Selectivity of inhibitors of endocannabinoid biosynthesis evaluated by activity-based protein profiling

The endocannabinoid 2-arachidonoylglycerol (2-AG) has been implicated as a key retrograde mediator in the nervous system based on pharmacological studies using inhibitors of the 2-AG biosynthetic enzymes diacyglycerol lipase alpha and beta (DAGL-alpha/beta). Here, we show by competitive activity-based protein profiling that the DAGL-alpha/beta inhibitors, tetrahydrolipstatin (THL) and RHC80267, block several brain serine hydrolases with potencies equal to or greater than their inhibitory activity against DAGL enzymes. Interestingly, a minimal overlap in target profiles was observed for THL and RHC80267, suggesting that pharmacological effects observed with both agents may be viewed as good initial evidence for DAGL-dependent events.     

Microbial Community Profiling to Investigate Transmission of Bacteria Between Life Stages of the Wood-Boring Beetle, Anoplophora glabripennis

Many insects harbor specific bacteria in their digestive tract, and these gut microbiota often play important roles in digestion and nutrient provisioning. While it is common for a given insect species to harbor a representative gut microbial community as a population, how this community is acquired and maintained from generation to generation is not known for most xylophagous insects, except termites. In this study, we examined acquisition of gut microbiota by the wood-feeding beetle, Anoplophora glabripennis, by identifying and comparing microbial community members among different life stages of the insect and with microbes it encounters in the environment. Automated ribosomal intergenic spacer analysis was employed to compare bacterial communities present in the egg and larval stages of A. glabripennis as well as with microbes found in the oviposition site and the surrounding woody tissue. Multivariate analyses were used to identify relationships between sample type and specific bacterial types (operational taxonomic units). From this analysis, bacteria that were derived from the environment, the oviposition site, and/or the egg were identified and compared with taxa found in larvae. Results showed that while some larval microbes were derived from environmental sources, other members of the larval microbial community appear to be vertically transmitted. These findings could lead to a better understanding of which microbial species are critical for the survival of this insect and to development of techniques that could be used to alter this community to disrupt the digestive physiology of the host insect as a biological control measure.     

A scalable, fully automated process for construction of sequence-ready barcoded libraries for 454

We present an automated, high throughput library construction process for 454 technology. Sample handling errors and cross-contamination are minimized via end-to-end barcoding of plasticware, along with molecular DNA barcoding of constructs. Automation-friendly magnetic bead-based size selection and cleanup steps have been devised, eliminating major bottlenecks and significant sources of error. Using this methodology, one technician can create 96 sequence-ready 454 libraries in 2 days, a dramatic improvement over the standard method.     

Strain fidelity of chronic wasting disease upon murine adaptation

Chronic wasting disease (CWD), a prion disease of deer and elk, is highly prevalent in some regions of North America. The establishment of mouse-adapted CWD prions has proven difficult due to the strong species barrier between mice and deer. Here we report the efficient transmission of CWD to transgenic mice overexpressing murine PrP. All mice developed disease 500 +/- 62 days after intracerebral CWD challenge. The incubation period decreased to 228 +/- 103 days on secondary passage and to 162 +/- 6 days on tertiary passage. Mice developed very large, radially structured cerebral amyloid plaques similar to those of CWD-infected deer and elk. PrP(Sc) was detected in spleen, indicating that murine CWD was lymphotropic. PrP(Sc) glycoform profiles maintained a predominantly diglycosylated PrP pattern, as seen with CWD in deer and elk, across all passages. Therefore, all pathological, biochemical, and histological strain characteristics of CWD appear to persist upon repetitive serial passage through mice. These findings indicate that the salient strain-specific properties of CWD are encoded by agent-intrinsic components rather than by host factors.     

A test of two mechanisms proposed to optimize grassland aboveground primary productivity in response to grazing

Aims Mesic grasslands have a long evolutionary history of grazing by large herbivores and as a consequence, grassland species have numerous adaptations allowing them to respond favourably to grazing. Although empirical evidence has been equivocal, theory predicts that such adaptations combined with alterations in resources can lead to grazing-induced overcompensation in aboveground net primary production (ANPP; grazed ANPP> ungrazed ANPP) under certain conditions. We tested two specific predictions from theory. First, overcompensation is more likely to occur in annually burned grasslands because limiting nutrients that would be lost with frequent fires are recycled through grazers and stimulate ANPP. Second, overcompensation of biomass lost to grazers is more likely to occur in unburned sites where grazing has the greatest effect on increasing light availability through alterations in canopy structure.Methods We tested these nutrient versus light-based predictions in grazed grasslands that had been annually burned or protected from fire for>20 years. We assessed responses in ANPP to grazing by large ungulates using both permanent and moveable grazing exclosures (252 exclosures from which biomass was harvested from 3192 quadrats) in a 2-year study. Study sites were located at the Konza Prairie Biological Station (KPBS) in North America and at Kruger National Park (KNP) in South Africa. At KPBS, sites were grazed by North American bison whereas in KNP sites were grazed either by a diverse suite of herbivores (e.g. blue wildebeest, Burchell's zebra, African buffalo) or by a single large ungulate (African buffalo).Important findings We found no evidence for overcompensation in either burned or unburned sites, regardless of grazer type. Thus, there was no support for either mechanism leading to overcompensation. Instead, complete compensation of total biomass lost to grazers was the most common response characterizing grazing–ANPP relationships with, in some cases, undercompensation of grass ANPP being offset by increased ANPP of forbs likely due to competitive release. The capability of these very different grass-dominated systems to maintain ANPP while being grazed has important implications for energy flow, ecosystem function and the trophic dynamics of grasslands.     

A hormone-dependent module regulating energy balance

Under fasting conditions, metazoans maintain energy balance by shifting from glucose to fat burning. In the fasted state, SIRT1 promotes catabolic gene expression by deacetylating the forkhead factor FOXO in response to stress and nutrient deprivation. The mechanisms by which hormonal signals regulate FOXO deacetylation remain unclear, however. We identified a hormone-dependent module, consisting of the Ser/Thr kinase SIK3 and the class IIa deacetylase HDAC4, which regulates FOXO activity in Drosophila. During feeding, HDAC4 is phosphorylated and sequestered in the cytoplasm by SIK3, whose activity is upregulated in response to insulin. SIK3 is inactivated during fasting, leading to the dephosphorylation and nuclear translocation of HDAC4 and to FOXO deacetylation. SIK3 mutant flies are starvation sensitive, reflecting FOXO-dependent increases in lipolysis that deplete triglyceride stores; reducing HDAC4 expression restored lipid accumulation. Our results reveal a hormone-regulated pathway that functions in parallel with the nutrient-sensing SIRT1 pathway to maintain energy balance.     

Bacterial production of free fatty acids from freshwater macroalgal cellulose

The predominant strategy for using algae to produce biofuels relies on the overproduction of lipids in microalgae with subsequent conversion to biodiesel (methyl-esters) or green diesel (alkanes). Conditions that both optimize algal growth and lipid accumulation rarely overlap, and differences in growth rates can lead to wild species outcompeting the desired lipid-rich strains. Here, we demonstrate an alternative strategy in which cellulose contained in the cell walls of multicellular algae is used as a feedstock for cultivating biofuel-producing microorganisms. Cellulose was extracted from an environmental sample of Cladophora glomerata-dominated periphyton that was collected from Lake Mendota, WI, USA. The resulting cellulose cake was hydrolyzed by commercial enzymes to release fermentable glucose. The hydrolysis mixture was used to formulate an undefined medium that was able to support the growth, without supplementation, of a free fatty acid (FFA)-overproducing strain of Escherichia coli (Lennen et. al 2010). To maximize free fatty acid production from glucose, an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible vector was constructed to express the Umbellularia californica acyl-acyl carrier protein (ACP) thioesterase. Thioesterase expression was optimized by inducing cultures with 50 μM IPTG. Cell density and FFA titers from cultures grown on algae-based media reached 50% of those (～90 μg/mL FFA) cultures grown on rich Luria-Bertani broth supplemented with 0.2% glucose. In comparison, cultures grown in two media based on AFEX-pretreated corn stover generated tenfold less FFA than cultures grown in algae-based media. This study demonstrates that macroalgal cellulose is a potential carbon source for the production of biofuels or other microbially synthesized compounds.