A parallel screening method has been developed to rapidly evaluate discrete library substrates of biomaterials using cell-based assays. The biomaterials used in these studies were surface-erodible polyanhydrides based on sebacic acid (SA), 1,6-bis(p-carboxyphenoxy)hexane (CPH), and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) that have been previously studied as carriers for drugs, proteins, and vaccines. Linearly varying compositional libraries of 25 different polyanhydride random copolymers (based on CPH:SA and CPTEG:CPH) were designed, fabricated, and synthesized using discrete (organic solvent-resistant) multi-sample substrates created using a novel rapid prototyping method. The combinatorial libraries were characterized at high throughput using infrared microscopy and validated using 1H NMR and size exclusion chromatography. The discrete libraries were rapidly screened for biocompatibility using standard SP2/0 myeloma, CHO and L929 fibroblasts, and J774 macrophage cell lines. At a concentration of 2.8 mg/mL, there was no appreciable cytotoxic effect on any of the four cell lines evaluated by any of the CPH:SA or CPTEG:CPH compositions. Furthermore, the activation of J774 macrophages was evaluated by incubating the cells with the polyanhydride libraries and quantifying the secreted cytokines (IL-6, IL-10, IL-12, and TNFalpha). The results indicated that copolymer compositions containing at least 50% CPH induced elevated amounts of TNFalpha. In summary, the results indicated that the methodologies described herein are amenable to the high throughput analysis of synthesized biomaterials and will facilitate the rapid and rational design of materials for use in biomedical applications.

with primers designed for barley has been attempted in the past (Li et al., 2000). Perennial ryegrass (Lolium Polymerase chain reaction (PCR)-based markers are generally perenne L.), tall fescue (Festuca arundinacea Schreb.), more rapid and less expensive to assay than hybridization-based markers (e.g., restriction fragment length polymorphisms [RFLP]), making and Avena, however, belong to the Poodae supertribe them useful for breeding applications, but few of such markers are and are therefore more closely related to each other available for oat (Avena sativa L.). Approaches to develop new markthan any are to Hordeum (Devos and Gale, 1997). Markers cheaply include using primer pairs designed for other species or ers developed for Lolium or Festuca may therefore crossusing publicly available sequence information. In this study we report amplify oat sequences more effectively than markers deon the design of 32 markers using publicly available oat sequence veloped for Hordeum. One study found 12% of primers data and on the map locations of 20 loci from 16 markers on the oat designed for Lolium amplified products from oat (Jones 'Ogle' ϫ 'TAM O-301' population.

Mycoplasmas are thought to control gene expression through simple mechanisms. The switching mechanisms needed to regulate transcription during significant environmental shifts do not seem to be required for these host-adapted organisms. Mycoplasma hyopneumoniae, a swine respiratory pathogen, undergoes differential gene expression, but as for all mycoplasmas, the mechanisms involved are still unknown. Since mycoplasmas contain only a single sigma factor and few regulator-type proteins, it is likely that other mechanisms control gene regulation, possibly involving intergenic (IG) regions. To study this further, we investigated whether IG regions are transcribed in M. hyopneumoniae, and measured transcription levels across five specific regions. Microarrays were constructed with probes covering 343 IG regions of the M. hyopneumoniae genome, and RNA isolated from laboratory-grown cells was used to interrogate the arrays. Transcriptional signals were identified in 321 (93.6 %) of the IG regions. Five large (.500 bp) IG regions were chosen for further analysis by qRT-PCR by designing primer sets whose products reside in flanking ORFs, bridge flanking ORFs and the IG region, or reside solely within the IG region. The results indicate that no single transcriptional start site can account for transcriptional activity within IG regions. Transcription can end abruptly at the end of an ORF, but this does not seem to occur at high frequency. Rather, transcription continues past the end of the ORF, with RNA polymerase gradually releasing the template. Transcription can also be initiated within IG regions in the absence of accepted promoter-like sequences.

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia and a major factor in the porcine respiratory disease complex. A clear understanding of the mechanisms of pathogenesis does not exist, although it is clear that M. hyopneumoniae adheres to porcine ciliated epithelium by action of a protein called P97. Previous studies have shown variation in the gene encoding the P97 cilium adhesin in different strains of M. hyopneumoniae, but the extent of genetic variation among field strains across the genome is not known. Since M. hyopneumoniae is a worldwide problem, it is reasonable to expect that a wide range of genetic variability may exist given all of the different breeds and housing conditions. This variation may impact the overall virulence of a single strain. Using microarray technology, this study examined the potential variation of 14 field strains compared to strain 232, on which the array was based. Genomic DNA was obtained, amplified with TempliPhi, and labeled indirectly with Alexa dyes. After genomic hybridization, the arrays were scanned and data were analyzed using a linear statistical model. The results indicated that genetic variation could be detected in all 14 field strains but across different loci, suggesting that variation occurs throughout the genome. Fifty-nine percent of the variable loci were hypothetical genes. Twenty-two percent of the lipoprotein genes showed variation in at least one field strain. A permutation test identified a location in the M. hyopneumoniae genome where there is spatial clustering of variability between the field strains and strain 232.

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Canola (Brassica napus L.) is grown on >8 Mha in Canada and is sensitive to high temperatures; therefore, research on breeding methodologies to improve heat-stress tolerance is warranted. This study utilised a doubled-haploid population created from two parents (PB36 and PB56) that differed in their ability to set seed following growth at high temperatures. The experiment was designed to identify potential quantitative trait loci (QTLs) responsible for conferring tolerance to increased temperatures, and to utilise this population as a test case for evaluating the prospects of whole-genome prediction. The population was phenotyped in a split-plot, randomised complete block experimental design at three locations with two planting-date treatments. The first planting date was during the normal planting period (control), and the second planting was timed to experience increased average temperatures (1.7°C, 2.0°C and 1.2°C) and increased number of days with maximum temperatures above the critical temperature of 29.5°C (4, 12 and 3 days). The stress treatment reduced yield on average by 16.7%. There were 66 QTLs discovered across the nine traits collected. Given the quantitative nature of the traits collected, the ability to use whole-genome prediction was investigated. The prediction accuracies ranged from 0.14 (yield) to 0.66 (1000-seed weight). Prediction had higher accuracy within the stress treatment than within the control treatment for seven of the nine traits, demonstrating that phenotyping within a stress environment can provide valuable data for whole-genome predictions.

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