Ophioviruses CPsV and MiLBVV movement protein is encoded in RNA 2 and interacts with the coat protein

Citrus psorosis virus (CPsV) and Mirafiori lettuce big-vein virus (MiLBVV), members of the Ophioviridae family, have segmented negative-sense single-stranded RNA genomes. To date no reports have described how ophioviruses spread within host plants and/or the proteins involved in this process. Here we show that the 54K protein of CPsV is encoded by RNA 2 and describe its subcellular distribution. Upon transient expression in Nicotiana benthamiana epidermal cells the 54K protein, and also its 54K counterpart protein of MiLBVV, localize to plasmodesmata and enhance GFP cell-to-cell diffusion between cells. Both proteins, but not the coat proteins (CP) of the respective viruses, functionally trans-complement cell-to-cell movement-defective Potato virus X (PVX) and Tobacco mosaic virus (TMV) mutants. The 54K and 54K proteins interact with the virus-specific CP in the cytoplasm, suggesting a potential role of CP in ophiovirus movement. This is the first study characterizing the movement proteins (MP) of ophioviruses.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

Enhanced anticancer activity of a combination of docetaxel and Aneustat (OMN54) in a patient-derived, advanced prostate cancer tissue xenograft model.

The current first-line treatment for advanced metastatic prostate cancer, i.e. docetaxel-based therapy, is only marginally effective. The aim of the present study was to determine whether such therapy can be improved by combining docetaxel with Aneustat (OMN54), a multivalent botanical drug candidate shown to have anti-prostate cancer activity in preliminary in vitro experiments, which is currently undergoing a Phase-I Clinical Trial. Human metastatic, androgen-independent C4-2 prostate cancer cells and NOD-SCID mice bearing PTEN-deficient, metastatic and PSA-secreting, patient-derived subrenal capsule LTL-313H prostate cancer tissue xenografts were treated with docetaxel and Aneustat, alone and in combination. In vitro, Aneustat markedly inhibited C4-2 cell replication in a dose-dependent manner. When Aneustat was combined with docetaxel, the growth inhibitions of the drugs were essentially additive. In vivo, however, the combination of docetaxel and Aneustat enhanced anti-tumor activity synergistically and very markedly, without inducing major host toxicity. Complete growth inhibition and shrinkage of the xenografts could be obtained with the combined drugs as distinct from the drugs on their own. Analysis of the gene expression of the xenografts using microarray indicated that docetaxel + Aneustat led to expanded anticancer activity, in particular to targeting of cancer hallmarks that were not affected by the single drugs. Our findings, obtained with a highly clinically relevant prostate cancer model, suggest, for the first time, that docetaxel-based therapy of advanced human prostate cancer may be improved by combining docetaxel with Aneustat

Federal Forests and the Renewable Fuel Standard

On December 19, 2007, the President signed into law the Energy Independence and Security Act of 2007 (EISA). This law (PL 110-140) includes an increase in the national Renewable Fuel Standard (RFS) mandating the production of 36 billion gallons of renewable fuels by 2022. Within the total mandate, 21 billion gallons must qualify as advanced biofuels -- fuels made from renewable biomass other than corn starch. There are additional carve-outs for biomass-based diesel and fuels made from cellulosic feedstocks, such as wood, grasses, and agricultural residues. An important component of the RFS is a series of greenhouse gas emissions screens, essential safeguards that ensure renewable fuels will meet minimum verifiable reductions in greenhouse emissions. For renewable fuels (from new facilities) to qualify under the RFS, they must achieve at least a 20 percent reduction in direct and indirect lifecycle emissions compared to equivalent petroleum fuels. Advanced fuels and cellulosic fuels are subject to a 50 percent and 60 percent emissions screen, respectively. Because of these stringent safeguards and the large quantity of fuel mandated, it is paramount that we not rule out potentially important feedstocks without valid reasons. The definition of 'renewable biomass' included in the law, however, does rule out a number of feedstocks, including thinning materials and woody residues from federal forests

Master of Science

thesisIntrauterine growth restriction (IUGR) refers to the failure of a fetus to reach its genetic growth potential in utero. Each year in the United States, 5-12% of premature babies are born IUGR, which increases their risk for postnatal morbidities. One such postnatal morbidity is brochopulmonary dysplasia (BPD), in which males are more severely affected than females. Histologically, BPD is characterized by impaired alveolar development. One pathway contributing to alveolar formation is estrogen signaling. The predominate estrogen in the lung, estradiol, binds to estrogen receptors (ERs) in the cytosol, dimerizes, and translocates to the nucleus. In the nucleus, ERs bind to estrogen response elements on target genes and affect transcription, resulting in appropriate gene expression and lung development. Androgen signaling works in a similar way through the ligand testosterone and an androgen receptor (AR). Testosterone within the lung can be used to produce estradiol locally by the converting enzyme aromatase. Normal alveolar formation requires an appropriate estrogen to testosterone ratio. We hypothesize that IUGR alters lung sex steroids, estradiol to testosterone ratios, and ER/AR expression in a sex-specific manner in newborn rat lung. Uteroplacental insufficiency was induced in pregnant rats by uterine artery ligation on day 19 of gestation, producing IUGR pups. We examined 4 groups for this study: IUGR female, IUGR male, control female, and control male. Each group consisted of n=6 rat pups derived from different litters. ELISA was used to measure protein concentrations of serum and lung estradiol and testosterone, while Western blotting was used to determine ER?, ER?, and AR protein abundance relative to GAPDH. The serum and lung estrogen to testosterone ratios as well as ER? protein abundance is depressed in male but not female newborn IUGR rat pups when compared to sex-matched controls. ER? and AR expression are increased in IUGR males when compared to sex-matched controls. We conclude that IUGR alters lung sex steroids, estradiol to testosterone ratios, and ER/AR expression in a sex-specific manner in newborn rat lung. We speculate that depressed estrogen signaling in male IUGR lung may contribute to worse outcomes

Advanced Post-Processing and Correlation Analyses in High-Velocity Air-Water Flows. 2- Microscopic Properties

The on-going interest in air-water flows is accompanied sometimes by citations of outdated articles and some ignorance of key contributions. A basic issue is the inadequate, incomplete interpretation of air-water flow instrumentation by hydraulic engineers and researchers. This article focus on the bubbly flow structure of high-velocity air-water flow based upon measurements by means of intrusive phase detection probes. It is shown that some advanced post-processing techniques may yield expanded information on the air-water structures and particle clustering

Molecular studies on a complex of potyviruses infecting solanaceous crops, and some specific virus-host interactions

This thesis constitutes a comprehensive analysis of the molecular and biological characteristics of three potyviruses (genus Potyvirus, family Potyviridae) naturally occurring in cultivated and wild species of family Solanaceae: Peru tomato virus (PTV), Potato virus V (PVV) and Wild potato mosaic virus (WPMV). In addition, the studies presented in this thesis focus on the genetic variability of isolates of PTV and PVV and on the role of the Potato virus A (PVA) 6K2 protein as a host-specific determinant of virus movement and symptom induction. Determination of the complete genomic sequences of PVV, PTV and WPMV demonstrated that these viruses are typical members of the genus Potyvirus. Furthermore, comparison of the polyprotein amino acid sequences and the biological and serological characteristics of these three viruses supported their current taxonomic position as independent species of the genus Potyvirus. The nucleotide sequences of the P1 protein, coat protein and non-translated regions of European and South American PVV isolates were determined and compared. Results showed limited genetic variability among the European isolates, in contrast to the higher variability found among the South American isolates of PVV. Phylogenetic analysis defined two distinct clusters, grouping the European isolates together but placing two South American isolates to a different group; these two isolates of PVV did not induce a hypersensitive response in an Nv gene-carrying potato cultivar in contrast to the European PVV isolates. Thus, European and South American PVV isolates belong to different strain groups. In addition, great genetic variability was detected among PTV isolates. Analysis of phylogenetic relationships among PTV, PVV, WPMV and other members of the genus Potyvirus commonly found infecting solanaceous crop plants showed that PTV, PVV and WPMV are the most closely related viruses which together with Potato virus Y, Pepper mottle virus, Pepper severe mosaic virus and Pepper yellow mosaic virus constitute a group distinguishable from other potyviruses. Thus, members of this group seem to share a common ancestor. The 6K2 protein of PVA was modified by deleting various portions or by introducing six histidine residues (6xHis) into various positions of this protein. These modifications disturbed functions required for viral infection in Nicotiana tabacum. Furthermore, inoculation of the insertion constructs to N. benthamiana plants did not result in systemic infection with the exception of one plant. This plant lacked typical PVA symptoms but had virus titers similar to the plants infected with the wild type virus: a single point mutation (Gly2 ® Cys2) in the 6xHis-containing 6K2 had restored the viral movement functions. However, partial deletion of the 6xHis-tag to gain the original size of the 6K2 protein was required to restore the induction of symptoms in N. benthamiana and to enable systemic infection of N. tabacum. Taken together, these results indicate the 6K2 is a host-specific determinant for long-distance movement and exemplify that mutations that arise during viral propagation represent a mechanisms by which viruses can evolve and adapt to different hosts

Association of the P6 protein of cauliflower mosaic virus with plasmodesmata and plasmodesmal proteins

"May 2014."Dissertation Supervisor: Dr. James E. Schoelz.Includes vita.The P6 protein of Cauliflower mosaic virus (CaMV) is responsible for the formation of inclusion bodies (IBs), which are the site for viral gene expression, replication and virion assembly. Moreover, recent evidence indicates that ectopically expressed P6 IBs move in association with actin microfilaments. Since CaMV virions accumulate preferentially in P6 IBs, we hypothesized that P6 IBs have a role in delivering CaMV virions to the plasmodesmata. We recently discovered that the P6 protein interacted with a C2 calcium-dependent membrane targeting protein (designated AtSRC2-2) in a yeast two-hybrid screen and confirmed this interaction through co-immunoprecipitation and co-localization assays in the CaMV host, Nicotiana benthamiana. An AtSRC2-2 protein fused to RFP was localized to the plasma membrane and specifically associated with plasmodesmata. The AtSRC2-2-RFP fusion also co-localized with two proteins previously shown to associate with plasmodesmata: the host protein PDLP1 and the CaMV movement protein (MP). Since P6 IBs were found to co-localize with AtSCR2-2 and had previously been shown to interact with CaMV MP, we investigated whether a portion of the P6 IBs might also be associated with plasmodesmata. We examined the co-localization of P6-GFP IBs with PDLP1, the CaMV MP, and with aniline blue, a chemical stain for callose, and found that P6-GFP IBs were associated with each of these markers. Furthermore, a P6-RFP protein was co-immunoprecipitated with PDLP1-GFP. Our evidence that a portion of P6-GFP IBs associate with AtSRC2-2, PDLP1, and CaMV MP at plasmodesmata supports a model in which P6 IBs function to transfer CaMV virions directly to plasmodesmata.Includes bibliographical references