Diversity of an ectomycorrhizal fungal community studied by a root tip and total soil DNA approach

Molecular methods based on soil DNA extracts are increasingly being used to study the fungal diversity of ectomycorrhizal (EM) fungal communities in soil. Contrary to EM root tip identification, the use of molecular methods enables identification of extramatrical mycelia in soil. To compare fungal diversity as determined by root tip identification and mycelial identification, six soil samples were analysed. Root tips were extracted from the six samples and after amplification, the basidiomycete diversity on the root tips was analysed by denaturing gradient gel electrophoresis (DGGE). The soil from the six samples was sieved, total soil DNA was extracted and after amplification, the basidiomycete diversity in the soil fractions was analysed by DGGE. Fourteen different bands were excised from the DGGE gel and sequenced; fungal taxon names could be assigned to eight bands. Out of a total of 14 fungal taxa detected in soil, 11 fungal taxa were found on root tips, of which seven were EM fungal taxa. To examine whether the sieving treatment would affect EM species diversity, two different sieve mesh sizes were used and in addition, the organic soil fraction was analysed separately. DGGE analysis showed no differences in banding pattern for the different soil fractions. The organic fraction gave the highest DGGE band intensities. This work demonstrates that there is a high correspondence between basidiomycete diversity detected by molecular analysis of root tips and soil samples, irrespective of the soil fraction being analysed.     

Cell-free propagation of prion strains

Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrP(Sc)). Disease is transmitted by the autocatalytic propagation of PrP(Sc) misfolding at the expense of the normal prion protein. The biggest challenge of the prion hypothesis has been to explain the molecular mechanism by which prions can exist as different strains, producing diseases with distinguishable characteristics. Here, we show that PrP(Sc) generated in vitro by protein misfolding cyclic amplification from five different mouse prion strains maintains the strain-specific properties. Inoculation of wild-type mice with in vitro-generated PrP(Sc) caused a disease with indistinguishable incubation times as well as neuropathological and biochemical characteristics as the parental strains. Biochemical features were also maintained upon replication of four human prion strains. These results provide additional support for the prion hypothesis and indicate that strain characteristics can be faithfully propagated in the absence of living cells, suggesting that strain variation is dependent on PrP(Sc) properties.     

Genomics in a changing arctic: critical questions await the molecular ecologist

Molecular ecology is poised to tackle a host of interesting questions in the coming years. The Arctic provides a unique and rapidly changing environment with a suite of emerging research needs that can be addressed through genetics and genomics. Here we highlight recent research on boreal and tundra ecosystems and put forth a series of questions related to plant and microbial responses to climate change that can benefit from technologies and analytical approaches contained within the molecular ecologist's toolbox. These questions include understanding (i) the mechanisms of plant acquisition and uptake of N in cold soils, (ii) how these processes are mediated by root traits, (iii) the role played by the plant microbiome in cycling C and nutrients within high‐latitude ecosystems and (iv) plant adaptation to extreme Arctic climates. We highlight how contributions can be made in these areas through studies that target model and nonmodel organisms and emphasize that the sequencing of the Populus and Salix genomes provides a valuable resource for scientific discoveries related to the plant microbiome and plant adaptation in the Arctic. Moreover, there exists an exciting role to play in model development, including incorporating genetic and evolutionary knowledge into ecosystem and Earth System Models. In this regard, the molecular ecologist provides a valuable perspective on plant genetics as a driver for community biodiversity, and how ecological and evolutionary forces govern community dynamics in a rapidly changing climate.     

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.     

Effects of Partial Replacement of Corn with Glycerin on Ruminal Fermentation in a Dual-Flow Continuous Culture System

The objective of this study was to evaluate the effects of partially replacing dry ground corn with glycerin on ruminal fermentation using a dual-flow continuous culture system. Six fermenters (1,223 ± 21 ml) were used in a replicated 3x3 Latin square arrangement with three periods of 10 d each, with 7 d for diet adaptation and 3 d for sample collections. All diets contained 75% concentrate and three dietary glycerin levels (0, 15, and 30% on dry matter basis), totaling six replicates per treatment. Fermenters were fed 72 g of dry matter/d equally divided in two meals/d, at 0800 and 2000 h. Solid and liquid dilution rates were adjusted daily to 5.5 and 11%/h, respectively. On d 8, 9, and 10, samples of 500 ml of solid and liquid digesta effluent were mixed, homogenized, and stored at -20°C. Subsamples of 10 ml were collected and preserved with 0.2 mL of a 50% H₂SO₄ solution for later determination of NH₃-N and volatile fatty acids. Microbial biomass was isolated from fermenters for chemical analysis at the end of each experimental period. Data were analyzed using the MIXED procedure in SAS with α = 0.05. Glycerin levels did not affect apparent digestibility of DM (P _Lin. = 0.13; P _Quad. = 0.40), OM (P _Lin. = 0.72; P _Quad. = 0.15), NDF (P _Lin. = 0.38; P _Quad. = 0.50) and ADF (P _Lin. = 0.91; P _Quad. = 0.18). Also, glycerin inclusion did not affect true digestibility of DM (P _Lin. = 0.35; P _Quad. = 0.48), and OM (P _Lin. = 0.08; P _Quad. = 0.19). Concentrations of propionate (P < 0.01) and total volatile fatty acids (P < 0.01) increased linearly and concentrations of acetate (P < 0.01), butyrate (P = 0.01), iso-valerate (P < 0.01), and total branched-chain volatile fatty acids, as well as the acetate: propionate ratio (P < 0.01) decreased with glycerin inclusion. Linear increases on NH₃-N concentration in digesta effluent (P < 0.01) and on NH₃-N flow (P < 0.01) were observed due to glycerin inclusion in the diets. Crude protein digestibility (P = 0.04) and microbial N flow (P = 0.04) were greater in the control treatment compared with the other treatments and responded quadratically with glycerin inclusion. Furthermore, the inclusion of glycerin linearly decreased (P = 0.02) non-ammonia N flow. Glycerin levels did not affect the flows of total N (P _Lin. = 0.79; P _Quad. = 0.35), and dietary N (P _Lin. = 0.99; P _Quad. = 0.07), as well as microbial efficiency (P _Lin. = 0.09; P _Quad. = 0.07). These results suggest that partially replacing dry ground corn with glycerin may change ruminal fermentation, by increasing total volatile fatty acids, and propionate concentration without affecting microbial efficiency, which may improve glucogenic potential of beef cattle diets.     

The Fap1 fimbrial adhesin is a glycoprotein: antibodies specific for the glycan moiety block the adhesion of Streptococcus parasanguis in an in vitro tooth model

Streptococcus parasanguis is a primary colonizer of the tooth surface and plays a pivotal role in the formation of dental plaque. The fimbriae of S. parasanguis are important in mediating adhesion to saliva-coated hydroxylapatite (SHA), an in vitro tooth adhesion model. The Fap1 adhesin has been identified as the major fimbrial subunit, and recent studies suggest that Fap1 is a glycoprotein. Monosaccharide analysis of Fap1 purified from the culture supernatant of S. parasanguis indicated the presence of rhamnose, glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine. A glycopeptide moiety was isolated from a pronase digest of Fap1 and purified by immunoaffinity chromatography. The monosaccharide composition of the purified glycopeptide was similar to that of the intact molecule. The functionality of the glycan moiety was determined using monoclonal antibodies (MAbs) specific for the intact Fap1 glycoprotein. These antibodies were grouped into two categories based on their ability to block adhesion of S. parasanguis to SHA and their corresponding specificity for either protein or glycan epitopes of the Fap1 protein. 'Non-blocking' MAb epitopes were mapped to unique protein sequences in the N-terminus of the Fap1 protein using non-glycosylated recombinant Fap1 proteins (rFap1 and drFap1) expressed in Escherichia coli. In contrast, the 'blocking' antibodies did not bind to the recombinant Fap1 proteins, and were effectively competed by the binding to the purified glycopeptide. These data suggest that the 'blocking' antibodies are specific for the glycan moiety and that the adhesion of S. parasanguis is mediated by sugar residues associated with Fap1.     

The CCAAT binding factor can mediate interactions between CONSTANS-like proteins and DNA

CONSTANS-Like (COL) proteins are plant-specific nuclear regulators of gene expression but do not contain a known DNA-binding motif. We tested whether a common DNA-binding protein can deliver these proteins to specific cis-acting elements. We screened for proteins that interact with two members of a subgroup of COL proteins. These COL proteins were Tomato COL1 (TCOL1), which does not seem to be involved in the control of flowering time, and the Arabidopsis thaliana CONSTANS (AtCO) protein which mediates photoperiodic induction of flowering. We show that the C-terminal plant-specific CCT (CO, CO-like, TIMING OF CAB EXPRESSION 1) domain of both proteins binds the trimeric CCAAT binding factor (CBF) via its HAP5/NF-YC component. Chromatin immunoprecipitation demonstrated that TCOL is recruited to the CCAAT motifs of the yeast CYC1 and HEM1 promoters by HAP5. In Arabidopsis, each of the three CBF components is encoded by several different genes that are highly transcribed. Under warm long days, high levels of expression of a tomato HAP5 (THAP5a) gene can reduce the flowering time of Arabidopsis. A mutation in the CCT domain of TCOL1 disrupts the interaction with THAP5 and the analogous mutation in AtCO impairs its function and delays flowering. CBFs are therefore likely to recruit COL proteins to their DNA target motifs in planta.