Linkage and segregation analysis of black and brindle coat color in domestic dogs

Mutations of pigment type switching have provided basic insight into melanocortin physiology and evolutionary adaptation. In all vertebrates that have been studied to date, two key genes, Agouti and Melanocortin 1 receptor (Mc1r), encode a ligand-receptor system that controls the switch between synthesis of red-yellow pheomelanin vs. black-brown eumelanin. However, in domestic dogs, historical studies based on pedigree and segregation analysis have suggested that the pigment type-switching system is more complicated and fundamentally different from other mammals. Using a genomewide linkage scan on a Labrador x greyhound cross segregating for black, yellow, and brindle coat colors, we demonstrate that pigment type switching is controlled by an additional gene, the K locus. Our results reveal three alleles with a dominance order of black (K(B)) > brindle (k(br)) > yellow (k(y)), whose genetic map position on dog chromosome 16 is distinct from the predicted location of other pigmentation genes. Interaction studies reveal that Mc1r is epistatic to variation at Agouti or K and that the epistatic relationship between Agouti and K depends on the alleles being tested. These findings suggest a molecular model for a new component of the melanocortin signaling pathway and reveal how coat-color patterns and pigmentary diversity have been shaped by recent selection.     

TYRP1 is associated with dun coat colour in Dexter cattle or how now brown cow?

Tyrosinase related protein 1 (TYRP1), which is involved in the coat colour pathway, was mapped to BTA8 between microsatellites BL1080 and BM4006, using a microsatellite in intron 5 of TYRP1. The complete coding sequence of bovine TYRP1 was determined from cDNA derived from skin biopsies of cattle with various colours. Sequence data from exons 2-8 from cattle with diluted phenotypes was compared with that from non-diluted phenotypes. In addition, full-sib families of beef cattle generated by embryo transfer and half-sib families from traditional matings in which coat colour was segregating were used to correlate TYRP1 sequence variants with dilute coat colours. Two non-conservative amino acid changes were detected in Simmental, Charolais and Galloway cattle but these polymorphisms were not associated with diluted shades of black or red, nor with the dun coat colour of Galloway cattle or the taupe brown colour of Braunvieh and Brown Swiss cattle. However, in Dexter cattle all 25 cattle with a dun brown coat colour were homozygous for a H424Y change. One Dexter that was also homozygous Y434 was red because of an "E+/E+" genotype at MC1R which lead to the production of only phaeomelanin. None of the 70 remaining black or red Dexter cattle were homozygous for Y434. This tyrosine mutation was not found in any of the 121 cattle of other breeds that were examined.     

<Emphasis Type="Italic">Burkholderia</Emphasis> Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

Background  

The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei.     

Bone formation rather than inflammation reflects Ankylosing Spondylitis activity on PET-CT: a pilot study

Introduction

Positron Emission Tomography - Computer Tomography (PET-CT) is an interesting imaging technique to visualize Ankylosing Spondylitis (AS) activity using specific PET tracers. Previous studies have shown that the PET tracers [¹⁸F]FDG and [¹¹C](R)PK11195 can target inflammation (synovitis) in rheumatoid arthritis (RA) and may therefore be useful in AS. Another interesting tracer for AS is [¹⁸F]Fluoride, which targets bone formation. In a pilot setting, the potential of PET-CT in imaging AS activity was tested using different tracers, with Magnetic Resonance Imaging (MRI) and conventional radiographs as reference.

Methods

In a stepwise approach different PET tracers were investigated. First, whole body [¹⁸F]FDG and [¹¹C](R)PK11195 PET-CT scans were obtained of ten AS patients fulfilling the modified New York criteria. According to the BASDAI five of these patients had low and five had high disease activity. Secondly, an extra PET-CT scan using [¹⁸F]Fluoride was made of two additional AS patients with high disease activity. MRI scans of the total spine and sacroiliac joints were performed, and conventional radiographs of the total spine and sacroiliac joints were available for all patients. Scans and radiographs were visually scored by two observers blinded for clinical data.

Results

No increased [¹⁸F]FDG and [¹¹C](R)PK11195 uptake was noticed on PET-CT scans of the first 10 patients. In contrast, MRI demonstrated a total of five bone edema lesions in three out of 10 patients. In the two additional AS patients scanned with [¹⁸F]Fluoride PET-CT, [¹⁸F]Fluoride depicted 17 regions with increased uptake in both vertebral column and sacroiliac joints. In contrast, [¹⁸F]FDG depicted only three lesions, with an uptake of five times lower compared to [¹⁸F]Fluoride, and again no [¹¹C](R)PK11195 positive lesions were found. In these two patients, MRI detected nine lesions and six out of nine matched with the anatomical position of [¹⁸F]Fluoride uptake. Conventional radiographs showed structural bony changes in 11 out of 17 [¹⁸F]Fluoride PET positive lesions.

Conclusions

Our PET-CT data suggest that AS activity is reflected by bone activity (formation) rather than inflammation. The results also show the potential value of PET-CT for imaging AS activity using the bone tracer [¹⁸F]Fluoride. In contrast to active RA, inflammation tracers [¹⁸F]FDG and [¹¹C](R)PK11195 appeared to be less useful for AS imaging.     

Gene mapping from a bovine 1;29 DNA library prepared with chromosome microdissection

Bovine gene mapping is progressing rapidly using syntenic group mapping based on somatic cell hybrids and linkage, and to a lesser extent on in situ hybridization. Single chromosome DNA libraries are a logical next step, and this was, therefore, the aim of our laboratory. Since we have access to several cattle with t(1;29) and this chromosome is readily distinguishable, we chose this as our first target—recognizing that we would not produce a single chromosome library in the strict sense because two autosomes are represented. We utilized an inverted microscope and a micromanipulator fitted with glass instruments pulled specifically to dissect off approximately 100 t(1:29) chromosomes per microdrop. A glass chamber made to accommodate a hanging drop was used to extract the DNA under a dissecting microscope. The DNA was then cleaved with EcoRI and inserted in gtwes arms. Host cells were then infected with these phage and positive clones obtained. The first clone, isolated from this library by hybridization with a human collagen 6A1 cDNA, was mapped by in situ hybridization to bovine Chromosome (Chr) 1q12–q14, near the centromere. The second clone, an anonymous DNA fragment (D1S11), was mapped to 1q43–q46, near the terminal end.     

Evolutionary dynamics of methicillin-resistant Staphylococcus aureus within a healthcare system

BackgroundIn the past decade, several countries have seen gradual replacement of endemic multi-resistant healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) with clones that are more susceptible to antibiotic treatment. One example is Singapore, where MRSA ST239, the dominant clone since molecular profiling of MRSA began in the mid-1980s, has been replaced by ST22 isolates belonging to EMRSA-15, a recently emerged pandemic lineage originating from Europe.ResultsWe investigated the population structure of MRSA in Singaporean hospitals spanning three decades, using whole genome sequencing. Applying Bayesian phylogenetic methods we report that prior to the introduction of ST22, the ST239 MRSA population in Singapore originated from multiple introductions from the surrounding region; it was frequently transferred within the healthcare system resulting in a heterogeneous hospital population. Following the introduction of ST22 around the beginning of the millennium, this clone spread rapidly through Singaporean hospitals, supplanting the endemic ST239 population. Coalescent analysis revealed that although the genetic diversity of ST239 initially decreased as ST22 became more dominant, from 2007 onwards the genetic diversity of ST239 began to increase once more, which was not associated with the emergence of a sub-clone of ST239. Comparative genomic analysis of the accessory genome of the extant ST239 population identified that the Arginine Catabolic Mobile Element arose multiple times, thereby introducing genes associated with enhanced skin colonization into this population.ConclusionsOur results clearly demonstrate that, alongside clinical practice and antibiotic usage, competition between clones also has an important role in driving the evolution of nosocomial pathogen populations.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0643-z) contains supplementary material, which is available to authorized users.     

Single-molecule sequencing reveals the molecular basis of multidrug-resistance in ST772 methicillin-resistant Staphylococcus aureus

Background

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-associated infection, but there is growing awareness of the emergence of multidrug-resistant lineages in community settings around the world. One such lineage is ST772-MRSA-V, which has disseminated globally and is increasingly prevalent in India. Here, we present the complete genome sequence of DAR4145, a strain of the ST772-MRSA-V lineage from India, and investigate its genomic characteristics in regards to antibiotic resistance and virulence factors.

Results

Sequencing using single-molecule real-time technology resulted in the assembly of a single continuous chromosomal sequence, which was error-corrected, annotated and compared to nine draft genome assemblies of ST772-MRSA-V from Australia, Malaysia and India. We discovered numerous and redundant resistance genes associated with mobile genetic elements (MGEs) and known core genome mutations that explain the highly antibiotic resistant phenotype of DAR4145. Staphylococcal toxins and superantigens, including the leukotoxin Panton-Valentinin Leukocidin, were predominantly associated with genomic islands and the phage φ-IND772PVL. Some of these mobile resistance and virulence factors were variably present in other strains of the ST772-MRSA-V lineage.

Conclusions

The genomic characteristics presented here emphasize the contribution of MGEs to the emergence of multidrug-resistant and highly virulent strains of community-associated MRSA. Antibiotic resistance was further augmented by chromosomal mutations and redundancy of resistance genes. The complete genome of DAR4145 provides a valuable resource for future investigations into the global dissemination and phylogeography of ST772-MRSA-V.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1599-9) contains supplementary material, which is available to authorized users.     

Global remodelling of cellular microenvironment due to loss of collagen VII

The mammalian cellular microenvironment is shaped by soluble factors and structural components, the extracellular matrix, providing physical support, regulating adhesion and signalling. A global, quantitative mass spectrometry strategy, combined with bioinformatics data processing, was developed to assess proteome differences in the microenvironment of primary human fibroblasts. We studied secreted proteins of fibroblasts from normal and pathologically altered skin and their post‐translational modifications. The influence of collagen VII, an important structural component, which is lost in genetic skin fragility, was used as model. Loss of collagen VII had a global impact on the cellular microenvironment and was associated with proteome alterations highly relevant for disease pathogenesis including decrease in basement membrane components, increase in dermal matrix proteins, TGF‐β and metalloproteases, but not higher protease activity. The definition of the proteome of fibroblast microenvironment and its plasticity in health and disease identified novel disease mechanisms and potential targets of intervention.     

Association of an Agouti allele with fawn or sable coat color in domestic dogs

The type of pigment synthesized in mammalian hair, yellow–red pheomelanin or black–brown eumelanin, depends on the interaction between Agouti protein and the Melanocortin 1 receptor. Although the genetics of pigmentation is broadly conserved across most mammalian species, pigment type-switching in domestic dogs is unusual because a yellow–tan coat with variable amounts of dark hair is thought to be caused by an allele of the Agouti locus referred to as fawn or sable (a^y). In a large survey covering thirty seven breeds, we identified an Agouti allele with two missense alterations, A82S and R83H, which was present (heterozygous or homozygous) in 41 dogs (22 breeds) with a fawn or sable coat, but was absent from 16 dogs (8 breeds) with a black-and-tan or tricolor phenotype. In an additional 33 dogs (14 breeds) with a eumelanic coat, 8 (German Shepherd Dogs, Groenendaels, Schipperkes, or Shetland Sheepdogs) were homozygous for a previously reported mutation, non-agouti R96C; the remainder are likely to have carried dominant black, which is independent of and epistatic to Agouti. This work resolves some of the complexity in dog coat color genetics and provides diagnostic opportunities and practical guidelines for breeders.