A versatile transreplication-based system to identify cellular proteins involved in geminivirus replication

A versatile green fluorescent protein (GFP) expression cassette containing the replication origins of the monopartite begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV) is described. Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon. The expression of the reporter gene (the GFP gene) was thereby modified. Whereas GFP fluorescence was dimmed in the intercostal areas, an increase of green fluorescence in veins of all leaves placed above the inoculation site, as well as in transport tissues of roots and stems, was observed. The release of episomal trans replicons from the transgene and the increase in GFP expression were dependent on the cognate geminiviral replication-associated protein (Rep) and required interaction between Rep and the intergenic region of TYLCSV. This expression system is able to monitor the replication status of TYLCSV in plants, as induction of GFP expression is only produced in those tissues where Rep is present. To further confirm this notion, the expression of a host factor required for geminivirus replication, the proliferating cellular nuclear antigen (PCNA) was transiently silenced. Inhibition of PCNA prevented GFP induction in veins and reduced viral DNA. We propose that these plants could be widely used to easily identify host factors required for geminivirus replication by virus-induced gene silencing.     

Development of subcutaneous fat in Spanish and Latin American children and adolescents: Reference values for biceps,triceps, subscapular and suprailiac skinfolds

Subcutaneous fat skinfolds represent a reliable assessment instrument of adiposity status. This study provides current percentile references for four subcutaneous skinfolds (biceps, triceps, subscapular, suprailiac) applicable to children and adolescents in Spain and in Latin American countries where data are scarce.The design consisted of a cross-sectional multicenter study performed with identical methods in 5 countries (Argentina, Cuba, Mexico, Spain and Venezuela). Total sample comprised 9163 children and youths (boys 4615 - girls 4548) aged 6–18 years, healthy and without apparent pathologies. Percentiles 3, 5, 10, 25, 50, 75, 90, 95 and 97 were calculated by the LMS method. Sexual dimorphism was assessed using the t-test and age differences with ANOVA. Normalized growth percentile references were obtained according to sex and age for each skinfold. The mean values of four skinfolds were significantly greater in girls than boys (p < 0.001) and, in both sexes, all skinfolds show statistical differences through age (p < 0.001) with different magnitudes.Except triceps in girls, peaks between 11 and 12 years of age are more noticeable in boys than in girls. Although the general model of growth is known, the skinfold measurements show variability among populations and differences of magnitude are presented according to the analyzed population. Therefore, these age and sex-specific reference percentile values for biceps, triceps, subscapular and suprailiac skinfolds, derived from a large sample of Spanish and Latin American children and adolescents, are a useful tool for adiposity diagnosis in this population for which no reference values were available.     

Photoinduced accumulation of carotene in Phycomyces

Blue light stimulates the accumulation of beta-carotene (photocarotenogenesis) in the fungus Phycomyces blakesleeanus. To be effective, light must be given during a defined period of development, which immediately precedes the cessation of mycelial growth and the depletion of the glucose supply. The competence periods for photocarotenogenesis and photomorphogenesis in Phycomyces are the same when they are tested in the same mycelium. Photocarotenogenesis exhibits a two-step dependence on exposure, as if it resulted from the additon of two separate components with different thresholds and amplitudes. The low-exposure component produces a small beta-carotene accumulation, in comparison with that of dark-grown mycelia. The high-exposure component has a threshold of about 100 J· m^–2 blue light and produces a large beta-carotene accumulation, which is not saturated at 2·10⁶ J·m^–2. Exposure-response curves were obtained at 12 wavelengths from 347 to 567 nm. The action spectra of the two components share general similarities with one another and with those of other Phycomyces photoresponses. The small, but significant differences in the action spectra of the two components imply that the respective photosystems are not identical. Light stimulates the carotene pathway in the carB mutants, which contain the colourless precursor phytoene, but not beta-carotene. Carotenogenesis is not photoinducible in carA mutants, independently of their carotene content. This and other observations on various car mutants indicate that light prevents the normal inhibition of the pathway by the carA and carS gene products. The chromophore(s) for photocarotenogenesis are presumably flavins, and not carotenes.We thank Dr. A. Palit, C. Chmielewicz and D. Durant (same address as E.D.L.) and L.M. Corrochano, A. Fernández Estefane, and J. Córdoba López (same address as E.R.B.) for their help. This work was supported by grants from Comisión Asesora para Investigación Científica y Técnica and Comisión Interministerial para Ciencia y Tecnología to E.C.O.; from the National Institutes of Health and the National Science Foundation to E.D.L.; and from the U.S.-Spain Joint Committee for Scientific and Technological Cooperation to E.C.O. and E.D.L.     

Empirical relationships among resilience indicators on Micronesian reefs

A process-orientated understanding of ecosystems usually starts with an exploratory analysis of empirical relationships among potential drivers and state variables. While relationships among herbivory, algal cover, and coral recruitment, have been explored in the Caribbean, the nature of such relationships in the Pacific appears to be variable or unclear. Here, we examine potential drivers structuring the benthos and herbivorous fish assemblages of outer-shelf reefs in Micronesia (Palau, Guam and Pohnpei). Surveys were stratified by wave exposure and protection from fishing. High biomass of most herbivores was favoured by high wave exposure. High abundance of large-bodied scarids was associated with low turf abundance, high coral cover, and marine reserves. The remaining herbivores were more abundant in reefs with low coral cover, possibly because space and hence food limitation occur in high-coral-cover reefs. Rugosity had no detectable effect on herbivorous fish abundance once differences in exposure and coral cover were accounted for. At identical depths, high wave exposure was associated with greater volumes (cover × canopy height) of macroalgae and algal turfs, which most likely resulted from high primary productivity driven by flow. In exposed areas, macroalgal cover declined as the acanthurid biomass increased. The volume of algal turfs was negatively associated with coral cover and herbivore biomass. In turn, high coral cover and herbivore biomass are likely to intensify grazing. The density of juvenile corals was variable where macroalgal cover was low but was confined to lower densities where macroalgal cover was high. High coral cover and density of juvenile corals were favoured in sheltered habitats. While a weak positive relationship was found between scarid biomass and juvenile coral density, we hypothesise that high scarid densities may hinder juvenile density through increased corallivory. New hypotheses emerged that will help clarify the role of acanthurids, wave exposure, and corallivory in driving the recovery of Pacific coral communities.     

Genetic and epigenetic mechanisms regulating hedgehog expression in the Drosophila wing

Stable subdivision of Drosophila limbs into Anterior (A) and Posterior (P) compartments is a consequence of asymmetric signaling by Hedgehog (Hh) from P to A cells. The activity of the homeodomain protein Engrailed (En) in P cells has been reported to help to generate this asymmetry by inducing the expression of hedgehog and simultaneously repressing the expression of the essential downstream component of the Hh signaling pathway Cubitus interruptus (Ci). In A cells, Ci has a major role in the repression of hh. Here we have revised the genetic and epigenetic mechanisms involved in the regulation of hh in the P compartment. First, we present evidence that hh expression in P cells is a consequence of the repression of ci by the activity of En. Thus, in the absence of Ci and En activities, cells do express hh. We also present data supporting the maintenance of hh expression in P cells through epigenetic mechanisms, and a permissive role of Notch signaling in this process. Notch and Trithorax (TrxG) group of proteins exert their action through a previously defined hh Polycomb Responsive Element (PRE).     

Magnetic resonance contrast and biological effects of intracellular superparamagnetic iron oxides on human mesenchymal stem cells with long-term culture and hypoxic exposure

Background aimsHuman mesenchymal stem cells (hMSCs) have gained interest for treatment of stroke injury. Using in vitro culture, the purpose of this study was to investigate the long-term detectability of hMSCs by magnetic resonance imaging (MRI) after transfection with a superparamagnetic iron oxide (SPIO) and evaluate the effects of SPIO on cellular activity, particularly under an ischemic environment.MethodshMSCs were exposed to low doses of SPIOs. After a short incubation period, cells were cultured for additional 1, 7 and 14 d to evaluate proliferation, colony formation and multilinear potential. Labeled cells were imaged and evaluated in agarose to quantify R₂ and R₂1 contrast at each time point. Cells were placed in a low-oxygen, low-serum environment and tested for cytotoxicity. In addition, labeled cells were transplanted into an ischemic stroke model and evaluated with ex vivo MRI and histology.ResultsCellular events such as proliferation and differentiation were not affected at any of the exposures tested when cultured for 14 d. The low iron exposure and short incubation time are sufficient for detectability with MRI. However, the higher iron dosage results in higher calcification and cytotoxicity under in vitro ischemic conditions. Transplantation of the hMSCs labeled with an initial exposure of 22.4 μg of Fe showed excellent retention of contrast in stroke-induced rats.ConclusionsAlthough SPIO labeling is stable for long-term MRI detection and has limited effects on the multilineage potential of hMSCs, high-dose SPIO labeling may affect hMSC survival under serum and oxygen withdrawal.     

Interaction between geminivirus replication protein and the SUMO-conjugating enzyme is required for viral infection

Geminiviruses are small DNA viruses that replicate in nuclei of infected plant cells by using plant DNA polymerases. These viruses encode a protein designated AL1, Rep, or AC1 that is essential for viral replication. AL1 is an oligomeric protein that binds to double-stranded DNA, catalyzes the cleavage and ligation of single-stranded DNA, and induces the accumulation of host replication machinery. It also interacts with several host proteins, including the cell cycle regulator retinoblastoma-related protein (RBR), the DNA replication protein PCNA (proliferating cellular nuclear antigen), and the sumoylation enzyme that conjugates SUMO to target proteins (SUMO-conjugating enzyme [SCE1]). The SCE1-binding motif was mapped by deletion to a region encompassing AL1 amino acids 85 to 114. Alanine mutagenesis of lysine residues in the binding region either reduced or eliminated the interaction with SCE1, but no defects were observed for other AL1 functions, such as oligomerization, DNA binding, DNA cleavage, and interaction with AL3 or RBR. The lysine mutations reduced or abolished virus infectivity in plants and viral DNA accumulation in transient-replication assays, suggesting that the AL1-SCE1 interaction is required for viral DNA replication. Ectopic AL1 expression did not result in broad changes in the sumoylation pattern of plant cells, but specific changes were detected, indicating that AL1 modifies the sumoylation state of selected host proteins. These results established the importance of AL1-SCE1 interactions during geminivirus infection of plants and suggested that AL1 alters the sumoylation of selected host factors to create an environment suitable for viral infection.