RBR E3 ubiquitin ligases: new structures, new insights, new questions

The RBR (RING-BetweenRING-RING) or TRIAD [two RING fingers and a DRIL (double RING finger linked)] E3 ubiquitin ligases comprise a group of 12 complex multidomain enzymes. This unique family of E3 ligases includes parkin, whose dysfunction is linked to the pathogenesis of early-onset Parkinson's disease, and HOIP (HOIL-1-interacting protein) and HOIL-1 (haem-oxidized IRP2 ubiquitin ligase 1), members of the LUBAC (linear ubiquitin chain assembly complex). The RBR E3 ligases share common features with both the larger RING and HECT (homologous with E6-associated protein C-terminus) E3 ligase families, directly catalysing ubiquitin transfer from an intrinsic catalytic cysteine housed in the C-terminal domain, as well as recruiting thioester-bound E2 enzymes via a RING domain. Recent three-dimensional structures and biochemical findings of the RBRs have revealed novel protein domain folds not previously envisioned and some surprising modes of regulation that have raised many questions. This has required renaming two of the domains in the RBR E3 ligases to more accurately reflect their structures and functions: the C-terminal Rcat (required-for-catalysis) domain, essential for catalytic activity, and a central BRcat (benign-catalytic) domain that adopts the same fold as the Rcat, but lacks a catalytic cysteine residue and ubiquitination activity. The present review discusses how three-dimensional structures of RBR (RING1-BRcat-Rcat) E3 ligases have provided new insights into our understanding of the biochemical mechanisms of these important enzymes in ubiquitin biology. INTRODUCTIO

A palaeoecological investigation of long-term stand-scale ecological dynamics in semi-open native pine woods: Contributing to conservation management in east Glen Affric

This thesis investigates past structure and dynamics of native Caledonian pine woodland, representing part of the western fringes of the northern European boreal woodlands. The biogeographical extent and Holocene history of the Scottish pine woods are well studied, yet questions remain at finer scales. This thesis is concerned with two factors over the recent Holocene oceanic period; (i) the long-term ecology within the woods; the spatio-temporal dynamics, the canopy structure, and community composition and continuity; and (ii) the former extent of the woods, especially the temporal pattern of the inferred easterly contraction in woodland over recent history. The importance of these factors - to ecologists, challenged with understanding the theories of equilibrium and non-equilibrium processes in long-lived woodland communities - and to conservation managers - challenged, by policy directives, with implementing the restoration and expansion of native woodlands - is discussed. Equally the identification and quantification of ecological detail over ecologically relevant temporal and spatial scales is an important challenge for palaeoecology. The thesis therefore applies fine spatial resolution pollen analyses over a network of sites, within, and at the western edge of, the extant woodland zone in east Glen Affric. Correlation between these individual site histories develops a detailed view of the grain and extent of woodland within the landscape, previously missing from western pine woods. Careful attention is paid to the interpretive potential and limitations of fine resolution palaeoecology; especially with regard (i) to techniques that can identify and spatially quantify stand-scale structure and community composition via reference to modern analogues; and (ii) to defining chronologies and elucidating rates and patterns of temporal change. The opportunities and limitations of the technique are explored and discussed, to ensure an understanding of the rigour and potential of the palaeoecological contribution to ecological research and to provide an evidence base for conservation. The application of 210Pb dating using the CRS and CIC models is explored. The CRS model is confirmed as suitable for peat deposits, but its use may mask fluctuations in peat sedimentation rate, which may be illustrated by the CIC model. The value of multiple 14C assays for each core and a need for a new approach to chronologies for application to fine-scale palaeecological studies is discussed. The relevant source area around the small basins in this semi-open pinewood is tentatively confirmed at 20 m from the pollen source. Tentative pollen productivity estimates for five key taxa in this ecosystem are presented. The temporal stability of native woodland in Glen Affric is confirmed to the eastern part of the extant woodland zone; but challenged to the west, where the open and semi-open landscape has a long history. Woodland diversity decreased over the last c. 200 years, and past woodland also shows a greater ground flora diversity. The western extent of the Caledonian woodland in this landscape may have changed little in the last c. 4000 years. Fluctuations, some clearly cyclical, in heath, and in woodland, communities are identified in the pollen record. The former may be aligned to changes in grazing regime or climatic shifts; and the latter to autochthonous shifts important in the maintenance of suitable edaphic conditions for the continuity of woodland. Former woodland is confirmed as likely to have been open in structure and mixed in tree species composition. The results presented here suggest that some caution should be applied to use of the term Caledonian, or native ‘pine’ forest: ‘Caledonian forest’ may better reflect the heterogeneity of past forests, particularly the importance, and persistence, of birch. The implications for conservation management and restoration are discussed. It may be difficult to establish a sustainable woodland to the west of the extant stands, and any pine woodland here may need to be mixed with stands of broadleaved trees to maintain or restore soil structure and ecological function. The landscape to the west may have been open for several thousands of years, and consideration of this is required when managing for the future to prevent loss of biodiversity.University of Stirling Forest Researc

Predicting Future Special Education Eligibility from Developmental Assessment of Young Children (DAYC) Scores

This quantitative non-experimental correlational study used logistic regression and archival data to examine the relationship between scores obtained by children at age three on the Developmental Assessment of Young Children (DAYC) rating scale and later special education eligibility status. The purpose of this study was to determine if DAYC scores can predict future special education eligibility as defined by the criteria of the State of Tennessee Department of Education and thereby provide a tactic for early identification and intervention. The 74 participants were students from a small, rural, high-poverty school district who were initially evaluated at age three using the DAYC and who were subsequently re-evaluated three years later. Students were originally rated by parents and teachers and later evaluated by licensed school psychologists using state-approved testing procedures and special education eligibility standards

Adapting upland ecosystem services to climate change: planning for the future at community level

The purpose of this workshop was to initiate participatory discussions on climate change adaptation among a wide range of stakeholders from upland environments. The focus was on: the identification of vulnerabilities; the adaptation options based on scenarios of an average 2°C temperature rise; capacity for uptake of adaptation options based on integration with other land management and community pressures; the role of governance in adaptation; the development of demonstration projects and the sharing of case studies. In particular, the workshop allowed for an international comparison of adaptation actions relevant to uplands and mountains and international comparisons of the role of differing forms of governance in the development and success of these actions. The discussions were aimed at knowledge sharing, consensus building and developing a shared base for the initiation of international adaptation projects and knowledge sharing networks on resilience and adaptation options

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation

The E3 ligase parkin ubiquitinates outer mitochondrial membrane proteins during oxidative stress and is linked to early-onset Parkinson’s disease. Parkin is autoinhibited but is activated by the kinase PINK1 that phosphorylates ubiquitin leading to parkin recruitment, and stimulates phosphorylation of parkin’s N-terminal ubiquitin-like (pUbl) domain. How these events alter the structure of parkin to allow recruitment of an E2~Ub conjugate and enhanced ubiquitination is an unresolved question. We present a model of an E2~Ub conjugate bound to the phosphoubiquitin-loaded C-terminus of parkin, derived from NMR chemical shift perturbation experiments. We show the UbcH7~Ub conjugate binds in the open state whereby conjugated ubiquitin binds to the RING1/IBR interface. Further, NMR and mass spectrometry experiments indicate the RING0/RING2 interface is re-modelled, remote from the E2 binding site, and this alters the reactivity of the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer. Our experiments provide evidence that parkin phosphorylation and E2~Ub recruitment act synergistically to enhance a weak interaction of the pUbl domain with the RING0 domain and rearrange the location of the RING2(Rcat) domain to drive parkin activity

Methodological challenges when carrying out research on CKD and AKI using routine electronic health records.

Research regarding chronic kidney disease (CKD) and acute kidney injury (AKI) using routinely collected data presents particular challenges. The availability, consistency, and quality of renal data in electronic health records has changed over time with developments in policy, practice incentives, clinical knowledge, and associated guideline changes. Epidemiologic research may be affected by patchy data resulting in an unrepresentative sample, selection bias, misclassification, and confounding by factors associated with testing for and recognition of reduced kidney function. We systematically explore the issues that may arise in study design and interpretation when using routine data sources for CKD and AKI research. First, we discuss how access to health care and management of patients with CKD may have an impact on defining the target population for epidemiologic study. We then consider how testing and recognition of CKD and AKI may lead to biases and how to potentially mitigate against these. Illustrative examples from our own research within the UK are used to clarify key points. Any research using routine renal data has to consider the local clinical context to achieve meaningful interpretation of the study findings

A palaeoecological investigation of long-term stand-scale ecological dynamics in semi-open native pine woods : contributing to conservation management in east Glen Affric

This thesis investigates past structure and dynamics of native Caledonian pine woodland, representing part of the western fringes of the northern European boreal woodlands. The biogeographical extent and Holocene history of the Scottish pine woods are well studied, yet questions remain at finer scales. This thesis is concerned with two factors over the recent Holocene oceanic period; (i) the long-term ecology within the woods; the spatio-temporal dynamics, the canopy structure, and community composition and continuity; and (ii) the former extent of the woods, especially the temporal pattern of the inferred easterly contraction in woodland over recent history. The importance of these factors - to ecologists, challenged with understanding the theories of equilibrium and non-equilibrium processes in long-lived woodland communities - and to conservation managers - challenged, by policy directives, with implementing the restoration and expansion of native woodlands - is discussed. Equally the identification and quantification of ecological detail over ecologically relevant temporal and spatial scales is an important challenge for palaeoecology. The thesis therefore applies fine spatial resolution pollen analyses over a network of sites, within, and at the western edge of, the extant woodland zone in east Glen Affric. Correlation between these individual site histories develops a detailed view of the grain and extent of woodland within the landscape, previously missing from western pine woods. Careful attention is paid to the interpretive potential and limitations of fine resolution palaeoecology; especially with regard (i) to techniques that can identify and spatially quantify stand-scale structure and community composition via reference to modern analogues; and (ii) to defining chronologies and elucidating rates and patterns of temporal change. The opportunities and limitations of the technique are explored and discussed, to ensure an understanding of the rigour and potential of the palaeoecological contribution to ecological research and to provide an evidence base for conservation. The application of 210Pb dating using the CRS and CIC models is explored. The CRS model is confirmed as suitable for peat deposits, but its use may mask fluctuations in peat sedimentation rate, which may be illustrated by the CIC model. The value of multiple 14C assays for each core and a need for a new approach to chronologies for application to fine-scale palaeecological studies is discussed. The relevant source area around the small basins in this semi-open pinewood is tentatively confirmed at 20 m from the pollen source. Tentative pollen productivity estimates for five key taxa in this ecosystem are presented. The temporal stability of native woodland in Glen Affric is confirmed to the eastern part of the extant woodland zone; but challenged to the west, where the open and semi-open landscape has a long history. Woodland diversity decreased over the last c. 200 years, and past woodland also shows a greater ground flora diversity. The western extent of the Caledonian woodland in this landscape may have changed little in the last c. 4000 years. Fluctuations, some clearly cyclical, in heath, and in woodland, communities are identified in the pollen record. The former may be aligned to changes in grazing regime or climatic shifts; and the latter to autochthonous shifts important in the maintenance of suitable edaphic conditions for the continuity of woodland. Former woodland is confirmed as likely to have been open in structure and mixed in tree species composition. The results presented here suggest that some caution should be applied to use of the term Caledonian, or native ‘pine’ forest: ‘Caledonian forest’ may better reflect the heterogeneity of past forests, particularly the importance, and persistence, of birch. The implications for conservation management and restoration are discussed. It may be difficult to establish a sustainable woodland to the west of the extant stands, and any pine woodland here may need to be mixed with stands of broadleaved trees to maintain or restore soil structure and ecological function. The landscape to the west may have been open for several thousands of years, and consideration of this is required when managing for the future to prevent loss of biodiversity.EThOS - Electronic Theses Online ServiceUniversity of Stirling : Forest ResearchGBUnited Kingdo

Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis

The PARK2 gene is mutated in 50% of autosomal recessive juvenile parkinsonism (ARJP) cases. It encodes parkin, an E3 ubiquitin ligase of the RBR family. Parkin exists in an autoinhibited state that is activated by phosphorylation of its N‐terminal ubiquitin‐like (Ubl) domain and binding of phosphoubiquitin. We describe the 1.8 Å crystal structure of human parkin in its fully inhibited state and identify the key interfaces to maintain parkin inhibition. We identify the phosphoubiquitin‐binding interface, provide a model for the phosphoubiquitin–parkin complex and show how phosphorylation of the Ubl domain primes parkin for optimal phosphoubiquitin binding. Furthermore, we demonstrate that the addition of phosphoubiquitin leads to displacement of the Ubl domain through loss of structure, unveiling a ubiquitin‐binding site used by the E2~Ub conjugate, thus leading to active parkin. We find the role of the Ubl domain is to prevent parkin activity in the absence of the phosphorylation signals, and propose a model for parkin inhibition, optimization for phosphoubiquitin recruitment, release of inhibition by the Ubl domain and engagement with an E2~Ub conjugate. Taken together, this model provides a mechanistic framework for activating parkin

Relating dung fungal spore influx rates to animal density in a temperate environment: Implications for palaeoecological studies

The management of the remainder of Europe’s once extensive forests is hampered by a poor understanding of the character of the vegetation and drivers of change before the onset of clearance for farming. Pollen data indicate a closed-canopy, mixed-deciduous forest, contrasting with the assertion that large herbivores would have maintained a mosaic of open grassland, regenerating scrub and forested groves. Coprophilous fungal spores from sedimentary sequences are increasingly used as a proxy for past herbivore impact on vegetation, but the method faces methodological and taphonomical issues. Using pollen trap data from a long-running experiment in Chillingham Wild Cattle Park, UK, we investigate the first steps in the mechanisms connecting herbivore density to the incorporation of fungal spores in sediments and assess the effects of environmental variables on this relationship. Herbivore utilisation levels correlate with dung fungal spore abundance. Chillingham is densely populated by large herbivores, but dung fungal spore influx is low. Herbivores may thus be present on the landscape but go undetected. The absence of dung fungal spores is therefore less informative than their presence. Dung fungal spores likely enter the sediment record through a different pathway from wind-borne pollen and thus dung fungal abundance is better expressed as influx rates than as percentage of total pollen. Landscape openness, vegetation type and site wetness do not distort the impact of utilisation levels on dung fungal spore representation. However, dung fungal spore influx varies markedly between seasons and years. Spores travel, leading to a background level of spore deposition across the landscape, and at times a depletion of spores, especially under wet weather conditions. Animal behaviour, as well as husbandry practices, can lead to the accumulation of dung, and thus fungal spores, in specific locations on the landscape that do not directly reflect grazing pressure