Visualizing Interactions along the Escherichia coli Twin-Arginine Translocation Pathway Using Protein Fragment Complementation

The twin-arginine translocation (Tat) pathway is well known for its ability to export fully folded substrate proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Studies of this mechanism in Escherichia coli have identified numerous transient protein-protein interactions that guide export-competent proteins through the Tat pathway. To visualize these interactions, we have adapted bimolecular fluorescence complementation (BiFC) to detect protein-protein interactions along the Tat pathway of living cells. Fragments of the yellow fluorescent protein (YFP) were fused to soluble and transmembrane factors that participate in the translocation process including Tat substrates, Tat-specific proofreading chaperones and the integral membrane proteins TatABC that form the translocase. Fluorescence analysis of these YFP chimeras revealed a wide range of interactions such as the one between the Tat substrate dimethyl sulfoxide reductase (DmsA) and its dedicated proofreading chaperone DmsD. In addition, BiFC analysis illuminated homo- and hetero-oligomeric complexes of the TatA, TatB and TatC integral membrane proteins that were consistent with the current model of translocase assembly. In the case of TatBC assemblies, we provide the first evidence that these complexes are co-localized at the cell poles. Finally, we used this BiFC approach to capture interactions between the putative Tat receptor complex formed by TatBC and the DmsA substrate or its dedicated chaperone DmsD. Our results demonstrate that BiFC is a powerful approach for studying cytoplasmic and inner membrane interactions underlying bacterial secretory pathways.     

Roles of vaccinia virus genes E3L and K3L and host genes PKR and RNase L during intratracheal infection of C57BL/6 mice

The importance of the 2'-5' oligoadenylate synthetase (OAS)/RNase L and double-stranded RNA (dsRNA)-dependent protein kinase (PKR) pathways in host interferon induction resulting from virus infection in response to dsRNA has been well documented. In poxvirus infections, the interactions between the vaccinia virus (VV) genes E3L and K3L, which target RNase L and PKR, respectively, serve to prevent the induction of the dsRNA-dependent induced interferon response in cell culture. To determine the importance of these host genes in controlling VV infections, mouse single-gene knockouts of RNase L and PKR and double-knockout mice were studied following intratracheal infection with VV, VVΔK3L, or VVΔE3L. VV caused lethal disease in all mouse strains. The single-knockout animals were more susceptible than wild-type animals, while the RNase L(-/-) PKR(-/-) mice were the most susceptible. VVΔE3L infections of wild-type mice were asymptomatic, demonstrating that E3L plays a critical role in controlling the host immune response. RNase L(-/-) mice showed no disease, whereas 20% of the PKR(-/-) mice succumbed at a dose of 10(8) PFU. Lethal disease was routinely observed in RNase L(-/-) PKR(-/-) mice inoculated with 10(8) PFU of VVΔE3L, with a distinct pathology. VVΔK3L infections exhibited no differences in virulence among any of the mouse constructs, suggesting that PKR is not the exclusive target of K3L. Surprisingly, VVΔK3L did not disseminate to other tissues from the lung. Hence, the cause of death in this model is respiratory disease. These results also suggest that an unanticipated role of the K3L gene is to facilitate virus dissemination.     

Controlling chirality

New strategies are continually being developed for using enzymes to efficiently catalyse the enantioselective synthesis of chiral non-racemic compounds. Alternatives to asymmetric synthesis or kinetic resolution include dynamic kinetic resolution, deracemisation and enantioconvergent transformations. Moreover, a much greater understanding is being developed of the parameters that can affect the stereochemical outcome of the reaction (e.g. solvent, substrate design, immobilization and directed evolution).     

Biophysical Variability and Pastoral Rights to Resources: West African Transhumance Revisited

This paper focuses on a conundrum that has dominated the literature on pastoral mobility and institutions in dryland regions of the world, where livestock production is the main livelihood system. High spatiotemporal variability of rainfall and forage resources are seen to require flexible rules and porous social boundaries to facilitate pastoral mobility—characteristics that run counter to conventional views of the requirements for effective common property institutions. We seek to explore this paradox by investigating the spatiotemporal variability of forage availability (using satellite derived vegetation indices as a proxy for green forage) in four transhumance zones (“transhumance sheds”) in Mali, West Africa. For each transhumance shed, three characteristics with important institutional implications are evaluated over an eleven-year period between 2000 and 2010: the inter-annual variability of forage phenology, seasonal changes in connectivity of green forage patches, and the degree to which key forage locations exist in the form of consistently early green-up and/or late senescence. Periods of vegetation green-up and senescence, which determine the timing of transhumant livestock movements, are found to be sufficiently regular from year to year to be governed by conventional institutions. Seasonal changes in the north-south connectivity of green patches are sufficiently rapid for customary systems of sharing of pasture information to be effective (rather than more technologically sophisticated systems of pasture information). Moreover, transhumance sheds contain key pastoral forage sites, which because of their consistently early greening or late senescence, are strong candidates for territorial protection from alternative land uses such as agriculture. These findings support local herders’ views of transhumance as composed of regular patterns of herd movements along prescribed corridors between key pastoral sites. The seasonal regularity of key pastoral resources has been obscured by an overemphasis on environmental unpredictability that characterizes dryland systems at certain spatial and temporal scales. This paper suggests that policies directed at improving pastoral resource governance must focus instead on securing pastoralists’ access rights to movement corridors, specific pastures and water points.     

Representation of aquatic vegetation change by plant macrofossils in a small and shallow freshwater lake

We explored spatial and temporal relationships between contemporary aquatic vegetation and surface sediment macrofossil remains in a small, shallow, English lake (Green Plantation Pond). The aquatic vegetation of Green Plantation Pond underwent a marked compositional change after 2005 with a shift from Elodea spp.-Potamogeton pusillus-Chara spp. to Ceratophyllum spp.-Chara spp.-Potamogeton crispus dominance. By comparing macrophyte and plant macrofossil distributions at multiple, closely spaced points in Green Plantation Pond for 2000 and 2008–2009, we studied the ability of macrofossils to track this major aquatic vegetation change. Representation of macrophytes by macrofossils was high with 63 and 76 % of extant plant species recorded by macro-remains in the 2000 and 2009 sediment surveys respectively. Nevertheless, plants were both over-represented (Nitella flexilis, Chara spp. and Zannichellia palustris) and under-represented (Ranunculus sect. Batrachium, Potamogeton spp.) in the sediment record in terms of relative macrofossil abundances and the number of occupied sample points. The study also revealed a lack of preservation of Elodea spp. leaf remains in the second (2009) survey compared to the first (2000) probably due to a longer time interval (5 vs. 10 months) between macrophyte and sediment sampling. Nevertheless, the macrofossils reliably recorded both the main shift in the contemporary vegetation (e.g. especially increases in Ceratophyllum spp. and P. crispus abundance) and other more subtle floristic changes (e.g. increases in Myriophyllum spicatum and Lemna spp.) exceptionally well. This study highlights the huge potential of macrofossils for tracking sub-decadal changes in the aquatic vegetation of small, shallow lakes.     

Cl? and K+ channels and their role in primary brain tumour biology

Profound cell volume changes occur in primary brain tumours as they proliferate, invade surrounding tissue or undergo apoptosis. These volume changes are regulated by the flux of Cl⁻ and K⁺ ions and concomitant movement of water across the membrane, making ion channels pivotal to tumour biology. We discuss which specific Cl⁻ and K⁺ channels are involved in defined aspects of glioma biology and how these channels are regulated. Cl⁻ is accumulated to unusually high concentrations in gliomas by the activity of the NKCC1 transporter and serves as an osmolyte and energetic driving force for volume changes. Cell volume condensation is required as cells enter M phase of the cell cycle and this pre-mitotic condensation is caused by channel-mediated ion efflux. Similarly, Cl⁻ and K⁺ channels dynamically regulate volume in invading glioma cells allowing them to adjust to small extracellular brain spaces. Finally, cell condensation is a hallmark of apoptosis and requires the concerted activation of Cl⁻ and Ca²⁺-activated K⁺ channels. Given the frequency of mutation and high importance of ion channels in tumour biology, the opportunity exists to target them for treatment.     

Genetic associations with micronutrient levels identified in immune and gastrointestinal networks

The discovery of vitamins and clarification of their role in preventing frank essential nutrient deficiencies occurred in the early 1900s. Much vitamin research has understandably focused on public health and the effects of single nutrients to alleviate acute conditions. The physiological processes for maintaining health, however, are complex systems that depend upon interactions between multiple nutrients, environmental factors, and genetic makeup. To analyze the relationship between these factors and nutritional health, data were obtained from an observational, community-based participatory research program of children and teens (age 6–14) enrolled in a summer day camp in the Delta region of Arkansas. Assessments of erythrocyte S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), plasma homocysteine (Hcy) and 6 organic micronutrients (retinol, 25-hydroxy vitamin D3, pyridoxal, thiamin, riboflavin, and vitamin E), and 1,129 plasma proteins were performed at 3 time points in each of 2 years. Genetic makeup was analyzed with 1 M SNP genotyping arrays, and nutrient status was assessed with 24-h dietary intake questionnaires. A pattern of metabolites (met_PC1) that included the ratio of erythrocyte SAM/SAH, Hcy, and 5 vitamins were identified by principal component analysis. Met_PC1 levels were significantly associated with (1) single-nucleotide polymorphisms, (2) levels of plasma proteins, and (3) multilocus genotypes coding for gastrointestinal and immune functions, as identified in a global network of metabolic/protein–protein interactions. Subsequent mining of data from curated pathway, network, and genome-wide association studies identified genetic and functional relationships that may be explained by gene–nutrient interactions. The systems nutrition strategy described here has thus associated a multivariate metabolite pattern in blood with genes involved in immune and gastrointestinal functions.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0408-4) contains supplementary material, which is available to authorized users.