The cyclin-dependent kinase inhibitor Roughex is involved in mitotic exit in Drosophila

Background: Exit from mitosis is a tightly regulated event. This process has been studied in greatest detail in budding yeast, where several activities have been identified that cooperate to downregulate activity of the cyclin-dependent kinase (CDK) Cdc28 and force an exit from mitosis. Cdc28 is inactivated through proteolysis of B-type cyclins by the multisubunit ubiquitin ligase termed the anaphase promoting complex/cyclosome (APC/C) and inhibition by the cyclin-dependent kinase inhibitor (CKI) Sic1. In contrast, the only mechanism known to be essential for CDK inactivation during mitosis in higher eukaryotes is cyclin destruction.Results: We now present evidence that the Drosophila CKI Roughex (Rux) contributes to exit from mitosis. Observations of fixed and living embryos show that metaphase is significantly longer in rux mutants than in wild-type embryos. In addition, Rux overexpression is sufficient to drive cells experimentally arrested in metaphase into interphase. Furthermore, rux mutant embryos are impaired in their ability to overcome a transient metaphase arrest induced by expression of a stable cyclin A. Rux has numerous functional similarities with Sic1. While these proteins share no sequence similarity, we show that Sic1 inhibits mitotic Cdk1-cyclin complexes from Drosophila in vitro and in vivo.Conclusions: Rux inhibits Cdk1-cyclin A kinase activity during metaphase, thereby contributing to exit from mitosis. To our knowledge, this is the first mitotic function ascribed to a CKI in a multicellular organism and indicates the existence of a novel regulatory mechanism for the metaphase to anaphase transition during development.     

Comparison of ERLIC-TiO2, HILIC-TiO2, and SCX-TiO2 for global phosphoproteomics approaches

Reversible phosphorylations play a critical role in most biological pathways. Hence, in signaling studies great effort has been put into identification of a maximum number of phosphosites per experiment. Mass spectrometry (MS)-based phosphoproteomics approaches have been proven to be an ideal analytical method for mapping of phosphosites. However, because of sample complexity, fractionation of phosphopeptides prior to MS analysis is a crucial step. In the current study, we compare the chromatographic strategies electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), hydrophilic interaction liquid chromatography (HILIC), and strong cation exchange chromatography (SCX) for their fractionation behavior of phosphopeptides. In addition, we investigate the use of repetitive TiO(2)-based enrichment steps for a maximum identification of phosphopeptides. On the basis of our results, SCX yields the highest number of identified phosphopeptides, whereas ERLIC is optimal for the identification of multiphosphorylated peptides. Consecutive incubations of fractions and flow-through by TiO(2) beads enrich qualitatively different sets of phosphopeptides, increasing the number of identified phosphopeptides per analysis.     

Combinatorial Use of Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) and Strong Cation Exchange (SCX) Chromatography for In-Depth Phosphoproteome Analysis

In large-scale phosphoproteomics studies, fractionation by strong cation exchange (SCX) or electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) is commonly used to reduce sample complexity, fractionate phosphopeptides from their unmodified counterparts, and increase the dynamic range for phosphopeptide identification. However, these procedures do not succeed to separate, both singly and multiply phosphorylated peptides due to their inverse physicochemical characteristics. Hence, depending on the chosen method only one of the two peptide classes can be efficiently separated. Here, we present a novel strategy based on the combinatorial separation of singly and multiply phosphorylated peptides by SCX and ERLIC for in-depth phosphoproteome analysis. In SCX, mostly singly phosphorylated peptides are retained and fractionated while not-retained multiply phosphorylated peptides are fractionated in a subsequent ERLIC approach (SCX-ERLIC). In ERLIC, multiply phosphorylated peptides are fractionated, while not-retained singly phosphorylated peptides are separated by SCX (ERLIC-SCX). Compared to single step fractionations by SCX, the combinatorial strategies, SCX-ERLIC and ERLIC-SCX, yield up to 48% more phosphopeptide identifications as well as a strong increase in the number of detected multiphosphorylated peptides. Phosphopeptides identified in two subsequent, complementary fractionations had little overlap (5%) indicating that ERLIC and SCX are orthogonal methods ideally suited for in-depth phosphoproteome studies.     

Microbial stratification in low pH oxic and suboxic macroscopic growths along an acid mine drainage

Macroscopic growths at geographically separated acid mine drainages (AMDs) exhibit distinct populations. Yet, local heterogeneities are poorly understood. To gain novel mechanistic insights into this, we used OMICs tools to profile microbial populations coexisting in a single pyrite gallery AMD (pH ∼2) in three distinct compartments: two from a stratified streamer (uppermost oxic and lowermost anoxic sediment-attached strata) and one from a submerged anoxic non-stratified mat biofilm. The communities colonising pyrite and those in the mature formations appear to be populated by the greatest diversity of bacteria and archaea (including ‘ARMAN'' (archaeal Richmond Mine acidophilic nano-organisms)-related), as compared with the known AMD, with ∼44.9% unclassified sequences. We propose that the thick polymeric matrix may provide a safety shield against the prevailing extreme condition and also a massive carbon source, enabling non-typical acidophiles to develop more easily. Only 1 of 39 species were shared, suggesting a high metabolic heterogeneity in local microenvironments, defined by the O₂ concentration, spatial location and biofilm architecture. The suboxic mats, compositionally most similar to each other, are more diverse and active for S, CO₂, CH₄, fatty acid and lipopolysaccharide metabolism. The oxic stratum of the streamer, displaying a higher diversity of the so-called ‘ARMAN''-related Euryarchaeota, shows a higher expression level of proteins involved in signal transduction, cell growth and N, H₂, Fe, aromatic amino acids, sphingolipid and peptidoglycan metabolism. Our study is the first to highlight profound taxonomic and functional shifts in single AMD formations, as well as new microbial species and the importance of H₂ in acidic suboxic macroscopic growths.     

Parallelized small-scale production of uniformly C-labeled cell extract for quantitative metabolome analysis

The need for quantitative intracellular metabolome information is central to modern applied biotechnology and systems biology. In most cases, sample preparation and metabolite analysis result in degradation of metabolites and signal suppression due to metabolite instability and matrix effects during LC–MS analysis. Therefore the application of uniformly (U) ¹³C-labeled cell extract as an internal standard has gained interest in recent years. In this study a multiple-step protocol has been developed for efficient preparation of U-¹³C-labeled Escherichia coli cell extracts in stirred-tank bioreactors on a milliliter scale with a minimal supply of costly ¹³C-labeled substrate. Significant reduction of fermentation medium salt concentration in the U-¹³C-labeled cell extract was achieved to reduce ion-suppression effects during mass-spectrometric analysis. Additionally, variation of reaction conditions in parallel-operated stirred-tank bioreactors on a milliliter scale enables the simultaneous preparation of U-¹³C-labeled cell extracts with varying metabolite concentrations, which is shown by an example of the labeled phosphoenolpyruvate level in E. coli.     

PROFICS: A bacterial selection system for directed evolution of proteases

Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial selection system, which enables the optimization of proteases for biotechnology, therapeutics or diagnosis in a simple overnight process. During the PROFICS process, proteases are selected for their ability to specifically cut a tag from a reporter enzyme and leave a native N-terminus. Precise and efficient cleavage after the recognition sequence reverses the phenotype of an Escherichia coli knockout strain deficient in an essential enzyme of pyrimidine synthesis. A toolbox was generated to select for proteases with different preferences for P1′ residues (the residue immediately following the cleavage site). The functionality of PROFICS is demonstrated with viral proteases and human caspase-2. PROFICS improved caspase-2 activity up to 25-fold after only one round of mutation and selection. Additionally, we found a significantly improved tolerance for all P1′ residues caused by a mutation in a substrate interaction site. We showed that this improved activity enables cells containing the new variant to outgrow cells containing all other mutants, facilitating its straightforward selection. Apart from optimizing enzymatic activity and P1′ tolerance, PROFICS can be used to reprogram specificities, erase off-target activity, optimize expression via tags/codon usage, or even to screen for potential drug-resistance-conferring mutations in therapeutic targets such as viral proteases in an unbiased manner.     

Probiotic modulation of symbiotic gut microbial–host metabolic interactions in a humanized microbiome mouse model

The transgenomic metabolic effects of exposure to either Lactobacillus paracasei or Lactobacillus rhamnosus probiotics have been measured and mapped in humanized extended genome mice (germ‐free mice colonized with human baby flora). Statistical analysis of the compartmental fluctuations in diverse metabolic compartments, including biofluids, tissue and cecal short‐chain fatty acids (SCFAs) in relation to microbial population modulation generated a novel top‐down systems biology view of the host response to probiotic intervention. Probiotic exposure exerted microbiome modification and resulted in altered hepatic lipid metabolism coupled with lowered plasma lipoprotein levels and apparent stimulated glycolysis. Probiotic treatments also altered a diverse range of pathways outcomes, including amino‐acid metabolism, methylamines and SCFAs. The novel application of hierarchical‐principal component analysis allowed visualization of multicompartmental transgenomic metabolic interactions that could also be resolved at the compartment and pathway level. These integrated system investigations demonstrate the potential of metabolic profiling as a top‐down systems biology driver for investigating the mechanistic basis of probiotic action and the therapeutic surveillance of the gut microbial activity related to dietary supplementation of probiotics.     

Larva-adult relationships in an ancestral dipteran: A re-examination of sensillar pathways across the antenna and leg anlagen of Chaoborus crystallinus (DeGeer, 1776; Chaoboridae)

 In one of his classical studies on insect metamorphosis, Weismann compared the imaginal anlagen of the ancestral phantom midge, Chaoborus, with those of advanced brachycerans. We have expanded his findings on the relationships between larval and imaginal organs using electron microscopy and cobalt backfilling of the antenna and leg anlagen and the axonal trajectories of corresponding larval sensilla. We show that both primordia are confluent with the larval antennae and ”leg” sensilla (an ancestral Keilin organ), respectively. These fully developed larval organs represent the distal tips of the imaginal anlagen rather than separate cell clusters. The axons of the larval antenna and leg sensilla project across the corresponding anlagen to their target neuromeres within the central nervous system (CNS). Within the discs, nerves composed of these larval axons, developing afferent fibres and efferences ascending from the CNS are found. Both the structure of the primordia and the axonal trajectories thus relate the situation found in advanced brachycerans with that seen in more ancestral insects. In addition, the larval antennae, legs, wings and even the eyes possess very similar afferent pioneer trajectories supporting the idea that the described pattern is generally used in the ontogeny of sensory systems. Received: 30 June 1998 / Accepted: 27 September 1998     

Process control for enhanced L-phenylalanine production using different recombinant Escherichia coli strains

A novel fed-batch approach for the production of L-phenylalanine (L-Phe) with recombinant E. coli is presented concerning the on-line control of the key fermentation parameters glucose and tyrosine. Two different production strains possessing either the tyrosine feedback resistant aroF(fbr) (encoding tyrosine feedback resistant DAHP-synthase (3-desoxy-D-arabino-heptusonate-7-phosphate)) or the wild-type aroF(wt) were used as model systems to elucidate the necessity of finding an individual process optimum for each genotype. With the aid of tyrosine control, wild-type aroF(wt) could be used for L-Phe production achieving higher final L-Phe titers (34 g/L) than the aroF(fbr) strain (28 g/L) and providing higher DAHP-synthase activities. With on-line glucose control, an optimum glucose concentration of 5 g/L could be identified that allowed a sufficient carbon supply for L-Phe production while at the same time an overflow metabolism leading to acetate by-product formation was avoided. The process approach is suitable for other production strains not only in lab-scale but also in pilot-scale bioreactors.