Minoxidil exerts different inhibitory effects on gene expression of lysyl hydroxylase 1, 2, and 3: implications for collagen cross-linking and treatment of fibrosis.

Collagen deposits in fibrotic lesions often display elevated levels of hydroxyallysine (pyridinoline) cross-links. The relation between the occurrence of pyridinoline cross-links and the irreversibility of fibrosis suggests that these cross-links contribute to the aberrant accumulation of collagen. Based on its inhibitory effect on lysyl hydroxylase activity minoxidil has been postulated to possess anti-fibrotic properties by limiting the hydroxylysine supply for hydroxyallysine cross-linking. However, to interfere with hydroxyallysine cross-linking specifically lysyl hydroxylation of the collagen telopeptide should be inhibited, a reaction predominantly catalysed by lysyl hydroxylase (LH) 2b. In this study, we demonstrate that minoxidil treatment of cultured fibroblasts reduces LH1>LH2b>LH3 mRNA levels dose-and time-dependently, but has essentially no effect on the total number of pyridinoline cross-links in the collagen matrix. Still the collagen produced in the presence of minoxidil displays some remarkable features: hydroxylation of triple helical lysine residues is reduced to 50% and lysylpyridinoline cross-linking is increased at the expense of hydroxylysylpyridinoline cross-linking. These observations can be explained by our finding that LH1 mRNA levels are the most sensitive to minoxidil treatment, corroborating that LH1 has a preference for triple helical lysine residues as substrate. In addition, the non-proportional increase in cross-links (20-fold) with respect to the decrease in lysyl hydroxylation state of the triple helix (2-fold) even suggests that LH1 preferentially hydroxylates triple helical lysine residues at the cross-link positions. We conclude that minoxidil is unlikely to serve as an anti-fibroticum, but confers features to the collagen matrix, which provide insight into the substrate specificity of LH1.     

High-resolution bioactivity profiling of mixtures toward the acetylcholine binding protein using a nanofractionation spotter technology

This study describes the evaluation, validation, and use of contactless postcolumn fractionation of bioactive mixtures with acetylcholine binding protein (AChBP) affinity analysis with help of a spotter technology. The high-resolution fractionation tailors the fractionation frequency to the chromatographic peaks. Postcolumn reagents for AChBP bioaffinity profiling are mixed prior to droplet ejection into 1536-well plates. After an incubation step, microplate reader analysis is used to determine bioactive compounds in a mixture. For ligands tested, a good correlation was found for IC(50)s determined in flow injection analysis mode when compared with traditional radioligand binding assays. After the evaluation and validation, bioaffinity profiling of actual mixtures was performed. The advantage of this "atline" technology using postcolumn bioaffinity analysis when compared to continuous flow online postcolumn bioaffinity profiling is the possibility to choose postcolumn incubation times freely without compromising resolution due to diffusion effects.     

Vegetation characteristics and eco-hydrological processes in a pristine mire in the Ob River valley (Western Siberia)

Relations between vegetation characteristics and eco-hydrological processes were assessed in a pristine mire in the valley of the Ob River (Western Siberia). Along a transect from the terrace scarp to the river, field data were collected on vegetation composition, peat stratigraphy, peat chemistry, hydrology and hydrochemistry. Based on floristic composition, eight vegetation communities were distinguished. Hydraulic head measurements were used to obtain an indication of groundwater flow directions. The water balance of the mire was calculated with a two-dimensional steady-state numerical groundwater model. Water types were defined based on cluster analysis of hydrochemical data. The results revealed that the dominant hydrological factor in the Ob mire is the discharge of groundwater, which supplies about threefold more water than net precipitation. Although the discharge flux decreases with increasing distance from the terrace scarp, high water levels and a “groundwater-like” mire water composition were observed in the major part of the study site. Precipitation and river water play only a minor role. Despite dilution of discharging groundwater with rainwater, spatial differences in pH and solute concentrations of the surficial mire water are small and not reflected in the vegetation composition. Although small amounts of silt and clay were found in the peat in the proximity of the river, indicating the occurrence of river floods in former times, no river-flood zone could be recognized based on hydrochemical characteristics or vegetation composition. A comparison of the Ob mire with well-studied and near-natural mires in the Biebrza River valley (Poland) revealed substantial differences in both vegetation characteristics and the intensity and spatial pattern of eco-hydrological processes. Differences in the origin and ratios of water fluxes as well as a dissimilar land use history would seem to be key factors explaining the differences observed.     

Diversity analysis of dairy and nondairy Lactococcus lactis isolates, using a novel multilocus sequence analysis scheme and (GTG)5-PCR fingerprinting

The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG)(5)-PCR fingerprinting and a novel multilocus sequence analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both L. lactis subsp. cremoris and L. lactis subsp. lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of nondairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only. MLSA of six partial genes (atpA, encoding ATP synthase alpha subunit; pheS, encoding phenylalanine tRNA synthetase; rpoA, encoding RNA polymerase alpha chain; bcaT, encoding branched chain amino acid aminotransferase; pepN, encoding aminopeptidase N; and pepX, encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. lactis isolates with unique sequence types for most isolates. This allowed high-resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis-based lineages, indicating a disparate acquisition route for this gene.     

R-Ras fills another GAP in semaphorin signalling

Plexins are cell-surface receptors for the semaphorin family of neuronal guidance cues. Following semaphorin binding, the plexin cytoplasmic region initiates poorly understood signal-transduction events that lead to modifications of the cytoskeleton. Recent findings shed new light on the signalling network downstream of semaphorins and plexins by demonstrating that one of the plexins, plexin-B1, possesses an intrinsic GTPase-activating protein (GAP) activity towards R-Ras. Inactivation of R-Ras by the plexin-B1 GAP domains is required for plexin-B1-mediated effects on the cytoskeleton. These results indicate that plexins not only bind to but also regulate directly the activity of some of their downstream effectors.     

Life cycle assessment of aquaculture systems—a review of methodologies

Purpose  

As capture fishery production has reached its limits and global demand for aquatic products is still increasing, aquaculture has become the world’s fastest growing animal production sector. In attempts to evaluate the environmental consequences of this rapid expansion, life cycle assessment (LCA) has become a frequently used method. The present review of current peer-reviewed literature focusing on LCA of aquaculture systems is intended to clarify the methodological choices made, identify possible data gaps, and provide recommendations for future development within this field of research. The results of this review will also serve as a start-up activity of the EU FP7 SEAT (Sustaining Ethical Aquaculture Trade) project, which aims to perform several LCA studies on aquaculture systems in Asia over the next few years.     

Prediction and identification of sequences coding for orphan enzymes using genomic and metagenomic neighbours

Despite the current wealth of sequencing data, one‐third of all biochemically characterized metabolic enzymes lack a corresponding gene or protein sequence, and as such can be considered orphan enzymes. They represent a major gap between our molecular and biochemical knowledge, and consequently are not amenable to modern systemic analyses. As 555 of these orphan enzymes have metabolic pathway neighbours, we developed a global framework that utilizes the pathway and (meta)genomic neighbour information to assign candidate sequences to orphan enzymes. For 131 orphan enzymes (37% of those for which (meta)genomic neighbours are available), we associate sequences to them using scoring parameters with an estimated accuracy of 70%, implying functional annotation of 16 345 gene sequences in numerous (meta)genomes. As a case in point, two of these candidate sequences were experimentally validated to encode the predicted activity. In addition, we augmented the currently available genome‐scale metabolic models with these new sequence–function associations and were able to expand the models by on average 8%, with a considerable change in the flux connectivity patterns and improved essentiality prediction.     

Hepsin regulates TGFβ signaling via fibronectin proteolysis

Transforming growth factor‐beta (TGFβ) is a multifunctional cytokine with a well‐established role in mammary gland development and both oncogenic and tumor‐suppressive functions. The extracellular matrix (ECM) indirectly regulates TGFβ activity by acting as a storage compartment of latent‐TGFβ, but how TGFβ is released from the ECM via proteolytic mechanisms remains largely unknown. In this study, we demonstrate that hepsin, a type II transmembrane protease overexpressed in 70% of breast tumors, promotes canonical TGFβ signaling through the release of latent‐TGFβ from the ECM storage compartment. Mammary glands in hepsin CRISPR knockout mice showed reduced TGFβ signaling and increased epithelial branching, accompanied by increased levels of fibronectin and latent‐TGFβ1, while overexpression of hepsin in mammary tumors increased TGFβ signaling. Cell‐free and cell‐based experiments showed that hepsin is capable of direct proteolytic cleavage of fibronectin but not latent‐TGFβ and, importantly, that the ability of hepsin to activate TGFβ signaling is dependent on fibronectin. Altogether, this study demonstrates a role for hepsin as a regulator of the TGFβ pathway in the mammary gland via a novel mechanism involving proteolytic downmodulation of fibronectin.