Using chimeric piggyBac transposase to achieve directed interplasmid transposition in silkworm Bombyx mori and fruit fly Drosophila cells

The piggyBac transposon has been long used to integrate foreign DNA into insect genomes. However, undesirable transgene expression can result from random insertions into the genome. In this study, the efficiency of chimeric Gal4-piggyBac transposase in directing integration onto a DNA target plasmid was evaluated in cultured silkworm Bombyx mori Bm-12 and fruit fly Drosophila Schneider 2 (S2) cells. The Gal4-piggyBac transposase has a Gal4 DNA-binding domain (DBD), and the target plasmid has upstream activating sequences (UAS) to which the Gal4 DBD can bind with high affinity. The results indicate that, in the Bm-12 and S2 cells, transpositional activity of Gal4-piggyBac transposase was increased by 4.0 and 7.5 times, respectively, compared to controls, where Gal4-UAS interaction was absent. Moreover, the Gal4-piggyBac transposase had the ability of directing piggyBac element integration to certain sites of the target plasmid, although the target-directing specificity was not as high as expected. The chimeric piggyBac transposase has the potential for use in site-directed transgenesis and gene function research in B. mori.     

bmo-miR-0001 and bmo-miR-0015 down-regulate expression of <Emphasis Type="Italic">Bombyx mori</Emphasis> fibroin light chain gene in vitro

Based on bioinformatic analysis, we selected two novel microRNAs (miRNAs), bmo-miR-0001 and bmomiR-0015, from high-throughput sequencing of the Bombyx mori larval posterior silk gland (PSG). Firstly, we examined the expression of bmo-miR-0001 and bmo-miR-0015 in 12 different tissues of the 5th instar Day-3 larvae of the silkworm. The results showed that the expression levels of both bmo-miR-0001 and bmo-miR-0015 were obviously higher in the PSG than in other tissues, implying there is a spatio-temporal condition for bmo-miR-0001 and bmo-miR-0015 to regulate the expression of BmFib-L. To test this hypothesis, we constructed pri-bmo-miR-0001 expressing the plasmid pcDNA3.0 [ie1-egfp-pri-bmo-miR-0001-SV40] and pri-bmo-miR-0015 expressing the plasmid pcDNA3.0 [ie1-egfp-pribmo-miR-0015-SV40]. Finally, the BmN cells were harvested and luciferase activity was detected. The results showed that luciferase activity was reduced significantly (P<0.05) in BmN cells co-transfected by pcDNA3.0 [ie1-egfp-pri-bmomiR-0001-SV40] or pcDNA3.0 [ie1-egfp-pri-bmo-miR-0015-SV40] with pGL3.0 [A3-luc-Fib-L-3′UTR-SV40], suggesting that both bmo-miR-0001 and bmo-miR-0015 can down-regulate the expression of BmFib-L in vitro.     

Decontamination of Bacillus subtilis var. niger spores on selected surfaces by chlorine dioxide gas

Objective  

Chlorine dioxide (CD) gas has been used as a fumigant in the disinfection of biosafety laboratories. In this study, some experiments were conducted to assess the inactivation of spores inoculated on six materials [stainless steel (SS), painted steel (PS), polyvinyl chlorid (PVC), polyurethane (PU), glass (GS), and cotton cloth (CC)] by CD gas. The main aims of the study were to determine the sporicidal efficacy of CD gas and the effect of prehumidification before decontamination on sporicidal efficacy.     

Optimization of succinic acid fermentation with Actinobacillus succinogenes by response surface methodology (RSM)

Succinic acid is considered as an important platform chemical. Succinic acid fermentation with Actinobacillus succinogenes strain BE-1 was optimized by central composite design (CCD) using a response surface methodology (RSM). The optimized production of succinic acid was predicted and the interactive effects between glucose, yeast extract, and magnesium carbonate were investigated. As a result, a model for predicting the concentration of succinic acid production was developed. The accuracy of the model was confirmed by the analysis of variance (ANOVA), and the validity was further proved by verification experiments showing that percentage errors between actual and predicted values varied from 3.02% to 6.38%. In addition, it was observed that the interactive effect between yeast extract and magnesium carbonate was statistically significant. In conclusion, RSM is an effective and useful method for optimizing the medium components and investigating the interactive effects, and can provide valuable information for succinic acid scale-up fermentation using A. succinogenes strain BE-1.     

Effects of gas supersaturation on lethality and avoidance responses in juvenile rock carp (Procypris rabaudi Tchang)

Laboratory experiments were conducted to determine the effects of total dissolved gas (TDG) supersaturation on acute lethality and avoidance responses in juvenile rock carp (Procypris rabaudi Tchang). The juvenile rock carp were exposed to water with different levels of supersaturation (105%, 115%, 120%, 125%, 130%, 135%, 140%, and 145%) and depth of 0.20 m at 25°C for 60 h. Median lethal time (LT50) was used to assess the lethal responses corresponding to different levels of gas supersaturation. The results show that half of the juvenile rock carp died at the 120%, 125%, 130%, 135%, 140%, and 145% levels of supersaturation, and the LT50 corresponding to different levels of supersaturation was 18.7, 15.4, 8.2, 6.6, 3.5, and 1.7 h. When the level of supersaturated water is below 115%, the mortality is negligible. Avoidance responses were observed 5 min after the fish were put into equilibrated water (99%, 0.08 m deep) and water with different supersaturated levels (105%, 115%, 125%, 135%, and 145%, 0.08 m deep) at 25 °C. The fish exhibited strong avoidance responses in supersaturated water when the gas supersaturation was above 135%. However, they exhibited an obvious preference to supersaturated water when the gas supersaturation was below 115%. Thus, the juvenile rock carp can likely survive in water with a supersaturated level of 115%.     

In vitro assay for the anti-brucella activity of medicinal plants against tetracycline-resistant Brucella melitensis

Brucellosis, a zoonosis caused by four species of brucella, has a high morbidity. Brucella melitensis is the main causative agent of brucellosis in both human and small ruminants. As an alternative to conventional antibiotics, medicinal plants are valuable resources for new agents against antibiotic-resistant strains. The aim of this study was to investigate the usage of native plants for brucellosis treatment. For this purpose, the anti-brucella activities of ethanolic and methanolic extracts of Salvia sclarea, Oliveria decumbens, Ferulago angulata, Vitex pseudo-negundo, Teucrium polium, Plantago ovata, Cordia myxa, and Crocus sativus were assessed. The activity against a resistant Br. melitensis strain was determined by disc diffusion method at various concentrations from 50–400 mg/ml. Antibiotic discs were also used as a control. Among the evaluated herbs, six plant (Salvia sclarea, Oliveria decumbens, Ferulago angulata, Vitex pseudo-negundo, Teucrium polium, and Crocus sativus) showed anti-brucella activity. Oliveria decumbens was chosen as the most effective plant for further studies. A tested isolate exhibited resistance to tetracycline, nafcillin, oxacillin, methicillin, and colistin. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values for Oliveria decumbens against resistant Br. melitensis were the same (5 mg/ml), and for gentamicin they were both 2 mg/ml. Time-kill kinetics for a methanolic extract of Oliveria decumbens was 7 h whereas for an ethanolic extract it was 28 h. Also, Oliveria decumbens extracts showed a synergistic effect in combination with doxycycline and tetracycline. In general, the similar values of MIC and MBC for Oliveria decumbens suggest that these extracts could act as bactericidal agents against Br. melitensis. In addition to Oliveria decumbens, Crocus sativus and Salvia sclarea also had good anti-brucella activity and these should be considered for further study.     

Alternaria toxin-induced resistance against rose aphids and olfactory response of aphids to toxin-induced volatiles of rose plants

The search for active toxins for managing weeds or plant diseases is believed to be a promising avenue of investigation. However, the effects of Alternaria toxins on insects have just begun to be investigated. Bioactivities of toxins from four strains of Alternaria alternata on Rosa chinensis and rose aphid Macrosiphum rosivorum were tested in the present study. At a concentration of 50.0 μg/ml, the crude extract (toxin) of strain 7484 was found not to be harmful to rose plants with excised leaf-puncture method (P≥0.079), and rose plants showed enhanced resistance to rose aphids when this Alternaria toxin was sprayed on the plants (P≤0.001). However, this toxin caused no detrimental effects on aphids in insecticidal bioassay at a concentration of 10.0 to 160.0 μg/ml (P≥0.096). Therefore, the Alternaria toxin had significantly induced the resistance of rose plants against rose aphids, demonstrating that the resistance mechanism triggered by the Alternaria toxin in the rose plant may also be used by the plant to defend itself against insects. Further bioassays aimed to discover the olfactory responses of aphids to the toxin-induced volatiles of host plants. The aphids were significantly more attracted to both volatiles emitted and collected from control rose plants than to both volatiles emitted and collected from the toxin-treated rose plants (P≤0.014). This result showed that the toxin-induced resistance related to the volatile changes of host plants.     

A meta-analysis of association between acne vulgaris and Demodex infestation

Until now, etiology of acne vulgaris is still uncertain. Although clinicians usually deny the association between Demodex infestation and acne vulgaris, it has been proved in some clinical practices. To confirm the association between Demodex infestation and acne vulgaris, a meta-analysis was conducted. Predefined selection criteria were applied to search all published papers that analyzed the association between Demodex infestation and acne vulgaris (January 1950 to August 2011) in ISI Web of Knowledge, MEDLINE, and China National Knowledge Infrastructure (CNKI) databases. A meta-analysis was performed to calculate odds ratios (ORs) and 95% confidence intervals (CIs) based on fixed effects models or random effects models. We enrolled the 60 Chinese and 3 English papers in this meta-analysis, which covered Turkey and 25 different provinces/municipalities in China and 42 130 participants including students and residents, aged from 1 to 78 years. The pooled OR in random effects models is 2.80 (95% CI, 2.34–3.36). Stability is robust according to sensitivity analysis. The fail-safe number is 18 477, suggesting that at least 18 477 articles with negative conclusions would be needed to reverse the conclusion that acne vulgaris was related to Demodex infestation. So the effect of publication bias was insignificant and could be ignored. It was concluded that acne vulgaris is associated with Demodex infestation. This indicates that when regular treatments for acne vulgaris are ineffective, examination of Demodex mites and necessary acaricidal therapies should be considered.     

Purification, chemical modification and immunostimulating activity of polysaccharides from Tremella aurantialba fruit bodies

Ultrafiltration and a series of chromatographic steps were used to isolate and purify polysaccharides from Tremella aurantialba fruit bodies. Three crude fractions (TAP50w, TAP10-50w, and TAP1-10w), five semi-purified fractions (TAPA-TAPE), and one purified fraction (TAPA1) were obtained. A sulfated derivative of TAPA1 (TAPA1-s) was prepared by chemical modification. The immunostimulating activity of the polysaccharide fractions in vitro was determined using the mouse spleen lymphocyte proliferation assay. Of the three crude fractions tested, cell proliferation rates were increased most by TAP50w. Furthermore, TAPA1-s was markedly more stimulatory than TAPA1, indicating that sulfonation was an effective way to enhance the immunostimulating activity of polysaccharide.     

Identification of a heat shock cognate protein 70 gene in Chinese soft-shell turtle (Pelodiscus sinensis) and its expression profiles under thermal stress

The heat shock cognate protein 70 (Hsc70) is a member of a 70-kDa heat shock protein (HSP70) family that functions as molecular chaperones. In this study, a novel Hsc70 gene from Chinese soft-shelled turtle (Pelodiscus sinensis) (tHsc70) was identified. The tHsc70 full-length complementary DNA (cDNA) is 2 272 bp long with a 1 941-bp open reading frame (ORF) encoding 646 amino acids. Three characteristic signature regions of the HSP70 family, two major domains of an adenosine triphosphate (ATP)/guanosine triphosphate (GTP) binding domain (ABD), and a substrate-binding domain (SBD) were present in the predicted tHsc70 amino acid sequence. The tHsc70 gene was expressed in Escherichia coli BL21 and the expression product reacted with the anti-Hsc70 mouse monoclonal antibody by Western blotting. Homology analysis revealed that tHsc70 shared identity from 53.9% to 87.7% at the nucleotide level, and 49.1% to 99.5% at the amino acid level with the known Hsc70s. Phylogenetic analysis showed that tHsc70 was clustered together with the Hsc70 gene of another reptile species (Alligator mississippiensis). The tHsc70 was expressed in the liver, lung, heart, and skeletal muscle. The expression patterns of tHsc70 messenger RNA (mRNA) differed among different tissues under different durations of heat stress at 40 °C. Adaptation at 25 °C for 1 h after heat stress was also different among tissues and length of heat stress. Irrespective of different profiles of expression under heat stress, tHsc70 may play roles in protecting turtles from thermal stress.     

Nephroprotective effects of Zingiber zerumbet Smith ethyl acetate extract against paracetamol-induced nephrotoxicity and oxidative stress in rats

Paracetamol (PCM) overdose can cause nephrotoxicity with oxidative stress as one of the possible mechanisms mediating the event. In this study, the effects of ethyl acetate extract of Zingiber zerumbet rhizome [200 mg per kg of body weight (mg/kg) and 400 mg/kg] on PCM-induced nephrotoxicity were examined. Rats were divided into five groups containing 10 rats each. The control group received distilled water while other groups were treated with extract alone (400 mg/kg), PCM alone (750 mg/kg), 750 mg/kg PCM+200 mg/kg extract (PCM+ 200-extract), and 750 mg/kg PCM+400 mg/kg extract (PCM+400-extract), respectively, for seven consecutive days. The Z. zerumbet extract was given intraperitoneally concurrent with oral administration of PCM. Treatment with Z. zerumbet extract at doses of 200 and 400 mg/kg prevented the PCM-induced nephrotoxicity and oxidative impairments of the kidney, as evidenced by a significantly reduced (P<0.05) level of plasma creatinine, plasma and renal malondialdehyde (MDA), plasma protein carbonyl, and renal advanced oxidation protein product (AOPP). Furthermore, both doses were also able to induce a significant increment (P<0.05) of plasma and renal levels of glutathione (GSH) and plasma superoxide dismutase (SOD) activity. The nephroprotective effects of Z. zerumbet extract were confirmed by a reduced intensity of renal cellular damage, as evidenced by histological findings. Moreover, Z. zerumbet extract administered at 400 mg/kg was found to show greater protective effects than that at 200 mg/kg. In conclusion, ethyl acetate extract of Z. zerumbet rhizome has a protective role against PCM-induced nephrotoxicity and the process is probably mediated through its antioxidant properties.     

Evaluation of the dust and methanol extracts of Garcinia kolae for the control of Callosobruchus maculatus (F.) and Sitophilus zeamais (Mots)

Insecticidal effects of different doses of the dust and methanol extracts of Garcinia kolae on Collosobruchus maculatus and Sitophilus zeamais were tested. The dust had no significant effect on the two insects; none of them died even at 3 d after treatment. The methanol extracts, however, had rapid lethal effects on both C. maculatus and S. zeamais. The mortality of C. maculatus by the lowest concentration of methanol extracts ranged from 95%∼100% whereas in S. zeamais, the mortality ranged from 87.5%∼100% and 70%∼100% in concentrations of 1 g extract+3 ml methanol and 1 g extract+5 ml methanol, respectively, from 24 to 48 h. The least concentration of 1 g extract+15 ml methanol had no significant lethal effect on Sitophilus zeamais.      

Anethum graveolens Linn. (dill) extract enhances the mounting frequency and level of testicular tyrosine protein phosphorylation in rats

Objective:To investigate the effect of Anethum graveolens(AG) extracts on the mounting frequency,histology of testis and epididymis,and sperm physiology.Methods:Male rats induced by cold immobilization before treating with vehicle or AG extracts [50,150,and 450 mg/kg body weight(BW)] via gastric tube for consecutive 1,7,and 14 d were examined for mounting frequency,testicular phosphorylation level by immunoblotting,sperm concentration,sperm acrosome reaction,and histological structures of testis and epididymis,respectively.Results:AG(50 mg/kg BW) significantly increased the mounting frequency on Days 1 and 7 compared to the control group.Additionally,rat testis treated with 50 mg/kg BW AG showed high levels of phosphorylated proteins as compared with the control group.In histological analyses,AG extract did not affect the sperm concentration,acrosome reaction,and histological structures of testis and epididymis.Conclusions:AG extract enhances the aphrodisiac activity and is not harmful to sperm and male reproductive organs.     

A homolog of glyceraldehyde-3-phosphate dehydrogenase from Riemerella anatipestifer is an extracellular protein and exhibits biological activity

Riemerella anatipestifer is the causative agent of septicemia anserum exsudativa in ducks. Its pathogenesis and virulence factors are still unclear. The glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an anchorless and multifunctional protein on the surface of several pathogenic microorganisms, is involved in virulence and adhesion. Whether homologs of GAPDH exist, and display similar characteristics in R. anatipestifer (RaGAPDH) has not been determined. In our research, the RaGAPDH activity from various R. anatipestifer isolates was confirmed. Twenty-two gapdh genes from genomic DNA of R. anatipestifer isolates were cloned and sequenced for phylogenetic analysis. The distribution of RaGAPDH in R. anatipestifer CZ2 strain was confirmed by antisera to recombinant RaGAPDH. The ability of purified RaGAPDH to bind host proteins was analyzed by solid-phase ligand-binding assay. Results revealed that all R. anatipestifer isolates showed different levels of GAPDH activity except four strains, which contained a gapdh-like gene. The gapdh of R. anatipestifer, which is located phylogenetically in the same branch as enterohemorrhagic Escherichia coli (EHEC), belonged to class I GAPDH, and encoded a 36.7-kDa protein. All RaGAPDH-encoding gene sequences from field isolates of R. anatipestifer displayed 100% homology. The RaGAPDH localized on the extracellular membrane of several R. anatipestifer strains. Further, it was released into the culture medium, and exhibited GAPDH enzyme activity. We also confirmed the binding of RaGAPDH to plasminogen and fibrinogen. These results demonstrated that GAPDH was present in R. anatipestifer, although not in all strains, and that RaGAPDH might contribute to the microorganism’s virulence.     

Diversity arrays technology (DArT) for studying the genetic polymorphism of flue-cured tobacco (Nicotiana tabacum)

Diversity arrays technology (DArT) is a microarray-based marker system that achieves high throughput by reducing the complexity of the genome. A DArT chip has recently been developed for tobacco. In this study, we genotyped 267 flue-cured cultivars/landraces, including 121 Chinese accessions over five decades from widespread geographic regions in China, 103 from the Americas, and 43 other foreign cultivars, using the newly developed chip. Three hundred and thirty polymorphic DArT makers were selected and used for a phylogenetic analysis, which suggested that the 267 accessions could be classified into two subgroups, which could each be further divided into 2–4 sections. Eight elite cultivars, which account for 83% of the area of Chinese tobacco production, were all found in one subgroup. Two high-quality cultivars, HHDJY and Cuibi1, were grouped together in one section, while six other high-yield cultivars were grouped into another section. The 330 DArT marker clones were sequenced and close to 95% of them are within non-repetitive regions. Finally, the implications of this study for Chinese flue-cured tobacco breeding and production programs were discussed.     

Quantification of Helicobacter pylori levels in soil samples from public playgrounds in Spain

Helicobacter pylori are ubiquitous Gram-negative bacteria with a high estimated level of infection in the world populations, but a majority of the infected persons are asymptomatic. This pathogen has been classified by the World Health Organization as a class I carcinogen and recognized as the causal agent of most peptic ulcers and chronic gastritis that might lead to stomach cancer. Although not all the transmission pathways of these bacteria into humans have been properly identified, enough data have suggested that the oral-oral or fecal-oral ones are the main infection routes. Helicobacter pylori have been detected in non-treated water and in drinking water, which suggested that water might be an important infection source. As childhood is the critical period of infection, the aim of the present work was to examine the presence of Helicobacter pylori in soil samples from public playing areas of Spanish parks.