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.     

Immunogenicity of a promiscuous T cell epitope peptide based conjugate vaccine against benzo[a]pyrene: redirecting antibodies to the hapten

The prototype polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) is an environmental pollutant and food contaminant of epidemiological importance. To protect against adverse effects of this ubiquitous carcinogen, we developed an immunoprophylactic strategy based on a B[a]P-protein conjugate vaccine to induce B[a]P specific antibodies (Grova et al., Vaccine. 2009;27:4142-51). Here, we investigated in mice the efficacy of B[a]P-peptide conjugates based on promiscuous T cell epitopes (TCE) into further improve this approach. We showed that B[a]P-peptide conjugates induced very different levels of hapten-specific antibodies with variable functional efficacy, depending on the carrier. In some cases peptide carriers induced a more efficient antibody response against B[a]P than tetanus toxoid as a protein carrier, with the capacity to sequester more B[a]P in the blood. Reducing the carrier size to a single TCE can dramatically shift the antibody bias from the carrier to the B[a]P. Conjugates based on the TCE FIGITEL induced the best anti-hapten response and no antibodies against the carrier peptide. Some peptide conjugates increased the selectivity of the antibodies for the activated metabolite 7,8-diol-B[a]P and B[a]P by one or two orders of magnitude. The antibody efficacy was also demonstrated in their ability to sequester B[a]P in the blood and modulate its faecal excretion (15-56%). We further showed that pre-existing immunity to the carrier from which the TCE was derived did not reduce the immunogenicity of the peptide conjugate. In conclusion, we showed that a vaccination against B[a]P using promiscuous TCEs of tetanus toxin as carriers is feasible even in case of a pre-existing immunity to the toxoid and that some TCE epitopes dramatically redirect the antibody response to the hapten. Further studies to demonstrate a long-term protection of an immunoprophylactic immunisation against B[a]P are warranted.     

Transorbital Sonographic Evaluation of Normal Optic Nerve Sheath Diameter in Healthy Volunteers in Bangladesh

Introduction

Measurement of optic nerve sheath diameter (ONSD) by ultrasound is increasingly used as a marker to detect raised intracranial pressure (ICP). ONSD varies with age and there is no clear consensus between studies for an upper limit of normal. Knowledge of normal ONSD in a healthy population is essential to interpret this measurement.

Methods

In a prospective observational study, ONSD was measured using a 15 MHz ultrasound probe in healthy volunteers in Chittagong, Bangladesh. The aims were to determine the normal range of ONSD in healthy Bangladeshi adults and children, compare measurements in males and females, horizontal and vertical beam orientations and left and right eyes in the same individual and to determine whether ONSD varies with head circumference independent of age.

Results

136 subjects were enrolled, 12.5% of whom were age 16 or under. Median ONSD was 4.41 mm with 95% of subjects in the range 4.25–4.75 mm. ONSD was bimodally distributed. There was no relationship between ONSD and age (≥4 years), gender, head circumference, and no difference in left vs right eye or horizontal vs vertical beam.

Conclusions

Ultrasonographic ONSD in Bangladeshi healthy volunteers has a narrow bimodal distribution independent of age (≥4 years), gender and head circumference. ONSD >4.75 mm in this population should be considered abnormal.     

N-cadherin/p120 Catenin Association at Cell-Cell Contacts Occurs in Cholesterol-rich Membrane Domains and Is Required for RhoA Activation and Myogenesis

p120 catenin is a major regulator of cadherin stability at cell-cell contacts and a modulator of Rho GTPase activities. In C2C12 myoblasts, N-cadherin is stabilized at cell contacts through its association with cholesterol-rich membrane domains or lipid rafts (LR) and acts as an adhesion-activated receptor that activates RhoA, an event required for myogenesis induction. Here, we report that association of p120 catenin with N-cadherin at cell contacts occurs specifically in LR. We demonstrate that interaction of p120 catenin with N-cadherin is required for N-cadherin association with LR and for its stabilization at cell contacts. LR disruption inhibits myogenesis induction and N-cadherin-dependent RhoA activation as does the perturbation of the N-cadherin-p120 catenin complex after p120 catenin knockdown. Finally, we observe an N-cadherin-dependent accumulation of RhoA at phosphatidylinositol 4,5-bisphosphate-enriched cell contacts which is lost after LR disruption. Thus, a functional N-cadherin-catenin complex occurs in cholesterol-rich membrane microdomains which allows the recruitment of RhoA and the regulation of its activity during myogenesis induction.Skeletal myogenesis is a multistep process regulated by diffusible molecules and the interaction of muscle cell precursors with their neighbors and the extracellular matrix (1, 2). Particularly, N-cadherin-dependent intercellular adhesion has a major role in cell cycle exit and induction of skeletal muscle differentiation through activation of the Rho family GTPases. RhoA positively regulates MyoD expression and skeletal muscle differentiation because it is required for serum response factor-mediated activation of several muscle-specific gene promoters (3, 4).Dynamic association of cadherin complexes at the plasma membrane (PM)⁴ is crucial for cadherin-mediated signaling. Their extracellular domain mediates homophilic cell-cell adhesion, whereas the intracellular domain associates with catenins that produce attachment sites for the F-actin cytoskeleton (5–7). The juxtamembrane domain of the cadherin cytoplasmic tail binds to p120 catenin, which regulates cadherin stability at cell contacts and modulates Rho GTPase activities (8–11). Cadherin stability is directly dependent on p120 catenin, and in its absence most cadherins are internalized and often degraded, suggesting that p120 catenin controls cadherin turnover at the cell surface (11, 12). Moreover, mutations in the E-cadherin region that bind to p120 catenin dissociate the E-cadherin-p120 catenin complex and disrupt strong cell adhesion, although interaction with other catenins remains intact (13). Cadherin stability at cell-cell contacts is also regulated by homophilic binding between extracellular domains and association with the F-actin cytoskeleton (14, 15). Association of N-cadherin with cholesterol-enriched microdomains, called lipid rafts (LR), at cell contacts, also stabilizes N-cadherin (16). Because p120 catenin interaction with cadherins and N-cadherin association with LR at cell contact sites are both involved in cadherin stability at cell contact sites, we asked whether p120 catenin association with N-cadherin required LR. We observed that their association occurred specifically in these cholesterol-rich domains. Moreover, using an N-cadherin mutant unable to bind to p120 catenin, we showed that the N-cadherin/p120 catenin interaction was required for N-cadherin association with LR and its stabilization at cell contacts. Because N-cadherin is implicated in the commitment to myogenesis through RhoA activation, we questioned whether its association with p120 catenin in LR was a prerequisite for RhoA activation. LR disruption inhibited myogenesis induction, association of p120 catenin with N-cadherin, and N-cadherin-dependent RhoA activation, as did the perturbation of the N-cadherin-p120 catenin complex after p120 catenin knockdown. Together, these data suggest a crucial role for the N-cadherin/p120 catenin association in LR in the regulation of RhoA activity during myogenesis induction.     

Organization and evolution of transposable elements along the bread wheat chromosome 3B

Background

The 17 Gb bread wheat genome has massively expanded through the proliferation of transposable elements (TEs) and two recent rounds of polyploidization. The assembly of a 774 Mb reference sequence of wheat chromosome 3B provided us with the opportunity to explore the impact of TEs on the complex wheat genome structure and evolution at a resolution and scale not reached so far.

Results

We develop an automated workflow, CLARI-TE, for TE modeling in complex genomes. We delineate precisely 56,488 intact and 196,391 fragmented TEs along the 3B pseudomolecule, accounting for 85% of the sequence, and reconstruct 30,199 nested insertions. TEs have been mostly silent for the last one million years, and the 3B chromosome has been shaped by a succession of bursts that occurred between 1 to 3 million years ago. Accelerated TE elimination in the high-recombination distal regions is a driving force towards chromosome partitioning. CACTAs overrepresented in the high-recombination distal regions are significantly associated with recently duplicated genes. In addition, we identify 140 CACTA-mediated gene capture events with 17 genes potentially created by exon shuffling and show that 19 captured genes are transcribed and under selection pressure, suggesting the important role of CACTAs in the recent wheat adaptation.

Conclusion

Accurate TE modeling uncovers the dynamics of TEs in a highly complex and polyploid genome. It provides novel insights into chromosome partitioning and highlights the role of CACTA transposons in the high level of gene duplication in wheat.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0546-4) contains supplementary material, which is available to authorized users.     

The Calpain/Calpastatin System Has Opposing Roles in Growth and Metastatic Dissemination of Melanoma

Conventional calpains are ubiquitous cysteine proteases whose activity is promoted by calcium signaling and specifically limited by calpastatin. Calpain expression has been shown to be increased in human malignant cells, but the contribution of the calpain/calpastatin system in tumorigenesis remains unclear. It may play an important role in tumor cells themselves (cell growth, migration, and a contrario cell death) and/or in tumor niche (tissue infiltration by immune cells, neo-angiogenesis). In this study, we have used a mouse model of melanoma as a tool to gain further understanding of the role of calpains in tumor progression. To determine the respective importance of each target, we overexpressed calpastatin in tumor and/or host in isolation. Our data demonstrate that calpain inhibition in both tumor and host blunts tumor growth, while paradoxically increasing metastatic dissemination to regional lymph nodes. Specifically, calpain inhibition in melanoma cells limits tumor growth in vitro and in vivo but increases dissemination by amplifying cell resistance to apoptosis and accelerating migration process. Meanwhile, calpain inhibition restricted to host cells blunts tumor infiltration by immune cells and angiogenesis required for antitumor immunity, allowing tumor cells to escape tumor niche and disseminate. The development of highly specific calpain inhibitors with potential medical applications in cancer should take into account the opposing roles of the calpain/calpastatin system in initial tumor growth and subsequent metastatic dissemination.     

MyoD, Myogenin, and Desmin-nls-lacZ Transgene Emphasize the Distinct Patterns of Satellite Cell Activation in Growth and Regeneration

Although satellite cell differentiation is involved in postnatal myogenesis from growth to posttrauma regeneration, the early stages of this process remain unclear. This study investigatedpHuDes-nls-lacZtransgene activity, as revealed by X-gal staining and the accumulation of MyoD, myogenin, endogenous desmin, and myosin, in order to determine whether satellite cells share the same activation program during growth and regeneration. After birth, skeletal myonuclei in which myogenin expression was limited were briefly characterized by transgene activity. Satellite cells were only evidenced by MyoD and slow myosin accumulation, but failed to initiate transgene expression. After freeze trauma, satellite cell activation led to MyoD, myogenin, and desmin expression. Subsequently, when myosin expression occurred, transgene activation was apparent in regenerating structures, with more intense X-gal staining in mononucleated cells than regenerating myotubes. After the second week posttrauma, only desmin and myogenin expression were maintained in regenerating structures. In culture, the behavior of satellite cells showed that desmin expression was committed before transgene activation occurred, i.e., concurrently with MyoD, myogenin, myosin expression, and the first fusion events. Quantitative analysis confirmed the discrepancy between endogenous desmin and transgene expression and demonstrated the close correlation between transgene activation and the fusion index. Our results strongly suggest that satellite cells promote distinct pathways of myogenic response during growth and regeneration.