Serum hyperglycosylated human chorionic gonadotropin to predict the gestational outcome in in vitro fertilization/intracytoplasmic sperm injection pregnancies.

Hyperglycosylated human chorionic gonadotropin (H-hCG) is secreted by the placenta in early pregnancy. Decreased H-hCG levels have been associated with abortion in spontaneous pregnancy. We retrospectively measured H-hCG and dimeric hCG in the sera of 87 in vitro fertilization patients obtained in the 3 weeks following embryo transfer and set the results in relation to pregnancy outcome. H-hCG and dimeric hCG were correlated (r(2) = 0.89), and were significantly decreased in biochemical pregnancy (2 microg/l and 18 IU/l, respectively) compared to early pregnancy loss (22 microg/l and 331 IU/l) and ongoing pregnancy (32 microg/l and 353 IU/l). Only H-hCG tended to discriminate between these last two groups.

Characterization of Fas (Apo-1, CD95)-Fas ligand interaction.

The death-inducing receptor Fas is activated when cross-linked by the type II membrane protein Fas ligand (FasL). When human soluble FasL (sFasL, containing the extracellular portion) was expressed in human embryo kidney 293 cells, the three N-linked glycans of each FasL monomer were found to be essential for efficient secretion. Based on the structure of the closely related lymphotoxin alpha-tumor necrosis factor receptor I complex, a molecular model of the FasL homotrimer bound to three Fas molecules was generated using knowledge-based protein modeling methods. Point mutations of amino acid residues predicted to affect the receptor-ligand interaction were introduced at three sites. The F275L mutant, mimicking the loss of function murine gld mutation, exhibited a high propensity for aggregation and was unable to bind to Fas. Mutants P206R, P206D, and P206F displayed reduced cytotoxicity toward Fas-positive cells with a concomitant decrease in the binding affinity for the recombinant Fas-immunoglobulin Fc fusion proteins. Although the cytotoxic activity of mutant Y218D was unaltered, mutant Y218R was inactive, correlating with the prediction that Tyr-218 of FasL interacts with a cluster of three basic amino acid side chains of Fas. Interestingly, mutant Y218F could induce apoptosis in murine, but not human cells.

Comparative analysis of the transcriptome across distant species.

The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a 'universal model' based on a single set of organism-independent parameters.

Persisting atypical and cystic forms of Borrelia burgdorferi and local inflammation in Lyme neuroborreliosis

BACKGROUND: The long latent stage seen in syphilis, followed by chronic central nervous system infection and inflammation, can be explained by the persistence of atypical cystic and granular forms of Treponema pallidum. We investigated whether a similar situation may occur in Lyme neuroborreliosis. METHOD: Atypical forms of Borrelia burgdorferi spirochetes were induced exposing cultures of Borrelia burgdorferi (strains B31 and ADB1) to such unfavorable conditions as osmotic and heat shock, and exposure to the binding agents Thioflavin S and Congo red. We also analyzed whether these forms may be induced in vitro, following infection of primary chicken and rat neurons, as well as rat and human astrocytes. We further analyzed whether atypical forms similar to those induced in vitro may also occur in vivo, in brains of three patients with Lyme neuroborreliosis. We used immunohistochemical methods to detect evidence of neuroinflammation in the form of reactive microglia and astrocytes. RESULTS: Under these conditions we observed atypical cystic, rolled and granular forms of these spirochetes. We characterized these abnormal forms by histochemical, immunohistochemical, dark field and atomic force microscopy (AFM) methods. The atypical and cystic forms found in the brains of three patients with neuropathologically confirmed Lyme neuroborreliosis were identical to those induced in vitro. We also observed nuclear fragmentation of the infected astrocytes using the TUNEL method. Abundant HLA-DR positive microglia and GFAP positive reactive astrocytes were present in the cerebral cortex. CONCLUSION: The results indicate that atypical extra- and intracellular pleomorphic and cystic forms of Borrelia burgdorferi and local neuroinflammation occur in the brain in chronic Lyme neuroborreliosis. The persistence of these more resistant spirochete forms, and their intracellular location in neurons and glial cells, may explain the long latent stage and persistence of Borrelia infection. The results also suggest that Borrelia burgdorferi may induce cellular dysfunction and apoptosis. The detection and recognition of atypical, cystic and granular forms in infected tissues is essential for the diagnosis and the treatment as they can occur in the absence of the typical spiral Borrelia form.

Translation of polarity cues into asymmetric spindle positioning in "Caenorhabditis elegans"

Abstract: Asymmetric cell division is important to generate tissue diversity. The Caenorhabditis elegans embryo is well suited to study the mechanisms of asymmetric cell division. In wild type one-cell stage embryos, the spindle sets up along the anterior-posterior axis (AP). During anaphase, the spindle elongates. While the anterior spindle pole is relatively immobile, the posterior spindle pole moves towards the posterior cortex during anaphase leading to an asymmetric spindle position. As a result, the first cleavage gives rise to a large anterior blastomere and a smaller posterior one, which differs also in cell fate determinants. This posterior spindle displacement occurs in response to polarity cues set up along the AP axis by the PAR proteins and is due to imbalanced pulling forces acting on the two spindle poles, with net forces acting on the posterior spindle pole being more extensive than those at the anterior one. The project of my thesis was to characterize the involvement of two new components, gpr-1 and gpr-2, in spindle positioning. These genes encode essentially identical proteins containing a GoLoco motif characteristic of proteins interacting with α subunits of heterotrimeric G protein (Gα). In gpr-1/2(RNAi) embryos and in embryos lacking simultaneously two α subunits, goa-1 and gpa-16, (Ga(RNAi) embryos), there is a minimal posterior displacement of the spindle during anaphase, and the first division is equal. I found that the pulling forces acting on the two spindle poles is weak and equal in gpr-1/2(RNAi) and Gα (RNAi) embryos. I found that GPR-1/2 acts downstream of polarity cues for generation of pulling forces. Furthermore, I showed that GPR-1/2 distribution was enriched at the posterior cortex during metaphase whereas GOA-1 and GPA-16 were uniformly distributed at the cell cortex throughout the cell cycle. Gα subunits oscillate between GDP- and GTP-bound forms. Gα signaling is turned on by GDP/GTP exchange catalyzed by guanine nucleotide exchange factors (GEFs) and turned off by hydrolysis of GTP catalyzed by GTPase activating proteins (GAPs). A third class of proteins, the guanine dissociation inhibitors (GDIs), binds the GDP-bound form of Gα subunits and inhibits nucleotide exchange. I found that GPR-1/2 acts as a GDI for GOA-1. Taken together, my findings suggest a model in which differential activation of Gα subunits along the AP axis may translate into generation of differential pulling forces on the anterior and posterior spindle poles, and, thus, asymmetric cell division. Résumé L'embryon du nématode Caenorhabditis elegans est un modèle approprié pour étudier les mécanismes de la division asymétrique. Chez l'embryon précoce, le fuseau mitotique se forme le long de l'axe antéro-postérieur (A/P) et au centre de l'embryon, le pôle antérieur restant relativement immobile alors que le pôle postérieur du fuseau se déplace vers le cortex postérieur au cours de l'anaphase conduisant à une position excentrée du fuseau. 11 en résulte une première division qui génère un blastomère antérieur et postérieur de grande et petite taille respectivement et qui diffèrent en facteurs développementaux. Ce déplacement postérieur se produit en réponse de la polarité établie par la distribution polarisée des protéines PAR et est le résultat de la génération de forces inégales tirant sur les deux pôles du fuseau, les forces agissant sur le pôle postérieur du fuseau étant plus grandes. Le projet de ma thèse était d'identifier la fonction de deux nouveaux constituants, gpr-1 et gpr-2 dans le positionnement asymétrique du fuseau. Ces gènes codent essentiellement pour la même protéine qui contient un motif GoLoco, caractéristique des protéines interagissant avec la sous-unité alpha des protéines G hétérotrimériques. Chez l'embryon gpr-1/2(RNAi) et chez les embryons dépourvus d'activité de deux sous-unités alpha, goa-1 et gpa-16, (Gα(RNAi)), j'ai montré qu'il y avait un déplacement minimal du fuseau vers le pôle postérieur au cours de l'anaphase et la première division est symétrique en raison de forces faibles et égales agissant sur les deux pôles du fuseau. J'ai également montré que gpr-1/2 était requis en aval des signaux établissant la polarité pour générer les forces responsables du positionnement asymétrique du fuseau. De plus, j'ai montré que GPR-1/2 était enrichi au pôle postérieur lors de la métaphase alors que GOA-1 et GPA-16 étaient localisés de façon uniforme au cortex de l'embryon précoce. Gas oscillent entre une forme liée au GDP et une forme liée au GTP. La signalisation des Gas est activée par l'échange GDP/GTP qui est catalysé par des protéines GEFs. La signalisation des Gas est désactivée par l'hydrolyse du GTP qui est catalysée par des protéines GAPs. Une troisième classe de protéines, GDIs lie la forme GDP et inhibe l'échange de nucléotides. J'ai montré que GPR-1/2 agissait comme un GDI pour GOA-1. Mes résultats suggèrent un modèle dans lequel une activation différentielle des Gα le long de l'axe A/P pourrait générer des forces différentielles sur le pôle antérieur et postérieur du fuseau.

Primary root protophloem differentiation requires balanced phosphatidylinositol-4,5-biphosphate levels and systemically affects root branching.

Protophloem is a specialized vascular tissue in growing plant organs, such as root meristems. In Arabidopsis mutants with impaired primary root protophloem differentiation, brevis radix (brx) and octopus (ops), meristematic activity and consequently overall root growth are strongly reduced. Second site mutation in the protophloem-specific presumed phosphoinositide 5-phosphatase COTYLEDON VASCULAR PATTERN 2 (CVP2), but not in its homolog CVP2-LIKE 1 (CVL1), partially rescues brx defects. Consistent with this finding, CVP2 hyperactivity in a wild-type background recreates a brx phenotype. Paradoxically, however, while cvp2 or cvl1 single mutants display no apparent root defects, the root phenotype of cvp2 cvl1 double mutants is similar to brx or ops, although, as expected, cvp2 cvl1 seedlings contain more phosphatidylinositol-4,5-biphosphate. Thus, tightly balanced phosphatidylinositol-4,5-biphosphate levels appear essential for proper protophloem differentiation. Genetically, OPS acts downstream of phosphatidylinositol-4,5-biphosphate levels, as cvp2 mutation cannot rescue ops defects, whereas increased OPS dose rescues cvp2 cvl1 defects. Finally, all three mutants display higher density and accelerated emergence of lateral roots, which correlates with increased auxin response in the root differentiation zone. This phenotype is also created by application of peptides that suppress protophloem differentiation, CLAVATA3/EMBRYO SURROUNDING REGION 26 (CLE26) and CLE45. Thus, local changes in the primary root protophloem systemically shape overall root system architecture.

Declining diversity of egg-associated bacteria during development of naturally spawned whitefish embryos (Coregonus spp.)

Fish eggs are associated with microbes, whose roles range from mutualism to parasitism. Recent laboratory experiments have shown that the taxonomic composition of associated microbial communities on the egg influences embryonic development. Host genetics also plays an important role in determining the consequences for embryonic growth and survival in this interaction. Moreover, it has been found that the importance of host genetics increases during embryogenesis. These findings suggest that during embryogenesis, the host increasingly influences the composition of its associated microbial community. However, little is known about the composition of microbial communities associated with naturally spawned eggs in the wild. We sampled fertilized whitefish eggs (Coregonus spp.) of different developmental stages from six sub-Alpine lakes and used a universal primer pair and 454 pyrosequencing in order to describe the taxonomic composition of egg-associated bacterial communities. We found bacterial communities on early embryos to be very diverse and to resemble the bacterial composition of the surrounding water environment. The bacterial communities on late embryos were significantly less diverse than on early embryos and displayed a clear shift in taxonomic composition that corresponded poorly with the bacterial composition of the surrounding water environment. The main bacterial components on whitefish eggs in this study were Proteobacteria, Actinobacteria, and Firmicutes, while the five most common families were Leuconostocaceae, Streptococcaceae, Comamonadaceae, Oxalobacteraceae and Moraxellaceae. Their putative relationships with the host are discussed. We conclude that natural symbiotic bacterial communities become more specialized during embryogenesis because of specific interactions with their embryo host.

Cardiovascular dysfunction in children conceived by assisted reproductive technologies.

Epidemiological studies demonstrate a relationship between pathological events during foetal development and future cardiovascular risk and the term 'foetal programming of cardiovascular disease' has been coined to describe this phenomenon. The use of assisted reproductive technologies (ARTs) is growing exponentially and 2-5% of children are now born by this procedure. Emerging evidence indicates that ART represents a novel important example of foetal programming. Assisted reproductive technology may modify the cardiovascular phenotype in two ways: (i) ART involves manipulation of the early embryo which is exquisitely sensitive to environmental insults. In line with this concern, ART alters vascular and cardiac function in children and studies in mice show that ART alters the cardiovascular phenotype by epigenetic alterations related to suboptimal culture conditions. (ii) Assisted reproductive technology markedly increases the risk of foetal insults that augment cardiovascular risk in naturally conceived individuals and are expected to have similar consequences in the ART population. Given the young age of the ART population, it will take another 20-30 years before data on cardiovascular endpoints will be available. What is clear already, however, is that ART emerges as an important cardiovascular risk factor. This insight requires us to revise notions on ART's long-term safety and to engage on a debate on its future. There is an urgent need to better understand the mechanisms underpinning ART-induced alteration of the cardiovascular phenotype, improve the procedure and its long-term safety, and, while awaiting this aim, not to abandon medicine's fundamental principle of doing no harm (to future children) and use ART parsimoniously.

Embryonic gene expression of Coregonus palaea (whitefish) under pathogen stress as analyzed by high-throughput RNA-sequencing.

Most fishes produce free-living embryos that are exposed to environmental stressors immediately following fertilization, including pathogenic microorganisms. Initial immune protection of embryos involves the chorion, as a protective barrier, and maternally-allocated antimicrobial compounds. At later developmental stages, host-genetic effects influence susceptibility and tolerance, suggesting a direct interaction between embryo genes and pathogens. So far, only a few host genes could be identified that correlate with embryonic survival under pathogen stress in salmonids. Here, we utilized high-throughput RNA-sequencing in order to describe the transcriptional response of a non-model fish, the Alpine whitefish Coregonus palaea, to infection, both in terms of host genes that are likely manipulated by the pathogen, and those involved in an early putative immune response. Embryos were produced in vitro, raised individually, and exposed at the late-eyed stage to a virulent strain of the opportunistic fish pathogen Pseudomonas fluorescens. The pseudomonad increased embryonic mortality and affected gene expression substantially. For example, essential, upregulated metabolic pathways in embryos under pathogen stress included ion binding pathways, aminoacyl-tRNA-biosynthesis, and the production of arginine and proline, most probably mediated by the pathogen for its proliferation. Most prominently downregulated transcripts comprised the biosynthesis of unsaturated fatty acids, the citrate cycle, and various isoforms of b-cell transcription factors. These factors have been shown to play a significant role in host blood cell differentiation and renewal. With regard to specific immune functions, differentially expressed transcripts mapped to the complement cascade, MHC class I and II, TNF-alpha, and T-cell differentiation proteins. The results of this study reveal insights into how P. fluorescens impairs the development of whitefish embryos and set a foundation for future studies investigating host pathogen interactions in fish embryos.

Ectodysplasin/NF-κB Promotes Mammary Cell Fate via Wnt/β-catenin Pathway.

Mammary gland development commences during embryogenesis with the establishment of a species typical number of mammary primordia on each flank of the embryo. It is thought that mammary cell fate can only be induced along the mammary line, a narrow region of the ventro-lateral skin running from the axilla to the groin. Ectodysplasin (Eda) is a tumor necrosis factor family ligand that regulates morphogenesis of several ectodermal appendages. We have previously shown that transgenic overexpression of Eda (K14-Eda mice) induces formation of supernumerary mammary placodes along the mammary line. Here, we investigate in more detail the role of Eda and its downstream mediator transcription factor NF-κB in mammary cell fate specification. We report that K14-Eda mice harbor accessory mammary glands also in the neck region indicating wider epidermal cell plasticity that previously appreciated. We show that even though NF-κB is not required for formation of endogenous mammary placodes, it is indispensable for the ability of Eda to induce supernumerary placodes. A genome-wide profiling of Eda-induced genes in mammary buds identified several Wnt pathway components as potential transcriptional targets of Eda. Using an ex vivo culture system, we show that suppression of canonical Wnt signalling leads to a dose-dependent inhibition of supernumerary placodes in K14-Eda tissue explants.

The Association of H1N1 Pandemic Influenza with Congenital Anomaly Prevalence in Europe: An Ecological Time Series Study.

BACKGROUND: In the context of the European Surveillance of Congenital Anomalies (EUROCAT) surveillance response to the 2009 influenza pandemic, we sought to establish whether there was a detectable increase of congenital anomaly prevalence among pregnancies exposed to influenza seasons in general, and whether any increase was greater during the 2009 pandemic than during other seasons. METHODS: We performed an ecologic time series analysis based on 26,967 pregnancies with nonchromosomal congenital anomaly conceived from January 2007 to March 2011, reported by 15 EUROCAT registries. Analysis was performed for EUROCAT-defined anomaly subgroups, divided by whether there was a prior hypothesis of association with influenza. Influenza season exposure was based on World Health Organization data. Prevalence rate ratios were calculated comparing pregnancies exposed to influenza season during the congenital anomaly-specific critical period for embryo-fetal development to nonexposed pregnancies. RESULTS: There was no evidence for an increased overall prevalence of congenital anomalies among pregnancies exposed to influenza season. We detected an increased prevalence of ventricular septal defect and tricuspid atresia and stenosis during pandemic influenza season 2009, but not during 2007-2011 influenza seasons. For congenital anomalies, where there was no prior hypothesis, the prevalence of tetralogy of Fallot was strongly reduced during influenza seasons. CONCLUSIONS: Our data do not suggest an overall association of pandemic or seasonal influenza with congenital anomaly prevalence. One interpretation is that apparent influenza effects found in previous individual-based studies were confounded by or interacting with other risk factors. The associations of heart anomalies with pandemic influenza could be strain specific.

Compensatory embryonic response to allele-specific inactivation of the murine X-linked gene Hcfc1.

Early in female mammalian embryonic development, cells randomly inactivate one of the two X chromosomes to achieve overall equal inactivation of parental X-linked alleles. Hcfc1 is a highly conserved X-linked mouse gene that encodes HCF-1 - a transcriptional co-regulator implicated in cell proliferation in tissue culture cells. By generating a Cre-recombinase inducible Hcfc1 knock-out (Hcfc1(lox)) allele in mice, we have probed the role of HCF-1 in actively proliferating embryonic cells and in cell-cycle re-entry of resting differentiated adult cells using a liver regeneration model. HCF-1 function is required for both extraembryonic and embryonic development. In heterozygous Hcfc1(lox/+) female embryos, however, embryonic epiblast-specific Cre-induced Hcfc1 deletion (creating an Hcfc1(epiKO) allele) around E5.5 is well tolerated; it leads to a mixture of HCF-1-positive and -negative epiblast cells owing to random X-chromosome inactivation of the wild-type or Hcfc1(epiKO) mutant allele. At E6.5 and E7.5, both HCF-1-positive and -negative epiblast cells proliferate, but gradually by E8.5, HCF-1-negative cells disappear owing to cell-cycle exit and apoptosis. Although generating a temporary developmental retardation, the loss of HCF-1-negative cells is tolerated, leading to viable heterozygous offspring with 100% skewed inactivation of the X-linked Hcfc1(epiKO) allele. In resting adult liver cells, the requirement for HCF-1 in cell proliferation was more evident as hepatocytes lacking HCF-1 fail to re-enter the cell cycle and thus to proliferate during liver regeneration. The survival of the heterozygous Hcfc1(epiKO/+) female embryos, even with half the cells genetically compromised, illustrates the developmental plasticity of the post-implantation mouse embryo - in this instance, permitting survival of females heterozygous for an X-linked embryonic lethal allele.