An improved, optimised and robust keratin azure assay for accurate assessment of keratinase activity.

Keratin, in the form of coarse sheep wool, has been identified as an undervalued natural resource, which with the appropriate tools (e.g. a keratinase biocatalyst) can be repurposed for various textile and industrial biotechnology applications. For these purposes, we describe a novel method for identifying keratinase activity through the use of α-keratin azure (KA), an anthraquinone dyed substrate. A colourimetric method monitored the keratinase activity of Proteinase K (PK), which degrades the KA substrate and releases soluble products that are observed at 595 nm. Initially, the azure dye standard, Remazol Brilliant Blue R (RBBR), was used to calibrate the assay and allowed the kinetics of the keratinase-catalysed reaction to be determined. The assay was also used to investigate substrate pre-treatment, as well as different reaction quenching/work up conditions. Milling and washing of the KA substrate provided the best reproducibility and centrifugation was the most effective method for removing unreacted starting material. This assay was then applied to investigate the reduction of the keratin disulfide bond on keratinase-catalysed degradation. This optimised, improved and robust method will enable identification of keratinases ideally suited for application in the valorisation of the α-keratin found in natural wool fibres.

Axolemmal nanoruptures arising from paranodal membrane injury induce secondary axon degeneration in murine Guillain-Barré syndrome.

The major determinant of poor outcome in Guillain-Barré syndrome (GBS) is axonal degeneration. Pathways leading to primary axonal injury in the motor axonal variant are well established, whereas mechanisms of secondary axonal injury in acute inflammatory demyelinating polyneuropathy (AIDP) are unknown. We recently developed an autoantibody-and complement-mediated model of murine AIDP, in which prominent injury to glial membranes at the node of Ranvier results in severe disruption to paranodal components. Acutely, axonal integrity was maintained, but over time secondary axonal degeneration occurred. Herein, we describe the differential mechanisms underlying acute glial membrane injury and secondary axonal injury in this model. Ex vivo nerve-muscle explants were injured for either acute or extended periods with an autoantibody-and complement-mediated injury to glial paranodal membranes. This model was used to test several possible mechanisms of axon degeneration including calpain activation, and to monitor live axonal calcium signalling. Glial calpains induced acute disruption of paranodal membrane proteins in the absence of discernible axonal injury. Over time, we observed progressive axonal degeneration which was markedly attenuated by axon-specific calpain inhibition. Injury was unaffected by all other tested methods of protection. Trans-axolemmal diffusion of fluorescent proteins  and live calcium imaging studies indirectly demonstrated the presence of nanoruptures in the axon membrane. This study outlines one mechanism by which secondary axonal degeneration arises in the AIDP variant of GBS where acute paranodal loop injury is prominent. The data also support the development of calpain inhibitors to attenuate both primary and secondary axonal degeneration in GBS.

The endogenous calpain inhibitor calpastatin attenuates axon degeneration in murine Guillain-Barré syndrome.

Axon degeneration accounts for the poor clinical outcome in Guillain-Barré syndrome (GBS), yet no treatments target this key pathogenic stage. Animal models demonstrate anti-ganglioside antibodies (AGAb) induce axolemmal complement pore formation through which calcium flux activates the intra-axonal calcium-dependent proteases, calpains. We previously showed protection of axonal components using soluble calpain inhibitors in ex vivo GBS mouse models, and herein, we assess the potential of axonally-restricted calpain inhibition as a neuroprotective therapy operating in vivo. Using transgenic mice that over-express the endogenous human calpain inhibitor calpastatin (hCAST) neuronally, we assessed distal motor nerve integrity in our established GBS models. We induced immune-mediated injury with monoclonal AGAb plus a source of human complement. The calpain substrates neurofilament and AnkyrinG, nerve structural proteins, were assessed by immunolabelling and in the case of neurofilament, by single-molecule arrays (Simoa). As the distal intramuscular portion of the phrenic nerve is prominently targeted in our in vivo model, respiratory function was assessed by whole-body plethysmography as the functional output in the acute and extended models. hCAST expression protects distal nerve structural integrity both ex and in vivo, as shown by attenuation of neurofilament breakdown by immunolabelling and Simoa. In an extended in vivo model, while mice still initially undergo respiratory distress owing to acute conduction failure, the recovery phase was accelerated by hCAST expression. Axonal calpain inhibition can protect the axonal integrity of the nerve in an in vivo GBS paradigm and hasten recovery. These studies reinforce the strong justification for developing further animal and human clinical studies using exogenous calpain inhibitors.

Complement inhibition prevents glial nodal membrane injury in a GM1 antibody-mediated mouse model.

The involvement of the complement pathway in Guillain-Barré syndrome pathogenesis has been demonstrated in both patient biosamples and animal models. One proposed mechanism is that anti-ganglioside antibodies mediate neural membrane injury through the activation of complement and the formation of membrane attack complex pores, thereby allowing the uncontrolled influx of ions, including calcium, intracellularly. Calcium influx activates the calcium-dependent protease calpain, leading to the cleavage of neural cytoskeletal and transmembrane proteins and contributing to subsequent functional failure. Complement inhibition has been demonstrated to provide effective protection from injury in anti-ganglioside antibody-mediated mouse models of axonal variants of Guillain-Barré syndrome; however, the role of complement in the pathogenesis of demyelinating variants has yet to be established. Thus, it is currently unknown whether complement inhibition would be an effective therapeutic for Guillain-Barré syndrome patients with injuries to the Schwann cell membrane. To address this, we recently developed a mouse model whereby the Schwann cell membrane was selectively targeted with an anti-GM1 antibody resulting in significant disruption to the axo-glial junction and cytoplasmic paranodal loops, presenting as conduction block. Herein, we utilize this Schwann cell nodal membrane injury model to determine the relevance of inhibiting complement activation. We addressed the early complement component C2 as the therapeutic target within the complement cascade by using the anti-C2 humanized monoclonal antibody, ARGX-117. This anti-C2 antibody blocks the formation of C3 convertase, specifically inhibiting the classical and lectin complement pathways and preventing the production of downstream harmful anaphylatoxins (C3a and C5a) and membrane attack complexes. Here, we demonstrate that C2 inhibition significantly attenuates injury to paranodal proteins at the node of Ranvier and improves respiratory function in ex vivo and in vivo Schwann cell nodal membrane injury models. In parallel studies, C2 inhibition also protects axonal integrity in our well-established model of acute motor axonal neuropathy mediated by both mouse and human anti-GM1 antibodies. These data demonstrate that complement inhibition prevents injury in a Schwann cell nodal membrane injury model, which is representative of neuropathies associated with anti-GM1 antibodies, including Guillain-Barré syndrome and multifocal motor neuropathy. This outcome suggests that both the motor axonal and demyelinating variants of Guillain-Barré syndrome should be included in future complement inhibition clinical trials.

Schwann cell nodal membrane disruption triggers bystander axonal degeneration in a Guillain-Barré syndrome mouse model.

In Guillain-Barré syndrome (GBS), both axonal and demyelinating variants can be mediated by complement-fixing anti-GM1 ganglioside autoantibodies that target peripheral nerve axonal and Schwann cell (SC) membranes, respectively. Critically, the extent of axonal degeneration in both variants dictates long-term outcome. The differing pathomechanisms underlying direct axonal injury and the secondary bystander axonal degeneration following SC injury are unresolved. To investigate this, we generated glycosyltransferase-disrupted transgenic mice that express GM1 ganglioside either exclusively in neurons [GalNAcT-/--Tg(neuronal)] or glia [GalNAcT-/--Tg(glial)], thereby allowing anti-GM1 antibodies to solely target GM1 in either axonal or SC membranes, respectively. Myelinated-axon integrity in distal motor nerves was studied in transgenic mice exposed to anti-GM1 antibody and complement in ex vivo and in vivo injury paradigms. Axonal targeting induced catastrophic acute axonal disruption, as expected. When mice with GM1 in SC membranes were targeted, acute disruption of perisynaptic glia and SC membranes at nodes of Ranvier (NoRs) occurred. Following glial injury, axonal disruption at NoRs also developed subacutely, progressing to secondary axonal degeneration. These models differentiate the distinctly different axonopathic pathways under axonal and glial membrane targeting conditions, and provide insights into primary and secondary axonal injury, currently a major unsolved area in GBS research.

Can changes in spending on health and social care explain the recent mortality trends in Scotland? A protocol for an observational study.

INTRODUCTION: There have been steady reductions in mortality rates in the majority of high-income countries, including Scotland, since 1945. However, reductions in mortality rates have slowed down since 2012-2014 in these nations; and have reversed in some cases. Deaths among those aged 55+ explain a large amount of these changing mortality trends in Scotland. Increased pressures on health and social care services have been suggested as one factor explaining these changes. This paper outlines a protocol for the approach to testing the extent to which health and social care pressures can explain recent mortality trends in Scotland. Although a slower rate of mortality improvements have affected people of all ages, certain ages have been more negatively affected than the others. The current analyses will be run by age-band to test if the service pressure-mortality link varies across age-group. METHODS AND ANALYSIS: This will be an observational ecological study based on the Scottish population. The exposures of interest will be the absolute (primary outcome) and percentage (secondary outcome) change in real terms per capita spending on social and healthcare services between 2011 and 2017. The outcome of interest will be the absolute (primary outcome) and percentage (secondary outcome) change in age-standardised mortality rate between 2012 and 2018 for men and women separately. The units of analysis will be the 32 local authorities and the 14 territorial health boards. The analyses will be run for both all age-groups combined and for the following age bands: <1, 1-15, 16-44, 45-64, 65-74, 75-84 and 85+.A series of descriptive analyses will summarise the distribution of health and social care expenditure and mortality trends between 2011 and 2018. Linear regression analysis will be used to investigate the direct association between health care spending and mortality rates. ETHICS AND DISSEMINATION: The data used in this study will be publicly available and aggregated and will not be individually identifiable; therefore, ethical committee approval is not needed. This work will not result in the creation of a new data set. On completion, the study will be stored within the National Health Service research governance system. All of the results will be published once they have been shared with partner agencies.

In vitro evidence consistent with an interaction between wild-type and mutant SOD1 protein associated with canine degenerative myelopathy.

Canine degenerative myelopathy (DM) is a progressive neurological disorder that may be considered to be a large animal model for specific forms of the fatal human disease, familial amyotrophic lateral sclerosis (fALS). DM is associated with a c118G>A mutation of the superoxide dismutase 1 (Sod1) gene, and a significant proportion of cases are inherited in an autosomal recessive manner in contrast to the largely, but not exclusively, dominant mode of inheritance in fALS. The consensus view is that these Sod1/SOD1 mutations result in a toxic gain of function but the mechanisms remain unclear. Here we used an in vitro neuroblastoma cell line transfection system to monitor wild-type and mutant forms of SOD1 fusion proteins containing either a Cherry or an enhanced green fluorescent protein (EGFP) tag. These fusion proteins retained SOD1 enzymatic activity on a native gel assay system. We demonstrate that SOD1 aggregate density is significantly higher in DM transfectants compared to wild-type. In addition, we show by co-immunoprecipitation and confocal microscopy, evidence for a potential interaction between wild-type and mutant forms of SOD1 in co-transfected cells. While in vitro studies have shown SOD1 heterodimer formation in fALS models, this is the first report for DM SOD1. Therefore, despite for the majority of cases there is a difference in the mode of inheritance between fALS and DM, a similar interaction between wild-type and mutant SOD1 forms can occur. Clarifying the role of SOD1 in DM may also be of benefit to understanding the role of SOD1 in fALS.

Validity of the AUDIT-C screen for at-risk drinking among students utilizing university primary care.

OBJECTIVE: Research is needed to establish the psychometric properties of brief screens in university primary care settings. This study aimed to assess the construct validity of one such screen, the Alcohol Use Disorders Identification Test-Consumption (AUDIT-C), for detecting at-risk drinking among students who have utilized on-campus primary care. PARTICIPANTS: 389 students recently seen in university primary care completed a confidential online survey in December 2014. METHODS: Bivariate correlations between the AUDIT-C and measures of alcohol consumption and negative drinking consequences provided concurrent evidence for construct validity. Receiver Operating Characteristic curve analyses determined optimal cut-off scores for at-risk drinking. RESULTS: The AUDIT-C significantly correlated with measures of alcohol consumption and negative drinking consequences (p < .001). Analyses support optimal AUDIT-C cut-off scores of 5 for females and 7 for males. CONCLUSIONS: The AUDIT-C is a valid screen for at-risk drinking among students who utilize university primary care.

Conducting Systematic Outcome Assessment in Private Addictions Treatment Settings.

Systematic outcome assessment is central to ascertaining the impact of treatment services and to informing future treatment initiatives. This project was designed to be conducted within the clinical operations of 4 private addictions treatment centers. A structured interview was used to assess patients' alcohol and other drug use and related variables (on treatment entry and at 1, 3, and 6 months following treatment discharge). The primary outcomes were percentage of days abstinent (PDA) from alcohol and drugs, PDA from alcohol, and PDA from other drugs. Collateral reports during follow-up also were gathered. A total of 280 patients (56% men) across the 4 programs participated. Percentage of days abstinent for each outcome increased significantly from baseline to the 1-month follow-up assessment, and this change was maintained at the 3- and 6-month follow-up assessments. Collateral reports mirrored the patient follow-up reports. Secondary outcomes of patient ratings of urges/cravings, depression, anxiety, and general life functioning all indicated significant improvement from baseline over the course of the follow-up. The results suggest the feasibility of conducting systematic outcome assessment in freestanding private addictions treatment environments.

Modelling Population Viability of Three Independent Javan Gibbon (Hylobates moloch) Populations on Java, Indonesia.

Population viability analysis is a predictive procedure that uses a combination of different modelling approaches to estimate species vulnerability to extinction. Javan gibbons (Hylobates moloch) are vulnerable to local extinction primarily due to loss of habitat and hunting for the illegal pet trade. Using the modelling software VORTEX, we assessed the status of Javan gibbons in 3 areas (Ujung Kulon National Park, Halimun-Salak National Park, and Dieng Mountains) which hold over half of the remaining estimated number of gibbons on Java. Ujung Kulon and Halimun-Salak are long-time protected areas, whereas Dieng Mountains remain unprotected. For each area, we calculated the probability of extinction over a 100-year time period by testing different area-specific scenarios (e.g., hunting, deforestation, and increase in carrying capacity). Our modelling suggests each of the populations has a high chance of becoming extinct within the next 100 years if hunting and deforestation persist. If these threats are eliminated, the model shows each of the populations are large enough to persist in the long term whilst maintaining high levels of current genetic diversity. We conclude that specific actions should be implemented to develop more inclusive conservation management practices, especially improving awareness regarding the illegal wildlife trade and increased protection of wild populations and their habitats.

Clinical course of alcohol use in veterans following an AUDIT-C Positive Screen.

There is little known regarding the typical trajectory of alcohol use following a positive screen for hazardous alcohol use. This information would help primary care providers as they attempt to determine the best use of patient visits that might include brief alcohol interventions versus other competing medical demands. This longitudinal observational study included 98 Veterans who screened positive on the Alcohol Use Disorders Identification Test-Consumption (>3) and were asked to report on their alcohol use every 3 months for 1 year. Using latent class growth modeling, we identified the best fitting latent class structure for each outcome of high-risk and heavy drinking, respectively. There was a class of participants with increased probability of having a high-risk week or episode of heavy drinking as well as a group of participants who appeared to maintain their current drinking pattern. Although the latent class growth modeling suggested that none of the groups of participants reduced the likelihood of occurrence of heavy drinking days, two groups did significantly reduce the probability of having a hazardous alcohol use week. These results suggest that there are specific classes of patients who are less likely to change their alcohol use following a positive screen, especially those patients who report engaging in heavy drinking.

Longitudinal relationships of insomnia, nightmares, and PTSD severity in recent combat veterans.

OBJECTIVE: This observational, longitudinal study of veterans with recent combat exposure describes the prevalence, severity and associations of posttraumatic stress disorder (PTSD), insomnia, and nightmares over time. METHODS: Eighty recent combat veterans recruited from Veterans Health Administration primary care settings met inclusion criteria including hazardous alcohol use and at least subthreshold PTSD. Insomnia status and nightmare status were assigned based on the Insomnia Severity Index total score and the PTSD Checklist nightmare item, respectively. Participants were re-assessed six months following their baseline assessment. Analyses of variance compared insomnia and nightmare groups on PTSD, depression, and alcohol use severity. Analyses of covariance (controlling for baseline differences) examined whether insomnia and/or nightmares were associated with the clinical course of PTSD. Persistence of conditions was also examined. RESULTS: At baseline, 74% presented with insomnia and 61% endorsed distressing nightmares. Insomnia was associated with significantly higher PTSD and depression severity at both baseline and six months. The presence of nightmares was associated with significantly higher PTSD severity at both time points and with depression severity at baseline only. Despite decreases in PTSD and depression severity, insomnia severity was relatively unchanged after six months. The prevalence and severity of nightmare complaints diminished modestly over time. CONCLUSION: Among this sample of recent combat veterans, insomnia and nightmares were each strongly associated with the severity of both PTSD and depressive symptoms. Over time, insomnia in particular did not appear to resolve spontaneously and was associated with ongoing PTSD. Addressing insomnia early, therefore, may be a strategy to alter the course of PTSD.

Telephone consultations to manage requests for same-day appointments: a randomised controlled trial in two practices.

BACKGROUND: General practitioners (GPs) in the United Kingdom have recently begun to adopt the use of telephone consultation during daytime surgery as a means of managing demand, particularly requests for same-day appointments. However, it is not known whether the strategy actually reduces GP workload. AIM: To investigate how the use of telephone consultations impacts on the management of requests for same-day appointments, on resource use, indicators of clinical care, and patient perceptions of consultations. DESIGN OF STUDY: Randomised controlled trial. SETTING: All patients (n = 388) seeking same-day appointments in each surgery in two urban practices (total population = 10,420) over a four-week period. METHOD: The primary outcome measure was use of doctor time for the index telephone or face-to-face consultation. Secondary outcomes were subsequent use of investigations and of services in the two-week period following consultation, frequency of blood pressure measurement and antibiotic prescriptions, and number of problems considered at consultation. Patient perceptions were measured by the Patient Enablement Instrument (PEI) and reported willingness to use telephone consultations in the future. RESULTS: Telephone consultations took less time (8.2 minutes versus 6.7 minutes; diff = 1.5, 95% confidence interval [CI] = 0.6 to 2.4, P = 0.002). Patients consulting by telephone reconsulted the GP more frequently in the two weeks that followed (0.6 consultations versus 0.4 consultations; diff = 0.2, 95% CI = 0.0 to 0.3, P = 0.01). Blood pressure was measured more often in the group of patients managed face-to-face (25/188 [13.3%] versus 12/181 [6.6%]; diff = 6.7%, 95% CI = 0.6% to 12.7%). There was no significant difference in patient perceptions or other secondary outcomes. CONCLUSION: Use of telephone consultations for same-day appointments was associated with time saving, and did not result in lower PEI scores. Possibly, however, this short-term saving was offset by higher re-consultation and less use of opportunistic health promotion.