New features on the expression and trafficking of mglur1 splice variants exposed by two novel mutant mouse lines

Metabotropic glutamate receptors (mGluRs) couple to G-proteins to modulate slow synaptic transmission via intracellular second messengers. The first cloned mGluR, mGluR1, regulates motor coordination, synaptic plasticity and synapse elimination. mGluR1 undergoes alternative splicing giving rise to four translated variants that differ in their intracellular C-terminal domains. Our current knowledge about mGluR1 relates almost entirely to the long mGluR1α isoform, whereas little is known about the other shorter variants. To study the expression of mGluR1γ, we have generated by means of the CRISPR/Cas9 system a new knock-in (KI) mouse line in which the C-terminus of this variant carries two short tags. Using this mouse line, we could establish that mGluR1γ is either untranslated or in amounts that are undetectable in the mouse cerebellum, indicating that only mGluR1α and mGluR1β are present and active at cerebellar synapses. The trafficking and function of mGluR1 appear strongly influenced by adaptor proteins such as long Homers that bind to the C-terminus of mGluR1α. We generated a second transgenic (Tg) mouse line in which mGluR1α carries a point mutation in its Homer binding domain and studied whether disruption of this interaction influenced mGluR1 subcellular localization at cerebellar parallel fiber (PF)-Purkinje cell (PC) synapses by means of the freeze-fracture replica immunolabeling technique. These Tg animals did not show any overt behavioral phenotype, and despite the typical mGluR1 perisynaptic distribution was not significantly changed, we observed a higher probability of intrasynaptic diffusion suggesting that long Homers regulate the lateral mobility of mGluR1. We extended our ultrastructural analysis to other mouse lines in which only one mGluR1 variant was reintroduced in PC of mGluR1-knock out (KO) mice. This work revealed that mGluR1α preferentially accumulates closer to the edge of the postsynaptic density (PSD), whereas mGluR1β has a less pronounced perijunctional distribution and, in the absence of mGluR1α, its trafficking to the plasma membrane is impaired with an accumulation in intracellular organelles. In conclusion, our study sets several firm points on largely disputed matters, namely expression of mGluR1γ and role of the C-terminal domain of mGluR1 splice variants on their perisynaptic clustering

Sex-specific associations in multiparametric 3 T MRI measurements in adult livers

BackgroundMRI relaxometry mapping and proton density fat fraction (PDFF) have been proposed for the evaluation of hepatic fibrosis. However, sex-specific relationships of age and body fat with these MRI parameters have not been studied in detail among adults without clinically manifest hepatic disease. We aimed to determine the sex-specific correlation of multiparametric MRI parameters with age and body fat and to evaluate their interplay associations.Methods147 study participants (84 women, mean age 48±14 years, range 19-85 years) were prospectively enrolled. 3 T MRI including T1, T2 and T1ρ mapping and PDFF and R2* map were acquired. Visceral and subcutaneous fat were measured on the fat images from Dixon water-fat separation sequence.ResultsAll MRI parameters demonstrated sex difference except for T1ρ. PDFF was more related to visceral than subcutaneous fat. Per 100 ml gain of visceral or subcutaneous fat is associated with 1 or 0.4% accretion of liver fat, respectively. PDFF and R2* were higher in men (both P = 0.01) while T1 and T2 were higher in women (both P P P P ConclusionVisceral fat plays an essential role in the elevated liver fat. When using MRI parametric measures for liver disease evaluation, the interplay between these parameters should be considered.Radiolog

Lessons learned in developing family medicine residency training programs in Japan

BACKGROUND: While family medicine is not well established as a discipline in Japan, a growing number of Japanese medical schools and training hospitals have recently started sougoushinryoubu (general medicine departments). Some of these departments are incorporating a family medicine approach to residency training. We sought to learn from family medicine pioneers of these programs lessons for developing residency training. METHODS: This qualitative project utilized a long interview research design. Questions focused on four topics: 1) circumstances when becoming chair/faculty member; 2) approach to starting the program; 3) how Western ideas of family medicine were incorporated; and 4) future directions. We analyzed the data using immersion/crystallization to identify recurring themes. From the transcribed data, we selected representative quotations to illustrate them. We verified the findings by emailing the participants and obtaining feedback. RESULTS: Participants included: five chairpersons, two program directors, and three faculty members. We identified five lessons: 1) few people understand the basic concepts of family medicine; 2) developing a core curriculum is difficult; 3) start with undergraduates; 4) emphasize clinical skills; and 5) train in the community. CONCLUSION: While organizational change is difficult, the identified lessons suggest issues that merit consideration when developing a family medicine training program. Lessons from complexity science could inform application of these insights in other countries and settings newly developing residency training

Lrp4 Modulates Extracellular Integration of Cell Signaling Pathways in Development

The extent to which cell signaling is integrated outside the cell is not currently appreciated. We show that a member of the low-density receptor-related protein family, Lrp4 modulates and integrates Bmp and canonical Wnt signalling during tooth morphogenesis by binding the secreted Bmp antagonist protein Wise. Mouse mutants of Lrp4 and Wise exhibit identical tooth phenotypes that include supernumerary incisors and molars, and fused molars. We propose that the Lrp4/Wise interaction acts as an extracellular integrator of epithelial-mesenchymal cell signaling. Wise, secreted from mesenchyme cells binds to BMP's and also to Lrp4 that is expressed on epithelial cells. This binding then results in the modulation of Wnt activity in the epithelial cells. Thus in this context Wise acts as an extracellular signaling molecule linking two signaling pathways. We further show that a downstream mediator of this integration is the Shh signaling pathway

Morphometrics as an Insight Into Processes Beyond Tooth Shape Variation in a Bank Vole Population

Phenotype variation is a key feature in evolution, being produced by development and the target of the screening by selection. We focus here on a variable morphological feature: the third upper molar (UM3) of the bank vole, aiming at identifying the sources of this variation. Size and shape of the UM3 occlusal surface was quantified in successive samples of a bank vole population. The first source of variation was the season of trapping, due to differences in the age structure of the population in turn affecting the wear of the teeth. The second direction of variation corresponded to the occurrence, or not, of an additional triangle on the tooth. This intra-specific variation was attributed to the space available at the posterior end of the UM3, allowing or not the addition of a further triangle.This size variation triggering the shape polymorphism is not controlled by the developmental cascade along the molar row. This suggests that other sources of size variation, possibly epigenetic, might be involved. They would trigger an important shape variation as side-effect by affecting the termination of the sequential addition of triangles on the tooth

G Protein Subunit Dissociation and Translocation Regulate Cellular Response to Receptor Stimulation

We examined the role of G proteins in modulating the response of living cells to receptor activation. The response of an effector, phospholipase C-β to M3 muscarinic receptor activation was measured using sensors that detect the generation of inositol triphosphate or diacylglycerol. The recently discovered translocation of Gβγ from plasma membrane to endomembranes on receptor activation attenuated this response. A FRET based G protein sensor suggested that in contrast to translocating Gβγ, non-translocating Gβγ subunits do not dissociate from the αq subunit on receptor activation leading to prolonged retention of the heterotrimer state and an accentuated response. M3 receptors with tethered αq induced differential responses to receptor activation in cells with or without an endogenous translocation capable γ subunit. G protein heterotrimer dissociation and βγ translocation are thus unanticipated modulators of the intensity of a cell's response to an extracellular signal

Detailed analysis of X chromosome inactivation in a 49,XXXXX pentasomy

<p>Abstract</p> <p>Background</p> <p>Pentasomy X (49,XXXXX) has been associated with a severe clinical condition, presumably resulting from failure or disruption of X chromosome inactivation. Here we report that some human X chromosomes from a patient with 49,XXXXX pentasomy were functionally active following isolation in inter-specific (human-rodent) cell hybrids. A comparison with cytogenetic and molecular findings provided evidence that more than one active X chromosome was likely to be present in the cells of this patient, accounting for her abnormal phenotype.</p> <p>Results</p> <p>5-bromodeoxyuridine (BrdU)-pulsed cultures showed different patterns among late replicating X chromosomes suggesting that their replication was asynchronic and likely to result in irregular inactivation. Genotyping of the proband and her mother identified four maternal and one paternal X chromosomes in the proband. It also identified the paternal X chromosome haplotype (P), indicating that origin of this X pentasomy resulted from two maternal, meiotic non-disjunctions. Analysis of the <it>HUMANDREC </it>region of the androgen receptor (<it>AR</it>) gene in the patient's mother showed a skewed inactivation pattern, while a similar analysis in the proband showed an active paternal X chromosome and preferentially inactivated X chromosomes carrying the 173 <it>AR </it>allele. Analyses of 33 cell hybrid cell lines selected in medium containing hypoxanthine, aminopterin and thymidine (HAT) allowed for the identification of three maternal X haplotypes (M1, M2 and MR) and showed that X chromosomes with the M1, M2 and P haplotypes were functionally active. In 27 cell hybrids in which more than one X haplotype were detected, analysis of X inactivation patterns provided evidence of preferential inactivation.</p> <p>Conclusion</p> <p>Our findings indicated that 12% of X chromosomes with the M1 haplotype, 43.5% of X chromosomes with the M2 haplotype, and 100% of the paternal X chromosome (with the P haplotype) were likely to be functionally active in the proband's cells, a finding indicating that disruption of X inactivation was associated to her severe phenotype.</p

Gene Expression Profiling of a Mouse Model of Pancreatic Islet Dysmorphogenesis

In the past decade, several transcription factors critical for pancreas organogenesis have been identified. Despite this success, many of the factors necessary for proper islet morphogenesis and function remain uncharacterized. Previous studies have shown that transgenic over-expression of the transcription factor Hnf6 specifically in the pancreatic endocrine cell lineage resulted in disruptions in islet morphogenesis, including dysfunctional endocrine cell sorting, increased individual islet size, increased number of peripheral endocrine cell types, and failure of islets to migrate away from the ductal epithelium. The mechanisms whereby maintained Hnf6 causes defects in islet morphogenesis have yet to be elucidated.We exploited the dysmorphic islets in Hnf6 transgenic animals as a tool to identify factors important for islet morphogenesis. Genome-wide microarray analysis was used to identify differences in the gene expression profiles of late gestation and early postnatal total pancreas tissue from wild type and Hnf6 transgenic animals. Here we report the identification of genes with an altered expression in Hnf6 transgenic animals and highlight factors with potential importance in islet morphogenesis. Importantly, gene products involved in cell adhesion, cell migration, ECM remodeling and proliferation were found to be altered in Hnf6 transgenic pancreata, revealing specific candidates that can now be analyzed directly for their role in these processes during islet development.This study provides a unique dataset that can act as a starting point for other investigators to explore the role of the identified genes in pancreatogenesis, islet morphogenesis and mature beta cell function