Ecological succession of bacterial communities during conventionalization of germ-free mice

Little is known about the dynamics of early ecological succession during experimental conventionalization of the gastrointestinal (GI) tract; thus, we measured changes in bacterial communities over time, at two different mucosal sites (cecum and jejunum), with germfree C57BL/6 mice as the recipients of cecal contents (input community) from a C57BL/6 donor mouse. Bacterial communities were monitored using pyrosequencing of 16S rRNA gene amplicon libraries from the cecum and jejunum and analyzed by a variety of ecological metrics. Bacterial communities, at day 1 postconventionalization, in the cecum and jejunum had lower diversity and were distinct from the input community (dominated by either Escherichia or Bacteroides). However, by days 7 and 21, the recipient communities had become significantly diverse and the cecal communities resembled those of the donor and donor littermates, confirming that transfer of cecal contents results in reassembly of the community in the cecum 7 to 21 days later. However, bacterial communities in the recipient jejunum displayed significant structural heterogeneity compared to each other or the donor inoculum or the donor littermates, suggesting that the bacterial community of the jejunum is more dynamic during the first 21 days of conventionalization. This report demonstrates that (i) mature input communities do not simply reassemble at mucosal sites during conventionalization (they first transform into a "pioneering" community and over time take on the appearance, in membership and structure, of the original input community) and (ii) the specific mucosal environment plays a role in shaping the community.     

Baseline toxicity and field efficacy of metaflumizone on Colorado potato beetle (Coleoptera: Chrysomelidae)

Baseline toxicity levels to a novel semicarbazone insecticide, metaflumizone were established for 25 field populations of Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae),from North America. Excluding the susceptible laboratory strain, 50% lethal concentrations of metaflumizone ranged from 0.57 to 1.31 ppm, while response slopes ranged from 1.92 to 4.24 (average = 2.93), and were unrelated to the 50% lethal concentration (r = 0.06; P = 0.76). Beetle populations with known resistance to the neonicotinoid imidacloprid also exhibited the highest LC50 levels to metaflumizone suggesting at least the possibility of cross-resistance. Additional experiments using a potato leaf-dip bioassay as well as field efficacy evaluations confirmed the high level of toxicity of metaflumizone to L. decemlineata and demonstrated a potential benefit of tank mixing a low rate of the pyrethroid esfenvalerate with metaflumizone at one-tenth the recommended field rate. These research findings confirm that metaflumizone is highly active against L. decemlineata larvae and adults and could provide an effective alternative insecticide for potato pest management.     

Vitamin C deficiency activates the purine nucleotide cycle in zebrafish

Vitamin C (ascorbic acid, AA) is a cofactor for many important enzymatic reactions and a powerful antioxidant. AA provides protection against oxidative stress by acting as a scavenger of reactive oxygen species, either directly or indirectly by recycling of the lipid-soluble antioxidant, α-tocopherol (vitamin E). Only a few species, including humans, guinea pigs, and zebrafish, cannot synthesize AA. Using an untargeted metabolomics approach, we examined the effects of α-tocopherol and AA deficiency on the metabolic profiles of adult zebrafish. We found that AA deficiency, compared with subsequent AA repletion, led to oxidative stress (using malondialdehyde production as an index) and to major increases in the metabolites of the purine nucleotide cycle (PNC): IMP, adenylosuccinate, and AMP. The PNC acts as a temporary purine nucleotide reservoir to keep AMP levels low during times of high ATP utilization or impaired oxidative phosphorylation. The PNC promotes ATP regeneration by converting excess AMP into IMP, thereby driving forward the myokinase reaction (2ADP → AMP + ATP). On the basis of this finding, we investigated the activity of AMP deaminase, the enzyme that irreversibly deaminates AMP to form IMP. We found a 47% increase in AMP deaminase activity in the AA-deficient zebrafish, complementary to the 44-fold increase in IMP concentration. These results suggest that vitamin C is crucial for the maintenance of cellular energy metabolism.     

Genetically modified crops and aquatic ecosystems: considerations for environmental risk assessment and non-target organism testing

Environmental risk assessments (ERA) support regulatory decisions for the commercial cultivation of genetically modified (GM) crops. The ERA for terrestrial agroecosystems is well-developed, whereas guidance for ERA of GM crops in aquatic ecosystems is not as well-defined. The purpose of this document is to demonstrate how comprehensive problem formulation can be used to develop a conceptual model and to identify potential exposure pathways, using Bacillus thuringiensis (Bt) maize as a case study. Within problem formulation, the insecticidal trait, the crop, the receiving environment, and protection goals were characterized, and a conceptual model was developed to identify routes through which aquatic organisms may be exposed to insecticidal proteins in maize tissue. Following a tiered approach for exposure assessment, worst-case exposures were estimated using standardized models, and factors mitigating exposure were described. Based on exposure estimates, shredders were identified as the functional group most likely to be exposed to insecticidal proteins. However, even using worst-case assumptions, the exposure of shredders to Bt maize was low and studies supporting the current risk assessments were deemed adequate. Determining if early tier toxicity studies are necessary to inform the risk assessment for a specific GM crop should be done on a case by case basis, and should be guided by thorough problem formulation and exposure assessment. The processes used to develop the Bt maize case study are intended to serve as a model for performing risk assessments on future traits and crops.     

Adjacent Habitat Influence on Stink Bug (Hemiptera: Pentatomidae) Densities and the Associated Damage at Field Corn and Soybean Edges

The local dispersal of polyphagous, mobile insects within agricultural systems impacts pest management. In the mid-Atlantic region of the United States, stink bugs, especially the invasive Halyomorpha halys (Stål 1855), contribute to economic losses across a range of cropping systems. Here, we characterized the density of stink bugs along the field edges of field corn and soybean at different study sites. Specifically, we examined the influence of adjacent managed and natural habitats on the density of stink bugs in corn and soybean fields at different distances along transects from the field edge. We also quantified damage to corn grain, and to soybean pods and seeds, and measured yield in relation to the observed stink bug densities at different distances from field edge. Highest density of stink bugs was limited to the edge of both corn and soybean fields. Fields adjacent to wooded, crop and building habitats harbored higher densities of stink bugs than those adjacent to open habitats. Damage to corn kernels and to soybean pods and seeds increased with stink bug density in plots and was highest at the field edges. Stink bug density was also negatively associated with yield per plant in soybean. The spatial pattern of stink bugs in both corn and soybeans, with significant edge effects, suggests the use of pest management strategies for crop placement in the landscape, as well as spatially targeted pest suppression within fields.     

Random DNA fragmentation allows detection of single-copy,single-exon alterations of copy number by oligonucleotide array CGH in clinical FFPE samples

Genomic technologies, such as array comparative genomic hybridization (aCGH), increasingly offer definitive gene dosage profiles in clinical samples. Historically, copy number profiling was limited to large fresh-frozen tumors where intact DNA could be readily extracted. Genomic analyses of pre-neoplastic tumors and diagnostic biopsies are often limited to DNA processed by formalin-fixation and paraffin-embedding (FFPE). We present specialized protocols for DNA extraction and processing from FFPE tissues utilizing DNase processing to generate randomly fragmented DNA. The protocols are applied to FFPE clinical samples of varied tumor types, from multiple institutions and of varied block age. Direct comparative analyses with regression coefficient were calculated on split-sample (portion fresh/portion FFPE) of colorectal tumor samples. We show equal detection of a homozygous loss of SMAD4 at the exon-level in the SW480 cell line and gene-specific alterations in the split tumor samples. aCGH application to a set of archival FFPE samples of skin squamous cell carcinomas detected a novel hemizygous deletion in INPP5A on 10q26.3. Finally we present data on derivative of log ratio, a particular sensitive detector of measurement variance, for 216 sequential hybridizations to assess protocol reliability over a wide range of FFPE samples.     

Adenosine derivatives with N6-alkyl, -alkylamine or -alkyladenosine substituents as probes for the A1-receptor

Three series of N6-substituted adenosine derivatives were synthesized, having in common an unbranched alkyl chain with lengths varying from 2 to 12 methylene units, but differing in their omega-alkyl substituents: N6-n-alkyladenosines (I), N6-omega-amino-alkyladenosines (II) and alpha omega,di-(adenosin-N6-yl)alkanes (III). The compounds of the latter series combine two functional groups in one molecule. A1-receptor affinity of these compounds was measured as inhibition of [3H]PIA binding to calf brain membranes. With relatively short chain lengths, compounds in series I are the most potent. In this series, optimum activity is reached with N6-n-pentyladenosine (Ki = 0.50 nM). With short chain lengths, compounds in series II and III are 6-20-fold less potent than their congeners in series I. The potency order however is reversed with higher chain lengths. While affinity in series II and III increases strongly, reaching an optimum with the nonyl derivatives, affinity in series I decreases sharply with alkyl chains larger than 8 methylene units. Highest affinity is found with 9-amino-nonyladenosine (Ki = 0.32 nM). In general, the omega-aminoalkyl derivatives are somewhat more potent than the corresponding di-adenosinyl derivatives. Implications for the possible topography of the N6 region of the A1-receptor and the area further removed from N6 are discussed.