Ionotropic receptors (IRs): chemosensory ionotropic glutamate receptors in Drosophila and beyond.

Ionotropic Receptors (IRs) are a recently characterized family of olfactory receptors in the fruit fly, Drosophila melanogaster. IRs are not related to insect Odorant Receptors (ORs), but rather have evolved from ionotropic glutamate receptors (iGluRs), a conserved family of synaptic ligand-gated ion channels. Here, we review the expression and function of IRs in Drosophila, highlighting similarities and differences with iGluRs. We also briefly describe the organization of the neuronal circuits in which IRs function, comparing and contrasting them with the sensory pathways expressing ORs. Finally, we summarize the bioinformatic identification and initial characterization of IRs in other species, which imply an evolutionarily conserved role for these receptors in chemosensation in insects and other protostomes.

Adaptive changes in a radial maze task: Efficient selection of baited arms with reduced foraging in senescent hooded rats

Qualitative differences in strategy selection during foraging in a partially baited maze were assessed in young and old rats. The baited and non-baited arms were at a fixed position in space and marked by a specific olfactory cue. The senescent rats did more re-entries during the first four-trial block but were more rapid than the young rats in selecting the reinforced arms during the first visits. Dissociation between the olfactory spatial cue reference by rotating the maze revealed that only few old subjects relied on olfactory cues to select the baited arms and the remainder relied mainly on the visuo-spatial cues.

Epigenetic alterations and constitutive Wnt signaling are early events in the progression from Barrett's esophagus to esophageal adenocarcinoma

SUMMARY Barrett's esophagus (BE) is an acquired condition in which the normal squamous epithelium in the distal esophagus is replaced by a metaplastic columnar epithelium, as a complication of chronic gastroesophageal reflux. The clinical significance of this disease is its associated predisposition to esophageal adenocarcinoma (EAC). EAC is a highly lethal disease. Better understanding of the pathogenesis of columnar metaplasia and its progression to cancer might allow the identification of biomarkers that can be used for early diagnosis, which will improve the patient survival. In this study, an improved protocol for methylation-sensitive single-strand conformation analysis, which is used to analyze promoter methylation, is proposed and a methylation-sensitive dot blot assay is described, which allows a rapid, easy, and sensitive detection of promoter methylation. Both methods were applied to study the methylation pattern of the APC promoter in histologically normal appearing gastric mucosa. The APC promoter showed monoallelic methylation, and because the methylated allele differed between the different gastric cell types, this corresponded to allelic exclusion. The APC methylation pattern was frequently altered in noimal gastric mucosa associated with neoplastic lesions, indicating that changes in the pattern of promoter methylation might precede the development of neoplasia, without accompanying histological manifestations. An epigenetic profile of 10 genes important in EAC was obtained in this study; 5 promoter genes (APC, TIMP3, TERT, CDKN2A and SFRP1) were found to be hypermethylated in the tumors. Furthermore, the promoter of APC, TIMP3 and TERT was frequently methylated in BE samples from EAC patients, but rarely in BE samples that did not progress to EAC. These three biomarkers might therefore be considered as potential predictive markers for increased EAC risk. Analysis of Wnt pathway alterations indicated that WNT2 ligand is overexpressed as early as the low-grade dysplastic stage and downregulation by promoter methylation of the SFRP1 gene occurrs already in the metaplastic lesions. Moreover, loss of APC expression is not the only factor involved in the activation of the Wnt pathway. These results indicate that a variety of biologic, mostly epigenetic events occurs very early in the carcinogenesis of BE. This new information might lead to improved early diagnosis of EAC and thus open the way to a possible application of these biomarkers in the prediction of increased EAC risk progression. RESUME L'oesophage de Barrett est une lésion métaplasique définie par le remplacement de la muqueuse malpighienne du bas oesophage par une muqueuse cylindrique glandulaire, suite à une agression chronique par du reflux gastro-esophagien. La plus importante signification clinique de cette maladie est sa prédisposition au développement d'un adénocarcinome. Le pronostic de l'adénocarcinome sur oesophage de Barrett est sombre. Seule une meilleure compréhension de la pathogenèse de l'épithélium métaplasique et de sa progression néoplasique permettrait l'identification de biomarqueurs pouvant être utilisés pour un diagnostic précoce ; la survie du patient serait ainsi augmentée. Dans cette étude, un protocole amélioré pour l'analyse de la méthylation par conformation simple brin est proposé. De plus, une technique d'analyse par dot blot permettant une détection rapide, facile et sensible de la méthylation d'un promoteur est décrite. Les deux méthodes ont été appliquées à l'étude de la méthylation du promoteur du gène APC dans des muqueuses gastriques histologiquement normales. Le promoteur APC a montré une méthylation monoallélique et, parce que les allèles méthylés différaient entre les différents types de cellules gastriques, celle-ci correspondait à une méthylation allélique exclusive. La méthylation d'APC a été trouvée fréquemment altérée dans la muqueuse gastrique normale associée à des lésions néoplasiques. Ceci indique que des changements dans la méthylation d'un promoteur peuvent précéder le développement d'une tumeur, et cela sans modification histologique. Un profil épigénétique des adénocarcinomes sur oesophage de Barrett a été obtenu dans cette étude. Cinq promoteurs (APC, TIMP3, TERT, CDKN2A et SFRP1) ont été trouvés hyperméthylés dans les tumeurs. Les promoteurs d'APC, TIMP3 et TERT étaient fréquemment méthylés dans l'épithélium métaplasique proche d'un adénocarcinome et rarement dans l'épithélium sans évolution néoplasique. Ces trois biomarkers pourraient par conséquent être considérés comme marqueur prédicatif d'un risque accru de développer une tumeur. L'analyse des altérations de la voie Wnt a montré que WNT2 est surexprimé déjà dans des dysplasies de bas-grade et que la dérégulation de SFRP1 par méthylation de son promoteur intervenait dans les lésions métaplasiques. Une perte d'expression d'APC n'est pas le seul facteur impliqué dans l'activation de cette voie. Ces résultats montrent qu'une grande diversité d'événements biologiques, principalement épigénétiques, surviennent très tôt lors de la carcinogenèse de l'oesophage de Barrett. Ces nouveaux éléments pourraient améliorer le diagnostic précoce et rendre possible l'application de ces biomarqueurs dans la prédiction d'un risque accru de développer un adénocarcinome sur un oesophage de Barrett.

Hormonal regulation of (Na+,K+)-ATPase biosynthesis in the toad bladder. Effect of aldosterone and 3,5,3'-triiodo-L-thyronine.

Aldosterone stimulates transepithelial Na+ transport in the toad bladder, and thyroid hormone antagonizes this mineralocorticoid action. In the present study, we assessed the influence of these two hormones on the biosynthesis of (Na+,K+)ATPase, the major driving force of Na+ transport. Rates of enzyme synthesis were estimated by immunoprecipitation with monospecific alpha (96,000 daltons) and beta (60,000 daltons) subunit antibodies. After a 30-min pulse of intact tissue with [35S]methionine, the anti-alpha-serum recognized the 96,000-dalton alpha subunit and the anti-beta-serum, a 42,000-dalton protein, in total cell extracts. The biosynthesis rates of both these proteins were increased 2.8- and 2.4-fold respectively, over controls by 80 nM aldosterone after 18 h of hormone treatment. The hormonal effect was not apparent up to 3 h of incubation and was dose dependent between 0.2 and 20 nM aldosterone. The hormonal induction was antagonized by spironolactone (500-fold excess) but not by amiloride. The action of aldosterone thus seems to be a receptor-mediated process and a primary event independent of the Na+ permeability of the apical membrane. Thyroid hormone, on the other hand, had no effect on either basal or aldosterone-stimulated synthesis rates of both enzyme proteins. The results demonstrate a direct effect of aldosterone on gene expression of the (Na+,K+)-ATPase. Ultimately, this phenomenon could be linked to the late mineralocorticoid action of this hormone. On the other hand, thyroid hormone, in contrast to the situation in mammals, does not stimulate de novo enzyme synthesis in amphibia. Neither can the antimineralocorticoid action of thyroid hormone in the toad bladder be explained by an inhibition of the (Na+,K+)-ATPase synthesis.

Multigenic lentiviral vectors for combined and tissue-specific expression of miRNA- and protein-based antiangiogenic factors.

Lentivirus-based gene delivery vectors carrying multiple gene cassettes are powerful tools in gene transfer studies and gene therapy, allowing coexpression of multiple therapeutic factors and, if desired, fluorescent reporters. Current strategies to express transgenes and microRNA (miRNA) clusters from a single vector have certain limitations that affect transgene expression levels and/or vector titers. In this study, we describe a novel vector design that facilitates combined expression of therapeutic RNA- and protein-based antiangiogenic factors as well as a fluorescent reporter from back-to-back RNApolII-driven expression cassettes. This configuration allows effective production of intron-embedded miRNAs that are released upon transduction of target cells. Exploiting such multigenic lentiviral vectors, we demonstrate robust miRNA-directed downregulation of vascular endothelial growth factor (VEGF) expression, leading to reduced angiogenesis, and parallel impairment of angiogenic pathways by codelivering the gene encoding pigment epithelium-derived factor (PEDF). Notably, subretinal injections of lentiviral vectors reveal efficient retinal pigment epithelium-specific gene expression driven by the VMD2 promoter, verifying that multigenic lentiviral vectors can be produced with high titers sufficient for in vivo applications. Altogether, our results suggest the potential applicability of combined miRNA- and protein-encoding lentiviral vectors in antiangiogenic gene therapy, including new combination therapies for amelioration of age-related macular degeneration.

Comparison between Radiographic (2-dimensional and 3-dimensional) and Histologic Findings of Periapical Lesions Treated with Apical Surgery.

INTRODUCTION: The aim of this study was to evaluate the concordance of 2- and 3-dimensional radiography and histopathology in the diagnosis of periapical lesions. METHODS: Patients were consecutively enrolled in this study provided that preoperative periapical radiography (PR) and cone-beam computed tomographic imaging of the tooth to be treated with apical surgery were performed. The periapical lesional tissue was histologically analyzed by 2 blinded examiners. The final histologic diagnosis was compared with the radiographic assessments of 4 blinded observers. The initial study material included 62 teeth in the same number of patients. RESULTS: Four lesions had to be excluded during processing, resulting in a final number of 58 evaluated cases (31 women and 27 men, mean age = 55 years). The final histologic diagnosis of the periapical lesions included 55 granulomas (94.8%) and 3 cysts (5.2%). Histologic analysis of the tissue samples from the apical lesions exhibited an almost perfect agreement between the 2 experienced investigators with an overall agreement of 94.83% (kappa = 0.8011). Radiographic assessment overestimated cysts by 28.4% (cone-beam computed tomographic imaging) and 20.7% (periapical radiography), respectively. Comparing the correlation of the radiographic diagnosis of 4 observers with the final histologic diagnosis, 2-dimensional (kappa = 0.104) and 3-dimensional imaging (kappa = 0.111) provided only minimum agreement. CONCLUSIONS: To establish a final diagnosis of an apical radiolucency, the tissue specimen should be evaluated histologically and specified as a granuloma (with/without epithelium) or a cyst. Analysis of 2-dimensional and 3-dimensional radiographic images alike results only in a tentative diagnosis that should be confirmed with biopsy.

Les odeurs répugnantes des démons : une preuve de leur présence corporelle au sabbat ?

The present article examines the meaning and function of olfactory remnants, often repugnant, linked to demons in the context of late medieval witchcraft and demonology. This reflection is developed within the framework of a «make believe» logic sustained by the doctrinal, theological, narrative and judiciary constructions of the witches' Sabbath. Incorporated within the order of sensory perception, references to the fetid smell of demons - who are by nature devoid of odour because they are pure spirits - constitute further proofs bearing witness to demonic presence, and thus testifying to the ignominy of the crime of witchcraft and to the guiltiness of the accused. According to those who attacked demon worshippers, the devil truly revealed himself physically; human beings were able to touch, hear, see and smell him. Sensory faculties were therefore perceived as being instrumental in corroborating the existence and reality of the Sabbath and the presence of the devil in bodily form. These considerations bring us to examine the olfactory fields associated with the devil's odour: odour of corpses, hell, sin, deviance, but also of defilement, impurity, corruption and excrements. These fetid odours are embedded in a logic of moral, spiritual and religious inversion of positive odours, such as the «sweet fragrance» of the saints, the «pure odour» of Christ or the «soft perfume» of virtue.

The Bacterium Frischella perrara Causes Scab Formation in the Gut of its Honeybee Host.

UNLABELLED: Honeybees harbor well-defined bacterial communities in their guts. The major members of these communities appear to benefit the host, but little is known about how they interact with the host and specifically how they interface with the host immune system. In the pylorus, a short region between the midgut and hindgut, honeybees frequently exhibit scab-like structures on the epithelial gut surface. These structures are reminiscent of a melanization response of the insect immune system. Despite the wide distribution of this phenotype in honeybee populations, its cause has remained elusive. Here, we show that the presence of a common member of the bee gut microbiota, the gammaproteobacterium Frischella perrara, correlates with the appearance of the scab phenotype. Bacterial colonization precedes scab formation, and F. perrara specifically localizes to the melanized regions of the host epithelium. Under controlled laboratory conditions, we demonstrate that exposure of microbiota-free bees to F. perrara but not to other bacteria results in scab formation. This shows that F. perrara can become established in a spatially restricted niche in the gut and triggers a morphological change of the epithelial surface, potentially due to a host immune response. As an intermittent colonizer, this bacterium holds promise for addressing questions of community invasion in a simple yet relevant model system. Moreover, our results show that gut symbionts of bees engage in differential host interactions that are likely to affect gut homeostasis. Future studies should focus on how these different gut bacteria impact honeybee health. IMPORTANCE: As pollinators, honeybees are key species for agricultural and natural ecosystems. Their guts harbor simple communities composed of characteristic bacterial species. Because of these features, bees are ideal systems for studying fundamental aspects of gut microbiota-host interactions. However, little is known about how these bacteria interact with their host. Here, we show that a common member of the bee gut microbiota causes the formation of a scab-like structure on the gut epithelium of its host. This phenotype was first described in 1946, but since then it has not been much further characterized, despite being found in bee populations worldwide. The scab phenotype is reminiscent of melanization, a conserved innate immune response of insects. Our results show that high abundance of one member of the bee gut microbiota triggers this specific phenotype, suggesting that the gut microbiota composition can affect the immune status of this key pollinator species.

Central serous chorioretinopathy: Recent findings and new physiopathology hypothesis.

Central serous chorioretinopathy (CSCR) is a major cause of vision threat among middle-aged male individuals. Multimodal imaging led to the description of a wide range of CSCR manifestations, and highlighted the contribution of the choroid and pigment epithelium in CSCR pathogenesis. However, the exact molecular mechanisms of CSCR have remained uncertain. The aim of this review is to recapitulate the clinical understanding of CSCR, with an emphasis on the most recent findings on epidemiology, risk factors, clinical and imaging diagnosis, and treatments options. It also gives an overview of the novel mineralocorticoid pathway hypothesis, from animal data to clinical evidences of the biological efficacy of oral mineralocorticoid antagonists in acute and chronic CSCR patients. In rodents, activation of the mineralocorticoid pathway in ocular cells either by intravitreous injection of its specific ligand, aldosterone, or by over-expression of the receptor specifically in the vascular endothelium, induced ocular phenotypes carrying many features of acute CSCR. Molecular mechanisms include expression of the calcium-dependent potassium channel (KCa2.3) in the endothelium of choroidal vessels, inducing subsequent vasodilation. Inappropriate or over-activation of the mineralocorticoid receptor in ocular cells and other tissues (such as brain, vessels) could link CSCR with the known co-morbidities observed in CSCR patients, including hypertension, coronary disease and psychological stress.

Prenatal Exposure to DEHP Affects Spermatogenesis and Sperm DNA Methylation in a Strain-Dependent Manner.

Di-(2-ethylhexyl)phtalate (DEHP) is a plasticizer with endocrine disrupting properties found ubiquitously in the environment and altering reproduction in rodents. Here we investigated the impact of prenatal exposure to DEHP on spermatogenesis and DNA sperm methylation in two distinct, selected, and sequenced mice strains. FVB/N and C57BL/6J mice were orally exposed to 300 mg/kg/day of DEHP from gestation day 9 to 19. Prenatal DEHP exposure significantly decreased spermatogenesis in C57BL/6J (fold-change = 0.6, p-value = 8.7*10-4), but not in FVB/N (fold-change = 1, p-value = 0.9). The number of differentially methylated regions (DMRs) by DEHP-exposure across the entire genome showed increased hyper- and decreased hypo-methylation in C57BL/6J compared to FVB/N. At the promoter level, three important subsets of genes were massively affected. Promoters of vomeronasal and olfactory receptors coding genes globally followed the same trend, more pronounced in the C57BL/6J strain, of being hyper-methylated in DEHP related conditions. In contrast, a large set of micro-RNAs were hypo-methylated, with a trend more pronounced in the FVB/N strain. We additionally analyze both the presence of functional genetic variations within genes that were associated with the detected DMRs and that could be involved in spermatogenesis, and DMRs related with the DEHP exposure that affected both strains in an opposite manner. The major finding in this study indicates that prenatal exposure to DEHP can decrease spermatogenesis in a strain-dependent manner and affects sperm DNA methylation in promoters of large sets of genes putatively involved in both sperm chemotaxis and post-transcriptional regulatory mechanisms.

Different flavors of Toll guide olfaction.

Toll-like receptors are historically linked to immunity across animal phyla, but accumulating evidence suggests they play additional roles in neuronal networks and in cell-cell interactions. Ward and colleagues now identify Toll-6 and Toll-7 as instructive guidance cues during Drosophila olfactory development.

Identification of pyridine analogs as new predator-derived kairomones.

In the wild, animals have developed survival strategies relying on their senses. The individual ability to identify threatening situations is crucial and leads to increase in the overall fitness of the species. Rodents, for example have developed in their nasal cavities specialized olfactory neurons implicated in the detection of volatile cues encoding for impending danger such as predator scents or alarm pheromones. In particular, the neurons of the Grueneberg ganglion (GG), an olfactory subsystem, are implicated in the detection of danger cues sharing a similar chemical signature, a heterocyclic sulfur- or nitrogen-containing motif. Here we used a "from the wild to the lab" approach to identify new molecules that are involuntarily emitted by predators and that initiate fear-related responses in the recipient animal, the putative prey. We collected urines from carnivores as sources of predator scents and first verified their impact on the blood pressure of the mice. With this approach, the urine of the mountain lion emerged as the most potent source of chemical stress. We then identified in this biological fluid, new volatile cues with characteristic GG-related fingerprints, in particular the methylated pyridine structures, 2,4-lutidine and its analogs. We finally verified their encoded danger quality and demonstrated their ability to mimic the effects of the predator urine on GG neurons, on mice blood pressure and in behavioral experiments. In summary, we were able to identify here, with the use of an integrative approach, new relevant molecules, the pyridine analogs, implicated in interspecies danger communication.

Evaluating the impact of summer vacation on the visual acuity of AMD patients treated with ranibizumab.

PurposeTo evaluate the impact of traditional French summer vacation on visual acuity and spectral domain-optical coherence tomography (SD-OCT) of Wet AMD patients being treated with intravitreal Ranibizumab.MethodsThis was a consecutive, comparative, single-centre, prospective analysis. All patients who were being treated with intravitreal injection of 0.5 mg ranibizumab at Cergy Pontoise Hospital, Department of Ophthalmology between July 2013 and September 2014 were included. Patients were divided into two groups: (A) patients who skipped one ranibizumab intravitreal injection during holidays, and (B) patients who received injection during their holidays. Evaluations occurred prior to traditional holiday (baseline) and 2 months later, consisting of BCVA using ETDRS, and a complete ophthalmic examination that included slit-lamp biomicroscopy, fundus examination, fluorescein angiography (FA), indocyanine green angiography (ICGA), and spectral domain-optical coherence tomography (SD-OCT). All patients were being treated with PRN anti-VEGF regimen and criteria for reinjection included a visual acuity loss >5 ETDRS letters and/or an increase of central retinal thickness, presence of subretinal fluid, intraretinal fluid, or pigment epithelium detachment. If reinjection criteria were not met, patients were advised to return in 4 weeks.ResultsThe mean visual acuity change was -0.071±0.149 (LogMAR) in group A and +0.003±0.178 in group B (P=0.041). At the second visit (2 months after preholidays visit), 61.8% of patients in group A had SRF and/or intraretinal cysts, and only 27.6% of patients in group B. There was a significant difference in the persistence of fluid between the two groups (P=0.007, χ(2)-test).ConclusionThis cases series demonstrated the detrimental impact of holidays on visual acuity in patients treated with ranibizumab for AMD, which, in spite of their treatment regimen, still leave in vacation. Therefore, it is important to convey the message of treatment adherence to patients, despite their need of holidays.

Fluctuation-Driven Neural Dynamics Reproduce Drosophila Locomotor Patterns.

The neural mechanisms determining the timing of even simple actions, such as when to walk or rest, are largely mysterious. One intriguing, but untested, hypothesis posits a role for ongoing activity fluctuations in neurons of central action selection circuits that drive animal behavior from moment to moment. To examine how fluctuating activity can contribute to action timing, we paired high-resolution measurements of freely walking Drosophila melanogaster with data-driven neural network modeling and dynamical systems analysis. We generated fluctuation-driven network models whose outputs-locomotor bouts-matched those measured from sensory-deprived Drosophila. From these models, we identified those that could also reproduce a second, unrelated dataset: the complex time-course of odor-evoked walking for genetically diverse Drosophila strains. Dynamical models that best reproduced both Drosophila basal and odor-evoked locomotor patterns exhibited specific characteristics. First, ongoing fluctuations were required. In a stochastic resonance-like manner, these fluctuations allowed neural activity to escape stable equilibria and to exceed a threshold for locomotion. Second, odor-induced shifts of equilibria in these models caused a depression in locomotor frequency following olfactory stimulation. Our models predict that activity fluctuations in action selection circuits cause behavioral output to more closely match sensory drive and may therefore enhance navigation in complex sensory environments. Together these data reveal how simple neural dynamics, when coupled with activity fluctuations, can give rise to complex patterns of animal behavior.

DLL4 promotes continuous adult intestinal lacteal regeneration and dietary fat transport.

The small intestine is a dynamic and complex organ that is characterized by constant epithelium turnover and crosstalk among various cell types and the microbiota. Lymphatic capillaries of the small intestine, called lacteals, play key roles in dietary fat absorption and the gut immune response; however, little is known about the molecular regulation of lacteal function. Here, we performed a high-resolution analysis of the small intestinal stroma and determined that lacteals reside in a permanent regenerative, proliferative state that is distinct from embryonic lymphangiogenesis or quiescent lymphatic vessels observed in other tissues. We further demonstrated that this continuous regeneration process is mediated by Notch signaling and that the expression of the Notch ligand delta-like 4 (DLL4) in lacteals requires activation of VEGFR3 and VEGFR2. Moreover, genetic inactivation of Dll4 in lymphatic endothelial cells led to lacteal regression and impaired dietary fat uptake. We propose that such a slow lymphatic regeneration mode is necessary to match a unique need of intestinal lymphatic vessels for both continuous maintenance, due to the constant exposure to dietary fat and mechanical strain, and efficient uptake of fat and immune cells. Our work reveals how lymphatic vessel responses are shaped by tissue specialization and uncover a role for continuous DLL4 signaling in the function of adult lymphatic vasculature.