"My life isn't my life, it's the systems": A qualitative exploration of women's experiences of day-to-day restrictive practices as inpatients.

WHAT IS KNOWN ON THE SUBJECT?: Sometimes someone needs to be in hospital because they are struggling with their mental health and need some extra support but being in hospital can also be a difficult experience. There are a lot of restrictions in place in hospital, like locked doors, rules to follow and not much choice about what happens to you. Other research has found that these restrictions can feel difficult and stressful for people and so more research is needed about this. We wanted to know what being in hospital felt like for women in particular. WHAT THE PAPER ADDS TO EXISTING KNOWLEDGE?: We interviewed women who were in hospital because of their mental health about what it was like for them in hospital. The women told us that they felt powerless while they were in hospital. They sometimes felt like they were being punished and this could affect their mood and could lead to them hurting themselves. They also said that they were not always listened to by staff, and they found it difficult being away from their family and friends while they were in hospital. The women also told us that being in hospital could sometimes help them to feel safe. WHAT ARE THE IMPLICATIONS FOR PRACTICE?: Women should be looked after in hospital in a way that helps them to feel in control of what happens to them. They should be supported to be able to go outside the hospital on leave, to keep in touch with their family and friends, and they should be listened to by staff. A project called "safewards" has suggested some ways for helping to make hospital wards safer. They have suggested that everyone should be clear about what the rules are when they go into hospital and ways that staff could communicate more clearly with the people they are working with. Our research supports using these techniques. ABSTRACT: Introduction Inpatient care often involves restrictive interventions such as seclusion and restraint and restrictive practices that limit the person's freedom, rights and daily activities. Restrictive practice has not been the explicit focus in previous research; however, it often appears as an important theme, with participants identifying it can have a detrimental effect on their well-being. More research specifically on this topic in an inpatient setting is, therefore, needed. Women might be particularly vulnerable to adverse effects of restrictive practices compared to men as women generally occupy less powerful positions in society and more often experience abuse. Aims The study aimed to explore women's experiences of routine restrictive practices in mental health inpatient settings. Methods Twenty-two women who were currently inpatients on mental health wards were interviewed about their experiences of restrictive practices in hospital. Interviews were analysed using thematic analysis. Results An overarching theme emerged of powerlessness. Four key sub-themes were also identified: restrictions perceived as punitive, having no voice, impact of restrictions on relationships and restrictions providing safety and support. Discussion Although restrictive practices were found to provide the women with a sense of safety, they were also found to impact upon the women's well-being, leading to increases in self-harm and over-reliance on restrictions. Implications for practice This research highlights the importance of gender-informed inpatient services for women that foster independence, empowerment and allow women to have their voices heard. Safewards interventions such as clear mutual expectations and soft words could contribute to mitigating the impact of restrictive practices.

Case formulation-A vehicle for change? Exploring the impact of cognitive behavioural therapy formulation in first episode psychosis: A reflexive thematic analysis.

OBJECTIVES: Formulation is considered a fundamental process of cognitive behavioural therapy for psychosis (CBTp). However, an exploration into the personal impact of different levels of case formulation (CF) from a service user (SU) perspective is lacking, particularly for those experiencing a first episode of psychosis. DESIGN: This Big Q qualitative design used semi-structured interviews. METHODS: Reflexive thematic analysis (TA) was used to analyse 10 participant interviews. NVivo 12 computer-assisted qualitative data analysis software aided data organisation and analysis. RESULTS: One overarching theme 'CF - A vehicle for change?' was developed as a pattern of shared meaning across the data set. Three main themes related to the overarching theme: (1) Vicious cycles: 'I never really thought about it being me maintaining the problems' (including one subtheme - Self-empowerment: 'Only you can make the changes for yourself'); (2) Early life experiences: 'My experiences have shaped the person that I am, therefore, it's not my fault' (including one subtheme - Disempowerment: '[My] core beliefs have been damaged'); and (3) Keep it simple: 'Don't push it too far over the top in case it becomes like spaghetti'. CONCLUSIONS: Maintenance formulations may be experienced as self-blaming, but also self-empowering, which may help to facilitate change. Longitudinal formulations may be experienced as non-blaming, but also disempowering, which may inhibit change. Simple CF diagrams may also facilitate change, whereas overly complex CFs may inhibit change. How CBTp therapists might look to improve the impact of different levels of CF for service users (SUs) in first episode psychosis (FEP) are described.

What does recovery mean to young people with mental health difficulties? - "It's not this magical unspoken thing, it's just recovery".

Background: There is extensive literature documenting the nature of recovery in mental health in adult populations, but there is very little exploring its nature and meaning for young people.Aims: To gain a detailed understanding from the perspective of young people about the concept of recovery in young people's mental health.Method: Semi structured interviews were conducted with 23 young people. Interviews were transcribed verbatim and analysed using thematic analysis.Results: Themes emerging from the interviews included young people's dynamic conceptualisations of recovery, awareness of others views of recovery, polarised goals of recovery, and facilitators and barriers of recovery.Conclusions: To be relevant for young people, the mental health recovery model must incorporate individual needs, developmental considerations and fluctuations in goals. It must also be embedded within the young person's ecological system such as family, friends and school, and be focussed around an explicit and collaborative recovery discussion with the young person.

Cognitive-behavioural therapy for clozapine-resistant schizophrenia: the FOCUS RCT.

BACKGROUND: Clozapine (clozaril, Mylan Products Ltd) is a first-choice treatment for people with schizophrenia who have a poor response to standard antipsychotic medication. However, a significant number of patients who trial clozapine have an inadequate response and experience persistent symptoms, called clozapine-resistant schizophrenia (CRS). There is little evidence regarding the clinical effectiveness of pharmacological or psychological interventions for this population. OBJECTIVES: To evaluate the clinical effectiveness and cost-effectiveness of cognitive-behavioural therapy (CBT) for people with CRS and to identify factors predicting outcome. DESIGN: The Focusing on Clozapine Unresponsive Symptoms (FOCUS) trial was a parallel-group, randomised, outcome-blinded evaluation trial. Randomisation was undertaken using permuted blocks of random size via a web-based platform. Data were analysed on an intention-to-treat (ITT) basis, using random-effects regression adjusted for site, age, sex and baseline symptoms. Cost-effectiveness analyses were carried out to determine whether or not CBT was associated with a greater number of quality-adjusted life-years (QALYs) and higher costs than treatment as usual (TAU). SETTING: Secondary care mental health services in five cities in the UK. PARTICIPANTS: People with CRS aged ≥ 16 years, with an International Classification of Diseases, Tenth Revision (ICD-10) schizophrenia spectrum diagnoses and who are experiencing psychotic symptoms. INTERVENTIONS: Individual CBT included up to 30 hours of therapy delivered over 9 months. The comparator was TAU, which included care co-ordination from secondary care mental health services. MAIN OUTCOME MEASURES: The primary outcome was the Positive and Negative Syndrome Scale (PANSS) total score at 21 months and the primary secondary outcome was PANSS total score at the end of treatment (9 months post randomisation). The health benefit measure for the economic evaluation was the QALY, estimated from the EuroQol-5 Dimensions, five-level version (EQ-5D-5L), health status measure. Service use was measured to estimate costs. RESULTS: Participants were allocated to CBT (n = 242) or TAU (n = 245). There was no significant difference between groups on the prespecified primary outcome [PANSS total score at 21 months was 0.89 points lower in the CBT arm than in the TAU arm, 95% confidence interval (CI) -3.32 to 1.55 points; p = 0.475], although PANSS total score at the end of treatment (9 months) was significantly lower in the CBT arm (-2.40 points, 95% CI -4.79 to -0.02 points; p = 0.049). CBT was associated with a net cost of £5378 (95% CI -£13,010 to £23,766) and a net QALY gain of 0.052 (95% CI 0.003 to 0.103 QALYs) compared with TAU. The cost-effectiveness acceptability analysis indicated a low likelihood that CBT was cost-effective, in the primary and sensitivity analyses (probability < 50%). In the CBT arm, 107 participants reported at least one adverse event (AE), whereas 104 participants in the TAU arm reported at least one AE (odds ratio 1.09, 95% CI 0.81 to 1.46; p = 0.58). CONCLUSIONS: Cognitive-behavioural therapy for CRS was not superior to TAU on the primary outcome of total PANSS symptoms at 21 months, but was superior on total PANSS symptoms at 9 months (end of treatment). CBT was not found to be cost-effective in comparison with TAU. There was no suggestion that the addition of CBT to TAU caused adverse effects. Future work could investigate whether or not specific therapeutic techniques of CBT have value for some CRS individuals, how to identify those who may benefit and how to ensure that effects on symptoms can be sustained. TRIAL REGISTRATION: Current Controlled Trials ISRCTN99672552. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 23, No. 7. See the NIHR Journals Library website for further project information.

Assessment of metacognitive beliefs in an at risk mental state for psychosis: A validation study of the Metacognitions Questionnaire-30.

AIM: The Metacognitions Questionnaire-30 (MCQ-30) has been used to assess metacognitive beliefs in a range of mental health problems. The aim of this study is to assess the validity of the MCQ-30 in people at risk for psychosis. METHODS: One hundred eighty-five participants meeting criteria for an at risk mental state completed the MCQ-30 as part of their involvement in a randomized controlled trial. Confirmatory and exploratory factor analyses were conducted to assess factor structure and construct validity. RESULTS: Confirmatory factor analyses confirmed the original five-factor structure of the MCQ-30. Examination of principal component analysis and parallel analysis outputs also suggested a five-factor structure. Correlation analyses including measures of depression, social anxiety, and beliefs about paranoia showed evidence of convergent validity. Discriminant validity was supported using the normalizing subscale of the beliefs about paranoia tool. CONCLUSIONS: The MCQ-30 demonstrated good fit using the original five-factor model, acceptable to very good internal consistency of items was evident and clinical usefulness in those at risk for psychosis was demonstrated.

Attentional avoidance increases voice hearing in an analogue task in people with psychosis: An experimental study.

Cognitive models of psychosis suggest that unhelpful ways of responding to experiences can maintain such experiences and the associated distress. The response styles of attentional avoidance and attentional focusing were manipulated in an analogue voice-hearing task. Predictions were that both would increase detection of words in response to an ambiguous audio-recording but that attentional avoidance would lead to a greater increase than focusing. We also predicted that there would be a greater increase in anxiety and distress in the avoidance group. Predictions were tested in a sample of 44 participants with a diagnosis of schizophrenia. Participants were randomly assigned to either attentional avoidance or focusing while listening to an ambiguous auditory task. Number of words identified and anxiety and distress were recorded. As predicted, there was an increase in the number of words identified in both groups; however, this increase was greater in the avoidance group. The prediction that there would also be an increase in distress that would be greater in the avoidance group was not supported. It is possible that emotional reactions relate more closely to appraisals of the voice. The results suggest that avoidance of experiences is particularly counterproductive and can result in greater detection of experiences.

An exploration of the relationship between use of safety-seeking behaviours and psychosis: A systematic review and meta-analysis.

Safety-seeking behaviours are responses employed to protect against perceived threat. In relation to anxiety disorders, safety-seeking behaviours have been implicated in both the formation and maintenance of distress. Several studies have highlighted similar findings in relation to psychosis; however, this literature has not yet been synthesized. This review is, therefore, being conducted in order to synthesize the literature on safety seeking in people with psychosis to increase the understanding of this relationship. A systematic search identified and included 43 studies comprising 2,592 participants, published between 1995 and 2015. The results indicated that people experiencing psychosis commonly respond to their experiences with behavioural and cognitive strategies intended to manage their difficulties. In relation to safety seeking, avoidance, and resistance, there was a pattern that these responses are associated with increased distress and appraisals of threat. The results relating to engagement response styles showed the opposite pattern. These results provide support for cognitive models of safety seeking and psychosis with many of the meta-analyses reported here showing a clear pattern of association between behavioural responses and distress. However, the results reported within individual studies are mixed. This appears to be particularly true with the response style of distraction, with our analyses unable to clarify this relationship. It is possible that the mixed results could reflect the complexities in defining safety seeking and distinguishing it from coping in this population. The clinical implications of this are discussed.

'You've got your own demons that you've got to fight every day': A qualitative exploration of how people respond to the experience of psychosis.

OBJECTIVES: Cognitive models of psychosis implicate how people respond to their distressing experiences in the maintenance of such experiences. Safety-seeking behaviours, which are employed in response to a catastrophic misinterpretation of threat, are viewed as unhelpful maintenance factors. However, the concept of safety seeking was developed in relation to anxiety disorders, and there may be additional complexities that apply in relation to the experience of psychosis. The ways in which people respond to their distressing experiences of psychosis are complex, multifaceted, and changeable, and qualitative research is needed to further the understanding of this process. DESIGN: A qualitative study was conducted using grounded theory methodology. METHOD: In-depth interviews were conducted with fifteen participants who had experience of psychosis. RESULTS: A core category of fighting a daily battle to maintain functioning was identified. Related to this, three main themes also emerged. These were the perceived importance of responses, appraisal of threat, and perceived ability to control experiences. These categories are interrelated in that an increase in one is likely to result in increases in the others. CONCLUSION: Although these results provide partial support for the traditional view of safety-seeking behaviours, they also demonstrate further complexities in the way that distressing psychotic experiences are responded to. This has implications for cognitive behavioural therapy where emphasis is often placed on dropping safety-seeking behaviours. PRACTITIONER POINTS: The traditional emphasis in cognitive behavioural therapy on dropping safety-seeking behaviours may not always be appropriate. This approach could have an impact on engagement in therapy and have the effect of reducing the client's feelings of choice and control. A detailed assessment of strategies used historically and how these may have been helpful previously, for example, suspiciousness, and withdrawal as a survival strategy should be conducted. The therapist should support the client to evaluate the importance of their responses alongside the accuracy of both threat appraisals and perceptions of ability to control experiences.

The Beliefs about Paranoia Scale: Confirmatory factor analysis and tests of a metacognitive model of paranoia in a clinical sample.

This study aimed to confirm the factor structure of the Beliefs about Paranoia Scale (BaPS), a self-report measure to assess metacognitive beliefs about paranoia, and to test hypotheses of a metacognitive model. We hypothesised that positive and negative beliefs about paranoia would be associated with severity of suspiciousness, and that the co-occurrence of positive and negative beliefs would be associated with increased suspiciousness. A total of 335 patients meeting criteria for a schizophrenia spectrum disorder completed the BaPS, the Positive and Negative Syndromes Scale (PANSS), and the Psychotic Symptom Rating Scales (PSYRATS). Confirmatory factor analysis verified that the three BaPS subscales (negative beliefs about paranoia, paranoia as a survival strategy, and normalizing beliefs) were an adequate fit of the data. Ordinal regression showed that positive beliefs about paranoia as a survival strategy and negative beliefs were both associated with severity of suspiciousness. This was the first study to show that the co-occurrence of positive and negative beliefs was associated with increased suspiciousness. All hypotheses were confirmed, suggesting that a metacognitive approach has utility for the conceptualization of paranoia. Clinical implications suggest a role for metacognitive therapy, including strategies such as detached mindfulness and worry postponement.

Measuring common responses to psychosis: Assessing the psychometric properties of a new measure.

Responses to psychotic experiences are central to cognitive models of psychosis. The current study aimed to develop and validate a self-report measure of common responses to the experience of psychosis. This measure is needed as cognitive and behavioural responses are implicated in the maintenance of psychosis, but there is currently no measure that comprehensively assesses these maintaining factors. The Measure of Common Responses to psychosis (MCR) was developed and utilised in a sample of 487 participants who met criteria for treatment-resistant schizophrenia. Principal components analysis using data from 287 participants reduced the initial item pool of 31 items to 15 items with a three component structure. The components represented social control and reassurance seeking, threat monitoring and avoidance and conscious self-regulation attempts. Confirmatory factor analysis using data from the remaining 200 participants generally supported this three factor structure. The three subscales were found to have good internal consistency and convergent validity. The MCR, therefore, appears to be a useful tool to identify and monitor response styles, and could be utilised in further research to increase our understanding of the complex relationships between responses, symptoms and distress. It can also be used in clinical practice to elicit information that will be helpful in the psychological formulation and treatment of psychosis.

Quetiapine immediate release v. placebo for schizophrenia: systematic review, meta-analysis and reappraisal.

BACKGROUND: Immediate-release (IR) quetiapine has been used to treat schizophrenia since 1997, although all the principal placebo-controlled trials have >50% missing outcome data. New studies with relatively lower rates of participant withdrawal have since been published. AIMS: To assess the efficacy and adverse effects of quetiapine IR for schizophrenia, with consideration of outcome quality and clinical meaningfulness of results, and to examine the potential impact of missing data on the main efficacy findings. METHOD: We conducted a systematic review and meta-analysis of randomised controlled trials comparing quetiapine IR and placebo (or subtherapeutic dose in relapse prevention trials) for the treatment of schizophrenia (PROSPERO registration CRD4201100165). Primary outcomes were change in overall symptoms and response rates. We also examined whether high rates of participant withdrawal (≥50%) attenuated effect sizes, and assessed the impact of making different assumptions about these people's outcomes. RESULTS: We identified 15 relevant trials (including 2 unpublished), providing the first 12-week data for this drug and the first data on self-reported quality of life. We found quetiapine IR to have a weighted mean difference (WMD) of 6.5 points (95% CI -8.9 to -4) on Positive and Negative Syndrome Scale (PANSS) total scores, which corresponds to a standardised mean difference (SMD) of -0.33 (95% CI -0.46 to -0.21). Longer trials reported larger mean differences favouring quetiapine IR, but the overall estimate was smaller if more conservative assumptions about the outcomes of people who left the trial early were made. Approximately 21 people needed to take quetiapine IR for 1 person to experience at least a 50% improvement in PANSS score. No difference in quality of life was observed (two RCTs), although small to moderate improvements in social functioning were found (three RCTs). Quetiapine IR caused sedation and increased rates of clinically significant weight gain, but no extrapyramidal effects were observed. CONCLUSIONS: Quetiapine IR has a small beneficial effect on overall psychotic symptoms over 2-12 weeks, but also leads to weight gain and sedation.

Cognitive therapy for people with schizophrenia spectrum disorders not taking antipsychotic drugs: a single-blind randomised controlled trial.

BACKGROUND: Antipsychotic drugs are usually the first line of treatment for schizophrenia; however, many patients refuse or discontinue their pharmacological treatment. We aimed to establish whether cognitive therapy was effective in reducing psychiatric symptoms in people with schizophrenia spectrum disorders who had chosen not to take antipsychotic drugs. METHODS: We did a single-blind randomised controlled trial at two UK centres between Feb 15, 2010, and May 30, 2013. Participants aged 16-65 years with schizophrenia spectrum disorders, who had chosen not to take antipsychotic drugs for psychosis, were randomly assigned (1:1), by a computerised system with permuted block sizes of four or six, to receive cognitive therapy plus treatment as usual, or treatment as usual alone. Randomisation was stratified by study site. Outcome assessors were masked to group allocation. Our primary outcome was total score on the positive and negative syndrome scale (PANSS), which we assessed at baseline, and at months 3, 6, 9, 12, 15, and 18. Analysis was by intention to treat, with an ANCOVA model adjusted for site, age, sex, and baseline symptoms. This study is registered as an International Standard Randomised Controlled Trial, number 29607432. FINDINGS: 74 individuals were randomly assigned to receive either cognitive therapy plus treatment as usual (n=37), or treatment as usual alone (n=37). Mean PANSS total scores were consistently lower in the cognitive therapy group than in the treatment as usual group, with an estimated between-group effect size of -6.52 (95% CI -10.79 to -2.25; p=0.003). We recorded eight serious adverse events: two in patients in the cognitive therapy group (one attempted overdose and one patient presenting risk to others, both after therapy), and six in those in the treatment as usual group (two deaths, both of which were deemed unrelated to trial participation or mental health; three compulsory admissions to hospital for treatment under the mental health act; and one attempted overdose). INTERPRETATION: Cognitive therapy significantly reduced psychiatric symptoms and seems to be a safe and acceptable alternative for people with schizophrenia spectrum disorders who have chosen not to take antipsychotic drugs. Evidence-based treatments should be available to these individuals. A larger, definitive trial is needed. FUNDING: National Institute for Health Research.

Proteome-wide mapping of cholesterol-interacting proteins in mammalian cells.

Cholesterol is an essential structural component of cellular membranes and serves as a precursor for several classes of signaling molecules. Cholesterol exerts its effects and is, itself, regulated in large part by engagement in specific interactions with proteins. The full complement of sterol-binding proteins that exist in mammalian cells, however, remains unknown. Here we describe a chemoproteomic strategy that uses clickable, photoreactive sterol probes in combination with quantitative mass spectrometry to globally map cholesterol-protein interactions directly in living cells. We identified over 250 cholesterol-binding proteins, including receptors, channels and enzymes involved in many established and previously unreported interactions. Prominent among the newly identified interacting proteins were enzymes that regulate sugars, glycerolipids and cholesterol itself as well as proteins involved in vesicular transport and protein glycosylation and degradation, pointing to key nodes in biochemical pathways that may couple sterol concentrations to the control of other metabolites and protein localization and modification.

A sulfated carbohydrate epitope inhibits axon regeneration after injury.

Chondroitin sulfate proteoglycans (CSPGs) represent a major barrier to regenerating axons in the central nervous system (CNS), but the structural diversity of their polysaccharides has hampered efforts to dissect the structure-activity relationships underlying their physiological activity. By taking advantage of our ability to chemically synthesize specific oligosaccharides, we demonstrate that a sugar epitope on CSPGs, chondroitin sulfate-E (CS-E), potently inhibits axon growth. Removal of the CS-E motif significantly attenuates the inhibitory activity of CSPGs on axon growth. Furthermore, CS-E functions as a protein recognition element to engage receptors including the transmembrane protein tyrosine phosphatase PTPσ, thereby triggering downstream pathways that inhibit axon growth. Finally, masking the CS-E motif using a CS-E-specific antibody reversed the inhibitory activity of CSPGs and stimulated axon regeneration in vivo. These results demonstrate that a specific sugar epitope within chondroitin sulfate polysaccharides can direct important physiological processes and provide new therapeutic strategies to regenerate axons after CNS injury.

Elucidation and chemical modulation of sulfolipid-1 biosynthesis in Mycobacterium tuberculosis.

Mycobacterium tuberculosis possesses unique cell-surface lipids that have been implicated in virulence. One of the most abundant is sulfolipid-1 (SL-1), a tetraacyl-sulfotrehalose glycolipid. Although the early steps in SL-1 biosynthesis are known, the machinery underlying the final acylation reactions is not understood. We provide genetic and biochemical evidence for the activities of two proteins, Chp1 and Sap (corresponding to gene loci rv3822 and rv3821), that complete this pathway. The membrane-associated acyltransferase Chp1 accepts a synthetic diacyl sulfolipid and transfers an acyl group regioselectively from one donor substrate molecule to a second acceptor molecule in two successive reactions to yield a tetraacylated product. Chp1 is fully active in vitro, but in M. tuberculosis, its function is potentiated by the previously identified sulfolipid transporter MmpL8. We also show that the integral membrane protein Sap and MmpL8 are both essential for sulfolipid transport. Finally, the lipase inhibitor tetrahydrolipstatin disrupts Chp1 activity in M. tuberculosis, suggesting an avenue for perturbing SL-1 biosynthesis in vivo. These data complete the SL-1 biosynthetic pathway and corroborate a model in which lipid biosynthesis and transmembrane transport are coupled at the membrane-cytosol interface through the activity of multiple proteins, possibly as a macromolecular complex.

Global profiling of dynamic protein palmitoylation.

The reversible thioester linkage of palmitic acid on cysteines, known as protein S-palmitoylation, facilitates the membrane association and proper subcellular localization of proteins. Here we report the metabolic incorporation of the palmitic acid analog 17-octadecynoic acid (17-ODYA) in combination with stable-isotope labeling with amino acids in cell culture (SILAC) and pulse-chase methods to generate a global quantitative map of dynamic protein palmitoylation events in cells. We distinguished stably palmitoylated proteins from those that turn over rapidly. Treatment with a serine lipase-selective inhibitor identified a pool of dynamically palmitoylated proteins regulated by palmitoyl-protein thioesterases. This subset was enriched in oncoproteins and other proteins linked to aberrant cell growth, migration and cancer. Our method provides a straightforward way to characterize global palmitoylation dynamics in cells and confirms enzyme-mediated depalmitoylation as a critical regulatory mechanism for a specific subset of rapidly cycling palmitoylated proteins.

Elucidating glycosaminoglycan-protein-protein interactions using carbohydrate microarray and computational approaches.

Glycosaminoglycan polysaccharides play critical roles in many cellular processes, ranging from viral invasion and angiogenesis to spinal cord injury. Their diverse biological activities are derived from an ability to regulate a remarkable number of proteins. However, few methods exist for the rapid identification of glycosaminoglycan-protein interactions and for studying the potential of glycosaminoglycans to assemble multimeric protein complexes. Here, we report a multidisciplinary approach that combines new carbohydrate microarray and computational modeling methodologies to elucidate glycosaminoglycan-protein interactions. The approach was validated through the study of known protein partners for heparan and chondroitin sulfate, including fibroblast growth factor 2 (FGF2) and its receptor FGFR1, the malarial protein VAR2CSA, and tumor necrosis factor-α (TNF-α). We also applied the approach to identify previously undescribed interactions between a specific sulfated epitope on chondroitin sulfate, CS-E, and the neurotrophins, a critical family of growth factors involved in the development, maintenance, and survival of the vertebrate nervous system. Our studies show for the first time that CS is capable of assembling multimeric signaling complexes and modulating neurotrophin signaling pathways. In addition, we identify a contiguous CS-E-binding site by computational modeling that suggests a potential mechanism to explain how CS may promote neurotrophin-tyrosine receptor kinase (Trk) complex formation and neurotrophin signaling. Together, our combined microarray and computational modeling methodologies provide a general, facile means to identify new glycosaminoglycan-protein-protein interactions, as well as a molecular-level understanding of those complexes.

Activation of Snake in a serine protease cascade that defines the dorsoventral axis is atypical and pipe-independent in Drosophila embryos.

During Drosophila embryogenesis, establishment of ventral and lateral cell fates requires spatial regulation of an extracellular serine protease cascade composed of Nudel, Gastrulation Defective (GD), Snake, and Easter. Pipe, a sulfotransferase expressed ventrally during oogenesis, sulfates secreted targets that somehow confer positive spatial input to this cascade. Nudel and GD activation are pipe-independent, while Easter activation requires pipe. The effect of pipe on Snake activation has been unknown. Here we show that Snake activation is cascade-dependent but pipe-independent. These findings support a conclusion that Snake's activation of Easter is the first spatially regulated step in the dorsoventral protease cascade.

Activity-based probes that target functional subclasses of phospholipases in proteomes.

Phospholipases are a large and diverse set of enzymes that metabolize the phospholipid components of cell membranes and function in key lipid-signaling pathways. The molecular characterization of novel phospholipases would benefit from chemical probes that selectively target these enzymes on the basis of their distinct substrate specificities and catalytic properties. Here we present the synthesis and characterization of a set of activity-based protein profiling (ABPP) probes that contain key recognition and reactivity elements for targeting phospholipases of the serine hydrolase superfamily. We show that these probes accurately report on the sn-1 and sn-2 substrate specificities of phospholipases in cell and tissue proteomes, including the sn-1-selective phospholipase DDHD1 and a calcium-dependent transacylase activity implicated in endocannabinoid biosynthesis. We anticipate that these phospholipase-directed ABPP probes will facilitate the discovery of new lipid-metabolizing enzymes and provide valuable insights into their substrate preferences.

A chemical approach to immunoprotein engineering: chemoselective functionalization of thioester proteins in their native state.

Less than 6 feet under: Serum proteins C3, C4, and alpha(2)M each contain a thioester domain buried within a hydrophobic pocket, which is thought to shield the labile thioester from hydrolysis. Herein, we make use of the inherent reactivity of the hydrazide for thioester moieties to chemoselectively label these crucial serum regulators in their native conformation; this demonstrates that access to the thioester site is much greater than previously supposed.