Design, expression and characterisation of lysine-rich forms of the barley seed protein CI-2

Chymotrypsin inhibitor CI-2 is a small (84 residue) barley seed protein that has been used extensively to study protein folding. It also contains eight lysine residues, making it an attractive target for expression in transgenic plants to increase their lysine contents. We have designed three lysine-enriched forms of CI-2 and compared their structures and properties with that of the wild type protein. One mutant containing three additional lysine residues in the inhibitory loop shows high stability to denaturation and reduced inhibitory activity, indicating its suitability for use in genetic engineering.     

Chytridiomycosis impacts predator-prey interactions in larval amphibian communities

Despite ecologists increasingly recognizing pathogens as playing significant roles in community dynamics, few experimental studies have quantified patterns of disease impacts on natural systems. Amphibians are experiencing population declines, and a fungal pathogen (Batrachochytrium dendrobatidis; Chytridiomycota) is a suspected causal agent in many declines. We studied the effects of a pathogenic fungus on community interactions between the gray treefrog, Hyla chrysoscelis, and eastern newts, Notophthalmus viridescens. Recent studies have characterized chytridiomycosis as an emerging infectious disease, whose suspected rapid range expansion and widespread occurrence pose a significant risk for amphibian populations worldwide. We reared larvae in outdoor polyethylene experimental tanks and tested the effects of initial larval density, predator presence, and fungal exposure on Hyla recruitment and predator-prey interactions between Hyla and Notophthalmus. Newts reduced treefrog survival, and high intraspecific density decreased metamorphic body mass independent of B. dendrobatidis. The presence of fungi reduced treefrog body mass at metamorphosis by 34%, but had no significant main effect on survival or larval period length. B. dendrobatidis differentially affected larval development in the presence of predators; Hyla developed slower when reared with the pathogen, but only when newts were present. This significant predator-by-pathogen interaction suggests that the impact of chytridiomycosis on larval amphibians may be exacerbated in complex communities. Our data suggest that B. dendrobatidis effects on host life history may be complex and indirect. Direct measurements of the community-level effects of pathogens offer an important opportunity to understand a significant threat to global biodiversity—declining amphibian populations.     

The influence of temperature and precipitation on spring dispersal of Frankliniella fusca changes as the season progresses

Effects of temperature and precipitation on spring dispersal patterns of Frankliniella fusca (Hinds) (Thysanoptera: Thripidae) caught on yellow sticky traps were estimated in central and eastern North Carolina and eastern Virginia, USA, from 1997 to 2001, and in 2004 and 2007. Data were collected from 44 sites within 14 locations over 7 years, resulting in 30 location‐year data combinations. The following independent variables were examined to determine their relationship to the number of F. fusca caught on sticky traps during specified time intervals: cumulative degree days (base 10.5 °C) from 1 November to the start of each trapping interval (DD), number of days with temperatures favorable for flight during each trapping interval (DTFF), and an index of rainfall during specific intervals prior to and during the trapping interval (RI). Regression models that contained various combinations of these variables explained 62, 79, 74, and 68% of the variation in the number of dispersing F. fusca captured during 1–15 April, 16–30 April, 1–15 May, and 16–31 May, respectively. The results provide strong evidence that the suppressive effects of precipitation on growth of local populations developing during late winter and early spring are subsequently manifest at the landscape scale as reductions in the populations of dispersing adults that may persist for as long as 5–6 weeks after the precipitation occurs.     

Influence of spatial structure on effective nutrient diffusion in bacterial biofilms

The main contribution of this paper is to use homogenization techniques to compute diffusion coefficients from experimental images of microbial biofilms. Our approach requires the analysis of several experimental spatial structures of biofilms in order to derive from them a Representative Volume Element (RVE). Then, we apply a suitable numerical procedure to the RVE to derive the diffusion coefficients. We show that diffusion coefficients significantly vary with the biofilm structure. These results suggest that microbial biofilm structures can favour nutrient access in some cases.     

Management of winter weeds affects Frankliniella fusca (Thysanoptera: Thripidae) dispersal

Frankliniella fusca (Hinds) naturally disperses from winter weeds to crops in spring, causing direct and indirect damage. Field preparation before planting includes use of herbicides or cultivation to kill unwanted vegetation, which adversely affects F. fusca host plants and potentially influences F. fusca dispersal. Common chickweed, Stellaria media (L.), infested with F. fusca, was used as a model to study effects of timing and type of vegetation management on adult dispersal. Infested weeds were caged and F. fusca weekly dispersal was monitored using sticky traps. Weed management treatments performed at an early (14 April-11 May) or late (2 wk after early treatment) date consisted of glyphosate, paraquat, disking, hoeing, or untreated control. Late glyphosate and hoeing treatments resulted in cumulative dispersal statistically similar to or greater than from control plots. Compared with the control, significantly more F. fusca dispersed from the glyphosate and hoeing plots during the 3 wk after treatment. More thrips dispersed from the late paraquat treatment 1 wk post-application than from the control. Dispersal from the disked treatment and early paraquat treatment was similar to that of the control 1- to 3-wk post-treatment. Early treatments resulted in significantly smaller cumulative dispersal than the control in all but one instance. Late disking and paraquat treatments resulted in cumulative F. fusca captures that were statistically similar or less than that in the control. Winter weed management type and timing affect F. fusca dispersal magnitude and duration.     

Reconstitution of Plant Alkane Biosynthesis in Yeast Demonstrates That Arabidopsis ECERIFERUM1 and ECERIFERUM3 Are Core Components of a Very-Long-Chain Alkane Synthesis Complex

In land plants, very-long-chain (VLC) alkanes are major components of cuticular waxes that cover aerial organs, mainly acting as a waterproof barrier to prevent nonstomatal water loss. Although thoroughly investigated, plant alkane synthesis remains largely undiscovered. The Arabidopsis thaliana ECERIFERUM1 (CER1) protein has been recognized as an essential element of wax alkane synthesis; nevertheless, its function remains elusive. In this study, a screen for CER1 physical interaction partners was performed. The screen revealed that CER1 interacts with the wax-associated protein ECERIFERUM3 (CER3) and endoplasmic reticulum-localized cytochrome b5 isoforms (CYTB5s). The functional relevance of these interactions was assayed through an iterative approach using yeast as a heterologous expression system. In a yeast strain manipulated to produce VLC acyl-CoAs, a strict CER1 and CER3 coexpression resulted in VLC alkane synthesis. The additional presence of CYTB5s was found to enhance CER1/CER3 alkane production. Site-directed mutagenesis showed that CER1 His clusters are essential for alkane synthesis, whereas those of CER3 are not, suggesting that CYTB5s are specific CER1 cofactors. Collectively, our study reports the identification of plant alkane synthesis enzymatic components and supports a new model for alkane production in which CER1 interacts with both CER3 and CYTB5 to catalyze the redox-dependent synthesis of VLC alkanes from VLC acyl-CoAs.     

IL-6 Indirectly Modulates the Induction of Glyceroneogenic Enzymes in Adipose Tissue during Exercise

Background

Glyceroneogenesis is an important step in the control of fatty acid re-esterification with PEPCK and PDK4 being identified as key enzymes in this process. We have previously shown that glyceroneogenic enzymes such as PDK4 are rapidly induced in white adipose tissue during exercise. Recent studies have suggested that IL-6 regulates adipose tissue metabolism and gene expression during exercise. Interestingly, IL-6 has been reported to directly decrease PEPCK expression. The purpose of this investigation was to determine the role of IL-6 in modulating the effects of exercise on the expression of glyceroneogenic enzymes in mouse adipose tissue. We hypothesized that the exercise-mediated induction of PDK4 and PEPCK would be greater in adipose tissue from IL-6 deficient mice compared to wild type controls.

Methodology and Principle Findings

Treatment of cultured epididymal adipose tissue (eWAT) with IL-6 (150 ng/ml) increased the phosphorylation of AMPK, ACC and STAT3 and induced SOCS3 mRNA levels while decreasing PEPCK and PDK4 mRNA. AICAR decreased the expression of PDK4 and PEPCK. The activation of AMPK by IL-6 was independent of increases in lipolysis. An acute bout of treadmill running (15 meters/minute, 5% incline, 90 minutes) did not induce SOCS3 or increase phosphorylation of STAT3 in eWAT, indicating that IL-6 signalling was not activated. Exercise-induced increases in PEPCK and PDK4 mRNA expression were attenuated in eWAT from IL-6^−/− mice in parallel with a greater relative increase in AMPK phosphorylation compared to exercised WT mice. These changes occurred independent of alterations in beta-adrenergic signalling in adipose tissue from IL-6^−/− mice.

Conclusions and Significance

Our findings question the role of IL-6 signalling in adipose tissue during exercise and suggest an indirect effect of this cytokine in the regulation of adipose tissue gene expression during exercise.