Effects of mutated replicase and movement protein genes on attenuation of tobacco mosaic virus

Our previous reports showed that one opal mutation (UGA) and one ochre mutation (UAA) respectively located in the replicase and movement protein (MP) genes of the attenuated tomato mosaic virus K(ToMV-K) contribute to the viral attenuation. To explore a wider application of this attenuation pattern to other plant viruses, we have constructed three mutants which respectively contain one opal mutation of the replicase gene and/or one ochre mutation of the MP using PCR-mediated site-directed mutagenesis from a virulent tobacco mosaic virus isolated from China (TMV-Cv). Plant infection performed by in vitro transcripts revealed that the MP truncated mutant TMV-Cvmp and the replicase-MP truncated mutant TMV-Cvrase-mp were infectious on both local lesion (Nicotiana tabacum cv. Xanthi NC) and systemic (N. tabacum cv. K326) host plants, while the replicase truncated mutant TMV-Cvrase was non-infectious. The K326 plant infected by TMV- Cvrease-mp displayed only a little mild mosaic. By electronic microscopy (EM), plant re-inoculation, RNA Dot-blot, RT-PCR and sequencing we demonstrated that the progeny viruses of TMV-Cvmp and TMV-Cvrease-mp shared similar morphological character with TMV-Cv, owned the abilities to infect, replicate and propagate in the assayed plants, and maintained the mutated sites during infection. These data showed that both the opal and the ochre mutations are able to cooperatively induce the attenuated phenotypes of TMV-Cvrase-mp on plants, indicating that the mutation pattern of ToMV-K could be used to attenuate other virulent plant viruses.

Application of a recombinant replicase to localize the Trionyx sinensis hemorrhagic syndrome virus and evaluate its effects on antiviral genes of T.sinensis

Trionyx sinensis Hemorrhagic Syndrome Virus(TSHSV)is an arterivirus newly discovered in Chinese softshell turtles.Little is known about the effect of antibodies against the virus or the distribution of the virus in different organs of infected turtles.In this study,a partial protein of TSHSV-HP4 was produced using a prokaryotic expression system,and its polyclonal antibody was generated.The polyclonal antibody was confirmed by western blot and dot enzyme-linked immunosorbent assay(dot-ELISA).The distribution of TSHSV in different organs of T.sinensis was examined by immunohistochemistry(IHC)and the expression of immune-related genes was analyzed using quantitative real-time polymerase chain reaction(qRT-PCR).The results indicated that the recombinant TSHSV-HP4 protein was successfully expressed,and the generated polyclonal antibody showed specific binding to viral particles in the lung tissues of infected turtles.The IHC assay indicated that the virus was highly localized in various cells,including intestinal lymphocytes,enterocytes,kidney epithelial cells,spleen cells,lung macrophages,and cardiomyocytes.The qRT-PCR analysis revealed that TSHSV was detected in all organs tested,including the lungs,liver,kidneys,spleen,and heart.The numbers of viral mRNA copies in lung and heart tissues were significantly higher in the virus-antibody group than in the virus group.The interferonstimulated genes(ISGs),myxovirus resistance protein 2(MX2)and radical S-adenosyl methionine domain containing 2(RSAD2)were highly upregulated in all groups of infected turtles.Antibody-dependent enhancement(ADE)seemed to occur after stimulation by the polyclonal antibody,because significantly greater expression of the two genes was detected in the virus-antibody group than in the virus group.Overall,these results are important in understanding the cell localization of TSHSV and the immune response of infected turtles.

A novel method for geometric quality assurance of rock joint replicas in direct shear testing-Part 1:Derivation of quality assurance parameters and geometric reproducibility

Since each rock joint is unique by nature,the utilization of replicas in direct shear testing is required to carry out experimental parameter studies.However,information about the ability of the replicas to simulate the shear mechanical behavior of the rock joint and their dispersion in direct shear testing is lacking.With the aim to facilitate generation of high-quality direct shear test data from replicas,a novel component in the testing procedure is introduced by presenting two parameters for geometric quality assurance.The parameters are derived from surface comparisons of three-dimensional(3D)scanning data of the rock joint and its replicas.The first parameter,smf,captures morphological deviations between the replica and the rock joint surfaces.smf is derived as the standard deviation of the deviations between the coordinate points of the replica and the rock joint.Four sources of errors introduced in the replica manufacturing process employed in this study could be identified.These errors could be minimized,yielding replicas with smf
0.06 mm.The second parameter is a vector,VHp100,which describes deviations with respect to the shear direction.It is the projection of the 100 mm long normal vector of the best-fit plane of the replica joint surface to the corresponding plane of the rock joint.

VHp100

was found to be less than or equal to 0.36 mm in this study.Application of these two geometric quality assurance parameters demonstrates that it is possible to manufacture replicas with high geometric similarity to the rock joint.In a subsequent paper(part 2),smf and VHp100 are incorporated in a novel quality assurance method,in which the parameters shall be evaluated prior to direct shear testing.Replicas having parameter values below established thresholds shall have a known and narrow dispersion and imitate the shear mechanical behavior of the rock joint.

A novel method for geometric quality assurance of rock joint replicas in direct shear testing-Part 2:Validation and mechanical replicability

Each rock joint is unique by nature which means that utilization of replicas in direct shear tests is required in experimental parameter studies.However,a method to acquire knowledge about the ability of the replicas to imitate the shear mechanical behavior of the rock joint and their dispersion in direct shear testing is lacking.In this study,a novel method is presented for geometric quality assurance of replicas.The aim is to facilitate generation of high-quality direct shear testing data as a prerequisite for reliable subsequent analyses of the results.In Part 1 of this study,two quality assurance parameters,smf and V_(Hp100),are derived and their usefulness for evaluation of geometric deviations,i.e.geometric reproducibility,is shown.In Part 2,the parameters are validated by showing a correlation between the parameters and the shear mechanical behavior,which qualifies the parameters for usage in the quality assurance method.Unique results from direct shear tests presenting comparisons between replicas and the rock joint show that replicas fulfilling proposed threshold values of σ_(mf)<0.06 mm and
|V_(Hp100)|
<0.2 mm have a narrow dispersion and imitate the shear mechanical behavior of the rock joint in all aspects apart from having a slightly lower peak shear strength.The wear in these replicas,which have similar morphology as the rock joint,is in the same areas as in the rock joint.The wear is slightly larger in the rock joint and therefore the discrepancy in peak shear strength derives from differences in material properties,possibly from differences in toughness.It is shown by application of the suggested method that the quality assured replicas manufactured following the process employed in this study phenomenologically capture the shear strength characteristics,which makes them useful in parameter studies.

RNAi-Mediated Resistance to Cucumber Mosaic Virus (CMV) in Genetically Engineered Tomato

Cucumber mosaic virus is one of the most constraints to the production of tomato and other vegetable crops worldwide. Here, we generated an RNAi construct containing inverted repeat of 1138 bp fragment of a partial replicase gene of CMV-O and used it to produce transgenic tomato plants expressing CMV-specific dsRNA of the replicase gene. Inoculation of transgenic plants with CMV strain O discriminated three categories of plants: plants that showed complete resistance, which were free of symptoms;highly resistant plants, which had mild symptoms, but later recovered because new leaves that emerged were free of symptoms;and susceptible plants, which showed severe symptoms similar to wild-type plants. The completely resistant lines were selected and challenged with a closely related strain, CMV-Y. Interestingly, the transgenic plant lines either remained immune or showed high levels of resistance to the strain. No virus could be detected in uninoculated new leaves of the resistant lines after RT-PCR and Dot immunobinding assay (DIBA) analyses. We could show that the resistance is correlated with post-transcriptional gene silencing because of the production of transgenic specific siRNA.

Novel algorithm for distributed replicas management based on dynamic programming

Replicas can improve the data reliability in distributed system. However, the traditional algorithms for replica management are based on the assumption that all replicas have the uniform reliability, which is inaccurate in some actual systems. To address such problem, a novel algorithm is proposed based on dynamic programming to manage the number and distribution of replicas in different nodes. By using Markov model, replicas management is organized as a multi-phase process, and the recursion equations are provided. In this algorithm, the heterogeneity of nodes, the expense for maintaining replicas and the engaged space have been considered. Under these restricted conditions, this algorithm realizes high data reliability in a distributed system. The results of case analysis prove the feasibility of the algorithm.

Computer Science Tools for Manual Editing of Computed Tomographic Images: Impact on the Quality of 3D Printed Models

Background: Three-dimensional printing (3DP) technologies are a trendsetting topic, also in the field of surgery. Preoperative planning for maxillofacial and neurological surgery, for instance, increasingly involves skull models obtained by 3DP. However, the cranial replicas currently used in this context have been shown to not reproduce the exact anatomy of the individual patient undergoing surgery. Objective: The present study aimed at investigating the extent to which manual editing, using current computer software tools, can improve skull models derived from medical images. Methods: Skull computed tomography (CT) was obtained on three cadavers and?sent to three institutions that provide preoperative 3DP services. Each institute independently?performed 3D reconstructions, including routine manual editing, and subsequently produced the replicas. The models were then qualitatively compared with the respective original skull. For?quantitative comparison surface scans of particular regions of interest were made and the deviations assessed using 3-matic software (Materialise, Leuven, Belgium). Results: Routine manual editing of CT images resulted in replicas that were clear improvements over automatically generated reconstructions. This was particularly the case for teeth artefacts and thin-walled entities (e.g. paranasal sinuses). Conversely, however, many anatomical structures remained incorrectly rendered (e.g. orbitae, pterygoid processes, and sella turcica). Extraosseous calcifications had regularly not been removed. After extensive manual editing, however, replicas were able to provide largely submillimeter accuracy (mean deviation 0.2496 mm;standard deviation ±0.2276 mm). Conclusions: This study confirms that manual editing with current computer science tools does improve the quality of CT-based 3D printed skull models. But, it also demonstrates that a number of structures remain largely incorrectly rendered when edited in the presently used surgical framework. Conversely, it shows that highly accurate replicas are feasible, provided that extensive manual editing is performed.

A Stable and Consistent Document Model Suitable for Asynchronous Cooperative Edition

Complex structured documents can be intentionally represented as a tree structure decorated with attributes. Ignoring attributes (these are related to semantic aspects that can be treated separately from purely structural aspects which interest us here), in the context of a cooperative edition, legal structures are characterized by a document model (an abstract grammar) and each intentional representation can be manipulated independently and eventually asynchronously by several co-authors through various editing tools that operate on its “partial replicas”. For unsynchronized edition of a partial replica, considered co-author must have a syntactic document local model that constraints him to ensure minimum consistency of local representation that handles with respect to the global model. This consistency is synonymous with the existence of one or more (global) intentional representations towards the global model, assuming the current local representation as her/their partial replica. The purpose of this paper is to present the grammatical structures which are grammars that permit not only to specify a (global) model for documents published in a cooperative manner, but also to derive automatically via a so call projection operation, consistent (local) models for each co-authors involved in the cooperative edition. We also show some properties that meet these grammatical structures.

Tree Automata for Extracting Consensus from Partial Replicas of a Structured Document

In an asynchronous cooperative editing workflow of a structured document, each of the co-authors receives in the different phases of the editing process, a copy of the document to insert its contribution. For confidentiality reasons, this copy may be only a partial replica containing only parts of the (global) document which are of demonstrated interest for the considered co-author. Note that some parts may be a demonstrated interest over a co-author;they will therefore be accessible concurrently. When it’s synchronization time (e.g. at the end of an asynchronous editing phase of the process), we want to merge all contributions of all authors in a single document. Due to the asynchronism of edition and to the potential existence of the document parts offering concurrent access, conflicts may arise and make partial replicas unmergeable in their entirety: they are inconsistent, meaning that they contain conflictual parts. The purpose of this paper is to propose a merging approach said by consensus of such partial replicas using tree automata. Specifically, from the partial replicas updates, we build a tree automaton that accepts exactly the consensus documents. These documents are the maximum prefixes containing no conflict of partial replicas merged.

Observation of electron–phonon coupling and linear dichroism in PL spectra of ultra-small CsPbBr_(3) nanoparticle solution

Blue-emission(~480 nm)CsPbBr_(3) nanoparticles with ultra-small size(~2.1 nm)are synthesized using the liquid nitrogen freezing with the ligand of dodecylbenzene sulfonic acid(DBSA).Asymmetric narrow emissions at the low energy side,with the full width at half-maximum of~20 nm,are observed in solution and film at room temperature.The spectral asymmetry is mainly ascribed to phonon vibronic replica with averaged phonon energy of~40 meV.Moreover,exciting this CsPbBr_(3) nanoparticles solution using linearly polarized 6 ns pulsed laser at 355 nm,we observe polarized emission with polarization degree(P_(PL))of~7%,and P_(PL) decreases more than 20%in the vibronic progression.However,the P_(PL) goes to zero in frozen solutions as well as in films.Thus we speculate the polarized emission is due to the photoinduced re-alignment of nanoparticles,and the diminished P_(PL) at the phonon side band may be due to the non-adiabatic electronic-to-vibronic transitions.The novel phenomena from the ultra-small CsPbBr_(3) nanoparticle demonstrated in this work may provide fundamental insights into its photophysics with direct implications for optoelectronics.

The pathogenicity on legumes of Cucumber mosaic virus was determined by 243 nucleotides on 2a polymerase gene of viral RNA2

We had isolated and identified two Cucumber mosaic virus (CMV) isolates, the CMV red bean (CMV-RB) isolate and the CMV pea (CMV-P1) isolate. CMV-RB induces necrotic local lesions on inoculated leaves of broad bean, pea, cowpea and bean, and could not infect these hosts systemically. However, CMV-P1 was able to infect these legumes systemically. To study the difference of pathogenic-ity on the legumes induced by these two CMV isolates, the full-length infectious cDNA clones of CMV-Fny, which induced similar symptoms as CMV-RB in the four legumes, were used. The 243 nucleotides fragment, which encodes highly conserved GDD amino acid motif on 2a replicase gene of CMV-Fny RNA2, was replaced with that of CMV-P1. The constructed chimeric virus FP could infect these legumes systemically. The exchange of this region changes the virus symptoms on the legumes, indicating that this 243 nucleotides fragment has major effect on pathogenicity of CMV on the legumes.

Carbide precipitation and element distribution in high Co-Ni secondary hardening steel

As the increasing need of the steels with both high strength and hydrogen embrittlement resistance ability, carbide precipitation and element distribution in high Co-Ni secondary hardening steel were concerned. Carbide precipitation and element distribution in M54 were observed using carbon replicas method. Both simulation and observation results showed that MC and M2C formed in the steel. MC was round particle, which would act as grain refiners. And MzC was needle-like phase, which would be remarkable strengthening phases. Nb and V were main metallic elements in MC phase. Mo and Cr were main metallic elements in MzC phase. W, Co, and Ni were probably mainly dissolved in the matrix. As the carbide precipitation in AerMetl00 was M2C, which had similar size and shape with M2C in M54, the tensile strength and yield strength of AerMetl00 and M54 were similar. Compared with traditional high Co-Ni secondary hardening steel, M54 had higher hydrogen embrittlement resistance ability, probably because of element W in the matrix.

Strengthening mechanism of Fe-Cu alloy during aging

To give a further research on strengthening mechanism of binary Fe-Cu alloys dur- ing the aging process, the compositions, size distributions and volume fractions of precipitates were investigated by carbon extraction replicas. Both the size and the volume fraction of precipitates increase with the aging time prolonged. The precip- itate sizes are corresponding with Weibull distribution. The stability of the sample aged for 35 rain is worse than that aged for 1800 min and 18000 min. The precipi- tate is Cu-rich phase in the early aging process, and Cu content increases with the prolonging aging time. Before aging peak, the precipitation of Cu-rich phase plays a dominant role in strengthening. The growth and the change in crystal structure of the precipitate particles are the main factors leading to the decrease in strengthening after the aging peak.