| 1. |
- André, Ingemar, et al.
(author)
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Residue-specific pK(a) determination of lysine and arginine side chains by indirect N-15 and C-13 NMR spectroscopy: Application to apo calmodulin
- 2007
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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 129:51, s. 15805-15813
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Journal article (peer-reviewed)abstract
- Electrostatic interactions in proteins can be probed experimentally through determination of residue-specific acidity constants, We describe here triple-resonance NMR techniques for direct determination of lysine and arginine side-chain protonation states in proteins. The experiments are based on detection of nonexchangeable protons over the full range of pH and temperature and therefore are well suited for pK(a) determination of individual amino acid side chains. The experiments follow the side-chain N-15(zeta) (lysine) and N-15(epsilon) or C-13(zeta) (arginine) chemical shift, which changes due to sizable changes in the heteronuclear electron distribution upon (de)protonation. Since heteronuclear chemical shifts are overwhelmed by the charge state of the amino acid side chain itself, these methods supersede H-1-based NMR in terms of accuracy, sensitivity, and selectivity. Moreover, the N-15(zeta) and N-15(epsilon) nuclei may be used to probe changes in the local electrostatic environment. Applications to three proteins are described: apo calmodulin, calbindin D-9k, and FKBP12. For apo calmodulin, residue-specific pK(a) values of lysine side chains were determined to fall between 10.7 and 11.2 as a result of the high net negative charge on the protein surface. Ideal two-state titration behavior observed for all lysines indicates the absence of significant direct charge interactions between the basic residues. These results are compared with earlier studies based on chemical modification.
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| 2. |
- Burkhard, Benjamin, et al.
(author)
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Mapping and assessing ecosystem services in the EU - Lessons learned from the ESMERALDA approach of integration
- 2018
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In: One Ecosystem. - : Pensoft Publishers. - 2367-8194. ; 3
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Journal article (peer-reviewed)abstract
- The European Union (EU) Horizon 2020 Coordination and Support Action ESMERALDA aimed at developing guidance and a flexible methodology for Mapping and Assessment of Ecosystems and their Services (MAES) to support the EU member states in the implementation of the EU Biodiversity Strategy’s Target 2 Action 5. ESMERALDA’s key tasks included network creation, stakeholder engagement, enhancing ecosystem services mapping and assessment methods across various spatial scales and value domains, work in case studies and support of EU member states in MAES implementation. Thus ESMERALDA aimed at integrating various project outcomes around four major strands: i) Networking, ii) Policy, iii) Research and iv) Application. The objective was to provide guidance for integrated ecosystem service mapping and assessment that can be used for sustainable decision-making in policy, business, society, practice and science at EU, national and regional levels. This article presents the overall ESMERALDA approach of integrating the above-mentioned project components and outcomes and provides an overview of how the enhanced methods were applied and how they can be used to support MAES implementation in the EU member states. Experiences with implementing such a large pan-European Coordination and Support Action in the context of EU policy are discussed and recommendations for future actions are given.
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| 3. |
- Cataldo, Luis Rodrigo, et al.
(author)
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The MafA-target gene PPP1R1A regulates GLP1R-mediated amplification of glucose-stimulated insulin secretion in β-cells
- 2021
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In: Metabolism: Clinical and Experimental. - : Elsevier BV. - 1532-8600.
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Journal article (peer-reviewed)abstract
- The amplification of glucose-stimulated insulin secretion (GSIS) through incretin signaling is critical for maintaining physiological glucose levels. Incretins, like glucagon-like peptide 1 (GLP1), are a target of type 2 diabetes drugs aiming to enhance insulin secretion. Here we show that the protein phosphatase 1 inhibitor protein 1A (PPP1R1A), is expressed in β-cells and that its expression is reduced in dysfunctional β-cells lacking MafA and upon acute MafA knock down. MafA is a central regulator of GSIS and β-cell function. We observed a strong correlation of MAFA and PPP1R1A mRNA levels in human islets, moreover, PPP1R1A mRNA levels were reduced in type 2 diabetic islets and positively correlated with GLP1-mediated GSIS amplification. PPP1R1A silencing in β-cell lines impaired GSIS amplification, PKA-target protein phosphorylation, mitochondrial coupling efficiency and also the expression of critical β-cell marker genes like MafA, Pdx1, NeuroD1 and Pax6. Our results demonstrate that the β-cell transcription factor MafA is required for PPP1R1A expression and that reduced β-cell PPP1R1A levels impaired β-cell function and contributed to β-cell dedifferentiation during type 2 diabetes. Loss of PPP1R1A in type 2 diabetic β-cells may explains the unresponsiveness of type 2 diabetic patients to GLP1R-based treatments.
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| 4. |
- Geneletti, Davide, et al.
(author)
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Ecosystem services mapping and assessment for policy- and decision-making : Lessons learned from a comparative analysis of European case studies
- 2020
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In: One Ecosystem. - : Pensoft Publishers. - 2367-8194. ; 5, s. e53111-
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Journal article (peer-reviewed)abstract
- This paper analyses and compares a set of case studies on ecosystem services (ES) mapping and assessment with the purpose of formulating lessons learned and recommendations. Fourteen case studies were selected during the EU Horizon 2020 “Coordination and Support Action” ESMERALDA to represent different policy- and decision-making processes throughout the European Union, across a wide range of themes, biomes and scales. The analysis is based on a framework that addresses the key steps of an ES mapping and assessment process, namely policy questions, stakeholder identification and involvement, application of mapping and assessment methods, dissemination and communication and implementation. The analysis revealed that most case studies were policy-orientated or gave explicit suggestions for policy implementation in different contexts, including urban, rural and natural areas. Amongst the findings, the importance of starting stakeholder engagement early in the process was confirmed in order to generate interest and confidence in the project and to increase their willingness to cooperate. Concerning mapping and assessment methods, it was found that the integration of methods and results is essential for providing a comprehensive overview from different perspectives (e.g. social, economic). Finally, lessons learned for effective implementation of ES mapping and assessment results are presented and discussed.
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| 5. |
- Goehring, Isabel, et al.
(author)
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Plasma Membrane Potential Oscillations in Insulin Secreting Ins-1 832/13 Cells do not Require Glycolysis and are not Initiated by Fluctuations in Mitochondrial Bioenergetics.
- 2012
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In: Journal of Biological Chemistry. - 1083-351X. ; 287:19, s. 15706-15717
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Journal article (peer-reviewed)abstract
- Oscillations in plasma membrane potential play a central role in glucose-induced insulin secretion from pancreatic β-cells and related insulinoma cell lines. We have employed a novel fluorescent plasma membrane potential (ψp) indicator in combination with indicators of cytoplasmic free Ca2+ ([Ca2+]c), mitochondrial membrane potential (ψm), matrix ATP concentration and NAD(P)H fluorescence to investigate the role of mitochondria in the generation of plasma membrane potential oscillations in clonal INS-1 832/13 β-cells. Elevated glucose caused oscillations in plasma membrane potential and cytoplasmic free Ca2+ concentration over the same concentration range required for insulin release, although considerable cell-to-cell heterogeneity was observed. Exogenous pyruvate was as effective as glucose in inducing oscillations, both in the presence and absence of 2.8mM glucose. Increased glucose and pyruvate each produced a concentration-dependent mitochondrial hyperpolarization. The causal relationships between pairs of parameters - ψp and [Ca2+]c, ψp and NAD(P)H, matrix ATP and [Ca2+]c, and ψm and [Ca2+]c were investigated at single cell level. It is concluded that, in these β-cells, depolarizing oscillations in ψp are not initiated by mitochondrial bioenergetic changes. Instead, regardless of substrate, it appears that the mitochondria may simply be required to exceed a critical bioenergetic threshold to allow release of insulin. Once this threshold is exceeded an autonomous ψp oscillatory mechanism is initiated.
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| 6. |
- Lindman, Stina, et al.
(author)
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Electrostatic contributions to residue-specific protonation equilibria and proton binding capacitance for a small protein
- 2006
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In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 45:47, s. 13993-14002
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Journal article (peer-reviewed)abstract
- Charge-charge interactions in proteins are important in a host of biological processes. Here we use C-13 NMR chemical shift data for individual aspartate and glutamate side chain carboxylate groups to accurately detect site-specific protonation equilibria in a variant of the B1 domain of protein G (PGB1-QDD). Carbon chemical shifts are dominated by changes in the electron distribution within the side chain and therefore excellent reporters of the charge state of individual groups, and the data are of high precision. We demonstrate that it is possible to detect local charge interactions within this small protein domain that stretch and skew the chemical shift titration curves away from "ideal" behavior and introduce a framework for the analysis of such convoluted data to study local charge-charge interactions and electrostatic coupling. It is found that, due to changes in electrostatic potential, the proton binding affinity, K-a, of each carboxyl group changes throughout the titration process and results in a linearly pH dependent pK(a) value. This result could be readily explained by calculations of direct charge-charge interactions based on Coulomb's law. In addition, the slope of pK(a) versus pH was dependent on screening by salt, and this dependence allowed the selective study of charge-charge interactions. For PGB1-QDD, it was established that mainly differences in self-energy, and not direct charge-charge interactions, are responsible for shifted pK(a) values within the protein environment.
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| 7. |
- Lindman, Stina, et al.
(author)
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pK(a) values for side-chain carboxyl groups of a PGB1 variant explain salt and pH-dependent stability
- 2007
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In: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 92:1, s. 257-266
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Journal article (peer-reviewed)abstract
- Determination of pK(a) values of titrating residues in proteins provides a direct means of studying electrostatic coupling as well as pH-dependent stability. The B1 domain of protein G provides an excellent model system for such investigations. In this work, we analyze the observed pK(a) values of all carboxyl groups in a variant of PGB1 (T2Q, N8D, N37D) at low and high ionic strength as determined using H-1-C-13 heteronuclear NMR in a structural context. The pK(a) values are used to calculate the pH-dependent stability in low and high salt and to investigate electrostatic coupling in the system. The observed pK(a) values can explain the pH dependence of protein stability but require pKa shifts relative to model values in the unfolded state, consistent with persistent residual structure in the denatured state. In particular, we find that most of the deviations from the expected random coil values can be explained by a significantly upshifted pK(a) value. We show also that C-13 backbone carbonyl data can be used to study electrostatic coupling in proteins and provide specific information on hydrogen bonding and electrostatic potential at nontitrating sites.
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| 8. |
- Lindman, Stina, et al.
(author)
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pK(a) Values for the Unfolded State under Native Conditions Explain the pH-Dependent Stability of PGB1.
- 2010
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In: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 99:10, s. 3365-3373
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Journal article (peer-reviewed)abstract
- Understanding the role of electrostatics in protein stability requires knowledge of these interactions in both the folded and unfolded states. Electrostatic interactions can be probed experimentally by characterizing ionization equilibria of titrating groups, parameterized as pK(a) values. However, pK(a) values of the unfolded state are rarely accessible under native conditions, where the unfolded state has a very low population. Here, we report pK(a) values under nondenaturing conditions for two unfolded fragments of the protein G B1 domain that mimic the unfolded state of the intact protein. pK(a) values were determined for carboxyl groups by monitoring their pH-dependent (13)C chemical shifts. Monte Carlo simulations using a Gaussian chain model provide corrections for changes in electrostatic interactions that arise from fragmentation of the protein. Most pK(a) values for the unfolded state agree well with model values, but some residues show significant perturbations that can be rationalized by local electrostatic interactions. The pH-dependent stability was calculated from the experimental pK(a) values of the folded and unfolded states and compared to experimental stability data. The use of experimental pK(a) values for the unfolded state results in significantly improved agreement with experimental data, as compared to calculations based on model data alone.
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| 9. |
- Mezheyeuski, Artur, et al.
(author)
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Survival-associated heterogeneity of marker-defined perivascular cells in colorectal cancer
- 2016
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In: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 7:27, s. 41948-41958
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Journal article (peer-reviewed)abstract
- Perivascular cells (PC) were recently implied as regulators of metastasis and immune cell activity. Perivascular heterogeneity in clinical samples, and associations with other tumor features and outcome, remain largely unknown.Here we report a novel method for digital quantitative analyses of vessel characteristics and PC, which was applied to two collections of human metastatic colorectal cancer (mCRC).Initial analyses identified marker-defined subsets of PC, including cells expressing PDGFR-β or α-SMA or both markers. PC subsets were largely independently expressed in a manner unrelated to vessel density and size. Association studies implied specific oncogenic mutations in malignant cells as determinants of PC status. Semi-quantitative and digital-image-analyses-based scoring of the NORDIC-VII cohort identified significant associations between low expression of perivascular PDGFR-α and -β and shorter overall survival. Analyses of the SPCRC cohort confirmed these findings. Perivascular PDGFR-α and -β remained independent factors for survival in multivariate analyses.Overall, our study identified host vasculature and oncogenic status as determinants of tumor perivascular features. Perivascular PDGFR-α and -β were identified as novel independent markers predicting survival in mCRC. The novel methodology should be suitable for similar analyses in other tumor collections.
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| 10. |
- O'Malley, Tiernan T., et al.
(author)
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A beta dimers differ from monomers in structural propensity, aggregation paths and population of synaptotoxic assemblies
- 2014
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In: Biochemical Journal. - 0264-6021. ; 461, s. 413-426
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Journal article (peer-reviewed)abstract
- Dimers of A beta (amyloid beta-protein) are believed to play an important role in Alzheimer's disease. In the absence of sufficient brain-derived dimers, we studied one of the only possible dimers that could be produced in vivo, [A beta](DiY) (dityrosine cross-linked A beta). For comparison, we used the A beta monomer and a design dimer cross-linked by replacement of Ser(26) with cystine [A beta S26C](2). We showed that similar to monomers, unaggregated dimers lack appreciable structure and fail to alter long-term potentiation. Importantly, dimers exhibit subtly different structural propensities from monomers and each other, and can self-associate to form larger assemblies. Although [A beta](DiY) and [A beta S26C](2) have distinct aggregation pathways, they both populate bioactive soluble assemblies for longer durations than A beta monomers. Our results indicate that the link between A beta dimers and Alzheimer's disease results from the ability of dimers to further assemble and form synaptotoxic assemblies that persist for long periods of time.
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| 11. |
- Van Bulck, Liesbet, et al.
(author)
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Patient-reported outcomes of adults with congenital heart disease from eight European countries : scrutinising the association with healthcare system performance
- 2019
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In: European Journal of Cardiovascular Nursing. - : Sage Publications. - 1474-5151 .- 1873-1953. ; 18:6, s. 465-473
-
Journal article (peer-reviewed)abstract
- Background: Inter-country variation in patient-reported outcomes of adults with congenital heart disease has been observed. Country-specific characteristics may play a role. A previous study found an association between healthcare system performance and patient-reported outcomes. However, it remains unknown which specific components of the countries’ healthcare system performance are of importance for patient-reported outcomes.Aims: The aim of this study was to investigate the relationship between components of healthcare system performance and patient-reported outcomes in a large sample of adults with congenital heart disease.Methods: A total of 1591 adults with congenital heart disease (median age 34 years; 51% men; 32% simple, 48% moderate and 20% complex defects) from eight European countries were included in this cross-sectional study. The following patient-reported outcomes were measured: perceived physical and mental health, psychological distress, health behaviours and quality of life. The Euro Health Consumer Index 2015 and the Euro Heart Index 2016 were used as measures of healthcare system performance. General linear mixed models were conducted, adjusting for patient-specific variables and unmeasured country differences.Results: Health risk behaviours were associated with the Euro Health Consumer Index subdomains about patient rights and information, health outcomes and financing and access to pharmaceuticals. Perceived physical health was associated with the Euro Health Consumer Index subdomain about prevention of chronic diseases. Subscales of the Euro Heart Index were not associated with patient-reported outcomes.Conclusion: Several features of healthcare system performance are associated with perceived physical health and health risk behaviour in adults with congenital heart disease. Before recommendations for policy-makers and clinicians can be conducted, future research ought to investigate the impact of the healthcare system performance on outcomes further.
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