Markierte RNA : Synthese, Eigenschaften und Anwendungen

In dieser Arbeit wurden verschiedene Strategien zur Markierung von RNA mit modifizierten Nukleosiden oder Fluorophoren untersucht. Diese RNAs wurden für verschiedene Untersuchungen auf dem Gebiet der chemischen Biologie eingesetzt, um Riboschalter-Ligand-Wechselwirkungen, die Faltung von Aptameren, metabolische Markierungen und die Umwandlung von Nukleosiden für die RNA-Sequenzierung und Aufklärung der zellulären RNA-Dynamik zu erforschen. Im ersten Teil dieser Arbeit wurde die Wechselwirkung zwischen der Struktur und Wirkung von modifizierten Liganden mit preQ1 Klasse-I und Klasse-II Riboschaltern untersucht. Zudem wurde der Zusammenhang zwischen in vitro und in vivo Verhalten erforscht. Riboschalter regulieren die Genexpression durch die spezifische Bindung kleiner Metaboliten, das zur Änderung ihrer Konformation führt. Sie agieren auf der transkriptionellen oder der translatorischen Ebene der Genregulation und sind ein interessantes Forschungsgebiet im Hinblick auf antibakterielle Medikamente sowie als Biosensor. Verschiedene Liganden mit Modifikationen an der 7-Aminomethylgruppe des preQ1 wurden rational konzipiert und synthetisiert. Das in vitro Verhalten der Liganden wurde mittels 2-Aminopurin Fluoreszenz Assay getestet. Das in vivo Verhalten wurde mittels Fluoreszenz Assay mit eGFP als Reportergen untersucht. Die Ergebnisse der Messungen weisen darauf hin, dass das rationale Ligandendesign nicht notwendigerweise zu effizienten Liganden für eine erfolgreiche Herabregulation der Genexpression in vivo führt. Liganden mit einer Azidoalkyl-Modifikation führten überraschenderweise zu einer hohen Herabregulation der Genexpression in vivo, obwohl die in vitro Ergebnisse wenig vielversprechend waren. Im Gegensatz dazu zeigten die aminoalkyl-modifizierten Liganden positive in vitro Ergebnisse in Bezug auf die Bindungsthermodynamik und Bindungskinetik, wohingegen sich diese Ergebnisse nicht in eine erhöhte Herabregulation der Genexpression übersetzen ließen. Des Weiteren wurde im Laufe dieser Arbeit eine praktikable Synthese zum 2-Aminopurinphosphoramidit für die Festphasensynthese entwickelt. Die 6-stufige Syntheseroute beginnt bei der 2-Amino-6-chlorpurin Ribose und das Endprodukt hat eine N-(Di-n-butylamin)methylen Schutzgruppe am N2, geeignet für die Anwendung von milden Entschützungsbedingungen nach der Festphasensynthese. Im zweiten Teil wurden die Strukturen von Aptameren, die zuvor von Batey und Mitarbeitern beschrieben wurden, auf ihre Anwendung als Biosensoren für Serotonin untersucht. Dafür wurden Einzelmolekül und Bulk-FRET Messungen durchgeführt. Die synthetischen RNAs wurden mit den Fluorophoren Cy3 und Cy5 markiert und anschließend mittels der T4 DNA Ligase ligiert, um das gewünschte, zweifach markierte Konstrukt zu erhalten. Unterschiedliche Markierungsmethoden mittels Click Chemie oder NHS-Ester Chemie wurden untersucht. Dazu wurden verschiedene modifizierte Phosphoramidite, die entweder eine Propargylgruppe oder eine Aminopropylgruppe am 2′-OH aufweisen, synthetisiert und in die RNA eingebaut. Die darauffolgenden Messungen der verschiedenen Konstrukte zeigten die Wichtigkeit des langen Stammes von 10 Basenpaaren in einem Konstrukt des Hammerhead Ribozyms für die erfolgreiche Bindung von Serotonin. Im dritten Teil dieser Arbeit wurde die Umwandlung der Nucleoside 6-Thioguanosin (6sG), 6-Selenoguanosin (6SeG) und 4-Thiothymidin (4sT) in einem Oligonukleotid mittels TUC-Seq Chemie untersucht. TUC-Seq wurde für die Umwandlung von 4-Thiouridin (4sU) in Cytidin (C) mittels Osmiumtetroxid (OsO4) und Ammoniumchlorid (NH4Cl) etabliert. Der Einbau in die RNA und die Umwandlung von 4sU zu C wird kombiniert mit der Analyse durch RNA-Sequenzierung. Dieser Ansatz findet Anwendung in der Untersuchung der RNA Lebenszeit und des RNA Abbaus. 6-Thioguanosin wurde erfolgreich in einem 2-stufigen Verfahren in 6-Hydrazin-2-aminopurin (6h2Ap) umgewandelt. Dabei wurde die RNA zuerst mit OsO4/NH4Cl gefolgt von einem Hydrazin Buffer behandelt. Die neue Modifikation (6h2Ap) wurde kristallisiert, um die Struktur und Basenpaarung zu bestätigen und zu analysieren. Das 6h2Ap bildet ein kanonisches Watson-Crick Basenpaar mit Uridin und wird von der Reversen Transkriptase als Adenosin abgelesen. Zusätzlich wurden erste Experimente zur in vivo Anwendung von 6sG als metabolischer Marker durchgeführt. Außerdem wurden RNAs mit 6-Selenoguanosin synthetisiert und die Modifikation wurde in einem einstufigen Verfahren in 2,6-Diaminopurin (DAP) umgewandelt. Für die vollständige Umwandlung waren erhöhte Ammonium Konzentrationen und eine höhere Temperatur im Vergleich zum Standard TUC-Seq Verfahren notwendig. Die erfolgreiche Umwandlung wurde durch Massenspektrometrie sowie thermischen Denaturierungsstudien bestätigt. Beide Umwandlungen, 6sG zu 6h2Ap und 6SeG zu DAP, sind kombinierbar mit der 4sU zu C Transformation, wie in Doppelmarkierungsexperimenten gezeigt wurde. Zusätzlich wurde TUC-Seq Chemie auf DNA Ebene erfolgreich implementiert durch die Umwandlung von 4-Thiothymidine zu 5-Methylcytidin (5mC). Thermische Denaturierungsstudien wurden durchgeführt, um den Einfluss von 5mC und 4sT auf die Stabilität der DNA zu untersuchen. In einer laufenden Kollaboration wird TUC-seq in diesem Zusammenhang angewendet und ist vielversprechend, um spezifische Aspekte der dreidimensionalen Organisation von chromosomaler DNA während der Replikation aufzudecken.In this thesis different strategies for the labeling of RNA with modified nucleosides or fluorophores were explored. These RNAs were then applied for distinct investigations in the field of chemical biology, targeting riboswitch-ligand interactions, the folding of aptamers, metabolic labeling, and nucleoside conversions for RNA sequencing to shed light on cellular RNA dynamics. In the first part of this thesis the structure-activity relationship (SAR) between modified ligands and the preQ1 class-I and class-II riboswitches were investigated as well as the correlation of in vitro and in vivo performances. Riboswitches are gene regulatory elements specifically binding small metabolites and thereby changing their conformation. They operate either on the transcriptional or the translational level of gene control and are interesting targets for antibacterial drugs or as biosensors. A variety of ligands bearing an attachment at the 7-aminomethyl group of preQ1 were rationally designed and synthesized. They were tested for their in vitro binding behavior utilizing 2-aminopurine fluorescence assays followed by in vivo fluorescence assays employing eGFP as reporter gene. The findings suggested that rational ligand design does not necessarily lead to efficient ligands for the down-regulation of gene expression in vivo. The obtained in vitro parameters did not translate into the expected in vivo results. Surprisingly, the azidoalkyl bearing ligands were mediocre in vitro but exhibited a high down-regulation of gene expression in vivo. Ligands with aminoalkyl attachments showed the opposite effect that is that the positive evaluation of binding kinetics and thermodynamics in vitro did not translate into an enhanced gene regulation. Additionally, a practical synthesis towards 2-aminopurine phosphoramidite for solid-phase synthesis was developed in the course of this thesis. The six step route starts from 2-amino-6-chloropurine riboside and the final product contains a N-(di-n-butylamino)methylene protecting group at the N2 for the application of mild deprotection conditions. In the second part the structures of aptamers previously described by Batey and co-workers were investigated for their application as biosensors for serotonin. Single-molecule and bulk FRET measurements were employed for this purpose. The synthetic RNAs were labeled with Cy3 and Cy5 fluorophores and subsequently ligated with T4 DNA ligase to obtain the desired double-labeled full-length constructs. Different labeling strategies were explored utilizing click chemistry as well as NHS-ester chemistry. Therefore, a variety of modified phosphoramidites containing either a propargyl group or an aminopropyl functionality attached to the 2′-OH were synthesized and incorporated into RNAs. The subsequent measurements of the different structures revealed the importance of the full length stem of 10 base pairs of a construct derived from the hammerhead ribozyme for the successful binding of serotonin. In the third part the conversion of the nucleosides 6-thioguanosine (6sG), 6-selenoguanosine (6SeG) and 4-thiothymidine (4sT) in oligonucleotides utilizing TUC-seq chemistry was investigated. TUC-seq was established for the transformation of 4-thiouridine (4sU) to cytidine (C) employing osmium tetroxide (OsO4) in combination with ammonium chloride (NH4Cl). The incorporation into RNA and conversion of 4sU to C is followed by the analysis via RNA sequencing. The approach is applicable for the investigation of RNA life and decay. 6-Thioguanosine was successfully converted to 6-hydrazino-2-aminopurine (6h2Ap) in a two-step procedure by applying OsO4/NH4Cl and a hydrazine buffer. A crystal structure was obtained of the novel modification to confirm and analyze the structure and pairing mode of 6h2Ap. It forms a Watson-Crick canonical base pair with uridine and is read as an adenosine by the reverse transcriptase. Additionally, preliminary experiments for the in vivo application as metabolic label were performed. Moreover, 6-selenoguanosine containing RNAs were synthesized and the modification was converted in a one-step procedure to 2,6-diaminopurine (DAP). Elevated ammonia concentrations and a higher temperature compared to the standard TUC-seq conditions were necessary for the full conversion. The successful transformation was confirmed by mass spectrometry and thermal denaturation studies. Both conversions, 6sG to 6h2Ap and 6SeG to DAP, are combinable with the 4sU to C transformation as was shown in double labeling experiments. Furthermore, the effective conversion of 4-thiothymidine to 5-methylcytidine (5mC) displayed the successful implementation of TUC-seq chemistry for DNAs. Thermal denaturation studies were performed to explore the influence of 5mC and 4sT on the DNA stability. In this context, collaborative work using TUC-seq is ongoing and highly promising to reveal specific aspects of the three-dimensional organization of chromosomal DNA during replication.vorgelegt von Eva Neuner, MSc.Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersZusammenfassung in deutscher SpracheDissertation Universität Innsbruck 201

Markierte RNA : Synthese, Eigenschaften und Anwendungen

In dieser Arbeit wurden verschiedene Strategien zur Markierung von RNA mit modifizierten Nukleosiden oder Fluorophoren untersucht. Diese RNAs wurden f\ufcr verschiedene Untersuchungen auf dem Gebiet der chemischen Biologie eingesetzt, um Riboschalter-Ligand-Wechselwirkungen, die Faltung von Aptameren, metabolische Markierungen und die Umwandlung von Nukleosiden f\ufcr die RNA-Sequenzierung und Aufkl\ue4rung der zellul\ue4ren RNA-Dynamik zu erforschen. Im ersten Teil dieser Arbeit wurde die Wechselwirkung zwischen der Struktur und Wirkung von modifizierten Liganden mit preQ1 Klasse-I und Klasse-II Riboschaltern untersucht. Zudem wurde der Zusammenhang zwischen in vitro und in vivo Verhalten erforscht. Riboschalter regulieren die Genexpression durch die spezifische Bindung kleiner Metaboliten, das zur \uc4nderung ihrer Konformation f\ufchrt. Sie agieren auf der transkriptionellen oder der translatorischen Ebene der Genregulation und sind ein interessantes Forschungsgebiet im Hinblick auf antibakterielle Medikamente sowie als Biosensor. Verschiedene Liganden mit Modifikationen an der 7-Aminomethylgruppe des preQ1 wurden rational konzipiert und synthetisiert. Das in vitro Verhalten der Liganden wurde mittels 2-Aminopurin Fluoreszenz Assay getestet. Das in vivo Verhalten wurde mittels Fluoreszenz Assay mit eGFP als Reportergen untersucht. Die Ergebnisse der Messungen weisen darauf hin, dass das rationale Ligandendesign nicht notwendigerweise zu effizienten Liganden f\ufcr eine erfolgreiche Herabregulation der Genexpression in vivo f\ufchrt. Liganden mit einer Azidoalkyl-Modifikation f\ufchrten \ufcberraschenderweise zu einer hohen Herabregulation der Genexpression in vivo, obwohl die in vitro Ergebnisse wenig vielversprechend waren. Im Gegensatz dazu zeigten die aminoalkyl-modifizierten Liganden positive in vitro Ergebnisse in Bezug auf die Bindungsthermodynamik und Bindungskinetik, wohingegen sich diese Ergebnisse nicht in eine erh\uf6hte Herabregulation der Genexpression \ufcbersetzen lie fen. Des Weiteren wurde im Laufe dieser Arbeit eine praktikable Synthese zum 2-Aminopurinphosphoramidit f\ufcr die Festphasensynthese entwickelt. Die 6-stufige Syntheseroute beginnt bei der 2-Amino-6-chlorpurin Ribose und das Endprodukt hat eine N-(Di-n-butylamin)methylen Schutzgruppe am N2, geeignet f\ufcr die Anwendung von milden Entsch\ufctzungsbedingungen nach der Festphasensynthese. Im zweiten Teil wurden die Strukturen von Aptameren, die zuvor von Batey und Mitarbeitern beschrieben wurden, auf ihre Anwendung als Biosensoren f\ufcr Serotonin untersucht. Daf\ufcr wurden Einzelmolek\ufcl und Bulk-FRET Messungen durchgef\ufchrt. Die synthetischen RNAs wurden mit den Fluorophoren Cy3 und Cy5 markiert und anschlie fend mittels der T4 DNA Ligase ligiert, um das gew\ufcnschte, zweifach markierte Konstrukt zu erhalten. Unterschiedliche Markierungsmethoden mittels Click Chemie oder NHS-Ester Chemie wurden untersucht. Dazu wurden verschiedene modifizierte Phosphoramidite, die entweder eine Propargylgruppe oder eine Aminopropylgruppe am 2\u2032-OH aufweisen, synthetisiert und in die RNA eingebaut. Die darauffolgenden Messungen der verschiedenen Konstrukte zeigten die Wichtigkeit des langen Stammes von 10 Basenpaaren in einem Konstrukt des Hammerhead Ribozyms f\ufcr die erfolgreiche Bindung von Serotonin. Im dritten Teil dieser Arbeit wurde die Umwandlung der Nucleoside 6-Thioguanosin (6sG), 6-Selenoguanosin (6SeG) und 4-Thiothymidin (4sT) in einem Oligonukleotid mittels TUC-Seq Chemie untersucht. TUC-Seq wurde f\ufcr die Umwandlung von 4-Thiouridin (4sU) in Cytidin (C) mittels Osmiumtetroxid (OsO4) und Ammoniumchlorid (NH4Cl) etabliert. Der Einbau in die RNA und die Umwandlung von 4sU zu C wird kombiniert mit der Analyse durch RNA-Sequenzierung. Dieser Ansatz findet Anwendung in der Untersuchung der RNA Lebenszeit und des RNA Abbaus. 6-Thioguanosin wurde erfolgreich in einem 2-stufigen Verfahren in 6-Hydrazin-2-aminopurin (6h2Ap) umgewandelt. Dabei wurde die RNA zuerst mit OsO4/NH4Cl gefolgt von einem Hydrazin Buffer behandelt. Die neue Modifikation (6h2Ap) wurde kristallisiert, um die Struktur und Basenpaarung zu best\ue4tigen und zu analysieren. Das 6h2Ap bildet ein kanonisches Watson-Crick Basenpaar mit Uridin und wird von der Reversen Transkriptase als Adenosin abgelesen. Zus\ue4tzlich wurden erste Experimente zur in vivo Anwendung von 6sG als metabolischer Marker durchgef\ufchrt. Au ferdem wurden RNAs mit 6-Selenoguanosin synthetisiert und die Modifikation wurde in einem einstufigen Verfahren in 2,6-Diaminopurin (DAP) umgewandelt. F\ufcr die vollst\ue4ndige Umwandlung waren erh\uf6hte Ammonium Konzentrationen und eine h\uf6here Temperatur im Vergleich zum Standard TUC-Seq Verfahren notwendig. Die erfolgreiche Umwandlung wurde durch Massenspektrometrie sowie thermischen Denaturierungsstudien best\ue4tigt. Beide Umwandlungen, 6sG zu 6h2Ap und 6SeG zu DAP, sind kombinierbar mit der 4sU zu C Transformation, wie in Doppelmarkierungsexperimenten gezeigt wurde. Zus\ue4tzlich wurde TUC-Seq Chemie auf DNA Ebene erfolgreich implementiert durch die Umwandlung von 4-Thiothymidine zu 5-Methylcytidin (5mC). Thermische Denaturierungsstudien wurden durchgef\ufchrt, um den Einfluss von 5mC und 4sT auf die Stabilit\ue4t der DNA zu untersuchen. In einer laufenden Kollaboration wird TUC-seq in diesem Zusammenhang angewendet und ist vielversprechend, um spezifische Aspekte der dreidimensionalen Organisation von chromosomaler DNA w\ue4hrend der Replikation aufzudecken.In this thesis different strategies for the labeling of RNA with modified nucleosides or fluorophores were explored. These RNAs were then applied for distinct investigations in the field of chemical biology, targeting riboswitch-ligand interactions, the folding of aptamers, metabolic labeling, and nucleoside conversions for RNA sequencing to shed light on cellular RNA dynamics. In the first part of this thesis the structure-activity relationship (SAR) between modified ligands and the preQ1 class-I and class-II riboswitches were investigated as well as the correlation of in vitro and in vivo performances. Riboswitches are gene regulatory elements specifically binding small metabolites and thereby changing their conformation. They operate either on the transcriptional or the translational level of gene control and are interesting targets for antibacterial drugs or as biosensors. A variety of ligands bearing an attachment at the 7-aminomethyl group of preQ1 were rationally designed and synthesized. They were tested for their in vitro binding behavior utilizing 2-aminopurine fluorescence assays followed by in vivo fluorescence assays employing eGFP as reporter gene. The findings suggested that rational ligand design does not necessarily lead to efficient ligands for the down-regulation of gene expression in vivo. The obtained in vitro parameters did not translate into the expected in vivo results. Surprisingly, the azidoalkyl bearing ligands were mediocre in vitro but exhibited a high down-regulation of gene expression in vivo. Ligands with aminoalkyl attachments showed the opposite effect that is that the positive evaluation of binding kinetics and thermodynamics in vitro did not translate into an enhanced gene regulation. Additionally, a practical synthesis towards 2-aminopurine phosphoramidite for solid-phase synthesis was developed in the course of this thesis. The six step route starts from 2-amino-6-chloropurine riboside and the final product contains a N-(di-n-butylamino)methylene protecting group at the N2 for the application of mild deprotection conditions. In the second part the structures of aptamers previously described by Batey and co-workers were investigated for their application as biosensors for serotonin. Single-molecule and bulk FRET measurements were employed for this purpose. The synthetic RNAs were labeled with Cy3 and Cy5 fluorophores and subsequently ligated with T4 DNA ligase to obtain the desired double-labeled full-length constructs. Different labeling strategies were explored utilizing click chemistry as well as NHS-ester chemistry. Therefore, a variety of modified phosphoramidites containing either a propargyl group or an aminopropyl functionality attached to the 2\u2032-OH were synthesized and incorporated into RNAs. The subsequent measurements of the different structures revealed the importance of the full length stem of 10 base pairs of a construct derived from the hammerhead ribozyme for the successful binding of serotonin. In the third part the conversion of the nucleosides 6-thioguanosine (6sG), 6-selenoguanosine (6SeG) and 4-thiothymidine (4sT) in oligonucleotides utilizing TUC-seq chemistry was investigated. TUC-seq was established for the transformation of 4-thiouridine (4sU) to cytidine (C) employing osmium tetroxide (OsO4) in combination with ammonium chloride (NH4Cl). The incorporation into RNA and conversion of 4sU to C is followed by the analysis via RNA sequencing. The approach is applicable for the investigation of RNA life and decay. 6-Thioguanosine was successfully converted to 6-hydrazino-2-aminopurine (6h2Ap) in a two-step procedure by applying OsO4/NH4Cl and a hydrazine buffer. A crystal structure was obtained of the novel modification to confirm and analyze the structure and pairing mode of 6h2Ap. It forms a Watson-Crick canonical base pair with uridine and is read as an adenosine by the reverse transcriptase. Additionally, preliminary experiments for the in vivo application as metabolic label were performed. Moreover, 6-selenoguanosine containing RNAs were synthesized and the modification was converted in a one-step procedure to 2,6-diaminopurine (DAP). Elevated ammonia concentrations and a higher temperature compared to the standard TUC-seq conditions were necessary for the full conversion. The successful transformation was confirmed by mass spectrometry and thermal denaturation studies. Both conversions, 6sG to 6h2Ap and 6SeG to DAP, are combinable with the 4sU to C transformation as was shown in double labeling experiments. Furthermore, the effective conversion of 4-thiothymidine to 5-methylcytidine (5mC) displayed the successful implementation of TUC-seq chemistry for DNAs. Thermal denaturation studies were performed to explore the influence of 5mC and 4sT on the DNA stability. In this context, collaborative work using TUC-seq is ongoing and highly promising to reveal specific aspects of the three-dimensional organization of chromosomal DNA during replication.vorgelegt von Eva Neuner, MSc.Abweichender Titel laut cbersetzung der Verfasserin/des VerfassersZusammenfassung in deutscher SpracheDissertation Universit\ue4t Innsbruck 201

Practical synthesis of N-(di-n-butylamino)methylene-protected 2-aminopurine riboside phosphoramidite for RNA solid-phase synthesis

Abstract 2-Aminopurine (Ap) is a fluorescent nucleobase analog that is frequently used as structure-sensitive reporter to study the chemical and biophysical properties of nucleic acids. In particular, thermodynamics and kinetics of RNA folding and RNA–ligand binding, as well as RNA catalytic activity are addressable by pursuing the Ap fluorescence signal in response to external stimuli. Site-specific incorporation of Ap into RNA is usually achieved by RNA solid-phase synthesis and requires appropriately functionalized Ap riboside building blocks. Here, we introduce a robust synthetic path toward a 2-aminopurine riboside phosphoramidite whose N2 functionality is masked with the N-(di-n-butylamino)methylene group. This protection is considered advantageous over previously described N-(dimethylamino)methylene or acyl protection patterns needed for the fine-tuned deprotection conditions to achieve large synthetic RNAs. Graphic abstract</jats:p

Time trends in general practitioners’ home visits for older patients: a retrospective cross-sectional study from Switzerland

WHAT IS KNOWN ON THE SUBJECT, AND WHAT THE STUDY ADDS: The number of home visits by general practitioners (GPs) has decreased in recent years, in contrast to the increasing number of frail and older patients in western countries. Current data on GP home visit numbers and rates are lacking for Switzerland. Our study provides new data on GP home visit numbers and rates, and their associations with patient characteristics. AIM Our study aimed at investigating the time trend of GP home visits to older patients from 2014 to 2018 in Switzerland, and associations between GP home visits and patient characteristics including healthcare utilisation and living situation. METHODS Retrospective cross-sectional study of insurance claims data from 2014 to 2018 among patients aged ≥65 years (Nextrapolated = 2,095,102; Nraw = 339,301). We compared patient characteristics between patients with and without GP home visits using descriptive statistics. We performed logistic regression analyses to detect associations between patient characteristics and GP home visits, including subgroups of patients aged ≥80 and patients living in a nursing home. Regression models were adjusted for age and sex. RESULTS The yearly GP home visit rate declined from 10.7% to 9.3% from 2014 to 2018 (p <0.0001). Among patients aged ≥80, the rate declined from 26.1% to 23.1% (p <0.0001), and among patients living in a nursing home from 68.7% to 65.8% (p <0.0001). Regression analyses revealed increased health care utilisation and a higher burden of morbidity and mortality in patients receiving GP home visits. CONCLUSION There is an ongoing decline of GP home visits over the past years, with a potentially negative impact on the quality of care for older and frail patients

Self-enucleation of the right eye by a 38-year-old woman diagnosed with schizoaffective disorder: a case report

BackgroundAutoenucleation is a rare form of self-mutilation typically associated with psychiatric disorders such as schizophrenia, substance-induced psychosis and bipolar disorder. The act is usually unilateral, although bilateral attempts are also well documented in the literature.Case presentationIt is a case study involving a female patient (NN) diagnosed with schizoaffective disorder who self-enucleated her right eye following sexual intercourse with a fellow patient, and was forcefully prevented by staff from enucleating the second eye. We report recurrent episodes of her illness culminating in this severe act of self-mutilation. The motivational reasons behind this form of self-harm along with differential diagnosis and potential treatment options are discussed in the context of the available literature.ConclusionAutoenucleation is commonly associated with religious and sexual delusions, and patients are thought to be at a greater risk of further self-harm. Timely antipsychotic treatment is likely to reduce the risk of such extreme forms of self-harm, although they can occur despite robust therapeutic intervention and treatment attempts. While self-inflicted eye injuries are rare, their prevention in what is typically a difficult patient group is fraught with challenges

Self-enucleation of the right eye by a 38-year-old woman diagnosed with schizoaffective disorder: a case report

Abstract Background Autoenucleation is a rare form of self-mutilation typically associated with psychiatric disorders such as schizophrenia, substance-induced psychosis and bipolar disorder. The act is usually unilateral, although bilateral attempts are also well documented in the literature. Case presentation It is a case study involving a female patient (NN) diagnosed with schizoaffective disorder who self-enucleated her right eye following sexual intercourse with a fellow patient, and was forcefully prevented by staff from enucleating the second eye. We report recurrent episodes of her illness culminating in this severe act of self-mutilation. The motivational reasons behind this form of self-harm along with differential diagnosis and potential treatment options are discussed in the context of the available literature. Conclusion Autoenucleation is commonly associated with religious and sexual delusions, and patients are thought to be at a greater risk of further self-harm. Timely antipsychotic treatment is likely to reduce the risk of such extreme forms of self-harm, although they can occur despite robust therapeutic intervention and treatment attempts. While self-inflicted eye injuries are rare, their prevention in what is typically a difficult patient group is fraught with challenges. </jats:sec

Potentially inappropriate proton-pump inhibitor prescription in the general population: a claims-based retrospective time trend analysis

Background: Proton-pump inhibitors (PPI) are among the most prescribed drugs worldwide, and a large body of evidence raises concerns about their inappropriate use. Previous estimates of inappropriate use varied due to different definitions and study populations. Aims: We aimed to measure the population-based incidence and time trends of PPI and potentially inappropriate PPI prescriptions (PIPPI) with a novel method, continuously assessing excessive cumulative doses based on clinical practice guidelines. We also assessed association of patient characteristics with PPI prescriptions and PIPPI. Methods: This was an observational study based on a large insurance claims database of persons aged >18 years with continuous claims records of ⩾12 months. The observation period was January 2012 to December 2017. We assessed the incidence and time trends of PPI prescriptions and PIPPI based on doses prescribed, defining ⩾11.5 g of pantoprazole dose equivalents during any consecutive 365 days (average daily dose >31 mg) as inappropriate. Results: Among 1,726,491 eligible persons, the annual incidence of PPI prescriptions increased from 19.7% (2012) to 23.0% (2017), ( p = <0.001), and the incidence of PIPPI increased from 4.8% (2013) to 6.4% (2017), ( p = <0.001). Age, male gender, drugs with bleeding risk and multimorbidity were independent determinants of PIPPI ( p = <0.001 for all). Conclusions: This study provides evidence that one of the most prescribed drug groups is commonly prescribed inappropriately in the general population and that this trend is increasing. Multimorbidity and drugs with bleeding risks were strong determinants of PIPPI. Addressing PPI prescriptions exceeding guideline recommendations could reduce polypharmacy and improve patient safety

Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ₁ riboswitches in <i>E.coli</i>

For this study, we utilized class-I and class-II preQ1-sensing riboswitches as model systems to decipher the structure-activity relationship of rationally designed ligand derivatives in vitro and in vivo. We found that synthetic preQ1 ligands with amino-modified side chains that protrude from the ligand-encapsulating binding pocket, and thereby potentially interact with the phosphate backbone in their protonated form, retain or even increase binding affinity for the riboswitches in vitro. They, however, led to significantly lower riboswitch activities in a reporter system in vivo in E. coli. Importantly, when we substituted the amino- by azido-modified side chains, the cellular activities of the ligands were restored for the class-I conditional gene expression system and even improved for the class-II counterpart. Kinetic analysis of ligand binding in vitro revealed enhanced on-rates for amino-modified derivatives while they were attenuated for azido-modified variants. This shows that neither high affinities nor fast on-rates are necessarily translated into efficient cellular activities. Taken together, our comprehensive study interconnects in vitro kinetics and in vitro thermodynamics of RNA-ligand binding with the ligands’ in vivo performance and thereby encourages azido- rather than amino-functionalized design for enhanced cellular activity.</p

Efficacy of motivating short interventions for smokers in primary care (COSMOS trial): study protocol for a cluster-RCT

BACKGROUND Tobacco abuse is a frequent issue in general practitioners' (GPs') offices, with doctors playing a key role in promoting smoking cessation to their patients. However, not all smokers are ready and willing to give up smoking. Thus, a GP focusing on smoking cessation alone might waste the opportunity to improve his patient's health by supporting a change in another harmful behaviour pattern. The aim of this study is to determine whether multi-thematic coaching will lead to higher overall health benefits without resulting in a reduced rate of successful smoking cessations, compared with a monothematic smoking cessation approach. METHODS The study is designed as a two-armed, double-blinded, cluster-randomised trial. GPs will be randomly assigned to the intervention or control group. In the intervention group, GPs will undergo training in patient-centred coaching, shared decision-making and motivational interviewing. The control group will be trained in a state-of-the-art smoking cessation algorithm. GPs will approach adult cigarette-smoking patients and advise those included according to the GP's group affiliation. The primary outcome is the between-group difference in the proportion of participants who achieve a beneficial change in at least one of seven different health-related behavioural dimensions, 12 months post baseline. Secondary outcomes include smoking cessation rates and the patients' self-perceived smoking-related motivation, self-efficacy and planning behaviour. Additionally, covariates describing both GPs and patients will be collected before the start of the intervention, and process outcome measures in compliance with the RE-AIM (Reach Effectiveness Adoption Implementation Maintenance) framework will be recorded during the ongoing study. DISCUSSION Tobacco consumption is still highly prevalent in the general population and often goes hand in hand with other behaviour patterns with adverse health effects. This study will add to the literature regarding effective strategies available to GPs to address unhealthy behaviour among their smoking patients beyond mere smoking cessation counselling. The study will also establish a basis for decisions about further promotion and dissemination of the coaching under study