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Aims: Type 2 diabetes (T2D) is a risk factor for ischemic stroke (IS) and associated with an adverse prognosis. Both stroke and diabetes care has evolved substantially during the last decade. This study aimed to determine the prevalence of T2D among IS patients along with time trends in the risk profile, use of glucose‐lowering medications, quality‐of‐care and clinical outcomes, including stroke severity; length‐of‐stay; mortality, readmission and recurrent stroke in a large national cohort. Methods: Registry‐based cohort study including all IS events in Denmark from 2004 to 2020. IS with co‐morbid T2D were compared to IS without diabetes while adjusting for age, sex, stroke severity, co‐morbidity and socio‐economic factors. Results: The study included 169,262 IS events; 24,479 with co‐morbid T2D. The prevalence of T2D in IS increased from 12.0% (2004–2006) to 17.0% (2019–2020). The adjusted absolute 30‐day mortality risk in IS with T2D decreased from 9.9% (2004–2006) to 7.8% (2019–2020). The corresponding adjusted risk ratios (aRR) were 1.22 95% confidence interval (1.09–1.37) and 1.29 (1.11–1.50), respectively. The aRR of 365‐day mortality was in 2004–2006: 1.20 (1.12–1.29) and in 2019–2020: 1.34 (1.22–1.47). The 30‐ and 365‐day readmissions rates were also consistently higher in IS with T2D. Conclusions: The prevalence of T2D in IS increased over time. The 30‐ and 365‐day mortality rates decreased over the time‐period but were consistently higher in IS with co‐morbid T2D. Readmissions were also higher in IS with T2D. This highlights an urgent need for strategies to further improve the prognosis in IS patients with co‐morbid T2D. [ABSTRACT FROM AUTHOR]

Abstract The aim of this paper was to describe, and when possible compare, the multivariate methods used by the participants in the EADGENE WP1.4 workshop. The first approach was for class discovery and class prediction using evidence from the data at hand. Several teams used hierarchical clustering (HC) or principal component analysis (PCA) to identify groups of differentially expressed genes with a similar expression pattern over time points and infective agent (E. coli or S. aureus). The main result from these analyses was that HC and PCA were able to separate tissue samples taken at 24 h following E. coli infection from the other samples. The second approach identified groups of differentially co-expressed genes, by identifying clusters of genes highly correlated when animals were infected with E. coli but not correlated more than expected by chance when the infective pathogen was S. aureus. The third approach looked at differential expression of predefined gene sets. Gene sets were defined based on information retrieved from biological databases such as Gene Ontology. Based on these annotation sources the teams used either the GlobalTest or the Fisher exact test to identify differentially expressed gene sets. The main result from these analyses was that gene sets involved in immune defence responses were differentially expressed.

Abstract A large variety of methods has been proposed in the literature for microarray data analysis. The aim of this paper was to present techniques used by the EADGENE (European Animal Disease Genomics Network of Excellence) WP1.4 participants for data quality control, normalisation and statistical methods for the detection of differentially expressed genes in order to provide some more general data analysis guidelines. All the workshop participants were given a real data set obtained in an EADGENE funded microarray study looking at the gene expression changes following artificial infection with two different mastitis causing bacteria: Escherichia coli and Staphylococcus aureus. It was reassuring to see that most of the teams found the same main biological results. In fact, most of the differentially expressed genes were found for infection by E. coli between uninfected and 24 h challenged udder quarters. Very little transcriptional variation was observed for the bacteria S. aureus. Lists of differentially expressed genes found by the different research teams were, however, quite dependent on the method used, especially concerning the data quality control step. These analyses also emphasised a biological problem of cross-talk between infected and uninfected quarters which will have to be dealt with for further microarray studies.

Abstract Microarray analyses have become an important tool in animal genomics. While their use is becoming widespread, there is still a lot of ongoing research regarding the analysis of microarray data. In the context of a European Network of Excellence, 31 researchers representing 14 research groups from 10 countries performed and discussed the statistical analyses of real and simulated 2-colour microarray data that were distributed among participants. The real data consisted of 48 microarrays from a disease challenge experiment in dairy cattle, while the simulated data consisted of 10 microarrays from a direct comparison of two treatments (dye-balanced). While there was broader agreement with regards to methods of microarray normalisation and significance testing, there were major differences with regards to quality control. The quality control approaches varied from none, through using statistical weights, to omitting a large number of spots or omitting entire slides. Surprisingly, these very different approaches gave quite similar results when applied to the simulated data, although not all participating groups analysed both real and simulated data. The workshop was very successful in facilitating interaction between scientists with a diverse background but a common interest in microarray analyses.

Objectives: Cystic fibrosis (CF) is one of the most common severe autosomal recessive disorders. Prenatal or preconception CF screening is offered in some countries. A maternal blood sample in early pregnancy can provide circulating trophoblasts and offers a DNA source for genetic analysis of both the mother and the fetus. This study aimed to develop a cell‐based noninvasive prenatal test (NIPT) to screen for the 50 most common CF variants. Methods: Blood samples were collected from 30 pregnancies undergoing invasive diagnostics and circulating trophoblasts were harvested in 27. Cystic fibrosis testing was conducted using two different methods: by fragment length analysis and by our newly developed NGS‐based CF analysis. Results: In all 27 cases, cell‐based NIPT provided a result using both methods in agreement with the invasive test result. Conclusion: This study shows that cell‐based NIPT for CF screening provides a reliable result without the need for partner‐ and proband samples. Key points: What's already known about this topic?Many pregnant women are positive towards prenatal screening for Cystic fibrosis (CF).The only current pregnancy marker is echogenic bowel in the second trimester, but this test has low sensitivity and specificity. What does this study add?Prenatal screening for CF can be done using circulating trophoblasts isolated from maternal blood early in pregnancyCirculating trophoblasts can provide screening for CF as a simple single‐visit setup without the need for a paternal sample. [ABSTRACT FROM AUTHOR]

After parenchymal loss, the liver regenerates restoring normal mass and metabolic function. Prevailing theories on triggering events leading to regeneration include humoral, metabolic, and flow-mediated mechanisms, the latter emphasizing the importance of shear stress mediated nitric oxide regulation. We aimed to investigate whether the grade of resection and hence the portal venous pressure and sinusoidal shear stress increase would be reflected in the gene expression profiles in the liver remnant by using a global porcine cDNA microarray chip with ~23,000 genes represented. Six pig livers were resected with 62% (low portal pressure resection) and 75% (high portal pressure resection), resulting in a portal venous pressure increase from a baseline of 6.1-8.2 and 12 mmHg, respectively. By sampling consecutive biopsies from the liver remnants, we found differentially expressed genes in the high portal pressure resection group to have functions related primarily to apoptosis, nitric oxide metabolism and oxidative stress, whereas differentially expressed genes in the low portal pressure resection group potentially regulate the cell cycle. Common to both groups was the upregulation of genes regulating inflammation, transport, cell proliferation, development, and protein metabolism. Also common to both groups was both up- and downregulation of genes regulating cell-cell signaling, signal transduction, cell adhesion, and translation. Genes regulating the metabolism of lipids, hormones, amines, and alcohol were downregulated in both groups. In conclusion, the genetic regenerative response in the liver remnant to varies according to the level of resection. microarray; shear stress; partial hepatectomy; regeneration

DNA hybridization and infB gene analysis in 66 clinical isolates of Haemophilus indicate that human isolates are dependent on X and V factors. A true 'Haemophilus sensu stricto', as determined by 16S RRNA sequence comparison, does not exist in the phylogeny of Haemophilus genus.

Research has been conducted on the prokaryotic translation-initiation factor 2 infB gene. The use of the infB partial sequences in phylogenetic analyses of related to Streptococcus agalactiae species is discussed.

Abstract Background Integration of genomic variation with phenotypic information is an effective approach for uncovering genotype-phenotype associations. This requires an accurate identification of the different types of variation in individual genomes. Results We report the integration of the whole genome sequence of a single Holstein Friesian bull with data from single nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) array technologies to determine a comprehensive spectrum of genomic variation. The performance of resequencing SNP detection was assessed by combining SNPs that were identified to be either in identity by descent (IBD) or in copy number variation (CNV) with results from SNP array genotyping. Coding insertions and deletions (indels) were found to be enriched for size in multiples of 3 and were located near the N- and C-termini of proteins. For larger indels, a combination of split-read and read-pair approaches proved to be complementary in finding different signatures. CNVs were identified on the basis of the depth of sequenced reads, and by using SNP and CGH arrays. Conclusions Our results provide high resolution mapping of diverse classes of genomic variation in an individual bovine genome and demonstrate that structural variation surpasses sequence variation as the main component of genomic variability. Better accuracy of SNP detection was achieved with little loss of sensitivity when algorithms that implemented mapping quality were used. IBD regions were found to be instrumental for calculating resequencing SNP accuracy, while SNP detection within CNVs tended to be less reliable. CNV discovery was affected dramatically by platform resolution and coverage biases. The combined data for this study showed that at a moderate level of sequencing coverage, an ensemble of platforms and tools can be applied together to maximize the accurate detection of sequence and structural variants.

Abstract Background Aggressive behaviour is an important aspect in the daily lives of animals living in groups. Aggressive animals have advantages, such as better access to food or territories, and they produce more offspring than low ranking animals. The social hierarchy in chickens is measured using the 'pecking order' concept, which counts the number of aggressive pecks given and received. To date, little is known about the underlying genetics of the 'pecking order'. Results A total of 60 hens from a high feather pecking selection line were divided into three groups: only receivers (R), only peckers (P) and mixed peckers and receivers (P&R). In comparing the R and P groups, we observed that there were 40 differentially expressed genes [false discovery rate (FDR) P < 0.10]. It was not fully clear how the 40 genes regulated aggressive behaviour; however, gene set analysis detected a number of GO identifiers, which were potentially involved in aggressive behavioural processes. These genes code for synaptosomes (GO:0019797), and proteins involved in the regulation of the excitatory postsynaptic membrane potential (GO:0060079), the regulation of the membrane potential (GO:0042391), and glutamate receptor binding (GO:0035254). Conclusion In conclusion, our study provides new insights into which genes are involved in aggressive behaviours in chickens. Pecking and receiving hens exhibited different gene expression profiles in their brains. Following confirmation, the identification of differentially expressed genes may elucidate how the pecking order forms in laying hens at a molecular level.

Abstract Background The recent development within high-throughput technologies for expression profiling has allowed for parallel analysis of transcriptomes and proteomes in biological systems such as comparative analysis of transcript and protein levels of tissue regulated genes. Until now, such studies of have only included microarray or short length sequence tags for transcript profiling. Furthermore, most comparisons of transcript and protein levels have been based on absolute expression values from within the same tissue and not relative expression values based on tissue ratios. Results Presented here is a novel study of two porcine tissues based on integrative analysis of data from expression profiling of identical samples using cDNA microarray, 454-sequencing and iTRAQ-based proteomics. Sequence homology identified 2.541 unique transcripts that are detectable by both microarray hybridizations and 454-sequencing of 1.2 million cDNA tags. Both transcript-based technologies showed high reproducibility between sample replicates of the same tissue, but the correlation across these two technologies was modest. Thousands of genes being differentially expressed were identified with microarray. Out of the 306 differentially expressed genes, identified by 454-sequencing, 198 (65%) were also found by microarray. The relationship between the regulation of transcript and protein levels was analyzed by integrating iTRAQ-based proteomics data. Protein expression ratios were determined for 354 genes, of which 148 could be mapped to both microarray and 454-sequencing data. A comparison of the expression ratios from the three technologies revealed that differences in transcript and protein levels across heart and muscle tissues are positively correlated. Conclusion We show that the reproducibility within cDNA microarray and 454-sequencing is high, but that the agreement across these two technologies is modest. We demonstrate that the regulation of transcript and protein levels across identical tissue samples is positively correlated when the tissue expression ratios are used for comparison. The results presented are of interest in systems biology research in terms of integration and analysis of high-throughput expression data from mammalian tissues.

Abstract Background Boar taint is the unpleasant odour and flavour of the meat of uncastrated male pigs that is primarily caused by high levels of androstenone and skatole in adipose tissue. Androstenone is a steroid and its levels are mainly genetically determined. Studies on androstenone metabolism have, however, focused on a limited number of genes. Identification of additional genes influencing levels of androstenone may facilitate implementation of marker assisted breeding practices. In this study, microarrays were used to identify differentially expressed genes and pathways related to androstenone metabolism in the liver from boars with extreme levels of androstenone in adipose tissue. Results Liver tissue samples from 58 boars of the two breeds Duroc and Norwegian Landrace, 29 with extreme high and 29 with extreme low levels of androstenone, were selected from more than 2500 individuals. The samples were hybridised to porcine cDNA microarrays and the 1% most significant differentially expressed genes were considered significant. Among the differentially expressed genes were metabolic phase I related genes belonging to the cytochrome P450 family and the flavin-containing monooxygenase FMO1. Additionally, phase II conjugation genes including UDP-glucuronosyltransferases UGT1A5, UGT2A1 and UGT2B15, sulfotransferase STE, N-acetyltransferase NAT12 and glutathione S-transferase were identified. Phase I and phase II metabolic reactions increase the water solubility of steroids and play a key role in their elimination. Differential expression was also found for genes encoding 17beta-hydroxysteroid dehydrogenases (HSD17B2, HSD17B4, HSD17B11 and HSD17B13) and plasma proteins alpha-1-acid glycoprotein (AGP) and orosomucoid (ORM1). 17beta-hydroxysteroid dehydrogenases and plasma proteins regulate the availability of steroids by controlling the amount of active steroids accessible to receptors and available for metabolism. Differences in the expression of FMO1, NAT12, HSD17B2 and HSD17B13 were verified by quantitative real competitive PCR. Conclusion A number of genes and pathways related to metabolism of androstenone in liver were identified, including new candidate genes involved in phase I oxidation metabolism, phase II conjugation metabolism, and regulation of steroid availability. The study is a first step towards a deeper understanding of enzymes and regulators involved in pathways of androstenone metabolism and may ultimately lead to the discovery of markers to reduce boar taint.

Abstract Background The bacterium Actinobacillus pleuropneumoniae is responsible for porcine pleuropneumonia, a widespread, highly contagious and often fatal respiratory disease of pigs. The general porcine innate immune response after A. pleuropneumoniae infection is still not clarified. The objective of this study was hence to characterise the transcriptional response, measured by using cDNA microarrays, in pigs 24 hours after experimental inoculation with A. pleuropneumoniae. Methods Microarray analyses were conducted to reveal genes being differentially expressed in inflamed versus non-inflamed lung tissue sampled from inoculated animals as well as in liver and tracheobronchial lymph node tissue sampled from three inoculated animals versus two non-inoculated animals. The lung samples were studied using a porcine cDNA microarray with 5375 unique PCR products while liver tissue and tracheobronchial lymph node tissue were hybridised to an expanded version of the porcine microarray with 26879 unique PCR products. Results A total of 357 genes differed significantly in expression between infected and non-infected lung tissue, 713 genes differed in expression in liver tissue from infected versus non-infected animals and 130 genes differed in expression in tracheobronchial lymph node tissue from infected versus non-infected animals. Among these genes, several have previously been described to be part of a general host response to infections encoding immune response related proteins. In inflamed lung tissue, genes encoding immune activating proteins and other pro-inflammatory mediators of the innate immune response were found to be up-regulated. Genes encoding different acute phase reactants were found to be differentially expressed in the liver. Conclusion The obtained results are largely in accordance with previous studies of the mammalian immune response. Furthermore, a number of differentially expressed genes have not previously been associated with infection or are presently unidentified. Determination of their specific roles during infection may lead to a better understanding of innate immunity in pigs. Although additional work including more animals is clearly needed to elucidate host response to porcine pleuropneumonia, the results presented in this study demonstrate three subsets of genes consistently expressed at different levels depending upon infection status.