Solvent-induced expansion of dried-demineralized dentin matrix

Abstract no. 249published_or_final_versio

Solvent-induced shrinkage of moist demineralized dentin

Abstract no. 117published_or_final_versio

Haemogenic endocardium contributes to transient definitive haematopoiesis.

Haematopoietic cells arise from spatiotemporally restricted domains in the developing embryo. Although studies of non-mammalian animal and in vitro embryonic stem cell models suggest a close relationship among cardiac, endocardial and haematopoietic lineages, it remains unknown whether the mammalian heart tube serves as a haemogenic organ akin to the dorsal aorta. Here we examine the haemogenic activity of the developing endocardium. Mouse heart explants generate myeloid and erythroid colonies in the absence of circulation. Haemogenic activity arises from a subset of endocardial cells in the outflow cushion and atria earlier than in the aorta-gonad-mesonephros region, and is transient and definitive in nature. Interestingly, key cardiac transcription factors, Nkx2-5 and Isl1, are expressed in and required for the haemogenic population of the endocardium. Together, these data suggest that a subset of endocardial/endothelial cells serve as a de novo source for transient definitive haematopoietic progenitors

Clinical, ultrasound and molecular biomarkers for early prediction of large for gestational age infants in nulliparous women: an international prospective cohort study

Objective: To develop a prediction model for term infants born large for gestational age (LGA) by customised birthweight centiles. Methods: International prospective cohort of nulliparous women with singleton pregnancy recruited to the Screening for Pregnancy Endpoints (SCOPE) study. LGA was defined as birthweight above the 90th customised centile, including adjustment for parity, ethnicity, maternal height and weight, fetal gender and gestational age. Clinical risk factors, ultrasound parameters and biomarkers at 14–16 or 19–21 weeks were combined into a prediction model for LGA infants at term using stepwise logistic regression in a training dataset. Prediction performance was assessed in a validation dataset using area under the Receiver Operating Characteristics curve (AUC) and detection rate at fixed false positive rates. Results: The prevalence of LGA at term was 8.8% (n = 491/5628). Clinical and ultrasound factors selected in the prediction model for LGA infants were maternal birthweight, gestational weight gain between 14–16 and 19–21 weeks, and fetal abdominal circumference, head circumference and uterine artery Doppler resistance index at 19–21 weeks (AUC 0.67; 95%CI 0.63–0.71). Sensitivity of this model was 24% and 49% for a fixed false positive rate of 10% and 25%, respectively. The addition of biomarkers resulted in selection of random glucose, LDL-cholesterol, vascular endothelial growth factor receptor-1 (VEGFR1) and neutrophil gelatinase-associated lipocalin (NGAL), but with minimal improvement in model performance (AUC 0.69; 95%CI 0.65–0.73). Sensitivity of the full model was 26% and 50% for a fixed false positive rate of 10% and 25%, respectively. Conclusion: Prediction of LGA infants at term has limited diagnostic performance before 22 weeks but may have a role in contingency screening in later pregnancy

Network Analysis Identifies ELF3 as a QTL for the Shade Avoidance Response in Arabidopsis

Quantitative Trait Loci (QTL) analyses in immortal populations are a powerful method for exploring the genetic mechanisms that control interactions of organisms with their environment. However, QTL analyses frequently do not culminate in the identification of a causal gene due to the large chromosomal regions often underlying QTLs. A reasonable approach to inform the process of causal gene identification is to incorporate additional genome-wide information, which is becoming increasingly accessible. In this work, we perform QTL analysis of the shade avoidance response in the Bayreuth-0 (Bay-0, CS954) x Shahdara (Sha, CS929) recombinant inbred line population of Arabidopsis. We take advantage of the complex pleiotropic nature of this trait to perform network analysis using co-expression, eQTL and functional classification from publicly available datasets to help us find good candidate genes for our strongest QTL, SAR2. This novel network analysis detected EARLY FLOWERING 3 (ELF3; AT2G25930) as the most likely candidate gene affecting the shade avoidance response in our population. Further genetic and transgenic experiments confirmed ELF3 as the causative gene for SAR2. The Bay-0 and Sha alleles of ELF3 differentially regulate developmental time and circadian clock period length in Arabidopsis, and the extent of this regulation is dependent on the light environment. This is the first time that ELF3 has been implicated in the shade avoidance response and that different natural alleles of this gene are shown to have phenotypic effects. In summary, we show that development of networks to inform candidate gene identification for QTLs is a promising technique that can significantly accelerate the process of QTL cloning

DNA End Resection Controls the Balance between Homologous and Illegitimate Recombination in Escherichia coli

Even a partial loss of function of human RecQ helicase analogs causes adverse effects such as a cancer-prone Werner, Bloom or Rothmund-Thompson syndrome, whereas a complete RecQ deficiency in Escherichia coli is not deleterious for a cell. We show that this puzzling difference is due to different mechanisms of DNA double strand break (DSB) resection in E. coli and humans. Coupled helicase and RecA loading activities of RecBCD enzyme, which is found exclusively in bacteria, are shown to be responsible for channeling recombinogenic 3′ ending tails toward productive, homologous and away from nonproductive, aberrant recombination events. On the other hand, in recB1080/recB1067 mutants, lacking RecBCD’s RecA loading activity while preserving its helicase activity, DSB resection is mechanistically more alike that in eukaryotes (by its uncoupling from a recombinase polymerization step), and remarkably, the role of RecQ also becomes akin of its eukaryotic counterparts in a way of promoting homologous and suppressing illegitimate recombination. The sickly phenotype of recB1080 recQ mutant was further exacerbated by inactivation of an exonuclease I, which degrades the unwound 3′ tail. The respective recB1080 recQ xonA mutant showed poor viability, DNA repair and homologous recombination deficiency, and very increased illegitimate recombination. These findings demonstrate that the metabolism of the 3′ ending overhang is a decisive factor in tuning the balance of homologous and illegitimate recombination in E. coli, thus highlighting the importance of regulating DSB resection for preserving genome integrity. recB mutants used in this study, showing pronounced RecQ helicase and exonuclease I dependence, make up a suitable model system for studying mechanisms of DSB resection in bacteria. Also, these mutants might be useful for investigating functions of the conserved RecQ helicase family members, and congruently serve as a simpler, more defined model system for human oncogenesis

Vascular Dysfunction Induced in Offspring by Maternal Dietary Fat Involves Altered Arterial Polyunsaturated Fatty Acid Biosynthesis

Nutrition during development affects risk of future cardiovascular disease. Relatively little is known about whether the amount and type of fat in the maternal diet affect vascular function in the offspring. To investigate this, pregnant and lactating rats were fed either 7%(w/w) or 21%(w/w) fat enriched in either18:2n-6, trans fatty acids, saturated fatty acids, or fish oil. Their offspring were fed 4%(w/w) soybean oil from weaning until day 77. Type and amount of maternal dietary fat altered acetylcholine (ACh)-mediated vaso-relaxation in offspring aortae and mesenteric arteries, contingent on sex. Amount, but not type, of maternal dietary fat altered phenylephrine (Pe)-induced vasoconstriction in these arteries. Maternal 21% fat diet decreased 20:4n-6 concentration in offspring aortae. We investigated the role of Δ6 and Δ5 desaturases, showing that their inhibition in aortae and mesenteric arteries reduced vasoconstriction, but not vaso-relaxation, and the synthesis of specific pro-constriction eicosanoids. Removal of the aortic endothelium did not alter the effect of inhibition of Δ6 and Δ5 desaturases on Pe-mediated vasoconstriction. Thus arterial smooth muscle 20:4n-6 biosynthesis de novo appears to be important for Pe-mediated vasoconstriction. Next we studied genes encoding these desaturases, finding that maternal 21% fat reduced Fads2 mRNA expression and increased Fads1 in offspring aortae, indicating dysregulation of 20:4n-6 biosynthesis. Methylation at CpG −394 bp 5′ to the Fads2 transcription start site predicted its expression. This locus was hypermethylated in offspring of dams fed 21% fat. Pe treatment of aortae for 10 minutes increased Fads2, but not Fads1, mRNA expression (76%; P<0.05). This suggests that Fads2 may be an immediate early gene in the response of aortae to Pe. Thus both amount and type of maternal dietary fat induce altered regulation of vascular tone in offspring though differential effects on vaso-relaxation, and persistent changes in vasoconstriction via epigenetic processes controlling arterial polyunsaturated fatty acid biosynthesis

Chemerin regulates β-cell function in mice

Although various function of chemerin have been suggested, its physiological role remains to be elucidated. Here we show that chemerin-deficient mice are glucose intolerant irrespective of exhibiting reduced macrophage accumulation in adipose tissue. The glucose intolerance was mainly due to increased hepatic glucose production and impaired insulin secretion. Chemerin and its receptor ChemR23 were expressed in β-cell. Studies using isolated islets and perfused pancreas revealed impaired glucose-dependent insulin secretion (GSIS) in chemerin-deficient mice. Conversely, chemerin transgenic mice revealed enhanced GSIS and improved glucose tolerance. Expression of MafA, a pivotal transcriptional factor for β-cell function, was downregulated in chemerin-deficient islets and a chemerin-ablated β-cell line and rescue of MafA expression restored GSIS, indicating that chemerin regulates β-cell function via maintaining MafA expression. These results indicate that chemerin regulates β-cell function and plays an important role in glucose homeostasis in a tissue-dependent manner

Interaction between genetic and epigenetic variation defines gene expression patterns at the asthma-associated locus 17q12-q21 in lymphoblastoid cell lines

Phenotypic variation results from variation in gene expression, which is modulated by genetic and/or epigenetic factors. To understand the molecular basis of human disease, interaction between genetic and epigenetic factors needs to be taken into account. The asthma-associated region 17q12-q21 harbors three genes, the zona pellucida binding protein 2 (ZPBP2), gasdermin B (GSDMB) and ORM1-like 3 (ORMDL3), that show allele-specific differences in expression levels in lymphoblastoid cell lines (LCLs) and CD4+ T cells. Here, we report a molecular dissection of allele-specific transcriptional regulation of the genes within the chromosomal region 17q12-q21 combining in vitro transfection, formaldehyde-assisted isolation of regulatory elements, chromatin immunoprecipitation and DNA methylation assays in LCLs. We found that a single nucleotide polymorphism rs4795397 influences the activity of ZPBP2 promoter in vitro in an allele-dependent fashion, and also leads to nucleosome repositioning on the asthma-associated allele. However, variable methylation of exon 1 of ZPBP2 masks the strong genetic effect on ZPBP2 promoter activity in LCLs. In contrast, the ORMDL3 promoter is fully unmethylated, which allows detection of genetic effects on its transcription. We conclude that the cis-regulatory effects on 17q12-q21 gene expression result from interaction between several regulatory polymorphisms and epigenetic factors within the cis-regulatory haplotype region

Structure of a bacterial type III secretion system in contact with a host membrane in situ

Many bacterial pathogens of animals and plants use a conserved type III secretion system (T3SS) to inject virulence effector proteins directly into eukaryotic cells to subvert host functions. Contact with host membranes is critical for T3SS activation, yet little is known about T3SS architecture in this state or the conformational changes that drive effector translocation. Here we use cryo-electron tomography and sub-tomogram averaging to derive the intact structure of the primordial Chlamydia trachomatis T3SS in the presence and absence of host membrane contact. Comparison of the averaged structures demonstrates a marked compaction of the basal body (4 nm) occurs when the needle tip contacts the host cell membrane. This compaction is coupled to a stabilization of the cytosolic sorting platform– ATPase. Our findings reveal the first structure of a bacterial T3SS from a major human pathogen engaged with a eukaryotic host, and reveal striking ‘pump-action’ conformational changes that underpin effector injection