Genetic variability of Myzus persicae nicotianae densovirus based on partial NS and VP gene sequences.

We previously described a novel densovirus [Myzus persicae nicotianae densovirus (MpnDV)] infecting M. persicae nicotianae (Hemiptera: Aphididae) with 34% prevalence. This single-stranded DNA virus has a 5480-nucleotide ambisense genome and belongs to the Densovirinae subfamily within the family Parvoviridae. In the present study, we estimated the genetic diversity of MpnDV using partial nonstructural protein (NS) and capsid protein (VP) gene sequences from 10 locations in China. First, we identified MpnDV-positive samples by amplifying a 445-bp fragment with primers MpDVF/MpDVR. Subsequently, we amplified and sequenced COI genes with primers MpCOIF/ MpCOIR, and partial NS and VP sequences with primers MpnDVF1/MpnDVR1. The respective 655-, 1461-, and 423-bp COI, NS, and VP fragments were used to analyze the genetic diversity of MpnDV using MEGA 6.0 and DnaSP 5.0. The high level of identity shared by all COI sequences (>99%) suggested that the aphids sampled were of the same species, and indicated population homogeneity across the 10 locations investigated. The nucleotide diversity of MpnDV sequences (0.0020 ± 0.0025) was significantly higher than that of the COI genes (0.0002 ± 0.0005). The pairwise fixation index for MpnDV was 0.832, and the total gene flow was 0.05. Phylogenetic analysis revealed that the MpnDV haplotypes clustered according to geographical location, except for those from the Liaoning and Shanxi provinces. In conclusion, MpnDV demonstrated a low level of gene flow and high genetic diversity, suggesting that it is vertically transmitted, and implying that endosymbiotic viruses could be used as markers in studies of insect population genetics.

Relationship between genetic polymorphisms of methylenetetrahydrofolate reductase and breast cancer chemotherapy response.

Activity of methylenetetrahydrofolate reductase (MTHFR), an enzyme involved in folate metabolism, is influenced by mutations in the corresponding gene, contributing to a decrease in 5,10-MTHF. Due to such polymorphisms, individuals differ in MTHFR enzyme activity and plasma folate levels. We investigated the relationship between two common MTHFR polymorphisms (C677T and A1298C) and breast cancer (BC) chemotherapy response. From February 2013 to January 2016, 148 advanced BC patients at the Center Hospital of Cangzhou were enrolled and treated with six different chemotherapy regimens. Subjects were genotyped using polymerase chain reaction-restriction fragment length polymorphism. Forty-one (27.7%), 70 (47.3%), and 37 (25.0%) patients carried the C/C, C/T, and T/T C677T genotypes, respectively; 101 (68.2%), 42 (28.4%), and 5 (3.4%) had the A/A, A/C, and C/C genotypes of A1298C, respectively. Total chemotherapy efficacy was 66.9% (99/148), with 7 (4.7%), 92 (62.2%), 36 (24.3%), and 13 (8.8%) cases showing complete response, partial response, no change, and progressive disease, respectively. Chemotherapy regimens did not differ in effectiveness (P > 0.05). Efficacy rates associated with C677T C/C, C/T, and T/T genotypes were 58.5, 58.6, and 91.9%, respectively, with T/T carriers exhibiting significantly better responses than the C/C (P < 0.05) and C/T groups (P < 0.05). Effectiveness among A1298C A/A, A/C, and C/C carriers was 70.6, 64.3, and 0.0%, respectively, but no difference was established between these genotypes in this regard (P > 0.05). The MTHFR C677T genotype may be associated with BC chemotherapy response, and could be of great value in guiding individualized treatment for this disease.

Population genetic structure of Myzus persicae nicotianae (Hemiptera: Aphididae) in China by microsatellite analysis.

The tobacco aphid, Myzus persicae nicotianae (Hemiptera: Aphididae), is an important agricultural pest that feeds on host plants and transmits plant viruses in China. To effectively control this pest, we investigated the genetic variation and genetic structure of 54 populations of tobacco aphids collected in China, using five microsatellite loci. An average of 7 alleles with effective number ranging from 1.5 to 6.6 was detected using these five loci, and the average polymorphic information content (PIC) was 0.652, suggesting that the five selected microsatellite loci were polymorphic and suitable for the study of population genetics. The expected heterozygosities in the populations studied ranged from 0.128 and 0.653, with an average value of 0.464. However, the observed heterozygosities ranged from 0.250 and 0.942 (average = 0.735), revealing a high genetic variability and heterozygosity excess in the Chinese tobacco aphid populations. The global fixation index (F(ST)) and mean gene flow (N(m)) were 0.34 (P < 0.0001) and 0.50, respectively, suggesting the high genetic differentiation among Chinese populations. The 54 populations of tobacco aphids were classified into two groups. The populations did not cluster geographically, as populations from the same provinces were usually present in different clusters. This was also confirmed by the Mantel test, which showed no significant correlation between the genetic distance and geographical distance or altitude. Long distance migration might be responsible for the lack of distance-related isolation.

Role of salubrinal in protecting cardiomyocytes from doxorubicin-induced apoptosis.

We determined whether salubrinal can protect cardio-myocytes from doxorubicin-induced apoptosis and explored the related mechanisms to provide experimental evidence for exploring novel drug candidates to decrease cardiac toxicity. Neonatal rat cardiomyocytes were isolated, cultured in vitro, and pretreated with salubrinal (10, 20, or 40 µM) to observe their response to doxorubicin-induced cell apoptosis. Lactate dehydrogenase assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling staining, and flow cytometry were used to assess the extent of cardiomyocyte apoptosis. Fluorescent probes conjugated with 2',7'-dichlorofluorescein diacetate and a chemiluminescence assay were used to detect the pro-duction of reactive oxygen species. Western blotting was employed to quantify expression levels of cleaved caspase-3, cytosolic cytochrome c, and B-cell lymphoma-extra large (Bcl-xL). The mechanisms of salubrinal-related functions were also explored. Salubrinal effectively inhibited doxorubicin-induced reactive oxygen species production and nicotinamide adenine dinucleotide phosphate oxidase activation, decreased the levels of cleaved caspase-3 and cytosol cytochrome c, and increased Bcl-xL expression, thereby protecting cardiomyocytes from doxorubicin-induced apoptosis. Furthermore, salubrinal was found to protect cardiomyocytes by decreasing the dephosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Salubrinal can protect cardiomyocytes from doxorubicin-induced apoptosis through its effects on eIF2α. It possibly ameliorates cardiac toxicity and can be used in clinical practice.

Effect of Bcl-2-siRNA on proliferation and apoptosis of pediatric acute B lymphoblastic leukemia (A-BLL) cells.

This study analyzed the effect of small interfering RNA specific for the Bcl-2 gene (siRNA Bcl-2) on the proliferation and chemotherapeutic sensitivity of pediatric A-BLL cells. Marrow samples were obtained from sixty newly-diagnosed A-BLL pediatric patients. The Bcl-2 mRNA expression in these samples was quantified by real time polymerase chain reaction. The Bcl-2 mRNA re-expression was analyzed by RNA interference using Bcl-2-siRNA. Cellular proliferation was detected using the MTT (Thiazolyl Blue Tetrazolium Bromide) assay. The cell apoptosis was quantified by flow cytometry. The Bcl-2 mRNA expression was significantly higher in the drug-resistance group than in the chemotherapy sensitivity group prior to chemotherapy (P < 0.05). In addition, the Bcl-2 mRNA expression in the chemotherapy sensitivity group was significantly higher before chemotherapy than that after chemotherapy (P < 0.05). The Bcl-2 mRNA expression significantly decreased in the leukemic cells of the Bcl-2-siRNA transfection group. We observed statistically significant differences in the relative mRNA expression levels among the Bcl-2-siRNA transfection, blank control, liposome empty transfection, and unrelated sequence oligonucleotide groups (P < 0.05). The rate of apoptosis in pediatric A-BLL leukemic cells was observed to increase significantly after transfection with Bcl-2-siRNA compared to the control, liposome empty transfection, and unrelated sequence oligonucleotide groups (P < 0.05). Therefore, we concluded that Bcl-2-siRNA can successfully inhibit the multiplicative capacity of A-BLL leukemic cells and promote apoptosis.

Cut-and-paste-based cloning strategy for large gene site-directed mutagenesis.

Site-directed mutagenesis is an essential technique for investigating the mechanisms of gene regulation on a molecular level, as well as for exploring post-translational modifications and functional structure at the protein level. Polymerase chain reaction combining in vitro synthesis of oligonucleotide primers allows for site-directed mutation to be performed with ease. However, site-directed mutagenesis is difficult when larger plasmids are involved. Here, we present a novel method for generating large gene site-directed mutagenesis products based on a cut-and-paste-based cloning strategy. This method uses 4 primers, incorporating relevant mutations and restriction enzyme site sequences, to generate 2 DNA fragments by polymerase chain reaction. The fragments are then ligated into TA cloning vectors. Large genes containing mutations of interest were obtained by cutting and then pasting, and then inserting one fragment into another T-vector. We demonstrated the practicality of this method by creating a G59S mutation within the p150(Glued)-encoding gene.

Disease indicators for sepsis and analysis of sepsis treatment in children using the continuous blood purification technique.

We analyzed disease severity, inflammation markers, and dynamic changes in cartilage glycoprotein 39 (YKL-40) and C-reactive protein (CRP) levels in children with sepsis before and after treatment with continuous blood purification (CBP). Study participants were 30 children with severe sepsis who were cured from the disease (experimental group) in the Children's Serious Disease Center of In-ner Mongolia People's Hospital between June 2012 and October 2013. Symptomatic CBP treatment was performed after disease severity scoring. Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), YKL-40, and CRP levels were tested 0, 12, 24, and 48 h after CBP treatment. YKL-40 mRNA expression in whole blood was determined biochemically, and its expression in peripheral blood was determined with an immunochemical method. We found a significant difference in disease severity scores before and 48 h after CBP treatment (P < 0.05). IL-6, TNF-α, YKL-40, and CRP levels in children with sepsis at 12, 24, and 48 h after CBP treatment significantly differed from those before treatment (P < 0.05). The relative expression of YKL-40 mRNA in the experimental group before CBP treatment significantly increased from that of the control group (P < 0.05). We found a positive correlation between IL-6, TNF-α, YKL-40, and CRP levels 48 h after CBP treatment. In conclusion, CBP is an effective treatment strategy for pediatric sepsis. YKL-40 and CRP can be used to evaluate the effects of sepsis treatment.

Expression differences in TEL-AML1 fusion gene in leukemia glucocorticoid-sensitive and -resistant cell lines.

We investigated the expression differences of the TEL-AML1 fusion gene in a leukemia glucocorticoid (GC)-sensitive cell line (CEM) and a GC-resistant cell line (Jurkat). Changes in TEL-AML1 expression before and after GC exposure were analyzed. Expression of GC-sensitive and GC-resistant leukemia cells following initial diagnosis and during treatment was simulated. Leukemia cells were divided into a GC-unexposed or a GC-exposed group. A methyl thiazolyl tetrazolium assay was used to detect cell proliferation inhibition, flow cytometry was used to observe cell apoptosis, reverse transcription-polymerase chain reaction was used to detect the mRNA expression of TEL-AML1 before and after exposure, and western blotting was used to analyze protein levels of TEL-AML1 before and after exposure. Inhibitory concentrations of 50% of cells in the Jurkat and CEM cells at 24 h were 382 and 9 mM, respectively, and at 48 h they were 216 and 2 mM. The proliferation inhibition effect of dexamethasone sodium phosphate on Jurkat cells was much lower than that on CEM cells. Jurkat cells showed obvious apoptosis after exposure to 100 mM dexamethasone sodium phosphate for 48 h. In the exposed group, Jurkat cells showed higher TEL-AML1 expression than did CEM cells (P < 0.05). In the unexposed group, TEL-AML1 gene expression in Jurkat cells was not affected by GC exposure (P > 0.05), while the CEM cells presented significant differences before and after exposure (P < 0.05). Sustained high expression of TEL-AML1 participated in and maintained the occurrence of GC resistance. Inhibition of TEL-AML1 may provide a new therapeutic approach to reverse GC resistance.

Analysis of drug-resistant gene detection of blaOXA-like genes from Acinetobacter baumannii.

Our study determines the resistance gene profile of a set of Acinetobacter baumannii hospital isolates. A. baumannii is responsible for nosocomial outbreaks and sporadic infections. We extracted and PCR amplified bacterial DNA isolated from patients with ages below 60 years (23.36%) and above 60 years (76.64%). Most of the patients were admitted in the ICU (36.13%) and pneumology departments (28.47%). Of 164 isolated strains, 16 (9.75%) contained OXA-51, 8 (4.88%) contained OXA-58, and 140 (85.37%) contained both OXA-51 and OXA-23. Additionally, 8 (7.41%) strains containing OXA-58 and 100 (92.59%) strains containing both OXA-51 and OXA-23 showed multidrug-resistance. Drug resistance rates of A. baumannii to amikacin, tobramycin-levofloxacin, and cotrimoxazole were above 90%, while drug resistance rates to ampicillin, cefotetan, cefazolin, cefoperazone, and nitrofurantoin were 100%. In conclusion, we found that isolated strains containing OXA-51 and OXA-23 were more likely to be resistant or have decreased sensitivity to carbapenems.

MicroRNA network analysis identifies key microRNAs and genes associated with precancerous lesions of gastric cancer.

To identify potential targets for the early treatment and prevention of gastric cancer, microRNA (miRNA) expression profiles of precancerous lesions of gastric cancer were investigated. The miRNA microarray dataset GSE24839 was downloaded from Gene Expression Omnibus (GEO) and included 10 Helicobacter pylori-related gastritis samples and 10 gastric intestinal metaplasia samples. Differentially expressed miRNAs (DEMs) were screened using the Student t-test; P < 0.05 was considered to be statistically significant. Co-expression networks of total miRNAs and DEMs were constructed based on the Pearson correlation coefficient for the two diseases. Target genes of the DEMs were retrieved using miRecords and pathway-enrichment analysis was performed using a hypergeometric test. A total of 20 DEMs were obtained for H. pylori-related gastritis and gastric intestinal metaplasia samples, including 12 up-regulated and 8 down-regulated miRNAs. The identified DEMs appear to play key roles in gastric cancer, as the average degree of the DEM sub-network was higher than that of the total miRNA co-expression network. Furthermore, target genes for 6 DEMs (hsa-miR-106b, hsa-miR-193a-3p, hsa-miR-204, hsa-miR-30e, hsa-miR-519d, and hsa-miR-524-5p) are in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including signal transduction, cell growth and death, and transport and catabolism. Among the target genes, 5 (RAB22A, SOX4, IKZF2, PLAG1, and BTBD7) were of interest because they can be simultaneously regulated by several DEMs. These findings suggest that these genes may be targets for modulating gastric cancer progression.

Association between the c.1564A>T genetic polymorphism of the MDR1 gene and hepatocellular carcinoma in Chinese population.

The objective of this study was to evaluate the influence of c.1564A>T genetic polymorphisms in the multidrug resistance 1 gene (MDR1) on hepatocellular carcinoma (HCC) susceptibility through association analysis. A total of 632 HCC patients and 645 cancer-free controls were enrolled in this study. The c.1564A>T genetic polymorphisms were genotyped by created restriction site-polymerase chain reaction (CRS-PCR) and confirmed using DNA sequencing methods. The potential associations of c.1564A>T genetic polymorphisms with the risk of HCC were analyzed by different genetic models. Statistically significantly increased risks of HCC were detected in the homozygote comparison (TT versus AA: OR = 1.70, 95%CI = 1.17-2.45, χ(2) = 7.99, P = 0.005), recessive model (TT versus AT/AA: OR = 1.64, 95%CI = 1.15-2.33, χ(2) = 7.66, P = 0.006), and allele contrast (T versus A: OR = 1.23, 95%CI = 1.04-1.45, χ(2) = 6.09, P = 0.014). Our data suggest that the genotypes/alleles from c.1564A>T genetic polymorphisms are statistically associated with HCC risk. The allele-T and genotype TT may contribute to susceptibility to HCC in the Chinese Han population.

A duplex SYBR Green I real-time quantitative PCR assay for detecting Escherichia coli O157:H7.

PCR and hybridization assays are widely used for the detection and identification of Escherichia coli serogroups and serotypes. We used these techniques for the detection of E. coli O157:H7, a dominant serogroup among E. coli strains that are considered major public health problems worldwide. We developed a quantitative PCR assay using SYBR Green I, based on the published sequences of the rfbE and fliC genes from E. coli O157:H7. This method detected the E. coli O157:H7 O somatic antigen gene and the flagellar antigen gene simultaneously, with good specificity, sensitivity, and repeatability. The sensitivity of the assay was 2.95 x 10 copies/µL, which is 10(3) times more sensitive than obtained with a conventional PCR. The intra-assay and inter-assay coefficients of variation were less than 2%. We concluded that this duplex quantitative PCR assay is adequate for the identification and quantitative analysis of E. coli O157:H7. This provides a new identification method for clinical diagnosis of E. coli O157:H7 and for food safety analysis, as well as for molecular epidemiological studies of foodborne diseases.

Survey of simple sequence repeats in woodland strawberry (Fragaria vesca).

The use of simple sequence repeats (SSRs), or microsatellites, as genetic markers has become popular due to their abundance and variation in length among individuals. In this study, we investigated linkage groups (LGs) in the woodland strawberry (Fragaria vesca) and demonstrated variation in the abundances, densities, and relative densities of mononucleotide, dinucleotide, and trinucleotide repeats. Mononucleotide, dinucleotide, and trinucleotide repeats were more common than longer repeats in all LGs examined. Perfect SSRs were the predominant SSR type found and their abundance was extremely stable among LGs and chloroplasts. Abundances of mononucleotide, dinucleotide, and trinucleotide repeats were positively correlated with LG size, whereas those of tetranucleotide and hexanucleotide SSRs were not. Generally, in each LG, the abundance, relative abundance, relative density, and the proportion of each unique SSR all declined rapidly as the repeated unit increased. Furthermore, the lengths and frequencies of SSRs varied among different LGs.

Gene expression profile and enrichment pathways in different stages of bladder cancer.

Bladder cancer is a highly heterogeneous neoplasm. We examined the gene expression profile in 3 bladder cancer stages (Ta, T1, T2) using expression microarray analysis of 40 bladder tumors. Differentially expressed genes were found by the t-test, with <0.005 as the significance threshold. KEGG pathway-enrichment analysis was used to study the signaling pathways of the genes. We found 36 genes that could be used as molecular markers for predicting the transition from Ta-T1 to T1-T2. Among these, 11 overlapped between Ta-T1 and T1-T2 stages. Six genes were down-regulated at the Ta-T1 stage, but were up-regulated at the T1-T2 stage (ANXA5, ATP6V1B2, CTGF, GEM, IL13RA1, and LCP1); 5 genes were up-regulated at the Ta-T1 stage, but down-regulated at the T1-T2 stage (ACPP, GNL1, RIPK1, RAPGEF3, and ZER1). Another 25 genes changed relative expression levels at the T1-T2 stage. These genes (including COL1A1, COL1A2, FN1, ITGA5, LGALS1, SPP1, VIM, POSTN, and COL18A1) may be involved in bladder cancer progression by affecting extracellular matrix-receptor interaction and focal adhesion. The cytokine-cytokine receptor interaction, neuroactive ligand-receptor interaction, and calcium-signaling pathway were associated with bladder cancer progression at both the Ta-T1 and T1-T2 stages.

RNAi-mediated knockdown of pituitary tumor- transforming gene-1 (PTTG1) suppresses the proliferation and invasive potential of PC3 human prostate cancer cells

Pituitary tumor-transforming gene-1 (PTTG1) is a proto-oncogene that promotes tumorigenesis and metastasis in numerous cell types and is overexpressed in a variety of human tumors. We have demonstrated that PTTG1 expression was up-regulated in both human prostate cancer specimens and prostate cancer cell lines. For a more direct assessment of the function of PTTG1 in prostate tumorigenesis, RNAi-mediated knockdown was used to selectively decrease PTTG1 expression in PC3 human prostate tumor cells. After three weeks of selection, colonies stably transfected with PTTG1-targeted RNAi (the knockdown PC3 cell line) or empty vector (the control PC3 cell line) were selected and expanded to investigate the role of PTTG1 expression in PC3 cell growth and invasion. Cell proliferation rate was significantly slower (28%) in the PTTG1 knockdown line after 6 days of growth as indicated by an MTT cell viability assay (P < 0.05). Similarly, a soft agar colony formation assay revealed significantly fewer (66.7%) PTTG1 knockdown PC3 cell colonies than control colonies after three weeks of growth. In addition, PTTG1 knockdown resulted in cell cycle arrest at G1 as indicated by fluorescence-activated cell sorting. The PTTG1 knockdown PC3 cell line also exhibited significantly reduced migration through Matrigel in a transwell assay of invasive potential, and down-regulation of PTTG1 could lead to increased sensitivity of these prostate cancer cells to a commonly used anticancer drug, taxol. Thus, PTTG1 expression is crucial for PC3 cell proliferation and invasion, and could be a promising new target for prostate cancer therapy.

RNAi-mediated knockdown of pituitary tumor- transforming gene-1 (PTTG1) suppresses the proliferation and invasive potential of PC3 human prostate cancer cells.

Pituitary tumor-transforming gene-1 (PTTG1) is a proto-oncogene that promotes tumorigenesis and metastasis in numerous cell types and is overexpressed in a variety of human tumors. We have demonstrated that PTTG1 expression was up-regulated in both human prostate cancer specimens and prostate cancer cell lines. For a more direct assessment of the function of PTTG1 in prostate tumorigenesis, RNAi-mediated knockdown was used to selectively decrease PTTG1 expression in PC3 human prostate tumor cells. After three weeks of selection, colonies stably transfected with PTTG1-targeted RNAi (the knockdown PC3 cell line) or empty vector (the control PC3 cell line) were selected and expanded to investigate the role of PTTG1 expression in PC3 cell growth and invasion. Cell proliferation rate was significantly slower (28%) in the PTTG1 knockdown line after 6 days of growth as indicated by an MTT cell viability assay (P < 0.05). Similarly, a soft agar colony formation assay revealed significantly fewer (66.7%) PTTG1 knockdown PC3 cell colonies than control colonies after three weeks of growth. In addition, PTTG1 knockdown resulted in cell cycle arrest at G1 as indicated by fluorescence-activated cell sorting. The PTTG1 knockdown PC3 cell line also exhibited significantly reduced migration through Matrigel in a transwell assay of invasive potential, and down-regulation of PTTG1 could lead to increased sensitivity of these prostate cancer cells to a commonly used anticancer drug, taxol. Thus, PTTG1 expression is crucial for PC3 cell proliferation and invasion, and could be a promising new target for prostate cancer therapy.