Taxonomy and biogeography of <Emphasis Type="Italic">Niphargus steueri</Emphasis> (Crustacea: Amphipoda)

Niphargus steueri (Niphargidae) comprises a complex of four subspecies (N. s. steueri, N. s. kolombatovici, N. s. subtypicus, and N. s. liburnicus), the morphology and distribution of which have been poorly known until now. New diagnostic characters of the species and its four subspecies are presented and illustrated. The species is distributed along the major part of the Dinaric Karst, between Slovenia in the northwest and Bosnia and Herzegovina in the southeast. The distribution of the four subspecies approximately resembles the distribution of the evolutionary lineages of the subterranean amphibian Proteus anguinus and the Dinaric lineage of the cave shrimp Troglocaris agg. anophthalmus. Niphargus s. steueri is restricted to the Istran Peninsula; N. s. subtypicus is distributed in southeast Slovenia and northwest Croatia; N. s. liburnicus is known from two disjunctive localities, one on the island of Krk (Croatia) and the other in Gorizia (Italy); and N. s. kolombatovici is restricted to Dalmacija and Herzegovina. The somewhat variable putative synapomorphies of N. steueri probably suggest that the group is old and that the present distribution pattern is a result of historical events, possibly events in the Miocene Dinaride Lake system. Two populations of N. s. kolombatovici and one population of N. s. subtypicus deviate from the general distributional pattern and may belong to cryptic taxa that cannot be distinguished on the basis of morphology. Both hypotheses corroborate with the estimated times of divergence and with the number of independent lineages in the similarly distributed but unrelated stygobionts Proteus and Troglocaris.     

Iron enriched yeast biomass--a promising mineral feed supplement

Yeast biomass enriched with iron could represent a new and safer solution for prevention from anaemia development. Such an iron source is less toxic and has better absorbability in organisms. The purpose of our research was the determination of the most suitable iron source in the cultivation medium for the yeast Saccharomyces cerevisiae, regarding good growth and iron accumulation in cells. Iron(III) citrate, iron(III) chloride, iron(III) nitrate and Fe-EDTA complex were used. The uptake of the chosen iron compound, Fe(III) citrate, by the yeasts Candida intermedia and Kluyveromyces marxianus was also investigated. Different growth behaviour of the three yeast strains in the presence of Fe(III) citrate was observed. The highest amounts of accumulated iron in S. cerevisiae, C. intermedia and K. marxianus biomass were about 13, 20 and 34mgFeg(-1)dry wt., respectively. To optimise the accumulation of iron in K. marxianus and to characterise iron enriched yeast biomass, further experiments are needed.     

Modeling fatty acid delivery from intestinal fatty acid binding protein to a membrane

Intestinal fatty acid binding protein (IFABP) interacts with biological membranes and delivers fatty acid (FA) into them via a collisional mechanism. However, the membrane-bound structure of the protein and the pathway of FA transfer are not precisely known. We used molecular dynamics (MD) simulations with an implicit membrane model to determine the optimal orientation of apo- and holo-IFABP (bound with palmitate) on an anionic membrane. In this orientation, the helical portal region, delimited by the alphaII helix and the betaC-betaD and betaE-betaF turns, is oriented toward the membrane whereas the putative beta-strand portal, delimited by the betaB-betaC, betaF-betaG, betaH-betaI turns and the N terminus, is exposed to solvent. Starting from the MD structure of holo-IFABP in the optimal orientation relative to the membrane, we examined the release of palmitate via both pathways. Although the domains can widen enough to allow the passage of palmitate, fatty acid release through the helical portal region incurs smaller conformational changes and a lower energetic cost.     

The SPFH domain-containing proteins: more than lipid raft markers

Membrane microdomains with distinct lipid compositions, called lipid rafts, represent a potential mechanism for compartmentalizing cellular functions within the plane of biological membranes. SPFH domain-containing proteins are found in lipid raft microdomains in diverse cellular membranes. The functions of these proteins are just beginning to be elucidated. Recent advances in the understanding of structural features and their roles within lipid rafts include a potential function for SPFH proteins in the formation of membrane microdomains and lipid raft-associated processes, such as endocytosis and mechanosensation.     

Metabolomic profiling of CHO fed-batch growth phases at 10, 100, and 1,000 L

Established bioprocess monitoring is based on quick and reliable methods, including cell count and viability measurement, extracellular metabolite measurement, and the measurement of physicochemical qualities of the cultivation medium. These methods are sufficient for monitoring of process performance, but rarely give insight into the actual physiological states of the cell culture. However, understanding of the latter is essential for optimization of bioprocess development. Our study used LC-MS metabolomics as a tool for additional resolution of bioprocess monitoring and was designed at three bioreactors scales (10 L, 100 L, and 1,000 L) to gain insight into the basal metabolic states of the Chinese hamster ovary (CHO) cell culture during fed-batch. Metabolites characteristics of the four growth stages (early and late exponential phase, stationary phase, and the phase of decline) were identified by multivariate analysis. Enriched metabolic pathways were then established for each growth phase using the CHO metabolic network model. Biomass generation and nucleotide synthesis were enriched in early exponential phase, followed by increased protein production and imbalanced glutathione metabolism in late exponential phase. Glycolysis became downregulated in stationary phase and amino-acid metabolism increased. Phase of culture decline resulted in rise of oxidized glutathione and fatty acid concentrations. Intracellular metabolic profiles of the CHO fed-batch culture were also shown to be consistent with scale and thus demonstrate metabolomic profiling as an informative method to gain physiological insight into the cell culture states during bioprocess regardless of scale.     

Inactivation of Lgt allows systematic characterization of lipoproteins from Listeria monocytogenes

Lipoprotein anchoring in bacteria is mediated by the prolipoprotein diacylglyceryl transferase (Lgt), which catalyzes the transfer of a diacylglyceryl moiety to the prospective N-terminal cysteine of the mature lipoprotein. Deletion of the lgt gene in the gram-positive pathogen Listeria monocytogenes (i) impairs intracellular growth of the bacterium in different eukaryotic cell lines and (ii) leads to increased release of lipoproteins into the culture supernatant. Comparative extracellular proteome analyses of the EGDe wild-type strain and the Delta lgt mutant provided systematic insight into the relative expression of lipoproteins. Twenty-six of the 68 predicted lipoproteins were specifically released into the extracellular proteome of the Delta lgt strain, and this proved that deletion of lgt is an excellent approach for experimental verification of listerial lipoproteins. Consequently, we generated Delta lgt Delta prfA double mutants to detect lipoproteins belonging to the main virulence regulon that is controlled by PrfA. Overall, we identified three lipoproteins whose extracellular levels are regulated and one lipoprotein that is posttranslationally modified depending on PrfA. It is noteworthy that in contrast to previous studies of Escherichia coli, we unambiguously demonstrated that lipidation by Lgt is not a prerequisite for activity of the lipoprotein-specific signal peptidase II (Lsp) in Listeria.     

Pathway-based approaches for analysis of genomewide association studies

Published genomewide association (GWA) studies typically analyze and report single-nucleotide polymorphisms (SNPs) and their neighboring genes with the strongest evidence of association (the “most-significant SNPs/genes” approach), while paying little attention to the rest. Borrowing ideas from microarray data analysis, we demonstrate that pathway-based approaches, which jointly consider multiple contributing factors in the same pathway, might complement the most-significant SNPs/genes approach and provide additional insights into interpretation of GWA data on complex diseases.     

Discrimination and scoring using small sets of genes for two-sample microarray data

Comparison of gene expression for two groups of individuals form an important subclass of microarray experiments. We study multivariate procedures, in particular use of Hotelling's T2 for discrimination between the groups with a special emphasis on methods based on few genes only. We apply the methods to data from an experiment with a group of atopic dermatitis patients compared with a control group. We also compare our methodology to other recently proposed methods on publicly available datasets. It is found that (i) use of several genes gives a much improved discrimination of the groups as compared to one gene only, (ii) the genes that play the most important role in the multivariate analysis are not necessarily those that rank first in univariate comparisons of the groups, (iii) Linear Discriminant Analysis carried out with sets of 2-5 genes selected according to their Hotelling T2 give results comparable to state-of-the-art methods using many more genes, a feature of our method which might be crucial in clinical applications. Finding groups of genes that together give optimal multivariate discrimination (given the size of the group) can identify crucial pathways and networks of genes responsible for a disease. The computer code that we developed to make computations is available as an R package.