The effect of mutation on an aggregation-prone protein: An in vivo, in vitro, and in silico analysis.

Aggregation of initially stably structured proteins is involved in more than 20 human amyloid diseases. Despite intense research, however, how this class of proteins assembles into amyloid fibrils remains poorly understood, principally because of the complex effects of amino acid substitutions on protein stability, solubility, and aggregation propensity. We address this question using ß2-microglobulin (ß2m) as a model system, focusing on D76N-ß2m that is involved in hereditary amyloidosis. This amino acid substitution causes the aggregation-resilient wild-type protein to become highly aggregation prone in vitro, although the mechanism by which this occurs remained elusive. Here, we identify the residues key to protecting ß2m from aggregation by coupling aggregation with antibiotic resistance in E. coli using a tripartite ß-lactamase assay (TPBLA). By performing saturation mutagenesis at three different sites (D53X-, D76X-, and D98X-ß2m) we show that residue 76 has a unique ability to drive ß2m aggregation in vivo and in vitro. Using a randomly mutated D76N-ß2m variant library, we show that all of the mutations found to improve protein behavior involve residues in a single aggregation-prone region (APR) (residues 60 to 66). Surprisingly, no correlation was found between protein stability and protein aggregation rate or yield, with several mutations in the APR decreasing aggregation without affecting stability. Together, the results demonstrate the power of the TPBLA to develop proteins that are resilient to aggregation and suggest a model for D76N-ß2m aggregation involving the formation of long-range couplings between the APR and Asn76 in a nonnative state.

The role of ß-barrels 1 and 2 in the enzymatic activity of factor XIII A-subunit.

Essentials The roles of ß-barrels 1 and 2 in factor XIII (FXIII) are currently unknown. FXIII truncations lacking ß-barrel 2, both ß-barrels, or full length FXIII, were made. Removing ß-barrel 2 caused total loss of activity, removing both ß-barrels returned 30% activity. ß-barrel 2 is necessary for exposure of the active site cysteine during activation. SUMMARY: Background Factor XIII is composed of an activation peptide segment, a ß-sandwich domain, a catalytic core, and, finally, ß-barrels 1 and 2. FXIII is activated following cleavage of its A-subunits by thrombin. The resultant transglutaminase activity leads to increased resistance of fibrin clots to fibrinolysis. Objectives To assess the functional roles of ß-barrels 1 and 2 in FXIII, we expressed and characterized the full-length FXIII A-subunit (FXIII-A) and variants truncated to residue 628 (truncated to ß-barrel 1 [TB1]), residue 515 (truncated to catalytic core [TCC]), and residue 184 (truncated to ß-sandwich). Methods Proteins were analyzed by gel electrophoresis, circular dichroism, fluorometric assays, and colorimetric activity assays, clot structure was analyzed by turbidity measurements and confocal microscopy, and clot formation was analyzed with a Chandler loop system. Results and Conclusions Circular dichroism spectroscopy and tryptophan fluorometry indicated that full-length FXIII-A and the truncation variants TCC and TB1 retain their secondary and tertiary structure. Removal of ß-barrel 2 (TB1) resulted in total loss of transglutaminase activity, whereas the additional removal of ß-barrel 1 (TCC) restored enzymatic activity to ~ 30% of that of full-length FXIII-A. These activity trends were observed with physiological substrates and smaller model substrates. Our data suggest that the ß-barrel 1 domain protects the active site cysteine in the FXIII protransglutaminase, whereas the ß-barrel 2 domain is necessary for exposure of the active site cysteine during activation. This study demonstrates the importance of individual ß-barrel domains in modulating access to the FXIII active site region.

The epidemiology of in-hospital cardiac arrests in Australia and New Zealand.

BACKGROUND: The epidemiology of in-hospital cardiac arrests (IHCA) in Australia and New Zealand (ANZ) has not been systematically assessed. AIM: To conduct a systematic review of the frequency, characteristics and outcomes of adult IHCA in ANZ. METHODS: Medline search for studies published in 1964-2014 using MeSH terms 'arrest AND hospital AND Australia', 'arrest AND hospital AND New Zealand', 'inpatient AND arrest AND Australia' and 'inpatient AND arrest AND New Zealand'. RESULTS: We screened 934 studies, analysed 50 and included 30. Frequency of IHCA ranged from 1.31 to 6.11 per 1000 admissions in 4 population studies and 0.58 to 4.59 per 1000 in 16 cohort studies. The frequency was 4.11 versus 1.32 per 1000 admissions in hospitals with rapid response system (RRS) compared with those without (odds ratio: 0.32; 95% confidence interval 0.28-0.37; P < 0.001). On aggregate, the initial cardiac rhythm was ventricular tachycardia/fibrillation in 31.4% (range 19.0-48.8%) in 10 studies reporting such data. On aggregate, IHCA were witnessed in 80.2% cases (three studies) and monitored patients in 53.4% cases (four studies). Details of life support were poorly documented. On aggregate, return of spontaneous circulation occurred in 46.0% of patients. Overall, 74.6% (range 59.4-77.5%) died in-hospital but survival was higher among monitored or younger patients, in those with a shockable rhythm, or during working hours. CONCLUSION: IHCA are uncommon in ANZ and three quarters die in-hospital. However, their frequency varies markedly across institutions and may be affected by the presence of RRS. Where reported, the long-term outcomes survivors appear to have acceptable neurological outcomes.

Potential donor families' experiences of organ and tissue donation-related communication, processes and outcome.

We aimed to describe the experiences of families of potential organ and tissue donors eligible for donation after circulatory death or brain death. Forty-nine family members of potential donors from four Melbourne hospitals were interviewed to assess their experiences of communication, processes and the outcomes of donation. Interviews were recorded, transcribed verbatim and analysed thematically. Families expressed a range of perspectives on themes of communication, hospital processes and care, the processes of consent and donation and reflected on decisions and outcomes. They expressed satisfaction overall with communication when receiving bad news, discussing death and donation. Honest and frank communication and being kept up-to-date and prepared for potential outcomes were important aspects for families, especially those of post circulatory death donors. Participants reported high levels of trust in healthcare professionals and satisfaction with the level of care received. Many donor families indicated the process was lengthy and stressful, but not significantly enough to adversely affect their satisfaction with the outcome. Both the decision itself and knowing others' lives had been saved provided them with consolation. No consenting families, and only some non-consenting families, regretted their decisions. Many expressed they would benefit from a follow-up opportunity to ask questions and clarify possible misunderstandings. Overall, while experiences varied, Australian families valued frank communication, trusted health professionals, were satisfied with the care their family member received and with donation processes, despite some apparent difficulties. Family satisfaction, infrequently assessed, is an important outcome and these findings may assist education for Australian organ donation professionals.

Platypnoea-orthodeoxia syndrome post laparoscopic surgery in a patient with a patent foramen ovale.

Platypnoea-orthodeoxia syndrome has the pathognomonic clinical findings of dyspnoea and arterial hypoxaemia relieved by recumbency. We report on a patient who presented with platypnoea-orthodeoxia syndrome post laparoscopic surgery. Platypnoea-orthodeoxia syndrome is an important diagnosis to consider when investigating hypoxia without an obvious cause.

Advances in ion mobility spectrometry-mass spectrometry reveal key insights into amyloid assembly.

Interfacing ion mobility spectrometry to mass spectrometry (IMS-MS) has enabled mass spectrometric analyses to extend into an extra dimension, providing unrivalled separation and structural characterization of lowly populated species in heterogeneous mixtures. One biological system that has benefitted significantly from such advances is that of amyloid formation. Using IMS-MS, progress has been made into identifying transiently populated monomeric and oligomeric species for a number of different amyloid systems and has led to an enhanced understanding of the mechanism by which small molecules modulate amyloid formation. This review highlights recent advances in this field, which have been accelerated by the commercial availability of IMS-MS instruments. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.

The making of a nutrition professional: the Association for Nutrition register.

OBJECTIVE: Nutritionists in the UK are at the start of an exciting time of professional development. The establishment of the Association for Nutrition in 2010 has presented an opportunity to review, revitalize and expand the UK Voluntary Register of Nutritionists. In the UK and elsewhere, there is a need for a specialist register of nutritionists with title protection as a public safeguard. DESIGN: The new structure will base professional registration on demonstration of knowledge and application in five core competencies. Initially, there will be five specialist areas: animal; public health; nutritional scientist; food; sports and exercise. The wording and requirements linking the specialist areas to the competencies have been carefully defined by leading individuals currently on the existing register in these specialist areas. These have been evaluated by a random sample of existing registrants to check for accuracy of definitions and examples. Other work aims to establish a clear quality assurance framework in nutrition for workers in the health and social care sectors (UK Public Health Skills and Career Framework Levels 1-4) who contribute to nutrition activity, such as community food workers, nutrition assistants and pharmacists. Students, co-professional affiliates and senior fellows will also find a place in the new Association. The title 'nutritionist' is not currently legally protected in the UK and it is used freely to cover a range of unregulated practice. CONCLUSIONS: The establishment of a professional register to protect the public and to provide a clear identity for nutritionists is a vital step forward.

'It's quite hard to grasp the enormity of it': perceived needs of people upon diagnosis of rheumatoid arthritis.

OBJECTIVES: The diagnosis of rheumatoid arthritis (RA) brings rapid pharmacological and multidisciplinary team interventions to address inflammatory processes and symptom management. However, people may also need support on the journey to self-management. The aim of this study was to explore what professional support patients feel they receive upon diagnosis, and what support they feel would be most helpful. METHODS: Two focus groups comprised patients with at least five years'; disease duration (n = 7), and patients more recently diagnosed (5-18 months, n = 5). The latter had attended at least two appointments in a rheumatology nurse specialist clinic during the previous year, aimed at providing support upon diagnosis. Transcripts were subjected to thematic analysis to identify common issues regarding support needs, which were then grouped into themes. Interviewing and analysis was performed by researchers not involved in clinical care. RESULTS: Four overarching themes emerged. 'Information' was needed about the symptoms of RA, its management and personal outcome, while 'Support' related to emotional needs ('It's quite hard to grasp the enormity of it'). Information and Support overlapped, in that patients wanted someone to talk to, and to be listened to. These two themes were underpinned by issues of service delivery: 'Choice' (patient or professional to talk to, groups, one-to-one) and 'Involvement' (holistic care, partnership), which overlapped in terms of the opportunity to decide when and which interventions to access. CONCLUSIONS: People with RA report not only informational, but also emotional support needs at diagnosis. The potential for delivering emotional support to patients around the time of diagnosis warrants further exploration.

Nursing support at the onset of rheumatoid arthritis: Time and space for emotions, practicalities and self-management.

BACKGROUND: Following a diagnosis of rheumatoid arthritis (RA), patients have to adapt to lifelong, unpredictable but repeated episodes of pain and disability, potentially leading to permanent loss of function and its consequences on their lives. We established nurse clinics with the aim of supporting newly diagnosed RA patients in adapting to and managing their long-term condition. The aim of this study was to explore the content of clinic discussions in this new clinical service, in order to ascertain patients' needs upon diagnosis. METHODS: All clinic letters from the nurse to the family doctor were analysed. Every topic mentioned was systematically coded independently by a researcher and a patient research partner, who compared and agreed codes. Codes were organized into categories, and, finally, into overarching themes. RESULTS: Twenty-four patients had 74 appointment letters. A total of 79 codes were identified, from which ten categories emerged, and, finally, three overarching themes. The first theme related to 'Emotional support', which underpinned the other two themes and was discussed in almost all appointments. Issues included discussions about the emotional consequences of RA, needing time to adjust, frustration and fears for the future. The second theme 'Practicalities of the treatment of RA', included subordinate themes relating to the nature of RA, such as identity, cause, timeline, consequences and treatment. Medication issues were discussed and referrals to the multidisciplinary team were made. The final theme related to the 'Self-management of RA', and included discussions on physical symptoms and their management. CONCLUSIONS: The offer to attend a nurse clinic soon after diagnosis allowed RA patients to discuss a wide range of practical and self-management issues. However, most patients also took the opportunity and time to discuss emotional reactions and adaptations to diagnosis. The data suggest an unmet need for emotional support that a nurse clinic might be able to provide.

Specific glycosaminoglycans promote unseeded amyloid formation from beta2-microglobulin under physiological conditions.

Dialysis-related amyloidosis (DRA) is a complication of hemodialysis where beta2-microglobulin (beta2m) forms plaques mainly in cartilaginous tissues. The tissue-specific deposition, along with a known intransigence of pure beta2m to form fibrils in vitro at neutral pH in the absence of preformed fibrillar seeds, suggests a role for factors within cartilage in enhancing amyloid formation from this protein. To identify these factors, we determined the ability of a derivative lacking the N-terminal six amino acids found in ex vivo beta2m amyloid deposits to form amyloid fibrils at pH 7.4 in the absence of fibrillar seeds. We show that the addition of the glycosaminoglycans (GAGs) chrondroitin-4 or 6-sulfate to fibril growth assays results in the spontaneous generation of amyloid-like fibrils. By contrast, no fibrils are observed over the same time course in the presence of hyaluronic acid, a nonsulfated GAG that is abundant in cartilaginous joints. Based on the observation that hyaluronic acid has no effect on fibril stability, while chrondroitin-6-sulfate decreases the rate of fibril disassembly, we propose that the latter GAG enhances amyloid formation by stabilizing the rare fibrils that form spontaneously. This leads to the accumulation of beta2m in fibrillar deposits. Our data rationalize the joint-specific deposition of beta2m amyloid in DRA, suggesting mechanisms by which amyloid formation may be promoted.

Progressive glycosylation of albumin and its effect on the binding of homocysteine may be a key step in the pathogenesis of vascular damage in diabetes mellitus.

The majority of diabetes research to date has rightly focussed on the direct effects of hyperglycaemia on tissues and a number of theories relating to the pathogenesis of vascular disease have been proposed. This research is important as until methods are found to achieve glycaemic control in all diabetic patients, prophylactic interventions to prevent vasculopathy will be required. One of the major blood proteins, human albumin is known to be covalently modified by extended incubation with glucose, leading to an impairment of ligand binding. One of the important ligands bound by albumin is homocysteine. There is increasing and compelling clinical, experimental and epidemiological evidence that homocysteine, and in particular the free unbound fraction, is vasculotoxic. If homocysteine binding to albumin is impaired by increasing glycosylation of albumin then either drugs which reduce homocysteine levels (pyridoxine, folic acid and cobalamin) or inhibit glycosylation (aminoguanidines) may be of benefit in the prevention of vascular damage in diabetic patients.

Emotion processing in Alzheimer's disease.

To date, there have been few studies of emotion processing in those suffering from Alzheimer's disease, yet this may have an important effect on the quality of life of both sufferers and their families. This paper describes an investigation of the relative changes in cognition and in recognition and identification of non-verbal communicative signals of emotion in those suffering from Alzheimer's disease, and seeks to address the implications for clinical practice. Twelve adults with a diagnosis of "probable" Alzheimer's disease and 12 matched older adult healthy comparison participants undertook a series of tasks involving face and prosody discrimination. Facial stimuli were presented on cards, and prosodic stimuli on audiotape. Scores were compared with a measure of general cognitive ability. There was a significant difference between the Alzheimer's disease group and healthy older adult group on emotion and cognition tasks respectively. However, the ability to recognize and identify non-verbal affect cues in emotional facial expression and emotional prosody was preserved relative to general cognitive ability in those suffering from Alzheimer's disease. In addition, there were no differences found in the recognition of different emotions (happiness, sadness, anger, fear or neutral). This relative sparing of non-verbal emotional processing skills has implications for provision of assessment and interventions based on the creation of effective forms of communication that are less reliant on cognitive ability.

Beta(2)-microglobulin and its deamidated variant, N17D form amyloid fibrils with a range of morphologies in vitro.

Amyloid fibrils formed by incubation of recombinant wild-type human beta(2)-microglobulin (beta(2)M) ab initio in vitro at low pH and high ionic strength are short and highly curved. By contrast, fibrils extracted from patients suffering from haemodialysis-related amyloidosis and those formed by seeding growth of the wild-type protein in vitro with fibrils ex vivo are longer and straighter than those previously produced ab initio in vitro. Here we explore the effect of growth conditions on morphology of beta(2)M fibrils formed ab initio in vitro from the wild-type protein, as well as a variant form of beta(2)M in which Asn17 is deamidated to Asp (N17D). We show that deamidation results in significant destabilisation of beta(2)M at neutral pH. Despite this, acidification is still necessary to form amyloid from the mutant protein in vitro. Interestingly, at low pH and low ionic strength long, straight fibrils of recombinant beta(2)M are formed in vitro. The fibrils comprise three distinct morphological types when examined using electron microscopy (EM) and atomic force microscopy (AFM) that vary in periodicity and the number of constituent protofibrils. Using kinetic experiments we suggest that the immature fibrils observed previously do not represent intermediates in the assembly of fully mature amyloid, at least under the conditions studied here.

Acidic conditions stabilise intermediates populated during the folding of Im7 and Im9.

The helical bacterial immunity proteins Im7 and Im9 have been shown to fold via kinetic mechanisms of differing complexity, despite having 60 % sequence identity. At pH 7.0 and 10 degrees C, Im7 folds in a three-state mechanism involving an on-pathway intermediate, while Im9 folds in an apparent two-state transition. In order to examine the folding mechanisms of these proteins in more detail, the folding kinetics of both Im7 and Im9 (at 10 degrees C in 0.4 M sodium sulphate) have been examined as a function of pH. Kinetic modelling of the folding and unfolding data for Im7 between pH 5.0 and 8.0 shows that the on-pathway intermediate is stabilised by more acidic conditions, whilst the native state is destabilised. The opposing effect of pH on the stability of these states results in a significant population of the intermediate at equilibrium at pH 6.0 and below. At pH 7.0, the folding and unfolding kinetics for Im9 can be fitted adequately by a two-state model, in accord with previous results. However, under acidic conditions there is a clear change of slope in the plot of the logarithm of the folding rate constant versus denaturant concentration, consistent with the population of one or more intermediate(s) early during folding. The kinetic data for Im9 at these pH values can be fitted to a three-state model, where the intermediate ensemble is stabilised and the native state destabilised as the pH is reduced, rationalising previous results that showed that an intermediate is not observed experimentally at pH 7.0. The data suggest that intermediate formation is a general step in immunity protein folding and demonstrate that it is necessary to explore a wide range of refolding conditions in order to show that intermediates do not form in the folding of other small, single-domain proteins.

Role of the single disulphide bond of beta(2)-microglobulin in amyloidosis in vitro.

The aggregation of beta(2)-microglobulin (beta(2)m) into amyloid fibrils occurs in the condition known as dialysis-related amyloidosis (DRA). The protein has a beta-sandwich fold typical of the immunoglobulin family, which is stabilized by a highly conserved disulphide bond linking Cys25 and Cys80. Oxidized beta(2)m forms amyloid fibrils rapidly in vitro at acidic pH and high ionic strength. Here we investigate the role of the single disulphide bond of beta(2)m in amyloidosis in vitro. We show that reduction of the disulphide bond destabilizes the native protein such that non-native molecules are populated at neutral pH. These species are prone to oligomerization but do not form amyloid fibrils when incubated for up to 8 mo at pH 7.0 in 0.4 M NaCl. Over the pH range 4.0-1.5 in the presence of 0.4 M NaCl, however, amyloid fibrils of reduced beta(2)m are formed. These fibrils are approximately 10 nm wide, but are shorter and assemble more rapidly than those produced from the oxidized protein. These data show that population of non-native conformers of beta(2)m at neutral pH by reduction of its single disulphide bond is not sufficient for amyloid formation. Instead, association of one or more specific partially unfolded molecules formed at acid pH are necessary for the formation of beta(2)m amyloid in vitro. Further experiments will now be needed to determine the role of different oligomeric species of beta(2)m in the toxicity of the protein in vivo.

Regional myofascial pain syndrome and headache: principles of diagnosis and management.

Myofascial pain is frequently overlooked in dealing with headache pain. Myofascial pain is defined as pain and/or autonomic phenomena referred from active trigger points, with associated dysfunction. The trigger point is a focus of hyperirritability in the muscle, that when compressed, is locally tender, and if sensitized, gives rise to referred pain and tenderness. The therapy for myofascial pain requires enhancing central inhibition through pharmacology or behavioral techniques and simultaneously reducing peripheral inputs through physical therapies including exercises and trigger point-specific therapy.

A partially folded intermediate species of the beta-sheet protein apo-pseudoazurin is trapped during proline-limited folding.

The folding of apo-pseudoazurin, a 123-residue, predominantly beta-sheet protein with a complex Greek key topology, has been investigated using several biophysical techniques. Kinetic analysis of refolding using far- and near-ultraviolet circular dichroism (UV CD) shows that the protein folds slowly to the native state with rate constants of 0.04 and 0.03 min(-1), respectively, at pH 7.0 and at 15 degrees C. This process has an activation enthalpy of approximately 90 kJ/mole and is catalyzed by cyclophilin A, indicating that folding is limited by trans-cis proline isomerization, presumably around the Xaa-Pro 20 bond that is in the cis isomer in the native state. Before proline isomerization, an intermediate accumulates during folding. This species has a substantial signal in the far-UV CD, a nonnative signal in the near-UV CD, exposed hydrophobic surfaces (judged by 1-anilino naphthalenesulphonate binding), a noncooperative denaturation transition, and a dynamic structure (revealed by line broadening on the nuclear magnetic resonance time scale). We compare the properties of this intermediate with partially folded states of other proteins and discuss its role in folding of this complex Greek key protein.