Determination of hATG8 Binding Selectivity of AIM (Autophagy-Interacting Motif) Peptides Using Native Electrospray Ionization Mass Spectrometry.

Establishing the hATG8 binding selectivity of AIM (autophagy-interacting motif) sequences found within autophagy system proteins provides insights into their biological roles, and in the case of disease-associated AIM mutations, potential pathophysiological mechanisms. Given the sometimes small differences in affinity for an individual AIM amongst the six hATG8 proteins, establishing AIM preferences can be experimentally challenging. We describe a native mass spectrometry method that is suitable for detecting such differences, using synthetic AIM peptides and recombinant hATG8 proteins, to probe hATG8-AIM interactions in the gas phase. Binding preferences of a single AIM peptide against multiple hATG8s, or two AIM peptides against a single hATG8 (e.g., wild-type versus mutant AIM), may be determined.

A library-derived peptide inhibitor of the BZLF1 transcription factor.

Transcription factor dysregulation is associated with many diseases, including cancer. Peptide-based molecules are increasingly recognised as important modulators of difficult intracellular protein-protein interaction targets, with peptide library screening consequently proven to be a viable strategy in developing inhibitors against a wide range of transcription factors (TFs). However, current strategies simply select the highest affinity of binding to a target TF rather than the ability to inhibit TF function. Here, we utilise our Transcription Block Survival (TBS) screening platform to enable high-throughput identification of peptides that inhibit TFs from binding to cognate DNA sites, hence inhibiting functionality. In this study, we explore whether the TBS can be expanded to derive a potent and functional peptide inhibitor of the BZLF1 transcription factor. The library-derived peptide, AcidicW, is shown to form a more stable dimer with BZLF1 than the BZLF1 homodimer, with a thermal denaturation temperature exceeding 80°C. AcidicW can also functionally inhibit the BZLF1:TRE DNA interaction with high potency and an IC50 of 612 nM.

The effect of helix-inducing constraints and downsizing upon a transcription block survival-derived functional cJun antagonist.

Inhibition of cJun is established as a promising therapeutic approach, particularly in cancer. We recently developed the "transcription block survival" (TBS) screening platform to derive functional peptide antagonists of transcription factor activity by ablating their ability to bind to cognate DNA. Using TBS, we screened a >131,000-member peptide library to select a 63-mer peptide that bound cJun and prevented 12-O-tetradecanoylphorbol-13-acetate response element (TRE) DNA binding. Iterative truncation was next combined with a systematic exploration of side-chain cyclization to derive a minimal active sequence. The resulting dual lactamized sequence was >40% smaller and retained low nM target affinity (equilibrium binding constant [K D ] = 0.2 versus 9.7 nM), with 8 residues at the acidic region required for functional antagonism. However, even modest C-terminal truncation resulted in functional loss. The peptide functionally antagonizes cJun (half-maximal inhibitory concentration [IC50] = 13 versus 45 µM) and is considerably more stable in human serum relative to its non-lactamized counterpart and HingeW.

In vitro single molecule and bulk phase studies reveal the AP-1 transcription factor cFos binds to DNA without its partner cJun.

The AP-1 transcription factor family crucially regulates progression of the cell cycle, as well as playing roles in proliferation, differentiation, and the stress response. The two best described AP-1 family members, cFos and cJun, are known to dimerize to form a functional AP-1 heterodimer that binds to a consensus response element sequence. Although cJun can also homodimerize and bind to DNA, the canonical view is that cFos cannot bind DNA without heterodimerizing with cJun. Here, we show that cFos can actually bind to DNA in the absence of cJun in vitro. Using dual color single molecule imaging of cFos alone, we directly visualize binding to and movement on DNA. Of all these DNA-bound proteins, detailed analysis suggested 30 to 46% were homodimers. Furthermore, we constructed fluorescent protein fusions of cFos and cJun for Förster resonance energy transfer experiments. These constructs indicated complete dimerization of cJun, but although cFos could dimerize, its extent was reduced. Finally, to provide orthogonal confirmation of cFos binding to DNA, we performed bulk-phase circular dichroism experiments that showed clear structural changes in DNA; these were found to be specific to the AP-1 consensus sequence. Taken together, our results clearly show cFos can interact with DNA both as monomers and dimers independently of its archetypal partner, cJun.

An Approach to Derive Functional Peptide Inhibitors of Transcription Factor Activity.

We report the development of a high-throughput, intracellular "transcription block survival" (TBS) screening platform to derive functional transcription factor antagonists. TBS is demonstrated using the oncogenic transcriptional regulator cJun, with the development of antagonists that bind cJun and prevent both dimerization and, more importantly, DNA binding remaining a primary challenge. In TBS, cognate TRE sites are introduced into the coding region of the essential gene, dihydrofolate reductase (DHFR). Introduction of cJun leads to TRE binding, preventing DHFR expression by directly blocking RNA polymerase gene transcription to abrogate cell proliferation. Peptide library screening identified a sequence that both binds cJun and antagonizes function by preventing DNA binding, as demonstrated by restored cell viability and subsequent in vitro hit validation. TBS is an entirely tag-free genotype-to-phenotype approach, selecting desirable attributes such as high solubility, target specificity, and low toxicity within a complex cellular environment. TBS facilitates rapid library screening to accelerate the identification of therapeutically valuable sequences.

An ALS-associated variant of the autophagy receptor SQSTM1/p62 reprograms binding selectivity toward the autophagy-related hATG8 proteins.

Recognition of human autophagy-related 8 (hATG8) proteins by autophagy receptors represents a critical step within this cellular quality control system. Autophagy impairment is known to be a pathogenic mechanism in the motor neuron disorder amyotrophic lateral sclerosis (ALS). Overlapping but specific roles of hATG8 proteins belonging to the LC3 and GABARAP subfamilies are incompletely understood, and binding selectivity is typically overlooked. We previously showed that an ALS-associated variant of the SQSTM1/p62 (p62) autophagy receptor bearing an L341V mutation within its ATG8-interacting motif (AIM) impairs recognition of LC3B in vitro, yielding an autophagy-deficient phenotype. Improvements in understanding of hATG8 recognition by AIMs now distinguish LC3-interaction and GABARAP-interaction motifs and predict the effects of L341V substitution may extend beyond loss of function to biasing AIM binding preference. Through biophysical analyses, we confirm impaired binding of the L341V-AIM mutant to LC3A, LC3B, GABARAP, and GABARAPL1. In contrast, p62 AIM interactions with LC3C and GABARAPL2 are unaffected by this mutation. Isothermal titration calorimetry and NMR investigations provided insights into the entropy-driven GABARAPL2/p62 interaction and how the L341V mutation may be tolerated. Competition binding demonstrated reduced association of the L341V-AIM with one hATG8 manifests as a relative increase in association with alternate hATG8s, indicating effective reprogramming of hATG8 selectivity. These data highlight how a single AIM peptide might compete for binding with different hATG8s and suggest that the L341V-AIM mutation may be neomorphic, representative of a disease mechanism that likely extends into other human disorders.

Selective antagonism of cJun for cancer therapy.

The activator protein-1 (AP-1) family of transcription factors modulate a diverse range of cellular signalling pathways into outputs which can be oncogenic or anti-oncogenic. The transcription of relevant genes is controlled by the cellular context, and in particular by the dimeric composition of AP-1. Here, we describe the evidence linking cJun in particular to a range of cancers. This includes correlative studies of protein levels in patient tumour samples and mechanistic understanding of the role of cJun in cancer cell models. This develops an understanding of cJun as a focal point of cancer-altered signalling which has the potential for therapeutic antagonism. Significant work has produced a range of small molecules and peptides which have been summarised here and categorised according to the binding surface they target within the cJun-DNA complex. We highlight the importance of selectively targeting a single AP-1 family member to antagonise known oncogenic function and avoid antagonism of anti-oncogenic function.

Anti-NMDAR Encephalitis: A Multidisciplinary Approach to Identification of the Disorder and Management of Psychiatric Symptoms.

BACKGROUND: The novelty of anti-NMDA receptor encephalitis, for which somatic treatments have only recently been developed, has led to a lack of information on assessment and treatment of its variable behavioral manifestations. METHOD: In this article, we discuss 4 challenging cases of anti-NMDAR encephalitis, focusing on the importance of a multidisciplinary approach to identification and management of the disorder and the necessity of close collaboration in the acute hospital setting for management of the behavioral symptoms. CONCLUSION: The cases we discuss highlight some of the medication and nonpharmacologic treatment strategies that may facilitate management of psychiatric symptoms, both while the medical workup is ongoing and after the diagnosis has been confirmed.

G-quadruplex ligands mediate downregulation of DUX4 expression.

Abnormal DUX4 expression in skeletal muscles plays a key role in facioscapulohumeral muscular dystrophy (FSHD) pathogenesis, although the molecular mechanisms regulating DUX4 expression are not fully defined. Using bioinformatic analysis of the genomic DUX4 locus, we have identified a number of putative G-quadruplexes (GQs) forming sequences. Their presence was confirmed in synthetic oligonucleotiode sequences derived from the enhancer, promoter and transcript of DUX4 through circular dichroism and nuclear magnetic resonance analysis. We further examined the binding affinity of a naturally occurring GQ stabilizing compound, berberine, to these non-canonical genetic structures using UV-Vis and fluorescence spectroscopy. Subsequent in vitro study in FSHD patient myoblasts indicated that berberine treatment reduced DUX4 expression and also expression of genes normally switched on by DUX4. Further investigation in a mouse model overexpressing exogenous DUX4 confirmed the therapeutic effects of berberine in downregulating DUX4 protein expression, inhibiting muscle fibrosis, and consequently rescuing muscle function. Our data demonstrate for the first time that GQs are present in the DUX4 locus and that the GQ interactive ligand reduces DUX4 expression suggesting potential role of GQs in FSHD pathogenesis. Our work provides the basis of a novel therapeutic strategy for the treatment of FSHD.

A Method to Account for Variation in Congenital Heart Surgery Length of Stay.

OBJECTIVES: We sought to develop a risk-adjustment methodology for length of stay in congenital heart surgery, as none exist. DESIGN: Prospective cohort analysis combined with previously obtained retrospective cohort analysis of a Department of Cardiovascular Surgery clinical database. PATIENTS: Patients discharged from Boston Children's Hospital between October 1, 2006, and May 31, 2014, that underwent a congenital heart surgery procedure(s) linked to one of 103 surgical procedure types. MEASUREMENTS AND MAIN RESULTS: Six thousand two hundred nine discharges during the reporting period at Boston Children's Hospital comprised the cohort. Seven Surgical Length Categories were developed to group surgical procedure types. A multivariable model for outcome length of stay was built using a derivation cohort consisting of a 75% random sample, starting with Surgical Length Categories and considering additional a priori factors. Postoperative factors were then added to improve predictive performance. The remaining 25% of the cohort was used to validate the multivariable models. The coefficient of determination (R) was used to estimate the variability in length of stay explained by each factor. The Surgical Length Categories yielded an R of 42%. Model performance increased when the a priori factors preoperative status, noncardiac abnormality, genetic anomaly, preoperative catheterization during episode of care, weight less than 3 kg, and preoperative vasoactive support medication were introduced to the model (R = 60.8%). Model performance further improved when postoperative ventilation greater than 7 days, operating room time, postoperative catheterization during episode of care, postoperative reintubation, number of postoperative vasoactive support medications, postoperative ICU infection, and greater than or equal to one secondary surgical procedure were added (R = 76.7%). The validation cohort yielded an R of 76.5%. CONCLUSIONS: We developed a statistically valid procedure-based categorical variable and multivariable model for length of stay of congenital heart surgeries. The Surgical Length Categories and important a priori and postoperative factors may be used to pursue a predictive tool for length of stay to inform scheduling and bed management practices.

The Decay of Motor Memories Is Independent of Context Change Detection.

When the error signals that guide human motor learning are withheld following training, recently-learned motor memories systematically regress toward untrained performance. It has previously been hypothesized that this regression results from an intrinsic volatility in these memories, resulting in an inevitable decay in the absence of ongoing error signals. However, a recently-proposed alternative posits that even recently-acquired motor memories are intrinsically stable, decaying only if a change in context is detected. This new theory, the context-dependent decay hypothesis, makes two key predictions: (1) after error signals are withheld, decay onset should be systematically delayed until the context change is detected; and (2) manipulations that impair detection by masking context changes should result in prolonged delays in decay onset and reduced decay amplitude at any given time. Here we examine the decay of motor adaptation following the learning of novel environmental dynamics in order to carefully evaluate this hypothesis. To account for potential issues in previous work that supported the context-dependent decay hypothesis, we measured decay using a balanced and baseline-referenced experimental design that allowed for direct comparisons between analogous masked and unmasked context changes. Using both an unbiased variant of the previous decay onset analysis and a novel highly-powered group-level version of this analysis, we found no evidence for systematically delayed decay onset nor for the masked context change affecting decay amplitude or its onset time. We further show how previous estimates of decay onset latency can be substantially biased in the presence of noise, and even more so with correlated noise, explaining the discrepancy between the previous results and our findings. Our results suggest that the decay of motor memories is an intrinsic feature of error-based learning that does not depend on context change detection.

The course and impact of family optimism in the post-acute period after acquired brain injury.

OBJECTIVE: To investigate the course and impact of family optimism in the post-acute stage of acquired brain injury. METHODS: At Time 1, 30 family relatives of in-patients in rehabilitation units and 30 relatives of patients recently discharged from such units completed questionnaires relating to their emotional health, engagement in the rehabilitation process and expectations about the future consequences and controllability of the injury. At Time 2 (12-18 months later), 23 of the original sample completed questionnaires about their emotional health and actual consequences and controllability of the injury. RESULTS: At Time 1, optimism about future consequences and controllability was associated with greater engagement in the rehabilitation process and better emotional health. The two groups did not differ on any of the measures, which did not support the expectation that the patient's discharge home would trigger a loss of optimism and emotional upset for the family. At Time 2, the actual consequences were worse than had been expected at Time 1 and greater disappointment was associated with a greater decline in emotional wellbeing. CONCLUSION: Family expectations about recovery are linked with important variables such as emotional wellbeing and engagement in the rehabilitation process and need careful management by clinicians.

A method to account for variation in congenital heart surgery charges.

BACKGROUND: In response to societal pressure to reduce expenditures and increase quality, we sought to develop a methodology to predict hospital charges related to congenital heart surgery. METHODS: Patients undergoing congenital heart surgery at Boston Children's Hospital in fiscal years 2007 to 2009 comprised the derivation cohort. Clinical data, including Current Procedural Terminology coding of the primary surgical intervention, were collected prospectively and linked to total hospital charges for an episode of care. Surgical charge categories were developed to group surgical procedure types using empiric data and expert consensus. A multivariable model was built using surgical charge categories and additional patient and procedural characteristics to predict the outcome, total hospital charges. A contemporary cohort for fiscal years 2010 to 2012 was used to validate surgical charge categories and the multivariable model. RESULTS: In the derivation cohort, 2,105 cases met inclusion criteria. One hundred three surgical procedure types were categorized into seven surgical charge categories, yielding a grouper variable with an R(2) explanatory value of 47.3%. Explanatory value increased with consideration of patient age, admission status, and preoperative ventilator dependence (R(2) = 59.4%), as well as weight category, noncardiac abnormality, and genetic syndrome other than trisomy 21 (R(2) = 61.5%). Additional variability in charge was explained when extracorporeal membrane oxygenation utilization and greater than one operating room visit during the episode of care were added (R(2) = 74.3%). The contemporary cohort yielded an R(2) explanatory value of 67.7%. CONCLUSIONS: The combination of clinical data with resource utilization information resulted in a statistically valid predictive model for total hospital charges in congenital heart surgery.

Development of a charge adjustment model for cardiac catheterization.

A methodology that would allow for comparison of charges across institutions has not been developed for catheterization in congenital heart disease. A single institution catheterization database with prospectively collected case characteristics was linked to hospital charges related and limited to an episode of care in the catheterization laboratory for fiscal years 2008-2010. Catheterization charge categories (CCC) were developed to group types of catheterization procedures using a combination of empiric data and expert consensus. A multivariable model with outcome charges was created using CCC and additional patient and procedural characteristics. In 3 fiscal years, 3,839 cases were available for analysis. Forty catheterization procedure types were categorized into 7 CCC yielding a grouper variable with an R (2) explanatory value of 72.6%. In the final CCC, the largest proportion of cases was in CCC 2 (34%), which included diagnostic cases without intervention. Biopsy cases were isolated in CCC 1 (12%), and percutaneous pulmonary valve placement alone made up CCC 7 (2%). The final model included CCC, number of interventions, and cardiac diagnosis (R (2) = 74.2%). Additionally, current financial metrics such as APR-DRG severity of illness and case mix index demonstrated a lack of correlation with CCC. We have developed a catheterization procedure type financial grouper that accounts for the diverse case population encountered in catheterization for congenital heart disease. CCC and our multivariable model could be used to understand financial characteristics of a population at a single point in time, longitudinally, and to compare populations.

The cost of providing methadone maintenance treatment in Ontario, Canada.

OBJECTIVES: To estimate the cost of providing methadone maintenance treatment in Ontario, Canada, from the perspective of the public payer. METHODS: We analyzed a database of all patient clinic visits, laboratory tests for urine toxicology screening, and methadone scripts from a group of methadone clinics in Ontario. The database consisted of patient visits and visit information from 1 January 2003 to 31 December 2009. We estimated the cost of providing methadone maintenance treatment as the sum of physician costs, laboratory costs for urine samples (toxicology screens), methadone costs, and pharmacy costs. Pharmacy costs include dispensing fees and markups. All costs are expressed in 2010 Canadian dollars. RESULTS: The database consisted of 9479 unique patients. The average age on the date of the first recorded visit was 34.3, and among the patients 62.3% were male. There were 6,425,937 patient days of treatment and the total cost of all treatment-related services was approximately $99,491,000. The total cost was comprised of physician billing (9.8%), pharmacy costs (39.8%), methadone (3.8%), and performing urine toxicology screens (46.7%). The average cost per day for treatment was $15.48, corresponding to $5651per year if patients were to remain in treatment continuously. CONCLUSIONS: The cost of providing methadone maintenance treatment in Ontario is comparable to estimates from the United States and Australia. SCIENTIFIC SIGNIFICANCE: This information is important to policy makers for planning and budgeting purposes and as part of a full cost-benefit or cost-effectiveness analysis of methadone treatment.

Service users leading the way: focus group methodology in developing accessible information DVDs with people with learning disabilities.

The English government sees it important to view service users as active partners in the delivery of accessible resources. The current article follows a brief report which described an innovative project on developing an accessible DVD explaining the Birmingham Clinical Psychology Service to people with learning disabilities. The article describes three focus groups involving adults with learning disabilities that met to reflect and evaluate the accessibility of the DVD. This process formed the evaluative phase of the DVD development project where people with learning disabilities evaluated the accessibility, level of understanding, and clarity of the DVD content. The DVD was rated positively by the focus groups, and minor changes were made to the final version of the DVD. The article also reflects upon the use of focus groups as a methodological approach in researching the views of people with learning disabilities.

Threat appraisal and avoidance after traumatic brain injury: why and how often are activities avoided?

OBJECTIVE: Goldstein emphasized the anxiety-related avoidance of activities after brain injury, but such avoidance has rarely been systematically investigated. This study aimed to compile a list of specific threat appraisals that may lead to avoidance and to obtain data on the frequency with which these appraisals and consequent avoidance occur. DESIGN: Survey. METHOD: Qualitative methodology was used to obtain an account of threat appraisals. These data were then used to compile two questionnaires that asked about the experience of these appraisals and consequent avoidance. Fifty individuals with a TBI completed the questionnaires. RESULTS: A varied list of threat appraisals was obtained. Appraisals and consequent avoidance were frequent in the sample. Males and those whose TBI resulted from an assault reported more avoidance. CONCLUSIONS: A core aim of rehabilitation is to facilitate participation in valued roles and activities. Threat appraisals and avoidance deserve more attention in research and practice because they may constitute a significant obstacle to achieving this.