Transcription from a gene desert in a melanoma porcine model.

The genetic mechanisms underlying cutaneous melanoma onset and progression need to be further understood to improve patients' care. Several studies have focused on the genetic determinism of melanoma development in the MeLiM pig, a biomedical model of cutaneous melanoma. The objective of this study was to better describe the influence of a particular genomic region on melanoma progression in the MeliM model. Indeed, a large region of the Sus scrofa chromosome 1 has been identified by linkage and association analyses, but the causal mechanisms have remained elusive. To deepen the analysis of this candidate region, a dedicated SNP panel was used to fine map the locus, downsizing the interval to less than 2 Mb, in a genomic region located within a large gene desert. Transcription from this locus was addressed using a tiling array strategy and further validated by RT-PCR in a large panel of tissues. Overall, the gene desert showed an extensive transcriptional landscape, notably dominated by repeated element transcription in tumor and fetal tissues. The transcription of LINE-1 and PERVs has been confirmed in skin and tumor samples from MeLiM pigs. In conclusion, although this study still does not identify a candidate mutation for melanoma occurrence or progression, it highlights a potential role of repeated element transcriptional activity in the MeLiM model.

KIT and melanoma predisposition in pigs: sequence variants and association analysis.

KIT mutations have been detected in different cancer subtypes, including melanoma. The gene also has been extensively studied in farm animals for its prominent role in coat color. The present work aimed at detecting KIT variants in a porcine model of cutaneous melanoma, the melanoblastoma-bearing Libechov Minipig (MeLiM). By sequencing exons and intron borders, 36 SNPs and one indel were identified. Of 10 coding SNPs, three were non-synonymous mutations, likely to affect the protein conformation. A promising variant, located in exon 19 (p.Val870Ala), was genotyped in a MeLiM × Duroc cross, and an association analysis was conducted on several melanoma-related traits. This variant showed a significant association with melanoma development, tumor ulceration and cutaneous invasion. In conclusion, although the KIT gene would not be a major causal gene for melanoma development in pig, its genetic variation could be influencing this trait.

Transcription specificity of the class Ib genes SLA-6, SLA-7 and SLA-8 of the swine major histocompatibility complex and comparison with class Ia genes.

Our aim was to analyse the transcription levels of the three non-classical class Ib genes SLA-6, SLA-7 and SLA-8 of the swine major histocompatibility complex in various tissues and conditions and to compare them to the transcription levels of classical class Ia genes. Twenty-five adult tissues from two pig breeds, pig renal PK15 cells infected with the Pseudorabies virus, and peripheral blood mononuclear cells (PBMCs) stimulated by lipopolysaccharide or a mixture of phorbol myristate acetate and ionomycin were included in our study. Relative transcription was quantified by quantitative real-time PCR. On average, in adult tissues and PBMCs and compared to SLA-6, the transcription level of SLA-Ia genes was 100-1000 times higher, the level of SLA-8 was 10-20 times higher, and that of SLA-7 was five times higher. Thus, SLA-8 is the most transcribed SLA-Ib gene, followed by the SLA-7 and SLA-6 genes. The highest transcription levels of SLA-Ib transcripts were found in the lymphoid organs, followed by the lung and the digestive tract. The tissue variability of expression levels was widest for the SLA-6 gene, with a 1:32 ratio between the lowest and highest levels in contrast to a 1:12 ratio for the SLA-7 and SLA-8 genes and a 1:16 ratio for the SLA-Ia genes. During PK-15 infection and PBMC stimulation, SLA-Ia and SLA-8 genes were downregulated, whereas SLA-6 and SLA-7 were upregulated, downregulated or not significantly modified. Our overall results confirm the tissue-wide transcription of the three SLA-Ib genes and suggest that they have complementary roles.

RACK1 cooperates with NRASQ61K to promote melanoma in vivo.

Melanoma is the deadliest skin cancer. RACK1 (Receptor for activated protein kinase C) protein was proposed as a biological marker of melanoma in human and domestic animal species harboring spontaneous melanomas. As a scaffold protein, RACK1 is able to coordinate the interaction of key signaling molecules implicated in both physiological cellular functions and tumorigenesis. A role for RACK1 in rewiring ERK and JNK signaling pathways in melanoma cell lines had been proposed. Here, we used a genetic approach to test this hypothesis in vivo in the mouse. We show that Rack1 knock-down in the mouse melanoma cell line B16 reduces invasiveness and induces cell differentiation. We have developed the first mouse model for RACK1 gain of function, Tyr::Rack1-HA transgenic mice, targeting RACK1 to melanocytes in vivo. RACK1 overexpression was not sufficient to initiate melanomas despite activated ERK and AKT. However, in a context of melanoma predisposition, RACK1 overexpression reduced latency and increased incidence and metastatic rate. In primary melanoma cells from Tyr::Rack1-HA, Tyr::NRasQ61K mice, activated JNK (c-Jun N-terminal kinase) and activated STAT3 (signal transducer and activator of transcription 3) acted as RACK1 oncogenic partners in tumoral progression. A sequential and coordinated activation of ERK, JNK and STAT3 with RACK1 is shown to accelerate aggressive melanoma development in vivo.

Rapid Discovery of De Novo Deleterious Mutations in Cattle Enhances the Value of Livestock as Model Species.

In humans, the clinical and molecular characterization of sporadic syndromes is often hindered by the small number of patients and the difficulty in developing animal models for severe dominant conditions. Here we show that the availability of large data sets of whole-genome sequences, high-density SNP chip genotypes and extensive recording of phenotype offers an unprecedented opportunity to quickly dissect the genetic architecture of severe dominant conditions in livestock. We report on the identification of seven dominant de novo mutations in CHD7, COL1A1, COL2A1, COPA, and MITF and exploit the structure of cattle populations to describe their clinical consequences and map modifier loci. Moreover, we demonstrate that the emergence of recessive genetic defects can be monitored by detecting de novo deleterious mutations in the genome of bulls used for artificial insemination. These results demonstrate the attractiveness of cattle as a model species in the post genomic era, particularly to confirm the genetic aetiology of isolated clinical case reports in humans.

Gene expression of 3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase in relation to androstenone, testosterone, and estrone sulphate in gonadally intact male and castrated pigs.

Androstenone is one of the main compounds responsible for boar taint, and 3beta-hydroxysteroid dehydrogenase (3betaHSD) might be involved in its metabolism. In this study, the gene expression of 3betaHSD and 17beta-hydroxysteroid dehydrogenase (17betaHSD) were determined by real-time PCR analysis and related to the concentrations of androstenone, testosterone, and estrone sulphate (E1S). The experiments were performed on gonadally intact male pigs classified based on high or low fat androstenone concentrations, as predetermined by HPLC, as well as on immunocastrated and surgically castrated male pigs. The male pigs with high androstenone concentrations in fat had low 3betaHSD gene expression in liver and testis. Moreover, the 17betaHSD gene expression in liver, but not in testis, varied negatively with fat androstenone concentrations. Immunocastrated and surgically castrated male pigs had nondetectable concentrations of fat androstenone and plasma testosterone and E1S, and the castration procedure induced a significant increase of 3betaHSD and 17betaHSD gene expression. The mRNA expression was generally much greater from the 3betaHSD than from the 17betaHSD gene. Furthermore, fat androstenone was negatively correlated with liver 3betaHSD gene expression (Pearson correlation, r = -0.69; P < 0.05), and the 17betaHSD gene expression in liver was negatively correlated with plasma E1S (r = -0.95; P < 0.001), indicating an important role of liver 17betaHSD in the estrogen metabolism of gonadally intact male pigs. Another strong correlation was found between 3betaHSD and 17betaHSD gene expression in liver of the gonadally intact male pigs (r = 0.86; P < 0.01), possibly reflecting similar regulation mechanisms of these genes.

A matter of bacterial life and death.

Over 50 years ago, standard microbiological methods were established for determining whether bacterial cells were dead or alive. Recently there has been a flurry of reports suggesting that bacteria may exist in an eclipsed state, escaping detection by standard methods. Whether there really is such a state is of more than academic interest, considering the implications for public health. The ensuing debate has been unusually energetic for the normally cultured community of microbiologists.

Recovery of hydrogen peroxide-sensitive culturable cells of Vibrio vulnificus gives the appearance of resuscitation from a viable but nonculturable state.

The viabilities of five strains of Vibrio vulnificus were evaluated during the storage of the organisms in sterile seawater at 5 degrees C. The number of CFU was measured by plate count methods on rich media. The total cell numbers were determined by direct microscopic count methods. The titer of CFU declined logarithmically to undetectable levels over a period of 2 to 3 weeks, while the total cell numbers were unchanged. Midway through each study, higher culturable cell counts began to be observed on plates containing catalase or sodium pyruvate; during the latter stages of the study, the plate counts on such media were up to 1,000-fold higher than those on unsupplemented plates. Because autoclaving is known to generate hydrogen peroxide in rich media, and because catalase and sodium pyruvate are known to eliminate hydrogen peroxide, it appears that the conditions of the experiments led to the selection of a hydrogen peroxide-sensitive culturable cell subpopulation. At the time of the final stage of the decline in viability of each culture, hydrogen peroxide-sensitive cells were the only culturable cells present. Warming samples of the cultures to room temperature led to the growth of these residual culturable cells, utilizing nutrients provided by the nonculturable cells. The cells that grew recovered hydrogen peroxide resistance. When mixtures of culturable and nonculturable cells were diluted to the point where only nonculturable cells were present, or when the hydrogen peroxide-sensitive culturable cells had declined to undetectable levels, warming had no effect; no culturable cells were recovered. Warming has been reported to "resuscitate" nonculturable cells. Recognition of the existence of hydrogen peroxide-sensitive culturable cell populations, as well as their ability to grow to high levels in the warmed seawater microcosms, leads instead to the conclusion that while warming permits culturable cells to grow, it has no effect on nonculturable cells.