Anaerobic oxidation of methane at different temperature regimes in Guaymas Basin hydrothermal sediments

Anaerobic oxidation of methane (AOM) was investigated in hydrothermal sediments of Guaymas Basin based on δ¹³C signatures of CH₄, dissolved inorganic carbon and porewater concentration profiles of CH₄ and sulfate. Cool, warm and hot in-situ temperature regimes (15–20 °C, 30–35 °C and 70–95 °C) were selected from hydrothermal locations in Guaymas Basin to compare AOM geochemistry and 16S ribosomal RNA (rRNA), mcrA and dsrAB genes of the microbial communities. 16S rRNA gene clone libraries from the cool and hot AOM cores yielded similar archaeal types such as Miscellaneous Crenarchaeotal Group, Thermoproteales and anaerobic methane-oxidizing archaea (ANME)-1; some of the ANME-1 archaea formed a separate 16S rRNA lineage that at present seems to be limited to Guaymas Basin. Congruent results were obtained by mcrA gene analysis. The warm AOM core, chemically distinct by lower porewater sulfide concentrations, hosted a different archaeal community dominated by the two deep subsurface archaeal lineages Marine Benthic Group D and Marine Benthic Group B, and by members of the Methanosarcinales including ANME-2 archaea. This distinct composition of the methane-cycling archaeal community in the warm AOM core was confirmed by mcrA gene analysis. Functional genes of sulfate-reducing bacteria and archaea, dsrAB, showed more overlap between all cores, regardless of the core temperature. 16S rRNA gene clone libraries with Euryarchaeota-specific primers detected members of the Archaeoglobus clade in the cool and hot cores. A V6-tag high-throughput sequencing survey generally supported the clone library results while providing high-resolution detail on archaeal and bacterial community structure. These results indicate that AOM and the responsible archaeal communities persist over a wide temperature range.     

Protein enrichment by capture-release based on strain-promoted cycloaddition of azide with bicyclononyne (BCN)

An enrichment strategy was devised for azide derivatized macromolecules, based on strain-promoted alkyne-azide cycloaddition (SPAAC) and a cleavable linker. A ring-strained alkyne, bicyclo[6.1.0]non-4-yne (BCN), was covalently attached to agarose beads via a hydrazine-sensitive linker. Benchmark studies of the resulting 'azido-trap' beads were performed with a fluorogenic coumarin derivative, leading to efficient capture of the azidocoumarin with concomitant fluorescence staining of the beads via SPAAC. The versatility of the beads for specific protein enrichment was shown by an effective and highly specific capture-release strategy for enrichment of azido-containing Candida antarctica lipase B (CalB) from a mixture of proteins. This approach is suited for selective enrichment of (glyco)proteins after metabolic incorporation of azides for subsequent (glyco)proteomics studies.     

Attentional Stimulus Selection through Selective Synchronization between Monkey Visual Areas

A central motif in neuronal networks is convergence, linking several input neurons to one target neuron. In visual cortex, convergence renders target neurons responsive to complex stimuli. Yet, convergence typically sends multiple stimuli to a target, and the behaviorally relevant stimulus must be selected. We used two stimuli, activating separate electrocorticographic V1 sites, and both activating an electrocorticographic V4 site equally strongly. When one of those stimuli activated one V1 site, it gamma synchronized (60-80?Hz) to V4. When the two stimuli activated two V1 sites, primarily the relevant one gamma synchronized to V4. Frequency bands of gamma activities showed substantial overlap containing the band of interareal coherence. The relevant V1 site had its gamma peak frequency 2-3?Hz higher than the irrelevant V1 site and 4-6?Hz higher than V4. Gamma-mediated interareal influences were predominantly directed from V1 to V4. We propose that selective synchronization renders relevant input effective, thereby modulating effective connectivity.     

Synthesis of ‘cineole cassette’ monoterpenes in Nicotiana section Alatae: gene isolation, expression, functional characterization and phylogenetic analysis

The scent bouquets of flowers of Nicotiana species, particularly those of section Alatae, are rich in monoterpenes, including 1,8-cineole, limonene, β-myrcene, α- and β-pinene, sabinene, and α-terpineol. New terpene synthase genes were isolated from flowers of Nicotiana bonariensis, N. forgetiana, N. longiflora, and N. mutabilis. The recombinant enzymes synthesize simultaneously the characteristic 'cineole cassette' monoterpenes with 1,8-cineole as the dominant volatile product. Interestingly, amino acid sequence comparison and phylogenetic tree construction clustered the newly isolated cineole synthases (CIN) of section Alatae together with the catalytically similar CIN of N. suaveolens of section Suaveolentes, thus suggesting a common ancestor. These CIN genes of N. bonariensis, N. forgetiana, N. longiflora, and N. mutabilis are distinct from the terpineol synthases (TERs) of the taxonomically related N. alata and N. langsdorfii (both Alatae), thus indicating gene diversification of monoterpene synthases in section Alatae. Furthermore, the presence of CINs in species of the American section Alatae supports the hypothesis that one parent of the Australian section Suaveolentes was a member of the present section Alatae. Amino acid sequences of the Nicotiana CINs and TERs were compared to identify relevant amino acids of the cyclization reaction from α-terpineol to 1,8-cineole.     

TLR9 Mediates Remote Liver Injury following Severe Renal Ischemia Reperfusion

Ischemia reperfusion injury is a common cause of acute kidney injury and is characterized by tubular damage. Mitochondrial DNA is released upon severe tissue injury and can act as a damage-associated molecular pattern via the innate immune receptor TLR9. Here, we investigated the role of TLR9 in the context of moderate or severe renal ischemia reperfusion injury using wild-type C57BL/6 mice or TLR9KO mice. Moderate renal ischemia induced renal dysfunction but did not decrease animal well-being and was not regulated by TLR9. In contrast, severe renal ischemia decreased animal well-being and survival in wild-type mice after respectively one or five days of reperfusion. TLR9 deficiency improved animal well-being and survival. TLR9 deficiency did not reduce renal inflammation or tubular necrosis. Rather, severe renal ischemia induced hepatic injury as seen by increased plasma ALAT and ASAT levels and focal hepatic necrosis which was prevented by TLR9 deficiency and correlated with reduced circulating mitochondrial DNA levels and plasma LDH. We conclude that TLR9 does not mediate renal dysfunction following either moderate or severe renal ischemia. In contrast, our data indicates that TLR9 is an important mediator of hepatic injury secondary to ischemic acute kidney injury.     

Molecular Characterization of Sulfate-Reducing Bacteria in the Guaymas Basin

The Guaymas Basin (Gulf of California) is a hydrothermal vent site where thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material which supports diverse sulfate-reducing bacteria. We explored the phylogenetic and functional diversity of the sulfate-reducing bacteria by characterizing PCR-amplified dissimilatory sulfite reductase (dsrAB) and 16S rRNA genes from the upper 4 cm of the Guaymas sediment. The dsrAB sequences revealed that there was a major clade closely related to the acetate-oxidizing delta-proteobacterial genus Desulfobacter and a clade of novel, deeply branching dsr sequences related to environmental dsr sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark). Other dsr clones were affiliated with gram-positive thermophilic sulfate reducers (genus Desulfotomaculum) and the delta-proteobacterial species Desulforhabdus amnigena and Thermodesulforhabdus norvegica. Phylogenetic analysis of 16S rRNAs from the same environmental samples resulted in identification of four clones affiliated with Desulfobacterium niacini, a member of the acetate-oxidizing, nutritionally versatile genus Desulfobacterium, and one clone related to Desulfobacula toluolica and Desulfotignum balticum. Other bacterial 16S rRNA bacterial phylotypes were represented by non-sulfate reducers and uncultured lineages with unknown physiology, like OP9, OP8, as well as a group with no clear affiliation. In summary, analyses of both 16S rRNA and dsrAB clone libraries resulted in identification of members of the Desulfobacteriales in the Guaymas sediments. In addition, the dsrAB sequencing approach revealed a novel group of sulfate-reducing prokaryotes that could not be identified by 16S rRNA sequencing.     

Effects of Dissolved Sulfide, pH, and Temperature on Growth and Survival of Marine Hyperthermophilic Archaea

The ability of metabolically diverse hyperthermophilic archaea to withstand high temperatures, low pHs, high sulfide concentrations, and the absence of carbon and energy sources was investigated. Close relatives of our study organisms, Methanocaldococcus jannaschii, Archaeoglobus profundus, Thermococcus fumicolans, and Pyrococcus sp. strain GB-D, are commonly found in hydrothermal vent chimney walls and hot sediments and possibly deeper in the subsurface, where highly dynamic hydrothermal flow patterns and steep chemical and temperature gradients provide an ever-changing mosaic of microhabitats. These organisms (with the possible exception of Pyrococcus strain GB-D) tolerated greater extremes of low pH, high sulfide concentration, and high temperature when actively growing and metabolizing than when starved of carbon sources and electron donors/acceptors. Therefore these organisms must be actively metabolizing in the hydrothermal vent chimneys, sediments, and subsurface in order to withstand at least 24 h of exposure to extremes of pH, sulfide, and temperature that occur in these environments.     

Conservation implications of significant population differentiation in an endangered estuarine seahorse

The spatial distribution of a species’ genetic diversity can provide insights into underlying evolutionary, ecological and environmental processes, and can contribute information towards the delineation of conservation units. The Knysna seahorse, Hippocampus capensis, is endangered and occurs in only three estuaries on the warm-temperate south coast of South Africa: Knsyna, Keurbooms and Swartvlei. Population sizes in the latter two estuaries have been very small for a prolonged period of time, and the populations residing in them may thus benefit from translocations as a means of increasing population sizes and possibly also genetic diversity. However, information on whether these three estuaries constitute distinct conservation units that warrant separate management is presently lacking. Here, we used genetic information from mitochondrial (control region) and nuclear microsatellite loci to assess the genetic diversity and spatial structure across the three estuaries, and also whether translocations should be included in the management plan for the Knysna seahorse. Although each population had a unique combination of alleles, and clustering methods identified the Swartvlei Estuary as being distinct from the others, levels of genetic admixture were high, and there was no evidence for reciprocal monophyly that would indicate that each estuary has a unique demographic history. On these grounds, we suggest recognising the three populations as a single evolutionarily significant unit (ESU), and encourage translocations between them to ensure the species’ long-term survival.     

Site-specific modification of Candida antarctica lipase B via residue-specific incorporation of a non-canonical amino acid

In order to modify proteins in a controlled way, new functionalities need to be introduced in a defined manner. One way to accomplish this is by the incorporation of a non-natural amino acid of which the side chain can selectively be reacted to other molecules. We have investigated whether the relatively simple method of residue-specific replacement of methionine by azidohomoalanine can be used to achieve monofunctionalization of the model enzyme Candida antarctica lipase B. A protein variant was engineered with one additional methionine residue. Due to the high hydrophobicity and low abundance of methionine, this was the only residue out of five that was exposed to the solvent. The use of the Cu (I)-catalyzed [3 + 2] cycloaddition under native conditions resulted in a monofunctionalized enzyme which retained hydrolytic activity. The strategy can be considered a convenient tool to modify proteins at a single position as long as one solvent-exposed methionine is available.