Facioscapulohumeral Disease (FSHD) is a dominantly inherited progressive myopathy associated with aberrant production of the transcription factor, Double Homeobox Protein 4 (DUX4). The expression of DUX4 depends on an open chromatin conformation of the D4Z4 macrosatellite array and a specific haplotype on chromosome 4. Even when these requirements are met, DUX4 transcripts and protein are only detectable in a subset of cells indicating that additional constraints govern DUX4 production. Since the direction of transcription, along with the production of non-coding antisense transcripts is an important regulatory feature of other macrosatellite repeats, we developed constructs that contain the non-coding region of a single D4Z4 unit flanked by genes that report transcriptional activity in the sense and antisense directions. We found that D4Z4 contains two promoters that initiate sense and antisense transcription within the array, and that antisense transcription predominates. Transcriptional start sites for the antisense transcripts, as well as D4Z4 regions that regulate the balance of sense and antisense transcripts were identified. We show that the choice of transcriptional direction is reversible but not mutually exclusive, since sense and antisense reporter activity was often present in the same cell and simultaneously upregulated during myotube formation. Similarly, levels of endogenous sense and antisense D4Z4 transcripts were upregulated in FSHD myotubes. These studies offer insight into the autonomous distribution of muscle weakness that is characteristic of FSHD.

CTCF is a highly conserved, multifunctional zinc finger protein involved in critical aspects of gene regulation including transcription regulation, chromatin insulation, genomic imprinting, X-chromosome inactivation, and higher order chromatin organization. Such multifunctional properties of CTCF suggest an essential role in development. Indeed, a previous report on maternal depletion of CTCF suggested that CTCF is essential for pre-implantation development. To distinguish between the effects of maternal and zygotic expression of CTCF, we studied pre-implantation development in mice harboring a complete loss of function Ctcf knockout allele. Although we demonstrated that homozygous deletion of Ctcf is early embryonically lethal, in contrast to previous observations, we showed that the Ctcf nullizygous embryos developed up to the blastocyst stage (E3.5) followed by peri-implantation lethality (E4.5-E5.5). Moreover, one-cell stage Ctcf nullizygous embryos cultured ex vivo developed to the 16-32 cell stage with no obvious abnormalities. Using a single embryo assay that allowed both genotype and mRNA expression analyses of the same embryo, we demonstrated that pre-implantation development of the Ctcf nullizygous embryos was associated with the retention of the maternal wild type Ctcf mRNA. Loss of this stable maternal transcript was temporally associated with loss of CTCF protein expression, apoptosis of the developing embryo, and failure to further develop an inner cell mass and trophoectoderm ex vivo. This indicates that CTCF expression is critical to early embryogenesis and loss of its expression rapidly leads to apoptosis at a very early developmental stage. This is the first study documenting the presence of the stable maternal Ctcf transcript in the blastocyst stage embryos. Furthermore, in the presence of maternal CTCF, zygotic CTCF expression does not seem to be required for pre-implantation development.

Obtaining NMR assignments for slowly tumbling molecules such as detergent-solubilized membrane proteins is often compromised by low sensitivity as well as spectral overlap. Both problems can be addressed by amino-acid specific isotope labeling in conjunction with (15)N-(1)H correlation experiments. In this work an extended combinatorial selective in vitro labeling scheme is proposed that seeks to reduce the number of samples required for assignment. Including three different species of amino acids in each sample,(15)N, 1-(13)C, and fully (13)C/(15)N labeled, permits identification of more amino acid types and sequential pairs than would be possible with previously published combinatorial methods. The new protocol involves recording of up to five 2D triple-resonance experiments to distinguish the various isotopomeric dipeptide species. The pattern of backbone NH cross peaks in this series of spectra adds a new dimension to the combinatorial grid, which otherwise mostly relies on comparison of [(15)N,(1)H]-HSQC and possibly 2D HN(CO) spectra of samples with different labeled amino acid compositions. Application to two α-helical membrane proteins shows that using no more than three samples information can be accumulated such that backbone assignments can be completed solely based on 3D HNCA/HN(CO)CA experiments. Alternatively, in the case of severe signal overlap in certain regions of the standard suite of triple-resonance spectra acquired on uniformly labeled protein, or missing signals due to a lack of efficiency of 3D experiments, the remaining gaps can be filled.

The molecular mechanisms that underlie poor birth outcomes in malaria during pregnancy remain poorly defined. To assess the role of host immune responses, mice known to respond differentially to Plasmodium chabaudi AS infection were studied. Following infection at day 0 of pregnancy, A/J mice developed significantly higher parasitemia than C57BL/6 (B6) mice and succumbed to infection. Both strains had evidence of parasite accumulation in the placenta at mid-gestation and aborted, with significantly higher embryo loss in infected A/J mice on day 9. While infection-induced systemic tumour necrosis factor (TNF) and interleukin (IL)-1β in the latter were significantly higher at day 11, day 10 IL-10 levels were higher in B6 mice. No differences in the levels of splenic lymphocyte subsets, neutrophils or monocytes between infected pregnant A/J and B6 mice were observed, with most cell types expanding in response to infection regardless of pregnancy. Antibody ablation of TNF exacerbated infection in A/J mice and did not ameliorate pregnancy outcome. Thus, malaria induces poor pregnancy outcome in both the mouse strains in the context of quantitatively different systemic inflammatory responses. Further evaluation of the roles of soluble and cellular immune components, particularly at the uteroplacental level, will be required to define the most critical pregnancy-compromising mechanisms.

An efficacious Chlamydia vaccine is urgently needed to control Chlamydia infections. Heterologous prime-boost vaccination regimens are emerging as a promising strategy for preventing intracellular viral and bacterial infections. However, it remains to be determined if this regimen would be a feasible and effective approach for Chlamydia infection. In this study, we examined the immune response and the protective efficacy induced by various vaccination regimens using a recombinant adenovirus vector expressing the Chlamydia antigen CPAF (AdCPAF) and recombinant CPAF (rCPAF) subunit vaccines formulated with CpG oligodeoxynucleotides and/or a synthetic immunomodulatory peptide HH2 as adjuvants. A single dose of AdCPAF stimulated potent antibody production but weak cellular immune responses in mice. A booster rCPAF vaccine formulated with both CpG and HH2, but not CpG alone or HH2 alone, showed robust adjuvant effects on induction of Th1-biased cellular immune responses in mice primed with AdCPAF. In contrast, a homologous regimen using rCPAF/CpG/HH2 subunit vaccine for both priming and boosting induced a weak antibody response, but potent cellular immunity with a mixed Th1/Th17 profile. Despite the disparities observed in humoral and cellular immune responses, both the heterologous and homologous prime-boost regimens conferred significant immune protection against genital Chlamydia muridarum challenge in C3H/HeN and BALB/c mice.

Behavioral specializations are frequently associated with expansions of the brain regions controlling them. This principle of proper mass spans sensory, motor, and cognitive abilities and has been observed in a wide variety of vertebrate species. Yet, it is unknown if this concept extrapolates to entire neural pathways or how selection on a behavioral capacity might otherwise shape circuit structure. We investigate these questions by comparing the songs and neuroanatomy of 49 species from 17 families of songbirds, which vary immensely in the number of unique song components they produce and possess a conserved neural network dedicated to this behavior. We find that syllable repertoire size is strongly related to the degree of song motor pathway convergence. Repertoire size is more accurately predicted by the number of neurons in higher motor areas relative to that in their downstream targets than by the overall number of neurons in the song motor pathway. Additionally, the convergence values along serial premotor and primary motor projections account for distinct portions of the behavioral variation. These findings suggest that selection on song has independently shaped different components of this hierarchical pathway, and they elucidate how changes in pathway structure could have underlain elaborations of this learned motor behavior.

Copy-number variations (CNVs) constitute very common differences between individual humans and possibly all genomes and may therefore be important fuel for evolution, yet how they form remains elusive. In starving Escherichia coli, gene amplification is induced by stress, controlled by the general stress response. Amplification has been detected only encompassing genes that confer a growth advantage when amplified. We studied the structure of stress-induced gene amplification in starving cells in the Lac assay in Escherichia coli by array comparative genomic hybridization (aCGH), with polymerase chain reaction (pcr) and DNA sequencing to establish the structures generated. About 10% of 300 amplified isolates carried other chromosomal structural change in addition to amplification. Most of these were inversions and duplications associated with the amplification event. This complexity supports a mechanism similar to that seen in human non-recurrent copy number variants. We interpret these complex events in terms of repeated template switching during DNA replication. Importantly, we found a significant occurrence (6 out of 300) of chromosomal structural changes that were apparently not involved in the amplification event. These secondary changes were absent from 240 samples derived from starved cells not carrying amplification, suggesting that amplification happens in a differentiated subpopulation of stressed cells licensed for global chromosomal structural change and genomic instability. These data imply that chromosomal structural changes occur in bursts or showers of instability that may have the potential to drive rapid evolution.

In plants, gametes, along with accessory cells, are formed by the haploid gametophytes through a series of mitotic divisions, cell specification and differentiation events. How the cells in the female gametophyte of flowering plants differentiate into gametes (the egg and central cell) and accessory cells remains largely unknown. In a screen for mutations that affect egg cell differentiation in Arabidopsis, we identified the wyrd (wyr) mutant, which produces additional egg cells at the expense of the accessory synergids. WYR not only restricts gametic fate in the egg apparatus, but is also necessary for central cell differentiation. In addition, wyr mutants impair mitotic divisions in the male gametophyte and endosperm, and have a parental effect on embryo cytokinesis, consistent with a function of WYR in cell cycle regulation. WYR is upregulated in gametic cells and encodes a putative plant ortholog of the inner centromere protein (INCENP), which is implicated in the control of chromosome segregation and cytokinesis in yeast and animals. Our data reveal a novel developmental function of the conserved cell cycle-associated INCENP protein in plant reproduction, in particular in the regulation of egg and central cell fate and differentiation.

BACKGROUND Placental malaria is associated with local accumulation of parasitized erythrocytes, deposition of the parasite hemoglobin metabolite, hemozoin, and accumulation of mononuclear cells in the intervillous space. Fetal syncytiotrophoblast cells in contact with maternal blood are known to respond immunologically to cytoadherent Plasmodium falciparum-infected erythrocytes, but their responsiveness to hemozoin, a potent pro-inflammatory stimulator of monocytes, macrophages and dendritic cells, is not known. METHODS The biochemical and immunological changes induced in primary syncytiotrophoblast by natural hemozoin was assessed. Changes in syncytiotrophoblast mitogen-activated protein kinase activation was assessed by immunoblotting and secreted cytokine and chemokine proteins were assayed by ELISA. Chemotaxis of peripheral blood mononuclear cells was assessed using a two-chamber assay system and flow cytometry was used to assess the activation of primary monocytes by hemozoin-stimulated syncytiotrophoblast conditioned medium. RESULTS Hemozoin stimulation induced ERK1/2 phosphorylation. Treated cells secreted CXCL8, CCL3, CCL4, and tumor necrosis factor and released soluble intercellular adhesion molecule-1. Furthermore, the dependence of the hemozoin responses on ERK1/2 stimulation was confirmed by inhibition of chemokine release in syncytiotrophoblast treated with an ERK pathway inhibitor. Hemozoin-stimulated cells elicited the specific migration of PBMCs, and conditioned medium from the cells induced the upregulation of intercellular adhesion molecule-1 on primary monocytes. CONCLUSIONS These findings confirm an immunostimulatory role for hemozoin and expand the cell types known to be responsive to hemozoin to include fetal syncytiotrophoblast. The results provide further evidence that syncytiotrophoblast cells can influence the local maternal immune response to placental malaria.

Communication is a strong selective pressure on brain evolution because the exchange of information between individuals is crucial for fitness-related behaviors, such as mating. Given the importance of communication, the brains of signal senders and receivers are likely to be functionally coordinated. We study vocal behavior and auditory processing in multiple species of estrildid finches with the goal of understanding how species identity and early experience interact to shape the neural systems that subserve communication. Male finches learn to produce species-specific songs, and both sexes learn to recognize songs. Our studies indicate that closely related species exhibit different auditory coding properties in the midbrain and forebrain and that early life experience of vocalizations contributes to these differences. Moreover, birds that naturally sing tonal songs can learn broadband songs from heterospecific tutors, providing an opportunity to examine the interplay between species identity and early experience in the development of vocal behavior and auditory tuning.