Soil Methane Uptake Increases under Continuous Throughfall Reduction in a Temperate Evergreen,Broadleaved Eucalypt Forest

Soils in temperate forests ecosystems are the greatest terrestrial CH₄ sink globally. Global and regional circulation models predict decreased average rainfall, increased extreme rainfall events and increased temperatures for many temperate ecosystems. However, most studies of soil CH₄ uptake have only considered extended periods of drought rather than an overall decrease in rainfall amount. We measured soil CH₄ uptake from March 2010 to March 2012 after installing passive rainfall reduction systems to intercept approximately 40% of throughfall in a temperate broadleaf evergreen eucalypt forest in south-eastern Australia. Throughfall reduction caused an average reduction of 15.1 ± 6.4% (SE) in soil volumetric water content, a reduction of 19.8 ± 6.9% in soil water-filled pore space (%WFPS) and a 20.1 ± 6.8% increase in soil air-filled porosity. In response to these changes, soil CH₄ uptake increased by 54.7 ± 19.3%. The increase in soil CH₄ uptake could be explained by increased diffusivity in drier soils, whilst the activity of methanotrophs remained relatively unchanged. It is likely that soil CH₄ uptake will increase if rainfall reduces in temperate broadleaf evergreen forests of Australia as a consequence of climate change.     

A novel cytosolic class I antigen-processing pathway for endoplasmic-reticulum-targeted proteins

Proteins bearing an endoplasmic reticulum (ER) leader are inserted into the ER followed by cleavage of the signal peptide. Major histocompatibility complex class I-restricted T-cell epitopes can be generated from these proteins by the proteasome after retrotranslocation into the cytosol. Here, we show that an HLA-A(*)0201-restricted epitope from prostate stem cell antigen contains the cleavage site of the ER signal peptidase. The resulting cleavage products fail to bind to HLA-A(*)0201 and are not recognized by T lymphocytes. As processing of prostate stem cell antigen by signal peptidase occurs immediately after co-translational insertion, the epitope must be processed from polypeptides that have never reached the ER. The processing of this epitope depends on the proteasome and the transporter associated with antigen processing and shows a novel pathway of class I processing that relies on the failure of ER-targeted proteins to reach their target compartment.     

��ChopNSpice,�� a Mass Spectrometric Approach That Allows Identification of Endogenous Small Ubiquitin-like Modifier-conjugated Peptides

Conjugation of small ubiquitin-like modifier (SUMO) to substrates is involved in a large number of cellular processes. Typically, SUMO is conjugated to lysine residues within a SUMO consensus site; however, an increasing number of proteins are sumoylated on non-consensus sites. To appreciate the functional consequences of sumoylation, the identification of SUMO attachment sites is of critical importance. Discovery of SUMO acceptor sites is usually performed by a laborious mutagenesis approach or using MS. In MS, identification of SUMO acceptor sites in higher eukaryotes is hampered by the large tryptic fragments of SUMO1 and SUMO2/3. MS search engines in combination with known databases lack the possibility to search MSMS spectra for larger modifications, such as sumoylation. Therefore, we developed a simple and straightforward database search tool (“ChopNSpice”) that successfully allows identification of SUMO acceptor sites from proteins sumoylated in vivo and in vitro. By applying this approach we identified SUMO acceptor sites in, among others, endogenous SUMO1, SUMO2, RanBP2, and Ubc9.Post-translational modification with ubiquitin and ubiquitin-like modifiers (Ubls)1 such as SUMO plays an important role in most, if not all, cellular processes (1–6). Conjugation of Ubls to their targets involves an isopeptide bond between the carboxyl group of the modifier and the ε-amino group of a lysine residue within the targets. Attachment of Ubls to specific targets involves an enzymatic cascade. First the Ubls are processed to expose their C-terminal diglycine motif. The mature Ubl is then transferred to its target via a cascade of E1 (activating), E2 (conjugating), and E3 (ligase) enzymes. The conjugation system for SUMO consists of a heterodimeric activating enzyme, Aos1/Uba2; a conjugating enzyme, Ubc9; and E3 ligases, such as RanBP2 or members of the PIAS family. The conjugation status undergoes perpetual change and is governed by a small family of SUMO proteases that hydrolyze the isopeptide bond between SUMO and its target (7, 8). Although in lower eukaryotes only one SUMO is present, vertebrates express at least three different SUMO paralogs: SUMO1, SUMO2, and SUMO3. Mature SUMO2 and SUMO3 (referred to as SUMO2/3) are 97% identical but differ substantially from SUMO1 (∼50% identity).Although the list of known SUMO substrates is growing rapidly, our understanding of the functional consequences for many of these targets is lagging behind. At a molecular level, the functional consequences of SUMO conjugation can be explained by a gain or loss of interaction with other macromolecules (3, 4). SUMO-dependent intramolecular conformational changes have also been described (9, 10). Thus, to appreciate the role that SUMO plays in the regulation of specific substrates, identification of the acceptor site(s) for SUMO conjugation is of key importance.So far, identification of SUMO acceptor sites has relied largely on mutation of the SUMO consensus site, which consists of a short motif with the sequence ψKXE (ψ represents a bulky hydrophobic residue, and X represents any amino acid). This motif is recognized by Ubc9 if presented in an extended conformation (11–13). However, an increasing number of proteins, such as PCNA, E2-25K, Daxx, and USP25, turned out to be sumoylated on lysine residues that do not conform to the SUMO consensus site (14–17). For this category of proteins, as well as for proteins that contain a large number of SUMO consensus sites, the identification of acceptor lysines is a burdensome task that often involves mutagenesis of each lysine residue within the substrate in turn.MS is currently one of the state-of-the-art technologies to identify protein factors and their post-translational modifications in an unbiased and sensitive manner. Several groups have shown that, using overexpressed tagged SUMO, MS can be efficiently exploited to identify endogenous substrates for SUMO conjugation (18–20). However, the identification of SUMO acceptor lysines using MS has remained a more challenging task (18, 21, 23, 24). So far, using tagged SUMO, unbiased identification of acceptor lysines for endogenous substrates has only been observed in Saccharomyces cerevisiae (18). The identification of substrates in higher eukaryotes has been hampered by the large conjugated SUMO peptide that arises upon tryptic digestion (>2154 Da with human SUMO1 and >3568 Da with human SUMO2/3 compared with 484 Da for Smt3 in S. cerevisiae). Such large fragments, in addition to the mass of the conjugated peptide, can impede their in-gel digestion, extraction, detection, and sequencing in MS. To overcome some of these limitations, several different strategies have been developed: 1) mutation of the tryptic fragment of SUMO, yielding a smaller tryptic fragment (23), 2) development of an automated recognition pattern tool (SUMmOn) (24), and 3) identification of targets using an in vitro to in vivo approach (21). Although these approaches have been applied successfully for the identification of SUMO conjugates in vitro and in vivo, unbiased identification of SUMO conjugates in vivo has not been achieved in higher eukaryotes. Another hurdle to such identification of SUMO conjugates is the variety of masses that can theoretically arise for just one SUMO-conjugated lysine in a given protein because of tryptic miscleavages. Thus, the unambiguous identification of SUMO acceptor sites requires the mass of the modified peptide carrying the conjugated SUMO (fragment) to be measured with high accuracy, and most importantly, it requires sequence analysis of the modified peptides. Because available proteomics search engines lack the possibility to search MSMS spectra for larger modifications, e.g. those that occur upon sumoylation, we developed a novel, simple, and straightforward database search tool (“ChopNSpice”) that, in combination with current proteomics search engines (such as MASCOT (25) or SEQUEST (26)), allows one to identify SUMO1 and SUMO2/3 acceptor sites unambiguously. We confirmed this strategy in vitro on various substrates and demonstrate the power of this technique by the identification of acceptor lysines within several endogenous targets from HeLa cells.     

Seasonal variation of cyanobacteria and microcystins in the Nui Coc Reservoir, Northern Vietnam

In order to understand the environmental variables which promote the proliferation of cyanobacteria and variation in microcystin concentrations in the Nui Coc reservoir, Vietnam, physicochemical parameters, the occurrence, and abundance of phytoplankton, cyanobacteria, and microcystin concentration were monitored monthly through the year 2009–2010. The relationships between these parameters were explored using principal component analysis (PCA) and Pearson correlation analysis. The phytoplankton community was mainly dominated by the cyanobacterium Microcystis with higher cyanobacteria abundance during summer and autumn season. PCA and Pearson correlation results showed that water temperature and phosphate concentration were the most important variables accounting for cyanobacteria, Microcystis, and microcystin occurrence. Analysis of the toxins by high-performance liquid chromatography demonstrated the presence of two microcystin variants: microcystin-LR (MC-RR) and microcystin-ddRR (MC-ddRR) with total concentrations of the toxins in filtered samples from surface water ranging from 0.11 to 1.52 μg MC-LR equiv L^?1. The high concentrations of microcystin in the Nui Coc reservoir highlighted the potential risk for human health in the basin. Our study underlined the need for regular monitoring of cyanobacteria and toxins in lakes and reservoirs, which are used for drinking water supplies, not only in Vietnam but also in tropical countries.     

Treatment of HNSCC cell lines with the EGFR-specific inhibitor cetuximab (Erbitux) results in paradox phosphorylation of tyrosine 1173 in the receptor

Overexpression of the epidermal growth factor receptor (EGFR, ErbB1, HER1) is frequent in head and neck squamous cell carcinomas (HNSCCs) and correlates with disease progression. Inhibition of EGFR with the kinase inhibitor AG1478 abolished receptor phosphorylation and reduced cell proliferation. However, treatment of HNSCC cells with cetuximab (Erbitux), a monoclonal antibody designed to block the EGFR ligand binding site, led to paradox EGFR activation due to hyperphosphorylation of tyrosine 1173, however, with a concomitant reduction in Erk1/2 phosphorylation levels. No pronounced influence on cell proliferation levels could be observed after treatment with this antibody. Since cetuximab appears able to activate EGFR in HNSCC cell lines, it is necessary to rethink the exact mechanisms by which cetuximab that recently was approved for the treatment of advanced head and neck cancer, inhibits tumor growth.     

The transferability of distribution models across regions: an amphibian case study

Aim Predicting species distribution is of fundamental importance for ecology and conservation. However, distribution models are usually established for only one region and it is unknown whether they can be transferred to other geographical regions. We studied the distribution of six amphibian species in five regions to address the question of whether the effect of landscape variables varied among regions. We analysed the effect of 10 variables extracted in six concentric buffers (from 100 m to 3 km) describing landscape composition around breeding ponds at different spatial scales. We used data on the occurrence of amphibian species in a total of 655 breeding ponds. We accounted for proximity to neighbouring populations by including a connectivity index to our models. We used logistic regression and information‐theoretic model selection to evaluate candidate models for each species. Location Switzerland. Results The explained deviance of each species’ best models varied between 5% and 32%. Models that included interactions between a region and a landscape variable were always included in the most parsimonious models. For all species, models including region‐by‐landscape interactions had similar support (Akaike weights) as models that did not include interaction terms. The spatial scale at which landscape variables affected species distribution varied from 100 m to 1000 m, which was in agreement with several recent studies suggesting that land use far away from the ponds can affect pond occupancy. Main conclusions Different species are affected by different landscape variables at different spatial scales and these effects may vary geographically, resulting in a generally low transferability of distribution models across regions. We also found that connectivity seems generally more important than landscape variables. This suggests that metapopulation processes may play a more important role in species distribution than habitat characteristics.     

Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli

Red recombination using PCR-amplified selectable markers is a well-established technique for mutagenesis of large DNA molecules in Escherichia coli. The system has limited efficacy and versatility, however, for markerless modifications including point mutations, deletions, and particularly insertions of longer sequences. Here we describe a procedure that combines Red recombination and cleavage with the homing endonuclease I-SceI to allow highly efficient, PCR-based DNA engineering without retention of unwanted foreign sequences. We applied the method to modification of bacterial artificial chromosome (BAC) constructs harboring an infectious herpesvirus clone to demonstrate the potential of the mutagenesis technique, which was used for the insertion of long sequences such as coding regions or promoters, introduction of point mutations, scarless deletions, and insertion of short sequences such as an epitope tag. The system proved to be highly reliable and efficient and can be adapted for a variety of different modifications of BAC clones, which are fundamental tools for applications as diverse as the generation of transgenic animals and the construction of gene therapy or vaccine vectors.     

The functional role of GABA and glycine in monaural and binaural processing in the inferior colliculus of horseshoe bats

Summary The functional role of GABA and glycine in monaural and binaural signal analysis was studied in single unit recordings from the central nucleus of the inferior colliculus (IC) of horseshoe bats (Rhinolophus rouxi) employing microiontophoresis of the putative neurotransmitters and their antagonists bicuculline and strychnine.Most neurons were inhibited by GABA (98%; N=107) and glycine (92%; N=118). Both neurotransmitters appear involved in several functional contexts, but to different degrees.Bicuculline-induced increases of discharge activity (99% of cells; N=191) were accompanied by changes of temporal response patterns in 35% of neurons distributed throughout the IC. Strychnine enhanced activity in only 53% of neurons (N=147); cells exhibiting response pattern changes were rare (9%) and confined to greater recording depths. In individual cells, the effects of both antagonists could markedly differ, suggesting a differential supply by GABAergic and glycinergic networks.Bicuculline changed the shape of the excitatory tuning curve by antagonizing lateral inhibition at neighboring frequencies and/or inhibition at high stimulation levels. Such effects were rarely observed with strychnine.Binaural response properties of single units were influenced either by antagonization of inhibition mediated by ipsilateral stimulation (bicuculline) or by changing the strength of the main excitatory input (bicuculline and strychnine).Abbreviations BF best frequency - Bic bicuculline - C control - CF constant frequency - CN cochlear nucleus - DNLL dorsal nucleus of the lateral lemniscus - FM frequency modulation - GABA gamma amino butyric acid - IC inferior colliculus - LSO lateral superior olive - Str strychnine