Influence of Dopaminergically Mediated Reward on Somatosensory Decision-Making

Reward-related dopaminergic influences on learning and overt behaviour are well established, but any influence on sensory decision-making is largely unknown. We used functional magnetic resonance imaging (fMRI) while participants judged electric somatosensory stimuli on one hand or other, before being rewarded for correct performance at trial end via a visual signal, at one of four anticipated financial levels. Prior to the procedure, participants received either placebo (saline), a dopamine agonist (levodopa), or an antagonist (haloperidol). Principal findings: higher anticipated reward improved tactile decisions. Visually signalled reward reactivated primary somatosensory cortex for the judged hand, more strongly for higher reward. After receiving a higher reward on one trial, somatosensory activations and decisions were enhanced on the next trial. These behavioural and neural effects were all enhanced by levodopa and attenuated by haloperidol, indicating dopaminergic dependency. Dopaminergic reward-related influences extend even to early somatosensory cortex and sensory decision-making.     

A Novel Diagnostic Target in the Hepatitis C Virus Genome

Background

Detection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5′-noncoding region (5′-NCR), and all show genotype-dependent variation of sensitivities and viral load results. Non-western HCV genotypes have been under-represented in evaluation studies. An alternative diagnostic target region within the HCV genome could facilitate a new generation of assays.

Methods and Findings

In this study we determined by de novo sequencing that the 3′-X-tail element, characterized significantly later than the rest of the genome, is highly conserved across genotypes. To prove its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test was developed and evaluated multicentrically on a large and complete panel of 725 clinical plasma samples, covering HCV genotypes 1–6, from four continents (Germany, UK, Brazil, South Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3–24.1 IU/ml), suggesting applicability in donor blood screening. The upper LOD exceeded 10⁻⁹ IU/ml, facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples, quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail NAT, for genotypes 1–6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based viral loads deviated by more than 0.5 log10 from 5′-NCR-based viral loads in only 12% of samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based protocol. The assay was implemented at low reaction costs (US$8.70 per sample), short turnover times (2.5 h for up to 96 samples), and without technical difficulties.

Conclusion

This study indicates a way to fundamentally improve HCV viral load monitoring and infection screening. Our prototype assay can serve as a template for a new generation of viral load assays. Additionally, to our knowledge this study provides the first open protocol to permit industry-grade HCV detection and quantification in resource-limited settings.     

Presence of yeasts in floral nectar is consistent with the hypothesis of microbial-mediated signaling in plant-pollinator interactions

Olfactory floral signals are significant factors in plant-pollinator mutualisms. Recently, unusual fermentation odors have been described in the nectar and flowers of some species. Since yeasts are common inhabitants of many angiosperms nectars, this raises the possibility that nectar yeasts may act as causal agents of fermentation odors in flowers and, therefore, as possible intermediate agents in plant signaling to pollinators. A recent field study has reported that nectar yeasts were quite frequent in floral nectar across three different regions in Europe and America, where they reached high densities (up to 10⁵ cells/mm³). Yeast incidence in floral nectar differed widely across plant host species in all sampling sites. A detailed study currently in progress on one of the species surveyed in that study (Helleborus foetidus, Ranunculaceae) has detected that, in addition to interespecific differences in yeast incidence, there is also a strong component of variance in yeast abundance that takes place at the subindividual level (among flowers of the same plant, among nectaries of the same flower). If yeast metabolism is eventually proved to contribute significantly to floral scent, then multilevel patchiness in the distribution of nectar yeasts (among species, among individuals within species, and among flowers and nectaries of the same individual) might contribute to concomitant multilevel variation in plant signaling and, eventually, also in pollination success, pollen flow and plant fitness.Key words: nectar, yeast, scent, plant-animal interaction, plant signalingPollinators forage on a wide range of flowers that differ in morphology, color, scent and quality and quantity of reward. The majority of these floral features are important visual and olfactory cues that are directly related to plant-pollinators signaling and the pollination process.^1–12 Recently, the intriguing possibility has been raised that microbial communities (especially nectarivorous yeasts) inhabiting flowers could explain better than, or in addition to, plant physiology itself, certain floral features that participate in plant-pollinators signaling, like yeasty nectar or floral scent.^13,14 However, some of these suggestions are based on circumstantial or indirect evidence indicative of the presence of microbes in flowers. For example, fermentation odors have been described in a number of Angiosperms,^14–16 in which different compounds found in nectar were not shared with any other floral parts.¹³ In addition, yeasty odors (ketones and shortchain alcohols) have only been observed in mature flowers that were already visited by pollinators and thus potentially contaminated with microbes, in contrast, for example, to the sesquiterpenes isolated in immature flowers that are also common in the foliage of many plants.¹⁴ Yeasty odors were found in species whose flowers are long-lived, produce large amounts of nectar, and are visited by flies and beetles, which are known to act as yeast vectors to flowers.^17–19 In spite of these plausible suggestions, studies indicating a potential role of microbes in the origin of floral scents generally have not looked directly for their presence or abundance in floral nectar, which clearly would provide critical empirical evidence in support of the hypothesis of microbial-mediated signaling in plant-pollinator interactions.That yeasts are common inhabitants of floral nectars was well known to microbiologists more than a century ago^20,21 and has been recently corroborated by Herrera et al.²² This study was conducted at three widely separated areas, which differed greatly in ecological features and biogeographical affinities: two study sites were located in the Southern Iberian Peninsula, about 350 km apart, and one in Yucatán Peninsula, eastern Mexico. Floral nectar samples from 40, 63 and 37 species, belonging to 21, 23 and 21 families, were examined microscopically for yeast cells at these three areas. Yeasts occurred very frequently in floral nectar at all areas, as revealed by the high proportion of nectar samples that contained them (31.8%, 42.3% and 54.4%; samples from all species at each site combined). In addition to being quite frequent in nectar samples, yeast cells often reached extraordinarily high densities in floral nectar at the three areas, which reached roughly 4 × 10⁵ cells/mm³. When plant species, rather than individual nectar samples, were considered as the units for analyses, Herrera et al.²² found wide variation among species in both the frequency of occurrence and the density of yeasts in nectar samples. A significant fraction of such variation was found to be correlated with differences in pollinator composition, a link between pollination ecology and floral nectar microbiology that has remained unexplored until now. Similar results showing high densities and frequency of occurrence of yeasts in nectar, and interespecific differences in these magnitudes related to variation in pollinator composition, have been also reported by de Vega et al.²³ for 40 South African plant species, which further supports the generality of the phenomenon. In addition to interespecific differences in the prevalence of nectar yeasts, the data examined by Herrera et al.²² and de Vega et al.²³ revealed also considerable intraespecific variability (i.e., among individuals plants of the same species), although this aspect of results was not explicitly considered in their studies.A study currently in progress has documented patterns of intraespecific variability in yeast occurrence in the nectaries of Helleborus foetidus (Ranunculaceae), a winter-flowering, bumble bee-pollinated perennial herb whose long-lived flowers last for roughly two weeks. Frequency of occurrence and cell density of yeasts in nectar were studied at six populations of this species from Sierra de Cazorla (SE Spain). Helleborus foetidus flowers have five separated horn-shaped nectaries hidden at the corolla base, each of which produces up to 5 µl of nectar. This enabled us to study patterns of yeast occurrence also at the within-flower level. At each population, total variance in yeast cell density on a per-nectary basis was partitioned into components due to differences between individual plants, flowers within plants and nectaries within flowers. We found extreme differences concerning the abundance and frequency of yeasts in H. foetidus nectar, the magnitude of intraespecific variation being similar or even greater than variation found in interespecific comparisons in the same study area (Pozo MI, et al. unpublished results). Our data suggest that temporal and spatial factors may explain differences regarding yeast abundance in H. foetidus nectar, and possibly other species as well. The largest component of intraespecific variance in yeast abundance occurred at the subindividual level, and was mainly accounted for by the variance between nectaries in the same flower (Fig. 1). This intraespecific variation in nectarivorous yeast incidence can have some important implications related to plant-pollinators interactions and, more specifically, to plant signaling, as outlined below.Open in a separate windowFigure 1Hierarchical dissection of variance in yeast abundance in single-nectary nectar samples of Helleborus foetidus. (A) Temporal patterns. Collection dates and plant, flower within plant and nectary within flower as hierarchical levels of variance analyzed. (B) Spatial patterns. Population, plant within populations and flower within plants as hierarchical levels of variance analyzed.Nectar-inhabiting yeasts modify certain flower characteristics linked to pollinator foraging behavior, such as nectar sugar composition and energetic value, by reducing total sugar concentration and altering the relative proportions of constituent sugars (sucrose, glucose and fructose) and the sucrose:hexose ratio.^23–26 Furthermore, as noted above, yeasts could be also implicated in floral volatiles emission.^13,14 Consequently, yeast incidence (measured both by frequency and abundance of yeast cells in nectar samples) may have been modifying signaling cues which have been postulated to be intrinsic plant species-specific. Although an empirical connection between yeast presence and fermentation nectar odor is needed, the fact that nectarivorous yeast presence would be as variable as described by our studies could imply the same variability for plant species signaling aspects, along with potential consequences for pollinators, since variance was mainly accounted for by variation below individual plant level. For example, in H. foetidus study variance in yeast abundance occurs mainly at the single nectary, which matches with the smallest scale that is perceived by a foraging insect. The fact that nectar is an important floral reward that plays a decisive role in the establishment of plant-pollinator mutualisms, together with the recently confirmed ubiquity of nectarivorous yeasts which could be acting as parasites of such mutualisms, open up new and exciting avenues to explore their effect on pollination success and pollen flow^27–30 and finally on plant fitness.^31–35     

Extracellular Tumor-Related mRNA in Plasma of Lymphoma Patients and Survival Implications

Background

We studied anomalous extracellular mRNAs in plasma from patients with diffuse large B-cell lymphoma (DLBCL) and their survival implications. mRNAs studied have been reported in the literature as markers of poor (BCL2, CCND2, MYC) and favorable outcome (LMO2, BCL6, FN1) in tumors. These markers were also analyzed in lymphoma tissues to test possible associations with their presence in plasma.

Methodology/Principal Findings

mRNA from 42 plasma samples and 12 tumors from patients with DLBCL was analyzed by real-time PCR. Samples post-treatment were studied. The immunohistochemistry of BCL2 and BCL6 was defined. Presence of circulating tumor cells was determined by analyzing the clonality of the immunoglobulin heavy-chain genes by PCR. In DLBCL, MYC mRNA was associated with short overall survival. mRNA targets with unfavorable outcome in tumors were associated with characteristics indicative of poor prognosis, with partial treatment response and with short progression-free survival in patients with complete response. In patients with low IPI score, unfavorable mRNA targets were related to shorter overall survival, partial response, high LDH levels and death. mRNA disappeared in post-treatment samples of patients with complete response, and persisted in those with partial response or death. No associations were found between circulating tumor cells and plasma mRNA. Absence of BCL6 protein in tumors was associated with presence of unfavorable plasma mRNA.

Conclusions/Significance

Through a non-invasive procedure, tumor-derived mRNAs can be obtained in plasma. mRNA detected in plasma did not proceed from circulating tumor cells. In our study, unfavorable targets in plasma were associated with poor prognosis in B-cell lymphomas, mainly MYC mRNA. Moreover, the unfavorable targets in plasma could help us to classify patients with poor outcome within the good prognosis group according to IPI.     

Filamins Regulate Cell Spreading and Initiation of Cell Migration

Mammalian filamins (FLNs) are a family of three large actin-binding proteins. FLNa, the founding member of the family, was implicated in migration by cell biological analyses and the identification of FLNA mutations in the neuronal migration disorder periventricular heterotopia. However, recent knockout studies have questioned the relevance of FLNa to cell migration. Here we have used shRNA-mediated knockdown of FLNa, FLNb or FLNa and FLNb, or, alternatively, acute proteasomal degradation of all three FLNs, to generate FLN-deficient cells and assess their ability to migrate. We report that loss of FLNa or FLNb has little effect on migration but that knockdown of FLNa and FLNb, or proteolysis of all three FLNs, impairs migration. The observed defect is primarily a deficiency in initiation of motility rather than a problem with maintenance of locomotion speed. FLN-deficient cells are also impaired in spreading. Re-expression of full length FLNa, but not re-expression of a mutated FLNa lacking immunoglobulin domains 19 to 21, reverts both the spreading and the inhibition of initiation of migration.Our results establish a role for FLNs in cell migration and spreading and suggest that compensation by other FLNs may mask phenotypes in single knockout or knockdown cells. We propose that interactions between FLNs and transmembrane or signalling proteins, mediated at least in part by immunoglobulin domains 19 to 21 are important for both cell spreading and initiation of migration.     

Genetic variation in the Solanaceae fruit bearing species lulo and tree tomato revealed by Conserved Ortholog (COSII) markers

The Lulo or naranjilla (Solanum quitoense Lam.) and the tree tomato or tamarillo (Solanum betaceum Cav. Sendt.) are both Andean tropical fruit species with high nutritional value and the potential for becoming premium products in local and export markets. Herein, we present a report on the genetic characterization of 62 accessions of lulos (n = 32) and tree tomatoes (n = 30) through the use of PCR-based markers developed from single-copy conserved orthologous genes (COSII) in other Solanaceae (Asterid) species. We successfully PCR amplified a set of these markers for lulos (34 out of 46 initially tested) and tree tomatoes (26 out of 41) for molecular studies. Six polymorphic COSII markers were found in lulo with a total of 47 alleles and five polymorphic markers in tree tomato with a total of 39 alleles in the two populations. Further genetic analyses indicated a high population structure (with F(ST) > 0.90), which may be a result of low migration between populations, adaptation to various niches and the number of markers evaluated. We propose COSII markers as sound tools for molecular studies, conservation and the breeding of these two fruit species.     

Diversity and vertical distribution of epiphytic macrolichens in lowland rain forest and lowland cloud forest of French Guiana

Recent work on bryophyte diversity in lowland forests of northern South America has suggested the existence of a new type of cloud forest, the “tropical lowland cloud forest” (LCF). LCF occurs in river valleys with high air humidity and radiation fog, and is rich in epiphytes. We explored the lichenological characteristics of putative LCF in a lowland area (200–400 m a.s.l.) near Saül, central French Guiana, using macrolichens (including large crustose species) as indicator taxa. We analyzed macrolichen diversity on 16 trees in two 1 ha plots, in LCF and in lowland rain forest without fog (LRF). Sampling efficiency was ca. 80% in both forest types. Canopies of both LRF and LCF were richer in lichen species than understory trunks. Species richness of macrolichens was rather similar in the two forest types but species composition was significantly different. Cyanolichen richness in LCF was ca. 2.5 times higher than in LRF; in contrast, LRF had 4 times more species of green-algal Parmeliaceae. Our study suggests that cyanolichens except for Coccocarpiaceae serve as indicators of LCF. We explain the detected diversity patterns by differences in water availability due to fog precipitation and higher humidity. This is indicated by the higher relative air humidity in the lowland cloud forest, which was >6% higher than in the rain forest.     

The import competence of a peroxisomal membrane protein is determined by Pex19p before the docking step

Biogenesis of the mammalian peroxisomal membrane requires the action of Pex3p and Pex16p, two proteins present in the organelle membrane, and Pex19p, a protein that displays a dual subcellular distribution (peroxisomal and cytosolic). Pex19p interacts with most peroxisomal intrinsic membrane proteins, but whether this property reflects its role as an import receptor for this class of proteins or a chaperone-like function in the assembly/disassembly of peroxisomal membrane proteins has been the subject of much controversy. Here, we describe an in vitro system particularly suited to address this issue. It is shown that insertion of a reporter protein into the peroxisomal membrane is a Pex3p-dependent process that does not require ATP/GTP hydrolysis. The system can be programmed with recombinant versions of Pex19p, allowing us to demonstrate that Pex19p-cargo protein complexes formed in the absence of peroxisomes are the substrates for the peroxisomal docking/insertion machinery. Data suggesting that cargo-loaded Pex19p displays a much higher affinity for Pex3p than Pex19p alone are also provided. These results suggest that soluble Pex19p participates in the targeting of newly synthesized peroxisomal membrane proteins to the organelle membrane and support the existence of a cargo-induced peroxisomal targeting mechanism for Pex19p.     

Peptide aptamer-mediated inhibition of target proteins by sequestration into aggresomes

Peptide aptamers (PAs) can be employed to block the intracellular function of target proteins. Little is known about the mechanism of PA-mediated protein inhibition. Here, we generated PAs that specifically bound to the duck hepatitis B virus (HBV) core protein. Among them, PA34 strongly blocked duck HBV replication by inhibiting viral capsid formation. We found that PA34 led to a dramatic intracellular redistribution of its target protein into perinuclear inclusion bodies, which exhibit the typical characteristics of aggresomes. As a result, the core protein is efficiently removed from the viral life cycle. Corresponding findings were obtained for bioactive PAs that bind to the HBV core protein or to the human papillomavirus-16 (HPV16) E6 protein, respectively. The observation that PAs induce the specific sequestration of bound proteins into aggresomes defines a novel mechanism as to how this new class of intracellular inhibitors blocks the function of their target proteins.     

Interdomain communication in DNA topoisomerase II. DNA binding and enzyme activation

Topoisomerase II catalyzes the ATP-dependent transport of a DNA segment (T-DNA) through a transient double strand break in another DNA segment (G-DNA). A fundamental mechanistic question is how the individual steps in this process are coordinated. We probed communication between the DNA binding sites and the individual enzymatic activities, ATP hydrolysis, and DNA cleavage. We employed short DNA duplexes to control occupancy at the two binding sites of wild-type enzyme and a variant with a G-DNA site mutation. The DNA concentration dependence of ATP hydrolysis and a fluorescence anisotropy assay provided thermodynamic information about DNA binding. The results suggest that G-DNA binds with higher affinity than T-DNA. Enzyme with only G-DNA bound is competent to cleave DNA, indicating that T-DNA is dispensable for DNA cleavage. The ATPase activity of enzyme bound solely to G-DNA is partially stimulated. Full stimulation requires binding of T-DNA. Both DNA binding sites therefore signal to the ATPase domains. The results support and extend current mechanistic models for topoisomerase II-catalyzed DNA transport and provide a framework for future mechanistic dissection.