The emergence of combinatorial strategies in the development of RNA oncolytic virus therapies

Oncolytic viruses (OVs) represent an exciting new biological approach to cancer therapy. In particular, RNA viruses have emerged as potent agents for oncolytic virotherapy because of their capacity to specifically target and destroy tumour cells while sparing normal cells and tissues. Several barriers remain in the development of OV therapy, including poor penetration into the tumour mass, inefficient virus replication in primary cancers, and tumour-specific resistance to OV-mediated killing. The combination of OVs with cytotoxic agents, such as small molecule inhibitors of signalling or immunomodulators, as well as stealth delivery of therapeutic viruses have shown promise as novel experimental strategies to overcome resistance to viral oncolysis. These agents complement OV therapy by unblocking host pathways, delivering viruses with greater efficiency and/or increasing virus proliferation at the tumour site. In this review, we summarize recent development of these concepts, the potential obstacles, and future prospects for the clinical utilization of RNA OVs in cancer therapy.     

Characterization of the role of the receptors PEX5 and PEX7 in the import of proteins into glycosomes of Trypanosoma brucei

Peroxins 5 and 7 are receptors for protein import into the peroxisomal matrix. We studied the involvement of these peroxins in the biogenesis of glycosomes in the protozoan parasite Trypanosoma brucei. Glycosomes are peroxisome-like organelles in which a major part of the glycolytic pathway is sequestered. We here report the characterization of the T. brucei homologue of PEX7 and provide several data strongly suggesting that it can bind to PEX5. Depletion of PEX5 or PEX7 by RNA interference had a severe effect on the growth of both the bloodstream-form of the parasite, that relies entirely on glycolysis for its ATP supply, and the procyclic form representative of the parasite living in the tsetse-fly midgut and in which also other metabolic pathways play a prominent role. The role of the two receptors in import of glycosomal matrix proteins with different types of peroxisome/glycosome-targeting signals (PTS) was analyzed by immunofluorescence and subcellular fractionation studies. Knocking down the expression of either receptor gene resulted, in procyclic cells, in the mislocalization of proteins with both a type 1 or 2 targeting motif (PTS1, PTS2) located at the C- and N-termini, respectively, and proteins with a sequence-internal signal (I-PTS) to the cytosol. Electron microscopy confirmed the apparent integrity of glycosomes in these procyclic cells. In bloodstream-form trypanosomes, PEX7 depletion seemed to affect only the subcellular distribution of PTS2-proteins. Western blot analysis suggested that, in both life-cycle stages of the trypanosome, the levels of both receptors are controlled in a coordinated fashion, by a mechanism that remains to be determined. The observation that both PEX5 and PEX7 are essential for the viability of the parasite indicates that the respective branches of the glycosome-import pathway in which each receptor acts might be interesting drug targets.     

Conformational stabilization at the active site of molluskan (Rapana thomasiana) hemocyanin by a cysteine-histidine thioether bridge A study by mass spectrometry and molecular modeling

In some type-3 copper proteins (molluskan hemocyanin, catechol oxidase and fungal tyrosinase) one of the histidine residues, liganding the Cu(A) atom of the dinuclear copper active site, is covalently linked to a cysteine residue by a thioether bridge. The purpose of this study was to disclose the function of this bridge. Mass spectral analysis of a peptide, isolated from Rapana thomasiana (gastropodan mollusk) hemocyanin, indicated a stabilization of the peptide structure in the region of the bridge. Molecular modeling of three thioether containing type-3 copper proteins using the dead-end elimination method showed that the concerned histidine would be very flexible if not linked to the cysteine. Also, the side chain orientation of the histidine is rather exceptional, as evidenced by statistical data from the protein databank. It is suggested that the role of the bridge is to fix the histidine in an orientation that is optimal for coordination of the Cu(A) atom.     

Local and relational judgements of surface colour: constancy indices and discrimination performance

Colour constancy is generally assumed to arise from a combination of perceptual constancy mechanisms operating to partially discount illumination changes and relational mechanisms involved in judging the colour relationships between object surfaces. Here we examined the characteristics of these mechanisms using a 'yes/no' task. Subjects judged whether a target colour patch embedded in an array of coloured patches (a) stayed the same across a simulated temporal illuminant change (local colour judgement), or (b) changed in a manner consistent with the illuminant change (relational colour judgement). The colour of the target patch remained constant in one-third of the trials, changed in accord with the illuminant shift in another third, and shifted partially with the illuminant change in the remaining third. We found that perceptual constancy was relatively weak and relational constancy strong, as assessed using a modified colour constancy index. Randomising the spatial positions of coloured patches across the illuminant change did not affect subjects' constancy indices. Application of signal detection analysis revealed some otherwise hidden effects. In the case of relational judgements, subjects adopted more conservative criteria (fewer true and false positives) with randomisation, maintaining a constant level of discrimination performance (d'). For local judgements, randomisation led to small increases in performance but no changes in criteria. We conclude that signal detection theory provides a useful tool to supplement conventional approaches to understanding colour constancy.     

Codling moth cytogenetics: karyotype, chromosomal location of rDNA, and molecular differentiation of sex chromosomes.

We performed a detailed karyotype analysis in the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), the key pest of pome fruit in the temperate regions of the world. The codling moth karyotype consisted of 2n = 56 chromosomes of a holokinetic type. The chromosomes were classified into 5 groups according to their sizes: extra large (3 pairs), large (3 pairs), medium (15 pairs), small (5 pairs), and dot-like (2 pairs). In pachytene nuclei of both sexes, a curious NOR (nucleolar organizer region) bivalent was observed. It carried 2 nucleoli, each associated with one end of the bivalent. FISH with an 18S ribosomal DNA probe confirmed the presence of 2 clusters of rRNA genes at the opposite ends of the bivalent. In accordance with this finding, 2 homologous NOR chromosomes were identified in mitotic metaphase, each showing hybridization signals at both ends. In highly polyploid somatic nuclei, females showed a large heterochromatin body, the so-called sex chromatin or W chromatin. The heterochromatin body was absent in male nuclei, indicating a WZ/ZZ (female/male) sex chromosome system. In keeping with the sex chromatin status, pachytene oocytes showed a sex chromosome bivalent (WZ) that was easily discernible by its heterochromatic W thread. To study molecular differentiation of the sex chromosomes, we employed genomic in situ hybridization (GISH) and comparative genomic hybridization (CGH). GISH detected the W chromosome by strong binding of the Cy3-labelled, female-derived DNA probe. With CGH, both the Cy3-labelled female-derived probe and Fluor-X labelled male-derived probe evenly bound to the W chromosome. This suggested that the W chromosome is predominantly composed of repetitive DNA sequences occurring scattered in other chromosomes but accumulated in the W chromosome. The demonstrated ways of W chromosome identification will facilitate the development of genetic sexing strains desirable for pest control using the sterile insect technique.     

The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.     

Disturbance and climate effects on carbon stocks and fluxes across Western Oregon USA

We used a spatially nested hierarchy of field and remote‐sensing observations and a process model, Biome‐BGC, to produce a carbon budget for the forested region of Oregon, and to determine the relative influence of differences in climate and disturbance among the ecoregions on carbon stocks and fluxes. The simulations suggest that annual net uptake (net ecosystem production (NEP)) for the whole forested region (8.2 million hectares) was 13.8 Tg C (168 g C m^?2 yr^?1), with the highest mean uptake in the Coast Range ecoregion (226 g C m^?2 yr^?1), and the lowest mean NEP in the East Cascades (EC) ecoregion (88 g C m^?2 yr^?1). Carbon stocks totaled 2765 Tg C (33 700 g C m^?2), with wide variability among ecoregions in the mean stock and in the partitioning above‐ and belowground. The flux of carbon from the land to the atmosphere that is driven by wildfire was relatively low during the late 1990s (～0.1 Tg C yr^?1), however, wildfires in 2002 generated a much larger C source (～4.1 Tg C). Annual harvest removals from the study area over the period 1995–2000 were ～5.5 Tg C yr^?1. The removals were disproportionately from the Coast Range, which is heavily managed for timber production (approximately 50% of all of Oregon's forest land has been managed for timber in the past 5 years). The estimate for the annual increase in C stored in long‐lived forest products and land fills was 1.4 Tg C yr^?1. Net biome production (NBP) on the land, the net effect of NEP, harvest removals, and wildfire emissions indicates that the study area was a sink (8.2 Tg C yr^?1). NBP of the study area, which is the more heavily forested half of the state, compensated for ～52% of Oregon's fossil carbon dioxide emissions of 15.6 Tg C yr^?1 in 2000. The Biscuit Fire in 2002 reduced NBP dramatically, exacerbating net emissions that year. The regional total reflects the strong east–west gradient in potential productivity associated with the climatic gradient, and a disturbance regime that has been dominated in recent decades by commercial forestry.     

Effects of an experimental drought on the functioning of a cacao agroforestry system,Sulawesi, Indonesia

Agroforestry systems may play a critical role in reducing the vulnerability of farmers' livelihood to droughts as tree‐based systems provide several mechanisms that can mitigate the impacts from extreme weather events. Here, we use a replicated throughfall reduction experiment to study the drought response of a cacao/Gliricidia stand over a 13‐month period. Soil water content was successfully reduced down to a soil depth of at least 2.5 m. Contrary to our expectations we measured only relatively small nonsignificant changes in cacao (?11%) and Gliricidia (?12%) sap flux densities, cacao leaf litterfall (+8%), Gliricidia leaf litterfall (?2%), soil carbon dioxide efflux (?14%), and cacao yield (?10%) during roof closure. However, cacao bean yield in roof plots was substantially lower (?45%) compared with control plots during the main harvest following the period when soil water content was lowest. This indicates that cacao bean yield was more sensitive to drought than other ecosystem functions. We found evidence in this agroforest that there is complementary use of soil water resources through vertical partitioning of water uptake between cacao and Gliricidia. This, in combination with acclimation may have helped cacao trees to cope with the induced drought. Cacao agroforests may thus play an important role as a drought‐tolerant land use in those (sub‐) tropical regions where the frequency and severity of droughts is projected to increase.