Charakterisierung eines Mausmodells zur BH4-sensitiven Phenylketonurie

Phenylketonurie (PKU) ist eine angeborene Störung des Aminosäurestoffwechsels, die unbehandelt zu schweren neurologischen Schäden führt. In vielen Fällen ist die einzige mögliche Behandlungsoption eine streng eiweißarme Diät. Im Rahmen klinischer Studien konnte gezeigt werden, dass etwa ein Drittel der Patienten auf die Gabe pharmakologischer Dosen des natürlichen Kofaktors der Phenylalaninhydroxylase (PAH), Tetrahydrobiopterin (BH4), ansprechen (Muntau et al. 2002). Mit der Hilfe von BH4 ergab sich eine Behandlungsalternative, die den Patienten erlaubte ihre Diät zu lockern und mehr natürliches Protein mit der Nahrung aufzunehmen (Muntau et al. 2002). Zentrale Fragestellung der vorliegenden Doktorarbeit war die Identifikation und eingehende Charakterisierung BH4-sensitiver Mausmodelle. Das homozygote Mausmodell Pahenu1/enu1 trägt die Variante p.Val106Ala der murinen Phenylalaninhydroxylase (Pah). Der Aminosäureaustausch beeinträchtigt die regulatorische Domäne und führt zu einem milden PKU-Phänotyp. Das homozygote Mausmodell Pahenu2/enu2 ist Träger der Mutation p.Phe263Ser, die in der katalytischen Domäne des Enzyms gelegen ist und zum klinischen Bild einer schweren PKU führt. Pahenu1/enu2 zeigt als compound-heterozygotes Mausmodell einen intermediären klinischen Phänotyp. Die Aminosäuren Phe 263 und Val 106 sind zwischen der murinen Pah und der humanen PAH konserviert. Wie im entsprechenden Mausmodell führt die Variante p.Val106Ala beim Menschen zu einem milden Phänotyp und p.Phe263Ser zu einem schweren Phänotyp. Bei Analyse der enzymkinetischen Parameter am rekombinanten Protein (Wildtyp, p.Val106Ala, p.Phe263Ser) konnte eine gute Vergleichbarkeit zwischen der murinen und humanen PAH gezeigt werden. Unterschiede in den Enzymaktivitäten zwischen Proben von murinen und humanen Leberlysaten beruhen auf unterschiedlichen hepatischen PAH-Mengen. Die im Rahmen der Arbeit erhobenen Daten zeigten, dass die murine Pah ein gut geeignetes Modell für das PAH-System des Menschen ist. Sowohl für Pahenu1/enu1 als auch für Pahenu1/enu2 konnten wir nachweisen, dass diese Mausmodelle von einer pharmakologischen Therapie mit BH4 profitieren und somit eine BH4-Sensitivität vorliegt. Pahenu2/enu2 zeigte kein Ansprechen auf die BH4-Therapie. Am rekombinant hergestellten Protein führte die Variante p.Val106Ala in vitro weder zu einer Verminderung der Affinität zum Kofaktor noch zu einer Reduktion der spezifischen Enzymaktivität. Somit kann die Wirkung von BH4 nicht durch eine direkte Wirkung als Kofaktor der enzymatischen Reaktion erklärt werden. In biophysikalischen und biochemischen Experimenten an gereinigten Proteinen und im zellulären Modell konnte gezeigt werden, dass die Variante p.Val106Ala zu vermehrter Aggregation und Degradation neigt und damit zu einer Verminderung des intakten Enzyms führt. Auf molekularer Ebene induzierte BH4 eine Korrektur der p.Val106Ala-Proteinfehlfaltung und führte somit, als sogenanntes pharmakologisches Chaperon, zu einer Erhöhung der effektiven intrazellulären Konzentration von korrekt gefalteter und funktionaler Pah. Beim Vergleich der pharmakokinetischen und -dynamischen Parameter einer BH4-Therapie von Pahenu1/enu1 und Pahenu1/enu2 haben wir Unterschiede zwischen diesen beiden BH4-sensitiven Mausmodellen festgestellt. Dies zeigt, dass die BH4-Wirkung nicht nur von der BH4-sensitiven Variante (hier p.Val106Ala) bestimmt wird, sondern auch vom zweiten Allel beeinflusst wird. Dieser Effekt der interallelischen Komplementation sollte bei den compound-heterozygoten Patienten berücksichtigt werden und könnte erweiterte Testprotokolle notwendig machen. Im Rahmen der ex vivo Experimente wurden bei nicht diätetisch oder pharmakologisch therapierten Mäusen Unterschiede in den freien, intrazellulären BH4-Konzentrationen zwischen den verschiedenen Mausgenotypen beobachtet. Interessanterweise waren diese Unterschiede der BH4- Konzentration nicht im Blut zu beobachten. Die intrazelluläre BH4-Konzentration von Hepatozyten war bei Pahenu1/enu1 und Pahenu1/enu2 im Vergleich zum Wildtyp sowie zu Pahenu2/enu2 erniedrigt. Es konnten keine Unterschiede in der Expression des Schlüsselenzyms der Tetrahydrobiopterin- Biosynthese, Gch1, nachgewiesen werden. Darüber hinaus konnte ein ungehemmter BH4-Verbrauch im Rahmen eines enzymatischen Uncoupling für die Pahenu1-Allel tragenden Mäuse ausgeschlossen werden. Wir konnten in vitro zeigen, dass zwar kein Unterschied in der absoluten BH4-Konzentration vorliegt, jedoch eine Verschiebung innerhalb des BH4-Pools stattfindet, was zu einer Reduktion des freien BH4 führte. Bei Pahenu1/enu1 und Pahenu1/enu2, die das Allel p.V106Ala tragen, scheint es in den Hepatozyten zu einer verstärkten Bindung von BH4 an die Pah (trapping) zu kommen, so dass BH4 nicht mehr frei verfügbar in der Zelle vorliegt. Somit kommt es neben dem mutationsbedingten Funktionsverlust der p.Val106Ala-Pah auch zu einem sekundären BH4-Mangel, der zu einer weiteren Verminderung der Enzymaktivität beitragen könnte. Die Gabe von pharmakologischen Dosen von BH4 könnte neben der Korrektur der Proteinfehlfaltung als pharmakologisches Chaperon somit auch einen Ausgleich des sekundären intrazellulären BH4-Mangels bewirken. Ein besseres Verständnis der molekularen Mechanismen der Krankheitsentstehung und der pharmakologischen Korrektur bei den BH4-sensitiven Mausmodellen Pahenu1/enu1 und Pahenu1/enu2 kann dazu beitragen, die pharmakologische Therapie, zum Beispiel durch die Entwicklung von optimierten oder neuen Wirkstoffen, zu verbessern und effizienter zu machen. Darüber hinaus können diese Mausmodelle auch genutzt werden, um grundsätzliche Fragestellungen zu Proteinfehlfaltungserkrankungen und Degradationsmechanismen zu adressieren. So könnten die Mausmodelle bei Untersuchungen der zellulären Degradationsmaschinerie, des Einflusses von oxidativem Stress auf Proteinfaltung und der Korrektur der gestörten Proteinhomöostase mittels Proteostase-Regulatoren verwendet werden.Phenylketonuria (PKU) is an inborn error of amino acid metabolism which causes severe neurological impairment, if untreated. For most patients, a strict low-phenylalanine diet is the only treatment option. In clinical studies, it was shown that approximately one third of the patients respond to supplementation with pharmacological dosages of tetrahydrobiopterin (BH4), which is the natural cofactor of the enzyme phenylalanine hydroxylase (PAH). Treatment with BH4 gave rise to a pharmacological therapeutic alternative that allows patients to relax their diet and tolerate more natural protein (Muntau et al. 2002). The main topic of this thesis was the identification and the detailed characterization of a BH4-responsive mouse model. The homozygous Pahenu1/enu1 mouse carries the allelic variant p.Val106Ala of murine Pah. The amino acid side chain replacement affects the regulatory domain leading to a mild PKU phenotype. The homozygous Pahenu2/enu2 mouse carries the allelic variant p.Phe263Ser, which maps to the catalytic domain of the enzyme and causes a clinical picture of severe PKU. The compound heterozygous Pahenu1/enu2 mouse shows an intermediate clinical phenotype. The amino acids Phe 263 and Val 106 of the PAH protein are conserved between humans and mice. According to phenotypes observed in mice, p.Val106Ala leads to a mild phenotype in humans and p.Phe263Ser induces classical PKU. Enzyme kinetics analyses using recombinantly expressed protein (wild-type, p.Val106Ala, p.Phe263Ser) showed good comparability between human and murine PAH. The differences between the activity of human and murine PAH in the liver tissues seemed to be due to different intrahepatic PAH amounts. The data presented here shows that murine Pah is an appropriate model for the human phenylalanine hydroxylating system. Both Pahenu1/enu1 and Pahenu1/enu2 showed BH4 responsiveness and benefit from treatment with BH4, whereas Pahenu2/enu2 showed no response to BH4 treatment. In vitro, the recombinantly expressed p.Val106Ala protein showed neither a reduction of affinity to its cofactor BH4 nor a marked reduction in specific enzyme activity. Thus, BH4 responsiveness cannot be explained by a direct cofactor effect on the enzymatic reaction. Detailed biophysical and biochemical investigation using purified protein and eukaryotically expressed protein showed propensity towards degradation and aggregation for p.Val106Ala, leading to a decrease in the available amount of functional protein. On a molecular level, BH4 rescued protein misfolding of p.Val106Ala, acting as a pharmacological chaperone. BH4 enhanced the amount of correctly folded and enzymatically active Pah. Comparison of pharmacokinetics and pharmacodynamics of BH4 therapy in Pahenu1/enu1 and Pahenu1/enu2 showed differences between genotypes. The BH4 effect is not only determined by one allele, e.g. a BH4- responsive variant (here p.Val106Ala), but also by the second allele (p.Val106Ala or null mutation p.Phe263Ser). These differences in response to the BH4 therapy may lead to the need for expanded testing in compound heterozygous patients. In ex vivo analyses, we observed differences in hepatic free intracellular BH4 between untreated genotypes. Interestingly, this was not congruent with the BH4 pattern in blood. The BH4 concentration in hepatocytes was decreased for Pahenu1/enu1 and Pahenu1/enu2 in comparison to the wild- type and Pahenu2/enu2. No differences were found in the expression of Gch1, the key enzyme of tetrahydrobiopterin biosynthesis. Moreover, no increase in the consumption of BH4 due to enzymatic uncoupling was observed for genotypes carrying the Pahenu1 allele. We demonstrated in vitro that the total amount of BH4 was not altered, but a shift in BH4 pools led to a decrease in free BH4. Misfolded p.Val106Ala Pah protein trapped BH4 making it unavailable for the enzymatic reaction. In addition to the loss of function due to the mutation p.Val106Ala, this mechanism may lead to secondary intracellular BH4 deficiency, which induces a further decrease in enzyme function. Therefore, therapeutic BH4 may not only exert a pharmacological chaperone effect on the misfolded p.Val106Ala protein, the treatment may also correct intrahepatic secondary BH4 deficiency. A better understanding of the molecular mechanisms of BH4-responsive PKU could lead to optimization of the current therapy with BH4 and might enable the identification of new and more efficient pharmacological compounds. Moreover, mice carrying the Pahenu1 allele could be used to address more general questions regarding diseases with protein misfolding and degradation. For example, the mouse model could help to elucidate mechanisms of intracellular protein degradation, the impact of oxidative stress on protein misfolding and therapeutic routes to correct protein homeostasis by proteostasis regulation

Charakterisierung eines Mausmodells zur BH4-sensitiven Phenylketonurie

Phenylketonurie (PKU) ist eine angeborene Störung des Aminosäurestoffwechsels, die unbehandelt zu schweren neurologischen Schäden führt. In vielen Fällen ist die einzige mögliche Behandlungsoption eine streng eiweißarme Diät. Im Rahmen klinischer Studien konnte gezeigt werden, dass etwa ein Drittel der Patienten auf die Gabe pharmakologischer Dosen des natürlichen Kofaktors der Phenylalaninhydroxylase (PAH), Tetrahydrobiopterin (BH4), ansprechen (Muntau et al. 2002). Mit der Hilfe von BH4 ergab sich eine Behandlungsalternative, die den Patienten erlaubte ihre Diät zu lockern und mehr natürliches Protein mit der Nahrung aufzunehmen (Muntau et al. 2002). Zentrale Fragestellung der vorliegenden Doktorarbeit war die Identifikation und eingehende Charakterisierung BH4-sensitiver Mausmodelle. Das homozygote Mausmodell Pahenu1/enu1 trägt die Variante p.Val106Ala der murinen Phenylalaninhydroxylase (Pah). Der Aminosäureaustausch beeinträchtigt die regulatorische Domäne und führt zu einem milden PKU-Phänotyp. Das homozygote Mausmodell Pahenu2/enu2 ist Träger der Mutation p.Phe263Ser, die in der katalytischen Domäne des Enzyms gelegen ist und zum klinischen Bild einer schweren PKU führt. Pahenu1/enu2 zeigt als compound-heterozygotes Mausmodell einen intermediären klinischen Phänotyp. Die Aminosäuren Phe 263 und Val 106 sind zwischen der murinen Pah und der humanen PAH konserviert. Wie im entsprechenden Mausmodell führt die Variante p.Val106Ala beim Menschen zu einem milden Phänotyp und p.Phe263Ser zu einem schweren Phänotyp. Bei Analyse der enzymkinetischen Parameter am rekombinanten Protein (Wildtyp, p.Val106Ala, p.Phe263Ser) konnte eine gute Vergleichbarkeit zwischen der murinen und humanen PAH gezeigt werden. Unterschiede in den Enzymaktivitäten zwischen Proben von murinen und humanen Leberlysaten beruhen auf unterschiedlichen hepatischen PAH-Mengen. Die im Rahmen der Arbeit erhobenen Daten zeigten, dass die murine Pah ein gut geeignetes Modell für das PAH-System des Menschen ist. Sowohl für Pahenu1/enu1 als auch für Pahenu1/enu2 konnten wir nachweisen, dass diese Mausmodelle von einer pharmakologischen Therapie mit BH4 profitieren und somit eine BH4-Sensitivität vorliegt. Pahenu2/enu2 zeigte kein Ansprechen auf die BH4-Therapie. Am rekombinant hergestellten Protein führte die Variante p.Val106Ala in vitro weder zu einer Verminderung der Affinität zum Kofaktor noch zu einer Reduktion der spezifischen Enzymaktivität. Somit kann die Wirkung von BH4 nicht durch eine direkte Wirkung als Kofaktor der enzymatischen Reaktion erklärt werden. In biophysikalischen und biochemischen Experimenten an gereinigten Proteinen und im zellulären Modell konnte gezeigt werden, dass die Variante p.Val106Ala zu vermehrter Aggregation und Degradation neigt und damit zu einer Verminderung des intakten Enzyms führt. Auf molekularer Ebene induzierte BH4 eine Korrektur der p.Val106Ala-Proteinfehlfaltung und führte somit, als sogenanntes pharmakologisches Chaperon, zu einer Erhöhung der effektiven intrazellulären Konzentration von korrekt gefalteter und funktionaler Pah. Beim Vergleich der pharmakokinetischen und -dynamischen Parameter einer BH4-Therapie von Pahenu1/enu1 und Pahenu1/enu2 haben wir Unterschiede zwischen diesen beiden BH4-sensitiven Mausmodellen festgestellt. Dies zeigt, dass die BH4-Wirkung nicht nur von der BH4-sensitiven Variante (hier p.Val106Ala) bestimmt wird, sondern auch vom zweiten Allel beeinflusst wird. Dieser Effekt der interallelischen Komplementation sollte bei den compound-heterozygoten Patienten berücksichtigt werden und könnte erweiterte Testprotokolle notwendig machen. Im Rahmen der ex vivo Experimente wurden bei nicht diätetisch oder pharmakologisch therapierten Mäusen Unterschiede in den freien, intrazellulären BH4-Konzentrationen zwischen den verschiedenen Mausgenotypen beobachtet. Interessanterweise waren diese Unterschiede der BH4- Konzentration nicht im Blut zu beobachten. Die intrazelluläre BH4-Konzentration von Hepatozyten war bei Pahenu1/enu1 und Pahenu1/enu2 im Vergleich zum Wildtyp sowie zu Pahenu2/enu2 erniedrigt. Es konnten keine Unterschiede in der Expression des Schlüsselenzyms der Tetrahydrobiopterin- Biosynthese, Gch1, nachgewiesen werden. Darüber hinaus konnte ein ungehemmter BH4-Verbrauch im Rahmen eines enzymatischen Uncoupling für die Pahenu1-Allel tragenden Mäuse ausgeschlossen werden. Wir konnten in vitro zeigen, dass zwar kein Unterschied in der absoluten BH4-Konzentration vorliegt, jedoch eine Verschiebung innerhalb des BH4-Pools stattfindet, was zu einer Reduktion des freien BH4 führte. Bei Pahenu1/enu1 und Pahenu1/enu2, die das Allel p.V106Ala tragen, scheint es in den Hepatozyten zu einer verstärkten Bindung von BH4 an die Pah (trapping) zu kommen, so dass BH4 nicht mehr frei verfügbar in der Zelle vorliegt. Somit kommt es neben dem mutationsbedingten Funktionsverlust der p.Val106Ala-Pah auch zu einem sekundären BH4-Mangel, der zu einer weiteren Verminderung der Enzymaktivität beitragen könnte. Die Gabe von pharmakologischen Dosen von BH4 könnte neben der Korrektur der Proteinfehlfaltung als pharmakologisches Chaperon somit auch einen Ausgleich des sekundären intrazellulären BH4-Mangels bewirken. Ein besseres Verständnis der molekularen Mechanismen der Krankheitsentstehung und der pharmakologischen Korrektur bei den BH4-sensitiven Mausmodellen Pahenu1/enu1 und Pahenu1/enu2 kann dazu beitragen, die pharmakologische Therapie, zum Beispiel durch die Entwicklung von optimierten oder neuen Wirkstoffen, zu verbessern und effizienter zu machen. Darüber hinaus können diese Mausmodelle auch genutzt werden, um grundsätzliche Fragestellungen zu Proteinfehlfaltungserkrankungen und Degradationsmechanismen zu adressieren. So könnten die Mausmodelle bei Untersuchungen der zellulären Degradationsmaschinerie, des Einflusses von oxidativem Stress auf Proteinfaltung und der Korrektur der gestörten Proteinhomöostase mittels Proteostase-Regulatoren verwendet werden.Phenylketonuria (PKU) is an inborn error of amino acid metabolism which causes severe neurological impairment, if untreated. For most patients, a strict low-phenylalanine diet is the only treatment option. In clinical studies, it was shown that approximately one third of the patients respond to supplementation with pharmacological dosages of tetrahydrobiopterin (BH4), which is the natural cofactor of the enzyme phenylalanine hydroxylase (PAH). Treatment with BH4 gave rise to a pharmacological therapeutic alternative that allows patients to relax their diet and tolerate more natural protein (Muntau et al. 2002). The main topic of this thesis was the identification and the detailed characterization of a BH4-responsive mouse model. The homozygous Pahenu1/enu1 mouse carries the allelic variant p.Val106Ala of murine Pah. The amino acid side chain replacement affects the regulatory domain leading to a mild PKU phenotype. The homozygous Pahenu2/enu2 mouse carries the allelic variant p.Phe263Ser, which maps to the catalytic domain of the enzyme and causes a clinical picture of severe PKU. The compound heterozygous Pahenu1/enu2 mouse shows an intermediate clinical phenotype. The amino acids Phe 263 and Val 106 of the PAH protein are conserved between humans and mice. According to phenotypes observed in mice, p.Val106Ala leads to a mild phenotype in humans and p.Phe263Ser induces classical PKU. Enzyme kinetics analyses using recombinantly expressed protein (wild-type, p.Val106Ala, p.Phe263Ser) showed good comparability between human and murine PAH. The differences between the activity of human and murine PAH in the liver tissues seemed to be due to different intrahepatic PAH amounts. The data presented here shows that murine Pah is an appropriate model for the human phenylalanine hydroxylating system. Both Pahenu1/enu1 and Pahenu1/enu2 showed BH4 responsiveness and benefit from treatment with BH4, whereas Pahenu2/enu2 showed no response to BH4 treatment. In vitro, the recombinantly expressed p.Val106Ala protein showed neither a reduction of affinity to its cofactor BH4 nor a marked reduction in specific enzyme activity. Thus, BH4 responsiveness cannot be explained by a direct cofactor effect on the enzymatic reaction. Detailed biophysical and biochemical investigation using purified protein and eukaryotically expressed protein showed propensity towards degradation and aggregation for p.Val106Ala, leading to a decrease in the available amount of functional protein. On a molecular level, BH4 rescued protein misfolding of p.Val106Ala, acting as a pharmacological chaperone. BH4 enhanced the amount of correctly folded and enzymatically active Pah. Comparison of pharmacokinetics and pharmacodynamics of BH4 therapy in Pahenu1/enu1 and Pahenu1/enu2 showed differences between genotypes. The BH4 effect is not only determined by one allele, e.g. a BH4- responsive variant (here p.Val106Ala), but also by the second allele (p.Val106Ala or null mutation p.Phe263Ser). These differences in response to the BH4 therapy may lead to the need for expanded testing in compound heterozygous patients. In ex vivo analyses, we observed differences in hepatic free intracellular BH4 between untreated genotypes. Interestingly, this was not congruent with the BH4 pattern in blood. The BH4 concentration in hepatocytes was decreased for Pahenu1/enu1 and Pahenu1/enu2 in comparison to the wild- type and Pahenu2/enu2. No differences were found in the expression of Gch1, the key enzyme of tetrahydrobiopterin biosynthesis. Moreover, no increase in the consumption of BH4 due to enzymatic uncoupling was observed for genotypes carrying the Pahenu1 allele. We demonstrated in vitro that the total amount of BH4 was not altered, but a shift in BH4 pools led to a decrease in free BH4. Misfolded p.Val106Ala Pah protein trapped BH4 making it unavailable for the enzymatic reaction. In addition to the loss of function due to the mutation p.Val106Ala, this mechanism may lead to secondary intracellular BH4 deficiency, which induces a further decrease in enzyme function. Therefore, therapeutic BH4 may not only exert a pharmacological chaperone effect on the misfolded p.Val106Ala protein, the treatment may also correct intrahepatic secondary BH4 deficiency. A better understanding of the molecular mechanisms of BH4-responsive PKU could lead to optimization of the current therapy with BH4 and might enable the identification of new and more efficient pharmacological compounds. Moreover, mice carrying the Pahenu1 allele could be used to address more general questions regarding diseases with protein misfolding and degradation. For example, the mouse model could help to elucidate mechanisms of intracellular protein degradation, the impact of oxidative stress on protein misfolding and therapeutic routes to correct protein homeostasis by proteostasis regulation

The COVID-19 Vaccine: Trust, doubt, and hope for a future beyond the pandemic in Germany

Public perceptions of COVID-19 vaccines are critical in reaching protective levels of herd immunity. Vaccine skepticism has always been relatively high in Germany, and surveys suggest that over the course of the pandemic, enthusiasm for the COVID-19 vaccine has dropped. Looking at the period just prior to the approval of the Pfizer/BioNTech and Moderna vaccines in Germany in the latter half of 2020, this paper aims to assess the reasons for and against COVID-19 vaccine uptake among residents of Germany, and to provide in-depth qualitative data to better understand and address concerns surrounding the safety and efficacy of a COVID-19 vaccine. Our findings indicate that there is widespread trust in German institutions and health experts to provide a safe vaccine for those who need it most. However, interviewees also point to the need for more information and the centrality of support from trusted medical authorities in making individual vaccination decisions. We also present the complexity of individual positions on vaccination, and suggest that vaccine hesitancy in relation to COVID-19 needs to be understood as a nuanced, and socially malleable, territory. This indicates that the goal of a vaccination campaign is not only achieving ‘herd immunity,’ but also a social endorsement of the collaborative effort that is required for a vaccine to be successful

Pahenu1 is a mouse model for tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency and promotes analysis of the pharmacological chaperone mechanism in vivo

The recent approval of sapropterin dihydrochloride, the synthetic form of 6[R]-l-erythro-5,6,7,8-tetrahydrobiopterin (BH4), for the treatment of phenylketonuria (PKU) as the first pharmacological chaperone drug initiated a paradigm change in the treatment of monogenetic diseases. Symptomatic treatment is now replaced by a causal pharmacological therapy correcting misfolding of the defective phenylalanine hydroxylase (PAH) in numerous patients. Here, we disclose BH4 responsiveness in Pahenu1, a mouse model for PAH deficiency. Loss of function resulted from loss of PAH, a consequence of misfolding, aggregation, and accelerated degradation of the enzyme. BH4 attenuated this triad by conformational stabilization augmenting the effective PAH concentration. This led to the rescue of the biochemical phenotype and enzyme function in vivo. Combined in vitro and in vivo analyses revealed a selective pharmaceutical action of BH4 confined to the pathological metabolic state. Our data provide new molecular-level insights into the mechanisms underlying protein misfolding with loss of function and support a general model of pharmacological chaperone-induced stabilization of protein conformation to correct this intracellular phenotype. Pahenu1 will be essential for pharmaceutical drug optimization and to design individually tailored therapie

Consensus guidelines for improving quality of assessment and training for neuromuscular diseases

Critical components of successful evaluation of clinical outcome assessments (COAs) in multisite clinical trials and clinical practice are standardized training, administration, and documented reliability of scoring. Experiences of evaluators, alongside patient differences from regional standards of care, may contribute to heterogeneity in clinical center\u27s expertise. Achieving low variability and high reliability of COA is fundamental to clinical research and to give confidence in our ability to draw rational, interpretable conclusions from the data collected. The objective of this manuscript is to provide a framework to guide the learning process for COAs for use in clinics and clinical trials to maximize reliability and validity of COAs in neuromuscular disease (NMD). This is a consensus-based guideline with contributions from fourteen leading experts in clinical outcomes and the field of clinical outcome training in NMD. This framework should guide reliable and valid assessments in NMD specialty clinics and clinical trials. This consensus aims to expedite study start up with a progressive training pathway ranging from research naïve to highly experienced clinical evaluators. This document includes recommendations for education guidelines and roles and responsibilities of key stakeholders in COA assessment and implementation to ensure quality and consistency of outcome administration across different settings

Effects of Lumacaftor/Ivacaftor on Cystic Fibrosis Disease Progression in Children 2 through 5 Years of Age Homozygous for F508del-CFTR: A Phase 2 Placebo-controlled Clinical Trial.

RATIONALE Lumacaftor/ivacaftor (LUM/IVA) was shown to be safe and well tolerated in children 2 through 5 years of age with cystic fibrosis (CF) homozygous for F508del-CFTR in a phase 3 open-label study. Improvements in sweat chloride concentration, markers of pancreatic function, and lung clearance index2.5 (LCI2.5), along with increases in growth parameters, suggested the potential for early disease modification with LUM/IVA treatment. OBJECTIVE To further assess the effects of LUM/IVA on CF disease progression in children 2 through 5 years of age using chest magnetic resonance imaging (MRI). METHODS This phase 2 study had two parts: a 48-week, randomized, double-blind, placebo-controlled treatment period in which children 2 through 5 years of age with CF homozygous for F508del-CFTR received either LUM/IVA or placebo (Part 1) followed by an open-label period in which all children received LUM/IVA for an additional 48 weeks (Part 2). We report results from Part 1. The primary endpoint was absolute change from baseline in chest MRI global score at Week 48. Secondary endpoints included absolute change in LCI2.5 through Week 48 and absolute changes in weight-for-age, stature-for-age, and body mass index-for-age z-scores at Week 48. Additional endpoints included absolute changes in sweat chloride concentration, fecal elastase-1 levels, serum immunoreactive trypsinogen, and fecal calprotectin through Week 48. The primary endpoint was analyzed using Bayesian methods, where the actual Bayesian posterior probability of LUM/IVA being superior to placebo in the MRI global chest score at Week 48 was calculated using a vague normal prior distribution; secondary and additional endpoints were analyzed using descriptive summary statistics. RESULTS Fifty-one children were enrolled and received LUM/IVA (n=35) or placebo (n=16). For the change in MRI global chest score at Week 48, the Bayesian posterior probability of LUM/IVA being better than placebo (treatment difference <0; higher score indicating greater abnormality) was 76%; the mean treatment difference was -1.5 (95% credible interval, -5.5 to 2.6). Treatment with LUM/IVA also led to within-group numerical improvements in LCI2.5, growth parameters, and biomarkers of pancreatic function as well as greater decreases in sweat chloride concentration compared with placebo from baseline through Week 48. Safety data were consistent with the established safety profile of LUM/IVA. CONCLUSIONS This placebo-controlled study suggests the potential for early disease modification with LUM/IVA treatment, including that assessed by chest MRI, in children as young as 2 years. Clinical trial registered with ClinicalTrials.gov (NCT03625466)

Biology of a widespread uncultivated archaeon that contributes to carbon fixation in the subsurface

Subsurface microbial life contributes significantly to biogeochemical cycling, yet it remains largely uncharacterized, especially its archaeal members. This 'microbial dark matter' has been explored by recent studies that were, however, mostly based on DNA sequence information only. Here, we use diverse techniques including ultrastuctural analyses to link genomics to biology for the SM1 Euryarchaeon lineage, an uncultivated group of subsurface archaea. Phylogenomic analyses reveal this lineage to belong to a widespread group of archaea that we propose to classify as a new euryarchaeal order ('Candidatus Altiarchaeales'). The representative, double-membraned species 'Candidatus Altiarchaeum hamiconexum' has an autotrophic metabolism that uses a not-yet-reported Factor(420)-free reductive acetyl-CoA pathway, confirmed by stable carbon isotopic measurements of archaeal lipids. Our results indicate that this lineage has evolved specific metabolic and structural features like nano-grappling hooks empowering this widely distributed archaeon to predominate anaerobic groundwater, where it may represent an important carbon dioxide sink

Citizen science’s transformative impact on science, citizen empowerment and socio-political processes

Citizen science (CS) can foster transformative impact for science, citizen empowerment and socio-political processes. To unleash this impact, a clearer understanding of its current status and challenges for its development is needed. Using quantitative indicators developed in a collaborative stakeholder process, our study provides a comprehensive overview of the current status of CS in Germany, Austria and Switzerland. Our online survey with 340 responses focused on CS impact through (1) scientific practices, (2) participant learning and empowerment, and (3) socio-political processes. With regard to scientific impact, we found that data quality control is an established component of CS practice, while publication of CS data and results has not yet been achieved by all project coordinators (55%). Key benefits for citizen scientists were the experience of collective impact (“making a difference together with others”) as well as gaining new knowledge. For the citizen scientists’ learning outcomes, different forms of social learning, such as systematic feedback or personal mentoring, were essential. While the majority of respondents attributed an important value to CS for decision-making, only few were confident that CS data were indeed utilized as evidence by decision-makers. Based on these results, we recommend (1) that project coordinators and researchers strengthen scientific impact by fostering data management and publications, (2) that project coordinators and citizen scientists enhance participant impact by promoting social learning opportunities and (3) that project initiators and CS networks foster socio-political impact through early engagement with decision-makers and alignment with ongoing policy processes. In this way, CS can evolve its transformative impact

Ty1 integrase overexpression leads to integration of non-Ty1 DNA fragments into the genome of Saccharomyces cerevisiae

The integrase of the Saccharomyces cerevisiae retrotransposon Ty1 integrates Ty1 cDNA into genomic DNA likely via a transesterification reaction. Little is known about the mechanisms ensuring that integrase does not integrate non-Ty DNA fragments. In an effort to elucidate the conditions under which Ty1 integrase accepts non-Ty DNA as substrate, PCR fragments encompassing a selectable marker gene were transformed into yeast strains overexpressing Ty1 integrase. These fragments do not exhibit similarity to Ty1 cDNA except for the presence of the conserved terminal dinucleotide 5′-TG-CA-3′. The frequency of fragment insertion events increased upon integrase overexpression. Characterization of insertion events by genomic sequencing revealed that most insertion events exhibited clear hallmarks of integrase-mediated reactions, such as 5 bp target site duplication and target site preferences. Alteration of the terminal dinucleotide abolished the suitability of the PCR fragments to serve as substrates. We hypothesize that substrate specificity under normal conditions is mainly due to compartmentalization of integrase and Ty cDNA, which meet in virus-like particles. In contrast, recombinant integrase, which is not confined to virus-like particles, is able to accept non-Ty DNA, provided that it terminates in the proper dinucleotide sequence