Cirp function and characterisation in RNA and microRNAs

In order to search for novel genes involved in cell proliferation, the hypothesis was that by infecting primary cells with a cDNA library of immortal cells would render immortalizing genes. Consequently it has been discovered CIRP (Cold inducible RNA-binding protein). Mammalian cells exposed to mild hypothermia show a general inhibition of protein synthesis and a concomitant increase in the expression of a small number of cold-shock mRNAs and proteins. Rbm3, another RNA binding protein belonging to the same family, has been postulated to facilitate protein synthesis at mild cold shock. To investigate if the same occurs for CIRP, CIRP was overexpressed in primary cells and protein sintesis was measured. Interestingly, CIRP increased protein synthesis, however, such increase did not involve an increase in the polysome fraction or affected the ribosome profile. In addition, the effect caused by CIRP inhibition or knockdown was also analyzed. Different siRNAs against CIRP were tested. Once checked their efficiency by decreasing CIRP at mRNA and protein levels, proliferation was tested by BrdU, cell number (DAPI) and proliferation curves were performed. Interestingly, CIRP provoke a decreased proliferation in primary cells: MEFs, HMEC; and cancer cells: TERA2 and HeLa. In conclusion, we describe for the first time that CIRP bypasses replicative senescence when over-expressed at physiological temperature (37ºC) by increasing a general protein synthesis. This effect is achieved through ERK1/2 activation in MEFs.The decrease in growth rate found in mammalian cells treated with mild cold stress is not entirely attributable to arrested metabolism. This decrease may also involve an active process in which CIRP and other stress-responsive proteins play a fundamental role in stimulating proliferation. Although most cell proteins are down-regulated or inhibited with cold stress, CIRP is activated to maintain cells in an active proliferative status and its overexpression at 37°C might be potentially oncogenic.

E2F1 mediates DNA damage and apoptosis through HCF-1 and the MLL family of histone methyltransferases.

E2F1 is a key positive regulator of human cell proliferation and its activity is altered in essentially all human cancers. Deregulation of E2F1 leads to oncogenic DNA damage and anti-oncogenic apoptosis. The molecular mechanisms by which E2F1 mediates these two processes are poorly understood but are important for understanding cancer progression. During the G1-to-S phase transition, E2F1 associates through a short DHQY sequence with the cell-cycle regulator HCF-1 together with the mixed-lineage leukaemia (MLL) family of histone H3 lysine 4 (H3K4) methyltransferases. We show here that the DHQY HCF-1-binding sequence permits E2F1 to stimulate both DNA damage and apoptosis, and that HCF-1 and the MLL family of H3K4 methyltransferases have important functions in these processes. Thus, HCF-1 has a broader role in E2F1 function than appreciated earlier. Indeed, sequence changes in the E2F1 HCF-1-binding site can modulate both up and down the ability of E2F1 to induce apoptosis indicating that HCF-1 association with E2F1 is a regulator of E2F1-induced apoptosis.

Extracellular matrix and platelet function in patients with musculocontractural Ehlers-Danlos syndrome caused by mutations in the CHST14 gene.

We report on a consanguineous, Afghani family with two sisters affected with characteristic facial features, multiple contractures, progressive joint and skin laxity, hemorrhagic diathesis following minor trauma and multisystem fragility-related manifestations suggestive of a diagnosis of musculocontractural Ehlers-Danlos syndrome (EDS). This novel form of connective tissue disorder was recently reported in patients of Japanese, Turkish, and Indian descent who were formerly classified as having EDS type VIB and has now been recognized to be a part of spectrum including patients previously classified as having adducted thumb-clubfoot syndrome. We identified a previously unreported mutation in the CHST14 gene, which codes for the enzyme dermatan 4-O-sulfotransferase. We discuss the prenatal presentation, detailed clinical manifestations, and neurological findings in two sisters with this newly described musculocontractural EDS-CHST14 type. We demonstrate that fibroblasts from one of our patients produce more chondroitin sulfate than normal and show lower than normal deposition of collagens I and II and fibrillin 1-containing microfibrills. These findings suggest that the imbalance in the glycosaminoglycan content in developing tissues might interfere with normal deposition of other extracellular matrix components and ultimately contribute to the development of the phenotype observed in these patients. Furthermore, we ruled out the contribution of intrinsic platelet factors to the bleeding diathesis observed in some affected individuals. © 2012 Wiley Periodicals, Inc.

Family-based affirmative action? Subversion and resilience strategies of gay father families

The present doctoral dissertation is aimed at analyzing how and with what consequences gay father families and their children’s schools negotiate possible differences in the construction of family and gender at home and in the families’ social milieus. This objective fits in with the broader goal of researching how family-school interactons are influenced by the social context such as hegemonic masculinity (Connell, 2002). The thesis is based on qualitative fieldwork carried out with 18 nonheterosexual parent families in Spain, comprising 30 interviews with 44 people. The principal participant group were 14 de novo (adoptive and surrogacy) gay father families with resident preadolescent children. The findings revealed that all the de novo families assumed open communication strategies at school with inclusive consequences: apart from incidental questions and reactions of surprise, the children did not suffer homophobic bullying. The analisis showed that the necessary condition for inclusion was not the open communication but rather illocutionary orientation (Habermas, 1984; Soler & Flecha, 2010), understood as the parents’ sensitivity to the attitudes of their children and schools. The schools received the families in an inclusive manner, which, however, was only receptive and not proactive, therefore some of the families (reconstituted ones), coerced by the social context, got excluded. Gender relations at home were predominantly androgynous, and outside home predominantly traditional, yet the children negotiated this difference with inclusive consequences. They participated in hegemonic collective practices, thus confirming the thesis on the similarity between homo- and heterosexual-parent families (Golombok, 2006). Consistently, also the families’ identity politics was “assimilationist” and non-queer. Admittedly, the analisis showed that such a politics was increased by social expectations. Still, the findings suggest that educational and other family policies should draw on broad agendas of gender and family diversity rather than on the politics of difference and the unique status of LGB families.

Participation of connexin-based channels in the control of vascular function

SUMMARYIntercellular communication is achieved at specialized regions of the plasma membrane by gap junctions. The proteins constituting the gap junctions are called connexins and are encoded by a family of genes highly conserved during evolution. In adult mouse, four connexins (Cxs) are known to be expressed in the vasculature: Cx37, Cx40, Cx43 and Cx45. Several recent studies have provided evidences that vascular connexins expression and blood pressure regulation are closely linked, suggesting a role for connexins in the control of blood pressure. However, the precise function that each vascular connexin plays under physiological and pathophysiological conditions is still not elucidated. In this context, this work was dedicated to evaluate the contribution of each of the four vascular connexins in the control of the vascular function and in the blood pressure regulation.In the present work, we first demonstrated that vascular connexins are differently regulated by hypertension in the mouse aorta. We also observed that endothelial connexins play a regulatory role on eNOS expression levels and function in the aorta, therefore in the control of vascular tone. Then, we demonstrated that Cx40 plays a pivotal role in the kidney by regulating the renal levels of COX-2 and nNOS, two key enzymes of the macula densa known to participate in the control of renin secreting cells. We also found that Cx43 forms the functional gap junction involved in intercellular Ca2+ wave propagation between vascular smooth muscle cells. Finally, we have started to generate transgenic mice expressing specifically Cx40 in the endothelium to investigate the involvement of Cx40 in the vasomotor tone, or in the renin secreting cells to evaluate the role of Cx40 in the control of renin secretion.In conclusion, this work has allowed us to identify new roles for connexins in the vasculature. Our results suggest that vascular connexins could be interesting targets for new therapies caring hypertension and vascular diseases.

Persistence of retinal function after intravitreal melphalan injection for retinoblastoma.

BACKGROUND: The risk/benefit profile of intravitreal melphalan injection for treatment of active vitreous seeds in retinoblastoma remains uncertain. We report clinical and electroretinography results after 6 months of one patient who has shown a favorable initial clinical response to intravitreal melphalan injections for treatment of refractory vitreous seeds. METHODS: Clinical case report. PATIENT: The patient presented at age 17 months with bilateral retinoblastoma [OD: International Classification (ICRB) group E, Reese-Ellsworth (R-E) class Vb; OS: ICRB D, R-E Vb] with no known prior family history. The right eye was enucleated primarily. The patient received systemic chemotherapy and extensive local treatment to the left eye. Ten months later, she presented with recurrent disease, including fine, diffuse vitreous seeds. Tumor control was established with intra-arterial chemotherapy and local treatment. Subsequent recurrence was treated with further intra-arterial chemotherapy, local treatment, and plaque radiotherapy with iodine-125. Persistent free-floating spherical vitreous seeds were treated with 4 cycles of intravitreal melphalan injection via the pars plana, with doses of 30, 30, 30, and 20 μg. RESULTS: After 6 months of follow-up, the left eye remained free of active tumor. Visual acuity was 20/40. Photopic ERGs amplitudes were unchanged compared with those recorded prior to the intravitreal injection treatments. CONCLUSIONS: Intravitreal melphalan injection for refractory spherical vitreous seeds of retinoblastoma with favorable tumor response is compatible with good central visual acuity and preservation of retinal function as indicated by photopic ERG recordings.

Analysis of the regulation of Nodal function by SPC-mediated cleavage during early mammalian development

RÉSUMÉ Après implantation dans l'utérus, le foetus de mammifère est composé de trois populations différentes de cellules: l'epiblast, l'ectoderme extraembryonnaire et l'endoderme viscéral. Pendant la gastrulation, les cellules de l'epiblast donnent naissance aux trois lignées germinales: l'ectoderme, le mésoderme et l'endodermes. Les lignées germinales produisent par la suite les différents tissus et organes du corps embryonnaire et adulte. Les cellules de l'ectoderme extraembryonnaire donnent par la suite le composant foetal du placenta qui est essentiel à la survie de l'embryon dans l'utérus. L'épiblast et l'ectoderme extraembryonnaire sont entourés par l'endoderme viscéral et forment une structure connue sous le nom de bouton embryonnaire. L'endoderme viscéral joue un rôle important dans l'embryogenèse car il comporte une sous-population de cellules appelées l'endoderme viscéral antérieur dont les signaux influencent l'épiblast adjacent et déterminent le futur axe antéro-postérieur de l'embryon. La protéine de signalisation Nodal de la famille des TGFß est essentielle dans l'épiblast pour spécifier le mésendoderme, l'endoderme viscéral antérieur, ainsi que pour maintenir les cellules souche de l'ectoderme extraembryonnaire. Ainsi, dans les embryons mutants pour Nodal, aucun axe antéro-postérieur n'est établi, les lignées germinales ne sont pas spécifiés et le placenta ne se développe pas. Au niveau moléculaire, comme pour les protéines de la famille des TGFß, Nodal est initialement synthétisée sous forme de précurseur avant d'être clivée de façon endoproteolytique par des protéanes sécrétées, les proprotéines convertases du type subtilisin (SPC), qui suppriment la partie inhibitrice N-terminale du pro peptide. Dans ce contexte, le projet de ma thèse a été d'analyser l'influence des SPC sur la fonction de Nodal en employant une combinaison d'approches génétiques et biochimiques. Premièrement, nous avons constaté que le clivage du précurseur par les protéases active Nodal, mais en même temps augmente son turn-over et diminue la portée de son action. Deuxièmement, dans l'embryon, il apparaît que Nodal est activé par l'action combinée de Furin et de PACE4, deux protéases sécrétées qui sont spécifiquement exprimées dans les cellules de l'ectoderme extraembryonnaire, donc adjacentes au domaine d'expression de Nodal. De manière similaire aux mutants de Nodal, les embryons mutants pour les deux protéases ne forment pas d'endoderme viscéral antérieur et ne gastrulent pas. Cependant, certains gènes cible de Nodal restent exprimés, suggérant que toutes les activités de Nodal ne sont pas dépendent du clivage par les SPCs. En effet, la génération et l'analyse de mutants portant un allèle knock-in qui code pour une forme mutante de Nodal résistante aux SPC, ont montré que ces mutants ont les caractères phénotypique des mutants de Nodal seulement de façon partielle. La formation de mésoderme est partiellement induite, et de façon remarquable, la forme de Nodal résistante aux SPC est capable d'agir à une distance de sa source, maintenant l'expression de ses propres protéases et d'autres gènes essentiels pour la spécification de l'ectoderme extraembryonnaire. Ensemble, ces résultats prouvent que par leur action directe les protéases extraembryonnaire modulent la signalisation de Nodal pendant le développement mammifère précoce. SUMMARY : Early after implantation in the uterus, the mammalian conceptus is composed of three different cell populations: the epiblast, the extraembryonic ectoderm and the visceral endoderm. During gastrulation, epiblast cells give rise to the three embryonic germ layers: the ectoderm, the mesoderm and the endoderm. These germ layers then generate the different tissues and organs of the embryonic and adult bodies. In parallel, extraembryonic ectoderm cells give rise to the fetal component of the placenta, which is essential for the survival of the embryo in the uterus. Both the epiblast and extraembryonic ectoderm are surrounded by the visceral endoderm to form a structure known as the egg cylinder. The visceral endoderm plays an important role as it harbours a subpopulation of cells called the anterior visceral endoderm, from which signals influence the adjacent epiblast and determine the future antero-posterior embryonic axis. The TGFß-related signalling protein Nodal is required within the epiblast to specify the mesoderm, the endoderm,the anterior visceral endoderm and is also essential to maintain stem cells in the extraembryonic ectoderm. Thus, in Nodal null conceptuses, no antero-posterior axis is established, the germ layers are not specified and the placenta does not develop. At the molecular level, Nodal, like related proteins of the TGFß family, is initially synthesized as a precursor and undergoes endoproteolytic cleavage by secreted proteases of the subtilisin-like proprotein convertases (SPC) to remove an inhibitory N-terminal pro peptide. In the embryo, Nodal is activated by the combined action of Furin and PACE4, two secreted SPCs that are specifically expressed in cells of the extraembryonic ectoderm, thus adjacent to the Nodal expression domain. Similar to Nodal null .embryos, mutant embryos lacking both these proteases fail to specify the anterior visceral endoderm and to undergo gastrulation. However, these mutants still express a subset of Nodal target genes, suggesting that part of Nodal activity is independent on cleavage by SPCs. Indeed, by generating and analyzing mutants with a knock-in allele that encodes an SPC-resistant mutant form of Nodal, I could show that they retain a subset of Nodal activities. Mesoderm formation is partially induced, but most remarkably, SPC-resistant Nodal form is able to act at a distance from its source, maintaining the expression of its proteases and of other genes essential for maintenance of the extraembryonic ectoderm.

A direct approach to the discounted penalty function

This paper provides a new and accessible approach to establishing certain results concerning the discounted penalty function. The direct approach consists of two steps. In the first step, closed-form expressions are obtained in the special case in which the claim amount distribution is a combination of exponential distributions. A rational function is useful in this context. For the second step, one observes that the family of combinations of exponential distributions is dense. Hence, it suffices to reformulate the results of the first step to obtain general results. The surplus process has downward and upward jumps, modeled by two independent compound Poisson processes. If the distribution of the upward jumps is exponential, a series of new results can be obtained with ease. Subsequently, certain results of Gerber and Shiu [H. U. Gerber and E. S. W. Shiu, North American Actuarial Journal 2(1): 48–78 (1998)] can be reproduced. The two-step approach is also applied when an independent Wiener process is added to the surplus process. Certain results are related to Zhang et al. [Z. Zhang, H. Yang, and S. Li, Journal of Computational and Applied Mathematics 233: 1773–1 784 (2010)], which uses different methods.

Valeur ajoutée de la supervision du médecin-assistant par un médecin de famille dans une permanence [Added value of family practitioners' supervision of junior doctors in a walk-in clinic].

The pending workforce crisis in family medicine has triggered various initiatives. This article describes the PMU-FLON walk-in clinic, a project of the Institute of General Medicine University of Lausanne. The working conditions in this clinic are close to that of a family practice. Doctors in training are supervised by family doctors who work part-time in the clinic. The objective is to improve training in the various fields of family medicine, from technical skills (improving optimal use of diagnostic tools), to integrating patients' requests in a more global patient-centered approach. This new educational model allows doctors in training to benefit from the specific approaches of different trainers. It will contribute to promoting quality family medicine in the future.

The Thioredoxin TRX-1 Modulates the Function of the Insulin-Like Neuropeptide DAF-28 during Dauer Formation in Caenorhabditis elegans

Thioredoxins comprise a conserved family of redox regulators involved in many biological processes, including stress resistance and aging. We report that the C. elegans thioredoxin TRX-1 acts in ASJ head sensory neurons as a novel modulator of the insulin-like neuropeptide DAF-28 during dauer formation. We show that increased formation of stress-resistant, long-lived dauer larvae in mutants for the gene encoding the insulin-like neuropeptide DAF-28 requires TRX-1 acting in ASJ neurons, upstream of the insulin-like receptor DAF-2. Genetic rescue experiments demonstrate that redox-independent functions of TRX-1 specifically in ASJ neurons are needed for the dauer formation constitutive (Daf-c) phenotype of daf-28 mutants. GFP reporters of trx-1 and daf-28 show opposing expression patterns in dauers (i.e. trx-1 is up-regulated and daf-28 is down-regulated), an effect that is not observed in growing L2/L3 larvae. In addition, functional TRX-1 is required for the down-regulation of a GFP reporter of daf-28 during dauer formation, a process that is likely subject to DAF-28-mediated feedback regulation. Our findings demonstrate that TRX-1 modulates DAF-28 signaling by contributing to the down-regulation of daf-28 expression during dauer formation. We propose that TRX-1 acts as a fluctuating neuronal signaling modulator within ASJ neurons to monitor the adjustment of neuropeptide expression, including insulin-like proteins, during dauer formation in response to adverse environmental conditions.

Molecular dissection of Arabidopsis thaliana endodermis development, structure and function

In vascular plants, the endodermis establishes a protective diffusion barrier surrounding the vasculature preventing the passive, uncontrolled entry of nutrients absorbed by the plant. It does so by means of a differentiation feature, the "Casparian Strip" (CS), a highly localized cell wall impregnation made of lignin, which seals the extracellular space. Although the existence of this differentiation feature has been intensively described, the mechanisms establishing this hallmark remain obscure. In this work I report, the developmental sequence of events that leads to a differentiated endodermis, in the plant model Arabidopsis thaliana. In addition, my descriptive approach gave important insights as to how these cells define membrane domains involved in the directional transport of nutrients. I also participated in characterizing a new transmembrane protein family, the CASPs, localized to the membrane domain underlying the CS, which we accordingly named the Casparian Strip membrane Domain (CSD). Our molecular analysis indicates that these proteins drive CS establishment. To identify more molecular factors of CS establishment, I performed a forward genetic screen. This screen led to the identification of 11 endodermis permissive mutants, which we named schengen (sgn) mutants. The causative mutations have been mapped to 5 independent loci: SGN1 to SGN5. SGN1 and SGN3 encode Receptor Like Kinases involved in the correct establishment of the CSD. A lack of those kinases leads to an incomplete CSD, which gives rise to interrupted CS barriers. Interestingly, SGN1 seems to also regulate CSD positioning to the middle of endodermal transversal walls. SGN4 encodes an NADPH oxidase involved in lignin polymerization essential for CS formation. The sgn5 mutant induces extra divisions of cortical cells strongly affecting the cell identity, but also leading to incorrect differentiation. A thorough characterization of the sgn2 mutant will follow elsewhere, yet preliminary results indicate that SGN2 encodes an Acyl-CoA N-acyltransferase. . In summary, with my work I have contributed a first set of molecular players of Casparian strip formation and initiated their characterization. Eventually, this might lead to an understanding of the molecular mechanisms of CS establishment in A.thaliana . This in turn will hopefully help to better understand nutrient uptake in higher plants and their response to environmental stresses. - Au sein des plantes vasculaires, l'endoderme représente un tissu protecteur mettant en place une barrière imperméable, empêchant n'importe quel élément de rejoindre les tissus conducteurs par simple diffusion. Cette barrière, appelée « Cadre de Caspary », correspond à une lignification de la paroi de l'endoderme et donne lieu à un cloisonnement de l'espace intercellulaire. Bien que cet élément de différenciation soit décrit en détail, sa mise en place reste incomprise. Cette étude indique la suite d'événements aboutissant à l'établissement du cadre de Caspary chez la plante modèle Arabidopsis thaliana. De plus, ce travail apporte de nouvelles connaissances expliquant comment ces cellules définissent des domaines membranaires importants pour le transport des nutriments. Nous décrivons une nouvelle famille de protéines membranaires, les CASPs (« CAparian Strip membrane domain Proteins »), localisées dans un domaine membranaire longeant le cadre de Caspary : le domaine de Caspary (CSD). L'analyse moléculaire des CASPs indique qu'elles dirigent la formation du cadre de Caspary. Par ailleurs, une approche génétique directe nous a permis d'identifier 11 mutants ayant un endoderme perméable. Nous avons nommé ces mutants Schengen, en référence à la zone de libre échange européenne. Les mutations impliquées dans ces mutants affectent 5 gènes désignés de SGN1 à SGN5. SGN1 et SGN3 produisent des protéines de type kinases (« Receptor-like Kinases », RLK) qui participent à la délimitation du CSD. L'absence de ces kinases aboutit à un domaine CSD incomplet, se traduisant par un cadre de Caspary discontinu. De plus, SGN1 semble réguler le positionnement du CSD au milieu de la paroi transversale de l'endoderme. SGN4 produit une enzyme de type NADPH oxydase impliquée dans la polymérisation du cadre de Caspary. Dans le mutant sgn5, on observe une division anormale des cellules du cortex créant ainsi une nouvelle couche cellulaire incapable d'achever sa différenciation en endoderme. Quant à la mutation sgn2, bien que nous pensons qu'elle affecte une Acyl-CoA N-acyltransferase, sa caractérisation ne sera réalisée que prochainement. Au final, ce travail procure de nouveaux éléments sur l'établissement du cadre de Caspary qui pourraient être importants afin de comprendre comment les plantes sélectionnent leurs nutriments et résistent à des conditions environnementales parfois hostiles. - De par leur immobilité, les plantes terrestres n'ont pas d'autre choix que de puiser leurs ressources dans leur environnement direct. La plante extrait du sol les nutriments qui lui sont nécessaires et les redistribue grâce à des tissus conducteurs. Afin de ne pas s'intoxiquer, il est donc essentiel de pouvoir sélectionner les éléments entrant dans la racine. Etonnement, ce n'est pas la surface des racines qui permet ce contrôle mais un tissu interne appelé endoderme. Ce dernier forme une barrière imperméable qui entoure chaque cellule et crée une jointure permettant de bloquer le passage des éléments entre les cellules. Cette structure, appelée « cadre de Caspary », oblige les éléments à entrer dans les cellules de l'endoderme et à être ainsi sélectionnés. Bien que cette structure soit décrite en détail, sa mise en place reste incomprise. Cette étude indique la suite d'événements qui aboutit à la formation du cadre de Caspary chez la plante modèle Arabidopsis thaliana. Ce travail apporte également de nouvelles connaissances expliquant comment ces cellules définissent, organisent et dirigent le transport des nutriments. Nous décrivons comment certains éléments de la cellule, les protéines CASPs (CAsparian Strip membrane domain Proteins), sont organisées un domaine particulier des membranes afin de créer une plateforme de construction longeant le cadre de Caspary : le domaine de Caspary (CSD). Afin de déterminer ce qu'il se passerait si une plante ne possédait pas de cadre de Caspary, nous avons réalisé une mutagénèse, ou approche génétique directe, et identifié 11 mutants (individu ayant un gène défectueux conduisant à la perte d'une fonction) ayant un endoderme perméable. Nous avons nommé ces mutants schengen, en référence à la zone de libre échange européenne. Les mutations impliquées dans ces mutants affectent 5 gènes désignés de SGN1 à SGN5. Les gènes SGN1 et SGN3 produisent des protéines de type kinases (« Receptor-like Kinases », RLK) servant à l'établissement de la plateforme de construction. L'absence de ces kinases aboutit à une base incomplète, se traduisant par un cadre de Caspary discontinu. Qui plus est, la kinase SGN1 semble réguler le positionnement de la plateforme au milieu de l'endoderme. Le gène SGN4 est par contre, impliqué dans la construction à proprement dite du cadre de Caspary. Dans le mutant sgn5, on observe une nouvelle couche de cellules ressemblant à de l'endoderme mais incapable de former correctement une barrière identique au cadre de Caspary. Quant au dernier mutant, sgn2, bien que cette étude fournisse des indices permettant de comprendre pourquoi le mutant sgn2 est défectueux, nous n'expliquerons ce cas que prochainement. En résumé, ce travail procure de nouvelles connaissances sur l'établissement du cadre de Caspary qui pourraient être importantes afin de comprendre comment les plantes sélectionnent leurs nutriments et résistent à des conditions environnementales parfois hostiles.

Combinatorial effects of splice variants modulate function of Aiolos

The transcription factor Aiolos (also known as IKZF3), a member of the Ikaros family of zinc-finger proteins, plays an important role in the control of B lymphocyte differentiation and proliferation. Previously, multiple isoforms of Ikaros family members arising from differential splicing have been described and we now report a number of novel isoforms of Aiolos. It has been demonstrated that full-length Ikaros family isoforms localize to heterochromatin and that they can associate with complexes containing histone deacetylase (HDAC). In this study, for the first time we directly investigate the cellular localization of various Aiolos isoforms, their ability to heterodimerize with Ikaros and associate with HDAC-containing complexes, and the effects on histone modification and binding to putative targets. Our work demonstrates that the cellular activities of Aiolos isoforms are dependent on combinations of various functional domains arising from the differential splicing of mRNA transcripts. These data support the general principle that the function of an individual protein is modulated through alternative splicing, and highlight a number of potential implications for Aiolos in normal and aberrant lymphocyte function.

ALDH1A3 loss of function causes bilateral anophthalmia/microphthalmia and hypoplasia of the optic nerve and optic chiasm.

The major active retinoid, all-trans retinoic acid, has long been recognized as critical for the development of several organs, including the eye. Mutations in STRA6, the gene encoding the cellular receptor for vitamin A, in patients with Matthew-Wood syndrome and anophthalmia/microphthalmia (A/M), have previously demonstrated the importance of retinol metabolism in human eye disease. We used homozygosity mapping combined with next-generation sequencing to interrogate patients with anophthalmia and microphthalmia for new causative genes. We used whole-exome and whole-genome sequencing to study a family with two affected brothers with bilateral A/M and a simplex case with bilateral anophthalmia and hypoplasia of the optic nerve and optic chiasm. Analysis of novel sequence variants revealed homozygosity for two nonsense mutations in ALDH1A3, c.568A>G, predicting p.Lys190*, in the familial cases, and c.1165A>T, predicting p.Lys389*, in the simplex case. Both mutations predict nonsense-mediated decay and complete loss of function. We performed antisense morpholino (MO) studies in Danio rerio to characterize the developmental effects of loss of Aldh1a3 function. MO-injected larvae showed a significant reduction in eye size, and aberrant axonal projections to the tectum were noted. We conclude that ALDH1A3 loss of function causes anophthalmia and aberrant eye development in humans and in animal model systems.

Survival-independent function of NF-kappaB/Rel during late stages of thymocyte differentiation.

Transcription factors of the NF-kappaB/Rel family are important mediators of extracellular signals. Their implication in positive selection of thymocytes is suggested by a defective thymic development in transgenic mice that over-express IkappaB in thymocytes. These mice exhibit an accumulation of an unusually prominent population of TCRhigh/CD4/CD8 double positive cells in the thymus and a dramatic reduction of CD4+ and CD8+ cells in the periphery. The present study addresses the role of NF-kappaB in survival and differentiation processes of maturing thymocytes using IkappaB/bcl-2 and IkappaB/HY double-transgenic mice. Neither the introduction of the anti-apoptosis gene bcl-2 nor the positively selecting background in female HY transgenic mice resulted in a rescue of the maturational defects observed in the thymus of IkappaB transgenic mice. Thus, rather than promoting survival the main role of NF-kappaB/Rel proteins during positive selection of thymocytes appears to be the mediation of differentiation signals.

Functional Characterization of a Novel Frameshift Mutation in the C-terminus of the Nav1.5 Channel Underlying a Brugada Syndrome with Variable Expression in a Spanish Family.

INTRODUCTION We functionally analyzed a frameshift mutation in the SCN5A gene encoding cardiac Na(+) channels (Nav1.5) found in a proband with repeated episodes of ventricular fibrillation who presented bradycardia and paroxysmal atrial fibrillation. Seven relatives also carry the mutation and showed a Brugada syndrome with an incomplete and variable expression. The mutation (p.D1816VfsX7) resulted in a severe truncation (201 residues) of the Nav1.5 C-terminus. METHODS AND RESULTS Wild-type (WT) and mutated Nav1.5 channels together with hNavβ1 were expressed in CHO cells and currents were recorded at room temperature using the whole-cell patch-clamp. Expression of p.D1816VfsX7 alone resulted in a marked reduction (≈90%) in peak Na(+) current density compared with WT channels. Peak current density generated by p.D1816VfsX7+WT was ≈50% of that generated by WT channels. p.D1816VfsX7 positively shifted activation and inactivation curves, leading to a significant reduction of the window current. The mutation accelerated current activation and reactivation kinetics and increased the fraction of channels developing slow inactivation with prolonged depolarizations. However, late INa was not modified by the mutation. p.D1816VfsX7 produced a marked reduction of channel trafficking toward the membrane that was not restored by decreasing incubation temperature during cell culture or by incubation with 300 μM mexiletine and 5 mM 4-phenylbutirate. CONCLUSION Despite a severe truncation of the C-terminus, the resulting mutated channels generate currents, albeit with reduced amplitude and altered biophysical properties, confirming the key role of the C-terminal domain in the expression and function of the cardiac Na(+) channel.