Alcohol Exposure Decreases CREB Binding Protein Expression and Histone Acetylation in the Developing Cerebellum

Fetal alcohol exposure affects 1 in 100 children making it the leading cause of mental retardation in the US. It has long been known that alcohol affects cerebellum development and function. However, the underlying molecular mechanism is unclear.We demonstrate that CREB binding protein (CBP) is widely expressed in granule and Purkinje neurons of the developing cerebellar cortex of naïve rats. We also show that exposure to ethanol during the 3(rd) trimester-equivalent of human pregnancy reduces CBP levels. CBP is a histone acetyltransferase, a component of the epigenetic mechanism controlling neuronal gene expression. We further demonstrate that the acetylation of both histone H3 and H4 is reduced in the cerebellum of ethanol-treated rats.These findings indicate that ethanol exposure decreases the expression and function of CBP in the developing cerebellum. This effect of ethanol may be responsible for the motor coordination deficits that characterize fetal alcohol spectrum disorders

Identification of an Immunogenic Mimic of a Conserved Epitope on the <i>Plasmodium falciparum</i> Blood Stage Antigen AMA1 Using Virus-Like Particle (VLP) Peptide Display

<div><p>We have developed a peptide display platform based on VLPs of the RNA bacteriophage MS2 that combines the high immunogenicity of VLP display with affinity selection capabilities. Random peptides can be displayed on the VLP surface by genetically inserting sequences into a surface-exposed loop of the viral coat protein. VLP-displayed peptides can then be isolated by selection using antibodies, and the VLP selectants can then be used directly as immunogens. Here, we investigated the ability of this platform to identify mimotopes of a highly conserved conformational epitope present on the <i>Plasmodium falciparum</i> blood-stage protein AMA1. Using 4G2, a monoclonal antibody that binds to this epitope and is a potent inhibitor of erythrocyte invasion, we screened three different VLP-peptide libraries and identified specific VLPs that bound strongly to the selecting mAb. We then tested the ability of a handful of selected VLPs to elicit anti-AMA1 antibody responses in mice. Most of the selected VLPs failed to reliably elicit AMA1 specific antibodies. However, one VLP consistently induced antibodies that cross-reacted with AMA1. Surprisingly, this VLP bound to 4G2 more weakly than the other selectants we identified. Taken together, these data demonstrate that VLP-peptide display can identify immunogenic mimics of a complex conformational epitope and illustrate the promise and challenges of this approach.</p></div

A subset of selectant VLPs elicit AMA1-reactive antibody responses.

<p>Groups of mice were immunized with VLPs displaying the listed peptide sequences. Each group consisted of three mice, except for group H, in which six mice were immunized. Anti-AMA1 IgG responses were measured by ELISA. Mean ODs and SEM are shown for each group.</p

Antibody responses in VLP-immunized mice boosted with recombinant AMA1.

<p>(A) Anti-peptide responses in responder mice are enhanced by boosting with 25μg recombinant rAMA1 (with incomplete Freund’s adjuvant). Mice were immunized with selectant VLPs and then boosted with recombinant AMA1. Mice were segregated into responders (red circles) and non-responders (blue circles). Also shown are the anti-peptide responses in mice immunized with selectant VLPs prior to boosting with rAMA1 (grey circles). As controls, groups of mice were immunized with wild-type MS2 VLPs (open circles) or recombinant AMA1 alone (black circles). Immunization with recombinant AMA1 boosts anti-peptide titers, but only in animals that had AMA1-cross-reactive titers present after VLP-selectant immunization. Mean and SEM are shown for each group. (B) Competition of immune sera with 4G2 for AMA1 binding. Sera from immunized mice were incubated with 250 ng rAMA1 on an ELISA plate in the presence of 100ng mAb 4G2. Serum was pooled from mice immunized with selectant VLPs boosted with rAMA1 (responders and non-responders), rAMA1 alone, or, as a control, wild-type MS2 VLPs. Data represent triplicate wells, normalized to the value of the no serum control. Mean OD₄₀₅ and SEM are shown at each dilution.</p

Relative binding of VLP affinity selectants to 4G2.

<p>ELISA plates were coated with 500ng of selected VLPs or rAMA1 and different amounts of 4G2 were applied. End-point dilution titers were determined as the reciprocal of the highest sera dilution with an OD greater than 2-fold higher than the no antibody control. The error bars represent the standard error of the mean of triplicate measurements.</p

Peptide families identified after 4G2 selection of a mixed VLP library.

<p>After 3 rounds of selection VLPs were deep sequenced. The top 1,450 unique peptide selectants were sorted into families using the online Immune Epitope Database analysis resource [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132560#pone.0132560.ref032" target="_blank">32</a>]. This analysis identified 10 peptide families with at least 10 members. This chart shows the consensus sequences and relative frequencies of each of these families.</p

Selectant VLPs elicit high-titer peptide-specific antibody responses but variable AMA1-reactive IgG titers.

<p>Groups of mice were immunized twice with 10mer selectant VLPs or wild-type MS2 VLPs. Serum was taken two-weeks following the boost. (A) Vaccine-elicited peptide titers were assessed by ELISA using recombinant PP7 bacteriophage VLPs displaying the NWDPTQFPGK peptide (in a surface-exposed AB loop similar to that of MS2) as the target antigen. (B) Antibody responses against native AMA1. In 3/10 vaccinated mice we observed low-titer cross-reactivity; these are referred to as “responders”. The remaining mice were characterized as “non-responders”. Mean OD₄₀₅ values are shown for each group. Error bars represent standard error of the mean, SEM.</p

Relative binding of selectant VLPs libraries was monitored by capture ELISA.

<p>Pooled crude VLP lysates, in duplicate, were applied to wells coated with 4G2 or an isotype control. VLP binding was detected using rabbit anti-MS2 polyclonal antibody. Relative binding was calculated by using the ratio of mean binding to 4G2 with the isotype control. The orange bar represents the final library from a selection using only a 10-mer insert VLP library (i.e. the library from which NWDPTQFPGT was identified). The pink bars represent all the stages of selection, including the original mutagenic library (round 0), using a VLP library with insert sequences randomized from the 10-mer NWDPTQFPGK selectant. The blue bars show the results of the mixed length VLP library selection. This experiment was repeated three times using different dilutions of the VLP libraries; similar ratios were observed in each experiment.</p

X Chromosome Contribution to the Genetic Architecture of Primary Biliary Cholangitis.

BACKGROUND & AIMS: Genome-wide association studies in primary biliary cholangitis (PBC) have failed to find X chromosome (chrX) variants associated with the disease. Here, we specifically explore the chrX contribution to PBC, a sexually dimorphic complex autoimmune disease. METHODS: We performed a chrX-wide association study, including genotype data from 5 genome-wide association studies (from Italy, United Kingdom, Canada, China, and Japan; 5244 case patients and 11,875 control individuals). RESULTS: Single-marker association analyses found approximately 100 loci displaying P < 5 × 10(-4), with the most significant being a signal within the OTUD5 gene (rs3027490; P = 4.80 × 10(-6); odds ratio [OR], 1.39; 95% confidence interval [CI], 1.028-1.88; Japanese cohort). Although the transethnic meta-analysis evidenced only a suggestive signal (rs2239452, mapping within the PIM2 gene; OR, 1.17; 95% CI, 1.09-1.26; P = 9.93 × 10(-8)), the population-specific meta-analysis showed a genome-wide significant locus in East Asian individuals pointing to the same region (rs7059064, mapping within the GRIPAP1 gene; P = 6.2 × 10(-9); OR, 1.33; 95% CI, 1.21-1.46). Indeed, rs7059064 tags a unique linkage disequilibrium block including 7 genes: TIMM17B, PQBP1, PIM2, SLC35A2, OTUD5, KCND1, and GRIPAP1, as well as a superenhancer (GH0XJ048933 within OTUD5) targeting all these genes. GH0XJ048933 is also predicted to target FOXP3, the main T-regulatory cell lineage specification factor. Consistently, OTUD5 and FOXP3 RNA levels were up-regulated in PBC case patients (1.75- and 1.64-fold, respectively). CONCLUSIONS: This work represents the first comprehensive study, to our knowledge, of the chrX contribution to the genetics of an autoimmune liver disease and shows a novel PBC-related genome-wide significant locus.The article is available via Open Access. Click on the 'Additional link' above to access the full-text.Published version, accepted versio