Functional identification of optimized RNAi triggers using a massively parallel sensor assay

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Highlights? The Sensor assay reliably identifies potent single-copy shRNAs ? Potent shRNAs are rare and generally not predicted by existing algorithms ? Analyses of ～20,000 shRNAs reveal insights into shRNA biogenesis and function ? Sensor-based rules provide a criteria framework for rational shRNA design     

Mapping of quantitative trait loci associated with protein expression variation in barley grains

Barley (Hordeum vulgare) is an important cereal crop grown for both the feed and malting industries. Hence, there is great interest to gain deeper insight into the determinants of grain nutritional quality in order to improve the assessment of new traits. Two-dimensional gel electrophoresis was employed for the characterization of the grain proteome of doubled-haploid introgression lines (IL) representing a wild barley genome (Hordeum spontaneum Hs213) within a modern cultivar background (H. vulgare cv. Brenda). Proteome maps were subjected to differential cluster analysis and revealed ILs with similar or different protein expression patterns compared to the Brenda parent. A total of 51 quantitative trait loci for protein expression (pQTL) were detected, and proteins underlying these pQTL were further examined by mass spectrometry. Identification was successful for 49 of the segregating spots and functional annotation of proteins revealed that most proteins are involved in metabolism and disease/defence-related processes. Among those, multigene families of glyceraldehyde-3-phosphate dehydrogenases, heat shock proteins, peroxidases, and serpins were identified. Overall, eight pQTL signals were discovered in two independently grown sets of plants. The mapped spots included protein disulfide isomerase, α-amylase inhibitor BDAI, NADP malic enzyme, adenosine kinase and peroxidase BP1. Specific marker information of proteins involved in developmental events and protein storage as well as in disease- and defence-related processes now allows for targeted breeding approaches to improve the grain quality in barley.     

Proteome analysis of wheat leaf rust fungus, Puccinia triticina, infection structures enriched for haustoria

Puccinia triticina (Pt) is a representative of several cereal-infecting rust fungal pathogens of major economic importance world wide. Upon entry through leaf stomata, these fungi establish intracellular haustoria, crucial feeding structures. We report the first proteome of infection structures from parasitized wheat leaves, enriched for haustoria through filtration and sucrose density centrifugation. 2-D PAGE MS/MS and gel-based LC-MS (GeLC-MS) were used to separate proteins. Generated spectra were compared with a partial proteome predicted from a preliminary Pt genome and generated ESTs, to a comprehensive genome-predicted protein complement from the related wheat stem rust fungus, Puccinia graminis f. sp. tritici (Pgt) and to various plant resources. We identified over 260 fungal proteins, 16 of which matched peptides from Pgt. Based on bioinformatic analyses and/or the presence of a signal peptide, at least 50 proteins were predicted to be secreted. Among those, six have effector protein signatures, some are related and the respective genes of several seem to belong to clusters. Many ribosomal structural proteins, proteins involved in energy, general metabolism and transport were detected. Measuring gene expression over several life cycle stages of ten representative candidates using quantitative RT-PCR, all were shown to be strongly upregulated and four expressed solely upon infection.     

Dissociable effects of valence and arousal in adaptive executive control

Background

Based on introspectionist, semantic, and psychophysiological experimental frameworks, it has long been assumed that all affective states derive from two independent basic dimensions, valence and arousal. However, until now, no study has investigated whether valence and arousal are also dissociable at the level of affect-related changes in cognitive processing.

Methodology/Principal Findings

We examined how changes in both valence (negative vs. positive) and arousal (low vs. high) influence performance in tasks requiring executive control because recent research indicates that two dissociable cognitive components are involved in the regulation of task performance: amount of current control (i.e., strength of filtering goal-irrelevant signals) and control adaptation (i.e., strength of maintaining current goals over time). Using a visual pop-out distractor task, we found that control is exclusively modulated by arousal because interference by goal-irrelevant signals was largest in high arousal states, independently of valence. By contrast, control adaptation is exclusively modulated by valence because the increase in control after trials in which goal-irrelevant signals were present was largest in negative states, independent of arousal. A Monte Carlo simulation revealed that differential effects of two experimental factors on control and control adaptation can be dissociated if there is no correlation between empirical interference and conflict-driven modulation of interference, which was the case in the present data. Consequently, the observed effects of valence and arousal on adaptive executive control are indeed dissociable.

Conclusions/Significance

These findings indicate that affective influences on cognitive processes can be driven by independent effects of variations in valence and arousal, which may resolve several heterogeneous findings observed in previous studies on affect-cognition interactions.     

Monitoring CD27 expression to evaluate Mycobacterium tuberculosis activity in HIV-1 infected individuals in vivo

The level of bacterial activity is only poorly defined during asymptomatic Mycobacterium tuberculosis (MTB) infection. The objective was to study the capacity of a new biomarker, the expression of the T cell maturation marker CD27 on MTB-specific CD4 T cells, to identify active tuberculosis (TB) disease in subjects from a MTB and HIV endemic region. The frequency and CD27 expression of circulating MTB-specific CD4 T cells was determined in 96 study participants after stimulation with purified protein derivative (PPD) using intracellular cytokine staining for IFNgamma (IFNγ). Subjects were then stratified by their TB and HIV status. Within PPD responders, a CD27⁻ phenotype was associated with active TB in HIV⁻ (p = 0.0003) and HIV⁺ (p = 0.057) subjects, respectively. In addition, loss of CD27 expression preceded development of active TB in one HIV seroconverter. Interestingly, in contrast to HIV⁻ subjects, MTB-specific CD4 T cell populations from HIV⁺ TB-asymptomatic subjects were often dominated by CD27⁻ cells. These data indicate that down-regulation of CD27 on MTB-specific CD4 T cell could be used as a biomarker of active TB, potentially preceding clinical TB disease. Furthermore, these data are consistent with the hypothesis that late, chronic HIV infection is frequently associated with increased mycobacterial activity in vivo. The analysis of T cell maturation and activation markers might thus be a useful tool to monitor TB disease progression.     

An RNA interference phenotypic screen identifies a role for FGF signals in colon cancer progression

In tumor cells, stepwise oncogenic deregulation of signaling cascades induces alterations of cellular morphology and promotes the acquisition of malignant traits. Here, we identified a set of 21 genes, including FGF9, as determinants of tumor cell morphology by an RNA interference phenotypic screen in SW480 colon cancer cells. Using a panel of small molecular inhibitors, we subsequently established phenotypic effects, downstream signaling cascades, and associated gene expression signatures of FGF receptor signals. We found that inhibition of FGF signals induces epithelial cell adhesion and loss of motility in colon cancer cells. These effects are mediated via the mitogen-activated protein kinase (MAPK) and Rho GTPase cascades. In agreement with these findings, inhibition of the MEK1/2 or JNK cascades, but not of the PI3K-AKT signaling axis also induced epithelial cell morphology. Finally, we found that expression of FGF9 was strong in a subset of advanced colon cancers, and overexpression negatively correlated with patients' survival. Our functional and expression analyses suggest that FGF receptor signals can contribute to colon cancer progression.     

Stable expression of a defense-related gene in wheat epidermis under transcriptional control of a novel promoter confers pathogen resistance

Tissue-specific or regulated expression of transgenes is desirable in order to prevent pleiotropic side effects of putatively harmful transgene products as well as loss of energy resources due to unnecessary accumulation of transgene products. Epidermis-specific expression would be useful for many defense-related genes directed against attack by fungal pathogens that enter the plant body by direct penetration through the epidermis. In an approach to enhance resistance of wheat to the powdery mildew fungus Blumeria graminis f.sp. tritici, a novel epidermis-specific promoter was developed and used for expression of two defense-related genes. A 2.3 kb fragment of the wheat GstA1 promoter in combination with an intron-containing part of the wheat WIR1a gene was found to drive strong and constitutive transient expression in wheat epidermis. This promoter-intron combination was used for overexpression of oxalate oxidase 9f-2.8 and TaPERO peroxidase, two defense-related wheat genes expressed in inner leaf tissues. Expression studies of several transgenic lines by in situ oxalate-oxidase staining, RNA and protein blot analyses, as well as real-time PCR, demonstrated strong and constitutive transgene expression in the shoot epidermis. Transient as well as stable over-expression of the TaPERO peroxidase gene in wheat epidermis under the control of the GstA1i promoter resulted in enhanced resistance against Blumeria graminis f.sp. tritici, whereas oxalate-oxidase overexpression had no effect in either system. The data suggest that the wheat GstA1 promoter in combination with the WIR1a intron is useful for transgenic approaches to fungal disease resistance in cereals.     

Face adaptation depends on seeing the face

Retinal input that is suppressed from visual awareness can nevertheless produce measurable aftereffects, revealing neural processes that do not directly result in a conscious percept. We here report that the face identity-specific aftereffect requires a visible face; it is effectively cancelled by binocular suppression or by inattentional blindness of the inducing face. Conversely, the same suppression does not interfere with the orientation-specific aftereffect. Thus, the competition between incompatible or interfering visual inputs to reach awareness is resolved before those aspects of information that are exploited in face identification are processed. We also found that the face aftereffect remained intact when the visual distracters in the inattention experiment were replaced with auditory distracters. Thus, cross-modal or cognitive interference that does not affect the visibility of the face does not interfere with the face aftereffect. We conclude that adaptation to face identity depends on seeing the face.     

Global gene expression profiling and cluster analysis in Xenopus laevis

We have undertaken a large-scale microarray gene expression analysis using cDNAs corresponding to 21,000 Xenopus laevis ESTs. mRNAs from 37 samples, including embryos and adult organs, were profiled. Cluster analysis of embryos of different stages was carried out and revealed expected affinities between gastrulae and neurulae, as well as between advanced neurulae and tadpoles, while egg and feeding larvae were clearly separated. Cluster analysis of adult organs showed some unexpected tissue-relatedness, e.g. kidney is more related to endodermal than to mesodermal tissues and the brain is separated from other neuroectodermal derivatives. Cluster analysis of genes revealed major phases of co-ordinate gene expression between egg and adult stages. During the maternal-early embryonic phase, genes maintaining a rapidly dividing cell state are predominantly expressed (cell cycle regulators, chromatin proteins). Genes involved in protein biosynthesis are progressively induced from mid-embryogenesis onwards. The larval-adult phase is characterised by expression of genes involved in metabolism and terminal differentiation. Thirteen potential synexpression groups were identified, which encompass components of diverse molecular processes or supra-molecular structures, including chromatin, RNA processing and nucleolar function, cell cycle, respiratory chain/Krebs cycle, protein biosynthesis, endoplasmic reticulum, vesicle transport, synaptic vesicle, microtubule, intermediate filament, epithelial proteins and collagen. Data filtering identified genes with potential stage-, region- and organ-specific expression. The dataset was assembled in the iChip microarray database, , which allows user-defined queries. The study provides insights into the higher order of vertebrate gene expression, identifies synexpression groups and marker genes, and makes predictions for the biological role of numerous uncharacterized genes.