Fluphenazine reduces proteotoxicity in C. elegans and mammalian models of alpha-1-antitrypsin deficiency

The classical form of α1-antitrypsin deficiency (ATD) is associated with hepatic fibrosis and hepatocellular carcinoma. It is caused by the proteotoxic effect of a mutant secretory protein that aberrantly accumulates in the endoplasmic reticulum of liver cells. Recently we developed a model of this deficiency in C. Elegans and adapted it for high-content drug screening using an automated, image-based array scanning. Screening of the Library of Pharmacologically Active Compounds identified fluphenazine (Flu) among several other compounds as a drug which reduced intracellular accumulation of mutant α1-antitrypsin Z (ATZ). Because it is representative of the phenothiazine drug class that appears to have autophagy enhancer properties in addition to mood stabilizing activity, and can be relatively easily re-purposed, we further investigated its effects on mutant ATZ. The results indicate that Flu reverses the phenotypic effects of ATZ accumulation in the C. elegans model of ATD at doses which increase the number of autophagosomes in vivo . Furthermore, in nanomolar concentrations, Flu enhances the rate of intracellular degradation of ATZ and reduces the cellular ATZ load in mammalian cell line models. In the PiZ mouse model Flu reduces the accumulation of ATZ in the liver and mediates a decrease in hepatic fibrosis. These results show that Flu can reduce the proteotoxicity of ATZ accumulation in vivo and, because it has been used safely in humans, this drug can be moved rapidly into trials for liver disease due to ATD. The results also provide further validation for drug discovery using C. elegans models that can be adapted to high-content drug screening platforms and used together with mammalian cell line and animal models. © 2014 Li et al

Deficient and null variants of SERPINA1 are proteotoxic in a Caenorhabditis elegans model of α1-antitrypsin deficiency

α1-antitrypsin deficiency (ATD) predisposes patients to both loss-of-function (emphysema) and gain-of-function (liver cirrhosis) phenotypes depending on the type of mutation. Although the Z mutation (ATZ) is the most prevalent cause of ATD, >120 mutant alleles have been identified. In general, these mutations are classified as deficient (<20% normal plasma levels) or null (<1% normal levels) alleles. The deficient alleles, like ATZ, misfold in the ER where they accumulate as toxic monomers, oligomers and aggregates. Thus, deficient alleles may predispose to both gain- and loss-of-function phenotypes. Null variants, if translated, typically yield truncated proteins that are efficiently degraded after being transiently retained in the ER. Clinically, null alleles are only associated with the loss-of-function phenotype. We recently developed a C. elegans model of ATD in order to further elucidate the mechanisms of proteotoxicity (gain-of-function phenotype) induced by the aggregationprone deficient allele, ATZ. The goal of this study was to use this C. elegans model to determine whether different types of deficient and null alleles, which differentially affect polymerization and secretion rates, correlated to any extent with proteotoxicity. Animals expressing the deficient alleles, Mmalton, Siiyama and S (ATS), showed overall toxicity comparable to that observed in patients. Interestingly, Siiyama expressing animals had smaller intracellular inclusions than ATZ yet appeared to have a greater negative effect on animal fitness. Surprisingly, the null mutants, although efficiently degraded, showed a relatively mild gainoffunction proteotoxic phenotype. However, since null variant proteins are degraded differently and do not appear to accumulate, their mechanism of proteotoxicity is likely to be different to that of polymerizing, deficient mutants. Taken together, these studies showed that C. elegans is an inexpensive tool to assess the proteotoxicity of different AT variants using a transgenic approach

Behavior therapy for pediatric trichotillomania: Exploring the effects of age on treatment outcome

<p>Abstract</p> <p>Background</p> <p>A randomized controlled trial examining the efficacy of behavior therapy for pediatric trichotillomania was recently completed with 24 participants ranging in age from 7 - 17. The broad age range raised a question about whether young children, older children, and adolescents would respond similarly to intervention. In particular, it is unclear whether the younger children have the cognitive capacity to understand concepts like "urges" and whether they are able to introspect enough to be able to benefit from awareness training, which is a key aspect of behavior therapy for trichotillomania.</p> <p>Methods</p> <p>Participants were randomly assigned to receive either behavior therapy (N = 12) or minimal attention control (N = 12), which was included to control for repeated assessments and the passage of time. Primary outcome measures were the independent evaluator-rated NIMH-Trichotillomania Severity Scale, a semi-structured interview often used in trichotillomania treatment trials, and a post-treatment clinical global impression improvement rating (CGI-I).</p> <p>Results</p> <p>The correlation between age and change in symptom severity for all patients treated in the trial was small and not statistically significant. A 2 (group: behavioral therapy, minimal attention control) × 2 (time: week 0, 8) × 2 (children < 9 yrs., children > 10) ANOVA with independent evaluator-rated symptom severity scores as the continuous dependent variable also detected no main effects for age or for any interactions involving age. In light of the small sample size, the mean symptom severity scores at weeks 0 and 8 for younger and older patients randomized to behavioral therapy were also plotted. Visual inspection of these data indicated that although the groups appeared to have started at similar levels of severity for children ≤ 9 vs. children ≥ 10; the week 8 data show that the three younger children did at least as well as if not slightly better than the nine older children and adolescents.</p> <p>Conclusions</p> <p>Behavior therapy for pediatric trichotillomania appears to be efficacious even in young children. The developmental and clinical implications of these findings will be discussed.</p> <p>Trial Registration</p> <p>Clinicaltrials.gov NCT00043563.</p

Update of the human and mouse SERPIN gene superfamily

The serpin family comprises a structurally similar, yet functionally diverse, set of proteins. Named originally for their function as serine proteinase inhibitors, many of its members are not inhibitors but rather chaperones, involved in storage, transport, and other roles. Serpins are found in genomes of all kingdoms, with 36 human protein-coding genes and five pseudogenes. The mouse has 60 Serpin functional genes, many of which are orthologous to human SERPIN genes and some of which have expanded into multiple paralogous genes. Serpins are found in tissues throughout the body; whereas most are extracellular, there is a class of intracellular serpins. Serpins appear to have roles in inflammation, immune function, tumorigenesis, blood clotting, dementia, and cancer metastasis. Further characterization of these proteins will likely reveal potential biomarkers and therapeutic targets for disease. © 2013 Heit et al

Prediction of photoperiodic regulators from quantitative gene circuit models

Photoperiod sensors allow physiological adaptation to the changing seasons. The external coincidence hypothesis postulates that a light-responsive regulator is modulated by a circadian rhythm. Sufficient data are available to test this quantitatively in plants, though not yet in animals. In Arabidopsis, the clock-regulated genes CONSTANS (CO) and FLAVIN, KELCH, F-BOX (FKF1) and their lightsensitive proteins are thought to form an external coincidence sensor. We use 40 timeseries of molecular data to model the integration of light and timing information by CO, its target gene FLOWERING LOCUS T (FT), and the circadian clock. Among other predictions, the models show that FKF1 activates FT. We demonstrate experimentally that this effect is independent of the known activation of CO by FKF1, thus we locate a major, novel controller of photoperiodism. External coincidence is part of a complex photoperiod sensor: modelling makes this complexity explicit and may thus contribute to crop improvement

In silico assessment of potential druggable pockets on the surface of α1-Antitrypsin conformers

The search for druggable pockets on the surface of a protein is often performed on a single conformer, treated as a rigid body. Transient druggable pockets may be missed in this approach. Here, we describe a methodology for systematic in silico analysis of surface clefts across multiple conformers of the metastable protein α1-antitrypsin (A1AT). Pathological mutations disturb the conformational landscape of A1AT, triggering polymerisation that leads to emphysema and hepatic cirrhosis. Computational screens for small molecule inhibitors of polymerisation have generally focused on one major druggable site visible in all crystal structures of native A1AT. In an alternative approach, we scan all surface clefts observed in crystal structures of A1AT and in 100 computationally produced conformers, mimicking the native solution ensemble. We assess the persistence, variability and druggability of these pockets. Finally, we employ molecular docking using publicly available libraries of small molecules to explore scaffold preferences for each site. Our approach identifies a number of novel target sites for drug design. In particular one transient site shows favourable characteristics for druggability due to high enclosure and hydrophobicity. Hits against this and other druggable sites achieve docking scores corresponding to a Kd in the µM–nM range, comparing favourably with a recently identified promising lead. Preliminary ThermoFluor studies support the docking predictions. In conclusion, our strategy shows considerable promise compared with the conventional single pocket/single conformer approach to in silico screening. Our best-scoring ligands warrant further experimental investigation

From the Eye of the Albatrosses: A Bird-Borne Camera Shows an Association between Albatrosses and a Killer Whale in the Southern Ocean

Albatrosses fly many hundreds of kilometers across the open ocean to find and feed upon their prey. Despite the growing number of studies concerning their foraging behaviour, relatively little is known about how albatrosses actually locate their prey. Here, we present our results from the first deployments of a combined animal-borne camera and depth data logger on free-ranging black-browed albatrosses (Thalassarche melanophrys). The still images recorded from these cameras showed that some albatrosses actively followed a killer whale (Orcinus orca), possibly to feed on food scraps left by this diving predator. The camera images together with the depth profiles showed that the birds dived only occasionally, but that they actively dived when other birds or the killer whale were present. This association with diving predators or other birds may partially explain how albatrosses find their prey more efficiently in the apparently ‘featureless’ ocean, with a minimal requirement for energetically costly diving or landing activities

Validation of the Edinburgh Claudication Questionnaire in 1st generation Black African-Caribbean and South Asian UK migrants: A sub-study to the Ethnic-Echocardiographic Heart of England Screening (E-ECHOES) study

<p>Abstract</p> <p>Background</p> <p>We determined the diagnostic accuracy of the Edinburgh Claudication Questionnaire (ECQ) in 1^stgeneration Black African-Caribbean UK migrants as previous diagnostic questionnaires have been found to be less accurate in this population. We also determined the diagnostic accuracy of translated versions of the ECQ in 1^stgeneration South Asian UK migrants, as this has not been investigated before.</p> <p>Methods</p> <p>Subjects were recruited from the Ethnic-Echocardiographic Heart of England Screening (E-ECHOES) study, a community based screening survey for heart failure in minority ethnic groups. Translated versions of the ECQ were prepared following a recognised protocol. All participants attending screening between October 2007 and February 2009 were asked to complete the ECQ in the language of their choice (English, Punjabi, Bengali, Urdu, Hindi or Gujarati). Subjects answering positively to experiencing leg pain or discomfort on walking were asked to return to have Ankle Brachial Pressure Index (ABPI) measured.</p> <p>Results</p> <p>154 out of 2831 subjects participating in E-ECHOES (5.4%) were eligible to participate in this sub-study, for which 74.3% returned for ABPI assessment. Non-responders were younger than participants (59[9] vs. 65[11] years; p = 0.015). Punjabi, English and Bengali questionnaires identified participants with Intermittent Claudication, so these questionnaires were assessed. The sensitivities (SN), specificities (SP), positive (PPV) and negative (NPV) predictive values were calculated. English: SN: 50%; SP: 68%; PPV: 43%; NPV: 74%. Punjabi: SN: 50%; SP: 87%; PPV: 43%; NPV: 90%. Bengali: SN: 33%; SP: 50%; PPV: 13%; NPV: 73%. There were significant differences in diagnostic accuracy between the 3 versions (Punjabi: 83.8%; Bengali: 45%; English: 62.2%; p < 0.0001). No significant differences were found in sensitivity and specificity between illiterate and literate participants in any of the questionnaires and there was no significant different difference between those under and over 60 years of age.</p> <p>Conclusions</p> <p>Our findings suggest that the ECQ is not as sensitive or specific a diagnostic tool in 1^stgeneration Black African-Caribbean and South Asian UK migrants than in the Edinburgh Artery Study, reflecting the findings of other diagnostic questionnaires in these minority ethnic groups. However this study is limited by sample size so conclusions should be interpreted with caution.</p

Unstated factors in orthopaedic decision-making: a qualitative study

<p>Abstract</p> <p>Background</p> <p>Total joint replacement (TJR) of the hip or knee for osteoarthritis is among the most common elective surgical procedures. There is some inequity in provision of TJR. How decisions are made about who will have surgery may contribute to disparities in provision. The model of shared decision-making between patients and clinicians is advocated as an ideal by national bodies and guidelines. However, we do not know what happens within orthopaedic practice and whether this reflects the shared model. Our study examined how decisions are made about TJR in orthopaedic consultations.</p> <p>Methods</p> <p>The study used a qualitative research design comprising semi-structured interviews and observations. Participants were recruited from three hospital sites and provided their time free of charge. Seven clinicians involved in decision-making about TJR were approached to take part in the study, and six agreed to do so. Seventy-seven patients due to see these clinicians about TJR were approached to take part and 26 agreed to do so. The patients' outpatient appointments ('consultations') were observed and audio-recorded. Subsequent interviews with patients and clinicians examined decisions that were made at the appointments. Data were analysed using thematic analysis.</p> <p>Results</p> <p>Clinical and lifestyle factors were central components of the decision-making process. In addition, the roles that patients assigned to clinicians were key, as were communication styles. Patients saw clinicians as occupying expert roles and they deferred to clinicians' expertise. There was evidence that patients modified their behaviour within consultations to complement that of clinicians. Clinicians acknowledged the complexity of decision-making and provided descriptions of their own decision-making and communication styles. Patients and clinicians were aware of the use of clinical and lifestyle factors in decision-making and agreed in their description of clinicians' styles. Decisions were usually reached during consultations, but patients and clinicians sometimes said that treatment decisions had been made beforehand. Some patients expressed surprise about the decisions made in their consultations, but this did not necessarily imply dissatisfaction.</p> <p>Conclusions</p> <p>The way in which roles and communication are played out in decision-making for TJR may affect the opportunity for shared decisions. This may contribute to variation in the provision of TJR. Making the importance of these factors explicit and highlighting the existence of patients' 'surprise' about consultation outcomes could empower patients within the decision-making process and enhance communication in orthopaedic consultations.</p