Investigation into the role of the SUMO-like domains of the Schizosaccharomyces pombe DNA repair protein Rad60

Ubiquitin and ubiquitin-like (UBLs) proteins are post-translational modifiers that share a characteristic ββαββαβ fold. SUMO (small ubiquitin-like modifier) is one of a number of ubiquitin-like proteins. Unlike ubiquitin, SUMO does not appear to have a role in protein degradation. Instead it has been shown to have roles in facilitating protein-protein interactions, altering protein localisation and modulating protein activity. Analysis of protein databases indicates the existence of ubiquitin-fusion proteins, which act to functionally mimic ubiquitination by interacting with the proteosome. During the course of this project a family of SUMO-like domain (SLD) proteins has been identified and termed the RENi family after its best-studied members S. pombe Rad60, S. cerevisiae Esc2 and M. musculus Nip45. I have initiated an investigation into the importance of the two SUMO-like domains for S. pombe Rad60 function. A rad60 mutant deleted for SLD1 (rad60-SLD1Δ) is not viable suggesting that SLD1 is required for the essential role of Rad60. A rad60 mutant deleted for SLD2 (rad60-ct) is viable but cells are sensitive to DNA damaging agents. This implies that SLD2 is not required for the essential function of Rad60 but is required for the response to DNA damage. The C-terminally truncated Rad60 protein (Rad60-ct) is mis-localised in rad60-ct cells. Provision of an NLS to the C-terminus of the Rad60-ct protein restores nuclear localisation but does not rescue the HU and MMS sensitivity of rad60-ct cells. Instead, expression of the Rad60-ctNLS protein has a dominant-negative effect in both wild-type and rad60-ct cells. The same phenomenon was observed when SLD2 was replaced with SUMO. This suggests that SLD2 is required not only to localise Rad60 to the nucleus, but also for the DNA damage response itself. Molecular modelling suggests that SLD1 and SLD2 can adopt the characteristic ββαββαβ fold. A novel ‘recombinase-mediated cassette-exchange’ system was used to initiate a structure/function study of Rad60 SLD2 by mutating residues predicted to help maintain the hydrophobic core. The DNA damage sensitive phenotype of L348G, L338G, L346G and I334G substitutions support the hypothesis that the SLD2 adopts a SUMO-like fold. Sumoylation of Rad60, in vitro, can be enhanced by the SUMO E3 ligase, Pli1 but not Nse2. Rad60 is sumoylated in a manner dependent on the C-terminus, which has is required to interact with the Hus5 conjugator, in vitro. This suggests that SLD2 may act to recruit Hus5 for sumoylation of itself and/or other proteins

Characterisation of the SUMO-like domains of Schizosaccharomyces pombe Rad60

The S. pombe Rad60 protein is required for the repair of DNA double strand breaks, recovery from replication arrest, and is essential for cell viability. It has two SUMO-like domains (SLDs) at its C-terminus, an SXS motif and three sequences that have been proposed to be SUMO-binding motifs (SBMs). SMB1 is located in the middle of the protein, SBM2 is in SLD1 and SBM3 is at the C-terminus of SLD2. We have probed the functions of the two SUMO-like domains, SLD1 and SLD2, and the putative SBMs. SLD1 is essential for viability, while SLD2 is not. rad60-SLD2Δ cells are sensitive to DNA damaging agents and hydroxyurea. Neither ubiquitin nor SUMO can replace SLD1 or SLD2. Cells in which either SBM1 or SBM2 has been mutated are viable and are wild type for response to MMS and HU. In contrast mutation of SBM3 results in significant sensitivity to MMS and HU. These results indicate that the lethality resulting from deletion of SLD1 is not due to loss of SBM2, but that mutation of SBM3 produces a more severe phenotype than does deletion of SLD2. Using chemical denaturation studies, FPLC and dynamic light scattering we show this is likely due to the destabilisation of SLD2. Thus we propose that the region corresponding to the putative SBM3 forms part of the hydrophobic core of SLD2 and is not a SUMO-interacting motif. Over-expression of Hus5, which is the SUMO conjugating enzyme and known to interact with Rad60, does not rescue rad60-SLD2Δ, implying that as well as having a role in the sumoylation process as previously described [1], Rad60 has a Hus5-independent function

Worthwhile work? Childcare, feminist ethics and cooperative research practices

Interdisciplinary research collaborations are often encouraged within higher education while the practicalities of such collaborations are glossed over. This project specifically addresses the praxis of research collaborations, exploring how feminist academics within different countries and disciplines came together to explore their mutual concern about the perceived worth and well-being of early childhood practitioners. Engaging in a formal methodological dialogue over eight months, seven academics discussed, analysed and dissected their different investments in research methods and intents, with the aim of agreeing to a common methodological framework. Unexpectedly, what emerged was not a product, but a process. We argue that this process offers much to those seeking deep collaboration in and through shared research. Building on a collective research interest, we found ourselves in a process of becoming, germinating the seed of a transnational research cooperative, based on trust and mutual respect, rather than the arid methodological contract originally envisioned

Observation of Amounts of Movement Practice Provided during Stroke Rehabilitation

Objective To investigate how much movement practice occurred during stroke rehabilitation, and what factors might influence doses of practice provided. Design Observational survey of stroke therapy sessions. Setting Seven inpatient and outpatient rehabilitation sites. Participants We observed a convenience sample of 312 physical and occupational therapy sessions for people with stroke. Interventions Not applicable. Main Outcome Measures We recorded numbers of repetitions in specific movement categories and data on potential modifying factors (patient age, side affected, time since stroke, FIM item scores, years of therapist experience). Descriptive statistics were used to characterize amounts of practice. Correlation and regression analyses were used to determine whether potential factors were related to the amount of practice in the 2 important categories of upper extremity functional movements and gait steps. Results Practice of task-specific, functional upper extremity movements occurred in 51% of the sessions that addressed upper limb rehabilitation, and the average number of repetitions/session was 32 (95% confidence interval [CI]=20–44). Practice of gait occurred in 84% of sessions that addressed lower limb rehabilitation and the average number of gait steps/session was 357 (95% CI=296–418). None of the potential factors listed accounted for significant variance in the amount of practice in either of these 2 categories. Conclusions The amount of practice provided during poststroke rehabilitation is small compared with animal models. It is possible that current doses of task-specific practice during rehabilitation are not adequate to drive the neural reorganization needed to promote function poststroke optimally

Asking new questions with old data: The Centralized Open-Access Rehabilitation database for Stroke

Background: This paper introduces a tool for streamlining data integration in rehabilitation science, the Centralized Open-Access Rehabilitation database for Stroke (SCOAR), which allows researchers to quickly visualize relationships among variables, efficiently share data, generate hypotheses, and enhance clinical trial design. Methods: Bibliographic databases were searched according to inclusion criteria leaving 2,892 titles that were further screened to 514 manuscripts to be screened by full text, leaving 215 randomized controlled trials in the database (489 independent groups representing 12,847 patients). Demographic, methodological, and statistical data were extracted by independent coders and entered into SCOAR. Results: Trial data came from 114 locations in 27 different countries and represented patients with a wide range of ages, 62 yr 41; 85, (shown as median range) and at various stages of recovery following their stroke, 141 d 1; 3372. There was considerable variation in the dose of therapy that patients received, 20 h 0; 221, over interventions of different durations, 28 d 10; 365. There was also a lack of common data elements (CDEs) across trials, but this lack of CDEs was most pronounced for baseline assessments of patient impairment and severity of stroke. Conclusions: Data integration across hundreds of RCTs allows clinicians and researchers to quickly visualize data from the history of the field and lays the foundation for making SCOAR a living database to which researchers can upload new data as trial results are published. SCOAR is a useful tool for clinicians and researchers that will facilitate data visualization, data sharing, the finding of relevant past studies, and the design of clinical trials by enabling more accurate and comprehensive power analyses. Furthermore, these data speak to the need for CDEs specific to stroke rehabilitation in randomized controlled trials.PROSPERO# CRD420140901

All My Buddies Was Male: Relationship Issues of Women With Addictions

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72776/1/j.1552-6909.1997.tb02729.x.pd

Frontal, Sensorimotor, and Posterior Parietal Regions Are Involved in Dual-Task Walking After Stroke

Background: Walking within the community requires the ability to walk while simultaneously completing other tasks. After a stroke, completing an additional task while walking is significantly impaired, and it is unclear how the functional activity of the brain may impact this. Methods: Twenty individual in the chronic stage post-stroke participated in this study. Functional near-infrared spectroscopy (fNIRS) was used to measure prefrontal, pre-motor, sensorimotor, and posterior parietal cortices during walking and walking while completing secondary verbal tasks of varying difficulty. Changes in brain activity during these tasks were measured and relationships were accessed between brain activation changes and cognitive or motor abilities. Results: Significantly larger activations were found for prefrontal, pre-motor, and posterior parietal cortices during dual-task walking. Increasing dual-task walking challenge did not result in an increase in brain activation in these regions. Higher general cognition related to lower increases in activation during the easier dual-task. With the harder dual-task, a trend was also found for higher activation and less motor impairment. Conclusions: This is the first study to show that executive function, motor preparation/planning, and sensorimotor integration areas are all important for dual-task walking post-stroke. A lack of further brain activation increase with increasing challenge suggests a point at which a trade-off between brain activation and performance occurs. Further research is needed to determine if training would result in further increases in brain activity or improved performance

Phase-dependent Brain Activation of the Frontal and Parietal Regions During Walking After Stroke - An fNIRS Study

Background: Recovery of walking post-stroke is highly variable. Accurately measuring and documenting functional brain activation characteristics during walking can help guide rehabilitation. Previous work in this area has been limited to investigations of frontal brain regions and have not utilized recent technological and analytical advances for more accurate measurements. There were three aims for this study: to characterize the hemodynamic profile during walking post-stroke, to investigate regional changes in brain activation during different phases of walking, and to related brain changes to clinical measures. Methods: Functional near-infrared spectroscopy (fNIRS) along the pre-frontal, premotor, sensorimotor, and posterior parietal cortices was used on twenty individuals greater than six months post-stroke. Individual fNIRS optodes were digitized and used to estimate channel locations on each participant and short separation channels were used to control for extracerebral hemodynamic changes. Participants walked at their comfortable pace several times along a hallway while brain activation was recorded. Exploratory cluster analysis was conducted to determine if there was a link between brain activation and clinical measures. Results: Sustained activation was observed in the pre-frontal cortex with the ipsilesional hemisphere showing greater activation compared to the contralesional side. Sensorimotor cortex was active during the early, acceleration stage of walking only. Posterior parietal cortex showed changes in activation during the later, steady-state stage of walking. Faster gait speeds also related to increased activation in contralesional sensorimotor and posterior parietal cortices. Exploratory analysis clustered participants into two distinct groups based on their brain activation profiles and generally showed that individuals with greater activation tended to have better physical outcomes. Conclusions: These findings can guide future research for obtaining adequate power and determining factors that can be used as effect modifiers to reduce inter-subject variability. Overall, this is the first study to report specific oxygenated and deoxygenated hemoglobin changes in frontal to parietal regions during walking in the stroke population. Our results shed light on the importance of measuring brain activation across the cortex and show the importance of pre-frontal, sensorimotor, and posterior parietal cortices in walking after a stroke

Unsteady Extinction of Opposed Jet Ethylene/Methane HIFiRE Surrogate Fuel Mixtures vs Air

A unique idealized study of the subject fuel vs. air systems was conducted using an Oscillatory-input Opposed Jet Burner (OOJB) system and a newly refined analysis. Extensive dynamic-extinction measurements were obtained on unanchored (free-floating) laminar Counter Flow Diffusion Flames (CFDFs) at 1-atm, stabilized by steady input velocities (e.g., U(sub air)) and perturbed by superimposed in-phase sinusoidal velocity inputs at fuel and air nozzle exits. Ethylene (C2H4) and methane (CH4), and intermediate 64/36 and 15/85 molar percent mixtures were studied. The latter gaseous surrogates were chosen earlier to mimic ignition and respective steady Flame Strengths (FS = U(sub air)) of vaporized and cracked, and un-cracked, JP-7 "like" kerosene for a Hypersonic International Flight Research Experimentation (HIFiRE) scramjet. For steady idealized flameholding, the 100% C2H4 flame is respectively approx. 1.3 and approx.2.7 times stronger than a 64/36 mix and CH4; but is still 12.0 times weaker than a 100% H2-air flame. Limited Hot-Wire (HW) measurements of velocity oscillations at convergent-nozzle exits, and more extensive Probe Microphone (PM) measurements of acoustic pressures, were used to normalize Dynamic FSs, which decayed linearly with pk/pk U(sub air) (velocity magnitude, HW), and also pk/pk P (pressure magnitude, PM). Thus Dynamic Flame Weakening (DFW) is defined as % decrease in FS per Pascal of pk/pk P oscillation, namely, DFW = -100 d(U(sub air)/U(sub air),0Hz)/d(pkpk P). Key findings are: (1) Ethylene flames are uniquely strong and resilient to extinction by oscillating inflows below 150 Hz; (2) Methane flames are uniquely weak; (3) Ethylene / methane surrogate flames are disproportionately strong with respect to ethylene content; and (4) Flame weakening is consistent with limited published results on forced unsteady CFDFs. Thus from 0 to approx. 10 Hz and slightly higher, lagging diffusive responses of key species led to progressive phase lags (relative to inputs) in the oscillating flames, and caused maximum weakening. At 20 to 150 Hz, diffusion-rate-limited effects diminished, causing flames to "regain strengnth," and eventually become completely insensitive beyond 300 Hz. Detailed mechanistic understanding is needed. Overall, ethylene flames are remarkably resilient to dynamic extinction by oscillating inflows. They are the strongest, with the notable exception of H2. For HIFiRE tests, the 64%/36% surrogate disproportionally retains the high dynamic FS of ethylene, so the potential for loss of scramjet flameholding (flameout) due to low frequency oscillations is significantly mitigated

Rotation of an oblate satellite: Chaos control

Aims. This paper investigates the chaotic rotation of an oblate satellite in the context of chaos control. Methods. A model of planar oscillations, described with the Beletskii equation, was investigated. The Hamiltonian formalism was utilized to employ a control method for suppressing chaos. Results. An additive control term, which is an order of magnitude smaller than the potential, is constructed. This allows not only for significantly diminished diffusion of the trajectory in the phase space, but turns the purely chaotic motion into strictly periodic motion.Comment: 9 pages, 4 figures; accepted in A&