Functional in vitro and in vivo reporter gene assays have recently been developed for the rapid determination of exposure to (xeno)estrogens. The in vitro estrogen receptor (ER)-mediated chemically ...activated luciferase gene expression (ER-CALUX) assay uses T47D human breast cancer cells stably transfected with an ER-mediated luciferase gene construct. In the in vivo assay, transgenic zebrafish are used in which the same luciferase construct has been stably introduced. In both assays, luciferase reporter gene activity can be easily quantified following short-term exposure to chemicals activating endogenous estrogen receptors. The objective of this study was to compare responses by known (xeno)estrogenic compounds in both assays. Exposure to the (xeno)estrogens estradiol (E2), estrone, ethynylestradiol (EE2), o,p‘-DDT, nonylphenol (NP), and di(2-ethylhexyl)phthalate (DEHP) revealed that EE2 was the most potent (xeno)estrogen tested and was 100 times more potent than E2 in the transgenic zebrafish assay, whereas in the in vitro ER-CALUX assay, EE2 and E2 were equipotent. Although the xenoestrogens o,p‘-DDT and NP were full estrogen agonists in the in vitro ER-CALUX assay, only o,p‘-DDT demonstrated weak dose-related estrogenic activity in vivo. To determine if differences in reporter gene activity may be explained by differential affinity of (xeno)estrogens to human and zebrafish ERs, full-length sequences of the zebrafish ER subtypes α, β, and γ were cloned, and transactivation by (xeno)estrogens was compared to human ERα and ERβ. Using transiently transfected recombinant ER and reporter gene constructs, EE2 also showed relatively potent activation of zebrafish ERα and ERβ compared to human ERα and ERβ. Zebrafish ERβ and ERγ showed higher transactivation by (xeno)estrogens relative to E2 than human ERβ.
In many organisms the allocation of primordial germ cells (PGCs) is determined by the inheritance of maternal factors deposited in the egg. However, in mammals, inductive cell interactions are ...required around gastrulation to establish the germ line. Here, we show that Bmp4 homozygous null embryos contain no PGCs. They also lack an allantois, an extraembryonic mesodermal tissue derived, like the PGCs, from precursors in the proximal epiblast. Heterozygotes have fewer PGCs than normal, due to a reduction in the size of the founding population and not to an effect on its subsequent expansion. Analysis of beta-galactosidase activity in Bmp4(lacZneo) embryos reveals that prior to gastrulation, Bmp4 is expressed in the extraembryonic ectoderm. Later, Bmp4 is expressed in the extraembryonic mesoderm, but not in PGCs. Chimera analysis indicates that it is the Bmp4 expression in the extraembryonic ectoderm that regulates the formation of allantois and primordial germ cell precursors, and the size of the founding population of PGCs. The initiation of the germ line in the mouse therefore depends on a secreted signal from the previously segregated, extraembryonic, trophectoderm lineage.
We describe the cloning and initial characterization of a novel cDNA from human embryonal carcinoma (EC) cells. This cDNA, which we named human growth differentiation factor 3 (hGDF3), encodes the ...homologue of mouse GDF3, a TGFbeta superfamily member belonging to the Growth/Differentiation Factors. We have analysed the expression of hGDF3 in human embryonal carcinoma cell lines and in primary testicular germ cell tumours of adolescents and adults (TGCTs). Expression of hGDF3 in human EC cell lines is stem cell-specific, is down-regulated upon RA-mediated differentiation and is increased upon culture of the cells in the presence of activin A. In TGCTs, hGDF3 expression is low in seminomas, while expression in non-seminomas is readily detectable and appears to be associated with the EC and yolk sac components in the tumours. We have also mapped the hGDF3 locus to the short arm of human chromosome 12, a region consistently overrepresented in human testicular germ cell tumours. Thus, hGDF3 represents an embryonal carcinoma stem cell-associated marker both in vitro and in vivo.
Objective
To determine consensus among an international, multidisciplinary group of experts regarding definitions of spinal osteoarthritis for research and for clinical practice.
Methods
A 15‐member, ...multidisciplinary steering committee generated 117 statements for a 3‐round Delphi study. Experts in back pain and/or osteoarthritis were identified and invited to participate. In round 1, participants could propose additional statements for voting. All statements were rated on a 1–9 Likert scale, and consensus was set at ≥70% of respondents agreeing or disagreeing with the statement and <15% of respondents providing the opposite response.
Results
In total, 255 experts from 11 different professional backgrounds were invited. From 173 available experts, 116 consented to participate. In round 1, 103 participants completed the survey, followed by 85 of 111 participants in round 2 (77%) and 87 of 101 participants in round 3 (86%). One‐third of participants were from Europe (30%), most were male (58%), one‐fifth were physical therapists (21%), and over one‐third had been in their profession for 11–20 years (35%). Of 131 statements, consensus was achieved for 71 statements (54%): 53 in agreement (75%) and 18 in disagreement (25%).
Conclusion
Although there was consensus for statements for definitions of spinal osteoarthritis that were analogous to definitions of osteoarthritis in appendicular joints, a future definition still needs refinement. Importantly, this Delphi highlighted that a future definition should be considered across a spectrum of structural changes and patient symptoms and expressed on a progressive scale.
Lgr5+ intestinal stem cells generate enterocytes and secretory cells. Secretory lineage commitment requires Notch silencing. The Notch ligand Dll1 is expressed by a subset of immediate stem cell ...daughters. Lineage tracing in Dll1(GFP-ires-CreERT2) knock-in mice reveals that single Dll1(high) cells generate small, short-lived clones containing all four secretory cell types. Lineage specification thus occurs in immediate stem cell daughters through Notch lateral inhibition. Cultured Dll1(high) cells form long-lived organoids (mini-guts) on brief Wnt3A exposure. When Dll1(high) cells are genetically marked before tissue damage, stem cell tracing events occur. Thus, secretory progenitors exhibit plasticity by regaining stemness on damage.
Aims
The palatability of a new paediatric formulation of valaciclovir was assessed in children and their parents: non‐inferiority of the new paediatric formulation (test formulation) compared to the ...reference formulation was investigated.
Methods
In vivo palatability testing was performed in a randomized, two‐period, multicentre, cross‐over study. Children and their parents scored the liking of the new paediatric valaciclovir formulation and the reference formulation on a 100 mm visual analogue scale (VAS). To support formulation development and palatability testing, electronic tongue measurements were applied.
Results
The electronic tongue measurement indicated taste‐masking capabilities for three different formulations in the developmental phase. A glycerol‐based formulation was further tested and compared to the reference formulation prepared out of crushed and suspended tablets. The mean difference (95% CI) in VAS scores between both formulations, as indicated by the children (n = 20), was 2.4 (−8.5, 13) mm, in favour of the new paediatric valaciclovir formulation. The mean (95% CI) difference in VAS scores indicated by the parents (n = 20) was −0.9 (−12, 9.8) mm.
Conclusion
The palatability of the new paediatric valaciclovir formulation was considered non‐inferior to the reference formulation prepared out of crushed tablets. We were able to optimize the study design and number of children to be included in the palatability testing by using electronic tongue measurements.
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