Proliferation inhibition and apoptosis promotion by dual silencing of VEGF and Survivin in human osteosarcoma

Simultaneous silencing of multiple upregulated genes is an attractive and viable treatment strat-egy for many incurable diseases including cancer.Herein we used a dual gene-targeted siRNA conjugate composed of VEGF and Survivin siRNA sequences in the same backbone to inhibit proliferation and angiogenesis in two human osteosarcoma cell lines.We synthesized siRNA sequences targeting the VEGF and Survivin genes individually (VEGF siRNA and Survivin siRNA) or simultaneously (one-chain-double-target siRNA:dual siRNA).VEGF and Survivin mRNA and protein expression levels in human osteosarcoma MG-63 and Saos-2 cells were detected by qRT- PCR and western blot analysis.VEGF and Survivin protein location and expression were evaluated by immunohistochemistry and immunofluorescence staining.MG-63 and Saos-2 cell migration,proliferation,apoptosis,and angiogenesis were detected by scratch test,MTT assay,flow cytometry,and capillary tube assay respectively.The dual siRNA induced similar downregulation of VEGF and Survivin mRNA and protein levels,compared with VEGF siRNA or Survivin siRNA alone.The dual siRNA caused greater suppression of MG-63 and Saos-2 cell migration,proliferation and angiogenesis,and promoted more cell apoptosis than VEGF siRNA or Survivin siRNA alone,suggesting that the effects of the dual siRNA on inhibiting cell proliferation,migration,and angiogenesis and promoting apoptosis were superior to those of the single-target siRNAs.Simultaneous silencing of VEGF and Survivin using the dual siRNA may be an advantageous alternative for the development of therapeutic strategies against human osteosarcoma.     

Discovering genes responsible for silk synthesis in Bombyx mori by piggyBac‐based random insertional mutagenesis

Silkworm mutants are valuable resources for both transgenic breeding and gene discovery. PiggyBac-based random insertional mutagenesis has been widely used in gene functional studies. In order to discover genes involved in silk synthesis, a piggyBac-based random insertional library was constructed using Bombyx mori, and the mutants with abnormal cocoon were particularly screened. By this means, a “thin cocoon” mutant was identified. This mutant revealed thinner cocoon shell and shorter posterior silk gland (PSG) compared with the wild type. The messenger RNA (mRNA) levels of all the three fibroin genes, including Fib-H, Fib-L and P25, were significantly down-regulated in the PSG of mutants. Four piggyBac insertion sites were identified in Aquaporin (AQP), Longitudinals lacking protein-like {Lola), Glutamyl aminopeptidase-like (GluAP) and Loc101744460. The mRNA levels of all the four genes were significantly altered in the silk gland of mutants. In particular, the mRNA amount of AQP, a gene responsible for the regulation of osmotic pressure, decreased dramatically immediately prior to the spinning stage in the anterior silk gland of mutants. The identification of the genes disrupted in the “thin cocoon” mutant in this study provided useful information for understanding silk production and transgenic breeding of silkworms in the future.     

Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus

Aedes (Stegomyia) albopictus, also known as the Asian tiger mosquito, is a mosquito which originated in Asia. In recent years, it has become increasingly rampant throughout the world. This mosquito can transmit several arboviruses, including dengue, Zika and chikungunya viruses, and is considered a public health threat. Despite the urgent need of genome engineering to analyze specific gene functions, progress in genetical manipulation of Ae. albopictus has been slow due to a lack of efficient methods and genetic markers. In the present study, we established targeted disruptions in two genes, kynurenine hydroxylase (kh) and dopachrome conversion enzyme (yellow), to analyze the feasibility of generating visible phenotypes with genome editing by the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) system in Ae. albopictus. Following Cas9 single guide RNA ribonucleoprotein injection into the posterior end of pre-blastoderm embryos, 30%-50% of fertile survivors produced alleles that failed to complement existing kh and yellow mutations. Complete eye and body pigmentation defects were readily observed in GI pupae and adults, indicating successful generation of highly heritable mutations. We conclude that the CRISPR/Cas9-mediated gene editing system can be used mAe. albopictus and that it can be adopted as an efficient tool for genome-scale analysis and biological study.     

A new genus and four new species of Eriophyidae from China (Acari: Eriophyoidea)

One new genus and four new species of eriophyoid mites from Hainan, China are described and illustrated in this paper: Dicoxiseta Wang, Tan & Yang, gen. nov., D. litsea Wang, Tan & Yang, sp. nov. infesting Litsea monopetala(Roxb.) Pers.(Lauraceae), Eriophyes casearius Wang, Yang & Tan, sp. nov. infesting Casearia glomerata Roxb.(Flacourtiaceae), Phyllocoptruta alstonia Wang, Gu & Tan, sp. nov. infesting Alstonia scholaris(L.) R. Br.(Apocynaceae) and Aculus paederius Wang, Gu & Tan, sp. nov. infesting Paederia scandens(Lour.) Merr.(Rubiaceae). Tegolophus artocarpi Keifer, 1977 is newly recorded from China. E. casearius Wang, Yang & Tan, sp. nov. distorts severely new foliage of its host plant. Three species, P. alstonia Wang, Gu & Tan, sp. nov., A. paederius Wang, Gu & Tan, sp. nov. and T. artocarpi, cause rust on the undersurfaces of host plants, while D. litsea Wang, Tan & Yang, sp. nov. is a vagrant on the undersurface of host plant leaves, without visible symptom.     

Investigation of bacterial community diversity in water of Zoige Alpine Wetland

Spatial isolation is currently thought to represent one of the major factors resulting in bacteria genetic variation and population abundance. The bacterial diversity in a distinct environment Zoige Alpine Wetland located in the northeast of the Qinghai-Tibetan Plateau with the altitude 3400 m on average aroused our great attention. This area belongs to Qinghai-Tibetan cold climate zone with the mean annual temperature about 1 °C. Although several studies on bacterial diversity in Qinghai-Tibetan Plateau had been reported, there is no report on wetland water in this area. In this work, six water samples were collected and the water qualities including COD_Cr, NH₄⁺-N, NO₃^--N, NO₂^--N, TN, TP, TOC were investigated, of which results indicated that more than 80% samples sorted as II–V class of surface water sources according to the National Water Quality Standard of China (GB3838-2002). Comparison of bacterial communities among the six samples was analyzed by DGGE of PCR-amplified 16S rDNA with universal bacterial primer sets. The profiles demonstrated that samples from the Flower Lake had more DNA bands than the Conservatory Station inferring higher diversity. In addition, the samples from the same environment shared similar compositions of bacterial communities. Bacterial community composition and predominant bacteria were analyzed by 16S rDNA clone library. The dominant group was Proteobacteria (51.6% of the total clones, which contained 24.2% alpha proteobacteria, 14.5% beta proteobacteria and 12.9% gamma proteobacteria). And the Bacteroidetes added to 17.7%, Verrucomicrobia to 4.8%. More than 24.2% of the total clones showed high similarity to uncultured bacteria. The above work provides some information on bacterial diversity for special site of spatial isolation.     

Expression of a new chimeric protein with a highly repeated sequence in tobacco cells

In wheat, the high-molecular weight (HMW) glutenin subunits are known to contribute to gluten viscoelasticity, and show some similarities to elastomeric animal proteins as elastin. When combining the sequence of a glutenin with that of elastin is a way to create new chimeric functional proteins, which could be expressed in plants. The sequence of a glutenin subunit was modified by the insertion of several hydrophobic and elastic motifs derived from elastin (elastin-like peptide, ELP) into the hydrophilic repetitive domain of the glutenin subunit to create a triblock protein, the objective being to improve the mechanical (elastomeric) properties of this wheat storage protein. In this study, we investigated an expression model system to analyze the expression and trafficking of the wild-type HMW glutenin subunit (GS_W) and an HMW glutenin subunit mutated by the insertion of elastin motifs (GS_M-ELP). For this purpose, a series of constructs was made to express wild-type subunits and subunits mutated by insertion of elastin motifs in fusion with green fluorescent protein (GFP) in tobacco BY-2 cells. Our results showed for the first time the expression of HMW glutenin fused with GFP in tobacco protoplasts. We also expressed and localized the chimeric protein composed of plant glutenin and animal elastin-like peptides (ELP) in BY-2 protoplasts, and demonstrated its presence in protein body-like structures in the endoplasmic reticulum. This work, therefore, provides a basis for heterologous production of the glutenin-ELP triblock protein to characterize its mechanical properties.     

The Arabidopsis Anaphase-Promoting Complex/Cyclosome Subunit 1 is Critical for Both Female Gametogenesis and Embryogenesis

Anaphase-promoting complex/cyclosome (APC/C), a multisubunit E3 ligase, plays a critical role in cell cycle control, but the functional characterization of each subunit has not yet been completed. To investigate the function of APC1 in Arabidopsis, we analyzed four mutant alleles of APC1, and found that mutation in APC1 resulted in significantly reduced plant fertility, accumulation of cyclin B, and disrupted auxin distribution in embryos. The three mutant alleles apc1-1, apc1-2 and apc1-3 shared variable defects in female gametogenesis including degradation, abnormal nuclear number, and disrupted polarity of nuclei in the embryo sac as well as in embryogenesis, in which embryos were arrested at multiple stages. All of these defects are similar to those previously identified in apc4. The mutant apc1-4, in which the T-DNA was inserted after the transmembrane domain at the C-terminus, showed much more severe phenotypes; that is, most of the ovules were arrested at the one-nucleate female gametophyte stage (stage FG1). In the apc1 apc4 double mutants, the fertility was further reduced by one-third in apc1-1/+ apc4-1/+, and in some cases no ovules even survived in siliques of apc1-4/+ apc4-1/+. Our data thus suggest that APC1, an essential component of APC/C, plays a synergistic role with APC4 both in female gametogenesis and in embryogenesis.     

The Mitochondrial Genome of Raphanus sativus and Gene Evolution of Cruciferous Mitochondrial Types

To explore the mitochondrial genes of the Cruciferae family, the mitochondrial genome of Raphanus sativus (sat) was sequenced and annotated. The circular mitochondrial genome of sat is 239,723 bp and includes 33 protein-coding genes, three rRNA genes and 17 tRNA genes. The mitochondrial genome also contains a pair of large repeat sequences 5.9 kb in length, which may mediate genome reorga-nization into two sub-genomic circles, with predicted sizes of 124.8 kb and 115.0 kb, respectively. Furthermore, gene evolution of mitochondrial genomes within the Cruciferae family was analyzed using sat mitochondrial type (mitotype), together with six other re-ported mitotypes. The cruciferous mitochondrial genomes have maintained almost the same set of functional genes. Compared with Cycas taitungensis (a representative gymnosperm), the mitochondrial genomes of the Cruciferae have lost nine protein-coding genes and seven mitochondrial-like tRNA genes, but acquired six chloroplast-like tRNAs. Among the Cruciferae, to maintain the same set of genes that are necessary for mitochondrial function, the exons of the genes have changed at the lowest rates, as indicated by the numbers of single nucleotide polymorphisms. The open reading frames (ORFs) of unknown function in the cruciferous genomes are not conserved. Evolutionary events, such as mutations, genome reorganizations and sequence insertions or deletions (indels), have resulted in the non- conserved ORFs in the cruciferous mitochondrial genomes, which is becoming significantly different among mitotypes. This work represents the first phylogenic explanation of the evolution of genes of known function in the Cruciferae family. It revealed significant variation in ORFs and the causes of such variation.     

Contaminant Removal of Domestic Wastewater by Constructed Wetlands： Effects of Plant Species

A comparative study of the efficiency of contaminant removal between five emergent plant species and between vegetated and unvegetated wetlands was conducted in small-scale （2.0 m×1.0 m×0.7 m, lengthxwidthxdepth） constructed wetlands for domestic wastewater treatment in order to evaluate the decontaminated effects of different wetland plants. There was generally a significant difference in the removal of total nitrogen （TN） and total phosphorus （TP）, but no significant difference in the removal of organic matter between vegetated and unvegetated wetlands. Wetlands planted with Canna indica Linn., Pennisetum purpureum Schum., and Phragmites communis Trin. had generally higher removal rates for TN and TP than wetlands planted with other species. Plant growth and fine root （root diameter ≤ 3 mm） biomass were related to removal efficiency. Fine root biomass rather than the mass of the entire root system played an important role in wastewater treatment. Removal efficiency varied with season and plant growth. Wetlands vegetated by P. purpureum significantly outperformed wetlands with other plants in May and June, whereas wetlands vegetated by P. communis and C. indica demonstrated higher removal efficiency from August to December. These findings suggest that abundance of fine roots is an important factor to consider in selecting for highly effective wetland plants. It also suggested that a plant community consisting of multiple plant species with different seasonal growth patterns and root characteristics may be able to enhance wetland performance.     

Eco-physiological responses of the declining population Spartina anglica to N and P fertilizer addition

Nitrogen and phosphorus are both important life elements. N, P and combined N-P fertilizers were added to the declining population Spartina anglica Hubbard in coastal China. Some growth parameters and eco-physiological responses of S. anglica to different fertilizer treatments (N, P and combined N-P fertilizer addition with high, medium and low levels, respectively) were measured. The fertilizer addition had a highly significant effect on the dynamics of its height-growth, number of leaves, number of roots and total biomass. Only N addition had a significant effect on leaf area and leaf thickness in all fertilizer treatments. On the dynamics of its height-growth, the effect of N addition was the most apparent, and the effect of N-P addition was not greater than those of N and P addition separately. The photosynthesis rate was enhanced and the yield was the highest with the highest N, the highest N-P and the medium P addition. The rates were higher than those of CK by 19.08 μmol·m^?2·s^?1, 15.47 μmol·m^?2·s^?1 and 11.23 μmol·m^?2·s^?1, respectively. The activity of SOD and POD increased with the treatments after freshwater stress for 14 days. Effects of medium N and P addition were significant for SOD activity. However, POD activity was significantly higher with the treatment of higher N and higher N-P addition. In a word, fertilizer addition improved the growth of the declining population S. anglica. The results indicated that the decline of S. anglica was correlated with the nutriment deficiency in soil, especially with the lack of N.