Mutant SOD1 aggregates formed in vitro and in cultured cells are polymorphic and differ from those arising in the CNS.

Mutations in the human Superoxide dismutase 1 (hSOD1) gene are well-established cause of the motor neuron disease ALS. Patients and transgenic (Tg) ALS model mice carrying mutant variants develop hSOD1 aggregates in the CNS. We have identified two hSOD1 aggregate strains, which both transmit spreading template-directed aggregation and premature fatal paralysis when inoculated into adult transgenic mice. This prion-like spread of aggregation could be a primary disease mechanism in SOD1-induced ALS. Human SOD1 aggregation has been studied extensively both in cultured cells and under various conditions in vitro. To determine how the structure of aggregates formed in these model systems related to disease-associated aggregates in the CNS, we used a binary epitope-mapping assay to examine aggregates of hSOD1 variants G93A, G85R, A4V, D90A, and G127X formed in vitro, in four different cell lines and in the CNS of Tg mice. We found considerable variability between replicate sets of in vitro-generated aggregates. In contrast, there was a high similarity between replicates of a given hSOD1 mutant in a given cell line, but pronounced variations between different hSOD1 mutants and different cell lines in both structures and amounts of aggregates formed. The aggregates formed in vitro or in cultured cells did not replicate the aggregate strains that arise in the CNS. Our findings suggest that the distinct aggregate morphologies in the CNS could result from a micro-environment with stringent quality control combined with second-order selection by spreading ability. Explorations of pathogenesis and development of therapeutics should be conducted in models that replicate aggregate structures forming in the CNS.

Effect of crown height on the screw joint stability of zirconia screw-retained crowns.

STATEMENT OF PROBLEM: Medium- to long-term data for the performance of zirconia crowns with titanium (Ti) bases are sparse, particularly when the crown height space and occlusal loads are high. PURPOSE: The purpose of this in vitro study was to assess the effect of the height of zirconia screw-retained implant crowns with a Ti base on the screw joint stability after cyclic loading. A secondary aim was to investigate the survival of zirconia crowns of different heights after cyclic loading. MATERIAL AND METHODS: Twenty-one internal connection implants were secured between fiberglass-reinforced epoxy resin sleeves. Mandibular first molar monolithic zirconia crowns with 3 different heights (6 mm, 10 mm, and 14 mm) were milled and bonded to the Ti bases (n=7). The screws were tightened to 30 Ncm, and a 30-degree 120-N cyclic load was applied to the crowns at 2 Hz for 5 million cycles. After 5 million cycles, the crowns were evaluated for stability, and the same protocol was repeated for 275-N and 435-N loads for 5 million cycles each. After loading, the detorque values were recorded. Failure was characterized based on whether the crown, screw, and/or implant fracture was observed. The detorque values were analyzed by using a 1-way-ANOVA with the restricted maximum likelihood estimation. The percentage of torque loss was calculated. The LIFETEST procedure was used to analyze the survival probability of the groups (α=.05). RESULTS: The effect of crown height on the detorque values of screws was not found to be statistically significant (P>.05). The mean detorque value for 6-mm crowns was 23.5 Ncm, 24.4 Ncm for 10-mm crowns, and 22.1 Ncm for 14-mm crowns. A significant effect of crown height was found on the survival (P=.006), and the time-to-failure survival of 14-mm crowns was significantly lower than the survival of 6 mm and 10 mm crowns (P=.020), where no failures were observed. Four 14-mm crowns failed between the 1 and 2 million cycles after the loads were increased to 435 N. The failure modes were the same for all the crowns, implants, and screws fractured. CONCLUSIONS: When the tested internal connection implant was used, the crown height did not affect the detorque values, and 14-mm crowns performed similarly to the shorter crowns in terms of torque loss after cyclic loading. However, survival of the 14-mm crown-implant complex was lower, resulting in screw and implant fractures.

Cathepsin A contributes to left ventricular remodeling by degrading extracellular superoxide dismutase in mice.

In the heart, the serine carboxypeptidase cathepsin A (CatA) is distributed between lysosomes and the extracellular matrix (ECM). CatA-mediated degradation of extracellular peptides may contribute to ECM remodeling and left ventricular (LV) dysfunction. Here, we aimed to evaluate the effects of CatA overexpression on LV remodeling. A proteomic analysis of the secretome of adult mouse cardiac fibroblasts upon digestion by CatA identified the extracellular antioxidant enzyme superoxide dismutase (EC-SOD) as a novel substrate of CatA, which decreased EC-SOD abundance 5-fold. In vitro, both cardiomyocytes and cardiac fibroblasts expressed and secreted CatA protein, and only cardiac fibroblasts expressed and secreted EC-SOD protein. Cardiomyocyte-specific CatA overexpression and increased CatA activity in the LV of transgenic mice (CatA-TG) reduced EC-SOD protein levels by 43%. Loss of EC-SOD-mediated antioxidative activity resulted in significant accumulation of superoxide radicals (WT, 4.54 µmol/mg tissue/min; CatA-TG, 8.62 µmol/mg tissue/min), increased inflammation, myocyte hypertrophy (WT, 19.8 µm; CatA-TG, 21.9 µm), cellular apoptosis, and elevated mRNA expression of hypertrophy-related and profibrotic marker genes, without affecting intracellular detoxifying proteins. In CatA-TG mice, LV interstitial fibrosis formation was enhanced by 19%, and the type I/type III collagen ratio was shifted toward higher abundance of collagen I fibers. Cardiac remodeling in CatA-TG was accompanied by an increased LV weight/body weight ratio and LV end diastolic volume (WT, 50.8 µl; CatA-TG, 61.9 µl). In conclusion, CatA-mediated EC-SOD reduction in the heart contributes to increased oxidative stress, myocyte hypertrophy, ECM remodeling, and inflammation, implicating CatA as a potential therapeutic target to prevent ventricular remodeling.

Weighted mandibular complete denture fabricated by using selective laser melting: A clinical report.

The rehabilitation of patients with severely resorbed mandibular ridges can be a clinical challenge when rehabilitation with endosteal implants is not the elected treatment. Historically, weighted mandibular complete dentures have been used successfully to manage patients with severely resorbed ridges, and the weight of their cast metal has been calculated by using the weight of the wax and the density of the alloy. This clinical report presents the management of an 87-year-old woman with a severely resorbed mandibular ridge by using a weighted mandibular complete denture fabricated by using selective laser melting (SLM) technology in which the weight of the metal base was calculated by using the volume of the digital file used for manufacture.

Effect of Crown Height on the Screw Stability of Titanium Screw-Retained Crowns.

PURPOSE: The aim of this in vitro study was to evaluate the effect of crown height on the screw stability of screw-retained titanium implant crowns subjected to cyclic loading conditions. MATERIALS AND METHODS: Twenty-one implants with internal hex connections were placed in epoxy resin holders. Mandibular first molar screw-retained titanium implant crowns with UCLA type, crown-abutment connections were CAD/CAM fabricated. Seven crowns of 3 different heights (6 mm, 10 mm, and 14 mm) were made. The crowns were seated onto the implants and screws were tightened to 30 Ncm. The implants were clamped into holders and stepwise cyclic loads were applied to the occlusal surface at 30-degree angles to the long axes of the crowns. The detorque values were measured after each 5 million cycles. Before increasing the applied load, the crowns were secured with new screws and tightened to 30 Ncm. Failure times, survival estimates and detorque values were then analyzed. (alpha = 0.05). RESULTS: Crown height did not significantly affect detorque values. However, five 14-mm crowns failed with varying fractures during the 475 N loading condition. Overall, a significantly lower survival for 14 mm crowns was found compared to 6 mm and 10 mm crowns (p = 0.004). CONCLUSIONS: Crown heights of one-piece screw-retained titanium implant crowns did not significantly affect detorque values. Screw fracture, however, was greater for crown height of 14 mm than those of 6 mm and 10 mm.

Physicochemical code for quinary protein interactions in Escherichia coli.

How proteins sense and navigate the cellular interior to find their functional partners remains poorly understood. An intriguing aspect of this search is that it relies on diffusive encounters with the crowded cellular background, made up of protein surfaces that are largely nonconserved. The question is then if/how this protein search is amenable to selection and biological control. To shed light on this issue, we examined the motions of three evolutionary divergent proteins in the Escherichia coli cytoplasm by in-cell NMR. The results show that the diffusive in-cell motions, after all, follow simplistic physical-chemical rules: The proteins reveal a common dependence on (i) net charge density, (ii) surface hydrophobicity, and (iii) the electric dipole moment. The bacterial protein is here biased to move relatively freely in the bacterial interior, whereas the human counterparts more easily stick. Even so, the in-cell motions respond predictably to surface mutation, allowing us to tune and intermix the protein's behavior at will. The findings show how evolution can swiftly optimize the diffuse background of protein encounter complexes by just single-point mutations, and provide a rational framework for adjusting the cytoplasmic motions of individual proteins, e.g., for rescuing poor in-cell NMR signals and for optimizing protein therapeutics.

Publisher Correction: In vitro investigation of chemical properties and biocompatibility of neurovascular braided implants.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Effect of coded healing abutment height and position on the trueness of digital intraoral implant scans.

STATEMENT OF PROBLEM: Information regarding the effect of the height and position of a coded healing abutment (CHA) on the trueness of intraoral digital scans is lacking. PURPOSE: The purpose of this in vitro study was to investigate the effect of the height and position of a scannable CHA on the trueness (distance and angular deviations) of intraoral digital scans. MATERIAL AND METHODS: Scannable CHAs (BellaTek Encode Impression system; Zimmer Biomet Dental) were used in 2 different height pairs (3 mm and 8 mm) on 2 implants at mandibular left second and first molar positions. Each pair was scanned 10 times by using 1 intraoral scanner (TRIOS; 3Shape) by 1 operator to generate a total of 20 intraoral scan files. Master standard tessellation language (STL) files were created for both 3-mm and 8-mm CHA pairs by using a structured blue light scanner (COMET L3D 8M 150 Precision Structured Blue Light Scanner; ZEISS). These master STL files were imported into a software program (PolyWorks Inspector) and were used as the reference for the inspection. Scans obtained by using the intraoral scanner were aligned to the reference scan by using a best-fit alignment to measure the distance and angular deviations. Two-way repeated-measures ANOVA was used to analyze the data, and the Tukey-Kramer test was used to determine significant differences among groups (α=.05). RESULTS: The CHA position had a significant effect on distance deviation (P<.001). However, no significant effect of CHA height on distance deviation was found. The interaction between CHA height and position had a significant effect on the angular deviation (P=.041). The 3-mm posterior CHA (P=.026) and 8-mm anterior CHA (P=.039) had significantly lower angular deviations than the 8-mm posterior CHA. CONCLUSIONS: The distance deviation of CHA was significantly influenced by position. CHAs in the anterior had lower distance deviations for both 3 mm and 8 mm. The effect of CHA height on distance deviation was found to be small and was affected by the location of the CHA. Height affected angular deviation depending on the position of the CHA. Both 3-mm posterior and 8-mm anterior CHAs showed lower angular deviations than the 8-mm posterior CHA.

Long-term outcome of adhesive fragment reattachment in crown-root fractured teeth.

BACKGROUND/AIMS: Adhesive fragment reattachment (AFR) is one treatment option for crown-root fractured teeth. However, there are no studies investigating the long-term outcome of this approach. The aim of this retrospective study was to evaluate the long-term outcome of AFR and periodontal health in crown-root fractured teeth by assessing complications and periodontal status. MATERIALS AND METHODS: Data regarding 41 patients with 51 traumatized teeth (TT) were included. Periodontal health was assessed by recording the pocket probing depth (PPD), clinical attachment level (CAL), bleeding on probing (BoP), gingival index (GI), and plaque index (PI) in the TT and in one unaffected control tooth (CT). Complications were classified as "restorative," "endodontic," and "additional root fracture." Based on these complications, the outcome was graded as "success," "partial success," "survival," and "failure." Statistics was performed by t test, chi-square test and logistic regression models. RESULTS: After 8.5 ± 4.6 years, 76.5% (39/51) of the TT had functionally survived. Functional survival of the reattached fragments was 66.7% (26/39) after 9.5 ± 3.7 years. PPD (TT: 4.11 ± 2.03; CT: 2.08 ± 0.65), CAL (TT: 4.78 ± 2.19; CT: 2.42 ± 1.03), and BoP values (TT: 77.4%; CT: 22.6%) were higher in TT than in CT. GI scores > 0 were found in 83.3% of the TT and in 27.8% of the CT. PI scores did not differ between TT and CT. Of the complications, 56.8% were "restorative," 22.7% "endodontic," and 20.5% "additional root fractures." Eleven (27.5%) TT were without complications and rated as "success." CONCLUSIONS: AFR in crown-root fractured teeth showed a high survival rate and occasionally compromised periodontal health. However, due to the high complication rate, it should be considered as a long-term temporary treatment to postpone other invasive therapy options. AFR can be a valuable way to avoid early loss of crown-root fractured teeth, especially in young patients. Moisture control and additional root fractures significantly influenced the outcome.

In vitro investigation of chemical properties and biocompatibility of neurovascular braided implants.

Braiding of Nitinol micro wires is an established technology for the manufacturing of fine-meshed neurovascular implants for tortuous vessel geometries. Electropolishing of wires before the braiding process has the potential to improve the in vitro behaviour in terms of thrombogenicity and endothelial cell proliferation. In this study, we present the first in vitro investigation of braided electropolished/blue oxide Nitinol samples in a blood flow loop, showing a significantly lower activation of the coagulation pathway (represented by the TAT III marker) and a tendency towards reduced platelet adhesion. Furthermore, we applied the same surface treatment on flat disks and measured protein adhesion as well as endothelial cell proliferation. We compared our results to non-electropolished samples with a native oxide surface. While platelet deposition was reduced on electropolished/blue oxide surface, a significant increase of endothelial cell seeding was observed. Investigation of inflammatory marker expression in endothelial cells provided divergent results depending on the marker tested, demanding closer investigation. Surface analysis using Auger electron spectroscopy revealed a thin layer mainly consisting of titanium oxynitride or titanium oxide + titanium nitride as a potential cause of the improved biological performance. Translated to the clinical field of intracranial aneurysm treatment, the improved biocompatibility has the potential to increase both safety (low thrombogenicity) and effectiveness (aneurysm neck reconstruction).

A comparison of implant complications and failures between the maxilla and the mandible.

STATEMENT OF PROBLEM: Identifying factors that affect the clinical outcomes of implant therapy is important. PURPOSE: The purpose of this retrospective study was to determine whether implant location was a factor affecting the complication and failure rates of single-tooth implant-supported restorations in a predoctoral setting. MATERIAL AND METHODS: The charts of 431 patients treated with a surgically placed dental implant and restored with a single crown in the predoctoral clinic were analyzed. Data on implant location, type of complication (surgical or prosthetic), and type of failure were collected and analyzed according to implant location using the Fisher Exact Test and Mantel-Haenszel Exact Chi Square Test analysis (α=.05). RESULTS: The charts revealed 158 complications (68 surgical and 90 prosthetic) in 110 patients, and 3.9% of the implants failed. No statistically significant difference was found between the number of surgical complications or prosthetic complications in the maxilla and the mandible (P=.469). CONCLUSIONS: Jaw location (maxilla compared with mandible) of the implant had no statistically significant impact on the incidence of surgically or prosthetically related complications. No statistically significant difference was found in overall implant failures, surgical failures, and prosthetic failures between maxillary and mandibular implants.

SOD1 aggregation in ALS mice shows simplistic test tube behavior.

A longstanding challenge in studies of neurodegenerative disease has been that the pathologic protein aggregates in live tissue are not amenable to structural and kinetic analysis by conventional methods. The situation is put in focus by the current progress in demarcating protein aggregation in vitro, exposing new mechanistic details that are now calling for quantitative in vivo comparison. In this study, we bridge this gap by presenting a direct comparison of the aggregation kinetics of the ALS-associated protein superoxide dismutase 1 (SOD1) in vitro and in transgenic mice. The results based on tissue sampling by quantitative antibody assays show that the SOD1 fibrillation kinetics in vitro mirror with remarkable accuracy the spinal cord aggregate buildup and disease progression in transgenic mice. This similarity between in vitro and in vivo data suggests that, despite the complexity of live tissue, SOD1 aggregation follows robust and simplistic rules, providing new mechanistic insights into the ALS pathology and organism-level manifestation of protein aggregation phenomena in general.

Thermodynamics of protein destabilization in live cells.

Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a ß-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.

Structural and kinetic analysis of protein-aggregate strains in vivo using binary epitope mapping.

Despite considerable progress in uncovering the molecular details of protein aggregation in vitro, the cause and mechanism of protein-aggregation disease remain poorly understood. One reason is that the amount of pathological aggregates in neural tissue is exceedingly low, precluding examination by conventional approaches. We present here a method for determination of the structure and quantity of aggregates in small tissue samples, circumventing the above problem. The method is based on binary epitope mapping using anti-peptide antibodies. We assessed the usefulness and versatility of the method in mice modeling the neurodegenerative disease amyotrophic lateral sclerosis, which accumulate intracellular aggregates of superoxide dismutase-1. Two strains of aggregates were identified with different structural architectures, molecular properties, and growth kinetics. Both were different from superoxide dismutase-1 aggregates generated in vitro under a variety of conditions. The strains, which seem kinetically under fragmentation control, are associated with different disease progressions, complying with and adding detail to the growing evidence that seeding, infectivity, and strain dependence are unifying principles of neurodegenerative disease.

Intestinal Barrier Function and the Gut Microbiome Are Differentially Affected in Mice Fed a Western-Style Diet or Drinking Water Supplemented with Fructose.

Background: The consumption of a Western-style diet (WSD) and high fructose intake are risk factors for metabolic diseases. The underlying mechanisms are largely unclear.Objective: To unravel the mechanisms by which a WSD and fructose promote metabolic disease, we investigated their effects on the gut microbiome and barrier function.Methods: Adult female C57BL/6J mice were fed a sugar- and fat-rich WSD or control diet (CD) for 12 wk and given access to tap water or fructose-supplemented water. The microbiota was analyzed with the use of 16S rRNA gene sequencing. Barrier function was studied with the use of permeability tests, and endotoxin, mucus thickness, and gene expressions were measured.Results: The WSD increased body weight gain but not endotoxin translocation compared with the CD. In contrast, high fructose intake increased endotoxin translocation 2.6- and 3.8-fold in the groups fed the CD + fructose and WSD + fructose, respectively, compared with the CD group. The WSD + fructose treatment also induced a loss of mucus thickness in the colon (-46%) and reduced defensin expression in the ileum and colon. The lactulose:mannitol ratio in the WSD + fructose mice was 1.8-fold higher than in the CD mice. Microbiota analysis revealed that fructose, but not the WSD, increased the Firmicutes:Bacteroidetes ratio by 88% for CD + fructose and 63% for WSD + fructose compared with the CD group. Bifidobacterium abundance was greater in the WSD mice than in the CD mice (63-fold) and in the WSD + fructose mice than in the CD + fructose mice (330-fold).Conclusions: The consumption of a WSD or high fructose intake differentially affects gut permeability and the microbiome. Whether these differences are related to the distinct clinical outcomes, whereby the WSD primarily promotes weight gain and high fructose intake causes barrier dysfunction, needs to be investigated in future studies.

Tricking a Protein To Swap Strands.

Despite continuing interest in partly unfolded proteins as precursors for aggregation and adverse gain-of-function in human disease, there is yet little known about the local transitions of native structures that possibly lead to such intermediate states. To target this problem, we present here a protein-design strategy that allows real-time detection of rupture and swapping of complete secondary-structure elements in globular proteins-molecular events that have previously been inaccessible experimental analysis. The approach is applied to the dynamic ß-barrel of SOD1, associated with pathologic aggregation in the neurodegenerative disease ALS. Data show that rupture and re-insertion of individual ß-strands do not take place locally but require the SOD1 barrel to unfold globally. The finding questions the very existence of partly unfolded intermediates in the SOD1 aggregation process and presents new clues to the mechanism by which hydrogen bonding maintains global structural integrity.

Improving the value of clinical research through the use of Common Data Elements.

The use of Common Data Elements can facilitate cross-study comparisons, data aggregation, and meta-analyses; simplify training and operations; improve overall efficiency; promote interoperability between different systems; and improve the quality of data collection. A Common Data Element is a combination of a precisely defined question (variable) paired with a specified set of responses to the question that is common to multiple datasets or used across different studies. Common Data Elements, especially when they conform to accepted standards, are identified by research communities from variable sets currently in use or are newly developed to address a designated data need. There are no formal international specifications governing the construction or use of Common Data Elements. Consequently, Common Data Elements tend to be made available by research communities on an empiric basis. Some limitations of Common Data Elements are that there may still be differences across studies in the interpretation and implementation of the Common Data Elements, variable validity in different populations, and inhibition by some existing research practices and the use of legacy data systems. Current National Institutes of Health efforts to support Common Data Element use are linked to the strengthening of National Institutes of Health Data Sharing policies and the investments in data repositories. Initiatives include cross-domain and domain-specific resources, construction of a Common Data Element Portal, and establishment of trans-National Institutes of Health working groups to address technical and implementation topics. The National Institutes of Health is seeking to lower the barriers to Common Data Element use through greater awareness and encourage the culture change necessary for their uptake and use. As National Institutes of Health, other agencies, professional societies, patient registries, and advocacy groups continue efforts to develop and promote the responsible use of Common Data Elements, particularly if linked to accepted data standards and terminologies, continued engagement with and feedback from the research community will remain important.

Global structural motions from the strain of a single hydrogen bond.

The origin and biological role of dynamic motions of folded enzymes is not yet fully understood. In this study, we examine the molecular determinants for the dynamic motions within the ß-barrel of superoxide dismutase 1 (SOD1), which previously were implicated in allosteric regulation of protein maturation and also pathological misfolding in the neurodegenerative disease amyotrophic lateral sclerosis. Relaxation-dispersion NMR, hydrogen/deuterium exchange, and crystallographic data show that the dynamic motions are induced by the buried H43 side chain, which connects the backbones of the Cu ligand H120 and T39 by a hydrogen-bond linkage through the hydrophobic core. The functional role of this highly conserved H120-H43-T39 linkage is to strain H120 into the correct geometry for Cu binding. Upon elimination of the strain by mutation H43F, the apo protein relaxes through hydrogen-bond swapping into a more stable structure and the dynamic motions freeze out completely. At the same time, the holo protein becomes energetically penalized because the twisting back of H120 into Cu-bound geometry leads to burial of an unmatched backbone carbonyl group. The question then is whether this coupling between metal binding and global structural motions in the SOD1 molecule is an adverse side effect of evolving viable Cu coordination or plays a key role in allosteric regulation of biological function, or both?

Investigation of Intra- and Inter-individual Variations of Mineralisation in Healthy Permanent Human Enamel by Raman Spectroscopy.

PURPOSE: To systematically examine mineralisation of healthy human enamel using Raman spectroscopy and provide an understanding of baseline variations that may be inherent in the healthy enamel from individual to individual as well as variations within a tooth. MATERIALS AND METHODS: Human teeth were obtained in compliance with the NIH guidelines. The teeth were collected fresh within the date of the extraction and kept moist at all times with wet tissue paper without any additional disinfecting treatment. The samples were individually wrapped in wet tissue paper and stored in a -20°C freezer. Prior to Raman analysis, the specimens were thawed at room temperature for 30 min. A Raman microscope was employed with a 10X objective used to focus the laser light (785 nm). Raman spectroscopy scores were validated by microcomputed tomography (µCT) on the two teeth which had the highest and lowest mineralisation found in the Raman scans. RESULTS: Mineralisation levels varied substantially between individuals. The highest Raman-based mineralisation intensity was about 5-fold greater than the lowest mineralisation score. Incisor mineralisation also varied dramatically depending on different sites on the tooth. CONCLUSIONS: Clinically applicable non-invasive techniques such as Raman spectroscopy that can quantify mineral content, such as Raman spectroscopy, may help answer whether or not mineralisation is associated with caries risk.

Fibrillation precursor of superoxide dismutase 1 revealed by gradual tuning of the protein-folding equilibrium.

Although superoxide dismutase 1 (SOD1) stands out as a relatively soluble protein in vitro, it can be made to fibrillate by mechanical agitation. The mechanism of this fibrillation process is yet poorly understood, but attains considerable interest due to SOD1's involvement in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). In this study, we map out the apoSOD1 fibrillation process from how it competes with the global folding events at increasing concentrations of urea: We determine how the fibrillation lag time (τ(lag)) and maximum growth rate (ν(max)) depend on gradual titration of the folding equilibrium, from the native to the unfolded state. The results show that the agitation-induced fibrillation of apoSOD1 uses globally unfolded precursors and relies on fragmentation-assisted growth. Mutational screening and fibrillation m-values (∂ log τ(lag)/∂[urea] and ∂ log ν(max)/∂[urea]) indicate moreover that the fibrillation pathway proceeds via a diffusely bound transient complex that responds to the global physiochemical properties of the SOD1 sequence. Fibrillation of apoSOD1, as it bifurcates from the denatured ensemble, seems thus mechanistically analogous to that of disordered peptides, save the competing folding transition to the native state. Finally, we examine by comparison with in vivo data to what extent this mode of fibrillation, originating from selective amplification of mechanically brittle aggregates by sample agitation, captures the mechanism of pathological SOD1 aggregation in ALS.