Cross-linked SecA dimers are not functional in protein translocation

The ATPase SecA is involved in post-translational protein translocation through the SecY channel across the bacterial inner membrane. SecA is a dimer that can dissociate into monomers with translocation activity. Here, we have addressed whether dissociation of the SecA dimer is required for translocation. We show that a dimer in which the two subunits are cross-linked by disulfide bridges is inactive in protein translocation, translocation ATPase, and binding to a lipid bilayer. In contrast, upon reduction of the disulfide bridges, the resulting monomers regain these activities. These data support the notion that dissociation of SecA dimers into monomers occurs during protein translocation.     

Identification of methylated flavonoid regioisomeric metabolites using enzymatic semisynthesis and liquid chromatography-tandem mass spectrometry

Discrimination of isomeric methylated metabolites is an important step toward identifying genes responsible for methylation, but presents substantial challenges because authentic standards are often unavailable and mass spectra of isomers have been considered indistinguishable. In this report, an approach is described for identifying methyl group positions in multiply methylated flavonoid metabolites using combinations of tandem mass spectrometry, liquid chromatography retention, and site-selective methylation by recombinant O-methyltransferases from Solanum habrochaites LA1777. The basis for observed fragment ions in tandem mass spectra of multiply methylated myricetin was further established using enzymatic incorporation of deuterium-labeled methyl groups using S-adenosylmethionine-d ₃ as precursor.     

Cell lineage analysis of the mammalian female germline

Fundamental aspects of embryonic and post-natal development, including maintenance of the mammalian female germline, are largely unknown. Here we employ a retrospective, phylogenetic-based method for reconstructing cell lineage trees utilizing somatic mutations accumulated in microsatellites, to study female germline dynamics in mice. Reconstructed cell lineage trees can be used to estimate lineage relationships between different cell types, as well as cell depth (number of cell divisions since the zygote). We show that, in the reconstructed mouse cell lineage trees, oocytes form clusters that are separate from hematopoietic and mesenchymal stem cells, both in young and old mice, indicating that these populations belong to distinct lineages. Furthermore, while cumulus cells sampled from different ovarian follicles are distinctly clustered on the reconstructed trees, oocytes from the left and right ovaries are not, suggesting a mixing of their progenitor pools. We also observed an increase in oocyte depth with mouse age, which can be explained either by depth-guided selection of oocytes for ovulation or by post-natal renewal. Overall, our study sheds light on substantial novel aspects of female germline preservation and development.     

Vulnerability of wetland soil carbon stocks to climate warming in the perhumid coastal temperate rainforest

The perhumid coastal temperate rainforest (PCTR) of southeast Alaska has some of the densest soil organic carbon (SOC) stocks in the world (>300 Mg C ha^?1) but the fate of this SOC with continued warming remains largely unknown. We quantified dissolved organic carbon (DOC) and carbon dioxide (CO₂) yields from four different wetland types (rich fen, poor fen, forested wetland and cedar wetland) using controlled laboratory incubations of surface (10 cm) and subsurface (25 cm) soils incubated at 8 and 15 °C for 37 weeks. Furthermore, we used fluorescence characterization of DOC and laboratory bioassays to assess how climate-induced soil warming may impact the quality and bioavailability of DOC delivered to fluvial systems. Soil temperature was the strongest control on SOC turnover, with wetland type and soil depth less important in controlling CO₂ flux and extractable DOC. The high temperature incubation increased average CO₂ yield by ~40 and ~25% for DOC suggesting PCTR soils contain a sizeable pool of readily biodegradable SOC that can be mineralized to DOC and CO₂ with future climate warming. Fluxes of CO₂ were positively correlated to both extractable DOC and percent bioavailable DOC during the last few months of the incubation suggesting mineralization of SOC to DOC is a strong control of soil respiration rates. Whether the net result is increased export of either carbon form will depend on the balance between the land to water transport of DOC and the ability of soil microbial communities to mineralize DOC to CO₂.     

Bidirectional communication system in katydids: the effect on chorus structure

Unlike most acoustic systems evolved for pair formation whereonly males signal, the katydidPhaneroptera nana has a bidirectional communication system where both males and females sing. Despiteextensive study on male chorusing behavior in different communicationsystems, this behavior has rarely been explored in duettingspecies. I examined how this bidirectional communication systemaffects the collective pattern of male signaling.P. nana malesalternate their songs, and in response to synthetic stimulidelay their calls, according to the phase of stimulation. Pairsof synthetic calls (simulating alternating males) presentedto females elicited equal female response, as long as the intercallinterval was 200 ms. Thus, male alternation is imposed by thefemale's responsiveness and may be interpreted as a "jammingavoidance reaction." Further evidence suggests that chorusstructure is not merely constrained by the female sensory temporalresolution, but rather is adaptively related to female choicein this species.     

Patterns of cell signaling pathway activation that characterize mammary development

Previous work has detailed the histological and biochemical changes associated with mammary development and remodeling. We have now made use of gene expression profiling, and in particular of the previously described signatures of cell signaling pathway activation, to explore the events associated with mammary gland development. We find that there is elevated E2F-specific pathway activity prior to lactation and relatively low levels of other important signaling pathways, such as RAS, MYC and SRC. Upon lactation and continuing into the involution phase, these patterns reverse with a dramatic increase in RAS, SRC and MYC pathway activity and a decline in E2F activity. At the end of involution, these patterns return to that of the adult non-lactating mammary gland. The importance of the changes in E2F pathway activity, particularly during the proliferative phase of mammary development, was confirmed through the analysis of mice deficient for various E2F proteins. Taken together, these results reveal a complex pattern of pathway activity in relation to the various phases of mammary gland development.     

Return of Salmon-Derived Nutrients from the Riparian Zone to the Stream during a Storm in Southeastern Alaska

Spawning salmon deliver nutrients (salmon-derived nutrients, SDN) to natal watersheds that can be incorporated into terrestrial and aquatic food webs, potentially increasing ecosystem productivity. Peterson Creek, a coastal watershed in southeast Alaska that supports several species of anadromous fish, was sampled over the course of a storm during September 2006 to test the hypothesis that stormflows re-introduce stored SDN into the stream. We used stable isotopes and PARAFAC modeling of fluorescence excitation–emission spectroscopy to detect flushing of DOM from salmon carcasses in the riparian zone back into a spawning stream. During the early storm hydrograph, streamwater concentrations of NH₄–N and total dissolved phosphorus (TDP), the fluorescent protein tyrosine and the δ¹⁵N content of DOM peaked, followed by a rapid decrease during maximum stormflow. Although δ¹⁵N has previously been used to track SDN in riparian zones, the use of fluorescence spectroscopy provides an independent indicator that SDN are being returned from the riparian zone to the stream after a period of intermediate storage outside the stream channel. Our findings further demonstrate the utility of using both δ¹⁵N of streamwater DOM and fluorescence spectroscopy with PARAFAC modeling to monitor how the pool of streamwater DOM changes in spawning salmon streams.     

The long-term genetic stability and individual specificity of the human gut microbiome

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Watershed ‘chemical cocktails’: forming novel elemental combinations in Anthropocene fresh waters

In the Anthropocene, watershed chemical transport is increasingly dominated by novel combinations of elements, which are hydrologically linked together as ‘chemical cocktails.’ Chemical cocktails are novel because human activities greatly enhance elemental concentrations and their probability for biogeochemical interactions and shared transport along hydrologic flowpaths. A new chemical cocktail approach advances our ability to: trace contaminant mixtures in watersheds, develop chemical proxies with high-resolution sensor data, and manage multiple water quality problems. We explore the following questions: (1) Can we classify elemental transport in watersheds as chemical cocktails using a new approach? (2) What is the role of climate and land use in enhancing the formation and transport of chemical cocktails in watersheds? To address these questions, we first analyze trends in concentrations of carbon, nutrients, metals, and salts in fresh waters over 100 years. Next, we explore how climate and land use enhance the probability of formation of chemical cocktails of carbon, nutrients, metals, and salts. Ultimately, we classify transport of chemical cocktails based on solubility, mobility, reactivity, and dominant phases: (1) sieved chemical cocktails (e.g., particulate forms of nutrients, metals and organic matter); (2) filtered chemical cocktails (e.g., dissolved organic matter and associated metal complexes); (3) chromatographic chemical cocktails (e.g., ions eluted from soil exchange sites); and (4) reactive chemical cocktails (e.g., limiting nutrients and redox sensitive elements). Typically, contaminants are regulated and managed one element at a time, even though combinations of elements interact to influence many water quality problems such as toxicity to life, eutrophication, infrastructure corrosion, and water treatment. A chemical cocktail approach significantly expands evaluations of water quality signatures and impacts beyond single elements to mixtures. High-frequency sensor data (pH, specific conductance, turbidity, etc.) can serve as proxies for chemical cocktails and improve real-time analyses of water quality violations, identify regulatory needs, and track water quality recovery following storms and extreme climate events. Ultimately, a watershed chemical cocktail approach is necessary for effectively co-managing groups of contaminants and provides a more holistic approach for studying, monitoring, and managing water quality in the Anthropocene.