Prolonged conversion of n-butyrate to n-butanol with Clostridium saccharoperbutylacetonicum in a two-stage continuous culture with in-situ product removal

n‐Butanol was produced continuously in a two‐stage fermentor system with integrated product removal from a co‐feed of n‐butyric acid and glucose. Glucose was always required as a source of ATP and electrons for the conversion of n‐butyrate to n‐butanol and for biomass growth; for the latter it also served as a carbon source. The first stage generated metabolically active planktonic cells of Clostridium saccharoperbutylacetonicum strain N1‐4 that were continuously fed into the second (production) stage; the volumetric ratio of the two fermentors was 1:10. n‐Butanol was removed continuously from the second stage via gas stripping. Implementing a two‐stage process was observed to dramatically dampen metabolic oscillations (i.e., periodical changes of solventogenic activity). Culture degeneration (i.e., an irreversible loss of solventogenic activity) was avoided by periodical heat shocking and re‐inoculating stage 1 and by maintaining the concentration of undissociated n‐butyric acid in stage 2 at 3.4 mM with a pH‐auxostat. The system was successfully operated for 42 days during which 93% of the fed n‐butyrate was converted to n‐butanol at a production rate of 0.39 g/(L × h). The molar yields Y_{n‐butanol/n‐butyrate} and Y_{n‐butanol/glucose} were 2.0, and 0.718, respectively. For the same run, the molar ratio of n‐butyrate to glucose consumed was 0.358. The molar yield of carbon in n‐butanol produced from carbon in n‐butyrate and glucose consumed (Y_{n‐butanol/carbon}) was 0.386. These data illustrate that conversion of n‐butyrate into n‐butanol by solventogenic Clostridium species is feasible and that this can be performed in a continuous system operating for longer than a month. However, our data also demonstrate that a relatively large amount of glucose is required to supply electrons and ATP for this conversion and for cell growth in a continuous culture. Biotechnol. Bioeng. 2012; 109:913–921. © 2011 Wiley Periodicals, Inc.     

Mass spectrometry-based proteomics for translational research: a technical overview

Mass spectrometry-based investigation of clinical samples enables the high-throughput identification of protein biomarkers. We provide an overview of mass spectrometry-based proteomic techniques that are applicable to the investigation of clinical samples. We address sample collection, protein extraction and fractionation, mass spectrometry modalities, and quantitative proteomics. Finally, we examine the limitations and further potential of such technologies. Liquid chromatography fractionation coupled with tandem mass spectrometry is well suited to handle mixtures of hundreds or thousands of proteins. Mass spectrometry-based proteome elucidation can reveal potential biomarkers and aid in the development of hypotheses for downstream investigation of the molecular mechanisms of disease.     

Evaporation effects as reflected in freshwaters and ostracod calcite from modern environments in Central and Northeast Yakutia (East Siberia,Russia)

Taxonomical and geochemical investigations on freshwater ostracods from 15 waters in Central and Northeast (NE) Yakutia have been undertaken in order to estimate their potential usefulness in palaeoenvironmental reconstructions based on regional fossil records. Higher variability in environmental factors such as pH, electrical conductivity, and ionic content was observed in thermokarst-affected lakes in Central Yakutia than in NE Yakutia lakes. Species diversity of freshwater ostracods reached up to eight taxa per lake, mostly dominated by Candona weltneri Hartwig 1899, in Central Yakutia, whereas in NE Yakutian waters the diversity was lower and Candona muelleri jakutica Pietrzeniuk 1977 or Fabaeformiscandona inaequivalvis (Sars 1898) had highest frequencies. Coupled analyses of stable isotopes (δ¹⁸O, δ¹³C) and element ratios (Sr/Ca, Mg/Ca) were performed on both host waters and ostracod calcite, aiming to estimate the modern relationships. Correlations between host waters and ostracod calcite of single species were found for δ¹⁸O, δ¹³C and Sr/Ca and Mg/Ca ratios. The relationships between δ¹⁸O, Mg/Ca and Sr/Ca ratios and electrical conductivity (salinity) as an expression of solute concentrations in the waters mainly controlled by evaporation are more complicated but evident, and may be useful in future interpretation of geochemical data from fossil Siberian ostracods. Handling editor: K. Martens     

Towards humane end points: behavioural changes precede clinical signs of disease in a Huntington's disease model

The number of animals used in science is increasing, bringing a concomitant obligation to minimize suffering. For animals with progressive conditions, euthanasia at a 'humane end point' is advised if the end point is scientifically valid, predictive and accurate. Our aim was to test the hypothesis that behavioural changes would reliably precede clinical signs of disease in a progressive neurological model, using retrospective analysis. We observed 100 pair-housed female R6/1 transgenic Huntington's disease (HD) mice and 28 pair-housed female wild-type (WT) mice in standard- or resource-enriched cages. Disease progression was monitored until one member of each HD pair reached a pre-defined end point based on pathological symptoms (HD end). This mouse was then euthanized together with its cage mate (HD other) and any matched WT pairs. At euthanasia, HD mice had significantly greater absolute and relative organ weights, and significantly higher alpha1 acid glycoprotein concentrations than WT mice, indicating reduced welfare. HD mice initially showed significantly greater use of cage resources than WT mice but this declined progressively. Steeper declines, and earlier cessation, in the use of some climbing and exploration resources occurred in the HD end mice compared with the HD other mice. Behavioural change can be an early indicator of disease onset.     

A phylogeny of the oil bee tribe Ctenoplectrini (Hymenoptera: Anthophila) based on mitochondrial and nuclear data: evidence for early Eocene divergence and repeated out-of-Africa dispersal

The bee tribe Ctenoplectrini, with two genera, comprises nine species in tropical Africa and ten in Asia and Australia. Most of them collect floral oil, pollen, and nectar from Cucurbitaceae, but three species are thought to be cleptoparasites. The unusual morphology of Ctenoplectrini has made it difficult to infer their closest relatives, in turn preventing an understanding of these bees' geographic and temporal origin. We used two mitochondrial and two nuclear markers (4741 nucleotides) generated for most of the species to test the monophyly of the tribe, its relationships to other Apidae, and its biogeographic history. Ctenoplectrini are strongly supported as monophyletic and closest to the Long-horned bees, Eucerini. The presumably cleptoparasitic species form a clade (Ctenoplectrina) that is sister to the remaining species (Ctenoplectra), confirming the independent evolution of cleptoparasitism in this tribe. Tree topology and molecular dating together suggest that Ctenoplectrini originated in Africa in the Early Eocene and that Ctenoplectra dispersed twice from Africa to Asia, sometime in the Late Eocene, 30-40 my ago, from where one species reached the Australian continent via Indonesia and New Guinea in the mid-Miocene, c. 13 my ago. Dry and cool mid-Miocene climates also coincide with the divergence between Ctenoplectra bequaerti from West Africa and Ctenoplectra terminalis from East and South Africa, perhaps related to fragmentation of the equatorial African rainforest belt.     

NITPICK: peak identification for mass spectrometry data

Background  

The reliable extraction of features from mass spectra is a fundamental step in the automated analysis of proteomic mass spectrometry (MS) experiments.     

Robust prediction of the MASCOT score for an improved quality assessment in mass spectrometric proteomics

Protein identification by tandem mass spectrometry is based on the reliable processing of the acquired data. Unfortunately, the generation of a large number of poor quality spectra is commonly observed in LC-MS/MS, and the processing of these mostly noninformative spectra with its associated costs should be avoided. We present a continuous quality score that can be computed very quickly and that can be considered an approximation of the MASCOT score in case of a correct identification. This score can be used to reject low quality spectra prior to database identification, or to draw attention to those spectra that exhibit a (supposedly) high information content, but could not be identified. The proposed quality score can be calibrated automatically on site without the need for a manually generated training set. When this score is turned into a classifier and when features are used that are independent of the instrument, the proposed approach performs equally to previously published classifiers and feature sets and also gives insights into the behavior of the MASCOT score.     

Spatio-temporal quantification of differential growth processes in root growth zones based on a novel combination of image sequence processing and refined concepts describing curvature production

Differential growth processes in root and shoot growth zones are governed by the transport kinetics of auxin and other plant hormones. While gene expression and protein localization of hormone transport facilitators are currently being unraveled using state-of-the-art techniques of live cell imaging, the quantitative analysis of growth reactions is lagging behind because of a lack of suitable methods. A noninvasive technique, based on digital image sequence processing, for visualizing and quantifying highly resolved spatio-temporal root growth processes was applied in the model plant Arabidopsis thaliana and was adapted to provide precise information on differential curvature production activity within the root growth zone. Comparison of root gravitropic curvature kinetics in wild-type and mutant plants altered in a facilitator for auxin translocation allowed the determination of differences in the location and in the temporal response of curvature along the growth zone between the investigated plant lines. The findings of the quantitative growth analysis performed here confirm the proposed action of the investigated transport facilitator. The procedure developed here for the investigation of differential growth processes is a valuable tool for characterizing the phenomenology of a wide range of shoot and root growth movements and hence facilitates elucidation of their molecular characterization.     

Photosynthesis can be enhanced by lateral CO2 diffusion inside leaves over distances of several millimeters

This study examines the extent to which lateral gas diffusion can influence intercellular CO(2) concentrations (c(i)) and thus photosynthesis in leaf areas with closed stomata. Leaves were partly greased to close stomata artificially, and effects of laterally diffusing CO(2) into the greased areas were studied by gas-exchange measurement and chlorophyll fluorescence imaging. Effective quantum yields (Delta F/F(m)') across the greased areas were analysed with an image-processing tool and transposed into c(i) profiles, and lateral CO(2) diffusion coefficients (D(C'lat)), directly proportional to lateral conductivities (), were estimated using a one-dimensional (1D) diffusion model. Effective CO(2) diffusion distances in Vicia faba (homobaric), Commelina vulgaris (homobaric) and Phaseolus vulgaris (heterobaric) leaves clearly differed, and were dependent on D(C'lat), light intensity, [CO(2)], and [O(2)]: largest distances were approx. 7.0 mm for homobaric leaves (with high D(C'lat)) and approx. 1.9 mm for heterobaric leaves (low D(C'lat)). Modeled lateral CO(2) fluxes indicate large support of photosynthesis over submillimeter distances for leaves with low D(C'lat), whereas in leaves with large D(C'lat), photosynthesis can be stimulated over distances of several millimeters. For the plant species investigated, the surplus CO(2) assimilation rates of the greased leaf areas (A(gr)) differed clearly, depending on lateral conductivities of the respective leaves.     

Tyrosine phosphorylation mapping of the epidermal growth factor receptor signaling pathway.

Phosphorylation is one of the most common forms of protein modification. The most frequent targets for protein phosphorylation in eukaryotes are serine and threonine residues, although tyrosine residues also undergo phosphorylation. Many of the currently applied methods for the detection and localization of protein phosphorylation sites are mass spectrometry-based and are biased against the analysis of tyrosine-phosphorylated residues because of the stability and low reactivity of phosphotyrosines. To overcome this lack of sensitive methods for the detection of phosphotyrosine-containing peptides, we have recently developed a method that is not affected by the more predominant threonine or serine phosphorylation within cells. It is based on the specific detection of immonium ion of phosphotyrosine at 216.043 Da and does not require prior knowledge of the protein sequence. In this report, we describe the first application of this new method in a proteomic strategy. Using anti-phosphotyrosine antibodies for immunoprecipitation and one-dimensional gel electrophoresis, we have identified 10 proteins in the epidermal growth factor receptor signaling pathway, of which 8 have been shown previously to be involved in epidermal growth factor signaling. Most importantly, in addition to several known tyrosine phosphorylation sites, we have identified five novel sites on SHIP-2, Hrs, Cbl, STAM, and STAM2, most of which were not predicted to be phosphorylated. Because of its sensitivity and selectivity, this approach will be useful in proteomic approaches to study tyrosine phosphorylation in a number of signal transduction pathways.