Identification of siRNA generating hot spots in multiple viral suppressors to generate broad-spectrum antiviral resistance in plants

Viruses are one of the most devastating plant pathogens causing severe economic losses worldwide. RNA silencing is a robust technology to knock down the expression of specific genes. This mechanism can be exploited to generate virus resistant plants through expression of the viral derived sequences. Viruses in turn have evolved to encode suppressors of RNA silencing to combat host defense. Mixed infection of plants is of common occurrence in nature and simultaneous targeting of suppressor(s) of multiple viruses offers an effective strategy. In this study, we have in silico designed siRNAs against suppressors of the two most devastating viruses of tomato, leaf curl causing tomato begomoviruses and Cucumber mosaic virus. Three different siRNA prediction programs were used to evaluate siRNAs generating capability of each sequence and common putative candidate siRNAs were selected fulfilling the stringent parameters. Our results indicated that in the case of each suppressor a particular region of 100–150 base pairs could be source of potent siRNAs referred as hotspots. Expression of these viral hot spots as a single construct in the plants would facilitate development of transgenic plants with a high degree of broad spectrum resistance against multiple viruses.

Electronic supplementary material

The online version of this article (doi:10.1007/s12298-014-0264-0) contains supplementary material, which is available to authorized users.     

Silver fir stand productivity is enhanced when mixed with Norway spruce: evidence based on large‐scale inventory data and a generic modelling approach

Questions: How to evaluate the mixture effect on basal area increment in two‐species forest stands? Is a mixed Norway spruce–silver fir stand more productive than pure adjacent stands of either species? How to develop generic modelling approaches to assess mixture effects in forest stands? Location: In addition to a case study on Norway spruce–silver fir stands in French mountain forests, the generic approach used goes beyond local applications. Methods: We took advantage of National Forest Inventory data to develop a unique stand basal‐area‐increment model for pure and mixed stands of Norway spruce and silver fir that responds to ecological site conditions. The database was made up of 284 pure Norway spruce stands, 196 pure silver fir stands, and 323 mixed stands of these species. Results: Pure silver fir basal area increment is strongly influenced by spring climatic conditions, whereas pure Norway spruce is more influenced by soil conditions. The mixture of these species has a positive effect on silver fir, which decreases as the proportion of fir increases. In contrast, the mixture has no noticeable effect on Norway spruce. Conclusion: We developed a stand basal‐area‐increment model evidencing an advantage of the mixture on silver fir basal area increment, but not on Norway spruce. The mathematical formulation of the model developed is generic and can be used in all two‐species mixture situations. It also makes it possible to compare different mixture situations with each other.     

Identification of enzyme inhibitory mechanisms from steady-state kinetics

Enzyme inhibitors are used in many areas of the life sciences, ranging from basic research to the combat of disease in the clinic. Inhibitors are traditionally characterized by how they affect the steady-state kinetics of enzymes, commonly analyzed on the assumption that enzyme-bound and free substrate molecules are in equilibrium. This assumption, implying that an enzyme-bound substrate molecule has near zero probability to form a product rather than dissociate, is valid only for very inefficient enzymes. When it is relaxed, more complex but also more information-rich steady-state kinetics emerges. Although solutions to the general steady-state kinetics problem exist, they are opaque and have been of limited help to experimentalists.Here we reformulate the steady-state kinetics of enzyme inhibition in terms of new parameters. These allow for assessment of ambiguities of interpretation due to kinetic scheme degeneracy and provide an intuitively simple way to analyze experimental data. We illustrate the method by concrete examples of how to assess scheme degeneracy and obtain experimental estimates of all available rate and equilibrium constants. We suggest simple, complementary experiments that can remove ambiguities and greatly enhance the accuracy of parameter estimation.     

Pathogenic bacteria prime the induction of Toll-like receptor signalling in human colonic cells by the Gal/GalNAc lectin Carbohydrate Recognition Domain of Entamoeba histolytica

In mixed intestinal infections with Entamoeba histolytica trophozoites and enteropathogenic bacteria, which are wide-spread in areas of endemic amoebiasis, interaction between the pathogens could be an important factor in the occurrence of invasive disease. It has been reported that exposure of human colonic cells to enteropathogenic bacteria increased trophozoite adherence to the cells and their subsequent damage. We report here that the Carbohydrate Recognition Domain (CRD) of the amoebic Gal/GalNAc lectin binds to Toll-like receptors TLR-2 and TLR-4 in human colonic cells, activating the “classic” signalling pathway of these receptors. Activation induced expression of TLR-2 and TLR-4 mRNAs and the mRNAs of pro-inflammatory cytokines, as well as an increase in the corresponding proteins. Direct correlation was observed between the increased expression of TLRs and pro-inflammatory cytokines, the enhanced adhesion of trophozoites to the cells and the inflicted cell damage. When cells were exposed to pathogenic bacteria Staphylococcus aureus (Gram+) or Shigella dysenteriae (Gram−), elements of an innate immune response were induced. CRD by itself elicited a similar cell response, while exposure to a commensal Escherichia coli had a null effect. Pre-exposure of the cells to pathogenic bacteria and then to CRD rendered an inflammatory-like microenvironment that after addition of trophozoites facilitated greater cell destruction. Our results suggest that CRD is recognised by human colonic cells as a pathogen-associated-molecular-pattern-like molecule and as such can induce the expression of elements of an innate immune response. In the human host, an exacerbated inflammatory environment, derived from pathogen interplay, may be an important factor for development of invasive disease.     

Electron transfer within C4 unsaturated spacers between terminal ferrocenyl moieties

The electrochemical and near-IR properties of a series of (bis)ferrocenyl complexes, in which the ferrocenyl moieties are spanned by enyne (E)-(-CHCH-CC-), diyne (-CC-)₂, or diene (E)-(-CHCH-)₂ C₄ unsaturated spacers, were investigated. The experiments performed on 1,4-diferrocenyl-(E)-1-buten-3-yne revealed behaviors indicative of through-bridge/metal-metal electron transfer. The enyne bridged (bis)ferrocenyl cation displays charge transfer properties intermediate between the dienyl and diynyl systems and can be assigned to weakly coupled Class II mixed valence species. The analysis of the collected data according to the classical two-state Hush theory revealed that the efficiency of the electron transfer increases from wholly sp-C to wholly sp²-C bridged complexes. Therefore, the degree of communication is modulated according to the structure of the bridging ligand.     

Detection of single and mixed covert baculovirus infections in eastern spruce budworm, Choristoneura fumiferana populations

We surveyed for covert baculovirus infections in the eastern spruce budworm, Choristoneura fumiferana (Clemens) and compared the prevalence of virus detected in a laboratory and a field population. DNA was extracted from budworm adults and then PCR with degenerate primers was used to identify individuals carrying baculovirus DNA. Multiplex PCR was then applied to the positive samples to distinguish between the multiple baculovirus types that could potentially be found in C. fumiferana populations. Covert infections were found in both the laboratory and the field population of C. fumiferana, although the frequency of infection and the composition of viruses found were very different. Overall 28% of insects from the laboratory population were positive for baculovirus DNA. Individual adults supported both single and mixed covert infections with CfMNPV plus CfDEFNPV, CfDEFNPV plus a GV and mixtures of all three viruses together. However, the majority of insects supported single virus infections, and surprisingly this virus was CfDEFNPV, a virus that is reported not to have per os activity in C. fumiferana larvae. Insects from field populations showed a very different pattern; 70.5% of individuals were baculovirus positive and all of these were positive for CfDEFNPV only.     

壳愈宁对混合痔术后肛缘水肿的临床疗效观察

目的:观察壳愈宁(壳聚糖生物喷膜剂)对混合痔术后肛缘水肿的临床疗效。方法:将我院肛肠外科2007年1月-2010年12月混合痔术后患者80例随机分为对照组、治疗组两组,每组40例,对照组常规换药,治疗组加用壳愈宁外喷治疗,疗程1周。结果:治疗组治疗后肛缘水肿的减轻程度与对照组比较有显著性差异(P<0.01),具有统计学意义。结论:壳愈宁对于混合痔术后水肿有确切的治疗效果,适合临床应用。     

壳愈宁对混合痔术后肛缘水肿的临床疗效观察

目的：观察壳愈宁（壳聚糖生物喷膜剂）对混合痔术后肛缘水肿的临床疗效。方法：将我院肛肠外科2007年1月．2010年12月混合痔术后患者80例随机分为对照组、治疗组两组,每组40例,对照组常规换药,治疗组加用壳愈宁外喷治疗,疗程1周。结果：治疗组治疗后肛缘水肿的减轻程度与对照组比较有显著性差异（P〈0．01）,具有统计学意义。结论：壳愈宁对于混合痔术后水肿有确切的治疗效果,适合临床应用。     

高阶混合中立型差分方程的振动性质

研究了一类高阶混合中立型差分方程的振动性质,得到了这类差分方程的振动判据.