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.

     

Expression of membrane-bound carbonic anhydrases IV, IX, and XIV in the mouse heart.

Expression of membrane-bound carbonic anhydrases (CAs) of CA IV, CA IX, CA XII, and CA XIV has been investigated in the mouse heart. Western blots using microsomal membranes of wild-type hearts demonstrate a 39-, 43-, and 54-kDa band representing CA IV, CA IX, and CA XIV, respectively, but CA XII could not be detected. Expression of CA IX in the CA IV/CA XIV knockout animals was further confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Cardiac cells were immunostained using anti-CA/FITC and anti-alpha-actinin/TRITC, as well as anti-CA/FITC and anti-SERCA2/TRITC. Subcellular CA localization was investigated by confocal laser scanning microscopy. CA localization in the sarcolemmal (SL) membrane was examined by double immunostaining using anti-CA/FITC and anti-MCT-1/TRITC. CAs showed a distinct distribution pattern in the sarcoplasmic reticulum (SR) membrane. CA XIV is predominantly localized in the longitudinal SR, whereas CA IX is mainly expressed in the terminal SR/t-tubular region. CA IV is present in both SR regions, whereas CA XII is not found in the SR. In the SL membrane, only CA IV and CA XIV are present. We conclude that CA IV and CA XIV are associated with the SR as well as with the SL membrane, CA IX is located in the terminal SR/t-tubular region, and CA XII is not present in the mouse heart. Therefore, the unique subcellular localization of CA IX and CA XIV in cardiac myocytes suggests different functions of both enzymes in excitation-contraction coupling.     

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.     

Single residue determines the specificity of neutrophil elastase for Shigella virulence factors

Human neutrophil elastase (NE) is a key host defense protease that cleaves virulence factors of Gram-negative bacteria. NE and cathepsin G (CG) are chymotrypsin-like serine proteases with sequence and structural similarities, and both are abundant in neutrophil granules. Unlike NE, CG does not cleave virulence factors of enteric bacteria. Through structure-function analysis, we identified regions in NE that are essential for cleaving Shigella virulence proteins. NE residues at eight different positions were replaced with analogous amino acids in CG or with alanine. Functional analysis of recombinant mutant proteins showed that a single residue at position 98 and multiple amino acid stretches in the three different regions 58A-61, 163-181, and 216-224 determine NE specificity. These NE mutants cleaved the CG-specific, but not the NE-specific, synthetic peptide substrate and did not degrade Shigella virulence factors. Interestingly, exchanging the amino acid at position 98 in CG for the NE equivalent enabled this CG mutant to cleave Shigella virulence factors. Analysis of the NE proteolytic products of the Shigella virulence factor IpaB shows that NE has specific cleavage sites. These results indicate that Shigella virulence factor specificity maps to a distinct region close to NE's active site.     

Repeat proliferation and partial endoreplication jointly shape the patterns of genome size evolution in orchids

Although the evolutionary drivers of genome size change are known, the general patterns and mechanisms of plant genome size evolution are yet to be established. Here we aim to assess the relative importance of proliferation of repetitive DNA, chromosomal variation (including polyploidy), and the type of endoreplication for genome size evolution of the Pleurothallidinae, the most species-rich orchid lineage. Phylogenetic relationships between 341 Pleurothallidinae representatives were refined using a target enrichment hybrid capture combined with high-throughput sequencing approach. Genome size and the type of endoreplication were assessed using flow cytometry supplemented with karyological analysis and low-coverage Illumina sequencing for repeatome analysis on a subset of samples. Data were analyzed using phylogeny-based models. Genome size diversity (0.2–5.1 Gbp) was mostly independent of profound chromosome count variation (2n = 12–90) but tightly linked with the overall content of repetitive DNA elements. Species with partial endoreplication (PE) had significantly greater genome sizes, and genomic repeat content was tightly correlated with the size of the non-endoreplicated part of the genome. In PE species, repetitive DNA is preferentially accumulated in the non-endoreplicated parts of their genomes. Our results demonstrate that proliferation of repetitive DNA elements and PE together shape the patterns of genome size diversity in orchids.     

Involvement of fibronectin during epiboly and gastrulation in embryos of the common carp,Cyprinus carpio

Summary The present report firstly describes a pilot study in which, during early development of embryos of the common carp, Cyprinus carpio, the cellular adhesion to fibronectin (FN) was blocked by administration of GRGDS peptide (which binds to the FN-receptor). As this treatment resulted in developmental aberrations, suggesting a functional role for FN, the major part of the work was focussed on the distribution of reactivity of anti-FN antibodies during epiboly and gastrulation. GRGDS treatment had a concentration dependent effect on development. Incubation of embryos in 1.5 mg/ml from the 32-cell stage onwards caused a retardation of epiboly, which did not proceed beyond 60%. The embryos did not show involution, as was confirmed by histological study. These preliminary results suggest that FN is involved in both epiboly and gastrulation of carp embryos. During cleavage, no specific extracellular binding of anti-FN antiserum could be observed. However, binding to a number of cell membranes took place from early epiboly onwards. After the onset of gastrulation, we observed a gradually increasing number of the deepest epiblast cells, showing immunostaining on part of their surface, facing the yolk syncytial layer (YSL) or the involuted cells. During early epiboly, anti-FN binding was restricted to areas in front of the migratory hypoblast cells. Later on, binding was found at the border of hypoblast and epiblast cells. At 100% epiboly, some contact areas of epiblast and hypoblast showed a discontinuous lining of reactivity, whilst other areas appeared devoid of anti-FN binding sites. The results indicate that FN is involved in the migration and guidance of hypoblast cells during gastrulation in carp. Correspondence to: P. Gevers