Microdomain Ca2+ Activation during Exocytosis in Paramecium Cells. Superposition of Local Subplasmalemmal Calcium Store Activation by Local Ca2+ Influx

In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of Ca2+-activated currents across the cell membrane (Erxleben, C., and H. Plattner. 1994. J. Cell Biol. 127:935– 945). We now show by voltage clamp and extracellular recordings that the product of current × time (As) closely parallels the number of exocytotic events. We suggest that Ca2+ mobilization from subplasmalemmal storage compartments, covering almost the entire cell surface, is a key event. In fact, after local stimulation, Ca2+ imaging with high time resolution reveals rapid, transient, local signals even when extracellular Ca2+ is quenched to or below resting intracellular Ca2+ concentration ([Ca2+]e ⩽ [Ca2+]i). Under these conditions, quenched-flow/freeze-fracture analysis shows that membrane fusion is only partially inhibited. Increasing [Ca2+]e alone, i.e., without secretagogue, causes rapid, strong cortical increase of [Ca2+]i but no exocytosis. In various cells, the ratio of maximal vs. minimal currents registered during maximal stimulation or single exocytotic events, respectively, correlate nicely with the number of Ca stores available. Since no quantal current steps could be observed, this is again compatible with the combined occurrence of Ca2+ mobilization from stores (providing close to threshold Ca2+ levels) and Ca2+ influx from the medium (which per se does not cause exocytosis). This implies that only the combination of Ca2+ flushes, primarily from internal and secondarily from external sources, can produce a signal triggering rapid, local exocytotic responses, as requested for Paramecium defense

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

International audienceLong non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA's family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co-and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That's the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest. This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest

C-fos and c-jun Proto-Oncogene Expression Is Decreased in Psoriasis: an In Situ Quantitative Analysis

Psoriasis is a common, sometimes sevcre, non-malignant skin disease characterized by hyperproliferation and abnormal differentiation of keratinocytes. Because proto-oncogenes are implicated in both cell proliferation and differentiation, their expression could be modified in skin diseases such as psoriasis. The c-fos and c-jun proto-oncogenes, whose products associate to form a heterodimeric transcription factor, are among the first genes to be expressed when certain cells are stimulated to either proliferate or differentiate. Recent studies in our laboratory have shown that the c-fos protooncogene is highly expressed in normal human adult skin. In the present study, we used in situ hybridization with RNA to compare the expression and localization of c-fos and c-jun transcripts in 15 lesional and non-lesional psoriatic skin samples. Two clinical variants of psoriasis were studied: the most severe and chronic form or plaque-type psoriasis (N = 10) and rapidly resolutive guttate-type psoriasis (N = 5). Quantitative analysis was performed using a semi-automatic image analyzer and the “Starwise grain” software program. Our control samples included 10 normal skins and eight specimens from other benign hyperproliferative non-psoriatic skin diseases, consisting of three with inflammation (seborrheic dermatitis and atopic dermatitis), and 5 without inflammation (seborrheic keratoses). Control genes we used for in situ hybridization and RNA integrity were keratin 14, which is expressed in the epidermis and was normally expressed in all tissue analyzed, and ribosomal RNA. Our data showed that c-fos and c-jun were expressed to an equivalent extent, both spatially and quantitatively, in all specimens tested. Expression was significantly decreased in plaque-type but not in guttate-type psoriasis. It was also decreased in the three other benign inflammatory cutaneous hyperproliferative disorders, but not in the five non-inflammatory cases. These results were surprising because hyperproliferation was here associated with a decrease in proto-oncogene expression, thus suggesting that c-fos and c-jun do not play a crucial role in the control of keratinocyte proliferation in vivo. However, their reduced expression in some abnormally differentiated skins indicates that both c-fos and c-jun proto-oncogenes may play a key role in keratinocyte differentiation. Their altered expression correlated with severity of the disease and the presence of an inflammatory infiltrate. These data offer a new insight into the role and regulation of these proto-oncogenes in vivo in humans

Diversité agro-morphologique précoce d’hybrides d’autofécondation F3 de cocotier (Cocos nucifera L.) impliquant l’écotype Nain Vert Sri Lanka (NVS) tolérant à la maladie du Jaunissement Mortel

Objectif : Appréhender précocement la diversité agro-morphologique de trois hybrides de cocotier issus d’autofécondation d’écotype Nain. En effet, la caractérisation agro-morphologique complète d’une plante pérenne comme le cocotier se fait sur une longue période qui peut couvrir 12 à 14 ans selon les variétés ; d’où l’utilisation des évaluations précoces pour prédire le potentiel agronomique à l’âge adulte des cocotiers.Méthodologie et résultats : Vingt et un caractères agro-morphologiques ont été mesurés sur les semences matures et jeunes plants des hybrides de 3ème génération d’autofécondation (F3) NRC x NVS et NVP05 x NVS et 2ème génération d’autofécondation (F2) NJM x NJM (témoin) créés en Côte d’Ivoire. Deux pools génétiques différents, dont l’un rassemble les hybrides F3 impliquant l’écotype Nain Vert Sri Lanka (NVS) tolérant à la maladie du Jaunissement Mortel (JM) et l’autre le témoin F2 NJM x NJM sensible au JM, ont été trouvés. De même, trois types de caractères agro-morphologiques ont été impliqués dans la différenciation des hybrides tolérant et sensible au JM.Conclusion et application des résultats : Les deux pools génétiques (tolérants et sensibles au JM) identifiés ainsi que leurs caractères agro-morphologiques (types I, II et III) spécifiques pourraient être exploités dans la recherche de QTLs associés à la tolérance au JM du cocotier.Mots clés : cocotier hybride Nain x Nain, caractérisation précoce, jaunissement mortel, QTL, Côte d’Ivoir

Development and validation of a real-time two-step RT-qPCR TaqMan (R) assay for quantitation of Sacbrood virus (SBV) and its application to a field survey of symptomatic honey bee colonies

Sacbrood virus (SBV) is the causal agent of a disease of honey bee larvae, resulting in failure to pupate and causing death. The typical clinical symptom of SBV is an accumulation of SBV-rich fluid in swollen subcuticular pouches, forming the characteristic fluid-filled sac that gives its name to the disease. Outbreaks of the disease have been reported in different countries, affecting the development of the brood and causing losses in honey bee colonies. Today, few data are available on the SBV viral load in the case of overt disease in larvae, or for the behavioural changes of SBV-infected adult bees. A two-step real-time RT-PCR assay, based on TaqMan (R) technology using a fluorescent probe (FAM-TAMRA) was therefore developed to quantify Sacbrood virus in larvae, pupae and adult bees from symptomatic apiaries. This assay was first validated according to the recent XP-U47-600 standard issued by the French Standards Institute, where the reliability and the repeatability of the results and the performance of the assay were confirmed. The performance of the qPCR assay was validated over the 6 log range of the standard curve (i.e. from 10(2) to 10(8) copies per well) with a measurement uncertainty evaluated at 0.11 log(10). The detection and quantitation limits were established respectively at 50 copies and 100 copies of SBV genome, for a template volume of 5 mu l of cDNA. The RT-qPCR assay was applied during a French SBV outbreak in 2012 where larvae with typical SBV signs were collected, along with individuals without clinical signs. The SBV quantitation revealed that, in symptomatic larvae, the virus load was significantly higher than in samples without clinical signs. Combining quantitation with clinical data, a threshold of SBV viral load related to an overt disease was proposed (10(10) SBV genome copies per individual)

The Logarithmic Sobolev Constant of The Lamplighter

We give estimates on the logarithmic Sobolev constant of some finite lamplighter graphs in terms of the spectral gap of the underlying base. Also, we give examples of application

État des lieux des bananeraies (Musa sp.) en zone de culture du cocotier, sur le littoral en Côte d’Ivoire : cas de la station Marc DELORME et des villages aux alentours

Cette étude avait pour objectif de mieux connaître la problématique liée à la production du plantain à la Station Marc DELORME et les localités aux alentours. Pour ce faire, une enquête impliquant 130 répondants a été effectuée. Les résultats ont révélé que la production de plantain sur le littoral est plus réalisée par des hommes (63,8). La tranche d’âge la plus active en ce qui concerne cette culture est celle de plus de 50 ans (45,5%). La variété la plus rencontrée dans les plantations était la variété CORNE (74,3%). La culture du plantain est faite en association (66,2%) ou en monoculture (33,8%). Au total, 62,3% des producteurs n’appliquent pas d’engrais, 77,7% ne pratiquent pas d’irrigation et 88,5% n’utilisent pas de pesticide au cours de la culture. Les bananes plantains récoltées sont plus destinées à la consommation domestique (53,8%) qu’à la vente. En période d’abondance, les coûts fluctuent entre 500 F à 3000 F CFA le régime. Par contre, En période de pénurie, les coûts des régimes varient entre 1000 et 4000 F CFA. La culture du plantain peut être envisagée en association avec le cocotier vu son importance au sein des ménages.   English title: Status of banana plantations (Musa sp.) in coconut cultivation areas on the coast of Côte d'Ivoire: case of the Marc DELORME station and surrounding villages The aim of this study is to better understand the problems related to plantain production at the Marc DELORME Station and the surrounding localities. To this end, a survey involving 130 respondents was conducted. The results revealed that plantain production is carried out more by men (63.8%). The most active age group with regard to this crop is the over 50s (45.5%). The variety most frequently found in the plantations was the CORNE variety (74.3%). Plantain is grown in association (66.2%) or as a monoculture (33.8%). In total, 62.3% of producers do not apply fertilizers, 77.7% do not use irrigation and 88.5% do not use pesticides during cultivation. The plantains harvested are more destined for domestic consumption (53.8%) than for sale. In periods of abundance, costs fluctuate between 500 F and 3000 F CFA per bunch. On the other hand, in periods of shortage, the costs of the bunch vary between 1000 and 4000 F CFA. The cultivation of plantain can be considered in association with the coconut tree given its importance in households

Prognostic significance of anti-p53 and anti-KRas circulating antibodies in esophageal cancer patients treated with chemoradiotherapy

<p>Abstract</p> <p>Background</p> <p>P53 mutations are an adverse prognostic factor in esophageal cancer. P53 and KRas mutations are involved in chemo-radioresistance. Circulating anti-p53 or anti-KRas antibodies are associated with gene mutations. We studied whether anti-p53 or anti-KRas auto-antibodies were prognostic factors for response to chemoradiotherapy (CRT) or survival in esophageal carcinoma.</p> <p>Methods</p> <p>Serum p53 and KRas antibodies (abs) were measured using an ELISA method in 97 consecutive patients treated at Saint Louis University Hospital between 1999 and 2002 with CRT for esophageal carcinoma (squamous cell carcinoma (SCCE) 57 patients, adenocarcinoma (ACE) 27 patients). Patient and tumor characteristics, response to treatment and the follow-up status of 84 patients were retrospectively collected. The association between antibodies and patient characteristics was studied. Univariate and multivariate survival analyses were conducted.</p> <p>Results</p> <p>Twenty-four patients (28%) had anti-p53 abs. Abs were found predominantly in SCCE (p = 0.003). Anti-p53 abs were associated with a shorter overall survival in the univariate analysis (HR 1.8 [1.03-2.9], p = 0.04). In the multivariate analysis, independent prognostic factors for overall and progression-free survival were an objective response to CRT, the CRT strategy (alone or combined with surgery [preoperative]) and anti-p53 abs. None of the long-term survivors had p53 abs. KRas abs were found in 19 patients (23%, no difference according to the histological type). There was no significant association between anti-KRas abs and survival neither in the univariate nor in the multivariate analysis. Neither anti-p53 nor anti-KRas abs were associated with response to CRT.</p> <p>Conclusions</p> <p>Anti-p53 abs are an independent prognostic factor for esophageal cancer patients treated with CRT. Individualized therapeutic approaches should be evaluated in this population.</p

Clinical spectrum of MTOR-related hypomelanosis of Ito with neurodevelopmental abnormalities.

PURPOSE: Hypomelanosis of Ito (HI) is a skin marker of somatic mosaicism. Mosaic MTOR pathogenic variants have been reported in HI with brain overgrowth. We sought to delineate further the pigmentary skin phenotype and clinical spectrum of neurodevelopmental manifestations of MTOR-related HI. METHODS: From two cohorts totaling 71 patients with pigmentary mosaicism, we identified 14 patients with Blaschko-linear and one with flag-like pigmentation abnormalities, psychomotor impairment or seizures, and a postzygotic MTOR variant in skin. Patient records, including brain magnetic resonance image (MRI) were reviewed. Immunostaining (n = 3) for melanocyte markers and ultrastructural studies (n = 2) were performed on skin biopsies. RESULTS: MTOR variants were present in skin, but absent from blood in half of cases. In a patient (p.[Glu2419Lys] variant), phosphorylation of p70S6K was constitutively increased. In hypopigmented skin of two patients, we found a decrease in stage 4 melanosomes in melanocytes and keratinocytes. Most patients (80%) had macrocephaly or (hemi)megalencephaly on MRI. CONCLUSION: MTOR-related HI is a recognizable neurocutaneous phenotype of patterned dyspigmentation, epilepsy, intellectual deficiency, and brain overgrowth, and a distinct subtype of hypomelanosis related to somatic mosaicism. Hypopigmentation may be due to a defect in melanogenesis, through mTORC1 activation, similar to hypochromic patches in tuberous sclerosis complex

LIF-Dependent Signaling: New Pieces in the Lego

LIF, a member of the IL6 family of cytokine, displays pleiotropic effects on various cell types and organs. Its critical role in stem cell models (e.g.: murine ES, human mesenchymal cells) and its essential non redundant function during the implantation process of embryos, in eutherian mammals, put this cytokine at the core of many studies aiming to understand its mechanisms of action, which could benefit to medical applications. In addition, its conservation upon evolution raised the challenging question concerning the function of LIF in species in which there is no implantation. We present the recent knowledge about the established and potential functions of LIF in different stem cell models, (embryonic, hematopoietic, mesenchymal, muscle, neural stem cells and iPSC). We will also discuss EVO-DEVO aspects of this multifaceted cytokine