Lack of Electricity Production by Pelobacter carbinolicus Indicates that the Capacity for Fe(III) Oxide Reduction Does Not Necessarily Confer Electron Transfer Ability to Fuel Cell Anodes

The ability of Pelobacter carbinolicus to oxidize electron donors with electron transfer to the anodes of microbial fuel cells was evaluated because microorganisms closely related to Pelobacter species are generally abundant on the anodes of microbial fuel cells harvesting electricity from aquatic sediments. P. carbinolicus could not produce current in a microbial fuel cell with electron donors which support Fe(III) oxide reduction by this organism. Current was produced using a coculture of P. carbinolicus and Geobacter sulfurreducens with ethanol as the fuel. Ethanol consumption was associated with the transitory accumulation of acetate and hydrogen. G. sulfurreducens alone could not metabolize ethanol, suggesting that P. carbinolicus grew in the fuel cell by converting ethanol to hydrogen and acetate, which G. sulfurreducens oxidized with electron transfer to the anode. Up to 83% of the electrons available in ethanol were recovered as electricity and in the metabolic intermediate acetate. Hydrogen consumption by G. sulfurreducens was important for ethanol metabolism by P. carbinolicus. Confocal microscopy and analysis of 16S rRNA genes revealed that half of the cells growing on the anode surface were P. carbinolicus, but there was a nearly equal number of planktonic cells of P. carbinolicus. In contrast, G. sulfurreducens was primarily attached to the anode. P. carbinolicus represents the first Fe(III) oxide-reducing microorganism found to be unable to produce current in a microbial fuel cell, providing the first suggestion that the mechanisms for extracellular electron transfer to Fe(III) oxides and fuel cell anodes may be different.     

A multi-locus chloroplast phylogeny for the Cucurbitaceae and its implications for character evolution and classification

Cucurbitaceae contain c. 800 species in 130 genera and are among the economically most important families of plants. We inferred their phylogeny based on chloroplast DNA sequences from two genes, one intron, and two spacers (rbcL, matK, trnL, trnL-trnF, rpl20-rps12) obtained for 171 species in 123 genera. Molecular data weakly support the traditional subfamilies Cucurbitoideae (111 genera) and Nhandiroboideae (19 genera, 60 species), and recover most of the eleven tribes, but almost none of the subtribes. Indofevillea khasiana is sister to all other Cucurbitoideae, and the genera of Joliffieae plus a few Trichosantheae form a grade near the base of Cucurbitoideae. A newly discovered large clade consists of the ancestrally Asian genera Nothoalsomitra, Luffa, Gymnopetalum, Hodgsonia, Trichosanthes, and the New World tribe Sicyeae. Genera that are poly- or paraphyletic include Ampelosicyos, Cucumis, Ibervillea, Neoachmandra, Psiguria, Trichosanthes, and Xerosicyos. Flower characters, especially number of free styles, fusion of filaments and/or anthers, tendril type, and pollen size, exine, and aperture number correlate well with the chloroplast phylogeny, while petal and fruit characters as well as karyotype exhibit much evolutionary flexibility.     

Co‐regulation proteomics reveals substrates and mechanisms of APC/C‐dependent degradation

Using multiplexed quantitative proteomics, we analyzed cell cycle‐dependent changes of the human proteome. We identified >4,400 proteins, each with a six‐point abundance profile across the cell cycle. Hypothesizing that proteins with similar abundance profiles are co‐regulated, we clustered the proteins with abundance profiles most similar to known Anaphase‐Promoting Complex/Cyclosome (APC/C) substrates to identify additional putative APC/C substrates. This protein profile similarity screening (PPSS) analysis resulted in a shortlist enriched in kinases and kinesins. Biochemical studies on the kinesins confirmed KIFC1, KIF18A, KIF2C, and KIF4A as APC/C substrates. Furthermore, we showed that the APC/C^CDH1‐dependent degradation of KIFC1 regulates the bipolar spindle formation and proper cell division. A targeted quantitative proteomics experiment showed that KIFC1 degradation is modulated by a stabilizing CDK1‐dependent phosphorylation site within the degradation motif of KIFC1. The regulation of KIFC1 (de‐)phosphorylation and degradation provides insights into the fidelity and proper ordering of substrate degradation by the APC/C during mitosis.     

FLEXIQinase, a mass spectrometry-based assay, to unveil multikinase mechanisms

We introduce a mass spectrometry-based method that provides residue-resolved quantitative information about protein phosphorylation. In this assay we combined our full-length expressed stable isotope-labeled protein for quantification strategy (FLEXIQuant) with a traditional kinase assay to determine the mechanisms of multikinase substrate phosphorylation such as priming-dependent kinase activities. The assay monitors the decrease in signal intensity of the substrate peptides and the concomitant increase in the (n × 80 Da)-shifted phosphorylated peptide. We analyzed the c-Jun N-terminal kinase (JNK)-dependent glycogen synthase kinase 3β (GSK3β) activity on doublecortin (DCX) revealing mechanistic details about the role of phosphorylation cross-talk in GSK3β activity and permitting an advanced model for GSK3β-mediated signaling.     

SCN5A Mutations in Brugada Syndrome Are Associated with Increased Cardiac Dimensions and Reduced Contractility

Background

The cardiac sodium channel (Na_v1.5) controls cardiac excitability. Accordingly, SCN5A mutations that result in loss-of-function of Na_v1.5 are associated with various inherited arrhythmia syndromes that revolve around reduced cardiac excitability, most notably Brugada syndrome (BrS). Experimental studies have indicated that Na_v1.5 interacts with the cytoskeleton and may also be involved in maintaining structural integrity of the heart. We aimed to determine whether clinical evidence may be obtained that Na_v1.5 is involved in maintaining cardiac structural integrity.

Methods

Using cardiac magnetic resonance (CMR) imaging, we compared right ventricular (RV) and left ventricular (LV) dimensions and ejection fractions between 40 BrS patients with SCN5A mutations (SCN5a-mut-positive) and 98 BrS patients without SCN5A mutations (SCN5a-mut-negative). We also studied 18 age/sex-matched healthy volunteers.

Results

SCN5a-mut-positive patients had significantly larger end-diastolic and end-systolic RV and LV volumes, and lower LV ejection fractions, than SCN5a-mut-negative patients or volunteers.

Conclusions

Loss-of-function SCN5A mutations are associated with dilatation and impairment in contractile function of both ventricles that can be detected by CMR analysis.     

Unveiling microbial life in the new deep-sea hypersaline Lake Thetis. Part II: a metagenomic study

So far only little is known about the microbial ecology of Mediterranean deep-sea hypersaline anoxic lakes (DHALs). These brine lakes were formed by evaporite dissolution/brine seeps and are important model environments to provide insights into possible metabolisms and distributions of microorganisms on the early Earth. Our study on the Lake Thetis, a new thalassohaline DHAL located South-East of the Medriff Corridor, has revealed microbial communities of contrasting compositions with a high number of novel prokaryotic candidate divisions. The major finding of our present work is co-occurrence of at least three autotrophic carbon dioxide fixation pathways in the brine-seawater interface that are likely fuelled by an active ramified sulphur cycle. Genes for the reductive acetyl-CoA and reductive TCA pathways were also found in the brine suggesting that these pathways are operational even at extremely elevated salinities and that autotrophy is more important in hypersaline environments than previously assumed. Surprisingly, genes coding for RuBisCo were found in the highly reduced brine. Three types of sulphide oxidation pathways were found in the interface. The first involves a multienzyme Sox complex catalysing the complete oxidation of reduced sulphur compounds to sulphate, the second type recruits SQR sulphide:quinone reductase for oxidation of sulphide to elemental sulphur, which, in the presence of sulphide, could further be reduced by polysulphide reductases in the third pathway. The presence of the latter two allows a maximal energy yield from the oxidation of sulphide and at the same time prevents the acidification and the accumulation of S(0) deposits. Amino acid composition analysis of deduced proteins revealed a significant overrepresentation of acidic residues in the brine compared with the interface. This trait is typical for halophilic organisms as an adaptation to the brine's extreme hypersalinity. This work presents the first metagenomic survey of the microbial communities of the recently discovered Lake Thetis whose brine constitutes one of saltiest water bodies ever reported.     

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.