Transgenic quails reveal dynamic TCF/ß-catenin signaling during avian embryonic development.

The Wnt/ß-catenin signaling pathway is highly conserved throughout evolution, playing crucial roles in several developmental and pathological processes. Wnt ligands can act at a considerable distance from their sources and it is therefore necessary to examine not only the Wnt-producing but also the Wnt-receiving cells and tissues to fully appreciate the many functions of this pathway. To monitor Wnt activity, multiple tools have been designed which consist of multimerized Wnt signaling response elements (TCF/LEF binding sites) driving the expression of fluorescent reporter proteins (e.g. GFP, RFP) or of LacZ. The high stability of those reporters leads to a considerable accumulation in cells activating the pathway, thereby making them easily detectable. However, this makes them unsuitable to follow temporal changes of the pathway's activity during dynamic biological events. Even though fluorescent transcriptional reporters can be destabilized to shorten their half-lives, this dramatically reduces signal intensities, particularly when applied in vivo. To alleviate these issues, we developed two transgenic quail lines in which high copy number (12× or 16×) of the TCF/LEF binding sites drive the expression of destabilized GFP variants. Translational enhancer sequences derived from viral mRNAs were used to increase signal intensity and specificity. This resulted in transgenic lines efficient for the characterization of TCF/ß-catenin transcriptional dynamic activities during embryogenesis, including using in vivo imaging. Our analyses demonstrate the use of this transcriptional reporter to unveil novel aspects of Wnt signaling, thus opening new routes of investigation into the role of this pathway during amniote embryonic development.

Mid-trimester microbial invasion of the amniotic cavity and the risk of preterm birth.

OBJECTIVE: To evaluate the rate of mid-trimester microbial invasion of the amniotic cavity (MIAC) in asymptomatic women and its association with preterm birth. STUDY DESIGN: This is a prospective cohort study of asymptomatic women undergoing mid-trimester amniocentesis for genetic testing between 14 and 24 weeks of gestation. For each participant, a sample of amniotic fluid was incubated in an aerobic and anaerobic facultative culture media and another sample was tested for the presence of specific Mycoplasma species (Ureaplasma urealyticum, Ureaplasma parvum, and Mycoplasma hominis) using quantitative-PCR. Results were not revealed to the participants or their health care providers. All participants were followed until delivery. MIAC was defined by a positive culture or a positive PCR for Mycoplasma species. The primary outcome was a spontaneous preterm birth or preterm premature rupture of membranes before 35 weeks of gestation. RESULTS: We included 812 women at a median gestational age of 16 5/7 (interquartile: 15 6/7-17 4/7) weeks. Twenty-six (3.2%) had a spontaneous delivery before 35 weeks. We observed no case of positive PCR for Mycoplasma species and 4 (0.5%) cases of positive culture that were all considered to be skin contaminants. None of those four cases was associated with preterm birth. Nulliparity, low family income and history of preterm birth were associated with spontaneous delivery before 35 weeks. CONCLUSION: We found no case of mid-trimester MIAC using a combination of culture and Mycoplasma-specific PCR techniques in a large cohort of low-risk asymptomatic pregnant women. We estimate that mid-trimester MIAC is rare in low-risk population but more sensitive and broad-range microbiologic techniques, such as 16S DNA detection by PCR, could be further evaluated.

TGFß signalling acts as a molecular brake of myoblast fusion.

Fusion of nascent myoblasts to pre-existing myofibres is critical for skeletal muscle growth and repair. The vast majority of molecules known to regulate myoblast fusion are necessary in this process. Here, we uncover, through high-throughput in vitro assays and in vivo studies in the chicken embryo, that TGFß (SMAD2/3-dependent) signalling acts specifically and uniquely as a molecular brake on muscle fusion. While constitutive activation of the pathway arrests fusion, its inhibition leads to a striking over-fusion phenotype. This dynamic control of TGFß signalling in the embryonic muscle relies on a receptor complementation mechanism, prompted by the merging of myoblasts with myofibres, each carrying one component of the heterodimer receptor complex. The competence of myofibres to fuse is likely restored through endocytic degradation of activated receptors. Altogether, this study shows that muscle fusion relies on TGFß signalling to regulate its pace.

CRISPR/Cas9 in the Chicken Embryo.

Genome editing is driving a revolution in the biomedical sciences that carries the promise for future treatments of genetic diseases. The CRISPR/Cas9 system of RNA-guided genome editing has been successfully applied to modify the genome of a wide spectrum of organisms. We recently showed that this technique can be combined with in vivo electroporation to inhibit the function of genes of interest in somatic cells of the developing chicken embryo. We present here a simplified version of the previously described technique that leads to effective gene loss-of-function.

Adherence to antimuscarinics in children with overactive bladder.

OBJECTIVES: Adherence to antimuscarinics for the treatment of overactive bladder is known to be low in adults but there is scarce data on adherence in paediatric patients. Our objectives were to evaluate the adherence of children to antimuscarinics and to identify influencing factors. METHODS: Children aged 5 to 18 years and treated with an antimuscarinic agent for at least 6 months were recruited at a routine visit and had to fill out a questionnaire. Their pharmacists were then contacted to inquire about prescription renewals since the beginning of treatment. The medication possession ratio was calculated and grouped by time blocks of 1, 3, 6 and 12 months. The pharmacists were contacted again 6 months after the recruitment visit. A medication possession ratio ≥ 80% was considered as good adherence. RESULTS: Seventy-two patients were recruited with a mean age of 10.1 years. The self-reported adherence was 93%. Prior to the questionnaire, the medication possession ratio was ≥ 80% in 36%, 57%, 64% and 74% of cases in blocks of 1, 3, 6 and 12 months, respectively. After the questionnaire, the medication possession ratio improved to 53%, 65% and 71% for blocks of 1, 3 and 6 months, respectively. No influencing factors were identified. CONCLUSIONS: Measured adherence to antimuscarinics in children with overactive bladder is higher than in adults but significantly lower than the self-reported adherence. Good self-reported adherence must be questioned to avoid unnecessary dose escalation or change of medication. Strategies to increase medication adherence are required to improve treatment efficacy.

Single-Cell-Based Analysis Highlights a Surge in Cell-to-Cell Molecular Variability Preceding Irreversible Commitment in a Differentiation Process.

In some recent studies, a view emerged that stochastic dynamics governing the switching of cells from one differentiation state to another could be characterized by a peak in gene expression variability at the point of fate commitment. We have tested this hypothesis at the single-cell level by analyzing primary chicken erythroid progenitors through their differentiation process and measuring the expression of selected genes at six sequential time-points after induction of differentiation. In contrast to population-based expression data, single-cell gene expression data revealed a high cell-to-cell variability, which was masked by averaging. We were able to show that the correlation network was a very dynamical entity and that a subgroup of genes tend to follow the predictions from the dynamical network biomarker (DNB) theory. In addition, we also identified a small group of functionally related genes encoding proteins involved in sterol synthesis that could act as the initial drivers of the differentiation. In order to assess quantitatively the cell-to-cell variability in gene expression and its evolution in time, we used Shannon entropy as a measure of the heterogeneity. Entropy values showed a significant increase in the first 8 h of the differentiation process, reaching a peak between 8 and 24 h, before decreasing to significantly lower values. Moreover, we observed that the previous point of maximum entropy precedes two paramount key points: an irreversible commitment to differentiation between 24 and 48 h followed by a significant increase in cell size variability at 48 h. In conclusion, when analyzed at the single cell level, the differentiation process looks very different from its classical population average view. New observables (like entropy) can be computed, the behavior of which is fully compatible with the idea that differentiation is not a "simple" program that all cells execute identically but results from the dynamical behavior of the underlying molecular network.

Vaginal Fluid Inflammatory Biomarkers and the Risk of Adverse Neonatal Outcomes in Women with PPROM.

Objective The purpose of this study was to evaluate the predictive value of vaginal fluid biomarkers for chorioamnionitis and adverse perinatal outcomes in women with preterm premature rupture of membranes (PPROM). Methods We recruited women with PPROM, without clinical chorioamnionitis, between 22 and 36 weeks' gestation. Vaginal fluid was collected on admission for the measurement of metalloproteinase-8 (MMP-8), interleukin-6 (IL-6), lactate, and glucose concentration. Placental pathology and neonatal charts were reviewed. Primary outcomes were histological chorioamnionitis and adverse neonatal neurological outcomes (intraventricular hemorrhage grade 2 or 3, periventricular leukomalacia, or hypoxic/ischemic encephalopathy). Linear regression analyses were used to adjust for gestational age at PPROM. Results Twenty-seven women were recruited at a mean gestational age of 31.6 ± 3.1 weeks, including 25 (93%) with successful collection of vaginal fluid sample. Histological chorioamnionitis and adverse neonatal neurological outcomes were observed in nine (33%) and four (15%) cases, respectively. In univariate analysis, MMP-8, IL-6, glucose, and lactate concentrations in vaginal fluid were associated with the risk of chorioamnionitis but not anymore after adjustment for gestational age at PPROM. MMP-8 concentration was the only biomarker associated with adverse neurological outcome, and it remained significant after adjustment for gestational age at PPROM (p = 0.02). Conclusion Vaginal fluid inflammatory biomarkers at admission for PPROM could predict adverse perinatal outcomes.

RAR/RXR binding dynamics distinguish pluripotency from differentiation associated cis-regulatory elements.

In mouse embryonic cells, ligand-activated retinoic acid receptors (RARs) play a key role in inhibiting pluripotency-maintaining genes and activating some major actors of cell differentiation. To investigate the mechanism underlying this dual regulation, we performed joint RAR/RXR ChIP-seq and mRNA-seq time series during the first 48 h of the RA-induced Primitive Endoderm (PrE) differentiation process in F9 embryonal carcinoma (EC) cells. We show here that this dual regulation is associated with RAR/RXR genomic redistribution during the differentiation process. In-depth analysis of RAR/RXR binding sites occupancy dynamics and composition show that in undifferentiated cells, RAR/RXR interact with genomic regions characterized by binding of pluripotency-associated factors and high prevalence of the non-canonical DR0-containing RA response element. By contrast, in differentiated cells, RAR/RXR bound regions are enriched in functional Sox17 binding sites and are characterized with a higher frequency of the canonical DR5 motif. Our data offer an unprecedentedly detailed view on the action of RA in triggering pluripotent cell differentiation and demonstrate that RAR/RXR action is mediated via two different sets of regulatory regions tightly associated with cell differentiation status.

Cytokinin signalling inhibitory fields provide robustness to phyllotaxis.

How biological systems generate reproducible patterns with high precision is a central question in science. The shoot apical meristem (SAM), a specialized tissue producing plant aerial organs, is a developmental system of choice to address this question. Organs are periodically initiated at the SAM at specific spatial positions and this spatiotemporal pattern defines phyllotaxis. Accumulation of the plant hormone auxin triggers organ initiation, whereas auxin depletion around organs generates inhibitory fields that are thought to be sufficient to maintain these patterns and their dynamics. Here we show that another type of hormone-based inhibitory fields, generated directly downstream of auxin by intercellular movement of the cytokinin signalling inhibitor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6), is involved in regulating phyllotactic patterns. We demonstrate that AHP6-based fields establish patterns of cytokinin signalling in the meristem that contribute to the robustness of phyllotaxis by imposing a temporal sequence on organ initiation. Our findings indicate that not one but two distinct hormone-based fields may be required for achieving temporal precision during formation of reiterative structures at the SAM, thus indicating an original mechanism for providing robustness to a dynamic developmental system.

Cost-effectiveness of the management of rh-negative pregnant women.

OBJECTIVE: The purpose of this study was to determine the most cost-effective option to prevent alloimmunization against the Rh factor. METHODS: A virtual population of Rh-negative pregnant women in Quebec was built to simulate the cost-effectiveness of preventing alloimmunization. The model considered four options: (1) systematic use of anti-D immunoglobulin; (2) fetal Rh(D) genotyping; (3) immunological determination of the father's Rh type; (4) mixed screening: immunological determination of the father's Rh type, followed if positive by fetal Rh(D) genotyping. Two outcomes were considered, in addition to the estimated costs: (1) the number of babies without hemolytic disease, and (2) the number of surviving infants. RESULTS: In a first pregnancy, two options emerged as the most cost-effective options: systematic prophylaxis and immunological Rh typing of the father, with overlapping confidence intervals between them. In a second pregnancy, the results were similar. In all cases (first or second pregnancy or a combination of the two) fetal genotyping was not found to be a cost-effective option. CONCLUSION: Routine prophylaxis and immunological Rh typing of the father are the most cost-effective options for the prevention of Rh alloimmunization. Considering that immunological typing of the father would probably not be carried out by the majority of clinicians, routine prophylaxis remains the preferred option. However, this could change if the cost of Rh(D) fetal genotyping fell below $140 per sample.

Objectif : Cette étude avait pour objectif d'identifier l'option la plus rentable pour la prévention de l'allo-immunisation contre le facteur Rh. Méthodes : Une population virtuelle québécoise de femmes enceintes séronégatives pour le facteur Rh a été créée pour simuler la rentabilité de la prévention de l'allo-immunisation. Ce modèle a pris en considération quatre options : (1) l'utilisation systématique d'immunoglobuline anti-D; (2) le génotypage Rh(D) fœtal; (3) la détermination immunologique du type Rh du père; (4) le dépistage mixte : détermination immunologique du type Rh du père, suivie (en présence de résultats positifs) du génotypage Rh(D) fœtal. Deux critères d'évaluation ont été pris en considération, en plus des coûts estimés : (1) le nombre d'enfants nés sans maladie hémolytique et (2) le nombre de nouveau-nés survivants. Résultats : Dans le cas d'une première grossesse, deux options se sont avérées les plus rentables : la prophylaxie systématique et la détermination immunologique du type Rh du père; leurs intervalles de confiance se chevauchaient. Dans le cas d'une deuxième grossesse, les résultats ont été semblables. Dans tous les cas (première ou deuxième grossesse, ou une combinaison des deux), nous avons constaté que le génotypage fœtal ne constituait pas une option rentable. Conclusion : La mise en œuvre systématique d'une prophylaxie et la détermination immunologique du type Rh du père constituent les options les plus rentables pour la prévention de l'allo-immunisation contre le facteur Rh. Puisqu'il est peu probable que la détermination immunologique du type Rh du père soit mise en œuvre par la majorité des cliniciens, la prophylaxie systématique demeure l'option à privilégier. Cependant, cela pourrait changer si le coût du génotypage Rh(D) fœtal chutait en deçà de 140 $ par prélèvement.

Quantifying the contribution of chromatin dynamics to stochastic gene expression reveals long, locus-dependent periods between transcriptional bursts.

BACKGROUND: A number of studies have established that stochasticity in gene expression may play an important role in many biological phenomena. This therefore calls for further investigations to identify the molecular mechanisms at stake, in order to understand and manipulate cell-to-cell variability. In this work, we explored the role played by chromatin dynamics in the regulation of stochastic gene expression in higher eukaryotic cells. RESULTS: For this purpose, we generated isogenic chicken-cell populations expressing a fluorescent reporter integrated in one copy per clone. Although the clones differed only in the genetic locus at which the reporter was inserted, they showed markedly different fluorescence distributions, revealing different levels of stochastic gene expression. Use of chromatin-modifying agents showed that direct manipulation of chromatin dynamics had a marked effect on the extent of stochastic gene expression. To better understand the molecular mechanism involved in these phenomena, we fitted these data to a two-state model describing the opening/closing process of the chromatin. We found that the differences between clones seemed to be due mainly to the duration of the closed state, and that the agents we used mainly seem to act on the opening probability. CONCLUSIONS: In this study, we report biological experiments combined with computational modeling, highlighting the importance of chromatin dynamics in stochastic gene expression. This work sheds a new light on the mechanisms of gene expression in higher eukaryotic cells, and argues in favor of relatively slow dynamics with long (hours to days) periods of quiet state.

The auxin signalling network translates dynamic input into robust patterning at the shoot apex.

The plant hormone auxin is thought to provide positional information for patterning during development. It is still unclear, however, precisely how auxin is distributed across tissues and how the hormone is sensed in space and time. The control of gene expression in response to auxin involves a complex network of over 50 potentially interacting transcriptional activators and repressors, the auxin response factors (ARFs) and Aux/IAAs. Here, we perform a large-scale analysis of the Aux/IAA-ARF pathway in the shoot apex of Arabidopsis, where dynamic auxin-based patterning controls organogenesis. A comprehensive expression map and full interactome uncovered an unexpectedly simple distribution and structure of this pathway in the shoot apex. A mathematical model of the Aux/IAA-ARF network predicted a strong buffering capacity along with spatial differences in auxin sensitivity. We then tested and confirmed these predictions using a novel auxin signalling sensor that reports input into the signalling pathway, in conjunction with the published DR5 transcriptional output reporter. Our results provide evidence that the auxin signalling network is essential to create robust patterns at the shoot apex.

Duplicate maize Wrinkled1 transcription factors activate target genes involved in seed oil biosynthesis.

WRINKLED1 (WRI1), a key regulator of seed oil biosynthesis in Arabidopsis (Arabidopsis thaliana), was duplicated during the genome amplification of the cereal ancestor genome 90 million years ago. Both maize (Zea mays) coorthologs ZmWri1a and ZmWri1b show a strong transcriptional induction during the early filling stage of the embryo and complement the reduced fatty acid content of Arabidopsis wri1-4 seeds, suggesting conservation of molecular function. Overexpression of ZmWri1a not only increases the fatty acid content of the mature maize grain but also the content of certain amino acids, of several compounds involved in amino acid biosynthesis, and of two intermediates of the tricarboxylic acid cycle. Transcriptomic experiments identified 18 putative target genes of this transcription factor, 12 of which contain in their upstream regions an AW box, the cis-element bound by AtWRI1. In addition to functions related to late glycolysis and fatty acid biosynthesis in plastids, the target genes also have functions related to coenzyme A biosynthesis in mitochondria and the production of glycerol backbones for triacylglycerol biosynthesis in the cytoplasm. Interestingly, the higher seed oil content in ZmWri1a overexpression lines is not accompanied by a reduction in starch, thus opening possibilities for the use of the transgenic maize lines in breeding programs.

Determination of glomerular filtration rate in anesthetized pigs by use of three-phase whole-kidney computed tomography and Patlak plot analysis.

OBJECTIVE: To develop a whole-kidney computed tomography (CT) technique that would allow 3-point Patlak plot determination of glomular filtration rate (GFR) and assess the correlation of GFR determined via CT (CT-GFR) with GFR determined via renal plasma clearance of inulin (Inu-GFR) in pigs. ANIMALS: 6 healthy anesthetized pigs. PROCEDURES: Each pig underwent 3-phase whole-kidney helical CT (arterial, early, and late parenchymal phases) before and after contrast medium administration. After contrast medium administration, corrected Hounsfield unit values were determined for each kidney and the aorta. A 3-point Patlak plot for each kidney was generated, and plasma clearance per unit volume was multiplied by renal volume to obtain whole-animal CT-GFR. Correlations of mean Inu-GFR for the left and right kidneys (and combined [total] values) with the corresponding CT-GFRs were assessed via linear regression and Bland-Altman analyses. RESULTS: Left kidney, right kidney, and total CT-GFRs were good predictors of the respective Inu-GFR values (r(2) = 92.3%, r(2) = 85.5%, and r(2) = 93.7%, respectively). For the left kidney, no significant bias between Inu-GFR and CT-GFR was detected. Right kidney and total CT-GFRs underestimated the corresponding Inu-GFRs (mean underestimation, -8.4 mL*min(1) and -12.6 mL*min(1), respectively). CONCLUSIONS AND CLINICAL RELEVANCE: Three-phase whole-kidney CT with Patlak plot analysis of GFR may underestimate right kidney and total Inu-GFRs in pigs. The Patlak plot generated may be sensitive to nonlinearity caused by temporal variation in GFR. Nonetheless, the 3-phase CT approach offers some practical advantages for simultaneous evaluation of renal morphology and measurement of GFR.

Evidence for non-linear pharmacokinetics of oxytocin in anesthetizetized rat.

PURPOSE: Because oxytocin (OT) is potentially useful in cardiovascular therapy but has hormonal roles on the cardiovascular and renal systems, we characterized its pharmacokinetic (PK) properties as a function of dose. METHODS: A single intravenous bolus of OT was given at doses of 200, 300, 500, 1000, 3000, 5000 and 10000 ng/kg to anesthetized male rats (n >= 4 per dose). Blood samples (6) were taken over 72 min to 150 min, depending on dose. The individual time-courses of plasma OT concentrations were analyzed with a one- or an open two-compartment PK model. Kruskal-Wallis tests (alpha=0.05) were used to compare the PK parameters among groups. RESULTS: At doses up to 500 ng/kg, OT showed a higher median systemic clearance (CLT = 0.0624 L/(min*kg); 0.0622 +/- 0.0228 as mean +/- SD value), a higher median central compartment volume of distribution (VC = 0.7906 L/kg; 0.6961 +/- 0.1754), and a lower median elimination half life (t(1/2)(lambdaz) 7.94 min; 9.08 +/- 4.3) with respect to the higher doses (CLT = 0.0266 L/(min*kg); 0.0284 +/- 0.0098, VC = 0.2213 L/kg; 0.2227 +/- 0.1142, and t(1/2)(lambdaz) 21.09 min; 28.36 +/- 21.8), all differences being significant (p 0.0008). Minimal differences were found for the estimates of these PK parameters among the 4 higher OT doses. CONCLUSION: The PK properties and persistence of exogenous OT are not proportional to dose, therefore this must be accounted for in dosing regimen design for potential cardiovascular therapy.

Single-slice dynamic computed tomographic determination of glomerular filtration rate by use of Patlak plot analysis in anesthetized pigs.

OBJECTIVE: To compare glomerular filtration rate (GFR) as estimated from Patlak plot analysis by use of single-slice computed tomography (CT) with that obtained from clearance of plasma inulin in pigs. ANIMALS: 8 healthy anesthetized juvenile pigs. PROCEDURES: All pigs underwent precontrast, whole-kidney, helical CT; postcontrast single-slice dynamic CT; and postcontrast, whole-kidney CT for volume determination. On dynamic images, corrected Hounsfield unit values were determined for each kidney and the aorta. A Patlak plot for each kidney was generated, and plasma clearance per unit volume was multiplied by renal volume to obtain whole-animal contrast clearance. Mean GFR determined via inulin clearance (Inu-GFR) was measured from each kidney and correlated to mean GFR determined via CT (CT-GFR) for the left kidney, right kidney, and both kidneys by use of linear regression and Bland-Altman analyses. RESULTS: CT-GFR results from 7 pigs were valid. Total and right kidney Inu-GFR were correlated with total and right kidney CT-GFR (total, R(2) = 0.85; right kidney, R(2) = 0.86). However, left kidney CT-GFR was poorly correlated with left kidney Inu-GFR (R(2) = 0.47). Bland-Altman analysis revealed no significant bias between Inu-GFR and CT-GFR for the left kidney, right kidney, or both kidneys. CONCLUSIONS AND CLINICAL RELEVANCE: CT-GFR as determined by use of a single-slice acquisition technique, low-dose of iohexol, and Patlak plot analysis correlated without bias with Inu-GFR for the right kidney and both kidneys (combined). This technique has promise as an accurate CT-GFR method that can be combined with renal morphologic evaluation.