Evolutionary history of the poly(ADP-ribose) polymerase gene family in eukaryotes

Abstract Background The Poly(ADP-ribose)polymerase (PARP) superfamily was originally identified as enzymes that catalyze the attachment of ADP-ribose subunits to target proteins using NAD+ as a substrate. The family is characterized by the catalytic site, termed the PARP signature. While these proteins can be found in a range of eukaryotes, they have been best studied in mammals. In these organisms, PARPs have key functions in DNA repair, genome integrity and epigenetic regulation. More recently it has been found that proteins within the PARP superfamily have altered catalytic sites, and have mono(ADP-ribose) transferase (mART) activity or are enzymatically inactive. These findings suggest that the PARP signature has a broader range of functions that initially predicted. In this study, we investigate the evolutionary history of PARP genes across the eukaryotes. Results We identified in silico 236 PARP proteins from 77 species across five of the six eukaryotic supergroups. We performed extensive phylogenetic analyses of the identified PARPs. They are found in all eukaryotic supergroups for which sequence is available, but some individual lineages within supergroups have independently lost these genes. The PARP superfamily can be subdivided into six clades. Two of these clades were likely found in the last common eukaryotic ancestor. In addition, we have identified PARPs in organisms in which they have not previously been described. Conclusions Three main conclusions can be drawn from our study. First, the broad distribution and pattern of representation of PARP genes indicates that the ancestor of all extant eukaryotes encoded proteins of this type. Second, the ancestral PARP proteins had different functions and activities. One of these proteins was similar to human PARP1 and likely functioned in DNA damage response. The second of the ancestral PARPs had already evolved differences in its catalytic domain that suggest that these proteins may not have possessed poly(ADP-ribosyl)ation activity. Third, the diversity of the PARP superfamily is larger than previously documented, suggesting as more eukaryotic genomes become available, this gene family will grow in both number and type.</p

The Achilles tendon total rupture score : a study of responsiveness, internal consistency and convergent validity on patients with acute Achilles tendon ruptures

Background The Achilles tendon Total Rupture Score was developed by a research group in 2007 in response to the need for a patient reported outcome measure for this patient population. Beyond this original development paper, no further validation studies have been published. Consequently the purpose of this study was to evaluate internal consistency, convergent validity and responsiveness of this newly developed patient reported outcome measure within patients who have sustained an isolated acute Achilles tendon rupture. Methods Sixty-four eligible patients with an acute rupture of their Achilles tendon completed the Achilles tendon Total Rupture Score alongside two further patient reported outcome measures (Disability Rating Index and EQ 5D). These were completed at baseline, six weeks, three months, six months and nine months post injury. The Achilles tendon Total Rupture Score was evaluated for internal consistency, using Cronbach's alpha, convergent validity, through correlation analysis and responsiveness, by analysing floor and ceiling effects and calculating its relative efficiency in comparison to the Disability Rating Index and EQ 5D scores. Results The Achilles tendon Total Rupture Score demonstrated high internal consistency (Cronbachs alpha > 0.8) and correlated significantly (p < 0.001) with the Disability Rating Index at five time points (pre-injury, six weeks, three, six and nine months) with correlation coefficients between -0.5 and -0.9. However, the confidence intervals were wide. Furthermore, the ability of the new score to detect clinically important changes over time (responsiveness) was shown to be greater than the Disability Rating Index and EQ 5D. Conclusions A universally accepted outcome measure is imperative to allow comparisons to be made across practice. This is the first study to evaluate aspects of validity of this newly developed outcome measure, outside of the developing centre. The ATRS demonstrated high internal consistency and responsiveness, with limited convergent validity. This research provides further support for the use of this outcome measure, however further research is required to advocate its universal use in patients with acute Achilles tendon ruptures. Such areas include inter-rater reliability and research to determine the minimally clinically important difference between scores

A Conserved Functional Domain of Drosophila Coracle Is Required for Localization at the Septate Junction and Has Membrane-organizing Activity

This is the published version, also available here: http://dx.doi.org/10.1083/jcb.140.6.1463.The protein 4.1 superfamily is comprised of a diverse group of cytoplasmic proteins, many of which have been shown to associate with the plasma membrane via binding to specific transmembrane proteins. Coracle, a Drosophila protein 4.1 homologue, is required during embryogenesis and is localized to the cytoplasmic face of the septate junction in epithelial cells. Using in vitro mutagenesis, we demonstrate that the amino-terminal 383 amino acids of Coracle define a functional domain that is both necessary and sufficient for proper septate junction localization in transgenic embryos. Genetic mutations within this domain disrupt the subcellular localization of Coracle and severely affect its genetic function, indicating that correct subcellular localization is essential for Coracle function. Furthermore, the localization of Coracle and the transmembrane protein Neurexin to the septate junction display an interdependent relationship, suggesting that Coracle and Neurexin interact with one another at the cytoplasmic face of the septate junction. Consistent with this notion, immunoprecipitation and in vitro binding studies demonstrate that the amino-terminal 383 amino acids of Coracle and cytoplasmic domain of Neurexin interact directly. Together these results indicate that Coracle provides essential membrane-organizing functions at the septate junction, and that these functions are carried out by an amino-terminal domain that is conserved in all protein 4.1 superfamily members

The bHLH transcription factor SPATULA regulates root growth by controlling the size of the root meristem

Abstract Background The Arabidopsis thaliana gene SPATULA (SPT), encoding a bHLH transcription factor, was originally identified for its role in pistil development. SPT is necessary for the growth and development of all carpel margin tissues including the style, stigma, septum and transmitting tract. Since then, it has been shown to have pleiotropic roles during development, including restricting the meristematic region of the leaf primordia and cotyledon expansion. Although SPT is expressed in roots, its role in this organ has not been investigated. Results An analysis of embryo and root development showed that loss of SPT function causes an increase in quiescent center size in both the embryonic and postembryonic stem cell niches. In addition, root meristem size is larger due to increased division, which leads to a longer primary root. spt mutants exhibit other pleiotropic developmental phenotypes, including more flowers, shorter internodes and an extended flowering period. Genetic and molecular analysis suggests that SPT regulates cell proliferation in parallel to gibberellic acid as well as affecting auxin accumulation or transport. Conclusions Our data suggest that SPT functions in growth control throughout sporophytic growth of Arabidopsis, but is not necessary for cell fate decisions except during carpel development. SPT functions independently of gibberellic acid during root development, but may play a role in regulating auxin transport or accumulation. Our data suggests that SPT plays a role in control of root growth, similar to its roles in above ground tissues.</p

Drosophila coracle, a Member of the Protein 4.1 Superfamily, Has Essential Structural Functions in the Septate Junctions and Developmental Functions in Embryonic and Adult Epithelial Cells

This is the published version, also available here: http://dx.doi.org/10.1091/mbc.9.12.3505.Although extensively studied biochemically, members of the Protein 4.1 superfamily have not been as well characterized genetically. Studies of coracle, a DrosophilaProtein 4.1 homologue, provide an opportunity to examine the genetic functions of this gene family. coracle was originally identified as a dominant suppressor ofEgfr Elp, a hypermorphic form of theDrosophila Epidermal growth factor receptor gene. In this article, we present a phenotypic analysis ofcoracle, one of the first for a member of the Protein 4.1 superfamily. Screens for new coracle alleles confirm the null coracle phenotype of embryonic lethality and failure in dorsal closure, and they identify additional defects in the embryonic epidermis and salivary glands. Hypomorphiccoracle alleles reveal functions in many imaginal tissues. Analysis of coracle mutant cells indicates that Coracle is a necessary structural component of the septate junction required for the maintenance of the transepithelial barrier but is not necessary for apical–basal polarity, epithelial integrity, or cytoskeletal integrity. In addition, coracle phenotypes suggest a specific role in cell signaling events. Finally, complementation analysis provides information regarding the functional organization of Coracle and possibly other Protein 4.1 superfamily members. These studies provide insights into a range of in vivo functions for coracle in developing embryos and adults

Plaster cast versus functional brace for non-surgical treatment of Achilles tendon rupture (UKSTAR) : a multicentre randomised controlled trial and economic evaluation

Background Patients with Achilles tendon rupture who have non-operative treatment have traditionally been treated with immobilisation of the tendon in plaster casts for several weeks. Functional bracing is an alternative non-operative treatment that allows earlier mobilisation, but evidence on its effectiveness and safety is scarce. The aim of the UKSTAR trial was to compare functional and quality-of-life outcomes and resource use in patients treated non-operatively with plaster cast versus functional brace. Methods UKSTAR was a pragmatic, superiority, multicentre, randomised controlled trial done at 39 hospitals in the UK. Patients (aged ≥16 years) who were being treated non-operatively for a primary Achilles tendon rupture at the participating centres were potentially eligible. The exclusion criteria were presenting more than 14 days after injury, previous rupture of the same Achilles tendon, or being unable to complete the questionnaires. Eligible participants were randomly assigned (1:1) to receive a plaster cast or functional brace using a centralised web-based system. Because the interventions were clearly visible, neither patients nor clinicians could be masked. Participants wore the intervention for 8 weeks. The primary outcome was patient-reported Achilles tendon rupture score (ATRS) at 9 months, analysed in the modified intention-to-treat population (all patients in the groups to which they were allocated, excluding participants who withdrew or died before providing any outcome data). The main safety outcome was the incidence of tendon re-rupture. Resource use was recorded from a health and personal social care perspective. The trial is registered with ISRCTN, ISRCTN62639639. Findings Between Aug 15, 2016, and May 31, 2018, 1451 patients were screened, of whom 540 participants (mean age 48·7 years, 79% male) were randomly allocated to receive plaster cast (n=266) or functional brace (n=274). 527 (98%) of 540 were included in the modified intention-to-treat population, and 13 (2%) were excluded because they withdrew or died before providing any outcome data. There was no difference in ATRS at 9 months post injury (cast group n=244, mean ATRS 74∙4 [SD 19∙8]; functional brace group n=259, ATRS 72∙8 [20∙4]; adjusted mean difference –1∙38 [95% CI –4∙9 to 2∙1], p=0·44). There was no difference in the rate of re-rupture of the tendon (17 [6%] of 266 in the plaster cast group vs 13 [5%] of 274 in the functional brace group, p=0·40). The mean total health and personal social care cost was £1181 for the plaster cast group and £1078 for the functional bract group (mean between-group difference –£103 [95% CI –289 to 84]). Interpretation Traditional plaster casting was not found to be superior to early weight-bearing in a functional brace, as measured by ATRS, in the management of patients treated non-surgically for Achilles tendon rupture. Clinicians may consider the use of early weight-bearing in a functional brace as a safe and cost-effective alternative to plaster casting

Cast versus functional brace in the rehabilitation of patients treated non-operatively for a rupture of the Achilles tendon: protocol for the UK study of tendo achilles rehabilitation (UK STAR) multi-centre randomised trial

INTRODUCTION Achilles tendon rupture affects over 11 000 people yearly in the UK, and the incidence is increasing. Controversy remains with regard to the best rehabilitation strategy for these patients. In operatively treated patients, functional bracing provides better outcomes compared with plaster casts. However, the role of functional bracing in non-operatively managed patients is unclear. This is the protocol for a multicentre randomised trial of plaster cast immobilisation versus functional bracing for patients with a non-operatively managed Achilles tendon rupture. METHODS AND ANALYSIS All adults presenting with a primary rupture of the Achilles tendon will be screened. Non-operatively treated patients will be eligible to take part in the trial. Broad eligibility criteria will ensure that the results of the study can be generalised to the wider patient population. Randomisation will be on a 1:1 basis. Both rehabilitation strategies are widely used within the National Health Service. Standardised protocols will be followed, and details of plaster material and brace will be as per the site's usual practice.A minimum of 330 patients will be randomised to obtain 90% power to detect a difference of 8 points in Achilles Tendon Total Rupture Score at 9 months. Quality of life and resource use will be collected at 3, 6 and 9 months. The differences between treatment groups will be assessed on an intention-to-treat basis. The results of the trial-based economic evaluation will be expressed in terms of incremental cost per quality-adjusted life-year gained. ETHICS AND DISSEMINATION The National Research Ethic Committee approved this study on 18 March 2016 (16/SC/0109).The National Institute for Health Research Health Technology Assessment monograph and a manuscript to a peer-reviewed journal will be submitted on completion of the trial (summer 2019). The results of this trial will substantially inform clinical practice on the clinical and cost-effectiveness of the treatment of this injury. This study has been registered on the International Standard Randomised Controlled Trial Number registry with reference no ISRCTN62639639

Optogenetic Apoptosis: Light-Triggered Cell Death

An optogenetic Bax has been designed that facilitates light-induced apoptosis. We demonstrate that mitochondrial recruitment of a genetically encoded light-responsive Bax results in the release of mitochondrial proteins, downstream caspase 3 cleavage, changes in cellular morphology, and ultimately cell death. Mutagenesis of a key phosphorylatable residue or modification of the C-terminus mitigates background levels (dark) of apoptosis due to Bax overexpression. The mechanism of optogenetic Bax-mediated apoptosis was explored using a series of small molecules known to interfere with various steps in programmed cell death. Optogenetic Bax appears to form a mitochondrial apoptosis-induced channel analogous to that of endogenous Bax

Activity Accumulation and Cardiometabolic Risk in Youth

Introduction This cross-sectional study aimed to: i) identify and characterize youth according to distinct physical activity (PA) and sedentary (SED) accumulation patterns; and ii) investigate associations of these derived patterns with cardiometabolic risk factors.Methods ActiGraph accelerometer data from 7-13 year olds from two studies were pooled (n=1,219; 843 [69%] with valid accelerometry included in analysis). Time accumulated in ≥5-min and ≥10-min SED bouts, ≥1-min and ≥5-min bouts of light (LPA), and ≥1-min bouts of moderate (MPA) and vigorous (VPA) PA were calculated. Frequency of breaks in SED were also obtained. Latent profile analysis was used to identify groups of participants based on their distinct accumulation patterns. Linear and logistic regression models were used to test associations of group accumulation patterns with cardiometabolic risk factors, including adiposity indicators, blood pressure and lipids. Total PA and SED time were also compared between groups.Results Three distinct groups were identified: “Prolonged sitters” had the most time in prolonged SED bouts and the least time in VPA bouts; “Breakers” had the highest frequency of SED breaks and lowest engagement in sustained bouts across most PA intensities; “Prolonged movers” had the least time accumulated in SED bouts and the most in PA bouts across most intensities. Whilst “Breakers” engaged in less time in PA bouts compared to other groups, they had the healthiest adiposity indicators. No associations with the remaining cardiometabolic risk factors were found.Conclusion Youth accumulate their daily activity in three distinct patterns (prolonged sitters, breakers and prolonger movers), with those breaking up sitting and most time in sporadic PA across the day having a lower adiposity risk. No relationships with other cardiometabolic risk factors were identified