Microbial community sequencing analysis of the calf eye microbiota and relationship to infectious bovine keratoconjunctivitis.

Infectious bovine keratoconjunctivitis (IBK) is an important production limiting disease in cattle. Moraxella bovis has historically been considered the primary causal agent; however, vaccines have not been consistently shown as effective in controlling disease incidence. The purpose of this study was to examine the bacterial community of calf eyes prior to disease onset using high-throughput sequencing of 16S ribosomal RNA and determine if it was associated with IBK occurrence. The study was designed as a case-control nested within a randomized controlled trial (RCT). Eye swabs were collected from all spring-born calves without clinical signs of IBK (t0 swabs) on a research farm with a previous history of IBK disease outbreaks. At follow-up or weaning, calves were diagnosed as IBK positive or negative. The lag time between enrollment swabs (t0) and IBK diagnosis ranged from approximately one to three months. Cases were randomly selected from IBK positive calves and controls were selected from IBK negative calves (i.e. calves that did not exhibit clinical signs of IBK throughout the course of the RCT). Analysis of the fold-change differences between cases and controls did not reveal large-scale distinctions in bacterial composition. However, principal component analysis suggested bacterial composition differences between calf management groups, which were based on dam parity. Moraxella was found to be among the top ten most abundant genera in our population; however, the difference in abundance was not significant between the cases and controls. No large-scale differences in the bacterial communities of calves that did or did not develop IBK were observed in our population. Nevertheless, it remains unclear whether the "natural" bacterial population of the calf might ultimately impact disease status. Further study is warranted to examine bacterial taxa that were observed to be significantly more abundant in the cases or controls as potential vaccines/therapeutic targets.

Genome-wide methylation profile following prenatal and postnatal dietary omega-3 fatty acid supplementation in pigs.

The objective of this study was to determine how prenatal and postnatal dietary omega-3 fatty acids alter white blood cell (leukocyte) DNA methylation of offspring. Fifteen gilts (n = 5 per treatment) were selected from one of three treatments: (i) control diet throughout gestation, lactation and nursery phase (CON); (ii) algal omega-3 fatty acid supplementation enriched in EPA and DHA (Gromega™ ) fed throughout gestation, lactation and nursery phase (Cn3); or (iii) Gromega™ supplementation maternally, during gestation and lactation only, and control diet during the nursery phase (Mn3). At 11 weeks of age and after 8 weeks of post-weaning nursery feeding, buffy coat genomic DNA was subjected to methyl CpG binding protein sequencing. The methylation enriched profile mapped to 26% of the porcine genome. On chromosome 4, a 27.7-kb differentially methylated region downstream of RUNX1T1 was hypomethylated in the Mn3 and Cn3 groups by 91.6% and 85.0% respectively compared to CON pigs. Conversely, hypermethylation was detected in intergenic regions of chromosomes 4 and 12. Regulatory impact factor and differential hubbing methods were used to identify pathways that were coordinately regulated by methylation due to feeding EPA and DHA during pregnancy. Despite limited ability to detect differential methylation, we describe methods that allow the identification of coordinated epigenetic regulation that could not otherwise be detected from subtle single locus changes in methylation. These data provide evidence of novel epigenetic regulation by maternal and early life supplementation of omega-3 fatty acids that may have implications to growth and inflammatory processes.

Investigation of the efficacy of albumin removal procedures on porcine serum proteome profile.

Improving the ability to predict livestock performance using biomarkers will provide a benefit for livestock genetic evaluation and improvement. The most practical biological sample to screen for development of biomarkers is serum due to the ease of collection. However, protein profiles in serum are complex and dynamic. Strategies are needed to manage variation in serum proteins used for biomarker identification. Albumin is the most abundant protein in serum, comprising over 50% of the overall protein content, and has historically been depleted from serum before biomarker identification. The objective of this study was to investigate the use of gel-based proteomic techniques to evaluate the need for porcine albumin depletion in biomarker identification. Albumin is known to bind many proteins in the blood, thus potential biomarkers could be removed during albumin depletion. Using two-dimensional difference in gel electrophoresis (2D-DIGE), we show whole serum can be used for biomarker discovery. The data obtained show that albumin removal methods are effective for porcine sera. Over 85% of the protein spots resolved on at least half of the gels were changed in abundance between whole and albumin depleted sera. Of the 204 protein spots significantly altered in abundance, 59 were changed over 400%. However, albumin removal also altered the serum proteome in an unpredictable manner; in the depleted sera, 86 protein spots were increased in abundance and 118 were decreased. Furthermore, the abundance of 59.4% of the protein spots in the albumin depleted samples had a larger standard error than whole sera. However, the resolution of albumin in 2D-DIGE analysis of whole sera permitted the detection and quantification of substantial numbers of proteins. Thus, it is proposed that whole serum can be used in a gel-based proteomics system for the identification of porcine biomarkers.

Integrating comparative expression profiling data and association of SNPs with Salmonella shedding for improved food safety and porcine disease resistance.

Salmonella in swine is a major food safety problem, as the majority of US swine herds are Salmonella-positive. Salmonella can be shed from colonized swine and contaminate (i) neighbouring pigs; (ii) slaughter plants and pork products; (iii) edible crops when swine manure is used as a fertilizer; and (iv) water supplies if manure used as crop fertilizer runs off into streams and waterways. A potentially powerful method of addressing pre-harvest food safety at the farm level is through genetic improvement of disease resistance in animals. In this research, we describe a successful strategy for discovering genetic variation at candidate genes associated with disease resistance in pigs. This involves integrating our recent global gene expression analysis of the porcine response to Salmonella with information from the literature about important candidate genes. We identified single-nucleotide polymorphisms (SNPs) in these functional candidate genes and genotyped three independent pig populations that had data on Salmonella faecal shedding or internal burden (total n = 377) at these loci. Of 31 SNPs genotyped, 21 SNPs segregated in at least two populations with a minor allele frequency of 15% or greater. Statistical analysis revealed thirteen SNPs associated with Salmonella faecal shedding or tissue colonization, with an estimated proportion of false positives (PFP) ≤0.2. The genes with associated SNPs included GNG3, NCF2, TAP1, VCL, AMT, CCR1, CD163, CCT7, EMP1 and ACP2. These associations provide new information about the mechanisms of porcine host response to Salmonella and may be useful in improving genetic resistance to this bacterium.

Use of bioinformatic SNP predictions in differentially expressed genes to find SNPs associated with Salmonella colonization in swine.

Asymptomatic Salmonella-carrier pigs present a major problem in preharvest food safety, with a recent survey indicating >50% of swine herds in the United States have Salmonella-positive animals. Salmonella-carrier pigs serve as a reservoir for contamination of neighbouring pigs, abattoir pens and pork products. In addition, fresh produce as well as water can be contaminated with Salmonella from manure used as fertilizer. Control of Salmonella at the farm level could be through genetic improvement of porcine disease resistance, a potentially powerful method of addressing preharvest pork safety. In this research, we integrate gene expression profiling data and sequence alignment-based prediction of single nucleotide polymorphisms (SNPs) to successfully identify SNPs in functional candidate genes to test for the associations with swine response to Salmonella. A list of 2527 genes that were differentially regulated in porcine whole blood in response to infection with Salmonella enterica serovar Typhimurium were selected. In those genes, SNPs were predicted using ANEXdb alignments based on stringent clustering of all publically available porcine cDNA and expressed sequence tag (EST) sequences. A set of 30 mostly non-synonymous SNPs were selected for genotype analysis of four independent populations (n = 750) with Salmonella faecal shedding or tissue colonization phenotypes. Nine SNPs segregated with minor allele frequency ≥15% in at least two populations. Statistical analysis revealed SNPs associated with Salmonella shedding, such as haptoglobin (HP, p = 0.001, q = 0.01), neutrophil cytosolic factor 2 (NCF2 #2, p = 0.04, q = 0.21) and phosphogluconate dehydrogenase (p = 0.066, q = 0.21). These associations may be useful in identifying and selecting pigs with improved resistance to this bacterium.

The effect of selection for residual feed intake on general behavioral activity and the occurrence of lesions in Yorkshire gilts.

The objectives of this study were to determine the effect of selection for improved residual feed intake on behavior, activity, and lesion scores in gilts in their home pen. A total of 192 gilts were used, 96 from a line that had been selected for decreased residual feed intake over 5 generations (LRFI) and 96 from a randomly bred control line. Gilts were housed in 12 pens (16 gilts/pen; 0.82 m(2)/gilt) containing 8 gilts from each line in a conventional grow-finish unit. Twelve hours of video footage were collected on the day of placement and then every 4 wk for 3 more observational periods. Video was scored using a 10-min instantaneous scan sampling technique for 4 postures (standing, lying, sitting, and locomotion) and 1 behavior (at drinker). Categories of active (standing, locomotion, and at drinker) and inactive (sitting and lying) were also created. Lesion scores were collected 24 h after behavior collection had begun. The body of a gilt was divided into 4 regions, with each region receiving a score of 0 (0 lesions) to 3 (5+ lesions). All statistical analyses used Proc Mixed of SAS. Data were analyzed separately for the day of placement and the subsequent 3 rounds. General activity was summarized on a percentage basis by each posture and behavior and subjected to an arcsine square root transformation to normalize data and stabilize variance. Analysis was performed on each behavior and posture. Lesion scores for each region of the body were analyzed as repeated measures. There were no differences (P > 0.05) between genetic lines for all postures and the behavior at drinker on the day of placement. However, over subsequent rounds it was observed that LRFI gilts spent less (P = 0.03) time standing, more time sitting (P = 0.05), and were less active (P = 0.03) overall. Gilts from the LRFI line had decreased (P < 0.045) lesion scores on the day after placement. However, over subsequent rounds there were no (P > 0.05) differences between the genetic lines. In conclusion, on the day of placement there were no postural, behavior, or general activity differences between genetic lines, but LRFI gilts had decreased lesion scores. Behavioral differences were observed between genetic lines over subsequent rounds, with LRFI gilts becoming less active, but there were no differences in lesion scores.

Effects of ad libitum and restricted feed intake on growth performance and body composition of Yorkshire pigs selected for reduced residual feed intake.

Residual feed intake (RFI), defined as the difference in the observed and expected feed intake while accounting for growth and backfat, has gained much attention, but little is known about why pigs selected for reduced RFI are more efficient. To this end, a line of Yorkshire pigs selected for reduced RFI was developed. The objective of this study was to evaluate the 5th generation of this select line against a randomly selected control line for performance, carcass and chemical carcass composition, and overall efficiency toward the later part of the growth phase. Eighty barrows, 40 from each line, were paired by age (~132 d, P < 0.60) and BW (74.8 ± 9.9 kg, P < 0.49) and randomly assigned to 1 of 4 feeding treatments in 10 replicates: 1) ad libitum, 2) 75% of ad libitum, 55% of ad libitum, and BW stasis, with weekly adjustments in intake to keep BW constant for each pig. Pigs were individually penned (group housing was used for selection) and on treatment for 6 wk. Initial BW did not differ between the lines (P < 0.49). The ad libitum select pigs consumed 10% less feed (P < 0.09) than the ad libitum control with no significant difference in BW (P < 0.80) and slight differences in carcass fat composition (P < 0.20) and backfat (P < 0.11), which resulted in significantly less carcass energy (P < 0.03). Under restricted feeding, the select line had an increase in BW (P = 0.10) while consuming the same ration of feed as the control line with no significant difference in chemical carcass composition and lighter visceral weights, which was significant for the 75% of ad libitum treatment (P < 0.01). Under BW stasis feeding the select line consumed 7.6% less feed overall (P = 0.21) and 18% less feed at the end of the 6 wk (P < 0.08), to maintain static BW with no significant difference in chemical carcass composition compared with the control line. Overall, the select line had lighter visceral weight (P < 0.02) and a greater dressing percentage (P < 0.03) compared with the control line. Using regression, the select line had reduced energy retention (P < 0.04) and feed energy utilization (P < 0.34); however, the select line appeared to have reduced maintenance requirements (P < 0.13). In conclusion, selection for reduced RFI decreases feed intake with no significant difference (P > 0.05) in growth performance, reduced backfat, increased dressing percentage, and reduced maintenance requirements. All of these traits are appealing to the producer and result in increased profits in the production setting.

Effects of ad libitum and restricted feeding on early production performance and body composition of Yorkshire pigs selected for reduced residual feed intake.

Residual feed intake (RFI), defined as the difference between observed and expected feed intake based on growth and backfat, has been used to investigate genetic variation in feed efficiency in cattle, poultry and pigs. However, little is known about the biological basis of differences in RFI in pigs. To this end, the objective of this study was to evaluate the fifth generation of a line of pigs selected for reduced RFI against a randomly selected Control line for performance, carcass and chemical carcass composition and overall efficiency. Here, emphasis was on the early grower phase. A total of 100 barrows, 50 from each line, were paired by age and weight (22.6 ± 3.9 kg) and randomly assigned to one of four feeding treatments in 11 replicates: ad libitum (Ad), 75% of Ad (Ad75), 55% of Ad (Ad55) and weight stasis (WS), which involved weekly adjustments in intake to keep body weight (BW) constant for each pig. Pigs were individually penned (group housing was used for selection) and were on treatment for 6 weeks. Initial BW did not significantly differ between the lines (P > 0.17). Under Ad feeding, the low RFI pigs consumed 8% less feed compared with Control line pigs (P < 0.06), had less carcass fat (P < 0.05), but with no significant difference in growth rate (P > 0.85). Under restricted feeding, low RFI pigs under the Ad75 treatment had a greater rate of gain while consuming the same amount of feed as Control pigs. Despite the greater gain, no significant line differences in carcass composition or carcass traits were observed. For the WS treatment, low RFI pigs had similar BW (P > 0.37) with no significant difference in feed consumption (P > 0.32). Overall, selection for reduced RFI has decreased feed intake, with limited differences in growth rate but reduced carcass fat, as seen under Ad feeding. Collectively, results indicate that the effects of selection for low RFI are evident during the early grower stage, which allows for greater savings to the producer.

Results from six generations of selection for intramuscular fat in Duroc swine using real-time ultrasound. I. Direct and correlated phenotypic responses to selection.

A study was conducted to evaluate the efficacy of selection for intramuscular fat (IMF) in a population of purebred Duroc swine using real-time ultrasound. Forty gilts were purchased from US breeders and randomly mated for 2 generations to boars available in regional boar studs, resulting in a base population of 56 litters. Littermate pairs of gilts from this population were randomly assigned to a select line (SL) or control line (CL) and mated to the same sire to establish genetic ties between lines. At an average BW of 114 kg, a minimum of 4 longitudinal ultrasound images were collected 7 cm off-midline across the 10th to 13th ribs of all pigs for the prediction of IMF (UIMF). At least 1 barrow or gilt was slaughtered from each litter, and carcass data were collected. A sample of the LM from the 10th to 11th rib interface was analyzed for carcass IMF (CIMF). Breeding values for IMF were estimated by fitting a 2-trait (UIMF and CIMF) animal model in MATVEC. In the SL, selection in each subsequent generation was based on EBV for IMF with the top 10 boars and top 75 gilts used to produce the next generation. One boar from each sire family and 50 to 60 gilts representing all sire families were randomly selected to maintain the CL. Through 6 generations of selection, an 88% improvement in IMF has been realized (4.53% in SL vs. 2.41% in CL). Results of this study revealed no significant correlated responses in measures of growth performance. However, 6 generations of selection for IMF have yielded correlated effects of decreased loin muscle area and increased backfat. Additionally, the SL obtained more desirable objective measures of tenderness and sensory evaluations of flavor and off-flavor. Meat quality characteristics of pH, water holding capacity, and percent cooking loss were not significantly affected by selection for IMF. Selection for IMF using real-time ultrasound is effective but may be associated with genetic ramifications for carcass composition traits. Intramuscular fat may be used in purebred Duroc swine breeding programs as an indicator trait for sensory traits that influence consumer acceptance; however, rapid improvement should not be expected when simultaneous improvement in other trait categories is also pursued.

Correlating blood immune parameters and a CCT7 genetic variant with the shedding of Salmonella enterica serovar Typhimurium in swine.

The porcine response to Salmonella infection is critical for control of Salmonella fecal shedding and the establishment of Salmonella carrier status. In this study, 40 crossbred pigs were intranasally inoculated with Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) and monitored for Salmonella fecal shedding and blood immune parameters at 2, 7, 14 and 20 days post-inoculation (dpi). Using a multivariate permutation test, a positive correlation was observed between Salmonella Typhimurium shedding levels at 2 and 7dpi and serum interferon-gamma (IFNgamma) levels at 2dpi (p<0.05), with Salmonella being shed in greater numbers from animals with higher IFNgamma levels. A positive correlation was also observed between IFNgamma levels and the number of banded neutrophils (2dpi), circulating neutrophils (7 and 14dpi), monocytes (7dpi), and white blood cells (WBCs) (7, 14 and 20dpi). We have further performed association studies on these immune response parameters as well as shedding status of the Salmonella-infected pigs with a single nucleotide polymorphism (SNP) in the porcine gene CCT7, previously shown by our group to be transcriptionally up-regulated in swine experimentally inoculated with Salmonella Typhimurium. Our analyses with the 40 pigs suggest a positive association (p=0.0012) of SNP genotype A/G at position AK240296.c1153G>A of the CCT7 gene with Salmonella shedding at 7dpi compared to the G/G homozygote genotype. Linking specific genes and genetic polymorphisms with the porcine immune response to Salmonella infection and shedding may identify potential markers for carrier pigs as well as targets for disease diagnosis, intervention and prevention.

Computational integration of structural and functional genomics data across species to develop information on the porcine inflammatory gene regulatory pathway.

We are investigating the porcine gut immune response to infection through gene expression profiling. Porcine Affymetrix GeneChip data was obtained from RNA prepared from mesenteric lymph node of swine infected with either Salmonella enterica serovar Typhimurium (ST) or S. Choleraesuis (SC) for 0, 8, 24, 48 or 504 hours post-inoculation (hpi). In total, 2365 genes with statistical evidence for differential expression (DE; p < 0.01, q < 0.26, fold-change > 2) between at least two time-points were identified. Comparative Gene Ontology analyses revealed that a high proportion of annotated DE genes in both infections are involved in immune and defence responses. Hierarchical clustering of expression patterns and annotations showed that 22 of the 83 genes upregulated from 8-24 hpi in the SC infection are known NF-kappaB targets. The promoter sequences of human genes orthologous to the DE genes were collected and TFM-Explorer was used to identify a set of 72 gene promoters with significant over-representation of NF-kappaB DNA-binding motifs. All 22 known NF-kappaB target genes are in this list; we hypothesize that the remaining 51 genes are un-recognized NF-kappaB targets. Integration of these results and verification of putative target genes will increase our understanding of the porcine response pathways responding to bacterial infection.

Evaluation of linkage disequilibrium measures between multi-allelic markers as predictors of linkage disequilibrium between single nucleotide polymorphisms.

Effectiveness of marker-assisted selection (MAS) and quantitative trait locus (QTL) mapping using population-wide linkage disequilibrium (LD) between markers and QTLs depends on the extent of LD and how it declines with distance between markers and QTLs in a population. Marker-QTL LD can be predicted from LD between markers. Our previous work evaluated LD measures between multi-allelic markers as predictors of usable LD of multi-allelic markers with QTLs. Since single nucleotide polymorphisms (SNPs) are the current marker of choice for high-density genotyping and LD-mapping of QTLs, the objective of this study was to use LD between multi-allelic markers to predict LD among biallelic SNPs or between SNPs and QTLs. Observable LD between multi-allelic markers was evaluated using nine measures. These included two pooled and standardized measures of LD between pairs of alleles at two markers based on Lewontin's LD measure, two pooled measures of squared correlations between alleles, one standardized measure using Hardy-Weinberg heterozygosities, and four measures based on the chi-square statistic for testing for association between alleles at two loci. The standardized chi-square measure that best predicted usable LD between multi-allelic markers and QTLs, based on our previous work, overestimated usable SNP-SNP or SNP-QTL LD. Instead, three other measures were found to be good predictors of usable SNP-SNP or SNP-QTL LD when LD is generated by drift. Therefore, the LD measure between multi-allelic markers that is best for predicting usable LD in a population depends on the type of markers (i.e. multi-allelic or biallelic) that will eventually be used for QTL mapping or MAS.

Selective transcriptional profiling for trait-based eQTL mapping.

There is currently great interest in identifying genetic loci associated with the expression of one or more genes by combining methods for the mapping of quantitative trait loci (QTL) with microarray technology. Because of the high cost of microarrays, it is useful to develop strategies for selecting a subset of individuals for expression profiling. We describe a method of selective transcriptional profiling that uses available information on individual quantitative traits and marker data to choose an optimal set of individuals for measurement with microarrays. We show how to analyse the resulting data to identify genes whose expression is associated with a QTL for a traditional trait or any other locus. Examples are used to illustrate the method and highlight some challenges associated with the analysis of selected data.

Evaluation of linkage disequilibrium measures between multi-allelic markers as predictors of linkage disequilibrium between markers and QTL.

Effectiveness of marker-assisted selection (MAS) and quantitative trait loci (QTL) mapping using population-wide linkage disequilibrium (LD) between markers and QTL depends on the extent of LD and how it declines with distance in a population. Because marker-QTL LD cannot be observed directly, the objective of this study was to evaluate alternative measures of observable LD between multi-allelic markers as predictors of usable LD of multi-allelic markers with presumed biallelic QTL. Observable LD between marker pairs was evaluated using eight existing measures and one new measure. These consisted of two pooled and standardized measures of LD between pairs of alleles at two markers based on Lewontin's LD measure, two pooled measures of squared correlations between alleles, one standardized measure using Hardy-Weinberg heterozygosities, and four measures based on the chi-square statistic for testing for association between alleles at two loci. In simulated populations with a range of LD generated by drift and a range of marker polymorphism, marker-marker LD measured by a standardized chi-square statistic (denoted chi(2')) was found to be the best predictor of useable marker-QTL LD for a group of multi-allelic markers. Estimates of the level and decline of marker-marker LD with distance obtained from chi(2') were linearly and highly correlated with usable LD of those markers with QTL across population structures and marker polymorphism. Corresponding relationships were poorer for the other marker-marker LD measures. Therefore, when LD is generated by drift, chi(2') is recommended to quantify the amount and extent of usable LD in a population for QTL mapping and MAS based on multi-allelic markers.

Controlling the proportion of false positives in multiple dependent tests.

Genome scan mapping experiments involve multiple tests of significance. Thus, controlling the error rate in such experiments is important. Simple extension of classical concepts results in attempts to control the genomewise error rate (GWER), i.e., the probability of even a single false positive among all tests. This results in very stringent comparisonwise error rates (CWER) and, consequently, low experimental power. We here present an approach based on controlling the proportion of false positives (PFP) among all positive test results. The CWER needed to attain a desired PFP level does not depend on the correlation among the tests or on the number of tests as in other approaches. To estimate the PFP it is necessary to estimate the proportion of true null hypotheses. Here we show how this can be estimated directly from experimental results. The PFP approach is similar to the false discovery rate (FDR) and positive false discovery rate (pFDR) approaches. For a fixed CWER, we have estimated PFP, FDR, pFDR, and GWER through simulation under a variety of models to illustrate practical and philosophical similarities and differences among the methods.

Complementary DNA macroarray analyses of differential gene expression in porcine fetal and postnatal muscle.

To study differential gene expression in porcine skeletal muscle, a porcine complementary DNA (cDNA) macroarray was produced that contained 327 expressed sequence tags (EST) derived from whole embryo and adult skeletal muscle, and differential display PCR products from fetal and postnatal muscle. Total RNA from four muscle samples, 75- and 105-d fetal hind limb muscles, and 1- and 7-wk postnatal semitendinosus muscle was used to make radiolabeled targets for duplicate hybridization to the macroarray membranes in an initial screen for expression. All EST that gave clear signals (n = 238) were then re-arrayed, and hybridization was conducted with additional biological replication of samples in the 75-d and 1-wk ages. Signal intensity for each gene was normalized to signal intensity measured at control spots on each membrane, which consisted of total cDNA from liver, lung, spleen, and skeletal muscle. Both normalized ratio levels and a mixed linear model analyses were used to identify genes differentially expressed among the muscle samples. Results showed 28 genes had differences in expression level greater than twofold between the 75-d fetal and 1-wk muscle RNA samples. All 28 genes were also identified as genes with significantly different (P < 0.01) expression using a mixed linear model analysis. Nineteen of these 28 genes had significant matches (basic local alignment search tool [BLAST] score > 100; P < 0.01) to known genes, two matched genes encoding human hypothetical proteins, and seven had no significant matches to Genbank nonredundant and dbEST (database of expressed sequence tags) entries. These results were confirmed for representative genes with RNA blot analysis of seven developmental time points, including RNA from the same muscle samples tested previously in the macroarray. The RNA blot results confirmed the macroarray results for all selected genes, demonstrating that the macroarray technique used in this study is accurate and reproducible. An unknown muscle clone (M218) with a slightly less than twofold increase in expression from the 75-d to the 1-wk age (1 wk/75 d = 1.94; P = 0.0114) was also shown to differ between these two ages using RNA blot analysis, demonstrating the methods used to identify differentially expressed genes may be conservative. The association between expression patterns of vimentin and desmin was also investigated. Results indicate the switch in intermediate filament protein from vimentin to desmin occurs primarily at the level of transcription and/or RNA processing.

Mining MEDLINE: abstracts, sentences, or phrases?

A growing body of works address automated mining of biochemical knowledge from digital repositories of scientific literature, such as MEDLINE. Some of these works use abstracts as the unit of text from which to extract facts. Others use sentences for this purpose, while still others use phrases. Here we compare abstracts, sentences, and phrases in MEDLINE using the standard information retrieval performance measures of recall, precision, and effectiveness, for the task of mining interactions among biochemical terms based on term co-occurrence. Results show statistically significant differences that can impact the choice of text unit.

Processing and representation of meta-data for sleep apnea diagnosis with an artificial intelligence approach.

In this article, we revise and try to resolve some of the problems inherent in questionnaire screening of sleep apnea cases and apnea diagnosis based on attributes which are relevant and reliable. We present a way of learning information about the relevance of the data, comparing this with the definition of the information by the medical expert. We generate a predictive data model using a data aggregation operator which takes relevance and reliability information about the data into account to produce a diagnosis for each case. We also introduce a grade of membership for each question response which allows the patient to indicate a level of confidence or doubt in their own judgement. The method is tested with data collected from patients in a Sleep Clinic using questionnaires specially designed for the study. Other artificial intelligence predictive modeling algorithms are also tested on the same data and their predictive accuracy compared to that of the aggregation operator.

Preclinical pharmacology of CP-424,391, an orally active pyrazolinone-piperidine [correction of pyrazolidinone-piperidine] growth hormone secretagogue.

Growth hormone secretagogues (GHSs) represent attractive therapeutic alternatives to recombinant growth hormone (GH), given their ability to amplify pulsatile hormone secretion in a relatively physiologic manner. CP-424,391 (391) is a novel, orally active pyrazolinone-piperidine [corrected] GHS. In rat pituitary cell cultures, 391 stimulated GH release with an EC50 = 3 nM. The addition of 391 to rat pituitary cells activated intracellular calcium signaling but did not elevate intracellular cyclic adenosine monophosphate (cAMP). 391 also modulated the effects of GH-releasing hormone and somatostatin on pituitary cell GH-release and intracellular signaling. In nonpituitary cell lines, the ability of 391 to stimulate intracellular signaling was dependent on the expression of recombinant human GHS receptor. Acute administration of 391 to anesthetized rats or to conscious dogs induced pulsatile release of G H in a dose-dependent manner. Plasma insulin-like growth factor-I (IGF-I) was elevated progressively over a 5-d course of daily oral dosing in dogs. Chronic oral administration of 391 augmented body weight gain in rats and dogs. Thus, the peptidomimetic GHS 391 has potential utility for the treatment of clinical conditions that could benefit from systemic augmentation of GH and IGF-I levels.

Biaryl diacid inhibitors of human s-PLA2 with anti-inflammatory activity.

Twenty-four hydrophobic dicarboxylic acids are described which were evaluated as inhibitors of 14 kDa human platelet phospholipase A2 (HP-PLA2). In general, biarylacetic acid derivatives were found to be more active than biaryl acids or biarylpropanoic acids. More potent inhibitors were obtained when hydrophobic groups were attached to the biaryl acid nucleus using an olefin linkage as compared to an ether linkage. Compounds with larger hydrophobic groups were usually more potent inhibitors of HP-PLA2. Five of the compounds disclosed in this report (2, 4, 28, 36b and 36i) were found to possess significant anti-inflammatory activity in a phorbol ester induced mouse ear edema model of chronic inflammation.