Methane-Carbon Flow into the Benthic Food Web at Cold Seeps – A Case Study from the Costa Rica Subduction Zone

Cold seep ecosystems can support enormous biomasses of free-living and symbiotic chemoautotrophic organisms that get their energy from the oxidation of methane or sulfide. Most of this biomass derives from animals that are associated with bacterial symbionts, which are able to metabolize the chemical resources provided by the seeping fluids. Often these systems also harbor dense accumulations of non-symbiotic megafauna, which can be relevant in exporting chemosynthetically fixed carbon from seeps to the surrounding deep sea. Here we investigated the carbon sources of lithodid crabs (Paralomis sp.) feeding on thiotrophic bacterial mats at an active mud volcano at the Costa Rica subduction zone. To evaluate the dietary carbon source of the crabs, we compared the microbial community in stomach contents with surface sediments covered by microbial mats. The stomach content analyses revealed a dominance of epsilonproteobacterial 16S rRNA gene sequences related to the free-living and epibiotic sulfur oxidiser Sulfurovum sp. We also found Sulfurovum sp. as well as members of the genera Arcobacter and Sulfurimonas in mat-covered surface sediments where Epsilonproteobacteria were highly abundant constituting 10% of total cells. Furthermore, we detected substantial amounts of bacterial fatty acids such as i-C15∶0 and C17∶1ω6c with stable carbon isotope compositions as low as −53‰ in the stomach and muscle tissue. These results indicate that the white microbial mats at Mound 12 are comprised of Epsilonproteobacteria and that microbial mat-derived carbon provides an important contribution to the crab''s nutrition. In addition, our lipid analyses also suggest that the crabs feed on other ¹³C-depleted organic matter sources, possibly symbiotic megafauna as well as on photosynthetic carbon sources such as sedimentary detritus.     

Contrasting Patterns of Genetic Differentiation among Blackcaps (Sylvia atricapilla) with Divergent Migratory Orientations in Europe

Migratory divides are thought to facilitate behavioral, ecological, and genetic divergence among populations with different migratory routes. However, it is currently contentious how much genetic divergence is needed to maintain distinct migratory behavior across migratory divides. Here we investigate patterns of neutral genetic differentiation among Blackcap (Sylvia atricapilla) populations with different migratory strategies across Europe. We compare the level of genetic divergence of populations migrating to southwestern (SW) or southeastern (SE) wintering areas with birds wintering in the British Isles following a recently established northwesterly (NW) migration route. The migratory divide between SW and SE wintering areas can be interpreted as a result of a re-colonization process after the last glaciation. Thus we predicted greater levels of genetic differentiation among the SW/SE populations. However, a lack of genetic differentiation was found between SW and SE populations, suggesting that interbreeding likely occurs among Blackcaps with different migratory orientations across a large area; therefore the SW/SE migratory divide can be seen as diffuse, broad band and is, at best, a weak isolating barrier. Conversely, weak, albeit significant genetic differentiation was evident between NW and SW migrants breeding sympatrically in southern Germany, suggesting a stronger isolating mechanism may be acting in this population. Populations located within/near the SW/SE contact zone were the least genetically divergent from NW migrants, confirming NW migrants likely originated from within the contact zone. Significant isolation-by-distance was found among eastern Blackcap populations (i.e. SE migrants), but not among western populations (i.e. NW and SW migrants), revealing different patterns of genetic divergence among Blackcap populations in Europe. We discuss possible explanations for the genetic structure of European Blackcaps and how gene flow influences the persistence of divergent migratory behaviors.     

ANOVA-Like Differential Expression (ALDEx) Analysis for Mixed Population RNA-Seq

Experimental variance is a major challenge when dealing with high-throughput sequencing data. This variance has several sources: sampling replication, technical replication, variability within biological conditions, and variability between biological conditions. The high per-sample cost of RNA-Seq often precludes the large number of experiments needed to partition observed variance into these categories as per standard ANOVA models. We show that the partitioning of within-condition to between-condition variation cannot reasonably be ignored, whether in single-organism RNA-Seq or in Meta-RNA-Seq experiments, and further find that commonly-used RNA-Seq analysis tools, as described in the literature, do not enforce the constraint that the sum of relative expression levels must be one, and thus report expression levels that are systematically distorted. These two factors lead to misleading inferences if not properly accommodated. As it is usually only the biological between-condition and within-condition differences that are of interest, we developed ALDEx, an ANOVA-like differential expression procedure, to identify genes with greater between- to within-condition differences. We show that the presence of differential expression and the magnitude of these comparative differences can be reasonably estimated with even very small sample sizes.     

A Whole-Cell Biosensor for the Detection of Gold

Geochemical exploration for gold (Au) is becoming increasingly important to the mining industry. Current processes for Au analyses require sampling materials to be taken from often remote localities. Samples are then transported to a laboratory equipped with suitable analytical facilities, such as Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) or Instrumental Neutron Activation Analysis (INAA). Determining the concentration of Au in samples may take several weeks, leading to long delays in exploration campaigns. Hence, a method for the on-site analysis of Au, such as a biosensor, will greatly benefit the exploration industry. The golTSB genes from Salmonella enterica serovar typhimurium are selectively induced by Au(I/III)-complexes. In the present study, the golTSB operon with a reporter gene, lacZ, was introduced into Escherichia coli. The induction of golTSB::lacZ with Au(I/III)-complexes was tested using a colorimetric β-galactosidase and an electrochemical assay. Measurements of the β-galactosidase activity for concentrations of both Au(I)- and Au(III)-complexes ranging from 0.1 to 5 µM (equivalent to 20 to 1000 ng g⁻¹ or parts-per-billion (ppb)) were accurately quantified. When testing the ability of the biosensor to detect Au(I/III)-complexes_(aq) in the presence of other metal ions (Ag(I), Cu(II), Fe(III), Ni(II), Co(II), Zn, As(III), Pb(II), Sb(III) or Bi(III)), cross-reactivity was observed, i.e. the amount of Au measured was either under- or over-estimated. To assess if the biosensor would work with natural samples, soils with different physiochemical properties were spiked with Au-complexes. Subsequently, a selective extraction using 1 M thiosulfate was applied to extract the Au. The results showed that Au could be measured in these extracts with the same accuracy as ICP-MS (P<0.05). This demonstrates that by combining selective extraction with the biosensor system the concentration of Au can be accurately measured, down to a quantification limit of 20 ppb (0.1 µM) and a detection limit of 2 ppb (0.01 µM).     

Relationship between Oscillatory Neuronal Activity during Reward Processing and Trait Impulsivity and Sensation Seeking

Background

The processing of reward and punishment stimuli in humans appears to involve brain oscillatory activity of several frequencies, probably each with a distinct function. The exact nature of associations of these electrophysiological measures with impulsive or risk-seeking personality traits is not completely clear. Thus, the aim of the present study was to investigate event-related oscillatory activity during reward processing across a wide spectrum of frequencies, and its associations with impulsivity and sensation seeking in healthy subjects.

Methods

During recording of a 32-channel EEG 22 healthy volunteers were characterized with the Barratt Impulsiveness and the Sensation Seeking Scale and performed a computerized two-choice gambling task comprising different feedback options with positive vs. negative valence (gain or loss) and high or low magnitude (5 vs. 25 points).

Results

We observed greater increases of amplitudes of the feedback-related negativity and of activity in the theta, alpha and low-beta frequency range following loss feedback and, in contrast, greater increase of activity in the high-beta frequency range following gain feedback. Significant magnitude effects were observed for theta and delta oscillations, indicating greater amplitudes upon feedback concerning large stakes. The theta amplitude changes during loss were negatively correlated with motor impulsivity scores, whereas alpha and low-beta increase upon loss and high-beta increase upon gain were positively correlated with various dimensions of sensation seeking.

Conclusions

The findings suggest that the processing of feedback information involves several distinct processes, which are subserved by oscillations of different frequencies and are associated with different personality traits.     

Donor–Acceptor Shape Matching Drives Performance in Photovoltaics

While the demonstrated power conversion efficiency of organic photovoltaics (OPVs) now exceeds 10%, new design rules are required to tailor interfaces at the molecular level for optimal exciton dissociation and charge transport in higher efficiency devices. We show that molecular shape‐complementarity between donors and acceptors can drive performance in OPV devices. Using core hole clock (CHC) X‐ray spectroscopy and density functional theory (DFT), we compare the electronic coupling, assembly, and charge transfer rates at the interface between C₆₀ acceptors and flat‐ or contorted‐hexabenzocorone (HBC) donors. The HBC donors have similar optoelectronic properties but differ in molecular contortion and shape matching to the fullerene acceptors. We show that shape‐complementarity drives self‐assembly of an intermixed morphology with a donor/acceptor (D/A) ball‐and‐socket interface, which enables faster electron transfer from HBC to C₆₀. The supramolecular assembly and faster electron transfer rates in the shape complementary heterojunction lead to a larger active volume and enhanced exciton dissociation rate. This work provides fundamental mechanistic insights on the improved efficiency of organic photovoltaic devices that incorporate these concave/convex D/A materials.     

Vibrio cholerae Evades Neutrophil Extracellular Traps by the Activity of Two Extracellular Nucleases

The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs) upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation.     

The platelet P2Y12 receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [3H]PSB-0413

Various radioligands have been used to characterize and quantify the platelet P2Y₁₂ receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y₁ and P2Y₁₂. We used the [³H]PSB-0413 selective P2Y₁₂ receptor antagonist radioligand to reevaluate the number of P2Y₁₂ receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [³H]PSB-0413 bound to 425 ± 50 sites/platelet (K_D = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y₁₂, with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y₁ ligand MRS2179 and the P2X₁ ligand α,β-Met-ATP did not displace [³H]PSB-0413 binding. Patients with severe P2Y₁₂ deficiency displayed virtually no binding of [³H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y₁₂ receptor had normal binding. Studies in mice showed that: (1) [³H]PSB-0413 bound to 634 ± 87 sites/platelet (K_D = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [³H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y₁₂ receptors, to identify patients with P2Y₁₂ deficiencies or quantify the effect of P2Y₁₂ targeting drugs.     

Characterization of new G protein-coupled adenine receptors in mouse and hamster

The nucleobase adenine has previously been reported to activate G protein-coupled receptors in rat and mouse. Adenine receptors (AdeR) thus constitute a new family of purine receptors, for which the designation “P0-receptors” has been suggested. We now describe the cloning and characterization of two new members of the AdeR family from mouse (MrgA10, termed mAde1R) and hamster (cAdeR). Both receptors were expressed in Sf9 insect cells, and radioligand binding studies were performed using [³H]adenine. Specific binding of the radioligand was detected in transfected, but not in untransfected cells, and K _D values of 286 nM (mAde1R, B _max 1.18 pmol/mg protein) and 301 nM (cAdeR, B _max 17.7 pmol/mg protein), respectively, were determined. A series of adenine derivatives was investigated in competition binding assays. Minor structural modifications generally led to a reduction or loss of affinity, with one exception: 2-fluoroadenine was at least as potent as adenine itself at the cAdeR. Structure–activity relationships at all AdeR orthologs and subtypes investigated so far were similar, but not identical. For functional analyses, the cAdeR was homologously expressed in Chinese hamster ovary (CHO) cells, while the mAde1R was heterologously expressed in 1321N1 astrocytoma cells. Like the previously described AdeRs from rat (rAdeR) and mouse (mAde2R), the mAde1R (EC₅₀ 9.77 nM) and the cAdeR (EC₅₀ 51.6 nM) were coupled to inhibition of adenylate cyclase. In addition, the cAdeR from hamster expressed in CHO cells produced an increase in intracellular calcium concentrations (EC₅₀ 6.24 nM) and was found to be additionally coupled to G_q proteins.     

Multiple Delivery of siRNA against Endoglin into Murine Mammary Adenocarcinoma Prevents Angiogenesis and Delays Tumor Growth

Endoglin is a transforming growth factor-β (TGF- β) co-receptor that participates in the activation of a signaling pathway that mediates endothelial cell proliferation and migration in angiogenic tumor vasculature. Therefore, silencing of endoglin expression is an attractive approach for antiangiogenic therapy of tumors. The aim of our study was to evaluate the therapeutic potential of small interfering RNA (siRNA) molecules against endoglin in vitro and in vivo. Therapeutic potential in vitro was assessed in human and murine endothelial cells (HMEC-1, 2H11) by determining endoglin expression level, cell proliferation and tube formation. In vivo, the therapeutic potential of siRNA molecules was evaluated in TS/A mammary adenocarcinoma growing in BALB/c mice. Results of our study showed that siRNA molecules against endoglin have a good antiangiogenic therapeutic potential in vitro, as expression of endoglin mRNA and protein levels in mouse and human microvascular endothelial cells after lipofection were efficiently reduced, which resulted in the inhibition of endothelial cell proliferation and tube formation. In vivo, silencing of endoglin with triple electrotransfer of siRNA molecules into TS/A mammary adenocarcinoma also significantly reduced the mRNA levels, number of tumor blood vessels and the growth of tumors. The obtained results demonstrate that silencing of endoglin is a promising antiangiogenic therapy of tumors that could not be used as single treatment, but as an adjunct to the established cytotoxic treatment approaches.