Arrayed adenoviral expression libraries for functional screening

With the publication of the sequence of the human genome, we are challenged to identify the functions of an estimated 70,000 human genes and the much larger number of proteins encoded by these genes. Of particular interest is the identification of gene products that play a role in human disease pathways, as these proteins include potential new targets that may lead to improved therapeutic strategies. This requires the direct measurement of gene function on a genomic scale in cell-based, functional assays. We have constructed and validated an individually arrayed, replication-defective adenoviral library harboring human cDNAs, termed PhenoSelect library. The adenoviral vector guarantees efficient transduction of diverse cell types, including primary cells. The arrayed format allows screening of this library in a variety of cellular assays in search for gene(s) that, by overexpression, induce a particular disease-related phenotype. The great majority of phenotypic assays, including morphological assays, can be screened with arrayed libraries. In contrast, pooled-library approaches often rely on phenotype-based isolation or selection of single cells by employing a flow cytometer or screening for cell survival. An arrayed placental PhenoSelect library was screened in cellular assays aimed at identifying regulators of osteogenesis, metastasis, and angiogenesis. This resulted in the identification of known regulators, as well as novel sequences that encode proteins hitherto not known to play a role in these pathways. These results establish the value of the PhenoSelect platform, in combination with cellular screens, for gene function discovery.     

Characterization of functional and structural integrity in experimental focal epilepsy: reduced network efficiency coincides with white matter changes

Background

Although focal epilepsies are increasingly recognized to affect multiple and remote neural systems, the underlying spatiotemporal pattern and the relationships between recurrent spontaneous seizures, global functional connectivity, and structural integrity remain largely unknown.

Methodology/Principal Findings

Here we utilized serial resting-state functional MRI, graph-theoretical analysis of complex brain networks and diffusion tensor imaging to characterize the evolution of global network topology, functional connectivity and structural changes in the interictal brain in relation to focal epilepsy in a rat model. Epileptic networks exhibited a more regular functional topology than controls, indicated by a significant increase in shortest path length and clustering coefficient. Interhemispheric functional connectivity in epileptic brains decreased, while intrahemispheric functional connectivity increased. Widespread reductions of fractional anisotropy were found in white matter regions not restricted to the vicinity of the epileptic focus, including the corpus callosum.

Conclusions/Significance

Our longitudinal study on the pathogenesis of network dynamics in epileptic brains reveals that, despite the locality of the epileptogenic area, epileptic brains differ in their global network topology, connectivity and structural integrity from healthy brains.     

Visual versus Automated Evaluation of Chest Computed Tomography for the Presence of Chronic Obstructive Pulmonary Disease

Background

Incidental CT findings may provide an opportunity for early detection of chronic obstructive pulmonary disease (COPD), which may prove important in CT-based lung cancer screening setting. We aimed to determine the diagnostic performance of human observers to visually evaluate COPD presence on CT images, in comparison to automated evaluation using quantitative CT measures.

Methods

This study was approved by the Dutch Ministry of Health and the institutional review board. All participants provided written informed consent. We studied 266 heavy smokers enrolled in a lung cancer screening trial. All subjects underwent volumetric inspiratory and expiratory chest computed tomography (CT). Pulmonary function testing was used as the reference standard for COPD. We evaluated the diagnostic performance of eight observers and one automated model based on quantitative CT measures.

Results

The prevalence of COPD in the study population was 44% (118/266), of whom 62% (73/118) had mild disease. The diagnostic accuracy was 74.1% in the automated evaluation, and ranged between 58.3% and 74.3% for the visual evaluation of CT images. The positive predictive value was 74.3% in the automated evaluation, and ranged between 52.9% and 74.7% for the visual evaluation. Interobserver variation was substantial, even within the subgroup of experienced observers. Agreement within observers yielded kappa values between 0.28 and 0.68, regardless of the level of expertise. The agreement between the observers and the automated CT model showed kappa values of 0.12–0.35.

Conclusions

Visual evaluation of COPD presence on chest CT images provides at best modest accuracy and is associated with substantial interobserver variation. Automated evaluation of COPD subjects using quantitative CT measures appears superior to visual evaluation by human observers.     

Modulation of photosynthetic energy conversion efficiency in nature: from seconds to seasons

Modulation of the efficiency with which leaves convert absorbed light to photochemical energy [intrinsic efficiency of open photosystem II (PSII) centers, as the ratio of variable to maximal chlorophyll fluorescence] as well as leaf xanthophyll composition (interconversions of the xanthophyll cycle pigments violaxanthin and zeaxanthin) were characterized throughout single days and nights to entire seasons in plants growing naturally in contrasting light and temperature environments. All pronounced decreases of intrinsic PSII efficiency took place in the presence of zeaxanthin. The reversibility of these PSII efficiency changes varied widely, ranging from reversible-within-seconds (in a vine experiencing multiple sunflecks under a eucalypt canopy) to apparently permanently locked-in for entire seasons (throughout the whole winter in a subalpine conifer forest at 3,000?m). While close association between low intrinsic PSII efficiency and zeaxanthin accumulation was ubiquitous, accompanying features (such as trans-thylakoid pH gradient, thylakoid protein composition, and phosphorylation) differed among contrasting conditions. The strongest and longest-lasting depressions in intrinsic PSII efficiency were seen in the most stress-tolerant species. Evergreens, in particular, showed the most pronounced modulation of PSII efficiency and thermal dissipation, and are therefore suggested as model species for the study of photoprotection. Implications of the responses of field-grown plants in nature for mechanistic models are discussed.     

Experimental warming studies on tree species and forest ecosystems: a literature review

Temperature affects a cascade of ecological processes and functions of forests. With future higher global temperatures being inevitable it is critical to understand and predict how forest ecosystems and tree species will respond. This paper reviews experimental warming studies in boreal and temperate forests or tree species beyond the direct effects of higher temperature on plant ecophysiology by scaling up to forest level responses and considering the indirect effects of higher temperature. In direct response to higher temperature (1) leaves emerged earlier and senesced later, resulting in a longer growing season (2) the abundance of herbivorous insects increased and their performance was enhanced and (3) soil nitrogen mineralization and leaf litter decomposition were accelerated. Besides these generalizations across species, plant ecophysiological traits were highly species-specific. Moreover, we showed that the effect of temperature on photosynthesis is strongly dependent on the position of the leaf or plant within the forest (canopy or understory) and the time of the year. Indirect effects of higher temperature included among others higher carbon storage in trees due to increased soil nitrogen availability and changes in insect performance due to alterations in plant ecophysiological traits. Unfortunately only a few studies extrapolated results to forest ecosystem level and considered the indirect effects of higher temperature. Thus more intensive, long-term studies are needed to further confirm the emerging trends shown in this review. Experimental warming studies provide us with a useful tool to examine the cascade of ecological processes in forest ecosystems that will change with future higher temperature.     

Geographic trends in flavone-glycosylation genes and seed morphology in EuropeanSilene pratensis (Caryophyllaceae)

Three of the loci controlling isovitexin glycosylation inSilene pratensis are polymorphic and show geographic trends which are compared with geographic trends in seed morphology (and other phenotypic characters) as demonstrated by multivariate analysis. Various lines of evidence support the hypothesis thatS. pratensis spread into Europe from at least two genetically differentiated sources.S. dioica, by contrast, shows little interpretable geographic variation in morphology or flavonoid content.     

A pattern analysis of the geographical distribution of flavone-glycosylating genes in Silene pratensis

The three flavone glycosylating genes in European Silene pratensis shows a distinct geographical variation. Three genetically different races can be distinguished on the basis of gene frequencies. Pattern recognition techniques and cluster analysis have been used on the data set of frequencies of the flavone-glycosylating genes in 285 European populations of S. pratensis. The combined results of these analyses confirm the previously recognized races and reveal a steep cline between races 7 and 2(+3) and a wide wide transition zone between races 2 and 3.     

A coenocline of the high-ranked syntaxa of ruderal vegetation

Two sets comprising 1419 and 1350 phytosociological relevés of ruderal vegetation classified into 9 and 7 orders, respectively, from an area in the Podunajská ní zina Lowland, western Slovakia were ordinated using correspondence and detrended correspondence analyses. The paper describes a coenocline of the high-ranked syntaxa contained in the data sets, and discusses some issues of the classification of the syntaxa involved.The Bidentetalia and Potentillo-Polygonetalia should be considered a special category of synanthropic vegetation as habitat moisture (flooding and waterlogging) play the controlling role in the formation of structural and dynamical patterns within these communities. The latter factor complex is responsible for the clear separation of these orders from the other syntaxa included in the ordinations. The coenocline of terrestrial ruderal vegetation units has the following sequence along the CA axis 1: Poo-Polygonetalia, Sisymbrietalia, Eragostrietalia, Onopordetalia, Agropyretalia repentis, Artemisietalia vulgaris and Glechometalia hederaceae.Various complexes of soil factors and anthropogenic disturbance are operational along the concatenation (a series of portions composing the coenocline). The factor complex includes soil compaction, trampling disturbance, nutrient status, soil texture and moisture, and solar irradiation. Step-by-step ordination and interpretation of concatenated portions of the coenocline proved to be useful in the analysis of complex data sets.Abbreviations CA = correspondence analysis - DCA = detrended correspondence analysis - PCA = principal components analysis     

Site-Specific Ubiquitination Determines Lysosomal Sorting and Signal Attenuation of the Granulocyte Colony-Stimulating Factor Receptor

Ubiquitination of cytokine receptors controls intracellular receptor routing and signal duration, but the underlying molecular determinants are unclear. The suppressor of cytokine signaling protein SOCS3 drives lysosomal degradation of the granulocyte colony-stimulating factor receptor (G-CSFR), depending on SOCS3-mediated ubiquitination of a specific lysine located in a conserved juxtamembrane motif. Here, we show that, despite ubiquitination of other lysines, positioning of a lysine within the membrane-proximal region is indispensable for this process. Neither reallocation of the motif nor fusion of ubiquitin to the C-terminus of the G-CSFR could drive lysosomal routing. However, within this region, the lysine could be shifted 12 amino acids toward the C-terminus without losing its function, arguing against the existence of a linear sorting motif and demonstrating that positioning of the lysine relative to the SOCS3 docking site is flexible. G-CSFR ubiquitination peaked after endocytosis, was inhibited by methyl-β-cyclodextrin as well as hyperosmotic sucrose and severely reduced in internalization-defective G-CSFR mutants, indicating that ubiquitination mainly occurs at endosomes. Apart from elucidating structural and spatio-temporal aspects of SOCS3-mediated ubiquitination, these findings have implications for the abnormal signaling function of G-CSFR mutants found in severe congenital neutropenia, a hematopoietic disorder with a high leukemia risk.