on the biological characteristics and repair potential of NSCs remain unclear. To examine some of these effects, NSCs were isolated from cortex of E14.5 mice and exposed to the conventional harvesting media [0.9% saline (Saline), phosphate-buffered saline (PBS) or artificial cerebrospinal fluid (ACSF)] or the proliferation culture medium (PCM) for different durations at 4°C. Treated NSCs were grafted by in situ injection into the lesion sites of traumatic brain injury (TBI) mice. In vitro, harvesting media-exposed NSCs displayed time-dependent reduction of viability and proliferation. S phase entry decreased in harvesting media-exposed cells, which was associated with upregulation of p53 protein and downregulation of cyclin E1 protein. Moreover, harvesting media exposure induced the necrosis and apoptosis of NSCs. The levels of Fas-L, cleaved caspase 3 and 8 were increased, which suggests that the death receptor signaling pathway is involved in the apoptosis of NSCs. In addition, exposure to Saline did not facilitate the neuronal differentiation of NSCs, suggesting that Saline exposure may be disadvantageous for neurogenesis. In vivo, NSC-mediated functional recovery in harvesting media-exposed NSC groups was notably attenuated in comparison with the PCM-exposed NSC group. In conclusion, harvesting media exposure modulates the biological characteristics and repair potential of NSCs after TBI. Our results suggest that insight of the effects of harvesting media exposure on NSCs is critical for developing strategies to assure the successful long-term engraftment of NSCs. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
9. Pharmacokinetics of Anti-HBV Polyoxometalate in Rats.
- Author
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Wang, Juan, Qu, Xiaofeng, Qi, Yanfei, Li, Jinhua, Song, Xiuling, Li, Li, Yin, Dehui, Xu, Kun, and Li, Juan
- Subjects
PHARMACOKINETICS ,POLYOXOMETALATES ,LABORATORY rats ,ANTIVIRAL agents ,NUCLEOSIDES ,BLOOD plasma - Abstract
Polyoxometalates are non-nucleoside analogs that have been proven to exhibit broad-spectrum antiviral activity. In particular, Cs
2 K4 Na[SiW9 Nb3 O40 ].H2 O 1 shows low toxicity and high activity against HBV. The preclinical pharmacokinetics of Compound 1 in rats were characterized by establishing and applying inductively coupled plasma-mass spectrometry method to determine the concentration of W in plasma, urine, feces, bile and organ samples. The quantitative ICP-MS method demonstrated good sensitivity and application in the pharmacokinetics study of polyoxometalates. The pharmacokinetic behavior of Compound 1 after intravenous or oral administration fit a two-compartment model. Tmax ranges from 0.1 h to 3 h and the T1/2 of Compound 1 is between 20 h and 30 h. The absolute bioavailability of Compound 1 at 45, 180 and 720 mg/kg groups were 23.68%, 14.67% and 11.93%, respectively. The rates of plasma protein binding of Compound 1 at 9, 18 and 36 mg/ml of Compound 1 are 62.13±9.41%, 71.20±24.98% and 49.00±25.59%, respectively. Compound 1 was widely distributed throughout the body, and high levels of compound 1 were found in the kidney and liver. The level of Compound 1 in excretion was lower: 30% for urine, 0.28% for feces and 0.42% for bile, respectively. For elaborate pharmacokinetic characteristics to be fully understood, the metabolism of Compound 1 needs to be studied further. [ABSTRACT FROM AUTHOR]- Published
- 2014
- Full Text
- View/download PDF
10. Genome-Wide Survey and Expression Analysis of the Putative Non-Specific Lipid Transfer Proteins in Brassica rapa L.
- Author
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Li, Jun, Gao, Guizhen, Xu, Kun, Chen, Biyun, Yan, Guixin, Li, Feng, Qiao, Jiangwei, Zhang, Tianyao, and Wu, Xiaoming
- Subjects
LIPID transfer protein ,GENE expression in plants ,PLANT physiology ,PHOSPHOLIPIDS ,PLANT defenses ,PLANT reproduction ,BRASSICA - Abstract
Background: Plant non-specific lipid transfer proteins (nsLtps) are small, basic proteins encoded by multigene families and have reported functions in many physiological processes such as mediating phospholipid transfer, defense reactions against phytopathogens, the adaptation of plants to various environmental conditions, and sexual reproduction. To date, no genome-wide overview of the Brassica rapa nsLtp (BrnsLtp) gene family has been performed. Therefore, as the first step and as a helpful strategy to elucidate the functions of BrnsLtps, a genome-wide study for this gene family is necessary. Methodology/Principal Finding: In this study, a total of 63 putative BrnsLtp genes were identified through a comprehensive in silico analysis of the whole genome of B. rapa. Based on the sequence similarities, these BrnsLtps was grouped into nine types (I, II, III, IV, V, VI, VIII, IX, and XI). There is no type VII nsLtps in B. rapa, and a new type, XI nsLtps, was identified in B. rapa. Furthermore, nine type II AtLtps have no homologous genes in B. rapa. Gene duplication analysis demonstrated that the conserved collinear block of each BrnsLtp is highly identical to those in Arabidopsis and that both segmental duplications and tandem duplications seem to play equal roles in the diversification of this gene family. Expression analysis indicated that 29 out of the 63 BrnsLtps showed specific expression patterns. After careful comparison and analysis, we hypothesize that some of the type I BrnsLtps may function like Arabidopsis pathogenesis-related-14 (PR-14) proteins to protect the plant from phytopathogen attack. Eleven BrnsLtps with inflorescence-specific expression may play important roles in sexual reproduction. Additionally, BrnsLtpI.3 may have functions similar to Arabidopsis LTP1. Conclusions/Significance: The genome-wide identification, bioinformatic analysis and expression analysis of BrnsLtp genes should facilitate research of this gene family and polyploidy evolution and provide new insight towards elucidating their biological functions in plants. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
11. Elucidation of How Cancer Cells Avoid Acidosis through Comparative Transcriptomic Data Analysis.
- Author
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Xu, Kun, Mao, Xizeng, Mehta, Minesh, Cui, Juan, Zhang, Chi, Mao, Fenglou, and Xu, Ying
- Subjects
- *
CANCER cells , *ACIDOSIS , *SQUAMOUS cell carcinoma , *ADENOCARCINOMA , *EXTRACELLULAR matrix , *COMPARATIVE studies - Abstract
The rapid growth of cancer cells fueled by glycolysis produces large amounts of protons in cancer cells, which tri mechanisms to transport them out, hence leading to increased acidity in their extracellular environments. It has been well established that the increased acidity will induce cell death of normal cells but not cancer cells. The main question we address here is: how cancer cells deal with the increased acidity to avoid the activation of apoptosis. We have carried out a comparative analysis of transcriptomic data of six solid cancer types, breast, colon, liver, two lung (adenocarcinoma, squamous cell carcinoma) and prostate cancers, and proposed a model of how cancer cells utilize a few mechanisms to keep the protons outside of the cells. The model consists of a number of previously, well or partially, studied mechanisms for transporting out the excess protons, such as through the monocarboxylate transporters, V-ATPases, NHEs and the one facilitated by carbonic anhydrases. In addition we propose a new mechanism that neutralizes protons through the conversion of glutamate to γ-aminobutyrate, which consumes one proton per reaction. We hypothesize that these processes are regulated by cancer related conditions such as hypoxia and growth factors and by the pH levels, making these encoded processes not available to normal cells under acidic conditions. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
12. Simultaneous Screening and Validation of Effective Zinc Finger Nucleases in Yeast
- Author
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Wang, Ling, Lin, Juan, Zhang, Tingting, Xu, Kun, Ren, Chonghua, and Zhang, Zhiying
- Subjects
ZINC-finger proteins ,NUCLEASES ,YEAST ,DNA-binding proteins ,GENETIC engineering ,GENE expression ,ANIMAL genetics - Abstract
Zinc finger nucleases (ZFNs) have been successfully used for genome modification in various cell types and species. However, construction of an effective ZFN remained challenging. Previous studies all focused on obtaining specific zinc finger proteins (ZFPs) first via bacterial 2-hybrid approach, and then fusing selected ZFPs to FokI nuclease domain. These assembled ZFNs have high rate of failing to cleave target sites in vivo. In this study, we developed a simultaneous screening and validation system to obtain effective ZFNs directly in yeast AH109. This system is based on Gal4 reporter system carrying a unique intermediate reporter plasmid with two 30-bp Gal4 homology arms and a ZFN target site. DNA double strand breaks introduced on target sequence by ZFNs were repaired by single strand annealing (SSA) mechanism, and the restored Gal4 drove reporter genes expression. Taking the advantage of OPEN (Oligomerized Pool ENgineering) selection, we constructed 3 randomized ZFNs libraries and 9 reporter strains for each target gene. We tested this system by taking goat α s1-casein as target gene following three-step selection. Consequently, 3 efficient pairs of ZFNs were obtained from positive colonies on selective medium. The ZFNs achieved a 15.9% disruption frequency in goat mammary epithelial cells. In conclusion, we created a novel system to obtain effective ZFNs directly with simultaneous screening and validation. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
13. A Novel Genetic System Based on Zinc Finger Nucleases for the Identification of Interactions between Proteins In Vivo.
- Author
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Wang, Ling, Xu, Kun, Lin, Juan, Shao, Simin, Zhang, Tingting, Xu, Huarong, Wei, Zehui, and Zhang, Zhiying
- Subjects
- *
ZINC-finger proteins , *NUCLEASES , *PROTEIN-protein interactions , *DNA-binding proteins , *GENE expression , *CHIMERIC proteins , *OPEN reading frames (Genetics) - Abstract
Yeast two-hybrid (Y2H) methods are powerful tools for detecting protein–protein interactions. The traditional Y2H method has been widely applied to screen novel protein interactions since it was established two decades ago. The high false-positive rate of the traditional method drove the development of modified Y2H systems. Here, we describe a novel Y2H system using zinc-finger nucleases (ZFNs). ZFNs contain two functional domains, a zinc-finger DNA-binding domain (ZFP) and a non-specific nuclease domain (FokI). In this system, the bait is expressed as a fusion protein with a specific ZFP, and the prey is fused to the FokI. A reporter vector is designed such that the ZFN target site disrupts the Gal4 open reading frame. By transforming the three plasmids into a yeast strain (AH109), the interaction between the bait and prey proteins reconstitutes ZFN function and generates the double-strand break (DSB) on its target site. The DNA DSB repair restores Gal4 function, which activates the expression of the four reporter genes. We used p53-SV40LT interacting proteins to prove the concept. In addition, 80% positive rate was observed in a cDNA screening test against WDSV orfA protein. Our results strongly suggested that this Y2H system could increase screening reliability and reproducibility, and provide a novel approach for interactomics research. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
14. A Novel Genetic System Based on Zinc Finger Nucleases for the Identification of Interactions between Proteins In Vivo.
- Author
-
Wang, Ling, Xu, Kun, Lin, Juan, Shao, Simin, Zhang, Tingting, Xu, Huarong, Wei, Zehui, and Zhang, Zhiying
- Subjects
ZINC-finger proteins ,NUCLEASES ,PROTEIN-protein interactions ,DNA-binding proteins ,GENE expression ,CHIMERIC proteins ,OPEN reading frames (Genetics) - Abstract
Yeast two-hybrid (Y2H) methods are powerful tools for detecting protein–protein interactions. The traditional Y2H method has been widely applied to screen novel protein interactions since it was established two decades ago. The high false-positive rate of the traditional method drove the development of modified Y2H systems. Here, we describe a novel Y2H system using zinc-finger nucleases (ZFNs). ZFNs contain two functional domains, a zinc-finger DNA-binding domain (ZFP) and a non-specific nuclease domain (FokI). In this system, the bait is expressed as a fusion protein with a specific ZFP, and the prey is fused to the FokI. A reporter vector is designed such that the ZFN target site disrupts the Gal4 open reading frame. By transforming the three plasmids into a yeast strain (AH109), the interaction between the bait and prey proteins reconstitutes ZFN function and generates the double-strand break (DSB) on its target site. The DNA DSB repair restores Gal4 function, which activates the expression of the four reporter genes. We used p53-SV40LT interacting proteins to prove the concept. In addition, 80% positive rate was observed in a cDNA screening test against WDSV orfA protein. Our results strongly suggested that this Y2H system could increase screening reliability and reproducibility, and provide a novel approach for interactomics research. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
15. Genome-wide survey and expression analysis of the putative non-specific lipid transfer proteins in Brassica rapa L.
- Author
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Li J, Gao G, Xu K, Chen B, Yan G, Li F, Qiao J, Zhang T, and Wu X
- Subjects
- Amino Acid Sequence, Base Sequence, Carrier Proteins chemistry, Carrier Proteins classification, Chromosome Mapping, Chromosomes, Plant genetics, Computational Biology, Genetic Variation, Models, Molecular, Molecular Sequence Data, Multigene Family genetics, Phylogeny, Plant Proteins chemistry, Plant Proteins classification, Protein Conformation, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Amino Acid, Sequence Homology, Nucleic Acid, Brassica rapa genetics, Carrier Proteins genetics, Gene Expression Regulation, Plant, Genome, Plant genetics, Plant Proteins genetics
- Abstract
Background: Plant non-specific lipid transfer proteins (nsLtps) are small, basic proteins encoded by multigene families and have reported functions in many physiological processes such as mediating phospholipid transfer, defense reactions against phytopathogens, the adaptation of plants to various environmental conditions, and sexual reproduction. To date, no genome-wide overview of the Brassica rapa nsLtp (BrnsLtp) gene family has been performed. Therefore, as the first step and as a helpful strategy to elucidate the functions of BrnsLtps, a genome-wide study for this gene family is necessary., Methodology/principal Finding: In this study, a total of 63 putative BrnsLtp genes were identified through a comprehensive in silico analysis of the whole genome of B. rapa. Based on the sequence similarities, these BrnsLtps was grouped into nine types (I, II, III, IV, V, VI, VIII, IX, and XI). There is no type VII nsLtps in B. rapa, and a new type, XI nsLtps, was identified in B. rapa. Furthermore, nine type II AtLtps have no homologous genes in B. rapa. Gene duplication analysis demonstrated that the conserved collinear block of each BrnsLtp is highly identical to those in Arabidopsis and that both segmental duplications and tandem duplications seem to play equal roles in the diversification of this gene family. Expression analysis indicated that 29 out of the 63 BrnsLtps showed specific expression patterns. After careful comparison and analysis, we hypothesize that some of the type I BrnsLtps may function like Arabidopsis pathogenesis-related-14 (PR-14) proteins to protect the plant from phytopathogen attack. Eleven BrnsLtps with inflorescence-specific expression may play important roles in sexual reproduction. Additionally, BrnsLtpI.3 may have functions similar to Arabidopsis LTP1., Conclusions/significance: The genome-wide identification, bioinformatic analysis and expression analysis of BrnsLtp genes should facilitate research of this gene family and polyploidy evolution and provide new insight towards elucidating their biological functions in plants.
- Published
- 2014
- Full Text
- View/download PDF
16. A novel genetic system based on zinc finger nucleases for the identification of interactions between proteins in vivo.
- Author
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Wang L, Xu K, Lin J, Shao S, Zhang T, Xu H, Wei Z, and Zhang Z
- Subjects
- Animals, Gene Library, Genes, Reporter genetics, Mice, Protein Binding, Deoxyribonucleases chemistry, Deoxyribonucleases metabolism, Two-Hybrid System Techniques, Zinc Fingers
- Abstract
Yeast two-hybrid (Y2H) methods are powerful tools for detecting protein-protein interactions. The traditional Y2H method has been widely applied to screen novel protein interactions since it was established two decades ago. The high false-positive rate of the traditional method drove the development of modified Y2H systems. Here, we describe a novel Y2H system using zinc-finger nucleases (ZFNs). ZFNs contain two functional domains, a zinc-finger DNA-binding domain (ZFP) and a non-specific nuclease domain (FokI). In this system, the bait is expressed as a fusion protein with a specific ZFP, and the prey is fused to the FokI. A reporter vector is designed such that the ZFN target site disrupts the Gal4 open reading frame. By transforming the three plasmids into a yeast strain (AH109), the interaction between the bait and prey proteins reconstitutes ZFN function and generates the double-strand break (DSB) on its target site. The DNA DSB repair restores Gal4 function, which activates the expression of the four reporter genes. We used p53-SV40LT interacting proteins to prove the concept. In addition, 80% positive rate was observed in a cDNA screening test against WDSV orfA protein. Our results strongly suggested that this Y2H system could increase screening reliability and reproducibility, and provide a novel approach for interactomics research.
- Published
- 2013
- Full Text
- View/download PDF
17. High-throughput discovery of chloroplast and mitochondrial DNA polymorphisms in Brassicaceae species by ORG-EcoTILLING.
- Author
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Zeng CL, Wang GY, Wang JB, Yan GX, Chen BY, Xu K, Li J, Gao GZ, Wu XM, Zhao B, and Liu L
- Subjects
- Base Sequence, Ecotype, Electrophoresis, Agar Gel, Genes, Chloroplast genetics, Genes, Plant genetics, Genome, Plant genetics, Molecular Sequence Data, Mutation genetics, Phylogeny, Reproducibility of Results, Species Specificity, Brassicaceae genetics, DNA, Chloroplast genetics, DNA, Mitochondrial genetics, High-Throughput Nucleotide Sequencing methods, Mutagenesis genetics, Organelles genetics, Polymorphism, Single Nucleotide genetics
- Abstract
Background: Information on polymorphic DNA in organelle genomes is essential for evolutionary and ecological studies. However, it is challenging to perform high-throughput investigations of chloroplast and mitochondrial DNA polymorphisms. In recent years, EcoTILLING stands out as one of the most universal, low-cost, and high-throughput reverse genetic methods, and the identification of natural genetic variants can provide much information about gene function, association mapping and linkage disequilibrium analysis and species evolution. Until now, no report exists on whether this method is applicable to organelle genomes and to what extent it can be used., Methodology/principal Findings: To address this problem, we adapted the CEL I-based heteroduplex cleavage strategy used in Targeting Induced Local Lesions in Genomes (TILLING) for the discovery of nucleotide polymorphisms in organelle genomes. To assess the applicability and accuracy of this technology, designated ORG-EcoTILLING, at different taxonomic levels, we sampled two sets of taxa representing accessions from the Brassicaceae with three chloroplast genes (accD, matK and rbcL) and one mitochondrial gene (atp6). The method successfully detected nine, six and one mutation sites in the accD, matK and rbcL genes, respectively, in 96 Brassica accessions. These mutations were confirmed by DNA sequencing, with 100% accuracy at both inter- and intraspecific levels. We also detected 44 putative mutations in accD in 91 accessions from 45 species and 29 genera of seven tribes. Compared with DNA sequencing results, the false negative rate was 36%. However, 17 SNPs detected in atp6 were completely identical to the sequencing results., Conclusions/significance: These results suggest that ORG-EcoTILLING is a powerful and cost-effective alternative method for high-throughput genome-wide assessment of inter- and intraspecific chloroplast and mitochondrial DNA polymorphisms. It will play an important role in evolutionary and ecological biology studies, in identification of related genes associated with agronomic importance such as high yield and improved cytoplasmic quality, and for identifying mitochondrial point mutations responsible for diseases in humans and other animals.
- Published
- 2012
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18. A comparative study of gene-expression data of basal cell carcinoma and melanoma reveals new insights about the two cancers.
- Author
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Xu K, Mao X, Mehta M, Cui J, Zhang C, and Xu Y
- Subjects
- Apoptosis genetics, Carcinoma, Basal Cell blood supply, Carcinoma, Basal Cell metabolism, Carcinoma, Basal Cell pathology, Cell Proliferation, Disease Progression, Energy Metabolism genetics, Genetic Markers genetics, Humans, Melanoma blood supply, Melanoma metabolism, Melanoma pathology, Neoplasm Invasiveness, Neoplasm Metastasis, Neovascularization, Pathologic genetics, Skin Neoplasms blood supply, Skin Neoplasms metabolism, Skin Neoplasms pathology, Carcinoma, Basal Cell genetics, Gene Expression Profiling, Melanoma genetics, Skin Neoplasms genetics
- Abstract
A comparative analysis of genome-scale transcriptomic data of two types of skin cancers, melanoma and basal cell carcinoma in comparison with other cancer types, was conducted with the aim of identifying key regulatory factors that either cause or contribute to the aggressiveness of melanoma, while basal cell carcinoma generally remains a mild disease. Multiple cancer-related pathways such as cell proliferation, apoptosis, angiogenesis, cell invasion and metastasis, are considered, but our focus is on energy metabolism, cell invasion and metastasis pathways. Our findings include the following. (a) Both types of skin cancers use both glycolysis and increased oxidative phosphorylation (electron transfer chain) for their energy supply. (b) Advanced melanoma shows substantial up-regulation of key genes involved in fatty acid metabolism (β-oxidation) and oxidative phosphorylation, with aerobic metabolism being far more efficient than anaerobic glycolysis, providing a source of the energetics necessary to support the rapid growth of this cancer. (c) While advanced melanoma is similar to pancreatic cancer in terms of the activity level of genes involved in promoting cell invasion and metastasis, the main metastatic form of basal cell carcinoma is substantially reduced in this activity, partially explaining why this cancer type has been considered as far less aggressive. Our method of using comparative analyses of transcriptomic data of multiple cancer types focused on specific pathways provides a novel and highly effective approach to cancer studies in general.
- Published
- 2012
- Full Text
- View/download PDF
19. Bmp7 functions via a polarity mechanism to promote cloacal septation.
- Author
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Xu K, Wu X, Shapiro E, Huang H, Zhang L, Hickling D, Deng Y, Lee P, Li J, Lepor H, and Grishina I
- Subjects
- Animals, Bone Morphogenetic Protein 7 deficiency, Cell Differentiation, Cell Division, Cell Proliferation, Cell Survival, Cloaca enzymology, Endoderm cytology, Epithelium embryology, Fetus embryology, Fetus enzymology, Humans, JNK Mitogen-Activated Protein Kinases metabolism, MAP Kinase Signaling System, Mesoderm cytology, Mesoderm metabolism, Mice, Models, Biological, Rectum embryology, Rectum enzymology, Smad Proteins metabolism, Urethra embryology, Urethra enzymology, Body Patterning, Bone Morphogenetic Protein 7 metabolism, Cell Polarity, Cloaca cytology, Cloaca embryology
- Abstract
Background: During normal development in human and other placental mammals, the embryonic cloacal cavity separates along the axial longitudinal plane to give rise to the urethral system, ventrally, and the rectum, dorsally. Defects in cloacal development are very common and present clinically as a rectourethral fistula in about 1 in 5,000 live human births. Yet, the cellular mechanisms of cloacal septation remain poorly understood., Methodology/principal Findings: We previously detected Bone morphogenetic protein 7 (Bmp7) expression in the urorectal mesenchyme (URM), and have shown that loss of Bmp7 function results in the arrest of cloacal septation. Here, we present evidence that cloacal partitioning is driven by Bmp7 signaling in the cloacal endoderm. We performed TUNEL and immunofluorescent analysis on cloacal sections from Bmp7 null and control littermate embryos. We found that loss of Bmp7 results in a dramatic decrease in the endoderm survival and a delay in differentiation. We used immunological methods to show that Bmp7 functions by activating the c-Jun N-terminal kinase (JNK) pathway. We carried out confocal and 3D imaging analysis of mitotic chromosome bundles to show that during normal septation cells in the cloacal endoderm divide predominantly in the apical-basal direction. Loss of Bmp7/JNK signaling results in randomization of mitotic angles in the cloacal endoderm. We also conducted immunohistochemical analysis of human fetal sections to show that BMP/phospho-SMAD and JNK pathways function in the human cloacal region similar as in the mouse., Conclusion/significance: Our results strongly indicate that Bmp7/JNK signaling regulates remodeling of the cloacal endoderm resulting in a topological separation of the urinary and digestive systems. Our study points to the importance of Bmp and JNK signaling in cloacal development and rectourethral malformations.
- Published
- 2012
- Full Text
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20. Familial CJD associated PrP mutants within transmembrane region induced Ctm-PrP retention in ER and triggered apoptosis by ER stress in SH-SY5Y cells.
- Author
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Wang X, Shi Q, Xu K, Gao C, Chen C, Li XL, Wang GR, Tian C, Han J, and Dong XP
- Subjects
- Cell Line, Tumor, Creutzfeldt-Jakob Syndrome pathology, Endoplasmic Reticulum pathology, Humans, PrPC Proteins genetics, PrPSc Proteins genetics, Proteins, Transfection, Apoptosis, Creutzfeldt-Jakob Syndrome genetics, Endoplasmic Reticulum metabolism, Mutant Proteins genetics, Prions metabolism
- Abstract
Background: Genetic prion diseases are linked to point and inserted mutations in the prion protein (PrP) gene that are presumed to favor conversion of the cellular isoform of PrP (PrP(C)) to the pathogenic one (PrP(Sc)). The pathogenic mechanisms and the subcellular sites of the conversion are not completely understood. Here we introduce several PRNP gene mutations (such as, PrP-KDEL, PrP-3AV, PrP-A117V, PrP-G114V, PrP-P102L and PrP-E200K) into the cultured cells in order to explore the pathogenic mechanism of familial prion disease., Methodology/principal Findings: To address the roles of aberrant retention of PrP in endoplasmic reticulum (ER), the recombinant plasmids expressing full-length human PrP tailed with an ER signal peptide at the COOH-terminal (PrP-KDEL) and PrP with three amino acids exchange in transmembrane region (PrP-3AV) were constructed. In the preparations of transient transfections, 18-kD COOH-terminal proteolytic resistant fragments (Ctm-PrP) were detected in the cells expressing PrP-KDEL and PrP-3AV. Analyses of the cell viabilities in the presences of tunicamycin and brefeldin A revealed that expressions of PrP-KDEL and PrP-3AV sensitized the transfected cells to ER stress stimuli. Western blots and RT-PCR identified the clear alternations of ER stress associated events in the cells expressing PrP-KDEL and PrP-3AV that induced ER mediated apoptosis by CHOP and caspase-12 apoptosis pathway. Moreover, several familial CJD related PrP mutants were transiently introduced into the cultured cells. Only the mutants within the transmembrane region (G114V and A117V) induced the formation of Ctm-PrP and caused the ER stress, while the mutants outside the transmembrane region (P102L and E200K) failed., Conclusions/significance: The data indicate that the retention of PrP in ER through formation of Ctm-PrP results in ER stress and cell apoptosis. The cytopathic activities caused by different familial CJD associated PrP mutants may vary, among them the mutants within the transmembrane region undergo an ER-stress mediated cell apoptosis.
- Published
- 2011
- Full Text
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21. A comparative analysis of gene-expression data of multiple cancer types.
- Author
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Xu K, Cui J, Olman V, Yang Q, Puett D, and Xu Y
- Subjects
- Humans, Oligonucleotide Array Sequence Analysis, Survival Rate, Gene Expression Profiling, Neoplasms genetics
- Abstract
A comparative study of public gene-expression data of seven types of cancers (breast, colon, kidney, lung, pancreatic, prostate and stomach cancers) was conducted with the aim of deriving marker genes, along with associated pathways, that are either common to multiple types of cancers or specific to individual cancers. The analysis results indicate that (a) each of the seven cancer types can be distinguished from its corresponding control tissue based on the expression patterns of a small number of genes, e.g., 2, 3 or 4; (b) the expression patterns of some genes can distinguish multiple cancer types from their corresponding control tissues, potentially serving as general markers for all or some groups of cancers; (c) the proteins encoded by some of these genes are predicted to be blood secretory, thus providing potential cancer markers in blood; (d) the numbers of differentially expressed genes across different cancer types in comparison with their control tissues correlate well with the five-year survival rates associated with the individual cancers; and (e) some metabolic and signaling pathways are abnormally activated or deactivated across all cancer types, while other pathways are more specific to certain cancers or groups of cancers. The novel findings of this study offer considerable insight into these seven cancer types and have the potential to provide exciting new directions for diagnostic and therapeutic development.
- Published
- 2010
- Full Text
- View/download PDF
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