Stimulating T cell responses against patient-derived breast cancer cells with neoantigen peptide-loaded peripheral blood mononuclear cells.

Breast cancer stands as a formidable global health challenge for women. While neoantigens exhibit efficacy in activating T cells specific to cancer and instigating anti-tumor immune responses, the accuracy of neoantigen prediction remains suboptimal. In this study, we identified neoantigens from the patient-derived breast cancer cells, PC-B-142CA and PC-B-148CA cells, utilizing whole-genome and RNA sequencing. The pVAC-Seq pipeline was employed, with minor modification incorporating criteria (1) binding affinity of mutant (MT) peptide with HLA (IC50 MT) ≤ 500 nm in 3 of 5 algorithms and (2) IC50 wild type (WT)/MT > 1. Sequencing results unveiled 2513 and 3490 somatic mutations, and 646 and 652 non-synonymous mutations in PC-B-142CA and PC-B-148CA, respectively. We selected the top 3 neoantigens to perform molecular dynamic simulation and synthesized 9-12 amino acid neoantigen peptides, which were then pulsed onto healthy donor peripheral blood mononuclear cells (PBMCs). Results demonstrated that T cells activated by ADGRL1E274K, PARP1E619K, and SEC14L2R43Q peptides identified from PC-B-142CA exhibited significantly increased production of interferon-gamma (IFN-γ), while PARP1E619K and SEC14L2R43Q peptides induced the expression of CD107a on T cells. The % tumor cell lysis was notably enhanced by T cells activated with MT peptides across all three healthy donors. Moreover, ALKBH6V83M and GAAI823T peptides from PC-B-148CA remarkably stimulated IFN-γ- and CD107a-positive T cells, displaying high cell-killing activity against target cancer cells. In summary, our findings underscore the successful identification of neoantigens with anti-tumor T cell functions and highlight the potential of personalized neoantigens as a promising avenue for breast cancer treatment.

The Atlantic salmon genome provides insights into rediploidization.

The whole-genome duplication 80 million years ago of the common ancestor of salmonids (salmonid-specific fourth vertebrate whole-genome duplication, Ss4R) provides unique opportunities to learn about the evolutionary fate of a duplicated vertebrate genome in 70 extant lineages. Here we present a high-quality genome assembly for Atlantic salmon (Salmo salar), and show that large genomic reorganizations, coinciding with bursts of transposon-mediated repeat expansions, were crucial for the post-Ss4R rediploidization process. Comparisons of duplicate gene expression patterns across a wide range of tissues with orthologous genes from a pre-Ss4R outgroup unexpectedly demonstrate far more instances of neofunctionalization than subfunctionalization. Surprisingly, we find that genes that were retained as duplicates after the teleost-specific whole-genome duplication 320 million years ago were not more likely to be retained after the Ss4R, and that the duplicate retention was not influenced to a great extent by the nature of the predicted protein interactions of the gene products. Finally, we demonstrate that the Atlantic salmon assembly can serve as a reference sequence for the study of other salmonids for a range of purposes.

Role of the intestinal microbiome in low-density polyethylene degradation by caterpillar larvae of the greater wax moth, Galleria mellonella.

Recently, a few insects, including the caterpillar larva of the greater wax moth Galleria mellonella, have been identified as avid 'plastivores'. These caterpillars are able to ingest and metabolize polyethylene at unprecedented rates. While it appears that G. mellonella plays an important role in the biodegradation process, the contribution of its intestinal microbiome remains poorly understood and contested. In a series of experiments, we present strong evidence of an intricate relationship between an intact microbiome, low-density polyethylene (LDPE) biodegradation and the production of glycol as a metabolic by-product. First, we biochemically confirmed that G. mellonella larvae consume and metabolize LDPE, as individual caterpillars fed on polyethylene excreted glycol, but those excretions were reduced by antibiotic treatment. Further, while the gut bacterial communities remained relatively stable regardless of diet, we showed that during the early phases of feeding on LDPE (24-72 h), caterpillars exhibited increased microbial abundance relative to those starved or fed on their natural honeycomb diet. Finally, by isolating and growing gut bacteria with polyethylene as their exclusive carbon source for over 1 year, we identified microorganisms in the genus Acinetobacter that appeared to be involved in this biodegradation process. Taken collectively, our study indicates that during short-term exposure, the intestinal microbiome of G. mellonella is intricately associated with polyethylene biodegradation in vivo.

A Very Hungry Caterpillar: Polyethylene Metabolism and Lipid Homeostasis in Larvae of the Greater Wax Moth (Galleria mellonella).

Larvae of the greater wax moth (Galleria mellonella) possess the remarkable ability to consume and rapidly degrade low-density polyethylene. Previous studies have investigated the involvement of the animal's microbiome, but little is known about the host's actual role and if it benefits from biodegradation of this synthetic polymer. We used a combination of RNA sequencing and biochemical approaches to assess caterpillars fed honeycomb, fed polyethylene (PE), or starved for up to 72 h. Sequencing of gut transcripts revealed PE-fed larvae retain an expression profile consistent with normal intestinal function but also show distinct molecular signatures indicative of enhanced fatty acid metabolism (FAM). Further, quantification of total lipid content validated the impact of a PE diet on FAM; in contrast to lipid-depleted starved animals, PE-fed caterpillars maintain lipid reserves similar to honeycomb-fed larvae. Additionally, we found the activity of putative enzymes involved in lipid oxidation (e.g., alcohol dehydrogenase) are considerably higher in PE-fed larvae, indicating that on a functional level, these caterpillars are inducing pathways to effectively metabolize PE. Overall, we put forward a hypothesized model where the similarity in chemical structure between PE and its natural honeycomb diet has endowed larvae of G. mellonella with the extraordinary capability to derive energy from PE as an exclusive food source through pre-existing metabolic pathways.

Strong selection pressures maintain divergence on genomic islands in Atlantic cod (Gadus morhua L.) populations.

BACKGROUND: Two distinct populations have been extensively studied in Atlantic cod (Gadus morhua L.): the Northeast Arctic cod (NEAC) population and the coastal cod (CC) population. The objectives of the current study were to identify genomic islands of divergence and to propose an approach to quantify the strength of selection pressures using whole-genome single nucleotide polymorphism (SNP) data. After applying filtering criteria, information on 93 animals (9 CC individuals, 50 NEAC animals and 34 CC × NEAC crossbred individuals) and 3,123,434 autosomal SNPs were used. RESULTS: Four genomic islands of divergence were identified on chromosomes 1, 2, 7 and 12, which were mapped accurately based on SNP data and which extended in size from 11 to 18 Mb. These regions differed considerably between the two populations although the differences in the rest of the genome were small due to considerable gene flow between the populations. The estimates of selection pressures showed that natural selection was substantially more important than genetic drift in shaping these genomic islands. Our data confirmed results from earlier publications that suggested that genomic islands are due to chromosomal rearrangements that are under strong selection and reduce recombination between rearranged and non-rearranged segments. CONCLUSIONS: Our findings further support the hypothesis that selection and reduced recombination in genomic islands may promote speciation between these two populations although their habitats overlap considerably and migrations occur between them.

Validation of genotype imputation in Southeast Asian populations and the effect of single nucleotide polymorphism annotation on imputation outcome.

BACKGROUND: Imputation involves the inference of untyped single nucleotide polymorphisms (SNPs) in genome-wide association studies. The haplotypic reference of choice for imputation in Southeast Asian populations is unclear. Moreover, the influence of SNP annotation on imputation results has not been examined. METHODS: This study was divided into two parts. In the first part, we applied imputation to genotyped SNPs from Southeast Asian populations from the Pan-Asian SNP database. Five percent of the total SNPs were removed. The remaining SNPs were applied to imputation with IMPUTE2. The imputed outcomes were verified with the removed SNPs. We compared imputation references from Chinese and Japanese haplotypes from the HapMap phase II (HMII) and the complete set of haplotypes from the 1000 Genomes Project (1000G). The second part was imputation accuracy and yield in Thai patient dataset. Half of the autosomal SNPs was removed to create Set 1. Another dataset, Set 2, was then created where we switched which half of the SNPs were removed. Both Set 1 and Set 2 were imputed with HMII to create a complete imputed SNPs dataset. The dataset was used to validate association testing, SNPs annotation and imputation outcome. RESULTS: The accuracy was highest for all populations when using the HMII reference, but at the cost of a lower yield. Thai genotypes showed the highest accuracy over other populations in both HMII and 1000G panels, although accuracy and yield varied across chromosomes. Imputation was tested in a clinical dataset to compare accuracy in gene-related regions, and coding regions were found to have a higher accuracy and yield. CONCLUSIONS: This work provides the first evidence of imputation reference selection for Southeast Asian studies and highlights the effects of SNP locations respective to genes on imputation outcome. Researchers will need to consider the trade-off between accuracy and yield in future imputation studies.

Genome-wide association study confirm major QTL for backfat fatty acid composition on SSC14 in Duroc pigs.

BACKGROUND: Fatty acid composition contributes importantly to meat quality and is essential to the nutritional value of the meat. Identification of genetic factors underlying levels of fatty acids can be used to breed for pigs with healthier meat. The aim of this study was to conduct genome-wide association studies (GWAS) to identify QTL regions affecting fatty acid composition in backfat from the pig breeds Duroc and Landrace. RESULTS: Using data from the Axiom porcine 660 K array, we performed GWAS on 454 Duroc and 659 Landrace boars for fatty acid phenotypes measured by near-infrared spectroscopy (NIRS) technology (C16:0, C16:1n-7, C18:0, C18:1n-9, C18:2n-6, C18:3n-3, total saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids). Two QTL regions on SSC4 and SSC14 were identified in Duroc for the de novo synthesized fatty acids traits, whereas one QTL on SSC8 was detected in Landrace for C16:1n-7. The QTL region on SSC14 has been reported in previous studies and a putative causative mutation has been suggested in the promoter region of the SCD gene. Whole genome re-sequencing data was used for genotype imputation and to fine map the SSC14 QTL region in Norwegian Duroc. This effort confirms the location of the QTL on this chromosome as well as suggesting other putative candidate genes in the region. The most significant single nucleotide polymorphisms (SNPs) located on SSC14 explain between 55 and 76% of the genetic variance and between 27 and 54% of the phenotypic variance for the de novo synthesized fatty acid traits in Norwegian Duroc. For the QTL region on SSC8 in Landrace, the most significant SNP explained 19% of the genetic variance and 5% of the phenotypic variance for C16:1n-7. CONCLUSIONS: This study confirms a major QTL affecting fatty acid composition on SSC14 in Duroc, which can be used in genetic selection to increase the level of fatty acid desaturation. The SSC14 QTL was not segregating in the Landrace population, but another QTL on SSC8 affecting C16:1n-7 was identified and might be used to increase the level of desaturation in meat products from this breed.

An improved genome assembly uncovers prolific tandem repeats in Atlantic cod.

BACKGROUND: The first Atlantic cod (Gadus morhua) genome assembly published in 2011 was one of the early genome assemblies exclusively based on high-throughput 454 pyrosequencing. Since then, rapid advances in sequencing technologies have led to a multitude of assemblies generated for complex genomes, although many of these are of a fragmented nature with a significant fraction of bases in gaps. The development of long-read sequencing and improved software now enable the generation of more contiguous genome assemblies. RESULTS: By combining data from Illumina, 454 and the longer PacBio sequencing technologies, as well as integrating the results of multiple assembly programs, we have created a substantially improved version of the Atlantic cod genome assembly. The sequence contiguity of this assembly is increased fifty-fold and the proportion of gap-bases has been reduced fifteen-fold. Compared to other vertebrates, the assembly contains an unusual high density of tandem repeats (TRs). Indeed, retrospective analyses reveal that gaps in the first genome assembly were largely associated with these TRs. We show that 21% of the TRs across the assembly, 19% in the promoter regions and 12% in the coding sequences are heterozygous in the sequenced individual. CONCLUSIONS: The inclusion of PacBio reads combined with the use of multiple assembly programs drastically improved the Atlantic cod genome assembly by successfully resolving long TRs. The high frequency of heterozygous TRs within or in the vicinity of genes in the genome indicate a considerable standing genomic variation in Atlantic cod populations, which is likely of evolutionary importance.

Fine mapping of a QTL affecting levels of skatole on pig chromosome 7.

BACKGROUND: Previous studies in the Norwegian pig breeds Landrace and Duroc have revealed a QTL for levels of skatole located in the region 74.7-80.5 Mb on SSC7. Skatole is one of the main components causing boar taint, which gives an undesirable smell and taste to the pig meat when heated. Surgical castration of boars is a common practice to reduce the risk of boar taint, however, a selection for boars genetically predisposed for low levels of taint would help eliminating the need for castration and be advantageous for both economic and welfare reasons. In order to identify the causal mutation(s) for the QTL and/or identify genetic markers for selection purposes we performed a fine mapping of the SSC7 skatole QTL region. RESULTS: A dense set of markers on SSC7 was obtained by whole genome re-sequencing of 24 Norwegian Landrace and 23 Duroc boars. Subsets of 126 and 157 SNPs were used for association analyses in Landrace and Duroc, respectively. Significant single markers associated with skatole spanned a large 4.4 Mb region from 75.9-80.3 Mb in Landrace, with the highest test scores found in a region between the genes NOVA1 and TGM1 (p < 0.001). The same QTL was obtained in Duroc and, although less significant, with associated SNPs spanning a 1.2 Mb region from 78.9-80.1 Mb (p < 0.01). The highest test scores in Duroc were found in genes of the granzyme family (GZMB and GZMH-like) and STXBP6. Haplotypes associated with levels of skatole were identified in Landrace but not in Duroc, and a haplotype block was found to explain 2.3% of the phenotypic variation for skatole. The SNPs in this region were not associated with levels of sex steroids. CONCLUSIONS: Fine mapping of a QTL for skatole on SSC7 confirmed associations of this region with skatole levels in pigs. The QTL region was narrowed down to 4.4 Mb in Landrace and haplotypes explaining 2.3% of the phenotypic variance for skatole levels were identified. Results confirmed that sex steroids are not affected by this QTL region, making these markers attractive for selection against boar taint.

Genome-wide association mapping for milk fat composition and fine mapping of a QTL for de novo synthesis of milk fatty acids on bovine chromosome 13.

BACKGROUND: Bovine milk is widely regarded as a nutritious food source for humans, although the effects of individual fatty acids on human health is a subject of debate. Based on the assumption that genomic selection offers potential to improve milk fat composition, there is strong interest to understand more about the genetic factors that influence the biosynthesis of bovine milk and the molecular mechanisms that regulate milk fat synthesis and secretion. For this reason, the work reported here aimed at identifying genetic variants that affect milk fatty acid composition in Norwegian Red cattle. Milk fatty acid composition was predicted from the nation-wide recording scheme using Fourier transform infrared spectroscopy data and applied to estimate heritabilities for 36 individual and combined fatty acid traits. The recordings were used to generate daughter yield deviations that were first applied in a genome-wide association (GWAS) study with 17,343 markers to identify quantitative trait loci (QTL) affecting fatty acid composition, and next on high-density and sequence-level datasets to fine-map the most significant QTL on BTA13 (BTA for Bos taurus chromosome). RESULTS: The initial GWAS revealed 200 significant associations, with the strongest signals on BTA1, 13 and 15. The BTA13 QTL highlighted a strong functional candidate gene for de novo synthesis of short- and medium-chained saturated fatty acids; acyl-CoA synthetase short-chain family member 2. However, subsequent fine-mapping using single nucleotide polymorphisms (SNPs) from a high-density chip and variants detected by resequencing showed that the effect was more likely caused by a second nearby gene; nuclear receptor coactivator 6 (NCOA6). These findings were confirmed with results from haplotype studies. NCOA6 is a nuclear receptor that interacts with transcription factors such as PPARγ, which is a major regulator of bovine milk fat synthesis. CONCLUSIONS: An initial GWAS revealed a highly significant QTL for de novo-synthesized fatty acids on BTA13 and was followed by fine-mapping of the QTL within NCOA6. The most significant SNPs were either synonymous or situated in introns; more research is needed to uncover the underlying causal DNA variation(s).

Two adjacent inversions maintain genomic differentiation between migratory and stationary ecotypes of Atlantic cod.

Atlantic cod is composed of multiple migratory and stationary populations widely distributed in the North Atlantic Ocean. The Northeast Arctic cod (NEAC) population in the Barents Sea undertakes annual spawning migrations to the northern Norwegian coast. Although spawning occurs sympatrically with the stationary Norwegian coastal cod (NCC), phenotypic and genetic differences between NEAC and NCC are maintained. In this study, we resolve the enigma by revealing the mechanisms underlying these differences. Extended linkage disequilibrium (LD) and population divergence were demonstrated in a 17.4-Mb region on linkage group 1 (LG1) based on genotypes of 494 SNPs from 192 parents of farmed families of NEAC, NCC or NEACxNCC crosses. Linkage analyses revealed two adjacent inversions within this region that repress meiotic recombination in NEACxNCC crosses. We identified a NEAC-specific haplotype consisting of 186 SNPs that was fixed in NEAC sampled from the Barents Sea, but segregating under Hardy-Weinberg equilibrium in eight NCC stocks. Comparative genomic analyses determine the NEAC configuration of the inversions to be the derived state and date it to ~1.6-2.0 Mya. The haplotype block harbours 763 genes, including candidates regulating swim bladder pressure, haem synthesis and skeletal muscle organization conferring adaptation to long-distance migrations and vertical movements down to large depths. Our results suggest that the migratory ecotype experiences strong directional selection for the two adjacent inversions on LG1. Despite interbreeding between NEAC and NCC, the inversions are maintaining genetic differentiation, and we hypothesize the co-occurrence of multiple adaptive alleles forming a 'supergene' in the NEAC population.

Fine mapping of a QTL on bovine chromosome 6 using imputed full sequence data suggests a key role for the group-specific component (GC) gene in clinical mastitis and milk production.

BACKGROUND: Clinical mastitis is an inflammation of the mammary gland and causes significant costs to dairy production. It is unfavourably genetically correlated to milk production, and, thus, knowledge of the mechanisms that underlie these traits would be valuable to improve both of them simultaneously through breeding. A quantitative trait locus (QTL) that affects both clinical mastitis and milk production has recently been fine-mapped to around 89 Mb on bovine chromosome 6 (BTA6), but identification of the gene that underlies this QTL was not possible due to the strong linkage disequilibrium between single nucleotide polymorphisms (SNPs) within this region. Our aim was to identify the gene and, if possible, the causal polymorphism(s) responsible for this QTL through association analysis of high-density SNPs and imputed full sequence data in combination with analyses of transcript and protein levels of the identified candidate gene. RESULTS: Associations between SNPs and the studied traits were strongest for SNPs that were located within and immediately upstream of the group-specific component (GC) gene. This gene encodes the vitamin D-binding protein (DBP) and has multiple roles in immune defense and milk production. A 12-kb duplication that was identified downstream of this gene covered its last exon and segregated with the QTL allele that is associated with increased mastitis susceptibility and milk production. However, analyses of GC mRNA levels on the available samples revealed no differences in expression between animals having or lacking this duplication. Moreover, we detected no differences in the concentrations of DBP and its ligand vitamin D between the animals with different GC genotypes that were available for this study. CONCLUSIONS: Our results suggest GC as the gene that underlies the QTL for clinical mastitis and milk production. However, since only healthy animals were sampled for transcription and expression analyses, we could not draw any final conclusion on the absence of quantitative differences between animals with different genotypes. Future studies should investigate GC RNA expression and protein levels in cows with different genotypes during an infection.

The KRAS-Mutant Consensus Molecular Subtype 3 Reveals an Immunosuppressive Tumor Microenvironment in Colorectal Cancer.

Colorectal cancers (CRC) with KRAS mutations (KRASmut) are frequently included in consensus molecular subtype 3 (CMS3) with profound metabolic deregulation. We explored the transcriptomic impact of KRASmut, focusing on the tumor microenvironment (TME) and pathways beyond metabolic deregulation. The status of KRASmut in patients with CRC was investigated and overall survival (OS) was compared with wild-type KRAS (KRASwt). Next, we identified CMS, and further investigated differentially expressed genes (DEG) of KRASmut and distinctive pathways. Lastly, we used spatially resolved gene expression profiling to define the effect of KRASmut in the TME regions of CMS3-classified CRC tissues. CRC patients with KRASmut were mainly enriched in CMS3. Their specific enrichments of immune gene signatures in immunosuppressive TME were associated with worse OS. Activation of TGFß signaling by KRASmut was related to reduced pro-inflammatory and cytokine gene signatures, leading to suppression of immune infiltration. Digital spatial profiling in TME regions of KRASmut CMS3-classified tissues suggested up-regulated genes, CD40, CTLA4, ARG1, STAT3, IDO, and CD274, that could be characteristic of immune suppression in TME. This study may help to depict the complex transcriptomic profile of KRASmut in immunosuppressive TME. Future studies and clinical trials in CRC patients with KRASmut should consider these transcriptional landscapes.

Fat on plastic: Metabolic consequences of an LDPE diet in the fat body of the greater wax moth larvae (Galleria mellonella).

The caterpillar larvae of the greater wax moth (Galleria mellonella) are avid plastivores, as when provided a diet of low-density polyethylene (LDPE) they actively feed on it. Recent work has highlighted the importance of their microbiome in the putative biodegradation of this plastic polymer, though the impact of plastic metabolism on the insect host is less clear. In the present study, we undertook an integrative approach spanning all levels of biological organization to explore the effects of a plastic diet on the metabolic physiology of this animal model of plastic biodegradation. We demonstrate that an LDPE diet is not sufficient to maintain optimal larval growth and survival. In addition, we confirm that plastic fed waxworms retain their fat body lipid stores in a manner proportional to their individual polyethylene consumption suggesting a direct effect of LDPE biodegradation. At the functional level, the oxidative capacity of the fat body of LDPE-fed larvae is maintained reflecting unaltered metabolic function of the tissue. Finally, metabolomic analyses confirmed fat body lipid stores maintenance in LDPE-fed worms, but uncovered various other nutritional deficiencies. Overall, this work unveils novel insights in the complex interplay between LDPE biodegradation and the metabolic physiology of this model plastivore.

Recent and historical recombination in the admixed Norwegian Red cattle breed.

BACKGROUND: Comparison of recent patterns of recombination derived from linkage maps to historical patterns of recombination from linkage disequilibrium (LD) could help identify genomic regions affected by strong artificial selection, appearing as reduced recent recombination. Norwegian Red cattle (NRF) make an interesting case study for investigating these patterns as it is an admixed breed with an extensively recorded pedigree. NRF have been under strong artificial selection for traits such as milk and meat production, fertility and health. While measures of LD is also crucial for determining the number of markers required for association mapping studies, estimates of recombination rate can be used to assess quality of genomic assemblies. RESULTS: A dataset containing more than 17,000 genome-wide distributed SNPs and 2600 animals was used to assess recombination rates and LD in NRF. Although low LD measured by r2 was observed in NRF relative to some of the breeds from which this breed originates, reports from breeds other than those assessed in this study have described more rapid decline in r2 at short distances than what was found in NRF. Rate of decline in r2 for NRF suggested that to obtain an expected r2 between markers and a causal polymorphism of at least 0.5 for genome-wide association studies, approximately one SNP every 15 kb or a total of 200,000 SNPs would be required. For well known quantitative trait loci (QTLs) for milk production traits on Bos Taurus chromosomes 1, 6 and 20, map length based on historic recombination was greater than map length based on recent recombination in NRF. Further, positions for 130 previously unpositioned contigs from assembly of the bovine genome sequence (Btau_4.0) found using comparative sequence analysis were validated by linkage analysis, and 28% of these positions corresponded to extreme values of population recombination rate. CONCLUSION: While LD is reduced in NRF compared to some of the breeds from which this admixed breed originated, it is elevated over short distances compared to some other cattle breeds. Genomic regions in NRF where map length based on historic recombination was greater than map length based on recent recombination coincided with some well known QTL regions for milk production traits. Linkage analysis in combination with comparative sequence analysis and detection of regions with extreme values of population recombination rate proved to be valuable for detecting problematic regions in the Btau_4.0 genome assembly.

Molecular characterization of a long range haplotype affecting protein yield and mastitis susceptibility in Norwegian Red cattle.

BACKGROUND: Previous fine mapping studies in Norwegian Red cattle (NRC) in the region 86-90.4 Mb on Bos taurus chromosome 6 (BTA6) has revealed a quantitative trait locus (QTL) for protein yield (PY) around 88 Mb and a QTL for clinical mastitis (CM) around 90 Mb. The close proximity of these QTLs may partly explain the unfavorable genetic correlation between these two traits in NRC. A long range haplotype covering this region was introduced into the NRC population through the importation of a Holstein-Friesian bull (1606 Frasse) from Sweden in the 1970s. It has been suggested that this haplotype has a favorable effect on milk protein content but an unfavorable effect on mastitis susceptibility. Selective breeding for milk production traits is likely to have increased the frequency of this haplotype in the NRC population. RESULTS: Association mapping for PY and CM in NRC was performed using genotypes from 556 SNPs throughout the region 86-97 Mb on BTA6 and daughter-yield-deviations (DYDs) from 2601 bulls made available from the Norwegian dairy herd recording system. Highest test scores for PY were found for single-nucleotide polymorphisms (SNPs) within and surrounding the genes CSN2 and CSN1S2, coding for the ß-casein and α(S2)-casein proteins. High coverage re-sequencing by high throughput sequencing technology enabled molecular characterization of a long range haplotype from 1606 Frasse encompassing these two genes. Haplotype analysis of a large number of descendants from this bull indicated that the haplotype was not markedly disrupted by recombination in this region. The haplotype was associated with both increased milk protein content and increased susceptibility to mastitis, which might explain parts of the observed genetic correlation between PY and CM in NRC. Plausible causal polymorphisms affecting PY were detected in the promoter region and in the 5'-flanking UTR of CSN1S2. These polymorphisms could affect transcription or translation of CSN1S2 and thereby affect the amount of α(S2)-casein in milk. Highest test scores for CM were found in the region 89-91 Mb on BTA6, very close to a cluster of genes coding for CXC chemokines. Expression levels of some of these CXC chemokines have previously been shown to increase in bovine mammary gland cell lines after exposure to bacterial cell wall components. CONCLUSION: Molecular characterization of the long range haplotype from the Holstein-Friesian bull 1606 Frasse, imported into NRC in the 1970s, revealed polymorphisms that could affect transcription or translation of the casein gene CSN1S2. Sires with this haplotype had daughters with significantly elevated milk protein content and selection for milk production traits is likely to have increased the frequency of this haplotype in the NRC population. The haplotype was also associated with increased mastitis susceptibility, which might explain parts of the genetic correlation between PY and CM in NRC.

Improved dynamic range of protein quantification in silver-stained gels by modelling gel images over time.

Silver staining is a commonly used protein stain to visualise proteins separated by 2-DE. Despite this, the technique suffers from a limited dynamic range, making the simultaneous quantification of high- and low-abundant proteins difficult. In this paper we take advantage of the fact that silver staining is not an end-point stain by photographing the gels during development. This procedure provides information about the change in measured absorbance for each pixel in the protein spots on the gel. The maximum rate of change was found to be correlated with the amount of applied protein, providing a new way of estimating protein amount in 2-DE gels. We observed an improvement in the dynamic range of silver staining by up to two orders of magnitude.

Comparison of muscle proteome profiles in pure breeds of Norwegian Landrace and Duroc at three different ages.

We have used proteomics as a tool to unravel the changes in protein composition between two pure pig breeds and three age groups. Forty two female pigs of Norwegian Landrace and Duroc breed slaughtered at 6, 9 and 12 months age were included in the study. Each of the breeds was raised in separate farms and was slaughtered at the same day in a commercial abattoir. A sample from the adductor muscle was collected approximately 45min postmortem. Proteome analyses of the water soluble proteins using 2D electrophoresis showed that of the 1125 analyzed protein spots, 94 and 41 proteins are changed in abundance according to breed and age, respectively. A total of 63 changed proteins were identified by mass spectrometry. The identified proteins were classified as structural proteins, metabolic proteins, stress/defense proteins and other proteins. This demonstrates a difference in metabolism and muscle composition between breeds and age groups and shows that proteomics is a useful tool to uncover the molecular basis for physiological differences in muscles between pig breeds and age groups.

"Islands of Divergence" in the Atlantic Cod Genome Represent Polymorphic Chromosomal Rearrangements.

In several species genetic differentiation across environmental gradients or between geographically separate populations has been reported to center at "genomic islands of divergence," resulting in heterogeneous differentiation patterns across genomes. Here, genomic regions of elevated divergence were observed on three chromosomes of the highly mobile fish Atlantic cod (Gadus morhua) within geographically fine-scaled coastal areas. The "genomic islands" extended at least 5, 9.5, and 13 megabases on linkage groups 2, 7, and 12, respectively, and coincided with large blocks of linkage disequilibrium. For each of these three chromosomes, pairs of segregating, highly divergent alleles were identified, with little or no gene exchange between them. These patterns of recombination and divergence mirror genomic signatures previously described for large polymorphic inversions, which have been shown to repress recombination across extensive chromosomal segments. The lack of genetic exchange permits divergence between noninverted and inverted chromosomes in spite of gene flow. For the rearrangements on linkage groups 2 and 12, allelic frequency shifts between coastal and oceanic environments suggest a role in ecological adaptation, in agreement with recently reported associations between molecular variation within these genomic regions and temperature, oxygen, and salinity levels. Elevated genetic differentiation in these genomic regions has previously been described on both sides of the Atlantic Ocean, and we therefore suggest that these polymorphisms are involved in adaptive divergence across the species distributional range.