Catalytic enzyme activity on a biosensor chip: combination of surface plasmon resonance and mass spectrometry

Surface plasmon resonance (SPR) as a label-free biosensor technique has become an important tool in drug discovery campaigns during the last couple of years. For good assay performance, it is of high interest to verify the functional activity on the immobilization of the target protein on the chip. This study illustrates the verification of the catalytic activity of the drug target protein PqsD by monitoring substrate conversion as a decrease in SPR signal and product detection by ultra high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS(2)). This assay would be applicable to control surface activity of immobilized ligands.     

Draft genome sequence of four coccolithoviruses: Emiliania huxleyi virus EhV-88, EhV-201, EhV-207, and EhV-208

The Coccolithoviridae are a group of viruses which infect the marine coccolithophorid microalga Emiliania huxleyi. The Emiliania huxleyi viruses (known as EhVs) described herein have 160- to 180-nm diameter icosahedral structures, have genomes of approximately 400 kbp, and consist of more than 450 predicted coding sequences (CDSs). Here, we describe the genomic features of four newly sequenced coccolithoviruses (EhV-88, EhV-201, EhV-207, and EhV-208) together with their draft genome sequences and their annotations, highlighting the homology and heterogeneity of these genomes to the EhV-86 model reference genome.     

Semi-automated image analysis for the assessment of megafaunal densities at the Arctic deep-sea observatory HAUSGARTEN

Megafauna play an important role in benthic ecosystem function and are sensitive indicators of environmental change. Non-invasive monitoring of benthic communities can be accomplished by seafloor imaging. However, manual quantification of megafauna in images is labor-intensive and therefore, this organism size class is often neglected in ecosystem studies. Automated image analysis has been proposed as a possible approach to such analysis, but the heterogeneity of megafaunal communities poses a non-trivial challenge for such automated techniques. Here, the potential of a generalized object detection architecture, referred to as iSIS (intelligent Screening of underwater Image Sequences), for the quantification of a heterogenous group of megafauna taxa is investigated. The iSIS system is tuned for a particular image sequence (i.e. a transect) using a small subset of the images, in which megafauna taxa positions were previously marked by an expert. To investigate the potential of iSIS and compare its results with those obtained from human experts, a group of eight different taxa from one camera transect of seafloor images taken at the Arctic deep-sea observatory HAUSGARTEN is used. The results show that inter- and intra-observer agreements of human experts exhibit considerable variation between the species, with a similar degree of variation apparent in the automatically derived results obtained by iSIS. Whilst some taxa (e. g. Bathycrinus stalks, Kolga hyalina, small white sea anemone) were well detected by iSIS (i. e. overall Sensitivity: 87%, overall Positive Predictive Value: 67%), some taxa such as the small sea cucumber Elpidia heckeri remain challenging, for both human observers and iSIS.     

Volatile Organic Compounds in Uremia

Background

Although “uremic fetor” has long been felt to be diagnostic of renal failure, the compounds exhaled in uremia remain largely unknown so far. The present work investigates whether breath analysis by ion mobility spectrometry can be used for the identification of volatile organic compounds retained in uremia.

Methods

Breath analysis was performed in 28 adults with an eGFR ≥60 ml/min per 1.73 m², 26 adults with chronic renal failure corresponding to an eGFR of 10–59 ml/min per 1.73 m², and 28 adults with end-stage renal disease (ESRD) before and after a hemodialysis session. Breath analysis was performed by ion mobility spectrometryafter gas-chromatographic preseparation. Identification of the compounds of interest was performed by thermal desorption gas chromatography/mass spectrometry.

Results

Breath analyses revealed significant differences in the spectra of patients with and without renal failure. Thirteen compounds were chosen for further evaluation. Some compounds including hydroxyacetone, 3-hydroxy-2-butanone and ammonia accumulated with decreasing renal function and were eliminated by dialysis. The concentrations of these compounds allowed a significant differentiation between healthy, chronic renal failure with an eGFR of 10–59 ml/min, and ESRD (p<0.05 each). Other compounds including 4-heptanal, 4-heptanone, and 2-heptanone preferentially or exclusively occurred in patients undergoing hemodialysis.

Conclusion

Impairment of renal function induces a characteristic fingerprint of volatile compounds in the breath. The technique of ion mobility spectrometry can be used for the identification of lipophilic uremic retention molecules.     

Sipunculan larvae and "cosmopolitan" species

Sipuncula is a relatively small taxon with roughly 150 recognized species. Many species are geographically widespread or "cosmopolitan." The pelagosphera larvae of some species are estimated to spend several months in the plankton. However, recent molecular evidence suggests that many of the "cosmopolitan" species actually represent species-complexes, some not even monophyletic. Herein, we present data on three sipunculan species with different developmental modes that occur both in the Sea of Japan and in the Northeast Pacific. The development of the three species-Phascolosoma agassizii, Thysanocardia nigra, and Themiste pyroides-is exceptionally well studied in both regions of the Pacific. Significant differences have been observed between the two regions with respect to egg size, developmental mode, and developmental timing. In general, eggs are larger and development slower in the Northeast Pacific when compared with the Sea of Japan. These differences have been explained as a result of phenotypic plasticity exhibited under different environmental conditions, in particular temperature, but we show that the populations of all three species are also remarkably distinct genetically and that gene flow between the two regions is extremely unlikely. In Thysanocardia nigra, we even found two very distinct genetic lineages within the same location in the Northeast Pacific. The amount of genetic divergence between populations from the Sea of Japan and those from the Northeast Pacific is not correlated with developmental mode. Themiste pyroides, the species with the most abbreviated development, actually has the least degree of genetic divergence between the regions. Analyses of molecular variance show that the majority of the observed variation in all three species is between the regions. We conclude that all three "cosmopolitan" species actually represent complexes of cryptic or pseudo-cryptic species. These examples demonstrate that a solid taxonomic framework based on molecular and morphological evidence is a prerequisite for evaluating relationships between dispersal capabilities, species' ranges, and the connectivity of populations.     

The ATPase cycle mechanism of the DEAD-box rRNA helicase, DbpA

DEAD-box proteins are ATPase enzymes that destabilize and unwind duplex RNA. Quantitative knowledge of the ATPase cycle parameters is critical for developing models of helicase activity. However, limited information regarding the rate and equilibrium constants defining the ATPase cycle of RNA helicases is available, including the distribution of populated biochemical intermediates, the catalytic step(s) that limits the enzymatic reaction cycle, and how ATP utilization and RNA interactions are linked. We present a quantitative kinetic and equilibrium characterization of the ribosomal RNA (rRNA)-activated ATPase cycle mechanism of DbpA, a DEAD-box rRNA helicase implicated in ribosome biogenesis. rRNA activates the ATPase activity of DbpA by promoting a conformational change after ATP binding that is associated with hydrolysis. Chemical cleavage of bound ATP is reversible and occurs via a γ-phosphate attack mechanism. ADP-P_i and RNA binding display strong thermodynamic coupling, which causes DbpA-ADP-P_i to bind rRNA with > 10-fold higher affinity than with bound ATP, ADP or in the absence of nucleotide. The rRNA-activated steady-state ATPase cycle of DbpA is limited both by ATP hydrolysis and by P_i release, which occur with comparable rates. Consequently, the predominantly populated biochemical states during steady-state cycling are the ATP- and ADP-P_i-bound intermediates. Thermodynamic linkage analysis of the ATPase cycle transitions favors a model in which rRNA duplex destabilization is linked to strong rRNA and nucleotide binding. The presented analysis of the DbpA ATPase cycle reaction mechanism provides a rigorous kinetic and thermodynamic foundation for developing testable hypotheses regarding the functions and molecular mechanisms of DEAD-box helicases.     

Investigating the mechanisms of photosynthetic proteins using continuum electrostatics

Computational methods based on continuum electrostatics are widely used in theoretical biochemistry to analyze the function of proteins. Continuum electrostatic methods in combination with quantum chemical and molecular mechanical methods can help to analyze even very complex biochemical systems. In this article, applications of these methods to proteins involved in photosynthesis are reviewed. After giving a short introduction to the basic concepts of the continuum electrostatic model based on the Poisson-Boltzmann equation, we describe the application of this approach to the docking of electron transfer proteins, to the comparison of isofunctional proteins, to the tuning of absorption spectra, to the analysis of the coupling of electron and proton transfer, to the analysis of the effect of membrane potentials on the energetics of membrane proteins, and to the kinetics of charge transfer reactions. Simulations as those reviewed in this article help to analyze molecular mechanisms on the basis of the structure of the protein, guide new experiments, and provide a better and deeper understanding of protein functions.     

Isolation and biological activity of new norhirsutanes from Creolophus cirrhatus

Five new norhirsutanes, named creolophins A-E, and complicatic acid were isolated from the culture broth of the rare tooth fungus Creolophus cirrhatus by solvent extraction, silica gel column chromatography and HPLC. In addition, neocreolophin, a complex dimerization product, was formed as an artefact during purification. The structures were elucidated by spectroscopic methods and are published in a separate paper. Two of the metabolites showed moderate antibacterial, antifungal and cytotoxic activities.     

Par-complex proteins promote proliferative progenitor divisions in the developing mouse cerebral cortex

The size of brain regions depends on the balance between proliferation and differentiation. During development of the mouse cerebral cortex, ventricular zone (VZ) progenitors, neuroepithelial and radial glial cells, enlarge the progenitor pool by proliferative divisions, while basal progenitors located in the subventricular zone (SVZ) mostly divide in a differentiative mode generating two neurons. These differences correlate to the existence of an apico-basal polarity in VZ, but not SVZ, progenitors. Only VZ progenitors possess an apical membrane domain at which proteins of the Par complex are strongly enriched. We describe a prominent decrease in the amount of Par-complex proteins at the apical surface during cortical development and examine the role of these proteins by gain- and loss-of-function experiments. Par3 (Pard3) loss-of-function led to premature cell cycle exit, reflected in reduced clone size in vitro and the restriction of the progeny to the lower cortical layers in vivo. By contrast, Par3 or Par6 (Pard6alpha) overexpression promoted the generation of Pax6+ self-renewing progenitors in vitro and in vivo and increased the clonal progeny of single progenitors in vitro. Time-lapse video microscopy revealed that a change in the mode of cell division, rather than an alteration of the cell cycle length, causes the Par-complex-mediated increase in progenitors. Taken together, our data demonstrate a key role for the apically located Par-complex proteins in promoting self-renewing progenitor cell divisions at the expense of neurogenic differentiation in the developing cerebral cortex.