Initiator elements function to determine the activity state of BX-C enhancers

A >300 kb cis-regulatory region is required for the proper expression of the three bithorax complex (BX-C) homeotic genes. Based on genetic and transgenic analysis, a model has been proposed in which the numerous BX-C cis-regulatory elements are spatially restricted through the activation or repression of parasegment-specific chromatin domains. Particular early embryonic enhancers, called initiators, have been proposed to control this complex process. Here, in order to better understand the process of domain activation, we have undertaken a systematic in situ dissection of the iab-6 cis-regulatory domain using a new method, called InSIRT. Using this method, we create and genetically characterize mutations affecting iab-6 function, including mutations specifically modifying the iab-6 initiator. Through our mutagenesis of the iab-6 initiator, we provide strong evidence that initiators function not to directly control homeotic gene expression but rather as domain control centers to determine the activity state of the enhancers and silencers within a cis-regulatory domain.     

Human Direct Actions May Alter Animal Welfare,a Study on Horses (Equus caballus)

Background

Back pain is the cause of bad welfare in humans and animals. Although vertebral problems are regularly reported on riding horses, these problems are not always identified nor noticed enough to prevent these horses to be used for work.

Methodology/Principal Findings

Nineteen horses from two riding centres were submitted to chiropractic examinations performed by an experienced chiropractor and both horses'' and riders'' postures were observed during a riding lesson. The results show that 74% of horses were severely affected by vertebral problems, while only 26% were mildly or not affected. The degree of vertebral problems identified at rest was statistically correlated with horses'' attitudes at work (neck height and curve), and horses'' attitudes at work were clearly correlated with riders'' positions. Clear differences appeared between schools concerning both riders'' and horses'' postures, and the analysis of the teachers'' speech content and duration highlighted differences in the attention devoted to the riders'' position.

Conclusion/Significance

These findings are to our knowledge the first to underline the impact of riding on horses'' back problems and the importance of teaching proper balance to beginner riders in order to increase animals'' welfare.     

Phase 1 Trial of the Plasmodium falciparum Blood Stage Vaccine MSP142-C1/Alhydrogel with and without CPG 7909 in Malaria Na?ve Adults

Background

Merozoite surface protein 1₄₂ (MSP1₄₂) is a leading blood stage malaria vaccine candidate. In order to induce immune responses that cover the major antigenic polymorphisms, FVO and 3D7 recombinant proteins of MSP1₄₂ were mixed (MSP1₄₂-C1). To improve the level of antibody response, MSP1₄₂-C1 was formulated with Alhydrogel plus the novel adjuvant CPG 7909.

Methods

A Phase 1 clinical trial was conducted in healthy malaria-naïve adults at the Center for Immunization Research in Washington, D.C., to evaluate the safety and immunogenicity of MSP1₄₂-C1/Alhydrogel +/− CPG 7909. Sixty volunteers were enrolled in dose escalating cohorts and randomized to receive three vaccinations of either 40 or 160 µg protein adsorbed to Alhydrogel +/− 560 µg CPG 7909 at 0, 1 and 2 months.

Results

Vaccinations were well tolerated, with only one related adverse event graded as severe (Grade 3 injection site erythema) and all other vaccine related adverse events graded as either mild or moderate. Local adverse events were more frequent and severe in the groups receiving CPG. The addition of CPG enhanced anti-MSP1₄₂ antibody responses following vaccination by up to 49-fold two weeks after second immunization and 8-fold two weeks after the third immunization when compared to MSP1₄₂-C1/Alhydrogel alone (p<0.0001). After the third immunization, functionality of the antibody was tested by an in vitro growth inhibition assay. Inhibition was a function of antibody titer, with an average of 3% (range −2 to 10%) in the non CPG groups versus 14% (3 to 32%) in the CPG groups.

Conclusion/Significance

The favorable safety profile and high antibody responses induced with MSP1₄₂-C1/Alhydrogel + CPG 7909 are encouraging. MSP1₄₂-C1/Alhydrogel is being combined with other blood stage antigens and will be taken forward in a formulation adjuvanted with CPG 7909.

Trial Registration

ClinicalTrials.gov Identifier: {"type":"clinical-trial","attrs":{"text":"NCT00320658","term_id":"NCT00320658"}}NCT00320658     

Mutations in MAP3K1 cause 46,XY disorders of sex development and implicate a common signal transduction pathway in human testis determination

Investigations of humans with disorders of sex development (DSDs) resulted in the discovery of many of the now-known mammalian sex-determining genes, including SRY, RSPO1, SOX9, NR5A1, WT1, NR0B1, and WNT4. Here, the locus for an autosomal sex-determining gene was mapped via linkage analysis in two families with 46,XY DSD to the long arm of chromosome 5 with a combined, multipoint parametric LOD score of 6.21. A splice-acceptor mutation (c.634-8T>A) in MAP3K1 segregated with the phenotype in the first family and disrupted RNA splicing. Mutations were demonstrated in the second family (p.Gly616Arg) and in two of 11 sporadic cases (p.Leu189Pro, p.Leu189Arg)-18% prevalence in this cohort of sporadic cases. In cultured primary lymphoblastoid cells from family 1 and the two sporadic cases, these mutations altered the phosphorylation of the downstream targets, p38 and ERK1/2, and enhanced binding of RHOA to the MAP3K1 complex. Map3k1 within the syntenic region was expressed in the embryonic mouse gonad prior to, and after, sex determination. Thus, mutations in MAP3K1 that result in 46,XY DSD with partial or complete gonadal dysgenesis implicate this pathway in normal human sex determination.     

Sumoylation of Drosophila SU(VAR)3-7 is required for its heterochromatic function

In Drosophila, SU(VAR)3-7 is an essential heterochromatin component. It is required for proper chromatin condensation, and changing its dose modifies position-effect variegation. Sumoylation is a post-translational modification shown to play a role in diverse biological processes. Here, we demonstrate that sumoylation is essential for proper heterochromatin function in Drosophila through modification of SU(VAR)3-7. Indeed, SU(VAR)3-7 is sumoylated at lysine K839; this modification is required for localization of SU(VAR)3-7 at pericentric heterochromatin, chromosome 4, and telomeres. In addition, sumoylation of SU(VAR)3-7 is a prerequisite for its ability to enhance position-effect variegation. Thus, these results show that the heterochromatic function of SU(VAR)3-7 depends on its own sumoylation, and unveil a role for sumoylation in Drosophila heterochromatin.     

Synthesis of novel cyclic NGR/RGD peptide analogs via on resin click chemistry

Targeted drug deliveries as well as high resolution imaging of cancerous tissues and organs via specific cancer cell markers have become important in chemotherapeutic interventions of cancer treatment. Short peptides such as RGD and NGR are showing promising results for targeted drug delivery and in vivo imaging. We have applied on resin Huisgen's 1,3-dipolar cycloaddition to synthesize new cyclic RGD and NGR peptide analogs. Preliminary binding assays of these new analogs by fluorescence polarization indicates specific binding to purified CD13 (Aminopeptidase N) and cell lysates from MCF-7 and SKOV-3 cancer cell lines.     

Biphasic effect of IL-1beta on the activity of argininosuccinate synthetase in Caco-2 cells. Involvement of nitric oxide production

The expression of the argininosuccinate synthetase gene (ASS), the limiting enzyme of arginine synthesis, was previously shown to be rapidly induced by a short-term (4 h) exposure to IL-1beta in Caco-2 cells [Biochimie, 2005, 403-409]. The present report shows that, by contrast, a long-term (24 h) exposure to IL-1beta inhibited the ASS activity despite an increase in both specific mRNA level and protein amount, demonstrating a post-translational effect. Concerning the mechanism involved, we demonstrate that the inhibiting effect is linked to the production of nitric oxide (NO) induced by IL-1beta. Indeed, the inhibiting effect of IL-1beta was totally blocked in the presence of l-NMMA, an inhibitor of the inducible nitric oxide synthase, or by culturing the cells in an arginine-deprived medium. Moreover, a decrease in the ASS activity was induced by culturing the cells in the presence of SNAP, a NO donor. Conversely, blocking the action of NO by antioxidant agents, the stimulatory effect of IL-1beta on ASS activity was restored, as measured at 24 h. Finally, such an inhibiting effect of NO on ASS activity may be related, at least in part, to S-nitrosylation of the protein. The physiological relevance of the antagonistic effects of IL-1beta and NO on ASS is discussed.     

Nitrite and Nitrous Oxide Reductase Regulation by Nitrogen Oxides in Rhodobacter sphaeroides f. sp. denitrificans IL106

We have cloned the nap locus encoding the periplasmic nitrate reductase in Rhodobacter sphaeroides f. sp. denitrificans IL106. A mutant with this enzyme deleted is unable to grow under denitrifying conditions. Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N(2)O reductases is not increased by the addition of nitrate. Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase. This demonstrates that R. sphaeroides f. sp. denitrificans IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N(2)O reductases in this species. In contrast, we show that nitrite induces the synthesis of the nitrate reductase.