Methanosarcina spp. drive vinyl chloride dechlorination via interspecies hydrogen transfer

Two highly enriched cultures containing Dehalococcoides spp. were used to study the effect of aceticlastic methanogens on reductive vinyl chloride (VC) dechlorination. In terms of aceticlastic methanogens, one culture was dominated by Methanosaeta, while the other culture was dominated by Methanosarcina, as determined by fluorescence in situ hybridization. Cultures amended with 2-bromoethanesulfonate (BES), an efficient inhibitor of methanogens, exhibited slow VC dechlorination when grown on acetate and VC. Methanogenic cultures dominated by Methanosaeta had no impact on dechlorination rates, compared to BES-amended controls. In contrast, methanogenic cultures dominated by Methanosarcina displayed up to sevenfold-higher rates of VC dechlorination than their BES-amended counterparts. Methanosarcina-dominated cultures converted a higher percentage of [2-(14)C]acetate to (14)CO(2) when concomitant VC dechlorination took place, compared to nondechlorinating controls. Respiratory indices increased from 0.12 in nondechlorinating cultures to 0.51 in actively dechlorinating cultures. During VC dechlorination, aqueous hydrogen (H(2)) concentrations dropped to 0.3 to 0.5 nM. However, upon complete VC consumption, H(2) levels increased by a factor of 10 to 100, indicating active hydrogen production from acetate oxidation. This process was thermodynamically favorable by means of the extremely low H(2) levels during dechlorination. VC degradation in nonmethanogenic cultures was not inhibited by BES but was limited by the availability of H(2) as electron donor, in cultures both with and without BES. These findings all indicate that Methanosarcina (but not Methanosaeta), while cleaving acetate to methane, simultaneously oxidizes acetate to CO(2) plus H(2), driving hydrogenotrophic dehalorespiration of VC to ethene by Dehalococcoides.     

Seasonal carbon allocation to arbuscular mycorrhizal fungi assessed by microscopic examination, stable isotope probing and fatty acid analysis

Background and Aim

Climate change models are limited by lack of baseline data, in particular carbon (C) allocation to – and dynamics within – soil microbial communities. We quantified seasonal C-assimilation and allocation by plants, and assessed how well this corresponds with intraradical arbuscular mycorrhizal fungal (AMF) storage and structural lipids (16:1ω5 NLFA and PLFA, respectively), as well as microscopic assessments of AMF root colonization.

Methods

Coastal Hypochoeris radicata plants were labeled with ¹³CO₂ in February, July and October, and ¹³C-allocation to fine roots and NLFA 16:1ω5, as well as overall lipid contents and AM colonization were quantified.

Results

C-allocation to fine roots and AMF storage lipids differed seasonally and mirrored plant C-assimilation, whereas AMF structural lipids and AM colonization showed no seasonal variation, and root colonization exceeded 80 % throughout the year. Molecular analyzes of the large subunit rDNA gene indicated no seasonal AMF community shifts.

Conclusions

Plants allocated C to AMF even at temperatures close to freezing, and fungal structures persisted in roots during times of low C-allocation. The lack of seasonal differences in PLFA and AM colonization indicates that NLFA analyses should be used to estimate fungal C-status. The implication of our findings for AM function is discussed.     

High-throughput multi-antigen microfluidic fluorescence immunoassays

Here we describe the development of a high-throughput multi-antigen microfluidic fluorescence immunoassay system. A 100-chamber polydimethylsiloxane (PDMS) chip performs up to 5 tests for each of 10 samples. In this particular study system, the specificity of detection was demonstrated, and calibration curves were produced for C-reactive protein (CRP), prostate-specific antigen (PSA), ferritin, and vascular endothelial growth factor (VEGF). The measurements show sensitivity at and below clinically normal levels (with a signal-to-noise ratio >8 at as low as 10 pM antigen concentration). The chip uses 100 nL per sample for all tests. The developed system is an important step toward derivative immunoassay applications in scientific research and "point-of-care" testing in medicine.     

Mitogen activated protein kinases SakAHOG1 and MpkC collaborate for Aspergillus fumigatus virulence

Here, we investigated which stress responses were influenced by the MpkC and SakA mitogen‐activated protein kinases of the high‐osmolarity glycerol (HOG) pathway in the fungal pathogen Aspergillus fumigatus. The ΔsakA and the double ΔmpkC ΔsakA mutants were more sensitive to osmotic and oxidative stresses, and to cell wall damaging agents. Both MpkC::GFP and SakA::GFP translocated to the nucleus upon osmotic stress and cell wall damage, with SakA::GFP showing a quicker response. The phosphorylation state of MpkA was determined post exposure to high concentrations of congo red and Sorbitol. In the wild‐type strain, MpkA phosphorylation levels progressively increased in both treatments. In contrast, the ΔsakA mutant had reduced MpkA phosphorylation, and surprisingly, the double ΔmpkC ΔsakA had no detectable MpkA phosphorylation. A. fumigatus ΔsakA and ΔmpkC were virulent in mouse survival experiments, but they had a 40% reduction in fungal burden. In contrast, the ΔmpkC ΔsakA double mutant showed highly attenuated virulence, with approximately 50% mice surviving and a 75% reduction in fungal burden. We propose that both cell wall integrity (CWI) and HOG pathways collaborate, and that MpkC could act by modulating SakA activity upon exposure to several types of stresses and during CW biosynthesis.     

A DNA barcode library for Germany′s mayflies,stoneflies and caddisflies (Ephemeroptera,Plecoptera and Trichoptera)

Mayflies, stoneflies and caddisflies (Ephemeroptera, Plecoptera and Trichoptera) are prominent representatives of aquatic macroinvertebrates, commonly used as indicator organisms for water quality and ecosystem assessments. However, unambiguous morphological identification of EPT species, especially their immature life stages, is a challenging, yet fundamental task. A comprehensive DNA barcode library based upon taxonomically well‐curated specimens is needed to overcome the problematic identification. Once available, this library will support the implementation of fast, cost‐efficient and reliable DNA‐based identifications and assessments of ecological status. This study represents a major step towards a DNA barcode reference library as it covers for two‐thirds of Germany's EPT species including 2,613 individuals belonging to 363 identified species. As such, it provides coverage for 38 of 44 families (86%) and practically all major bioindicator species. DNA barcode compliant sequences (≥500 bp) were recovered from 98.74% of the analysed specimens. Whereas most species (325, i.e., 89.53%) were unambiguously assigned to a single Barcode Index Number (BIN) by its COI sequence, 38 species (18 Ephemeroptera, nine Plecoptera and 11 Trichoptera) were assigned to a total of 89 BINs. Most of these additional BINs formed nearest neighbour clusters, reflecting the discrimination of geographical subclades of a currently recognized species. BIN sharing was uncommon, involving only two species pairs of Ephemeroptera. Interestingly, both maximum pairwise and nearest neighbour distances were substantially higher for Ephemeroptera compared to Plecoptera and Trichoptera, possibly indicating older speciation events, stronger positive selection or faster rate of molecular evolution.     

Intravital microscopy of the murine pulmonary microcirculation.

Intravital microscopy (IVM) is considered as the gold standard for in vivo investigations of dynamic microvascular regulation. The availability of transgenic and knockout animals has propelled the development of murine IVM models for various organs, but technical approaches to the pulmonary microcirculation are still scarce. In anesthetized and ventilated BALB/c mice, we established a microscopic access to the surface of the right upper lung lobe by surgical excision of a window of 7- to 10-mm diameter from the right thoracic wall. The window was covered by a transparent polyvinylidene membrane and sealed with alpha-cyanoacrylate. Removal of intrathoracic air via a trans-diaphragmal intrapleural catheter coupled the lung surface to the window membrane. IVM preparations were hemodynamically stable for at least 120 min, with mean arterial blood pressure above 70 mmHg, and mean arterial Po(2) and arterial Pco(2) in the range of 90-100 Torr and 30-40 Torr, respectively. Imaged lungs did not show any signs of acute lung injury or edema. Following infusion of FITC dextran, subpleural pulmonary arterioles and venules of up to 50-microm diameter and alveolar capillary networks could be visualized during successive expiratory plateau phases over a period of at least 2 h. Vasoconstrictive responses to hypoxia (11% O(2)) or infusion of the thromboxane analog U-46619 were prominent in medium-sized arterioles (30- to 50-microm diameter), minor in small arterioles <30 microm, and absent in venules. The presented IVM model may constitute a powerful new tool for investigations of pulmonary microvascular responses in mice.     

Single-molecule studies of synaptotagmin and complexin binding to the SNARE complex

The assembly of multiprotein complexes at the membrane interface governs many signaling processes in cells. However, very few methods exist for obtaining biophysical information about protein complex formation at the membrane. We used single molecule fluorescence resonance energy transfer to study complexin and synaptotagmin interactions with the SNARE complex in deposited lipid bilayers. Using total internal reflectance microscopy, individual binding events at the membrane could be resolved despite an excess of unbound protein in solution. Fluorescence resonance energy transfer (FRET)-efficiency derived distances for the complexin-SNARE interaction were consistent with the crystal structure of the complexin-SNARE complex. The unstructured N-terminal region of complexin showed broad distributions of FRET efficiencies to the SNARE complex, suggesting that information on conformational variability can be obtained from FRET efficiency distributions. The low-affinity interaction of synaptotagmin with the SNARE complex changed dramatically upon addition of Ca2+ with high FRET efficiency interactions appearing between the C2B domain and linker domains of synaptotagmin and the membrane proximal portion of the SNARE complex. These results demonstrate that single molecule FRET can be used as a "spectroscopic ruler" to simultaneously gain structural and kinetic information about transient multiprotein complexes at the membrane interface.     

Short bioactive Spiegelmers to migraine-associated calcitonin gene-related peptide rapidly identified by a novel approach: tailored-SELEX

We developed an integrated method to identify aptamers with only 10 fixed nucleotides through ligation and removal of primer binding sites within the systematic evolution of ligands by exponential enrichment (SELEX) process. This Tailored-SELEX approach was validated by identifying a Spiegelmer (‘mirror-image aptamer’) that inhibits the action of the migraine-associated target calcitonin gene-related peptide 1 (α-CGRP) with an IC₅₀ of 3 nM at 37°C in cell culture. Aptamers are oligonucleotide ligands that can be generated to bind to targets with high affinity and specificity. Stabilized aptamers and Spiegelmers have shown activity in vivo and may be used as therapeutics. Aptamers are isolated by in vitro selection from combinatorial nucleic acid libraries that are composed of a central randomized region and additional fixed primer binding sites with ~30–40 nt. The identified sequences are usually not short enough for efficient chemical Spiegelmer synthesis, post-SELEX stabilization of aptamers and economical production. If the terminal primer binding sites are part of the target recognizing domain, truncation of aptamers has proven difficult and laborious. Tailored-SELEX results in short sequences that can be tested more rapidly in biological systems. Currently, our identified CGRP binding Spiegelmer serves as a lead compound for in vivo studies.