Control of the peroxisomal beta-oxidation pathway by a novel family of nuclear hormone receptors.

Three novel members of the Xenopus nuclear hormone receptor superfamily have been cloned. They are related to each other and similar to the group of receptors that includes those for thyroid hormones, retinoids, and vitamin D3. Their transcriptional activity is regulated by agents causing peroxisome proliferation and carcinogenesis in rodent liver. All three Xenopus receptors activate the promoter of the acyl coenzyme A oxidase gene, which encodes the key enzyme of peroxisomal fatty acid beta-oxidation, via a cognate response element that has been identified. Therefore, peroxisome proliferators may exert their hypolipidemic effects through these receptors, which stimulate the peroxisomal degradation of fatty acids. Finally, the multiplicity of these receptors suggests the existence of hitherto unknown cellular signaling pathways for xenobiotics and putative endogenous ligands.

The neuronal adhesion protein D2 in differentiating aggregates of brain cells.

The D2-protein is a high molecular weight protein involved in interneuronal adhesion. The concentration of D2-protein was measured both in aggregates of fetal rat telencephalic cells cultured in a chemically defined medium and in developing forebrain. Both the concentration of the D2-protein and the degree of sialylation were changed in the cultures in parallel with the corresponding values obtained from postnatal forebrain. In the cultures the highest specific concentration of D2-protein was observed after 12 days in culture. This value was 2.7 times higher than the average value of adult rat forebrain. Antibodies to D2-protein have previously been shown to inhibit fasciculation of neuritic fibers extending from cultured explants of sympathetic ganglia. We investigated the effect of such antibodies on the differentiation of aggregating telencephalic cells. By adding surplus antibodies to the cultures from day 11 to day 16 we were able to decrease the specific concentration of D2-protein on the neurons by 53% measured at day 19. The decrease was not compensated fully even after further 10 days in the culture. Although the concentration of D2-protein was decreased during the period of synaptogenesis no change was found in the specific concentration of a marker of mature synapses, the D3-protein. Thus, in this culture system synaptogenesis could proceed to an unimpaired extent in the presence of a decreased concentration of a putatively involved adhesion molecule. However, the specific concentration of two markers of myelination, 2',3'-cyclic nucleotide 3'-phosphodiesterase and myelin basic protein, were both increased, suggesting an antibody-induced stimulation of myelination in the cultured aggregates.

Intérêt pronostique du grade histologique FNLCC dans les tumeurs stromales gastro-intestinales : étude anatomo-clinique et immunohistochimique de 68 cas

Résumé Les tumeurs stromales gastro-intestinales sont des tumeurs conjonctives de malignité variable du tube digestif. Leur évolution est souvent difficilement prévisible, certaines tumeurs d'aspect histologique rassurant pouvant parfois donner des métastases. Plusieurs systèmes de grade histologique ont été développés pour essayer de prédire au mieux l'évolution de ces tumeurs. Aucun d'entre eux, n'a, jusqu'à présent, fait preuve d'une valeur universellement reconnue. Le but de notre étude était d'évaluer la valeur pronostique du système de grade histologique de la Fédération Nationale des Centres de Lutte Contre le Cancer (« grade français FNCLCC») au sein d'une population de patients porteurs d'une tumeur gastro-intestinale à potentiel de malignité significatif (GIST maligne). Ce système qui a été initialement développé pour les sarcomes des tissus mous de l'adulte, est actuellement reconnu comme le plus performant et le plus discriminant pour séparer les sarcomes de bas grade de malignité de ceux de haut grade. La valeur pronostique de ce système n'a jamais été foimellement examinée dans les GIST malignes. Soixante-huit tumeurs stromales gastro-intestinales malignes ont été étudiées sur le plan anatomo-clinique et immunohistochimique. Elles ont fait l'objet d'un immunomarquage par des anticorps dirigés contre les épitopes suivants : c-kit (anti-CD117), CD34, protéine S100, actine musculaire lise, desmine, énolase spécifique des neurones (NSE), cycline A et cycline D3. Seules les GIST malignes qui exprimaient le CD117, et dont la taille était ≥ 5 cm ou bien qui comportaient un nombre de mitoses ≥ 5 pour 50 champs microscopiques au fort grossissement ont été sélectionnées pour cette étude. Les analyses univariées ont montré que les patients qui présentaient une GIST localisée au moment du diagnostic avaient un pronostic nettement meilleur que ceux qui présentaient une extension péritonéale de la maladie, quelle que soit son étendue, et/ou une GIST extradigestive ( 72% versus 14% de survie à 5 ans ; p<0.0001). Ainsi, ces derniers patients doivent être tous considérés comme métastatiques d'emblée. Pour les patients avec une GIST localisée au départ (n=48), un grade histologique élevé (grade 3) et une expression partielle ou une absence d'expression du CD34 étaient les seuls facteurs associés à une réduction significative de la survie globale et à une réduction significative de la survie sans récidive de la maladie. Un indice de marquage nucléaire par la cycline D3 ≤50% était aussi associé à une réduction significative de la survie sans récidive de la maladie. La valeur pronostique du grade FNCLCC a été comparée à celles des autres systèmes actuellement utilisés et notamment à celle du système consensuel de Fletcher et coll. Les études univariées et multivariées ont montré que le grade FNCLCC permettait de mieux 111 sélectionner les patients à haut risque (p=0.048, n=48), c'est à dire ceux qui pourraient potentiellement tirer bénéfice d'un traitement adjuvant par imatinib mésylate. En conclusion, le grade ENCLCC est applicable aux tumeurs stromales gastro-intestinales «malignes ». Comparativement aux autres systèmes de grading et notamment au système consensuel de Fletcher et coll., il semble être celui qui sélectionne le mieux les patients à haut risque, qui seraient potentiellement candidats à un traitement adjuvant par imatinib mésylate (Glivec?). Une absence d'expression ou une expression seulement focale du CD34 par les G1ST semble être un facteur additionnel de mauvais pronostic. Le marquage par la cycline D3 est inversement proportionnel à l'agressivité tumorale.

Development of neuronal markers in aggregating fetal rat telencephalon cells cultured in the presence of triiodothyronine.

The concentrations of the general neuronal markers D2-protein (N-CAM), D3-protein and neuron specific enolase (NSE) in reaggregating cultures of fetal rat telencephalon cells were affected by the presence of 30 nM triiodothyronine in the defined culture medium. The extent of normal developmental changes were enhanced by triiodothyronine, as demonstrated by crossed immunoelectrophoresis. From 13 to 19 days in culture, the concentration of D2-protein decreased, and the concentrations of both D3-protein and NSE increased. Nerve growth factor (NGF) was without effect on the development of these general neuronal markers. However, as shown previously both triiodothyronine and NGF increased the activity of choline acetyltransferase, a marker for cholinergic neurons. The results suggest an enhanced overall differentiation of several types of telencephalon neurons in the presence of triiodothyronine, and a specific stimulation of cholinergic telencephalon neurons by NGF.

Pharmacological inhibition of fibroblast growth factor (FGF) receptor signaling ameliorates FGF23-mediated hypophosphatemic rickets.

Fibroblast growth factor 23 (FGF23) is a circulating factor secreted by osteocytes that is essential for phosphate homeostasis. In kidney proximal tubular cells FGF23 inhibits phosphate reabsorption and leads to decreased synthesis and enhanced catabolism of 1,25-dihydroxyvitamin D3 (1,25[OH]2 D3 ). Excess levels of FGF23 cause renal phosphate wasting and suppression of circulating 1,25(OH)2 D3 levels and are associated with several hereditary hypophosphatemic disorders with skeletal abnormalities, including X-linked hypophosphatemic rickets (XLH) and autosomal recessive hypophosphatemic rickets (ARHR). Currently, therapeutic approaches to these diseases are limited to treatment with activated vitamin D analogues and phosphate supplementation, often merely resulting in partial correction of the skeletal aberrations. In this study, we evaluate the use of FGFR inhibitors for the treatment of FGF23-mediated hypophosphatemic disorders using NVP-BGJ398, a novel selective, pan-specific FGFR inhibitor currently in Phase I clinical trials for cancer therapy. In two different hypophosphatemic mouse models, Hyp and Dmp1-null mice, resembling the human diseases XLH and ARHR, we find that pharmacological inhibition of FGFRs efficiently abrogates aberrant FGF23 signaling and normalizes the hypophosphatemic and hypocalcemic conditions of these mice. Correspondingly, long-term FGFR inhibition in Hyp mice leads to enhanced bone growth, increased mineralization, and reorganization of the disturbed growth plate structure. We therefore propose NVP-BGJ398 treatment as a novel approach for the therapy of FGF23-mediated hypophosphatemic diseases.

Genetics of essential tremor.

Essential tremor (ET) is a prevalent condition manifesting with progressive action tremor. Although ET was traditionally viewed as a sporadic disease, a significant proportion of cases report a positive family history of tremor. Autosomal dominant inheritance can be demonstrated in many families. Previously, genome-wide linkage studies in families mapped three loci for ET, hereditary essential tremor-1 (ETM1), ETM2 and ETM3. However, no causal mutation has been replicated in candidate genes within these loci, including dopamine D3 receptor (DRD3) and HS1-binding protein 3 (HS1BP3). Recently, the first genome-wide association study in ET followed by replication studies conducted in diverse populations identified a significant association between the leucine-rich repeat and Ig domain containing 1 gene (LINGO1) SNP rs9652490 and risk for ET Although further novel variants were indentified in LINGO1 and its paralog LINGO2 that may be associated with risk for ET, the pathogenic mechanisms involved remain elusive. Given the possibility that ET as a complex trait may be influenced by the combined effects of rare variants, novel high-throughput technologies sequencing all exons across the genome (exome sequencing) or the whole genome (genome sequencing) may become crucial in understanding/deciphering the genetic background of ET.

Hypertension et vitamine D: retour sur la scène [Hypertension and vitamin D: not again].

Vitamin D deficiency , defined by a 25-OH vitamin D3 plasma level < 30 ng/ml, is highly prevalent in the population. Several observational studies have suggested that such a deficiency increases the risk of hypertension development. Vitamin D seems to have an inhibitory effect on renin secretion and might by this mechanism exert an antihypertensive effect. Recent randomized trials have failed however to demonstrate a blood pressure lowering effect of vitamin D supplementation.

Synergistic effect of calcitriol and interferon-α on hepatitis C virus replication in vitro

Background and aims: V itamin D is an important modulator o fnumerous c ellular processes, including innate and adaptive immunepathways. A recent large-scale genetic validation study performed withinthe framework of the Swiss Hepatitis C Cohort S tudy has demonstratedan association between t he 1α-hydroxylase promoter single nucleotidepolymorphism CYP27B1-1260 rs10877012 and sustained virologicresponse (SVR) after pegylated interferon-α ( PEG-IFN-α) plus ribavirintreatment of c hronic hepatitis C in patients w ith a p oor-response IL28Bgenotype. This suggests an intrinsic role o f vitamin D signaling in theresponse t o treatment of chronic hepatitis C, especially in patients withlimited sensitivity to IFN-α. In the present study, we investigated theeffect of 1,25-(OH)2 v itamin D3 (calcitriol) alone or in combination withIFN-α on the hepatitis C virus (HCV) life cycle in vitro.Methods: H uh-7.5 cells harboring Con1- or JFH-1-derived HCVreplicons or cell culture-derived HCV were exposed to 0.1-100 nMcalcitriol ± 1 -100 IU/ml IFN-α. The effect on HCV RNA replication andviral particle production was investigated by quantitative r eal-time PCR,immunoblot analyses, and infectivity titration analyses. The expression ofinterferon-stimulated genes (ISGs) and of calcitriol target genes wasassessed by quantitative real-time PCR.Results: Calcitriol had no relevant effect on the viability of Huh-7.5 cells.Calcitriol strongly induced and repressed the expression of the calcitrioltarget genes CYP24A1 and CCNC, respectively, confirming that Huh-7.5cells c an respond to c alcitriol signaling. P hysiological doses of calcitrioldid not significantly a ffect HCV RNA replication or i nfectious particleproduction in vitro, and calcitriol alone h ad no significant effect on theexpression of several ISGs. However, calcitriol in combination with IFN-αsubstantially increased the expression of ISGs compared to IFN-α alone.In addition, calcitriol plus IFN-α s ynergistically inhibited HCV RNAreplication.Conclusions: C alcitriol at physiological concentrations and IFN-α a ctsynergistically on the expression of I SGs and HCV RNA replication i nvitro. Experiments exploring the underlying mechanisms are underway.

Evaluation de l'activité fonctionnelle des membres supérieurs chez des patients atteints de maladies neuromusculaires par mesures cinématiques (Physilog®)

Contexte :¦Les affections neuromusculaires sont des maladies rares, le plus souvent d'origine génétique et dont le degré de sévérité est variable. Certaines sont progressives et conduisent à une perte de l'indépendance motrice et à une insuffisance respiratoire, tandis que d'autres permettent une vie proche de la normale.¦Les progrès des connaissances en génétique moléculaire ont permis d'améliorer significativement la connaissance des mécanismes physiopathologiques de ces maladies et ont également ouverts la voie à de nouvelles possibilités thérapeutiques. Concernant les maladies les plus graves, le but premier de ces thérapies sera de tenter de préserver la marche indépendante le plus longtemps possible. Toutefois, les patients ayant déjà perdu la marche et qui dépendent de la fonction de leur membres supérieurs pour garder une certaine indépendance devront également pouvoir être inclus dans des essais thérapeutiques et bénéficier des traitements reconnus efficaces.¦De nouveaux moyens de mesure de la fonction motrice des membres supérieurs doivent être développés non seulement dans la perspective de ces éventuels essais thérapeutiques pour ces patients limités à la chaise roulante, mais également dans le but de pouvoir évaluer plus précisément l'histoire naturelle des maladies même après la perte de la marche indépendante.¦Objectifs:¦Evaluer la fonction motrice des membres supérieurs chez des patients myopathes ayant perdu la marche indépendante par mesure cinématique.¦Méthode :¦Mesure de l'activité des membres supérieurs et le profil de cette activité par mesure cinématique (Physilog) chez:¦-Vingt-cinq patients atteints d'une myopathie en chaise roulante.¦-Vingt-cinq sujets contrôle assis¦Ces mesures seront comparées aux scores obtenus à l'échelle de Mesure de Fonction Motrice (Bérard et al, 2005) et plus précisément à l'axe D3 de cette échelle qui évalue les fonctions distales.¦Les patients seront vus à deux reprises pour une période de deux fois deux heures pendant laquelle ils porteront le Physilog. Il leur sera demandé d'effectuer une série d'exercices afin d'évaluer la fonction motrice des membres supérieurs (manger un yaourt avec une cuillère, verser un verre d'eau et le boire, se toucher selon les possibilités la tête, le front, la bouche, le cou, jouer à la console Wii) Le score de Brooke sera également testé. Chaque exercice sera répété plusieurs fois afin de d'assurer de la qualité des résultats.¦Système portable Physilog :¦Physilog est un système de mesure ambulatoire. La version utilisée dans cette étude est composée de 2 capteurs miniatures et d'une unité numérique portable légère (~215 g), qui filtre, amplifie et sauvegarde les signaux fournis par les capteurs sur une carte mémoire (Physilog-BioAGM, CH).¦Afin de pouvoir enregistrer l'activité des deux membres supérieurs, deux appareils Physilog (4 capteurs) seront utilisés par patient. Deux capteurs seront fixés sur chaque membre supérieur. Les capteurs sont fixés sur la peau à l'aide d'une bande autocollante.¦Les capteurs mis au point par le LMAM (laboratoire de mesure et d'analyse du mouvement) mesurent la rotation angulaire et l'accélération au niveau des membres supérieurs. L'algorithme utilisé dans cette étude a été développé dans le laboratoire LMAM à l'EPFL et a fait l'objet d'une thèse.¦Résultats escomptés :¦Les résultats de l'étude pilote montrent que le Physilog permet de quantifier de manière fiable et reproductible la fonction des membres supérieurs durant des activités courantes de la vie quotidienne et pourrait donc devenir un moyen d'évaluation fiable des nouveaux traitements.

Genetic analyses reveal a role for vitamin D insufficiency in HCV-associated hepatocellular carcinoma development

BACKGROUND: Vitamin D insufficiency has been associated with the occurrence of various types of cancer, but causal relationships remain elusive. We therefore aimed to determine the relationship between genetic determinants of vitamin D serum levels and the risk of developing hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). METHODOLOGY/PRINCIPAL FINDINGS: Associations between CYP2R1, GC, and DHCR7 genotypes that are determinants of reduced 25-hydroxyvitamin D (25[OH]D3) serum levels and the risk of HCV-related HCC development were investigated for 1279 chronic hepatitis C patients with HCC and 4325 without HCC, respectively. The well-known associations between CYP2R1 (rs1993116, rs10741657), GC (rs2282679), and DHCR7 (rs7944926, rs12785878) genotypes and 25(OH)D3 serum levels were also apparent in patients with chronic hepatitis C. The same genotypes of these single nucleotide polymorphisms (SNPs) that are associated with reduced 25(OH)D3 serum levels were found to be associated with HCV-related HCC (P = 0.07 [OR = 1.13, 95% CI = 0.99-1.28] for CYP2R1, P = 0.007 [OR = 1.56, 95% CI = 1.12-2.15] for GC, P = 0.003 [OR = 1.42, 95% CI = 1.13-1.78] for DHCR7; ORs for risk genotypes). In contrast, no association between these genetic variations and liver fibrosis progression rate (P>0.2 for each SNP) or outcome of standard therapy with pegylated interferon-α and ribavirin (P>0.2 for each SNP) was observed, suggesting a specific influence of the genetic determinants of 25(OH)D3 serum levels on hepatocarcinogenesis. CONCLUSIONS/SIGNIFICANCE: Our data suggest a relatively weak but functionally relevant role for vitamin D in the prevention of HCV-related hepatocarcinogenesis.

Establishment of models to study mouse and human retinogenesis "in vitro" : isolation and characterization of retinal stem cells

SUMMARY IN FRENCH Les cellules souches sont des cellules indifférenciées capables a) de proliférer, b) de s'auto¬renouveller, c) de produire des cellules différenciées, postmitotiques et fonctionnelles (multipotencialité), et d) de régénérer le tissu après des lésions. Par exemple, les cellules de souches hematopoiétiques, situées dans la moelle osseuse, peuvent s'amplifier, se diviser et produire diverses cellules différenciées au cours de la vie, les cellules souches restant dans la moelle osseuse et consentant leur propriété. Les cellules souches intestinales, situées dans la crypte des microvillosités peuvent également régénérer tout l'intestin au cours de la vie. La rétine se compose de six classes de neurones et d'un type de cellule gliale. Tous ces types de cellules sont produits par un progéniteur rétinien. Le pic de production des photorécepteurs se situe autour des premiers jours postnatals chez la souris. A cette période la rétine contient les cellules hautement prolifératives. Dans cette étude, nous avons voulu analyser le phénotype de ces cellules et leur potentiel en tant que cellules souches ou progénitrices. Nous nous sommes également concentrés sur l'effet de certains facteurs épigéniques sur leur destin cellulaire. Nous avons observé que toutes les cellules prolifératives isolées à partir de neurorétines postnatales de souris expriment le marqueur de glie radiaire RC2, ainsi que des facteurs de transcription habituellement trouvés dans la glie radiaire (Mash1, Pax6), et répondent aux critères des cellules souches : une capacité élevée d'expansion, un état indifférencié, la multipotencialité (démontrée par analyse clonale). Nous avons étudié la différentiation des cellules dans différents milieux de culture. En l'absence de sérum, l'EGF induit l'expression de la β-tubulin-III, un marqueur neuronal, et l'acquisition d'une morphologie neuronale, ceci dans 15% des cellules présentes. Nous avons également analysé la prolifération de cellules. Seulement 20% des cellules incorporent le bromodéoxyuridine (BrdU) qui est un marqueur de division cellulaire. Ceci démontre que l'EGF induit la formation des neurones sans une progression massive du cycle cellulaire. Par ailleurs, une stimulation de 2h d'EGF est suffisante pour induire la différentiation neuronale. Certains des neurones formés sont des cellules ganglionnaires rétiniennes (GR), comme l'indique l'expression de marqueurs de cellules ganglionnaires (Ath5, Brn3b et mélanopsine), et dans de rare cas d'autres neurones rétiniens ont été observés (photorécepteurs (PR) et cellules bipolaires). Nous avons confirmé que les cellules souches rétiniennes tardives n'étaient pas restreintes au cours du temps et qu'elles conservent leur multipotencialité en étant capables de générer des neurones dits précoces (GR) ou tardifs (PR). Nos résultats prouvent que l'EGF est non seulement un facteur contrôlant le développement glial, comme précédemment démontré, mais également un facteur efficace de différentiation pour les neurones rétiniens, du moins in vitro. D'autre part, nous avons voulu établir si l'oeil adulte humain contient des cellules souches rétiniennes (CSRs). L'oeil de certains poissons ou amphibiens continue de croître pendant l'âge adulte du fait de l'activité persistante des cellules souches rétiniennes. Chez les poissons, le CSRs se situe dans la marge ciliaire (CM) à la périphérie de la rétine. Bien que l'oeil des mammifères ne se développe plus pendant la vie d'adulte, plusieurs groupes ont prouvé que l'oeil de mammifères adultes contient des cellules souches rétiniennes également dans la marge ciliaire plus précisément dans l'épithélium pigmenté et non dans la neurorétine. Ces CSRs répondent à certains critères des cellules souches. Nous avons identifié et caractérisé les cellules souches rétiniennes résidant dans l'oeil adulte humain. Nous avons prouvé qu'elles partagent les mêmes propriétés que leurs homologues chez les rongeurs c.-à-d. auto-renouvellement, amplification, et différenciation en neurones rétiniens in vitro et in vivo (démontré par immunocoloration et microarray). D'autre part, ces cellules peuvent être considérablement amplifiées, tout en conservant leur potentiel de cellules souches, comme indiqué par l'analyse de leur profil d'expression génique (microarray). Elles expriment également des gènes communs à diverses cellules souches: nucleostemin, nestin, Brni1, Notch2, ABCG2, c-kit et son ligand, aussi bien que cyclin D3 qui agit en aval de c-kit. Nous avons pu montré que Bmi1et Oct4 sont nécessaires pour la prolifération des CSRs confortant leur propriété de cellules souches. Nos données indiquent que la neurorétine postnatale chez la souris et l'épithélium pigmenté de la marge ciliaire chez l'humain adulte contiennent les cellules souches rétiniennes. En outre, nous avons développé un système qui permet d'amplifier et de cultiver facilement les CSRs. Ce modèle permet de disséquer les mécanismes impliqués lors de la retinogenèse. Par exemple, ce système peut être employé pour l'étude des substances ou des facteurs impliqués, par exemple, dans la survie ou dans la génération des cellules rétiniennes. Il peut également aider à disséquer la fonction de gènes ou les facteurs impliqués dans la restriction ou la spécification du destin cellulaire. En outre, dans les pays occidentaux, la rétinite pigmentaire (RP) touche 1 individu sur 3500 et la dégénérescence maculaire liée à l'âge (DMLA) affecte 1 % à 3% de la population âgée de plus de 60 ans. La génération in vitro de cellules rétiniennes est aussi un outil prometteur pour fournir une source illimitée de cellules pour l'étude de transplantation cellulaire pour la rétine. SUMMARY IN ENGLISH Stem cells are defined as undifferentiated cells capable of a) proliferation, b) self maintenance (self-renewability), c) production of many differentiated functional postmitotic cells (multipotency), and d) regenerating tissue after injury. For instance, hematopoietic stem cells, located in bone marrow, can expand, divide and generate differentiated cells into the diverse lineages throughout life, the stem cells conserving their status. In the villi crypt, the intestinal stem cells are also able to regenerate the intestine during their life time. The retina is composed of six classes of neurons and one glial cell. All these cell types are produced by the retinal progenitor cell. The peak of photoreceptor production is reached around the first postnatal days in rodents. Thus, at this stage the retina contains highly proliferative cells. In our research, we analyzed the phenotype of these cells and their potential as possible progenitor or stem cells. We also focused on the effect of epigenic factor(s) and cell fate determination. All the proliferating cells isolated from mice postnatal neuroretina harbored the radial glia marker RC2, expressed transcription factors usually found in radial glia (Mash 1, Pax6), and met the criteria of stem cells: high capacity of expansion, maintenance of an undifferentiated state, and multipotency demonstrated by clonal analysis. We analyzed the differentiation seven days after the transfer of the cells in different culture media. In the absence of serum, EGF led to the expression of the neuronal marker β-tubulin-III, and the acquisition of neuronal morphology in 15% of the cells. Analysis of cell proliferation by bromodeoxyuridine incorporation revealed that EGF mainly induced the formation of neurons without stimulating massively cell cycle progression. Moreover, a pulse of 2h EGF stimulation was sufficient to induce neuronal differentiation. Some neurons were committed to the retinal ganglion cell (RGC) phenotype, as revealed by the expression of retinal ganglion markers (Ath5, Brn3b and melanopsin), and in few cases to other retinal phenotypes (photoreceptors (PRs) and bipolar cells). We confirmed that the late RSCs were not restricted over-time and conserved multipotentcy characteristics by generating retinal phenotypes that usually appear at early (RGC) or late (PRs) developmental stages. Our results show that EGF is not only a factor controlling glial development, as previously shown, but also a potent differentiation factor for retinal neurons, at least in vitro. On the other hand, we wanted to find out if the adult human eye contains retina stem cells. The eye of some fishes and amphibians continues to grow during adulthood due to the persistent activity of retinal stem cells (RSCs). In fish, the RSCs are located in the ciliary margin zone (CMZ) at the periphery of the retina. Although, the adult mammalian eye does not grow during adult life, several groups have shown that the adult mouse eye contains retinal stem cells in the homologous zone (i.e. the ciliary margin), in the pigmented epithelium and not in the neuroretina. These RSCs meet some criteria of stem cells. We identified and characterized the human retinal stem cells. We showed that they posses the same features as their rodent counterpart i.e. they self-renew, expand and differentiate into retinal neurons in vitro and in vivo (indicated by immunostaining and microarray analysis). Moreover, they can be greatly expanded while conserving their sternness potential as revealed by the gene expression profile analysis (microarray approach). They also expressed genes common to various stem cells: nucleostemin, nestin, Bmil , Notch2, ABCG2, c-kit and its ligand, as well as cyclin D3 which acts downstream of c-kit. Furthermore, Bmil and Oct-4 were required for RSC proliferation reinforcing their stem cell identity. Our data indicate that the mice postnatal neuroretina and the adult pigmented epithelium of adult human ciliary margin contain retinal stem cells. We developed a system to easily expand and culture RSCs that can be used to investigate the retinogenesis. For example, it can help to screen drugs or factors involved, for instance, in the survival or generation of retinal cells. This could help to dissect genes or factors involved in the restriction or specification of retinal cell fate. In Western countries, retinitis pigmentosa (RP) affects 1 out of 3'500 individuals and age-related macula degeneration (AMD) strikes 1 % to 3% of the population over 60. In vitro generation of retinal cells is thus a promising tool to provide an unlimited cell source for cellular transplantation studies in the retina.

Single Subject Finger Somatotopy Mapping at 7T.

Introduction: The primary somatosensory cortex (SI) contains Brodmann areas (BA) 1, 2, 3a, and 3b. Research in non-human primates showed that BAs 3b, 1, and 2 each contain one full representation of the hand with separate representations for each finger. This research also showed that the finger representation in BA3b has larger and clearer finger somatotopy than BA1 and 2. Although several efforts to map finger somatotopy in SI by fMRI have been made at 1.5 and 3T these studies have yielded variable results and were not able to detect single subject finger somatotopy, probably due to the limited spatial extent of the cortical areas representing a digit (close to the resolution in most fMRI experiments), complications due to acquisition of consistent maps for individual subjects (Schweizer et al 2008), or inter-individual variability in sulcal anatomy impeding group studies. Here, we used 7T fMRI to investigate finger somatotopy in SI, some of its functional characteristics, and its reproducibility. Methods: Eight right-handed male subjects were scanned on a 7T scanner (Siemens Medical, Germany) with an 8-channel Tx/Rx rf-coil (Rapid Biomedical, Germany). 1.3x1.3x1.3mm3 resolution fMRI data were acquired using a sinusoidal readout EPI sequence (Speck et al, 2008) and FOV=210mm, TE/TR=27ms/2.5s, GRAPPA=2. Each volume contained 28 transverse slices covering SI. A single EPI volume with 64 slices was acquired to aid coregistration. 1x1x1mm3 anatomical data were acquire using the MP2RAGE sequence (Marques et al, 2009; TE/TR/TI1,2/TRmprage=2.63ms/7.2ms/0.9,3.2s/5s). Subjects were positioned supine in the scanner with their right arm comfortably against the magnet bore. An experimenter was positioned at the entrance of the bore where he could easily reach and stroke successively the two distal phalanxes of each digit. The order of stroked digit was D1 (thumb)-D3-D5-D2-D4, with 20s ON, 10s OFF alternated. This sequence was repeated four times per run and two functional runs were acquired per subject. Realignment, smoothing (FWHM 2 mm), coregistration of the anatomical to the fMRI data and calculation of t-statistics were done using SPM8. An SI mask was obtained via an F-contrast (p<0.001) over all digits. Within the mask, voxels were labeled with the number of the digit demonstrating the highest t-value for that particular voxel. Results: For all subjects, areas corresponding to the five digits were identified in contralateral SI. BA3b showed the most consistent somatotopic finger representation (see an example in Fig.1). The five digits were localized in a consecutive order in the cortex, with D1 most anterior, inferior and distal and D5, most posterior, superior and medial (mean distance between centres of mass of digit representations ±stderr: 4.2±0.7mm; see Fig. 2). The analysis of average beta values within each finger representation region revealed the specificity of the somatotopic region to the tactile input for each tested finger (except digit 4 and 5). Five of these subjects also presented an orderly and consecutive representation of the five digits in BA1 and 2. Conclusions: Our data reveal that the increased BOLD sensitivity at 7T and the high spatial resolution used in this study allow consistent somatotopic mapping using human touch as a stimulus and that human SI contains at least three separate regions that contain five separate representations of all single contralateral fingers. Moreover, adjacent fingers were represented at adjacent cortical regions across the three SI regions. The spatial organization of SI as reflected in individual subject topography corresponds well with previous electrophysiological data in non-human primates. The small distance between digit representations highlights the need for the high spatial resolution available at 7T.

Effects of zoledronate versus placebo on spine bone mineral density and microarchitecture assessed by the trabecular bone score in postmenopausal women with osteoporosis: A three-year study.

The trabecular bone score (TBS) is an index of bone microarchitectural texture calculated from anteroposterior dual-energy X-ray absorptiometry (DXA) scans of the lumbar spine (LS) that predicts fracture risk, independent of bone mineral density (BMD). The aim of this study was to compare the effects of yearly intravenous zoledronate (ZOL) versus placebo (PLB) on LS BMD and TBS in postmenopausal women with osteoporosis. Changes in TBS were assessed in the subset of 107 patients recruited at the Department of Osteoporosis of the University Hospital of Berne, Switzerland, who were included in the HORIZON trial. All subjects received adequate calcium and vitamin D3. In these patients randomly assigned to either ZOL (n = 54) or PLB (n = 53) for 3 years, BMD was measured by DXA and TBS assessed by TBS iNsight (v1.9) at baseline and 6, 12, 24, and 36 months after treatment initiation. Baseline characteristics (mean ± SD) were similar between groups in terms of age, 76.8 ± 5.0 years; body mass index (BMI), 24.5 ± 3.6 kg/m(2) ; TBS, 1.178 ± 0.1 but for LS T-score (ZOL-2.9 ± 1.5 versus PLB-2.1 ± 1.5). Changes in LS BMD were significantly greater with ZOL than with PLB at all time points (p < 0.0001 for all), reaching +9.58% versus +1.38% at month 36. Change in TBS was significantly greater with ZOL than with PLB as of month 24, reaching +1.41 versus-0.49% at month 36; p = 0.031, respectively. LS BMD and TBS were weakly correlated (r = 0.20) and there were no correlations between changes in BMD and TBS from baseline at any visit. In postmenopausal women with osteoporosis, once-yearly intravenous ZOL therapy significantly increased LS BMD relative to PLB over 3 years and TBS as of 2 years. © 2013 American Society for Bone and Mineral Research.

Re-thinking cell cycle regulators: the cross-talk with metabolism.

Analysis of genetically engineered mice deficient in cell cycle regulators, including E2F1, cdk4, and pRB, showed that the major phenotypes are metabolic perturbations. These key cell cycle regulators contribute to lipid synthesis, glucose production, insulin secretion, and glycolytic metabolism. It has been shown that deregulation of these pathways can lead to metabolic perturbations and related metabolic diseases, such as obesity and type II diabetes. The cyclin-cdk-Rb-E2F1 pathway regulates adipogenesis in addition to its well-described roles in cell cycle regulation and cancer. It was also shown that E2F1 directly participates in the regulation of pancreatic growth and function. Similarly, cyclin D3, cdk4, and cdk9 are also adipogenic factors with strong effects on whole organism metabolism. These examples support the emerging notion that cell cycle regulatory proteins also modulate metabolic processes. These cell cycle regulators are activated by insulin and glucose, even in non-proliferating cells. Most importantly, these cell cycle regulators trigger the adaptive metabolic switch that normal and cancer cells require in order to proliferate. These changes include increased lipid synthesis, decreased oxidative metabolism, and increased glycolytic metabolism. In summary, these factors are essential regulators of anabolic biosynthetic processes, blocking at the same time oxidative and catabolic pathways, which is reminiscent of cancer cell metabolism.

Loss of Memo, a novel FGFR regulator, results in reduced lifespan.

Memo is a widely expressed 33-kDa protein required for heregulin (HRG)-, epidermal growth factor (EGF)-, and fibroblast growth factor (FGF)-induced cell motility. Studies in mouse embryonic fibroblasts, wild-type or knockout for Memo, were performed to further investigate the role of Memo downstream of FGFR. We demonstrated that Memo associates with the FGFR signalosome and is necessary for optimal activation of signaling. To uncover Memo's physiological role, Memo conditional-knockout mice were generated. These animals showed a reduced life span, increased insulin sensitivity, small stature, graying hair, alopecia, kyphosis, loss of subcutaneous fat, and loss of spermatozoa in the epididymis. Memo-knockout mice also have elevated serum levels of active vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D), and calcium compared to control littermates expressing Memo. In summary, the results from in vivo and in vitro models support the hypothesis that Memo is a novel regulator of FGFR signaling with a role in controlling 1,25(OH)2D production and normal calcium homeostasis.