The function of Notch signalling in segment formation in the crustacean Daphnia magna (Branchiopoda)

Ten years ago we showed for the first time that Notch signalling is required in segmentation in spiders, indicating the existence of similar mechanisms in arthropod and vertebrate segmentation. However, conflicting results in various arthropod groups hampered our understanding of the ancestral function of Notch in arthropod segmentation. Here we fill a crucial data gap in arthropods and analyse segmentation in a crustacean embryo. We analyse the expression of homologues of the Drosophila and vertebrate segmentation genes and show that members of the Notch signalling pathway are expressed at the same time as the pair-rule genes. Furthermore, inactivation of Notch signalling results in irregular boundaries of the odd-skipped-like expression domains and affects the formation of segments. In severe cases embryos appear unsegmented. We suggest two scenarios for the function of Notch signalling in segmentation. The first scenario agrees with a segmentation clock involving Notch signalling, while the second scenario discusses an alternative mechanism of Notch function which is integrated into a hierarchical segmentation cascade.     

Responses to iron deficiency in Arabidopsis thaliana: The Turbo iron reductase does not depend on the formation of root hairs and transfer cells

Arabidopsis thaliana (L.) Heynh. Columbia wild type and a root hair-less mutant RM57 were grown on iron-containing and iron-deficient nutrient solutions. In both genotypes, ferric chelate reductase (FCR) of intact roots was induced upon iron deficiency and followed a Michaelis-Menten kinetic with a K _m of 45 and 54 M Fe^III-EDTA and a V _max of 42 and 33 nmol Fe²⁺·(g FW)^–1·min^–1 for the wild type and the mutant, respectively. The pH optimum for the reaction was around pH 5.5. The approximately four fold stimulation of FCR activity was independent of formation of root hairs and/or transfer cells induced by iron deficiency. Iron-deficiency-induced chlorosis and the development of a rigid root habit disappeared when ferric chelate was applied to the leaves, while FCR activity remained unchanged. The time course of the responses to iron deficiency showed that morphological and physiological responses were controlled separately.Abbreviations FCR ferric chelate reductase - FW fresh weight Thanks are due to Klaas Sjollema (Department of Electronmicroscopy, University of Groningen, The Netherlands) for help with the electron microscopy sample preparation and especially to Dr. Uwe Santore (Heinrich-Heine-University for electron microscopy. This work was supported by the SCIENCE programm of the European community; P.R.M.) and a Personal Research Grant by the Ministerium für Wissenschaft und Forschung of Nordrhein-Westfalen (P.R.M.) and last, not least by the productive discussions in ECOTRANS B.V.     

Selective enrichment and isolation of Rhodopseudomonas palustris using trans-cinnamic acid as sole carbon source

Abstract Enrichment cultures for anoxygenic phototrophs capable of using cinnamic acid as sole organic carbon source consistently yielded the nonsulfur purple bacterium Rhodopseudomonas palustris . Pure cultures of R. palustris obtained from the enrichments grew photoheterotrophically on cinnamate and benzoate as well as on derivatives of these compounds. Photosynthetic growth on cinnamate was greatly stimulated by addition of exogenous CO₂, and resulted in breakage of the aromatic nucleus. Growth yield studies suggested that cinnamate was converted by R. palustris to intermediates that can be quantitatively assimilated into cell material.     

A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.     

Assessment of Metabolomic and Proteomic Biomarkers in Detection and Prognosis of Progression of Renal Function in Chronic Kidney Disease

Chronic kidney disease (CKD) is part of a number of systemic and renal diseases and may reach epidemic proportions over the next decade. Efforts have been made to improve diagnosis and management of CKD. We hypothesised that combining metabolomic and proteomic approaches could generate a more systemic and complete view of the disease mechanisms. To test this approach, we examined samples from a cohort of 49 patients representing different stages of CKD. Urine samples were analysed for proteomic changes using capillary electrophoresis-mass spectrometry and urine and plasma samples for metabolomic changes using different mass spectrometry-based techniques. The training set included 20 CKD patients selected according to their estimated glomerular filtration rate (eGFR) at mild (59.9±16.5 mL/min/1.73 m²; n = 10) or advanced (8.9±4.5 mL/min/1.73 m²; n = 10) CKD and the remaining 29 patients left for the test set. We identified a panel of 76 statistically significant metabolites and peptides that correlated with CKD in the training set. We combined these biomarkers in different classifiers and then performed correlation analyses with eGFR at baseline and follow-up after 2.8±0.8 years in the test set. A solely plasma metabolite biomarker-based classifier significantly correlated with the loss of kidney function in the test set at baseline and follow-up (ρ = −0.8031; p<0.0001 and ρ = −0.6009; p = 0.0019, respectively). Similarly, a urinary metabolite biomarker-based classifier did reveal significant association to kidney function (ρ = −0.6557; p = 0.0001 and ρ = −0.6574; p = 0.0005). A classifier utilising 46 identified urinary peptide biomarkers performed statistically equivalent to the urinary and plasma metabolite classifier (ρ = −0.7752; p<0.0001 and ρ = −0.8400; p<0.0001). The combination of both urinary proteomic and urinary and plasma metabolic biomarkers did not improve the correlation with eGFR. In conclusion, we found excellent association of plasma and urinary metabolites and urinary peptides with kidney function, and disease progression, but no added value in combining the different biomarkers data.     

Contours of Risk: Spatializing Human Behaviors to Understand Disease Dynamics in Changing Landscapes

We echo viewpoints presented in recent publications from EcoHealth and other journals arguing for the need to understand linkages between human health, disease ecology, and landscape change. We underscore the importance of incorporating spatialities of human behaviors and perceptions in such analyses to further understandings of socio-ecological interactions mediating human health. We use Buruli ulcer, an emerging necrotizing skin infection and serious health concern in central Ghana, to illustrate our argument.     

GAPDH enhances group II intron splicing in vitro

Group II introns are autocatalytic RNAs which self-splice in vitro. However, in vivo additional protein factors might be involved in the splicing process. We used an affinity chromatography method called 'StreptoTag' to identify group II intron binding proteins from Saccharomyces cerevisiae. This method uses a hybrid RNA consisting of a streptomycin-binding affinity tag and the RNA of interest, which is bound to a streptomycin column and incubated with yeast protein extract. After several washing steps the bound RNPs are eluted by addition of streptomycin. The eluted RNPs are separated and the proteins identified by mass-spectrometric analysis. Using crude extract from yeast in combination with a substructure of the bl1 group II intron (domains IV-VI) we were able to identify four glycolytic enzymes; glucose-6-phosphate isomerase (GPI), 3-phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI). From these proteins GAPDH increases in vitro splicing of the bl1 group II intron by up to three times. However, in vivo GAPDH is not a group II intron-splicing factor, since it is not localised in yeast mitochondria. Therefore, the observed activity reflects an unexpected property of GAPDH. Band shift experiments and UV cross linking demonstrated the interaction of GAPDH with the group II intron RNA. This novel activity expands the reaction repertoire of GAPDH to a new RNA species.     

Natural variation reveals contrasting abilities to cope with alkaline and saline soil among different <Emphasis Type="Italic">Medicago truncatula</Emphasis> genotypes

Aims

Urban soils are the basis of many ecosystem services in cities. Here, we examine formerly residential vacant lot soils in Cleveland, Ohio and Detroit, Michigan, USA for their potential to provide multiple ecosystem services. We examine two key contrasts: 1) differences between cities and 2) differences within vacant lots created during demolition, specifically pre-existing (i.e., prior to demolition) soils outside of the building footprint and fill soils added within the former building’s footprint.

Methods

Deep soil cores were collected from vacant lots in Cleveland and Detroit. Soil properties that are proxies for three ecosystem services were measured: hydraulic conductivity for stormwater retention, topsoil depth and soil nitrogen (N) level for support for plant growth, and soil carbon (C) content for C storage.

Results

Both city and soil group contrasts created distinct ecosystem service provisioning based on proxy measures. Cleveland soils had greater hydraulic conductivity and greater soil C and N levels but thinner topsoil layers than Detroit. Within vacant lots of both cities, pre-existing soils had greater soil C and N levels, but lower hydraulic conductivity values than fill soils.

Conclusions

Soil properties of vacant lots were generally suitable for providing multiple ecosystem services. City-level differences in soil properties created differences in ecosystem service potential between cities and these differences were evident in pre-existing and fill soils. When comparing between cities, though, fill soils were more similar than pre-existing soils indicating some homogenization of ecosystem service potential with greater redistribution of soil.

     

Zonal hierarchy of differentiation markers and nestin expression during oval cell mediated rat liver regeneration

Oval cells constitute a heterogeneous population of proliferating progenitors found in rat livers following carcinogenic treatment (2-acetylaminofluorene and 70% hepatectomy). The aim of this study was to investigate the cellular pattern of various differentiation and cell type markers in this model of liver regeneration. Immunophenotypic characterisation revealed at least two subtypes emerging from the portal field. First, a population of oval cells formed duct-like structures and expressed bile duct (CD49f) as well as hepatocytic markers (α-foetoprotein, CD26). Second, a population of non-ductular oval cells was detected between and distally from the ductules expressing the neural marker nestin and the haematopoietic marker Thy1. Following oval cell isolation, a subset of the nestin-positive cells was shown to co-express hepatocytic and epithelial markers (albumin, CD26, pancytokeratin) and could be clearly distinguished from anti-desmin reactive hepatic stellate cells. The gene expression profiles (RT-PCR) of isolated oval cells and oval cell liver tissue were found to be similar to foetal liver (ED14). The present results suggest that the two oval cell populations are organised in a zonal hierarchy with a marker gradient from the inner (displaying hepatocytic and biliary markers) to the outer zone (showing hepatocytic and extrahepatic progenitor markers) of the proliferating progeny clusters.