Engineering enzymes with non-canonical active site functionality

The combination of computational enzyme design and laboratory evolution provides an attractive platform for the creation of protein catalysts with new function. To date, designed mechanisms have relied upon Nature’s alphabet of 20 genetically encoded amino acids, which greatly restricts the range of functionality which can be installed into enzyme active sites. Here, we have exploited engineered components of the cellular translation machinery to create a protein catalyst which operates via a non-canonical catalytic nucleophile. We have subsequently shown that powerful laboratory evolution protocols can be readily adapted to allow optimization of enzymes containing non-canonical active site functionality. Crystal structures obtained along the evolutionary trajectory highlight the origins of improved activity. Thus our approach merges beneficial features of organo- and biocatalysis, by combining the intrinsic reactivities and greater versatility of small molecule catalysts with the rate enhancements, reaction selectivities and evolvability of proteins. Please click Additional Files below to see the full abstract

Structure and mechanism of a canonical poly(ADP-ribose) glycohydrolase

Poly(ADP-ribosyl)ation is a reversible post-translational protein modification involved in the regulation of a number of cellular processes including DNA repair, chromatin structure, mitosis, transcription, checkpoint activation, apoptosis and asexual development. The reversion of poly(ADP-ribosyl)ation is catalysed by poly(ADP-ribose) (PAR) glycohydrolase (PARG), which specifically targets the unique PAR (1''-2') ribose-ribose bonds. Here we report the structure and mechanism of the first canonical PARG from the protozoan Tetrahymena thermophila. In addition, we reveal the structure of T. thermophila PARG in a complex with a novel rhodanine-containing mammalian PARG inhibitor RBPI-3. Our data demonstrate that the protozoan PARG represents a good model for human PARG and is therefore likely to prove useful in guiding structure-based discovery of new classes of PARG inhibitors

Visualization of poly(ADP-ribose) bound to PARG reveals inherent balance between exo- and endo-glycohydrolase activities

Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo-glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo-mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo-glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo-mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios

Changes in physical activity during hospital admission for chronic respiratory disease: Inpatient step count does not recover

Background and objectiveEstablishing the amount of inpatient physical activity (PA) undertaken by individuals hospitalized for chronic respiratory disease is needed to inform interventions. This observational study investigated whether PA changes when a person is an inpatient, how long is required to obtain representative PA measures and whether PA varies within a day and between patients of differing lengths of stay.MethodsA total of 389 participants were recruited as early as possible into their hospitalization. Patients wore a PA monitor from recruitment until discharge. Step count was extracted for a range of wear time criteria. Single‐day intraclass correlation coefficients (ICC) were calculated, with an ICC ≥ 0.80 deemed acceptable.ResultsPA data were available for 259 participants. No changes in daily step count were observed during the inpatient stay (586 (95% CI: 427–744) vs 652 (95% CI: 493–812) steps/day for day 2 and 7, respectively). ICC across all wear time criteria were > 0.80. The most stringent wear time criterion, retaining 80% of the sample, was ≥11 h on ≥1 day. More steps were taken during the morning and afternoon than overnight and evening. After controlling for the Medical Research Council (MRC) grade or oxygen use, there was no difference in step count between patients admitted for 2–3 days (short stay) and those admitted for 7–14 days (long stay).ConclusionPatients move little during their hospitalization, and inpatient PA did not increase during their stay. A wear time criterion of 11 waking hours on any single day was representative of the entire admission whilst retaining an acceptable proportion of the initial sample size. Patients may need encouragement to move more during their hospital stay

Common mental disorders, neighbourhood income inequality and income deprivation: small-area multilevel analysis

Background. Common mental disorders are more prevalent in areas of high neighbourhood socioeconomic deprivation but whether the prevalence varies with neighbourhood income inequality is not known. Aims. To investigate the hypothesis that the interaction between small-area income deprivation and income inequality was associated with individual mental health. Method. Multilevel analysis of population data from the Welsh Health Survey, 2003/04–2010. A total of 88 623 respondents aged 18–74 years were nested within 50 587 households within 1887 lower super output areas (neighbourhoods) and 22 unitary authorities (regions), linked to the Gini coefficient (income inequality) and the per cent of households living in poverty (income deprivation). Mental health was measured using the Mental Health Inventory MHI-5 as a discrete variable and as a ‘case’ of common mental disorder. Results. High neighbourhood income inequality was associated with better mental health in low-deprivation neighbourhoods after adjusting for individual and household risk factors (parameter estimate +0.70 (s.e. = 0.33), P = 0.036; odds ratio (OR) for common mental disorder case 0.92, 95% CI 0.88–0.97). Income inequality at regional level was significantly associated with poorer mental health (parameter estimate -1.35 (s.e. = 0.54), P = 0.012; OR = 1.13, 95% CI 1.04–1.22). Conclusions. The associations between common mental disorders, income inequality and income deprivation are complex. Income inequality at neighbourhood level is less important than income deprivation as a risk factor for common mental disorders. The adverse effect of income inequality starts to operate at the larger regional level

Current knowledge of external sulfate attack

This paper offers an update of the current understanding of sulfate attack, with emphasis on the sulfates present in an external water source percolating through, and potentially reacting with, the cement matrix. The paper considers the explanations put forward to explain sulfate attack, both from a chemical and microstructural perspective. Similarly, this paper reviews work on the physical damage caused by the precipitation of sulfate salts in porous materials. With the increased use of binary and ternary blends, this paper also considers the impact of binder composition on sulfate resistance, and similarly reviews how the nature of the sulfate species can affect the nature and extent of any deterioration. This then leads on to the important consideration of differences between field- and lab-based studies; reviewing the effect of various experimental parameters on sulfate resistance. This latter topic is of great importance to anyone who wishes to carry out such experiments

Associations Between Screen-Based Sedentary Behavior and Cardiovascular Disease Risk Factors in Korean Youth

The purposes of this study were to: 1) describe the patterns of screen-based sedentary behaviors, and 2) examine the association between screen-based sedentary behavior and cardiovascular disease (CVD) risk factors in representative Korean children and adolescents, aged 12 to 18 yr, in the Korean National Health and Nutrition Examination Survey. Screen-based sedentary behavior was measured using self-report questionnaires that included items for time spent watching TV and playing PC/video games. Physical activity was measured using items for frequency and duration of moderate-to-vigorous physical activity (MVPA). CVD risk factors such as body mass index (BMI), waist circumference, LDL cholesterol, HDL cholesterol, total cholesterol, triglycerides, glucose, systolic blood pressure, and diastolic blood pressure were measured. Boys spent more time playing PC/video games, and girls spent more time watching TV. After adjusting for age, gender, annual household income, and MVPA, an additional hour of watching TV was significantly associated with the risk of overweight (OR 1.17 [95% CI 1.03-1.33]), high abdominal adiposity (OR 1.27 [1.06-1.51]), and low HDL cholesterol (OR 1.27 [1.10-1.47]). An additional hour spent playing PC/video games also increased the risk of high abdominal adiposity (OR 1.20 [1.03-1.40]). Prospective observations and interventions are needed to determine causal relationships between screen-based sedentary behavior and CVD risk profiles in Korean youth

Evaluating cutpoints for the MHI-5 and MCS using the GHQ-12: a comparison of five different methods

Background The Mental Health Inventory (MHI-5) and the Mental Health Component Summary score (MCS) derived from the Short Form 36 (SF-36) instrument are well validated and reliable scales. A drawback of their construction is that neither has a clinically validated cutpoint to define a case of common mental disorder (CMD). This paper aims to produce cutpoints for the MHI-5 and MCS by comparison with the General Health Questionnaire (GHQ-12). Methods Data were analysed from wave 9 of the British Household Panel Survey (2000), providing a sample size of 14,669 individuals. Receiver Operating Characteristic (ROC) curves were used to compare the scales and define cutpoints for the MHI-5 and MCS, using the following optimisation criteria: the Youden Index, the point closest to (0,1) on the ROC curve, minimising the misclassification rate, the minimax method, and prevalence matching. Results For the MHI-5, the Youden Index and the (0,1) methods both gave a cutpoint of 76, minimising the misclassification rate gave a cutpoint of 60 and the minimax method and prevalence matching gave a cutpoint of 68. For the MCS, the Youden Index and the (0,1) methods gave cutpoints of 51.7 and 52.1 respectively, minimising the error rate gave a cutpoint of 44.8 and both the minimax method and prevalence matching gave a cutpoint of 48.9. The correlation between the MHI-5 and the MCS was 0.88. Conclusion The Youden Index and (0,1) methods are most suitable for determining a cutpoint for the MHI-5, since they are least dependent on population prevalence. The choice of method is dependent on the intended application. The MHI-5 performs remarkably well against the longer MCS