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

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

Migration Patterns, Use of Stopover Areas, and Austral Summer Movements of Swainson\u27s Hawks

From 1995 to 1998, we tracked movements of adult Swainson’s Hawks (Buteo swainsoni), using satellite telemetry to characterize migration, important stopover areas, and movements in the austral summer. We tagged 46 hawks from July to September on their nesting grounds in seven U.S. states and two Canadian provinces. Swainson’s Hawks followed three basic routes south on a broad front, converged along the east coast of central Mexico, and followed a concentrated corridor to a communal area in central Argentina for the austral summer. North of 20°N, southward and northward tracks differed little for individuals from east of the continental divide but differed greatly (up to 1700 km) for individuals from west of the continental divide. Hawks left the breeding grounds mid-August to mid-October; departure dates did not differ by location, year, or sex. Southbound migration lasted 42 to 98 days, northbound migration 51 to 82 days. Southbound, 36% of the Swainson’s Hawks departed the nesting grounds nearly 3 weeks earlier than the other radio-marked hawks and made stopovers 9.0–26.0 days long in seven separate areas, mainly in the southern Great Plains, southern Arizona and New Mexico, and northcentral Mexico. The birds stayed in their nonbreeding range for 76 to 128 days. All used a core area in central Argentina within 23% of the 738 800-km2 austral summer range, where they frequently moved long distances (up to 1600 km). Conservation of Swainson’s Hawks must be an international effort that considers habitats used during nesting and non-nesting seasons, including migration stopovers

Identification of Tuberculosis Susceptibility Genes with Human Macrophage Gene Expression Profiles

Although host genetics influences susceptibility to tuberculosis (TB), few genes determining disease outcome have been identified. We hypothesized that macrophages from individuals with different clinical manifestations of Mycobacterium tuberculosis (Mtb) infection would have distinct gene expression profiles and that polymorphisms in these genes may also be associated with susceptibility to TB. We measured gene expression levels of >38,500 genes from ex vivo Mtb-stimulated macrophages in 12 subjects with 3 clinical phenotypes: latent, pulmonary, and meningeal TB (n = 4 per group). After identifying differentially expressed genes, we confirmed these results in 34 additional subjects by real-time PCR. We also used a case-control study design to examine whether polymorphisms in differentially regulated genes were associated with susceptibility to these different clinical forms of TB. We compared gene expression profiles in Mtb-stimulated and unstimulated macrophages and identified 1,608 and 199 genes that were differentially expressed by >2- and >5-fold, respectively. In an independent sample set of 34 individuals and a subset of highly regulated genes, 90% of the microarray results were confirmed by RT-PCR, including expression levels of CCL1, which distinguished the 3 clinical groups. Furthermore, 6 single nucleotide polymorphisms (SNPs) in CCL1 were found to be associated with TB in a case-control genetic association study with 273 TB cases and 188 controls. To our knowledge, this is the first identification of CCL1 as a gene involved in host susceptibility to TB and the first study to combine microarray and DNA polymorphism studies to identify genes associated with TB susceptibility. These results suggest that genome-wide studies can provide an unbiased method to identify critical macrophage response genes that are associated with different clinical outcomes and that variation in innate immune response genes regulate susceptibility to TB

Objective vs. Self-Reported Physical Activity and Sedentary Time: Effects of Measurement Method on Relationships with Risk Biomarkers

<p><b>Purpose:</b> Imprecise measurement of physical activity variables might attenuate estimates of the beneficial effects of activity on health-related outcomes. We aimed to compare the cardiometabolic risk factor dose-response relationships for physical activity and sedentary behaviour between accelerometer- and questionnaire-based activity measures.</p> <p><b>Methods:</b> Physical activity and sedentary behaviour were assessed in 317 adults by 7-day accelerometry and International Physical Activity Questionnaire (IPAQ). Fasting blood was taken to determine insulin, glucose, triglyceride and total, LDL and HDL cholesterol concentrations and homeostasis model-estimated insulin resistance (HOMAIR). Waist circumference, BMI, body fat percentage and blood pressure were also measured.</p> <p><b>Results:</b> For both accelerometer-derived sedentary time (<100 counts.min−1) and IPAQ-reported sitting time significant positive (negative for HDL cholesterol) relationships were observed with all measured risk factors – i.e. increased sedentary behaviour was associated with increased risk (all p≤0.01). However, for HOMAIR and insulin the regression coefficients were >50% lower for the IPAQ-reported compared to the accelerometer-derived measure (p<0.0001 for both interactions). The relationships for moderate-to-vigorous physical activity (MVPA) and risk factors were less strong than those observed for sedentary behaviours, but significant negative relationships were observed for both accelerometer and IPAQ MVPA measures with glucose, and insulin and HOMAIR values (all p<0.05). For accelerometer-derived MVPA only, additional negative relationships were seen with triglyceride, total cholesterol and LDL cholesterol concentrations, BMI, waist circumference and percentage body fat, and a positive relationship was evident with HDL cholesterol (p = 0.0002). Regression coefficients for HOMAIR, insulin and triglyceride were 43–50% lower for the IPAQ-reported compared to the accelerometer-derived MVPA measure (all p≤0.01).</p> <p><b>Conclusion:</b> Using the IPAQ to determine sitting time and MVPA reveals some, but not all, relationships between these activity measures and metabolic and vascular disease risk factors. Using this self-report method to quantify activity can therefore underestimate the strength of some relationships with risk factors.</p&gt

Creatine Monohydrate and Conjugated Linoleic Acid Improve Strength and Body Composition Following Resistance Exercise in Older Adults

Aging is associated with lower muscle mass and an increase in body fat. We examined whether creatine monohydrate (CrM) and conjugated linoleic acid (CLA) could enhance strength gains and improve body composition (i.e., increase fat-free mass (FFM); decrease body fat) following resistance exercise training in older adults (>65 y). Men (N = 19) and women (N = 20) completed six months of resistance exercise training with CrM (5g/d)+CLA (6g/d) or placebo with randomized, double blind, allocation. Outcomes included: strength and muscular endurance, functional tasks, body composition (DEXA scan), blood tests (lipids, liver function, CK, glucose, systemic inflammation markers (IL-6, C-reactive protein)), urinary markers of compliance (creatine/creatinine), oxidative stress (8-OH-2dG, 8-isoP) and bone resorption (Ν-telopeptides). Exercise training improved all measurements of functional capacity (P<0.05) and strength (P<0.001), with greater improvement for the CrM+CLA group in most measurements of muscular endurance, isokinetic knee extension strength, FFM, and lower fat mass (P<0.05). Plasma creatinine (P<0.05), but not creatinine clearance, increased for CrM+CLA, with no changes in serum CK activity or liver function tests. Together, this data confirms that supervised resistance exercise training is safe and effective for increasing strength in older adults and that a combination of CrM and CLA can enhance some of the beneficial effects of training over a six-month period. Trial Registration. ClinicalTrials.gov NCT0047390