Regulation of bZIP Transcription Factors by Phosphorylation

Genexpressionsmuster sind einer ständigen Veränderung unterworfen, einerseits aufgrund von Stresssituation, andererseits aber auch als Teil des normalen Entwicklungszyklus. Eine schnelle Änderung der Aktivität eines Transkriptionsfaktors (TF) ist notwendig, um schnelle Reaktion auf veränderte Umweltbedingungen oder Zustände in der Zelle zu ermöglichen. Ein Mechanismus um die Aktivität schnell anzupassen ist Phosphorylierung. Sie kann Einfluss auf die Lokalisierung, Stabilität, DNA Bindung oder Protein-Protein Interaktion des TF haben. BZIP (basic leucine zipper) TF sind an der Regulation diverser Prozesse beteiligt und wurden in allen Eukaryoten gefunden. Das Genom von Arabidopsis thaliana codiert für ca. 75 davon. C/S1 Gruppen Mitglieder könnten eine Rolle bei der Regulation der Genexpression als Reaktion auf Energie Mangel und Stress aber auch in der Samenreifung spielen. Im Zusammenhang mit Energie Mangel wurden sie bereits mit AKIN10 und AKIN11 in Verbindung gebracht, 2 Kinasen für die eine zentrale Rolle in der Energie-Mangel-Signaltransduktion vorgeschlagen wird. Das legt nahe, dass Phosphorylierung eine Rolle für die Aktivität des TF spielt. Hier zeige ich, dass bZIP63, ein Mitglied der C-Gruppe, in planta phosphoryliert ist. Dafür wurden bZIP63-GFP Überexpressor Pflanzen in zwei unterschiedlichen Ansätzen verwendet: 2D gel Elektrophorese in Kombination mit Western blots und Immunpräzipitation gefolgt von Massenspektrometrie (MS) Analyse. Auf Western blots von Protein Extrakten nach 2D Elektrophorese kann man einen Shift des Signals von bZIP63-GFP zum höheren pH Bereich beobachten, was auf in vivo Phosphorylierung hindeutet. Immunpräzipitation und MS Analyse von bZIP63-GFP aus im Dunklen geernteten Blättern führte zur Identifikation von 4 in vivo Phosphorylierungsstellen auf dem Protein unter diesen Bedingungen. In-Gel Kinase Assays wurden verwendet um potentielle bZIP63 spezifische Kinasen zu identifizieren. Es gab 3 starke Signale, was auf Beteiligung von mindestens 3 Kinasen hinweist. Zur Identifikation der Kinasen wurden Proteine mit einer hohen Affinität zu bZIP63 durch Affinitätsreinigung im Proteinextrakte angereichert, bevor dieser in In-Gel Kinase Assays verwendet wurde. Im Bereich des Signals wurden Gelstücke ausgeschnitten und darin enthaltene Proteine durch MS identifiziert. 3 Gruppen von Kinasen wurden gefunden. 2 Mitglieder der SnRK1 Familie, AKIN10 und AKIN11, wurden identifiziert. CDPKs (calcium dependent protein kinases) waren mit CPK3 und einigen anderen Mitgliedern vertreten, die jedoch aufgrund schlechter Proteinabdeckung nicht eindeutig identifiziert werden konnten. Die dritte Gruppe von Kinasen ist Casein Kinase II, von der 2 katalytische Untereinheiten, CKA1 und CKA2, gefunden wurden. Zusammengefasst war es mir möglich mehrere in vivo Phosphorylierungsstellen und potentielle bZIP63 spezifische Kinasen zu identifizieren und die Basis für weitere Untersuchungen über die Funktion der Phosphorylierung zu legen.Gene expression patterns change constantly in response to stress, but also as part of the normal developmental cycle. Rapid change in the activity of a TF (transcription factor) is essential to allow fast responses to changing environmental or intracellular conditions. One mechanism to modulate TF activity rapidly is phosphorylation, which can affect localisation, stability, DNA binding or protein–protein interaction of the TF. BZIP (basic leucine zipper) transcription factors are involved in regulation of numerous processes and can be found in all eukaryotes. The Arabidopsis thaliana genome encodes about 75 of them. The C/S1 group members are proposed to play a role in reprogramming gene expression in response to energy deprivation and stress as well as in seed maturation. In the context of energy deprivation they were already associated with AKIN10 and AKIN11, 2 kinases proposed to be central regulators of energy deprivation signalling. This suggested a role for protein phosphorylation in the activity of these transcription factors. Here I show that bZIP63, a member of the C–group, is phosphorylated in planta using bZIP63–GFP over–expressor plants in two different approaches: 2D gel electrophoresis in combination with Western blotting and Immunprecipitation followed by Mass spectrometry analysis. On Western blots of protein extracts after 2D separation the signal from bZIP63–GFP shifts to higher pH ranges when the extract is treated with phosphatase, indicating in vivo phosphorylation of the protein. Immunprecipitation and Mass spectrometry analysis of bZIP63–GFP from leaves collected in the dark revealed 4 in vivo phosphorylation sites on the protein under these conditions. In–gel kinase assays were used to identify potential bZIP63 specific kinases. 3 strong signals were obtained from total protein extracts, indicating a role for at least 3 kinases. For identification of the kinases protein extracts were enriched for proteins with a high affinity for bZIP63 via affinity purification before use in the in–gel kinase assay. Gel slices were cut out in the range of the signal and proteins identified by Mass spectrometry. 3 groups of protein kinases were found in the samples. Two members of the SnRK1 (SNF1 (Sucrose non–fermenting1) related kinases) family, AKIN10 and AKIN11 were identified. CDPKs (calcium dependent protein kinases) were represented by CPK3 and some other members, but protein coverage for these was too low for clear identification. The third group of kinases is Casein Kinase II, of which two catalytic subunits, CKA1 and CKA2 were found. Taken together, I could identify several in vivo phosphorylation sites on bZIP63 and potential bZIP63 specific kinases clearing the way for further investigation on function of bZIP63 phosphorylation

Interventions by healthcare professionals to improve management of physical long-term conditions in adults who are homeless: a systematic review protocol

Introduction People experiencing homelessness are at increased risk of, and have poorer outcomes from, a range of physical long-term conditions (LTCs). It is increasingly recognised that interventions targeting people who are homeless should be tailored to the specific needs of this population. This systematic review aims to identify, describe and appraise trials of interventions that aim to manage physical LTCs in homeless adults and are delivered by healthcare professionals. Methods and analysis Seven electronic databases (Medline, EMBASE, Cochrane Central Register of Controlled Trials, Assia, Scopus, PsycINFO and CINAHL) will be searched from 1960 (or inception) to October 2016 and supplemented by forward citation searching, handsearching of reference lists and searching grey literature. Two reviewers will independently review titles, abstract and full-texts using DistillerSR software. Inclusion criteria include (1) homeless adults with any physical LTC, (2) interventions delivered by a healthcare professional (any professional trained to provide any form of healthcare, but excluding social workers and professionals without health-related training), (3) comparison with usual care or an alternative intervention, (4) report outcomes such as healthcare usage, physical and psychological health or well-being or cost-effectiveness, (5) randomised controlled trials, non-randomised controlled trials, controlled before-after studies. Quality will be assessed using the Cochrane EPOC Risk of Bias Tool. A meta-analysis will be performed if sufficient data are identified; however, we anticipate a narrative synthesis will be performed. Ethics and dissemination This review will synthesise existing evidence for interventions delivered by healthcare professionals to manage physical LTCs in adults who are homeless. The findings will inform the development of future interventions and research aiming to improve the management of LTCs for people experiencing homelessness. Ethical approval will not be required for this systematic review as it does not contain individual patient data. We will disseminate the results of this systematic review via conference presentations, healthcare professional networks, social media and peer-reviewed publication

A systematic review of interventions by healthcare professionals to improve management of non-communicable diseases and communicable diseases requiring long-term care in adults who are homeless

Objective: Identify, describe and appraise trials of interventions delivered by healthcare professionals to manage non-communicable diseases (NCDs) and communicable diseases that require long-term care or treatment (LT-CDs), excluding mental health and substance use disorders, in homeless adults. Design: Systematic review of randomised controlled trials (RCTs), non-RCTs and controlled before–after studies. Interventions characterised using Effective Practice and Organisation of Care (EPOC) taxonomy. Quality assessed using EPOC risk of bias criteria. Data sources: Database searches (MEDLINE, Embase, PsycINFO, Scopus, Cumulative Index to Nursing and Allied Health Literature (CINAHL), Applied Social Sciences Index and Abstracts (ASSIA) and Cochrane Central Register of Controlled Trials), hand searching reference lists, citation searches, grey literature and contact with study authors. Setting: Community. Participants: Adults (≥18 years) fulfilling European Typology of Homelessness criteria. Intervention: Delivered by healthcare professionals managing NCD and LT-CDs. Outcomes: Primary outcome: unscheduled healthcare utilisation. Secondary outcomes: mortality, biological markers of disease control, adherence to treatment, engagement in care, patient satisfaction, knowledge, self-efficacy, quality of life and cost-effectiveness. Results: 11 studies were included (8 RCTs, 2 quasi-experimental and 1 feasibility) involving 9–520 participants (67%–94% male, median age 37–49 years). Ten from USA and one from UK. Studies included various NCDs (n=3); or focused on latent tuberculosis (n=4); HIV (n=2); hepatitis C (n=1) or type 2 diabetes (n=1). All interventions were complex with multiple components. Four described theories underpinning intervention. Three assessed unscheduled healthcare utilisation: none showed consistent reduction in hospitalisation or emergency department attendance. Six assessed adherence to specific treatments, of which four showed improved adherence to latent tuberculosis therapy. Three concerned education case management, all of which improved disease-specific knowledge. No improvements in biological markers of disease (two studies) and none assessed mortality. Conclusions: Evidence for management of NCD and LT-CDs in homeless adults is sparse. Educational case-management interventions may improve knowledge and medication adherence. Large trials of theory-based interventions are needed, assessing healthcare utilisation and outcomes as well as assessment of biological outcomes and cost-effectiveness

Building response to tunnelling

AbstractUnderstanding how buildings respond to tunnelling-induced ground movements is an area of great importance for urban tunnelling projects, particularly for risk management. In this paper, observations of building response to tunnelling, from both centrifuge modelling and a field study in Bologna, are used to identify mechanisms governing the soil–structure interaction. Centrifuge modelling was carried out on an 8-m-diameter beam centrifuge at Cambridge University, with buildings being modelled as highly simplified elastic and inelastic beams of varying stiffness and geometry. The Bologna case study presents the response of two different buildings to the construction of a sprayed concrete lining (SCL) tunnel, 12m in diameter, with jet grouting and face reinforcement.In both studies, a comparison of the building settlement and horizontal displacement profiles, with the greenfield ground movements, enables the soil structure interaction to be quantified. Encouraging agreement between the modification to the greenfield settlement profile, displayed by the buildings, and estimates made from existing predictive tools is observed. Similarly, both studies indicate that the horizontal strains, induced in the buildings, are typically at least an order of magnitude smaller than the greenfield values. This is consistent with observations in the literature. The potential modification to the settlement distortions is shown to have significant implications on the estimated level of damage. Potential issues for infrastructures connected to buildings, arising from the embedment of rigid buildings into the soil, are also highlighted

A systematic review of interventions by healthcare professionals to improve management of non-communicable diseases and communicable diseases requiring long-term care in adults who are homeless

AbstractObjective Identify, describe and appraise trials of interventions delivered by healthcare professionals to manage non-communicable diseases (NCDs) and communicable diseases that require long-term care or treatment (LT-CDs), excluding mental health and substance use disorders, in homeless adults.Design Systematic review of randomised controlled trials (RCTs), non-RCTs and controlled before–after studies. Interventions characterised using Effective Practice and Organisation of Care (EPOC) taxonomy. Quality assessed using EPOC risk of bias criteria.Data sources Database searches (MEDLINE, Embase, PsycINFO, Scopus, Cumulative Index to Nursing and Allied Health Literature (CINAHL), Applied Social Sciences Index and Abstracts (ASSIA) and Cochrane Central Register of Controlled Trials), hand searching reference lists, citation searches, grey literature and contact with study authors.Setting Community.Participants Adults (≥18 years) fulfilling European Typology of Homelessness criteria.Intervention Delivered by healthcare professionals managing NCD and LT-CDs.Outcomes Primary outcome: unscheduled healthcare utilisation. Secondary outcomes: mortality, biological markers of disease control, adherence to treatment, engagement in care, patient satisfaction, knowledge, self-efficacy, quality of life and cost-effectiveness.Results 11 studies were included (8 RCTs, 2 quasi-experimental and 1 feasibility) involving 9–520 participants (67%–94% male, median age 37–49 years). Ten from USA and one from UK. Studies included various NCDs (n=3); or focused on latent tuberculosis (n=4); HIV (n=2); hepatitis C (n=1) or type 2 diabetes (n=1). All interventions were complex with multiple components. Four described theories underpinning intervention. Three assessed unscheduled healthcare utilisation: none showed consistent reduction in hospitalisation or emergency department attendance. Six assessed adherence to specific treatments, of which four showed improved adherence to latent tuberculosis therapy. Three concerned education case management, all of which improved disease-specific knowledge. No improvements in biological markers of disease (two studies) and none assessed mortality.Conclusions Evidence for management of NCD and LT-CDs in homeless adults is sparse. Educational case-management interventions may improve knowledge and medication adherence. Large trials of theory-based interventions are needed, assessing healthcare utilisation and outcomes as well as assessment of biological outcomes and cost-effectiveness

Development of multi-purposes procedures and service tools for GNSS data processing finalized to monitor a deep-seated earthslide in the Dolomites (Italy)

The Corvara landslide is an active, large-scale, deep-seated and slow moving earthslide of about 30 Mm3 located in the Dolomites (Italy). It is frequently damaging a national road and, occasionally, isolated buildings and recre- ational ski facilities. In this work we present the analysis performed on data acquired thank to the installation of 3 DualFrequency GPS in permanent acquisition installed in the accumulation, track and source zone of the active portion of the landslide. In particular two years (2014 and 2015) of data were processed with several approaches and goals: daily time series were produced through Precise Point Positioning and Differential Positioning using both scientific packages and automatic on line tool based on open source libraries, specifically developed in order to provide a prototypal service. The achievable results based on single frequency (L1) data processing were also investigated in order to pave the way to the deployment of lowcost GPS receiver for this kind of application. Moreover, daily and sub-daily phenomena were analyzed. Different strategies were investigated in order to de- scribe the kinematics on the basis of 0.2 Hz data collected by the 3 permanent receivers. For particular events also the variometric approach, through the recent advances of VADASE, was applied, to detect significant movements. Finally, tropospheric parameters were estimated over the whole period in order to give a contribution to the SAR interferometry techniques. Also for this specific purpose and application, the possibilities of single frequency use were assessed

Porcine CD8αdim/-NKp46high NK cells are in a highly activated state

Natural Killer (NK) cells play a crucial role in the early phase of immune responses against various pathogens. In swine so far only little information about this lymphocyte population exists. Phenotypical analyses with newly developed monoclonal antibodies (mAbs) against porcine NKp46 recently revealed that in blood NKp46(-) and NKp46(+) cells with NK phenotype exist with comparable cytotoxic properties. In spleen a third NKp46-defined population with NK phenotype was observed that was characterised by a low to negative CD8α and increased NKp46 expression. In the current study it is shown that this NKp46(high) phenotype was correlated with an increased expression of CD16 and CD27 compared to the CD8α(+)NKp46(-) and NKp46(+) NK-cell subsets in spleen and blood. Additionally NKp46(high) NK cells expressed elevated levels of the chemokine receptor CXCR3 on mRNA level. Functional analyses revealed that splenic NKp46(high) NK cells produced much higher levels of Interferon-γ and Tumor Necrosis Factor-α upon stimulation with cytokines or phorbol-12-myristate-13-acetate/Ionomycin compared to the other two subsets. Furthermore, cross-linking of NKp46 by NKp46-specific mAbs led to a superior CD107a expression in the NKp46(high) NK cells, thus indicating a higher cytolytic capacity of this subset. Therefore porcine splenic NKp46(high) NK cells represent a highly activated subset of NK cells and may play a profound role in the immune surveillance of this organ

SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants

Metabolic adjustment to changing environmental conditions, particularly balancing of growth and defense responses, is crucial for all organisms to survive. The evolutionary conserved AMPK/Snf1/SnRK1 kinases are well-known metabolic master regulators in the low-energy response in animals, yeast and plants. They act at two different levels: by modulating the activity of key metabolic enzymes, and by massive transcriptional reprogramming. While the first part is well established, the latter function is only partially understood in animals and not at all in plants. Here we identified the Arabidopsis transcription factor bZIP63 as key regulator of the starvation response and direct target of the SnRK1 kinase. Phosphorylation of bZIP63 by SnRK1 changed its dimerization preference, thereby affecting target gene expression and ultimately primary metabolism. A bzip63 knock-out mutant exhibited starvation-related phenotypes, which could be functionally complemented by wild type bZIP63, but not by a version harboring point mutations in the identified SnRK1 target sites