Examination of ataxin-3 (atx-3) aggregation by structural mass spectrometry techniques: A rationale for expedited aggregation upon polyglutamine (polyQ) expansion

Expansion of polyglutamine stretches leads to the formation of polyglutamine-containing neuronal aggregates and neuronal death in nine diseases for which there currently are no treatments or cures. This is largely due to a lack in understanding of the mechanisms by which expanded polyglutamine regions contribute to aggregation and disease. To complicate matters further, several of the polyglutamine-disease related proteins, including ataxin-3, have a multistage aggregation mechanism in which flanking domain self-assembly precedes polyglutamine aggregation yet is influenced by polyglutamine expansion. How polyglutamine expansion influences flanking domain aggregation is poorly understood. Here, we use a combination of mass spectrometry and biophysical approaches to investigate this issue for ataxin-3. We show that the conformational dynamics of the flanking Josephin domain in ataxin-3 with an expanded polyglutamine tract are altered in comparison to those exhibited by its nonexpanded counterpart, specifically within the aggregation-prone region of the Josephin domain (amino acid residues 73-96). Expansion thus exposes this region more frequently in ataxin-3 containing an expanded polyglutamine tract, providing a molecular explanation of why aggregation is accelerated upon polyglutamine expansion. Here, harnessing the power of ion mobility spectrometry-mass spectrometry, oligomeric species formed during aggregation are characterized and a model for oligomer growth proposed. The results suggest that a conformational change occurs at the dimer level that initiates self-assembly. New insights into ataxin-3 fibril architecture are also described, revealing the region of the Josephin domain involved in protofibril formation and demonstrating that polyglutamine aggregation proceeds as a distinct second step after protofibril formation without requiring structural rearrangement of the protofibril core. Overall, the results enable the effect of polyglutamine expansion on every stage of ataxin-3 self-assembly, from monomer through to fibril, to be described and a rationale for expedited aggregation upon polyglutamine expansion to be provided

Kinetic investigation of different supported catalysts for the polymerization of propylene under industrially relevant conditions

Rahmen dieser Arbeit wird die Kinetik von zwei Ziegler-Natta Katalysatoren der vierten Generation sowie eines geträgerten Metallocen Katalysators für die Polymerisation von Propylen unter industrierelevanten Bedingungen untersucht. Darin wird der Einfluss verschiedener Reaktionsbedingungen (Temperatur, Druck, Wasserstoffkonzentration) und im Besonderen der Einfluss der Prepolymerisation auf den kinetischen Verlauf der Katalysatoren betrachtet sowie Polymereigenschaften untersucht. Desweiteren werden für jeden Katalysatortyp vereinfachte, phänomenologische kinetische Modelle abgeleitet, mit denen die Polymerisationen mit und ohne Prepolymerisation bei den verschiedenen Reaktionsbedingungen quantitativ beschrieben werden können. Die Polymerisationen mit Ziegler-Natta Katalysatoren w urden in der Gasphase in einem 5 l horizontal gelagerten Rührkesselreaktor durchgeführt, der in semi-batch Modus betrieben wurde. Beide Ziegler-Natta Katalysatoren zeigten ähnliche kinetische Verläufe sowie ein ähnliches Wasserstoffansprechverhalten. Bei Polymerisationen mit Prepolymerisation wurden deutlich höhere Katalysatoraktivitäten sowie eine verbesserte Polymermorphologie erzielt. Dabei ist der Einfluss der Prepolymerisation katalysatorspezifisch und abhängig von der Katalysatoraktivität. Die Polymerisation mit Metallocen Katalysator wurde in flüssigen Propylen in einem speziellen 250 ml Reaktionskalorimeter durchgeführt. Es wurden verschiedene Methoden entwickelt (insitu und externe Prepolymerisation), um definierte Prepolymerisationsbedingungen sowie eine frühe Messung d er Kinetik zu ermöglichen. Die kinetischen Untersuchungen wurden mit der entwickelten externen Prepolymerisationsmethode durchgeführt. Für die mathematische Beschreibung der Polymerisation wird ein quasi-homogenes Partikelmodell angenommen, bei dem Partikelwachstum sowie Partikelwärmebilanz berücksichtig werden. Dabei wird angenommen, dass eine Partikelüberhitzung zu Beginn der Rektion zu einer Verringerung der Katalysatoraktivität führt, wenn die Polymerisation ohne Prepolymerisation durchgeführt wird. Aufgrund der ähnlichen kinetischen Verläufe kann das gleiche Modell für beide Ziegler-Natta Katalysatoren verwendet werden. Unterschiede zwischen den Katalysatoren werden nur anhand der unterschiedlichen Menge an polymerisationsaktiven Zentren wiedergeben. Für die Modellierung der Flüssigphasenpolymerisation mit Metallocen Katalysator wird ein ähnliches kinetisches Modell hergeleitet, bei dem Unterschiede in der Monomer- sowie Wasserstoffkonzentrationen in der flüssigen Phase berücksichtigt werden. Mit den ermittelten kinetischen Parametern können gemittelte Katalysatoraktivitäten und Molmassen berechnet sowie die kinetischen Verläufe der Katalysatoren während der Polymerisation bei den verschiedenen Reaktionsbedingungen quantitativ beschrieben werden

Kinetic modeling of the gas-phase polymerization of propylene with a high active Ziegler-Natta catalyst under different injection conditions: Presentation held at 12th International Workshop on Polymer Reaction Engineering, 17th to 20th of May 2016, Hamburg/Germany

Since the development of the Ziegler-Natta catalysts in 1953, continous improvements were done concerning to higher catalyst activity and higher stereoselectivity. Especially at the reaction start, when the pure catalyst is injected under high rate conditions, the high activity of the catalyst can cause particle overheating and/or lead to an uncontrolled catalyst fragmentation resulting in lower catalyst activity and bad particle morphology. One possibility to solve the problem is to apply a prepolymerization step before the main polymerization reaction. In the prepolymerization, the reaction starts at low rate conditions (mild reaction temperatures, low monomer concentration) in order to realize a controlled catalyst fragmentation and to improve heat removal conditions by creating a higher heat transfer area. In previous studies, the gas-phase polymerization of propylene with a high active Ziegler-Natta catalyst was carried out with and without prepolymerization step. For polymerizations with prepolymerization the reached activities were much higher compared to polymerizations without prepolymerization. With prepolymerization the produced particles were spherical and have an even surface. In contrast, polymer particles produced without prepolymerization show non-spherical shapes and rough particle surfaces which could be a hint of the uncontrolled catalyst break up at the beginning of the reaction. Based on experimental derived activity profiles a phenomenological kinetic model was developed describing the polymerization of propylene with the different injection conditions (prepolymerization). Therein heat and mass balances were implemented in order to describe the particle temperature and particle growth during the reaction. A case study on particle overheating clearly shows a significant increase of the particle temperature when no prepolymerization is applied. An increasing reaction temperature (gas-phase) resulted in a strongly increase in particle temperature. When using a prepolymerization step the particle temperature only slightly increase with increasing reaction temperature. Less thermal deactivation of the catalyst species and therefore higher activities could be the consequence. The presented study will show the derived kinetic model for the propylene polymerization with and without prepolymerization. Modeling and estimation of the kinetic parameters of the used Ziegler-Natta catalyst were carried out using the software gPROMS ModelBuilder (Process Systems Enterprise). Weight average molecular weights were determined using the method of moments. With the derived kinetic model it is possible to calculate the activity profiles of the polymerization reaction at different injection conditions (prepolymerization) over an industrially relevant temperature range and for different hydrogen concentrations

Reaction calorimetry for studying kinetics in bulk phase polymerization of propene

Polypropylene is one of the commercially most important polymers and is produced via coordinative polymerization with supported metal-organic catalysts in different processes. While kinetic measurements for slurry and gas-phase polymerization of propene are well-established, for bulk phase polymerization of propene, often the only kinetic information obtained from an experiment is the yield. In this paper, two calorimetric methods and their application for measurement of kinetics of bulk phase polymerization of propene are discussed. On the one hand a special calibration-free heat flow calorimeter and on the other hand power compensation calorimetry coupled with a software sensor for online baseline correction.Publikationsfonds ML

Efficacy and safety of sparsentan versus irbesartan in patients with IgA nephropathy (PROTECT): 2-year results from a randomised, active-controlled, phase 3 trial

BackgroundSparsentan, a novel, non-immunosuppressive, single-molecule, dual endothelin angiotensin receptor antagonist, significantly reduced proteinuria versus irbesartan, an angiotensin II receptor blocker, at 36 weeks (primary endpoint) in patients with immunoglobulin A nephropathy in the phase 3 PROTECT trial's previously reported interim analysis. Here, we report kidney function and outcomes over 110 weeks from the double-blind final analysis.MethodsPROTECT, a double-blind, randomised, active-controlled, phase 3 study, was done across 134 clinical practice sites in 18 countries throughout the Americas, Asia, and Europe. Patients aged 18 years or older with biopsy-proven primary IgA nephropathy and proteinuria of at least 1·0 g per day despite maximised renin–angiotensin system inhibition for at least 12 weeks were randomly assigned (1:1) to receive sparsentan (target dose 400 mg oral sparsentan once daily) or irbesartan (target dose 300 mg oral irbesartan once daily) based on a permuted-block randomisation method. The primary endpoint was proteinuria change between treatment groups at 36 weeks. Secondary endpoints included rate of change (slope) of the estimated glomerular filtration rate (eGFR), changes in proteinuria, a composite of kidney failure (confirmed 40% eGFR reduction, end-stage kidney disease, or all-cause mortality), and safety and tolerability up to 110 weeks from randomisation. Secondary efficacy outcomes were assessed in the full analysis set and safety was assessed in the safety set, both of which were defined as all patients who were randomly assigned and received at least one dose of randomly assigned study drug. This trial is registered with ClinicalTrials.gov, NCT03762850.FindingsBetween Dec 20, 2018, and May 26, 2021, 203 patients were randomly assigned to the sparsentan group and 203 to the irbesartan group. One patient from each group did not receive the study drug and was excluded from the efficacy and safety analyses (282 [70%] of 404 included patients were male and 272 [67%] were White) . Patients in the sparsentan group had a slower rate of eGFR decline than those in the irbesartan group. eGFR chronic 2-year slope (weeks 6–110) was −2·7 mL/min per 1·73 m2 per year versus −3·8 mL/min per 1·73 m2 per year (difference 1·1 mL/min per 1·73 m2 per year, 95% CI 0·1 to 2·1; p=0·037); total 2-year slope (day 1–week 110) was −2·9 mL/min per 1·73 m2 per year versus −3·9 mL/min per 1·73 m2 per year (difference 1·0 mL/min per 1·73 m2 per year, 95% CI −0·03 to 1·94; p=0·058). The significant reduction in proteinuria at 36 weeks with sparsentan was maintained throughout the study period; at 110 weeks, proteinuria, as determined by the change from baseline in urine protein-to-creatinine ratio, was 40% lower in the sparsentan group than in the irbesartan group (−42·8%, 95% CI −49·8 to −35·0, with sparsentan versus −4·4%, −15·8 to 8·7, with irbesartan; geometric least-squares mean ratio 0·60, 95% CI 0·50 to 0·72). The composite kidney failure endpoint was reached by 18 (9%) of 202 patients in the sparsentan group versus 26 (13%) of 202 patients in the irbesartan group (relative risk 0·7, 95% CI 0·4 to 1·2). Treatment-emergent adverse events were well balanced between sparsentan and irbesartan, with no new safety signals.InterpretationOver 110 weeks, treatment with sparsentan versus maximally titrated irbesartan in patients with IgA nephropathy resulted in significant reductions in proteinuria and preservation of kidney function

Sparsentan in patients with IgA nephropathy: a prespecified interim analysis from a randomised, double-blind, active-controlled clinical trial

Background: Sparsentan is a novel, non-immunosuppressive, single-molecule, dual endothelin and angiotensin receptor antagonist being examined in an ongoing phase 3 trial in adults with IgA nephropathy. We report the prespecified interim analysis of the primary proteinuria efficacy endpoint, and safety. Methods: PROTECT is an international, randomised, double-blind, active-controlled study, being conducted in 134 clinical practice sites in 18 countries. The study examines sparsentan versus irbesartan in adults (aged ≥18 years) with biopsy-proven IgA nephropathy and proteinuria of 1·0 g/day or higher despite maximised renin-angiotensin system inhibitor treatment for at least 12 weeks. Participants were randomly assigned in a 1:1 ratio to receive sparsentan 400 mg once daily or irbesartan 300 mg once daily, stratified by estimated glomerular filtration rate at screening (30 to 1·75 g/day). The primary efficacy endpoint was change from baseline to week 36 in urine protein-creatinine ratio based on a 24-h urine sample, assessed using mixed model repeated measures. Treatment-emergent adverse events (TEAEs) were safety endpoints. All endpoints were examined in all participants who received at least one dose of randomised treatment. The study is ongoing and is registered with ClinicalTrials.gov, NCT03762850. Findings: Between Dec 20, 2018, and May 26, 2021, 404 participants were randomly assigned to sparsentan (n=202) or irbesartan (n=202) and received treatment. At week 36, the geometric least squares mean percent change from baseline in urine protein-creatinine ratio was statistically significantly greater in the sparsentan group (-49·8%) than the irbesartan group (-15·1%), resulting in a between-group relative reduction of 41% (least squares mean ratio=0·59; 95% CI 0·51-0·69; p<0·0001). TEAEs with sparsentan were similar to irbesartan. There were no cases of severe oedema, heart failure, hepatotoxicity, or oedema-related discontinuations. Bodyweight changes from baseline were not different between the sparsentan and irbesartan groups. Interpretation: Once-daily treatment with sparsentan produced meaningful reduction in proteinuria compared with irbesartan in adults with IgA nephropathy. Safety of sparsentan was similar to irbesartan. Future analyses after completion of the 2-year double-blind period will show whether these beneficial effects translate into a long-term nephroprotective potential of sparsentan. Funding: Travere Therapeutics

Efficacy and safety of sparsentan versus irbesartan in patients with IgA nephropathy (PROTECT): 2-year results from a randomised, active-controlled, phase 3 trial

Background Sparsentan, a novel, non-immunosuppressive, single-molecule, dual endothelin angiotensin receptor antagonist, significantly reduced proteinuria versus irbesartan, an angiotensin II receptor blocker, at 36 weeks (primary endpoint) in patients with immunoglobulin A nephropathy in the phase 3 PROTECT trial's previously reported interim analysis. Here, we report kidney function and outcomes over 110 weeks from the double-blind final analysis. Methods PROTECT, a double-blind, randomised, active-controlled, phase 3 study, was done across 134 clinical practice sites in 18 countries throughout the Americas, Asia, and Europe. Patients aged 18 years or older with biopsy-proven primary IgA nephropathy and proteinuria of at least 1·0 g per day despite maximised renin–angiotensin system inhibition for at least 12 weeks were randomly assigned (1:1) to receive sparsentan (target dose 400 mg oral sparsentan once daily) or irbesartan (target dose 300 mg oral irbesartan once daily) based on a permuted-block randomisation method. The primary endpoint was proteinuria change between treatment groups at 36 weeks. Secondary endpoints included rate of change (slope) of the estimated glomerular filtration rate (eGFR), changes in proteinuria, a composite of kidney failure (confirmed 40% eGFR reduction, end-stage kidney disease, or all-cause mortality), and safety and tolerability up to 110 weeks from randomisation. Secondary efficacy outcomes were assessed in the full analysis set and safety was assessed in the safety set, both of which were defined as all patients who were randomly assigned and received at least one dose of randomly assigned study drug. This trial is registered with ClinicalTrials.gov, NCT03762850. Findings Between Dec 20, 2018, and May 26, 2021, 203 patients were randomly assigned to the sparsentan group and 203 to the irbesartan group. One patient from each group did not receive the study drug and was excluded from the efficacy and safety analyses (282 [70%] of 404 included patients were male and 272 [67%] were White) . Patients in the sparsentan group had a slower rate of eGFR decline than those in the irbesartan group. eGFR chronic 2-year slope (weeks 6–110) was −2·7 mL/min per 1·73 m2 per year versus −3·8 mL/min per 1·73 m2 per year (difference 1·1 mL/min per 1·73 m2 per year, 95% CI 0·1 to 2·1; p=0·037); total 2-year slope (day 1–week 110) was −2·9 mL/min per 1·73 m2 per year versus −3·9 mL/min per 1·73 m2 per year (difference 1·0 mL/min per 1·73 m2 per year, 95% CI −0·03 to 1·94; p=0·058). The significant reduction in proteinuria at 36 weeks with sparsentan was maintained throughout the study period; at 110 weeks, proteinuria, as determined by the change from baseline in urine protein-to-creatinine ratio, was 40% lower in the sparsentan group than in the irbesartan group (−42·8%, 95% CI −49·8 to −35·0, with sparsentan versus −4·4%, −15·8 to 8·7, with irbesartan; geometric least-squares mean ratio 0·60, 95% CI 0·50 to 0·72). The composite kidney failure endpoint was reached by 18 (9%) of 202 patients in the sparsentan group versus 26 (13%) of 202 patients in the irbesartan group (relative risk 0·7, 95% CI 0·4 to 1·2). Treatment-emergent adverse events were well balanced between sparsentan and irbesartan, with no new safety signals. Interpretation Over 110 weeks, treatment with sparsentan versus maximally titrated irbesartan in patients with IgA nephropathy resulted in significant reductions in proteinuria and preservation of kidney function.</p