Synthesis and X-ray crystal structure of [2(phosphinomethyl)ferrocenyl]diphenyl-phosphine

[2-(Phosphinomethyl)ferrocenyl]diphenylphosphine 2, is an air stable primary phosphine bearing a 1,2-disubstituted ferrocene framework, which has been prepared by reduction of the corresponding phosphonate. Confirmation of its structure has been obtained by X-ray single-crystals diffraction analysis. Despite its high stability toward oxidation, phosphine 2 still displays a normal coordinative behaviour toward [(pi-cymene)RuCl2]2. The expected (pi-cymene)RuCl2(phosphine) complex is formed by coordination of the primary phosphine function, while the conceivably competitive complexation of the PPh2 group was not observed

Oligonucleotide−Oligospermine Conjugates (Zip Nucleic Acids): A Convenient Means of Finely Tuning Hybridization Temperatures

Synthesis of oligonucleotide probes and control of their hybridization temperature are key aspects of polymerase chain reaction (PCR)-based detection of genetic sequences. A straightforward means to approach the last goal is to decrease the repulsion between the polyanionic probe and target strands. To this end, we have developed a versatile automated synthesis of oligonucleotide−oligospermine derivatives that gave fast access to a large variety of compounds. Plots of their hybridization temperatures Tm vs overall charge provided a measure of the impact of interstrand phosphate repulsion (and of spermine-mediated attraction) on the main driving force of duplex formation, i.e., base pairing. It showed that stabilization brought about by excess cationic charges can be of larger absolute magnitude than interstrand repulsion, even in high salt media. Base sequence and conjugation site (3′ or 5′) hardly influenced the effect of spermine on Tm. In typical PCR probe conditions, the Tm increased linearly with the number of grafted spermines (e.g., 6.2 °C per spermine for a decanucleotide probe). The large data set of Tm vs number of spermines and oligonucleotide length allowed us to empirically derive a simple mathematical relation that is accurately predicting the Tm of any oligonucleotide−oligospermine derivative. Zip nucleic acids (ZNA) are thus providing an interesting alternative to locked nucleic acids (LNA) or minor groove binders (MGB) for raising the stability of 8−12-mer oligonucleotides up to ca. 70 °C, the level required for quantitative PCR experiments

Muscle Dystroglycan Organizes the Postsynapse and Regulates Presynaptic Neurotransmitter Release at the Drosophila Neuromuscular Junction

International audienceBACKGROUND: The Dystrophin-glycoprotein complex (DGC) comprises dystrophin, dystroglycan, sarcoglycan, dystrobrevin and syntrophin subunits. In muscle fibers, it is thought to provide an essential mechanical link between the intracellular cytoskeleton and the extracellular matrix and to protect the sarcolemma during muscle contraction. Mutations affecting the DGC cause muscular dystrophies. Most members of the DGC are also concentrated at the neuromuscular junction (NMJ), where their deficiency is often associated with NMJ structural defects. Hence, synaptic dysfunction may also intervene in the pathology of dystrophic muscles. Dystroglycan is a central component of the DGC because it establishes a link between the extracellular matrix and Dystrophin. In this study, we focused on the synaptic role of Dystroglycan (Dg) in Drosophila. METHODOLOGY/PRINCIPAL FINDINGS: We show that Dg was concentrated postsynaptically at the glutamatergic NMJ, where, like in vertebrates, it controls the concentration of synaptic Laminin and Dystrophin homologues. We also found that synaptic Dg controlled the amount of postsynaptic 4.1 protein Coracle and alpha-Spectrin, as well as the relative subunit composition of glutamate receptors. In addition, both Dystrophin and Coracle were required for normal Dg concentration at the synapse. In electrophysiological recordings, loss of postsynaptic Dg did not affect postsynaptic response, but, surprisingly, led to a decrease in glutamate release from the presynaptic site. CONCLUSION/SIGNIFICANCE: Altogether, our study illustrates a conservation of DGC composition and interactions between Drosophila and vertebrates at the synapse, highlights new proteins associated with this complex and suggests an unsuspected trans-synaptic function of Dg

Global Retinoblastoma Presentation and Analysis by National Income Level.

Importance: Early diagnosis of retinoblastoma, the most common intraocular cancer, can save both a child's life and vision. However, anecdotal evidence suggests that many children across the world are diagnosed late. To our knowledge, the clinical presentation of retinoblastoma has never been assessed on a global scale. Objectives: To report the retinoblastoma stage at diagnosis in patients across the world during a single year, to investigate associations between clinical variables and national income level, and to investigate risk factors for advanced disease at diagnosis. Design, Setting, and Participants: A total of 278 retinoblastoma treatment centers were recruited from June 2017 through December 2018 to participate in a cross-sectional analysis of treatment-naive patients with retinoblastoma who were diagnosed in 2017. Main Outcomes and Measures: Age at presentation, proportion of familial history of retinoblastoma, and tumor stage and metastasis. Results: The cohort included 4351 new patients from 153 countries; the median age at diagnosis was 30.5 (interquartile range, 18.3-45.9) months, and 1976 patients (45.4%) were female. Most patients (n = 3685 [84.7%]) were from low- and middle-income countries (LMICs). Globally, the most common indication for referral was leukocoria (n = 2638 [62.8%]), followed by strabismus (n = 429 [10.2%]) and proptosis (n = 309 [7.4%]). Patients from high-income countries (HICs) were diagnosed at a median age of 14.1 months, with 656 of 666 (98.5%) patients having intraocular retinoblastoma and 2 (0.3%) having metastasis. Patients from low-income countries were diagnosed at a median age of 30.5 months, with 256 of 521 (49.1%) having extraocular retinoblastoma and 94 of 498 (18.9%) having metastasis. Lower national income level was associated with older presentation age, higher proportion of locally advanced disease and distant metastasis, and smaller proportion of familial history of retinoblastoma. Advanced disease at diagnosis was more common in LMICs even after adjusting for age (odds ratio for low-income countries vs upper-middle-income countries and HICs, 17.92 [95% CI, 12.94-24.80], and for lower-middle-income countries vs upper-middle-income countries and HICs, 5.74 [95% CI, 4.30-7.68]). Conclusions and Relevance: This study is estimated to have included more than half of all new retinoblastoma cases worldwide in 2017. Children from LMICs, where the main global retinoblastoma burden lies, presented at an older age with more advanced disease and demonstrated a smaller proportion of familial history of retinoblastoma, likely because many do not reach a childbearing age. Given that retinoblastoma is curable, these data are concerning and mandate intervention at national and international levels. Further studies are needed to investigate factors, other than age at presentation, that may be associated with advanced disease in LMICs

Travel burden and clinical presentation of retinoblastoma: analysis of 1024 patients from 43 African countries and 518 patients from 40 European countries

BACKGROUND: The travel distance from home to a treatment centre, which may impact the stage at diagnosis, has not been investigated for retinoblastoma, the most common childhood eye cancer. We aimed to investigate the travel burden and its impact on clinical presentation in a large sample of patients with retinoblastoma from Africa and Europe. METHODS: A cross-sectional analysis including 518 treatment-naïve patients with retinoblastoma residing in 40 European countries and 1024 treatment-naïve patients with retinoblastoma residing in 43 African countries. RESULTS: Capture rate was 42.2% of expected patients from Africa and 108.8% from Europe. African patients were older (95% CI -12.4 to -5.4, p<0.001), had fewer cases of familial retinoblastoma (95% CI 2.0 to 5.3, p<0.001) and presented with more advanced disease (95% CI 6.0 to 9.8, p<0.001); 43.4% and 15.4% of Africans had extraocular retinoblastoma and distant metastasis at the time of diagnosis, respectively, compared to 2.9% and 1.0% of the Europeans. To reach a retinoblastoma centre, European patients travelled 421.8 km compared to Africans who travelled 185.7 km (p<0.001). On regression analysis, lower-national income level, African residence and older age (p<0.001), but not travel distance (p=0.19), were risk factors for advanced disease. CONCLUSIONS: Fewer than half the expected number of patients with retinoblastoma presented to African referral centres in 2017, suggesting poor awareness or other barriers to access. Despite the relatively shorter distance travelled by African patients, they presented with later-stage disease. Health education about retinoblastoma is needed for carers and health workers in Africa in order to increase capture rate and promote early referral

Cationic oligonucleotides,Synthesis and evaluation

Ce travail de thèse porte sur la synthèse et l'évaluation d'oligonucléotides cationiques obtenus par conjugaison de chaînes polyamines à l'extrémité 5 d'oligonucléotides naturels. Nous avons utilisé dans un premier temps des molécules hétérobifonctionnelThis work is based on the synthesis and evaluation of cationic oligonucleotides (ODN) prepared by conjugation of polyamine tails to the 5 -end of natural oligonucleotides. The conjugation was first performed with heterobifunctional molecules to link poly

Cationic oligonucleotides,Synthesis and evaluation

Ce travail de thèse porte sur la synthèse et l'évaluation d'oligonucléotides cationiques obtenus par conjugaison de chaînes polyamines à l'extrémité 5 d'oligonucléotides naturels. Nous avons utilisé dans un premier temps des molécules hétérobifonctionnelles pour conjuguer un oligonucléotide (ODN) à une polyéthylènimine et à des peptides contenant des résidus lysines et arginines. La préparation des oligonucléotides cationiques a par la suite été améliorée par la mise au point d'une synthèse automatique d'oligonucléotides conjugués à des polyspermines. L'hybridation de ces oligonucléotides sur un brin complémentaire a alors été évaluée en mesurant les températures de fusion des hétéroduplexes dans des conditions salines physiologiques, montrant une stabilisation croissante avec la charge positive du conjugué. L'électrophorèse nous a également permis d'observer que l'addition d'une queue cationique à un oligonucléotide lui permettait d'induire un déplacement de brin. L'internalisation cellulaire de nos molécules a de plus été démontrée en utilisant des conjugués ODN-peptide marqués en 3 par une fluorescéine, et des expériences de séquençage réalisées en utilisant les ODN-peptides comme amorces pour la Taq polymerase ont prouvé que ces derniers pouvaient être reconnus par des enzymes. En conclusion, nous avons montré que les oligonucléotides globalement cationiques pouvaient être synthétisés, qu ils ont une meilleure affinité d'hybridation et des propriétés de  vectorisation  intracellulaire tout en étant toujours reconnaissables et exploitables par des enzymes.This work is based on the synthesis and evaluation of cationic oligonucleotides (ODN) prepared by conjugation of polyamine tails to the 5 -end of natural oligonucleotides. The conjugation was first performed with heterobifunctional molecules to link polyethylenimine and lysine/arginine polypeptide tails to ODN. The design of the cationic moiety was then improved, leading to the automatic synthesis of polyspermine-ODN conjugates. After that, we began the study of the conjugates physico-chemical properties and thermal denaturation experiments were performed at physiological salt concentration to evaluate the stability of the heteroduplex formed between our conjugates and a complementary strand of DNA. Melting temperature measurements showed an increase in stability due to the global cationic charge of the conjugates. Moreover, polyacrylamide gel electrophoresis studies pointed out that the addition of a cationic tail allowed strand displacement. Finally, cellular entry of our conjugates was observed using fluorescently labeled ODN-peptide conjugates, and the automatic sequencing of plasmid using ODN-peptide as a primer for polymerase chain elongation proved that the conjugates were still recognized by enzymes such as Taq polymerase. In summary, we have showed that the synthesis of ODN-polyamine with overall cationic charges is possible, and that these conjugates have an increased affinity for DNA. They can also enter cells without assistance and are still able to be recruited by enzymatic complexes

Oligonucléotides cationiques: synthèse et évaluation

Ce travail de thèse porte sur la synthèse et l’évaluation d’oligonucléotides cationiques obtenus par conjugaison de chaînes polyamines à l’extrémité 5’ d’oligonucléotides naturels. Nous avons utilisé dans un premier temps des molécules hétérobifonctionnelles pour conjuguer un oligonucléotide (ODN) à une polyéthylènimine et à des peptides contenant des résidus lysines et arginines. La préparation des oligonucléotides cationiques a par la suite été améliorée par la mise au point d’une synthèse automatique d’oligonucléotides conjugués à des polyspermines. L’hybridation de ces oligonucléotides sur un brin complémentaire a alors été évaluée en mesurant les températures de fusion des hétéroduplexes dans des conditions salines physiologiques, montrant une stabilisation croissante avec la charge positive du conjugué. L’électrophorèse nous a également permis d’observer que l’addition d’une queue cationique à un oligonucléotide lui permettait d’induire un déplacement de brin. L’internalisation cellulaire de nos molécules a de plus été démontrée en utilisant des conjugués ODN-peptide marqués en 3’ par une fluorescéine, et des expériences de séquençage réalisées en utilisant les ODNpeptides comme amorces pour la Taq polymerase ont prouvé que ces derniers pouvaient être reconnus par des enzymes. En conclusion, nous avons montré que les oligonucléotides globalement cationiques pouvaient être synthétisés, qu’ils ont une meilleure affinité d’hybridation et des propriétés de « vectorisation » intracellulaire tout en étant toujours reconnaissables et exploitables par des enzymes.This work is based on the synthesis and evaluation of cationic oligonucleotides (ODN) prepared by conjugation of polyamine tails to the 5’-end of natural oligonucleotides. The conjugation was first performed with heterobifunctional molecules to link polyethylenimine and lysine/arginine polypeptide tails to ODN. The design of the cationic moiety was then improved, leading to the automatic synthesis of polyspermine-ODN conjugates. After that, we began the study of the conjugates’ physico-chemical properties and thermal denaturation experiments were performed at physiological salt concentration to evaluate the stability of the heteroduplex formed between our conjugates and a complementary strand of DNA. Melting temperature measurements showed an increase in stability due to the global cationic charge of the conjugates. Moreover, polyacrylamide gel electrophoresis studies pointed out that the addition of a cationic tail allowed strand displacement. Finally, cellular entry of our conjugates was observed using fluorescently labeled ODN-peptide conjugates, and the automatic sequencing of plasmid using ODN-peptide as a primer for polymerase chain elongation proved that the conjugates were still recognized by enzymes such as Taq polymerase. In summary, we have showed that the synthesis of ODN-polyamine with overall cationic charges is possible, and that these conjugates have an increased affinity for DNA. They can also enter cells without assistance and are still able to be recruited by enzymatic complexes

Oligonucléotides cationiques (Synthèse et évaluation)

Ce travail de thèse porte sur la synthèse et l évaluation d oligonucléotides cationiques obtenus par conjugaison de chaînes polyamines à l extrémité 5 d oligonucléotides naturels. Nous avons utilisé dans un premier temps des molécules hétérobifonctionnelles pour conjuguer un oligonucléotide (ODN) à une polyéthylènimine et à des peptides contenant des résidus lysines et arginines. La préparation des oligonucléotides cationiques a par la suite été améliorée par la mise au point d une synthèse automatique d oligonucle otides conjugués à des polyspermines. L hybridation de ces oligonucléotides sur un brin complémentaire a alors été évaluée en mesurant les températures de fusion des hétéroduplexes dans des conditions salines physiologiques, montrant une stabilisation croissante avec la charge positive du conjugué. L électrophorèse nous a également permis d observer que l addition d une queue cationique à un oligonucléotide lui permettait d induire un déplacement de brin. L internalisation cellulaire de nos molécules a de plus été démontrée en utilisant des conjugués ODN-peptide marqués en 3 par une fluorescéine, et des expériences de séquençage réalisées en utilisant les ODN-peptides comme amorces pour la Taq polymerase ont prouvé que ces derniers pouvaient être reconnus par des enzymes. En conclusion, nous avons montré que les oligonucléotides globalement cationiques pouvaient être synthétisés, qu ils ont une meilleure affinité d hybridation et des propriétés de vectorisation intracellulaire tout en étant toujours reconnaissables et exploitables par des enzymes.This work is based on the synthesis and evaluation of cationic oligonucleotides (ODN) prepared by conjugation of polyamine tails to the 5 -end of natural oligonucleotides. The conjugation was first performed with heterobifunctional molecules to link polyethylenimine and lysine/arginine polypeptide tails to ODN. The design of the cationic moiety was then improved, leading to the automatic synthesis of polyspermine-ODN conjugates. After that, we began the study of the conjugates physico-chemical properties and thermal denaturation experiments were performed at physiological salt concentration to evaluate the stability of the heteroduplex formed between our conjugates and a complementary strand of DNA. Melting temperature measurements showed an increase in stability due to the global cationic charge of the conjugates. Moreover, polyacrylamide gel electrophoresis studies pointed out that the addition of a cationic tail allowed strand displacement. Finally, cellular entry of our conjugates was observed using fluorescently labeled ODN-peptide conjugates, and the automatic sequencing of plasmid using ODN-peptide as a primer for polymerase chain elongation proved that the conjugates were still recognized by enzymes such as Taq polymerase. In summary, we have showed that the synthesis of ODN-polyamine with overall cationic charges is possible, and that these conjugates have an increased affinity for DNA. They can also enter cells without assistance and are still able to be recruited by enzymatic complexes.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Indole, a signaling molecule produced by the gut microbiota, negatively impacts emotional behaviors in rats

Gut microbiota produces a wide and diverse array of metabolites that are an integral part of the host metabolome. The emergence of the gut microbiome-brain axis concept has prompted investigations on the role of gut microbiota dysbioses in the pathophysiology of brain diseases. Specifically, the search for microbe-related metabolomic signatures in human patients and animal models of psychiatric disorders has pointed out the importance of the microbial metabolism of aromatic amino acids. Here, we investigated the effect of indole on brain and behavior in rats. Indole is produced by gut microbiota fromtryptophan, through the tryptophanase enzyme encoded by the tnaA gene. First, we mimicked an acute and high overproduction of indole by injecting this compound in the cecum of conventional rats. This treatment led to a dramatic decrease of motor activity. The neurodepressant oxidized derivatives of indole, oxindole and isatin, accumulated in the brain. In addition, increase in eye blinking frequency and in c-Fos protein expression in the dorsal vagal complex denoted a vagus nerve activation. Second, we mimicked a chronic and moderate overproduction of indole by colonizing germ-free rats with the indole-producing bacterial species Escherichia coli. We compared emotional behaviors of these rats with those of germ-free rats colonized with a genetically-engineered counterpart strain unable to produce indole. Rats overproducing indole displayed higher helplessness in the tail suspension test, and enhanced anxiety-like behavior in the novelty, elevated plus maze and open-field tests. Vagus nerve activation was suggested by an increase in eye blinking frequency. However, unlike the conventional rats dosed with a high amount of indole, the motor activity was not altered and neither oxindole nor isatin could be detected in the brain. Further studies are required for a comprehensive understanding of the mechanisms supporting indole effects on emotional behaviors. As our findings suggest that people whose gut microbiota is highly prone to produce indole could be more likely to develop anxiety and mood disorders, we addressed the issue of the inter-individual variability of indole producing potential in humans. An in silico investigation of metagenomic data focused on the tnaA gene products definitively proved this inter-individual variability