Silencing of lncRNA XIST suppresses proliferation and autophagy and enhances vincristine sensitivity in retinoblastoma cells by sponging miR-204-5p.

OBJECTIVE: Retinoblastoma (RB) is the most prevalent intraocular malignancy in childhood. Long non-coding RNAs (lncRNAs) have been found as critical oncogenic drivers and tumor suppressor in RB. The aim of the present work was to investigate the impact and mechanism of XIST on RB cell autophagy and vincristine (VCR) sensitivity. MATERIALS AND METHODS: The levels of XIST and miR-204-5p were assessed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Western blot analysis was used for the determination of related protein levels. Cell proliferation and IC50 value of VCR were detected using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Flow cytometry was performed to evaluate cell apoptosis. The activities of caspase-3 and caspase-9 were identified using a corresponding assay kit. The direct interaction between XIST and miR-204-5p was confirmed using Dual-Luciferase reporter assay. Xenograft model was established to observe the effect of XIST on RB in vivo. RESULTS: Our data indicated that XIST was highly expressed in RB tissues and cell lines. XIST knockdown weakened the proliferation and autophagy and enhanced VCR sensitivity in RB cells. XIST acted as a molecular sponge of miR-204-5p. Moreover, the regulatory effects of XIST silencing on RB cell proliferation, autophagy and VCR sensitivity were mediated by miR-204-5p. Additionally, XIST silencing weakened tumor growth and enhanced VCR sensitivity in vivo through up-regulating miR-204-5p. CONCLUSIONS: Our current study suggested that XIST silencing suppressed RB progression and promoted VCR sensitivity in vitro and in vivo at least partially by acting as a miR-204-5p sponge, highlighting a powerful therapeutic strategy for RB treatment.

Urea free synthesis of chitin-based acrylate superabsorbent polymers under homogeneous conditions: Effects of the degree of deacetylation and the molecular weight.

In order to understand the chemical structure of chitin-based acrylate superabsorbent polymers (SAP), chitin was dissolved in NaOH aqueous solution via freezing-thawing cyclic treatment without urea, subsequently, a transparent hydrogel was prepared by copolymerizing the alkali-chitin solution and acrylic acid directly. The effects of the degree of deacetylation (DDA) and the molecular weight (Mw) of chitin on the properties of SAP were investigated in detail. With increasing the DDA and Mw, the yield improved while the water absorbency decreased, yet the effect of DDA is insignificant if the Mw is smaller enough. The structures were characterized by FT-IR, XRD, TG, DSC, XPS, solid-state 13C NMR and elemental analyses. The results indicated that the poly(acrylic acid) chains were successfully grafted onto the chitin backbones, and the reaction sites were the NH2 on the chitosan units. The possible mechanism was further discussed, which was similar to that suggested for chitosan-g-poly(acrylic acid) SAP.

Reevaluation of Two Quantitative Trait Loci for Type II Resistance to Fusarium Head Blight in Wheat Germplasm PI 672538.

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, is a destructive disease in wheat. A population consisting of 229 F2 and F2:3 plants derived from the cross PI 672538 × L661 was used to evaluate the reactions to FHB. The FHB resistance data distribution in the F2 population indicates that some quantitative trait loci (QTLs) were controlling the FHB resistance in PI 672538. We further detected two major QTLs (Qfhs-2B, Qfhs-3B) from analysis of the resistance data and the PCR-amplified results using WinQTLCart 2.5 software. Qfhs-2B, flanked by Xbarc55-2B and Xbarc1155-2B, explained more than 11.6% of the phenotypic variation of the percentage of diseased spikelets (PDS), and Qfhs-3B, flanked by Xwmc54-3B and Xgwm566-3B, explained more than 10% of the PDS phenotypic variation in the F2:3 population. In addition, Qfhs-3B was different from Fhb1 in terms of the pedigree, inheritance, resistance response, chromosomal location, and marker diagnosis. We also detected QTLs for other disease resistance indices, including the percentage of damaged kernels and 1,000-grain weight, in similar chromosomal regions. Therefore, the FHB resistance of PI 672538 was mainly controlled by two major QTLs, mapped on 2B (FhbL693a) and 3B (FhbL693b). PI 672538 could be a useful germplasm for improving wheat FHB resistance.

Development of SCAR Markers and an SYBR Green Assay to Detect Puccinia striiformis f. sp. tritici in Infected Wheat Leaves.

Stripe rust, caused by the pathogenic fungus Puccinia striiformis f. sp. tritici, is an important disease of wheat worldwide. A rapid and reliable detection of the pathogen in latent infected wheat leaves is useful for accurate and early forecast of outbreaks and timely application of fungicides for managing the disease. Using the previously reported primer pair Bt2a/Bt2b, a 362-bp amplicon was obtained from P. striiformis f. sp. tritici and a 486-bp amplicon was obtained from both P. triticina (the leaf rust pathogen) and P. graminis f. sp. tritici (the stem rust pathogen). Based on the sequence of the 362-bp fragment, two pairs of sequence characterized amplified region (SCAR) primers were designed. PSTF117/PSTR363 produced a 274-bp amplicon and TF114/TR323 produced a 180-bp amplicon from P. striiformis f. sp. tritici, whereas they did not produce any amplicon from P. triticina, P. graminis f. sp. tritici, or any other wheat-infecting fungi. The detection limit of PSTF117/PSTR363 was 1 pg/µl and TF114/TR323 was 100 fg/µl. Both SCAR markers could be detected in wheat leaves 9 h post inoculation. An SYBR Green RT-PCR method was also developed to detect P. striiformis f. sp. tritici in infected leaves with the detection limit of 1.0 fg DNA from asymptomatic leaf samples of 6 h after inoculation. These methods should be useful for rapid diagnosis and accurate detection of P. striiformis f. sp. tritici in infected wheat leaves for timely control of the disease.

Multi-walled carbon nanotube-supported metal-doped ZnO nanoparticles and their photocatalytic property.

A simple and versatile approach has been developed to synthesize multi-walled carbon nanotubes/metal-doped ZnO nanohybrid materials (MWNT/M-doped ZnO) by means of the co-deposition method. The experimental results illuminate that MWNTs can be modified by metal-doped ZnO nanoparticles at 450 °C, such as Mn, Mg, and Co elements. Furthermore, the MWNT/Mg-doped ZnO hybrids have been proven to have a high photocatalytic ability for methyl orange (MO), in which the degraded rate for MO reaches 100 % in 60 min. The enhancement in photocatalytic activity is attributed to the excellent electriconal property of MWNTs and Mg-doping. The resultant MWNT/Mg-doped ZnO nanohybrids have potential applications in photocatalysis and environmental protection.

Identification of a specific scar marker for detection of Tilletia foetida (Wall) Liro pathogen of wheat.

Common bunt is one of the most important destructive diseases of wheat worldwide and is a domestic quarantined disease in China. However, a rapid and efficient method to identify the corresponding pathogens is currently limited. The objective of the present study was to develop a diagnostic molecular marker specific towards Tilletia foetida (Wall) Liro, a causal agent of the bunt disease. One specific DNA fragment for T. foetida (286 bp in length) was amplified using an Amplified Fragment Length Polymorphism (AFLP) assay and, this fragment was cloned and sequenced. One pair of specific primers (SC(286-1)/SC(286-2)), which was designed according to the sequence, could specifically amplify the corresponding fragment in all of the T. foetida isolates employed from both the People's Republic of China and United States, whereas this fragment could not be amplified by the other fungal species tested. Therefore, a specific Sequence Characterized Amplified Region (SCAR) marker was developed. This SCAR marker could distinguish T. foetida from related pathogenic fungi efficiently and could be used for the early diagnosis of the common bunt of wheat in the field, and provide an efficient way for disease surveillance and disease forecasting in cereal crop.

Multi-walled carbon nanotubes supported Cu-doped ZnO nanoparticles and their optical property.

Multi-walled carbon nanotubes (MWNTs)/Cu-doped ZnO composite powders were prepared by co-precipitation method, and were characterized by X-ray diffraction, electron microscopy, fluorescence spectrum, and ultraviolet spectrum. Experimental results show that the MWNTs can be modified by Cu-doped ZnO nanoparticles with hexagonal wurtzite structure after annealed at 450 °C, and the nanoparticle size is about 15 nm. Two ultraviolet (UV) peaks and a green band centered at about 510 nm are observed in the fluorescence spectrum of MWNTs/Cu-doped ZnO composite powder annealed at 450 °C. Furthermore, MWNTs and Cu doping significantly improve the UV absorption ability of ZnO.

Race and Virulence Dynamics of Puccinia triticina in China During 2000-2006.

Wheat leaf rust, caused by Puccinia triticina, is an important foliar disease of wheat in China. The dynamics of races and virulence in P. triticina populations in China during 2000 to 2006 were studied. Leaf rust samples were collected during surveys of wheat fields and trap nurseries in 16 provinces, and provided by coworkers throughout China. The virulence of single-pustule isolates was determined on near-isogenic Thatcher lines for leaf rust resistance genes Lr1, Lr2a, Lr2c, Lr3, Lr9, Lr16, Lr24, Lr26, Lr3ka, Lr11, Lr17, and Lr30, and races were denominated using the Prt code system. During 2000 to 2006, 79 races were identified from a total of 613 isolates. Races PHT (23.7%), THT (14.7%), PHJ (11.4%), and THJ (4.2%) were the four common races, all avirulent to Lr9 and Lr24. The frequency of isolates with virulence to Lr1, Lr2c, Lr3, Lr11, Lr16, Lr17, and Lr26 was over 80%, and these isolates were widely distributed in China, whereas the frequencies of virulence to Lr9, Lr19, Lr24, Lr25, Lr28, and Lr29 were 0.2 to 2.5%. The diversity of virulence phenotypes of Chinese P. triticina populations appeared to increase from 2000 to 2006. P. triticina races and virulences in China appear to be isolated from those in other countries.

Development of a SCAR marker by inter-simple sequence repeat for diagnosis of dwarf bunt of wheat and detection of Tilletia controversa KüHN.

Dwarf bunt of wheat, caused by Tilletia controversa KüHN: , is a destructive disease on wheat as well as an important internationally quarantined disease in many countries. The primer ISSR818 generated a polymorphic pattern displaying a 867-bp DNA fragment specific for T. controversa. The marker was converted into a sequence characterized amplified region (SCAR), and specific primers (TCKSF3/TCKSR3) designed for use in PCR detection assays; they amplified a unique DNA fragment in all isolates of T. controversa but not in the related pathogens. The detection limit with the primer set (TCKSF3/TCKSR3) was 5 ng of DNA which could be obtained from 5.5 microg of teliospores in a 25-microL PCR reaction mixture.

First Detection of Virulence in Puccinia striiformis f. sp. tritici in China to Resistance Genes Yr24 (=Yr26) Present in Wheat Cultivar Chuanmai 42.

Stripe rust disease of wheat caused by Puccinia striiformis f. sp. tritici was observed on previously resistant bread wheat (Triticum aestivum L.) cv. Chuanmai 42 during the 2008-2009 crop season in Pi County, Sichuan Province, China. More than 10 single pustules were isolated from the diseased leaf samples collected in the field and inoculated on 7-day-old susceptible wheat seedlings cv. Mingxian 169. After 18 to 24 h of incubation at 100% relative humidity in darkness, the plants were moved into the greenhouse, maintained at 15 to 18°C, and supplemented with 10,000 lx of fluorescent light for 10 h per day. The second leaves were clipped when chlorotic spots appeared on leaves (~7 days postinoculation), and plants were covered with glass cylinders to prevent cross contamination. Urediniospores of each isolate were collected 16 days after inoculation and temporarily kept in a dryer at low temperature (3 to 4°C). The virulence spectra of the isolates were tested on Chinese differentials and wheat lines with known Yr genes at the seedling stage (1). A new Yr24 (=Yr26) virulent pathotype, different from currently known pathotypes in China, was identified. To our knowledge, this is the first detection of Yr24 virulence in Puccinia striiformis f. sp. tritici populations on Chuanmai 42. In addition, the new pathotype was also virulent to Lantian 17, Guinong 22 (Chinese differential), and 92R137 derived wheat lines Nannong 04Y10 and Nannong 05Y628, known to carry Yr24 (2,3). The avirulence/virulence formula of the new pathotype is Yr1, 3, 4, H46, 5, 6, 15, 17, 18, 32, Sp, Sd/Yr2, 8, 9, 10, 12, 24 (=26), 31, and Su. Wheat cultivars and breeding materials, previously protected by Yr24 gene, are now vulnerable to stripe rust epidemics in the region. Pure isolates of the new pathotype (Accession No. 09-6-16-3) are stored in the Chinese Academy of Agricultural Sciences (CAAS; Beijing) stripe rust collection. References: (1) W. Q. Chen et al. Plant Dis. 93:1093, 2009. (2) G. Q. Li et al. Theor. Appl. Genet. 112:1434, 2006. (3) Z. F. Li et al. Plant Dis. 90:1302, 2006.

Development of a sequence-characterized amplified region marker for diagnosis of dwarf bunt of wheat and detection of Tilletia controversa Kühn.

AIMS: Dwarf bunt of wheat, caused by Tilletia controversa Kühn, is a destructive disease on wheat as well as an important international quarantined disease in many countries. The objective of this investigation was to develop a diagnostic molecular marker generated from amplified fragment length polymorphism (AFLP) for rapid identification of T. controversa. METHODS AND RESULTS: A total of 30 primer combinations were tested by AFLP to detect DNA polymorphisms between T. controversa and related species. The primer combination E08/M02 generated a polymorphic pattern displaying a 451-bp DNA fragment specific for T. controversa. The marker was converted into a sequence-characterized amplified region (SCAR), and specific primers (SC-01(49)/SC-02(415)), designed for use in PCR detection assays, amplified a unique DNA fragment in all isolates of T. controversa, but not in the related pathogens. The detection limit with the primer set SC-01(49)/SC-02(415) was 10 ng of DNA which could be obtained from 11 microg of teliospores in a 25-microl PCR reaction. CONCLUSIONS: An approach to distinguish T. controversa from similar pathogenic fungi has been developed based on the use of a SCAR marker. SIGNIFICANCE AND IMPACT OF THE STUDY: Development of the simple, high throughput assay kit for the rapid diagnosis of dwarf bunt of wheat and detection of T. controversa is anticipated in further studies.

Suppression of N-Ras by shRNA-expressing plasmid increases sensitivity of HepG2 cells to vincristine-induced growth inhibition.

Oncogenic ras genes relate to the development of human cancers. In this study, we used a plasmid-mediated short-hairpin RNA (shRNA) targeting N-ras gene to combine with clinical drug vincristine (VCR) for the treatment of human hepatoma cells. Our results showed that anti-N-Ras shRNA expression vector (pCSH1-shNR) knocked down the target mRNA and protein. Higher efficacy on growth inhibition was observed when pCSH1-shNR was combined with VCR. This synergistic effect was associated with abrogation of VCR-induced overexpressions of P-glycoprotein and multidrug resistance-associated protein 1 by pCSH1-shNR through downregulations of N-Ras and total Ras. Western blot analysis showed that pCSH1-shNR-induced downregulations of N-Ras and total Ras were potentiated by VCR. Following Ras downregulation, phosphorylations of ERK1/2 and Akt were dramatically inhibited by combinatory treatment. The data showed that pCSH1-shNR-induced inhibition of nuclear factor-kappaB was enhanced by VCR. In addition, the combination of pCSH1-shNR and VCR synergistically inhibited the growth of human hepatoma HepG2 in vivo. Our findings suggested that combination of gene-specific therapeutics and appropriate chemotherapeutic agents might be a promising approach for the treatment of human hepatocellular carcinoma.

Race Dynamics, Diversity, and Virulence Evolution in Puccinia striiformis f. sp. tritici, the Causal Agent of Wheat Stripe Rust in China from 2003 to 2007.

Stripe (or yellow) rust caused by Puccinia striiformis f. sp. tritici is the most destructive foliar disease of wheat in China. The pathogen populations were analyzed for virulence evolution, complexity, phenotypic dynamics, and diversity on temporal and spatial bases. A total of 41 races were identified and characterized from 4,714 stripe rust isolates collected during 2003 through 2007 from wheat growing areas in 15 provinces in China. The races were based on avirulence/virulence patterns to 19 differential host genotypes. Chinese stripe rust population exhibited high diversity with a complex virulence structure. Comparisons using the relative Shannon's index indicated that some differences in the richness and evenness of races were present in pathogen populations within years and between regions despite a national tendency to reduced diversity over time. A noticeably increased frequency of race CYR33 (Chinese yellow rust 33) with virulence for YrSu was the major virulence change recorded in this study compared to the results on an annual basis. Isolates of Puccinia striiformis f. sp. tritici from different regions showed differences in the composition of races, distribution frequency, and diversity. The uneven distribution of major races and comparatively greater diversity in the Northwest and Southwest regions than that in the Huang-Huai-Hai region suggest that long-distance migrations of the pathogen occur from one or more over-summering areas eastward into over-wintering areas. This supports the hypothesis that southern Gansu and northwestern Sichuan comprises a "center of origin for virulence". Mutation of virulence or avirulence for host resistance in the stripe rust fungus may be the basic cause of the occurrence of new virulent types. The subsequent dominance of certain races will vary with parasitic fitness and the opportunities to be selected through large-scale cultivation of varieties with matching resistance genes. Implications of the center of origin for virulence variation and diversity in the pathogen population and an alternative strategy for limiting virulence evolution are discussed.

Normalized fifth-order spherical aberration coefficients derived from a Gaussian trajectory on rotationally symmetrical magnetic lenses.

This paper reports normalized fifth-order spherical aberration coefficients C(5s)/R derived from Gaussian trajectory for rotationally symmetrical magnetic lenses, where S/D (the polepiece gap-width/bore-diameter ratio) is in the range 0.20-2.00, and k(2) (the lens-intensity parameter) is 0.10-100. It is noted that the normalized fifth-order spherical-aberration coefficient C(5s)/R changes its sign within the given parameter ranges.