Search

Background: The cost-effectiveness of using a mobile diary app as an adjunct in dialectical behavior therapy (DBT) in patientswith borderline personality disorder is unknown.Objective: This study aims to perform an economic evaluation of a mobile diary app compared with paper-based diary cardsin DBT treatment for patients with borderline personality disorder in a psychiatric outpatient facility.Methods: This study was conducted alongside a pragmatic, multicenter, randomized controlled trial. The participants wererecruited at 5 Danish psychiatric outpatient facilities and were randomized to register the emotions, urges, and skills used in amobile diary app or on paper-based diary cards. The participants in both groups received DBT delivered by the therapists. Acost-consequence analysis with a time horizon of 12 months was performed. Consequences included quality-adjusted life years(QALYs), depression severity, borderline severity, suicidal behavior, health care use, treatment compliance, and system usability.All relevant costs were included. Focus group interviews were conducted with patients, therapists, researchers, and industryrepresentatives to discuss the potential advantages and disadvantages of using a mobile diary app.Results: A total of 78 participants were included in the analysis. An insignificantly higher number of participants in the papergroup dropped out before the start of treatment (P=.07). Of those starting treatment, participants in the app group had an averageof 37.1 (SE 27.55) more days of treatment and recorded an average of 3.16 (SE 5.10) more skills per week than participants inthe paper group. Participants in both groups had a QALY gain and a decrease in depression severity, borderline severity, andsuicidal behavior. Significant differences were found in favor of the paper group for both QALY gain (adjusted difference −0.054;SE 0.03) and reduction in depression severity (adjusted difference −1.11; SE 1.57). The between-group difference in total costsranged from US $107.37 to US $322.10 per participant during the 12 months. The use of services in the health care sector wassimilar across both time points and groups (difference: psychiatric hospitalization and 2.02; SE 3.19). Overall, the patients showed high acceptability and considered the app as being easy to use. Therapists worriedabout potential negative influences on the therapist-patient interaction from new work tasks accompanying the introduction ofthe new technology but pointed at innovation potential from digital database registrations.Conclusions: This study suggests both positive and negative consequences of mobile diary apps as adjuncts to DBT comparedwith paper diary cards. More research is needed to draw conclusions regarding its cost-effectiveness. Background:The cost-effectiveness of using a mobile diary app as an adjunct in dialectical behavior therapy (DBT) in patients with borderline personality disorder is unknown.Objective:This study aims to perform an economic evaluation of a mobile diary app compared with paper-based diary cards in DBT treatment for patients with borderline personality disorder in a psychiatric outpatient facility.Methods:This study was conducted alongside a pragmatic, multicenter, randomized controlled trial. The participants were recruited at 5 Danish psychiatric outpatient facilities and were randomized to register the emotions, urges, and skills used in a mobile diary app or on paper-based diary cards. The participants in both groups received DBT delivered by the therapists. A cost-consequence analysis with a time horizon of 12 months was performed. Consequences included quality-adjusted life years (QALYs), depression severity, borderline severity, suicidal behavior, health care use, treatment compliance, and system usability. All relevant costs were included. Focus group interviews were conducted with patients, therapists, researchers, and industry representatives to discuss the potential advantages and disadvantages of using a mobile diary app.Results:A total of 78 participants were included in the analysis. An insignificantly higher number of participants in the paper group dropped out before the start of treatment (P=.07). Of those starting treatment, participants in the app group had an average of 37.1 (SE 27.55) more days of treatment and recorded an average of 3.16 (SE 5.10) more skills per week than participants in the paper group. Participants in both groups had a QALY gain and a decrease in depression severity, borderline severity, and suicidal behavior. Significant differences were found in favor of the paper group for both QALY gain (adjusted difference −0.054; SE 0.03) and reduction in depression severity (adjusted difference −1.11; SE 1.57). The between-group difference in total costs ranged from US $107.37 to US $322.10 per participant during the 12 months. The use of services in the health care sector was similar across both time points and groups (difference: psychiatric hospitalization Conclusions:This study suggests both positive and negative consequences of mobile diary apps as adjuncts to DBT compared with paper diary cards. More research is needed to draw conclusions regarding its cost-effectiveness.

Purpose Manufacturing processes for polypeptide/protein drugs are designed to ensure robust quality, efficacy and safety. Process differences introduced by follow-on manufacturers may result in changes in quality and clinical outcomes. This study investigated the impact of production methods on the stability and impurities of liraglutide and semaglutide drug substances/products, and the potential impact on drug quality, efficacy and safety. Methods State-of-the-art analytical methods were used to compare physical and chemical stability, and impurity profiles of drug substances/products from different suppliers. Identified polypeptide-related impurities were evaluated for immunogenicity potential by in silico T cell epitope prediction. Semaglutide immunogenicity in clinical trials (SUSTAIN) was evaluated using a tiered antibody analysis. Results Manufacturing scale and process strongly impacted the physical stability of the products. Trace metals increased high-molecular-weight protein formation for liraglutide and semaglutide. Synthetic and recombinant liraglutide produced by five suppliers had distinct impurity profiles compared with the originator. In silico evaluation suggested that new impurities could be immunogenic. Immunogenicity of semaglutide in clinical trials was lower than for liraglutide. Conclusions Differences in manufacturing processes affect chemical/physical stability and impurity profile, and may impact immunogenicity. Follow-on versions of liraglutide and semaglutide, and possibly other polypeptides, should be clinically evaluated for efficacy and safety.

Adult-onset neurodegeneration and other protein conformational diseases are associated with the appearance, persistence, and accumulation of misfolded and aggregation-prone proteins. To protect the proteome from long-term damage, the cell expresses a highly integrated protein homeostasis (proteostasis) machinery to ensure that proteins are properly expressed, folded, and cleared, and to recognize damaged proteins. Molecular chaperones have a central role in proteostasis as they have been shown to be essential to prevent the accumulation of alternate folded proteotoxic states as occurs in protein conformation diseases exemplified by neurodegeneration. Studies using invertebrate models expressing proteins associated with Huntington's disease, Alzheimer's disease, ALS, and Parkinson's disease have provided insights into the genetic networks and stress signaling pathways that regulate the proteostasis machinery to prevent cellular dysfunction, tissue pathology, and organismal failure. These events appear to be further amplified by aging and provide evidence that age-related failures in proteostasis may be a common element in many diseases.

Recent work suggests that the molecular structure of amyloid-like fibrils is determined by environmental conditions as well as amino acid sequence. To probe the involvement of side chains in fibrillation of the 29-residue hormone glucagon, we have measured fibrillation kinetics of 15 alanine mutants. At acidic pH, all of the mutants are able to form fibrils. However, substitution of hydrophobic residues in the N- and C-termini (in particular Phe6, Tyr10, Val23, and Met27) decelerates fibrillation dramatically. This indicates that the hydrophobicity and/or high beta-sheet propensity of these residues may be important for fibrillation. In contrast, substitution of Leu14 increases fibrillation propensity compared to that of the wild type. Nevertheless, despite identical fibrillation conditions, the thioflavin T and tryptophan fluorescence spectra of fibrils formed by mutants Tyr13, Leu14, and Asp15 are significantly different from those of other mutants, indicating that substitution of these residues may influence not only the fibrillation kinetics and fibril stability but also the preferred final structure of the fibrils that is formed, in line with the general structural polymorphism of glucagon fibrils. In contrast, under alkaline conditions, only a handful of the alanine mutants are capable of forming fibrils, suggesting that more side chains are involved in stabilizing interactions here. In addition, fibrils formed by wild-type glucagon at alkaline pH appear very stable, compared to fibrils formed at acidic pH. This suggests that the distribution of charges determines the number of different fibrillated states available to a peptide, since these can block formation of metastable fibrillated states.

Recent work suggests that protein fibrillation mechanisms and the structure of the resulting protein fibrils are very sensitive to environmental conditions such as temperature and ionic strength. Here we report the effect of several inorganic salts on the fibrillation of glucagon. At acidic pH, fibrillation is much less influenced by cations than anions, for which the effects follow the electroselectivity series; e.g., the effect of sulfate is approximately 65-fold higher than that of chloride per mole. Increased salt concentrations generally accelerate fibrillation, but result in formation of an alternate type of fibrils. Stability of these fibrils is highly affected by changes in anion concentration; the apparent melting temperature is increased by approximately 22 degrees C for any 10-fold concentration increase, indicating that the fibrils cannot exist without anions. In contrast, fibrillation under alkaline conditions is more affected by cations than anions. We conclude that ions interact directly as structural ligands with glucagon fibrils where they coordinate charges and assist in formation of new fibrils. As ex vivo amyloid plaques often contain large amounts of highly sulfated organic molecules, the specific effects of sulfate ions on glucagon may have general relevance in the study of amyloidosis and other protein deposition diseases.

The natively unfolded peptide hormone glucagon forms fibrillar structures with amyloid properties. Here, we summarize past advances in glucagon fibrillation and combine them with recent new unpublished data to provide some more general conclusions on how glucagon fibrillation adapts to different physicochemical conditions such as high temperature, pressure, mechanical and chemical stress. Factors such as peptide concentration, accessible surface area, surface hydration of the glucagon molecular state, contact surface, temperature and ionic strength all contribute to fibrillar structure and stability. In addition to fundamental changes in secondary structure, glucagon fibril morphology can vary at two macroscopic levels, namely, the degree of association (three-filament fibrils form under quiescent conditions, and two-filament fibrils form with vigorous shaking) and the type of association (twisted fibrils at low temperature, rod-like bundled fibrils at higher temperatures). Laterally bundled fibrils are presumably more stable because of the larger effective contact area. Polymorphism also opens up for interconversion of different fibril types: the low temperature fibrils can convert to a more stable fibril upon incubation at elevated temperatures (but not vice versa), indicating that fibrils are fundamentally malleable if they have not attained the most stable fibrillar state. While the effect of pressure on glucagon is complex (accelerating fibrillation at intermediate pressures and decelerating it at higher pressures), the thermal expansion coefficients obtained from these studies agree well with our previous calorimetric studies to reveal reduced or increased hydration of fibrils (leading to reduced or increased stability) depending on fibrillation conditions. Finally, we report that the cyclodextrin kleptose retards glucagon fibrillation but without interfering fundamentally with the monomer–oligomer equilibrium.

Protein amyloid formation proceeds through a number of different stages. Oligomeric species observed at early stages have aroused particular interest because of evidence for their involvement in cytotoxic processes such as membrane permeabilization. It is unclear whether these oligomers are obligate precursors to fibrils or represent "dead-end" species that impede fibrillation. Because of the many interconverting species present during amyloid formation, it is important to study the process as non-invasively as possible. Small angle X-ray scattering (SAXS) measurements allow us to monitor structural changes in solution for a population of different species over time. Here, SAXS was used to provide a detailed structural description of the fibrillation of the 29 residue peptide hormone glucagon at pH 2.5 from the monomer and early oligomers to mature fibers. Investigation of the pseudo-equilibrium behavior in the lag phase before fibrillation at several concentrations showed that glucagon is present in a monomeric form below about 5.1 mg/mL, while larger oligomers with average aggregation numbers of about three and seven, are formed at 6.4 and 10.7 mg/mL, respectively. Applying several modeling tools to the experimental data, it is shown that the early oligomerization states can be described as associations between glucagon molecules. After the lag phase, a short rod-like protofibril (radius of ~16 A and length >300 A) is formed and subsequently grows to N1000 A in length and assembles into long triple-bundled mature fibers. The protofibril shares many features with the elongated oligomer proposed to be the structural nucleus for insulin fibrils. We propose that on-pathway fibrillar intermediates share this elongated shape that easily allows them to be incorporated into mature fibrils. This contrasts with the annular shape, which is suggested to be involved in cytotoxic membrane permeabilization and may represent a dead-end species off the fibrillar pathway.

Udgivelsesdato: 2008-Apr-16 The 29-residue peptide hormone glucagon has been used as a model system for the study of amyloid-like fibrils. Atomic force microscopy (AFM) studies have detected putative oligomeric species during this lag phase, but this has not been confirmed by any spectroscopic technique. Here we use an attached pyrene group to detect association (excimer formation) between individual glucagon molecules. Our data show that excimer formation precedes fibrillation both at different pHs and with sulfate, and support our original proposal that glucagon fibril formation is preceded by oligomer formation. We suggest that pyrene-labelling may be a useful way to monitor oligomer formation during protein fibrillation.

We have established a time-resolved fluorescence assay to study fibrillation of the 29 residue peptide hormone glucagon under a variety of different conditions in a high-throughput format. Fibrils formed at pH 2.5 differ in fibrillation kinetics, morphology, thioflavin T staining and FTIR/CD spectra depending on salts, glucagon concentration and fibrillation temperature. Apparent fibrillar stability correlates with spectral and kinetic properties; generally, fibrils formed under conditions favourable for rapid fibrillation (ambient temperatures, high glucagon concentration or high salt concentration) appear less thermostable than those formed under more challenging conditions (high temperatures, low glucagon or low salt concentrations). Properties of preformed fibrils used for seeding are inherited in a prion-like manner. Thus, we conclude that the structure of fibrils formed by glucagon is not the result of the global energy minimization, but rather kinetically controlled by solvent conditions and seed-imprinting. Fibrillar polymorphism, which is being reported for an increasing number of proteins, probably reflects that fibrils have not been under evolutionary constraints to retain a single active conformation. Our results highlight the complexity of the fibrillation mechanism of glucagon, since even subtle changes in fibrillation conditions can alter the type of fibrils formed, or result in formation of mixtures of several types of fibrils.

We present a protein engineering analysis of the fibrillation of a protein from a thermophilic organism, the 101 residue S6 from Thermus thermophilus. When agitated, S6 fibrillates at pH 2.0 in 0.4 M NaCl. Under these solvent conditions, S6 has native-like secondary structure and also unfolds and refolds cooperatively. However, its tertiary structure appears to be more plastic than at neutral pH, and some regions of the protein may be partially unstructured. At 42 °C, there is a lag phase of several days after which fibrillation takes place over several hours. Data from the fibrillation behaviour of a comprehensive series of single and double mutants of S6 suggests that several factors control the onset of fibrillation. Firstly, there appears to be a contiguous region of “gatekeeper” residues that inhibit fibrillation, since their truncation significantly reduces the duration of the lag phase. This region overlaps extensively with the partially unstructured region of the protein, suggesting that residues with enhanced flexibility and solvent-accessibility are important for the initiation of fibrillation. Secondly, longer lag phases correlate with faster rates of unfolding. We interpret this to mean that kinetic stability also controls fibrillation but in the sense that the quasi-native state, rather than the denatured state, is the species that participates in nucleation. This implies that fibrillation can also occur from a quasi-native state as opposed to an ensemble of highly fluctuating structures, and highlights the delicate balance between flexibility and structure required to form organized assemblies of polypeptide chains.

We report the construction of a phage-displayed repertoire of mutants of the ribonuclease barnase from Bacillus amyloliquefaciens. The construction was guided by the natural variability between two closely related ribonucleases, barnase and binase from Bacillus intermedius. This repertoire was selected using a proteolytic selection method, allowing sorting of the library according to the resistance of the mutants toward proteolysis. Susceptibility toward proteolysis has been correlated with flexibility and unfolding, and is thus expected to yield mutants with increased thermal stability. Enrichment of barnase mutants with specific combinations of amino acid residues at four of the randomised positions was observed. Three of these enriched amino acid residues are present in neither barnase nor binase. For some of the mutations, the improvement in proteolytic stability does not lead to a pronounced improvement in thermodynamic stability, indicating that the factors governing the proteolytic stability in some cases may be different from those governing the thermodynamic stability, e.g. propensity to local unfolding.The results obtained add important knowledge to a novel use of phage display technology for selection of thermodynamically stable proteins. Only by carefully establishing the parameters that can be adjusted, and recognising the influence this will have on the outcome of selection, will it be possible to realise the powerful technique of proteolytic selection.