Normal X-inactivation mosaicism in corneas of heterozygous FlnaDilp2/+ female mice--a model of human Filamin A (FLNA) diseases

<p>Abstract</p> <p>Background</p> <p>Some abnormalities of mouse corneal epithelial maintenance can be identified by the atypical mosaic patterns they produce in X-chromosome inactivation mosaics and chimeras. Human <it>FLNA</it>/+ females, heterozygous for X-linked, filamin A gene (<it>FLNA</it>) mutations, display a range of disorders and X-inactivation mosaicism is sometimes quantitatively unbalanced. <it>Flna</it>^{<it>Dilp2/+ </it>}mice, heterozygous for an X-linked filamin A (<it>Flna</it>) nonsense mutation have variable eye, skeletal and other abnormalities, but X-inactivation mosaicism has not been investigated. The aim of this study was to determine whether X-inactivation mosaicism in the corneal epithelia of <it>Flna</it>^{<it>Dilp2/+ </it>}mice was affected in any way that might predict abnormal corneal epithelial maintenance.</p> <p>Results</p> <p>X-chromosome inactivation mosaicism was studied in the corneal epithelium and a control tissue (liver) of <it>Flna</it>^{<it>Dilp2/+ </it>}and wild-type (WT) female X-inactivation mosaics, hemizygous for the X-linked, <it>LacZ </it>reporter H253 transgene, using β-galactosidase histochemical staining. The corneal epithelia of <it>Flna</it>^{<it>Dilp2/+ </it>}and WT X-inactivation mosaics showed similar radial, striped patterns, implying epithelial cell movement was not disrupted in <it>Flna</it>^{<it>Dilp2/+ </it>}corneas. Corrected stripe numbers declined with age overall (but not significantly for either genotype individually), consistent with previous reports suggesting an age-related reduction in stem cell function. Corrected stripe numbers were not reduced in <it>Flna</it>^{<it>Dilp2/+ </it>}compared with WT X-inactivation mosaics and mosaicism was not significantly more unbalanced in the corneal epithelia or livers of <it>Flna</it>^{<it>Dilp2/+ </it>}than wild-type <it>Flna^+/+</it>X-inactivation mosaics.</p> <p>Conclusions</p> <p>Mosaic analysis identified no major effect of the mouse <it>Flna^Dilp2</it>mutation on corneal epithelial maintenance or the balance of X-inactivation mosaicism in the corneal epithelium or liver.</p

Effects of Aberrant Pax6 Gene Dosage on Mouse Corneal Pathophysiology and Corneal Epithelial Homeostasis

Background: Altered dosage of the transcription factor PAX6 causes multiple human eye pathophysiologies. PAX6(+/-) heterozygotes suffer from aniridia and aniridia-related keratopathy (ARK), a corneal deterioration that probably involves a limbal epithelial stem cell (LESC) deficiency. Heterozygous Pax6(+/Sey-Neu) (Pax6(+/-)) mice recapitulate the human disease and are a good model of ARK. Corneal pathologies also occur in other mouse Pax6 mutants and in PAX77(Tg/-) transgenics, which over-express Pax6 and model human PAX6 duplication. Methodology/Principal Findings: We used electron microscopy to investigate ocular defects in Pax6(+/-) heterozygotes (low Pax6 levels) and PAX77(Tg/-) transgenics (high Pax6 levels). As well as the well-documented epithelial defects, aberrant Pax6 dosage had profound effects on the corneal stroma and endothelium in both genotypes, including cellular vacuolation, similar to that reported for human macular corneal dystrophy. We used mosaic expression of an X-linked LacZ transgene in X-inactivation mosaic female (XLacZ(Tg/-)) mice to investigate corneal epithelial maintenance by LESC clones in Pax6(+/-) and PAX77(Tg/-) mosaic mice. PAX77(Tg/-) mosaics, over-expressing Pax6, produced normal corneal epithelial radial striped patterns (despite other corneal defects), suggesting that centripetal cell movement was unaffected. Moderately disrupted patterns in Pax6(+/-) mosaics were corrected by introducing the PAX77 transgene (in Pax6(+/-), PAX77(Tg/-) mosaics). Pax6(Leca4/+), XLacZ(Tg/-) mosaic mice (heterozygous for the Pax6(Leca4) missense mutation) showed more severely disrupted mosaic patterns. Corrected corneal epithelial stripe numbers (an indirect estimate of active LESC clone numbers) declined with age (between 15 and 30 weeks) in wild-type XLacZ(Tg/-) mosaics. In contrast, corrected stripe numbers were already low at 15 weeks in Pax6(+/-) and PAX77(Tg/-) mosaic corneas, suggesting Pax6 under-and over-expression both affect LESC clones. Conclusions/Significance: Pax6(+/-) and PAX77(Tg/-) genotypes have only relatively minor effects on LESC clone numbers but cause more severe corneal endothelial and stromal defects. This should prompt further investigations of the pathophysiology underlying human aniridia and ARK

The insecure airway: a comparison of knots and commercial devices for securing endotracheal tubes

BACKGROUND: Endotracheal Tubes (ETTs) are commonly secured using adhesive tape, cloth tape, or commercial devices. The objectives of the study were (1) To compare degrees of movement of ETTs secured with 6 different commercial devices and (2) To compare movement of ETTs secured with cloth tape tied with 3 different knots (hitches). METHODS: A 17 cm diameter PVC tube with 14 mm "mouth" hole in the side served as a mannequin. ETTs were subjected to repeated jerks, using a cable and pulley system. Measurements: (1) Total movement of ETTs relative to "mouth" (measure used for devices) (2) Slippage of ETT through securing knot (measure used for knots). RESULTS: Among commercial devices, the Dale(® )showed less movement than other devices, although some differences between devices did not reach significance. Among knots, Magnus and Clove Hitches produced less slippage than the Cow Hitch, but these differences did not reach statistical significance. CONCLUSION: Among devices tested, the Dale(® )was most secure. Within the scope offered by the small sample sizes, there were no statistically significant differences between the knots in this study

Interaction between drug and placebo effects: a cross-over balanced placebo design trial

<p>Abstract</p> <p>Background</p> <p>The total effect of a medication is the sum of its drug effect, placebo effect (meaning response), and their possible interaction. Current interpretation of clinical trials' results assumes no interaction. Demonstrating such an interaction has been difficult due to lack of an appropriate study design.</p> <p>Methods</p> <p>180 adults were randomized to caffeine (300 mg) or placebo groups. Each group received the assigned intervention described by the investigators as caffeine or placebo, in a randomized crossover design. 4-hour-area-under-the-curve of energy, sleepiness, nausea (on 100 mm visual analog scales), and systolic blood pressure levels as well as caffeine pharmacokinetics (in 22 volunteers nested in the caffeine group) were determined. Caffeine drug, placebo, placebo-plus-interaction, and total effects were estimated by comparing outcomes after, receiving caffeine described as placebo to receiving placebo described as placebo, receiving placebo described as caffeine or placebo, receiving caffeine described as caffeine or placebo, and receiving caffeine described as caffeine to receiving placebo described as placebo, respectively.</p> <p>Results</p> <p>The placebo effect on area-under-the-curve of energy (mean difference) and sleepiness (geometric mean ratio) was larger than placebo-plus-interaction effect (16.6 [95% CI, 4.1 to 29.0] vs. 8.4 [-4.2 to 21.0] mm*hr and 0.58 [0.39 to 0.86] vs. 0.69 [0.49 to 0.97], respectively), similar in size to drug effect (20.8 [3.8 to 37.8] mm*hr and 0.49 [0.30 to 0.91], respectively), and its combination with the later was larger than total caffeine effect (29.5 [11.9 to 47.1] mm*hr and 0.37 [0.22 to 0.64]). Placebo-plus-interaction effect increased caffeine terminal half-life by 0.40 [0.12 to 0.68] hr (P = 0.007).</p> <p>Conclusions</p> <p>Drug and placebo effects of a medication may be less than additive, which influences the interpretation of clinical trials. The placebo effect may increase active drug terminal half-life, a novel mechanism of placebo action.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov identification number - NCT00426010.</p

Three Dimensional Visualization and Fractal Analysis of Mosaic Patches in Rat Chimeras: Cell Assortment in Liver, Adrenal Cortex and Cornea

The production of organ parenchyma in a rapid and reproducible manner is critical to normal development. In chimeras produced by the combination of genetically distinguishable tissues, mosaic patterns of cells derived from the combined genotypes can be visualized. These patterns comprise patches of contiguously similar genotypes and are different in different organs but similar in a given organ from individual to individual. Thus, the processes that produce the patterns are regulated and conserved. We have previously established that mosaic patches in multiple tissues are fractal, consistent with an iterative, recursive growth model with simple stereotypical division rules. Fractal dimensions of various tissues are consistent with algorithmic models in which changing a single variable (e.g. daughter cell placement after division) switches the mosaic pattern from islands to stripes of cells. Here we show that the spiral pattern previously observed in mouse cornea can also be visualized in rat chimeras. While it is generally held that the pattern is induced by stem cell division dynamics, there is an unexplained discrepancy in the speed of cellular migration and the emergence of the pattern. We demonstrate in chimeric rat corneas both island and striped patterns exist depending on the age of the animal. The patches that comprise the pattern are fractal, and the fractal dimension changes with the age of the animal and indicates the constraint in patch complexity as the spiral pattern emerges. The spiral patterns are consistent with a loxodrome. Such data are likely to be relevant to growth and cell division in organ systems and will help in understanding how organ parenchyma are generated and maintained from multipotent stem cell populations located in specific topographical locations within the organ. Ultimately, understanding algorithmic growth is likely to be essential in achieving organ regeneration in vivo or in vitro from stem cell populations

Evidence That Descending Cortical Axons Are Essential for Thalamocortical Axons to Cross the Pallial-Subpallial Boundary in the Embryonic Forebrain

Developing thalamocortical axons traverse the subpallium to reach the cortex located in the pallium. We tested the hypothesis that descending corticofugal axons are important for guiding thalamocortical axons across the pallial-subpallial boundary, using conditional mutagenesis to assess the effects of blocking corticofugal axonal development without disrupting thalamus, subpallium or the pallial-subpallial boundary. We found that thalamic axons still traversed the subpallium in topographic order but did not cross the pallial-subpallial boundary. Co-culture experiments indicated that the inability of thalamic axons to cross the boundary was not explained by mutant cortex developing a long-range chemorepulsive action on thalamic axons. On the contrary, cortex from conditional mutants retained its thalamic axonal growth-promoting activity and continued to express Nrg-1, which is responsible for this stimulatory effect. When mutant cortex was replaced with control cortex, corticofugal efferents were restored and thalamic axons from conditional mutants associated with them and crossed the pallial-subpallial boundary. Our study provides the most compelling evidence to date that cortical efferents are required to guide thalamocortical axons across the pallial-subpallial boundary, which is otherwise hostile to thalamic axons. These results support the hypothesis that thalamic axons grow from subpallium to cortex guided by cortical efferents, with stimulation from diffusible cortical growth-promoting factors

LRIG1 is a gatekeeper to exit from quiescence in adult neural stem cells

Adult neural stem cells (NSCs) must tightly regulate quiescence and proliferation. Single-cell analysis has suggested a continuum of cell states as NSCs exit quiescence. Here we capture and characterize in vitro primed quiescent NSCs and identify LRIG1 as an important regulator. We show that BMP-4 signaling induces a dormant non-cycling quiescent state (d-qNSCs), whereas combined BMP-4/FGF-2 signaling induces a distinct primed quiescent state poised for cell cycle re-entry. Primed quiescent NSCs (p-qNSCs) are defined by high levels of LRIG1 and CD9, as well as an interferon response signature, and can efficiently engraft into the adult subventricular zone (SVZ) niche. Genetic disruption of Lrig1 in vivo within the SVZ NSCs leads an enhanced proliferation. Mechanistically, LRIG1 primes quiescent NSCs for cell cycle re-entry and EGFR responsiveness by enabling EGFR protein levels to increase but limiting signaling activation. LRIG1 is therefore an important functional regulator of NSC exit from quiescence

Increased Corneal Epithelial Turnover Contributes to Abnormal Homeostasis in the Pax6(+/-) Mouse Model of Aniridia

We aimed to test previous predictions that limbal epithelial stem cells (LESCs) are quantitatively deficient or qualitatively defective in Pax6(+/-) mice and decline with age in wild-type (WT) mice. Consistent with previous studies, corneal epithelial stripe patterns coarsened with age in WT mosaics. Mosaic patterns were also coarser in Pax6(+/-) mosaics than WT at 15 weeks but not at 3 weeks, which excludes a developmental explanation and strengthens the prediction that Pax6(+/-) mice have a LESC-deficiency. To investigate how Pax6 genotype and age affected corneal homeostasis, we compared corneal epithelial cell turnover and label-retaining cells (LRCs; putative LESCs) in Pax6(+/-) and WT mice at 15 and 30 weeks. Limbal BrdU-LRC numbers were not reduced in the older WT mice, so this analysis failed to support the predicted age-related decline in slow-cycling LESC numbers in WT corneas. Similarly, limbal BrdU-LRC numbers were not reduced in Pax6(+/-) heterozygotes but BrdU-LRCs were also present in Pax6(+/-) corneas. It seems likely that Pax6(+/-) LRCs are not exclusively stem cells and some may be terminally differentiated CD31-positive blood vessel cells, which invade the Pax6(+/-) cornea. It was not, therefore, possible to use this approach to test the prediction that Pax6(+/-) corneas had fewer LESCs than WT. However, short-term BrdU labelling showed that basal to suprabasal movement (leading to cell loss) occurred more rapidly in Pax6(+/-) than WT mice. This implies that epithelial cell loss is higher in Pax6(+/-) mice. If increased corneal epithelial cell loss exceeds the cell production capacity it could cause corneal homeostasis to become unstable, resulting in progressive corneal deterioration. Although it remains unclear whether Pax6(+/-) mice have LESC-deficiency, we suggest that features of corneal deterioration, that are often taken as evidence of LESC-deficiency, might occur in the absence of stem cell deficiency if corneal homeostasis is destabilised by excessive cell loss

Epigenetic remodelling licences adult cholangiocytes for organoid formation and liver regeneration.

Following severe or chronic liver injury, adult ductal cells (cholangiocytes) contribute to regeneration by restoring both hepatocytes and cholangiocytes. We recently showed that ductal cells clonally expand as self-renewing liver organoids that retain their differentiation capacity into both hepatocytes and ductal cells. However, the molecular mechanisms by which adult ductal-committed cells acquire cellular plasticity, initiate organoids and regenerate the damaged tissue remain largely unknown. Here, we describe that ductal cells undergo a transient, genome-wide, remodelling of their transcriptome and epigenome during organoid initiation and in vivo following tissue damage. TET1-mediated hydroxymethylation licences differentiated ductal cells to initiate organoids and activate the regenerative programme through the transcriptional regulation of stem-cell genes and regenerative pathways including the YAP-Hippo signalling. Our results argue in favour of the remodelling of genomic methylome/hydroxymethylome landscapes as a general mechanism by which differentiated cells exit a committed state in response to tissue damage.RCUK Cancer Research UK ERC H2020 Wellcome Trus

Genome-wide association analysis reveals QTL and candidate mutations involved in white spotting in cattle

International audienceAbstractBackgroundWhite spotting of the coat is a characteristic trait of various domestic species including cattle and other mammals. It is a hallmark of Holstein–Friesian cattle, and several previous studies have detected genetic loci with major effects for white spotting in animals with Holstein–Friesian ancestry. Here, our aim was to better understand the underlying genetic and molecular mechanisms of white spotting, by conducting the largest mapping study for this trait in cattle, to date.ResultsUsing imputed whole-genome sequence data, we conducted a genome-wide association analysis in 2973 mixed-breed cows and bulls. Highly significant quantitative trait loci (QTL) were found on chromosomes 6 and 22, highlighting the well-established coat color genes KIT and MITF as likely responsible for these effects. These results are in broad agreement with previous studies, although we also report a third significant QTL on chromosome 2 that appears to be novel. This signal maps immediately adjacent to the PAX3 gene, which encodes a known transcription factor that controls MITF expression and is the causal locus for white spotting in horses. More detailed examination of these loci revealed a candidate causal mutation in PAX3 (p.Thr424Met), and another candidate mutation (rs209784468) within a conserved element in intron 2 of MITF transcripts expressed in the skin. These analyses also revealed a mechanistic ambiguity at the chromosome 6 locus, where highly dispersed association signals suggested multiple or multiallelic QTL involving KIT and/or other genes in this region.ConclusionsOur findings extend those of previous studies that reported KIT as a likely causal gene for white spotting, and report novel associations between candidate causal mutations in both the MITF and PAX3 genes. The sizes of the effects of these QTL are substantial, and could be used to select animals with darker, or conversely whiter, coats depending on the desired characteristics