Transgenic expression of bean alpha-amylase inhibitor in peas results in altered structure and immunogenicity

The development of modern gene technologies allows for the expression of recombinant proteins in non-native hosts. Diversity in translational and post-translational modification pathways between species could potentially lead to discrete changes in the molecular architecture of the expressed protein and subsequent cellular function and antigenicity. Here, we show that transgenic expression of a plant protein (alpha-amylase inhibitor-1 from the common bean (Phaseolus vulgaris L. cv. Tendergreen)) in a non-native host (transgenic pea (Pisum sativum L.)) led to the synthesis of a structurally modified form of this inhibitor. Employing models of inflammation, we demonstrated in mice that consumption of the modified alphaAI and not the native form predisposed to antigen-specific CD4+ Th2-type inflammation. Furthermore, consumption of the modified alphaAI concurrently with other heterogeneous proteins promoted immunological cross priming, which then elicited specific immunoreactivity of these proteins. Thus, transgenic expression of non-native proteins in plants may lead to the synthesis of structural variants possessing altered immunogenicity.     

Design and evaluation of nematode 18S rDNA primers for PCR and denaturing gradient gel electrophoresis (DGGE) of soil community DNA

Consensus nematode 18S ribosomal DNA primers were designed by aligning available 18S sequences and identifying a variable region flanked by highly conserved regions. These primers were then used to amplify nematode 18S rDNA from whole soil community DNA extracted from a range of European grassland types. Cloning of the PCR amplicons (778 bp) followed by restriction digest analysis (RFLP) resulted in the recovery of 34 unique nematode sequences from the four grasslands studied. Comparison of these data with the limited number of 18S rDNA nematode sequences currently held in on-line databases revealed that all of the sequences could be assigned to known nematode taxa albeit tentatively in some cases. Two of the sequences recovered from the site in the Netherlands (wet, hay-grassland) were recovered in a clade that included a sequence of the genus Trichodorus whilst other sequences from this site showed similarity with 18S rDNA sequences of the genus Prismatolaimus (five sequences), Xiphinema (one sequence) and Enoplus (one sequence). Of the remaining sequences, two showed some affinity with Mylonchulus (UK, upland peat), four with Steinernema (UK) and one sequence with Mesorhabditis (Hungary, east European Steppe). Three sequences from the Netherlands and one from Hungary were recovered in a clade that included a sequence of the genus Pratylenchoides whilst three further sequences from the Netherlands and two from Hungary were recovered in a clade encompassing the genus Globodera. Of the remaining nine sequences, two (NL6, NL62) formed a distinct lineage within the Adenophorea with 90% bootstrap recovery in a paraphyletic clade that included sequences of Prismatolaimus and Trichodorus. Seven sequences (three from the Netherlands, three from the UK and one from Greece) were left unassigned though the tree topology suggested some relationship (58% bootstrap recovery) with the genus Cephalobus. To assess whether primers used to amplify 18S rDNA might be used to fingerprint genetic diversity in nematode communities in soil, the environmental sequence data were used to design a second set of primers carrying a GC-clamp. These primers amplified a 469 bp fragment internal to the region flanked by the primer set used to derive the nematode trees and were used to amplify 18S rDNA for subsequent analysis using denaturing gradient gel electrophoresis (DGGE). DGGE analysis of six major European grassland types revealed considerable genetic diversity between sites. However, the relationships seen with the DGGE data were inconsistent with previous studies where the same soils had been characterized with respect to functional and morphological diversity. To confirm that this second set of primers was amplifying nematode sequences, selected bands on the DGGE gels were extracted, PCR amplified and sequenced. The final alignment was 337 bases. These analyses revealed the presence of sequence signatures from the genera Paratrichodorus, Plectus, Steinernema, Globodera, Cephalobus and Pratylenchoides.     

Comparing and Blending Regional Climate Model Predictions for the American Southwest

We consider the problem of forecasting future regional climate. Our method is based on blending different members of an ensemble of regional climate model (RCM) simulations while accounting for the discrepancies between these simulations, under present day conditions, and observational records for the recent past. To this end, we develop Bayesian space-time models that assess the discrepancies between climate model simulations and observational records. Those discrepancies are then propagated into the future to obtain blended forecasts of 21st century climate. The model allows for location-dependent spatial heterogeneities, providing local comparisons between the different simulations. Additionally, we estimate the different modes of spatial variability, and use the climate model-specific coefficients of the spatial factors for comparisons. We focus on regional climate model simulations performed in the context of the North American Regional Climate Change Assessment Program (NARCCAP). We consider, in particular, simulations from RegCM3 using three different forcings: NCEP, GFDL and CGCM3. We use simulations for two time periods: current climate conditions, covering 1971 to 2000, and future climate conditions under the SRES A2 emissions scenario, covering 2041 to 2070. We investigate yearly mean summer temperature for a domain in the South West of the United States. The results indicated the RCM simulations underestimate the mean summer temperature increase for most of the domain compared to our model.     

Waterjet injection of powdered activated carbon for sediment remediation

Purpose  

In situ contaminated sediment remediation through the addition of activated carbon has been proven to be an effective remediation technique. An amendment delivery system was developed to accurately place and inject a powdered activated carbon slurry. The system was set up to deliver a series of discrete injections over a rectangular grid with the objective to deliver 3% carbon (C) by dry weight to an inundated saturated sediment at a maximum sediment depth of 30 cm.     

Evaluation of the zoonotic potential of Giardia duodenalis in fecal samples from dogs and cats in Ontario

This study determined the distribution and zoonotic potential of Giardia duodenalis assemblage types among canine and feline fecal samples from Ontario. The effectiveness of Giardia assemblage typing methods by sequencing the genes of small subunit ribosomal RNA (ssu-rRNA), β-giardin (bg), glutamate dehydrogenase (gdh), and triose phosphate isomerase (tpi) was evaluated simultaneously. From 2008 to 2010, 118 canine and 15 feline Giardia positive fecal samples were tested. The ssu-rRNA sequencing method typed 64% (75/118) and 87% (13/15) of the Giardia-positive canine and feline samples, respectively. Among the typeable samples, 68% (51/75) of canine samples contained G. duodenalis assemblage D and 31% (23/75) contained G. duodenalis assemblage C (both non-zoonotic assemblage types). Only 1% (1/75) of the typeable canine samples contained a potentially zoonotic assemblage B. In contrast, 100% (13/13) of the typeable feline samples contained potentially zoonotic assemblages A (n = 12) or B (n = 1).     

Evaluation of peripheral lymphocytes after weaning and vaccination for Mycoplasma hyopneumoniae

This study evaluated immune cell populations in pigs following weaning and vaccination for Mycoplasma hyopneumoniae. Piglets (n = 24) were weaned (day 0) at 16 (±1) days of age, and randomly assigned to the vaccination group (n = 16) or control group (n = 8). Complete blood cell counts, flow cytometry and serology were completed for blood samples collected on days 0 (within hours of weaning), 3, 7, 14, 30 and 60. The M. hyopneumoniae S:P ratios (sample optical density: positive control optical density) were negative in the vaccination group until days 30 and 60, when the S:P ratios were 1.3 and 1.0, respectively. Control animals remained serologically negative. The percentage of CD4⁺ T cells was less (P < 0.01) in control pigs than vaccinated pigs at day 3. In contrast, numbers of CD8⁺ and CD4⁺CD8⁺ T cells were greater (P < 0.01) in control pigs than in vaccinated pigs at days 3 and 7. After day 7, few differences in immune cell types were evident between the groups. Differences in lymphocyte populations could not be solely attributed to vaccination, due at least in part, to the confounding influence of weaning. It was difficult to distinguish the influence of vaccination from the impact of weaning on peripheral immune cell populations.