The CFTR Met 470 Allele Is Associated with Lower Birth Rates in Fertile Men from a Population Isolate

Although little is known about the role of the cystic fibrosis transmembrane regulator (CFTR) gene in reproductive physiology, numerous variants in this gene have been implicated in etiology of male infertility due to congenital bilateral absence of the vas deferens (CBAVD). Here, we studied the fertility effects of three CBAVD–associated CFTR polymorphisms, the (TG)m and polyT repeat polymorphisms in intron 8 and Met470Val in exon 10, in healthy men of European descent. Homozygosity for the Met470 allele was associated with lower birth rates, defined as the number of births per year of marriage (P = 0.0029). The Met470Val locus explained 4.36% of the phenotypic variance in birth rate, and men homozygous for the Met470 allele had 0.56 fewer children on average compared to Val470 carrier men. The derived Val470 allele occurs at high frequencies in non-African populations (allele frequency  = 0.51 in HapMap CEU), whereas it is very rare in African population (Fst  = 0.43 between HapMap CEU and YRI). In addition, haplotypes bearing Val470 show a lack of genetic diversity and are thus longer than haplotypes bearing Met470 (measured by an integrated haplotype score [iHS] of −1.93 in HapMap CEU). The fraction of SNPs in the HapMap Phase2 data set with more extreme Fst and iHS measures is 0.003, consistent with a selective sweep outside of Africa. The fertility advantage conferred by Val470 relative to Met470 may provide a selective mechanism for these population genetic observations.     

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.     

Polymers for improving the in vivo transduction efficiency of AAV2 vectors

Background

Adeno-associated virus has attracted great attention as vehicle for body-wide gene delivery. However, for the successful treatment of a disease such as Duchenne muscular dystrophy infusion of very large amounts of vectors is required. This not only raises questions about the technical feasibility of the large scale production but also about the overall safety of the approach. One way to overcome these problems would be to find strategies able to increase the in vivo efficiency.

Methodology

Here, we investigated whether polymers can act as adjuvants to increase the in vivo efficiency of AAV2. Our strategy consisted in the pre-injection of polymers before intravenous administration of mice with AAV2 encoding a murine secreted alkaline phosphatase (mSeAP). The transgene expression, vector biodistribution and tissue transduction were studied by quantification of the mSeAP protein and real time PCR. The injection of polyinosinic acid and polylysine resulted in an increase of plasmatic mSeAP of 2- and 12-fold, respectively. Interestingly, polyinosinic acid pre-injection significantly reduced the neutralizing antibody titer raised against AAV2.

Conclusions

Our results show that the pre-injection of polymers can improve the overall transduction efficiency of systemically administered AAV2 and reduce the humoral response against the capsid proteins.     

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     

Biochemical Basis of the Interaction between Cystic Fibrosis Transmembrane Conductance Regulator and Immunoglobulin-like Repeats of Filamin

Mutations in the chloride channel cystic fibrosis transmembrane regulator (CFTR) cause cystic fibrosis, a genetic disorder characterized by defects in CFTR biosynthesis, localization to the cell surface, or activation by regulatory factors. It was discovered recently that surface localization of CFTR is stabilized by an interaction between the CFTR N terminus and the multidomain cytoskeletal protein filamin. The details of the CFTR-filamin interaction, however, are unclear. Using x-ray crystallography, we show how the CFTR N terminus binds to immunoglobulin-like repeat 21 of filamin A (FlnA-Ig21). CFTR binds to β-strands C and D of FlnA-Ig21 using backbone-backbone hydrogen bonds, a linchpin serine residue, and hydrophobic side-chain packing. We use NMR to determine that the CFTR N terminus also binds to several other immunoglobulin-like repeats from filamin A in vitro. Our structural data explain why the cystic fibrosis-causing S13F mutation disrupts CFTR-filamin interaction. We show that FlnA-Ig repeats transfected into cultured Calu-3 cells disrupt CFTR-filamin interaction and reduce surface levels of CFTR. Our findings suggest that filamin A stabilizes surface CFTR by anchoring it to the actin cytoskeleton through interactions with multiple filamin Ig repeats. Such an interaction mode may allow filamins to cluster multiple CFTR molecules and to promote colocalization of CFTR and other filamin-binding proteins in the apical plasma membrane of epithelial cells.     

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.     

An in silico platform for the study of epithelial pre-invasive neoplastic development

To study the possible mechanisms of epithelial preneoplastic development we developed an integrated platform using a 3D agent-based model (ABM). This platform, named idefics, allows for the implementation of normal biological rules at the cell level that interact and generate the usual homeostatic equilibrium of epithelium as well as other abnormal processes that can disrupt this equilibrium. The structure of this model is based on data from multi-scale quantitative analysis of a unique collection of preneoplastic lesions of the lung, cervix, and oral epithelium, that have been collected in our lab in the last 25 years. In this paper, we are describing the different components of this platform and will present results from different simulations generated by the model.     

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.