bluuebunny/crossref_metadata_2025 huggingface dataset:
Fact and Fiction
Authors: Parrington John
Published: June 2021

This chapter assesses how much novels can reveal about the various mechanisms underlying human consciousness. Some might say, very little, and argue that only scientific study can uncover such mechanisms. However, because language plays such a key role in shaping human consciousness, the fictional explorations of the human condition that we find in novelistic literature can greatly add to our scientific understanding by concretizing that condition in its diverse forms. The chapter also explores a related question: how much do novels draw on new insights about the nature of consciousness, so increasing their ability to inform us about the human condition, and its relationship to changing forms of society? The best novels have a complexity and ambiguity of meaning that itself reflects the many contradictions in society and the individual psyche within that society. Importantly, this means there can be multiple readings of great novels, with different readers interpreting them in various ways. The chapter then examines several novels that have multiple interpretations and which also illuminate and enhance our understanding of different aspects of consciousness. These include Emily Brontë’s Wuthering Heights (1847), Henry James’s The Turn of the Screw (1898), William Golding’s Pincher Martin (1956), and Kazuo Ishiguro’s The Unconsoled (1995).
Pulling Apart
Authors: Casal Bértoa Fernando , Enyedi Zsolt
Published: June 2021

The ninth chapter considers polarization as a systemic property, manifested by the proportion of votes collected by anti-political-establishment parties. We find that virtually all party systems that collapsed had high levels of polarization. We also detect a clear tendency towards increasing polarization during the last half century, with important regional variations. According to our data, the degree of polarization is negatively related to closure, even though the systems that combined openness with high anti-political-establishment support have typically collapsed in the past in Europe. Low polarization appears to help the development of closure even in fragmented systems.
Conclusion
Authors: Casal Bértoa Fernando , Enyedi Zsolt
Published: June 2021

After comparing the different indicators (i.e. party institutionalization, fragmentation, polarization, volatility and closure) employed in the book, we show how the Cold War period (1945–1989) was exceptionally stable, especially in contrast to the inter-war and post-Berlin Wall periods. We also show how currently the West looks increasingly like the East in terms of the level of party institutionalization, while the East increasingly resembles the West in terms of closure. Actually, the West is becoming more polarized and fragmented than the East. In terms of parliamentary fragmentation, the party systems of the South and the East are converging towards each other, while the West is diverging from the rest of Europe with its increasingly high number of parties. Finally, the chapter summarizes the main findings of the book.
Quantum Field Theory and Critical Phenomena
Authors: Zinn-Justin Jean
Published: June 2021

Introduced as a quantum extension of Maxwell's classical theory, quantum electrodynamic (QED) has been the first example of a quantum field theory (QFT). Eventually, QFT has become the framework for the discussion of all fundamental interactions at the microscopic scale except, possibly, gravity. More surprisingly, it has also provided a framework for the understanding of second order phase transitions in statistical mechanics. In fact, as hopefully this work illustrates, QFT is the natural framework for the discussion of most systems involving an infinite number of degrees of freedom with local couplings. These systems range from cold Bose gases at the condensation temperature (about ten nanokelvin) to conventional phase transitions (from a few degrees to several hundred) and high energy particle physics up to a TeV, altogether more than twenty orders of magnitude in the energy scale. Therefore, although excellent textbooks about QFT had already been published, I thought, many years ago, that it might not be completely worthless to present a work in which the strong formal relations between particle physics and the theory of critical phenomena are systematically emphasized. This option explains some of the choices made in the presentation. A formulation in terms of field integrals has been adopted to study the properties of QFT. The language of partition and correlation functions has been used throughout, even in applications of QFT to particle physics. Renormalization and renormalization group (RG) properties are systematically discussed. The notion of effective field theory (EFT) and the emergence of renormalizable theories are described. The consequences for fine-tuning and triviality issue are emphasized. This fifth edition has been updated and fully revised.
Locus genii
Authors: Marr Alexander
Published: June 2021

Over the course of the early modern period, genius was transformed in criticism from a commonplace of ‘inclination’ into a powerful tool of aesthetic classification, signalling heightened creativity in a person or work of art. This chapter argues that genius as a concept emerges with particular strength in the writings of the Roger de Piles, specifically in his accounts of Peter Paul Rubens. Investigating the language, conceptual, and formal properties that de Piles associates with esprit and génie, it concludes that in his hands early modern ‘ingenuity’ was transformed into ‘genius’.
The Cultural Evolution of Creative Ideas and Social Innovations
Authors: Unrau Mike , Gabora Liane
Published: June 2021

We apply complex systems science to the study of social systems and show how a complex-systems-inspired theory of creativity, which is referred to as ‘honing theory’, provides insight into social innovation. We propose that creativity and social innovation are processes of self-organization that yield a lower-entropy state in worldviews, which are self-organizing webs of understanding. This allows us to offer a novel perspective on the evolution of technology, the role of creativity in cultural evolution and the manner in which creativity drives innovation in social systems, such as the economy. We also introduce creative destruction as having metaphoric relevance for a social system transition from entropy to negentropy, and offer a social innovation example addressing economic collapse and resilient reorganization. We conclude that concepts from complex systems theory, and particularly entropy, shed light on both creativity and social innovation and further our understanding of how innovation affects social systems, such as in cultural and economic change.
Superconducting State
Authors: Kresin Vladimir , Ovchinnikov Sergei , Wolf Stuart
Published: June 2021

For the past almost fifty years, scientists have been trying to explain the phenomenon of superconductivity. The mechanism is the key ingredient of microscopic theory, which was developed by Bardeen, Cooper, and Schrieffer in 1957. The theory also introduced the basic concepts of pairing, coherence length, energy gap, and so on. Since then, microscopic theory has undergone an intensive development. This book provides a very detailed theoretical treatment of the key mechanisms of superconductivity, including the current state of the art (phonons, magnons, plasmons). In addition, the book contains descriptions of the properties of the key superconducting compounds that are of the most interest for science and applications. For many years, there has been a search for new materials with higher values of the main parameters, such as the critical temperature and critical current. At present, the possibility of observing superconductivity at room temperature has become perfectly realistic. That is why the book is especially concerned with high-Tc systems such as high-Tc oxides, hydrides with record values for critical temperature under high pressure, nanoclusters, and so on. A number of interesting novel superconducting systems have been discovered recently, including topological materials, interface systems, and intercalated graphene. The book contains rigorous derivations based on statistical mechanics and many-body theory. The book also provides qualitative explanations of the main concepts and results. This makes the book accessible and interesting for a broad audience.
Normative Belief and Motivation
Authors: Gregory Alex
Published: June 2021

This chapter begins by explaining and endorsing the view that normative beliefs can motivate us to act. Views in this vicinity are normally labelled as versions of ‘judgement internalism’, but I argue that that view has often been understood in ways that render it less plausible. The chapter then combines this claim about the motivational power of normative belief with ODM (Only Desires Motivate) in order to show that desires and reasons beliefs are the same state of mind. This argument is often thought to be an argument for non-cognitivism, which reduces normative beliefs to desires, but the chapter shows that non-cognitivism makes poor sense of normative beliefs about other people, and so the argument instead strongly favours desire-as-belief. The chapter ends by briefly showing how this argument against non-cognitivism and in favour of desire-as-belief connects in a natural way to the Frege-Geach problem for non-cognitivism.
Fair Opportunity and Responsibility
Authors: Brink David O.
Published: June 2021

Fair Opportunity and Responsibility lies at the intersection of moral psychology and criminal jurisprudence and analyzes responsibility and its relations to desert, culpability, excuse, blame, and punishment. It links responsibility with the reactive attitudes but makes the justification of the reactive attitudes depend on a response-independent conception of responsibility. Responsibility and excuse are inversely related; an agent is responsible for misconduct if and only if it is not excused. Consequently, we can study responsibility by understanding excuses. We excuse misconduct when an agent’s capacities or opportunities are significantly impaired, because these capacities and opportunities are essential if agents are to have a fair opportunity to avoid wrongdoing. This conception of excuse tells us that responsibility itself consists in agents having suitable cognitive and volitional capacities—normative competence—and a fair opportunity to exercise these capacities free from undue interference—situational control. Because our reactive attitudes and practices presuppose the fair opportunity conception of responsibility, this supports a predominantly retributive conception of blame and punishment that treats culpable wrongdoing as the desert basis of blame and punishment. We can then apply the fair opportunity framework to assessing responsibility and excuse in circumstances of structural injustice, situational influences in ordinary circumstances and in wartime, insanity and psychopathy, immaturity, addiction, and crimes of passion. Though fair opportunity has important implications for each issue, treating them together allows us to explore common themes and appreciate the need to take partial responsibility and excuse seriously in our practices of blame and punishment.
Prolegomena
Authors: Brink David O.
Published: June 2021

The main themes about the reactive attitudes, fair opportunity, and desert are introduced. The potential fragility of fair opportunity is explored. Cases of partial responsibility and excuse are introduced. The chapter concludes with methodological remarks about combining moral psychology and criminal law perspectives, disclaimers, remarks about the origins of the book, and acknowledgements.
The Fair Opportunity Conception of Responsibility
Authors: Brink David O.
Published: June 2021

The fair opportunity conception of responsibility is articulated and defended by appeal to common assumptions about the nature of excuses. Fair opportunity requires agential capacities and opportunities in the form of normative competence and situational control. The cognitive and volitional capacities constitutive of normative competence require reasons-responsiveness, which comes in degrees. The person is the locus of responsibility
Fair Opportunity, Capacities, and Possibilities
Authors: Brink David O.
Published: June 2021

Though some compatibilists deny that responsibility requires alternate possibilities, fair opportunity requires the ability of wrongdoers to do otherwise. However, these alternate possibilities are not the ones precluded by determinism. Different kinds of capacities are distinguished—actual and potential, specific and general. We should be interested in actual capacities that are relatively specific. The relevant capacities can be identified via counterfactuals. In this chapter, Pereboom’s influential incompatibilist manipulation argument is examined and rejected.
Blame, Punishment, and Predominant Retributivism
Authors: Brink David O.
Published: June 2021

Blame is a directed attitude that involves a core aversive reaction based on a belief that the target is blameworthy but gives rise to a syndrome of familiar expressive and communicative functions. Punishment involves blame and sanctions. Retributivist essentials are explained and defended, and it is argued that retributivism must be part of the truth about the justification of punishment. Predominant retributivism should be preferred to pure retributivism.
Immaturity and Fair Opportunity
Authors: Brink David O.
Published: June 2021

There is a moral asymmetry between juvenile and adult offenders if, all else being equal, juveniles should be punished less for their offenses than their adult counterparts should be punished. The trend to transfer juveniles to adult criminal court denies this asymmetry. Developmental and democratic rationales for the asymmetry are distinguished, and the developmental rationale is shown to be more basic. The developmental rationale for the asymmetry is reflected in Supreme Court cases in the United States recognizing constitutional limitations on sentences for juvenile offenders. These cases pose interesting questions about the tension between categorical sentencing rules and individualized justice.
The Draughtsmen
Authors: Payk Marcus M.
Published: June 2021

Little is known about the formal crafting of the Paris Peace Treaties in 1919/20 and the set of international lawyers that was tasked to draft the hundreds and thousands of articles of one of the most contested peace settlements of all times. Taking a fresh look behind the scenes of the negotiations in Paris, this chapter unearths role and influence of the drafting committee, offers a characterization of the in-house lawyers involved, and examines how they transformed political bargaining into legally binding rules and treaty provisions. The broader aim is to explore what international legal advisors do in government service and how they operate along the blurred the boundary between law and politics.
Changing Hats
Authors: Priemel Kim Christian
Published: June 2021

The war crimes tribunals at Nuremberg are well known as key arenas of judicial retribution after the Second World War and for institutionalizing international criminal justice. Their personnel, the ‘Nuremberg lawyers’, have been credited with advancing the cause of international law and (re)building the post-war global order. Critics, though, have chastised what they conceive of as the mistaken recourse to legal ideas and legal language as an either naïve or outright hypocritical, but in any case inadequate way of addressing the challenges of power politics in the Cold War era and beyond. Common to both sides are sweeping, often implicit notions of who the ‘Nuremberg lawyers’ actually were, what drove them, and how they interacted. By categorizing academic training and practical experience, national and biographical contexts, normative inclinations, individual ambitions, and practical functions, this chapter offers a classification of Nuremberg’s lawyers which provides a helpful taxonomic tool for international tribunals more generally.
Agents of Constitutionalism
Authors: Rasmussen Morten
Published: June 2021

Why did European law experience a constitutional breakthrough in 1963–1964 when the political and institutional development of European integration had moved in the direction of an increasingly intergovernmental approach based on international treaties in the late 1950s? By employing a biographical approach to five key agents of European constitutionalism during the founding years of European law, the chapter argues that this handful of jurists from different countries was able to secure a constitutional breakthrough in the interpretation of European law despite the political trend. The key motivation shared by them all was grounded in the recent European past of the interwar crisis and the Second World War, which led them to support federalism as the best way to organize European cooperation. The five held very different positions both in the European institutions and at member state level, but managed to influence the development of European law through participation in the negotiations of the founding treaties, constitutional reforms at the national level and the agency of the European Commission and the European Court of Justice.
Introduction
Authors: Coady C. A. J.
Published: June 2021

This Introduction sets the background and scenario for the chapters that follow. It begins by sketching the public impact that terrorist acts have had in the past few decades and how that impact has been affected by the terrible coronavirus of 2020 with its staggering worldwide death toll. It then charts the development of philosophical responses to the dangers of terrorism that begin surprisingly slowly in the 1970s, increase in volume in the 1980s and 1990s, and accelerate dramatically in the wake of the September 11, 2001 hijacked plane attacks on New York and Washington. It also gives an indication of the broader sense of “meaning” in the book’s title that underpins much of the discussion in the later chapters that relies upon but goes beyond the very necessary conceptual clarifications developed in the early chapters. Finally, there are summaries of the themes and arguments in the book’s eight chapters, concluding with a note on the doctrine of double effect and the role of intention in the moral discussion of terrorist acts.
Further Objections
Authors: Coady C. A. J.
Published: June 2021

Chapter 2 discusses objections to the definition of terrorism from Chapter 1 that criticize it for being either too narrow or too broad. The narrowness criticisms object that (a) there are terrorist acts that target combatants, (b) there are terrorist acts that do not involve a political motive, such as certain criminal or religious acts, and (c) some non-intentional violence afflicting non-combatants should be encompassed by the definition. The “too broad” category of objections argues that (a) the tactical definition should include an ingredient of intentionally provoking fear, (b) the definition’s encompassing terrorist acts by states is mistaken, (c) including non-combatant property in the definition is mistaken, and (d) this approach simply defines terrorist acts as murder and loses what is distinctive of such acts. These objections are criticized and rejected for the most part, though some elicit concessional comments about their possible ancillary benefits in relation to the preferred tactical definition.
Counter-Terrorism and Its Ethical Hazards
Authors: Coady C. A. J.
Published: June 2021

Chapter 7 discusses ethical problems faced by contemporary counter-terrorism measures. The discussion is primarily focused on reactions of states to sub-state terrorism broadly understood. There is an initial discussion of whether, and in what circumstances, terrorists should be treated as combatants or criminals and the bearing of this upon military and non-military forms of counter-terrorism. Problems with military responses connected with the inflammatory slogan “the war on terror,” including “targeted killing,” are also discussed. Thereafter, the chapter deals mostly with non-military responses and their moral and political hazards. These are examined under the three categories of: (1) legal and regulatory measures; (2) diplomatic measures; and (3) measures to remove the grievance. Under (1), difficulties connected with legal definitions of terrorism, and their tendency to promote abuses of civil rights, are explored; under (2) and (3) the path of political diplomacy is supported, but obstacles to its success in practice are discussed.
C1‐ Tetrahydrofolate Synthase Contribution to 1C Metabolism
Authors: Blemur Danielle , Field Martha , Stover Patrick
Published: June 2021

Folate‐dependent one‐carbon (1C) metabolism is required for the de novo synthesis of thymidylate, purines, and methionine. Impairments in 1C metabolism result from inadequate intake of folate and other B‐vitamins, polymorphisms in folate‐utilizing genes, physiological states such as cancer and pregnancy, and the interaction among these factors. Although impaired 1C metabolism is associated with these pathologies, the mechanisms and causal pathways have not been established. Recent studies have shown that folate‐dependent de novo thymine (dTMP) biosynthesis takes place in the nucleus at the sites of DNA replication. Enzymes involved include serine hydroxymethyl‐transferase (SHMT1 and SHMT2α), thymidylate synthase (TYMS), dihydrofolate reductase (DHFR), and C1‐tetrahydrofolate (C1‐THF) synthase. Both C1‐THF synthase and SHMT generate 5,10‐methyleneTHF, the co‐factor required for the TYMS‐catalyzed conversion of dUMP to dTMP. Pull‐down experiments with C1‐THF synthase and SHMT will confirm the hypothesis that the dTMP biosynthesis multi‐enzyme complex includes folate‐dependent C1‐THF synthase, TYMS, DHFR, and Lamin B and A/C and affects nuclear folate levels and dTMP synthesis capacity. We anticipate that C1‐THF synthase will interact with SHMT1, TYMS, DHFR and lamin proteins, since it was identified as an interacting partner in a proteomics screen. If these interactions change as a result of SHMT1 knockdown, then that will help to show that SHMT is a scaffold protein in the replication complex. These studies were supported by NIH R37DK58144 to PJS.
Regulation of assembly of bipolar myosin II filaments
Authors: Kandel Avi , Frei Ryan , Prehoda Ken
Published: June 2021

Myosin is a motor protein that has many different functions, including muscle contraction, cell transport, and cell adhesion. The current project focuses on non‐muscle myosin II B in Drosophila. Myosin is composed of heavy chains and light chains. There are two identical heavy chains that each contain an N‐terminal globular head region, neck region, and a C‐terminal long α‐helical tail region. There are also two types of light chains, the regulatory light chain and essential light chain. The phosphorylation state of the regulatory light chain regulates whether myosin is in an active or inactive position. In myosin's inactive position, it is believed that part of the tail region binds to the neck region. To understand the mechanism underlying myosin activation and inactivation, it is essential to pinpoint the site where the neck region binds to the tail region. We approach this question by expressing varying lengths of the tail region and assay neck region binding to these tail region fragments, thereby systematically narrowing down the location of binding on the primary structure of the tail region. To accomplish this, we must first coexpress the neck region with the light chains. So far, it has been confirmed that the heavy chain can be coexpressed with the light chains and form the desired trimeric complex. The information gained from this experiment will help us better understand the regulation of non‐muscle myosin II B.
Metoclopramide is a Substrate but Not an Inactivator of CYP2D6
Authors: Bolles Amanda K. , Briggs Erran D. , Livezey Mara R. , Nagy Leslie D. , Furge Laura Lowe
Published: June 2021

CYP2D6 is a major drug metabolizing enzyme responsible for the metabolism of ~20% of pharmaceutical drugs. Inactivation of CYP2D6 is rare, but ‐ due to its importance in drug metabolism, can be clinically significant. Thus, understanding of the mechanisms of inactivation of CYP2D6 is important for devising strategies to avoid undesirable drug‐drug interactions. Metoclopramide is a drug that has previously been reported to be a mechanism‐based inactivator of CYP2D6. In order to better understand possible inactivation of CYP2D6 by metoclopramide, we expanded these initial findings by determining the spectral binding constant, partition ratio, metabolite profile, enzyme activity after an inactivation phase, and metabolite profile. While we did observe reversible competitive inhibition, our studies did not show metoclopramide to be an inactivator of CYP2D6 (Support: NIH 1R15‐GM086767–01 & ‐01S1).
Interaction of Mechanism‐Based Inactivators with Modified CYP2D6
Authors: Livezey Mara R. , Hicks Michael J. , Furge Laura Lowe
Published: June 2021

Mechanism‐based inactivators lead to covalent adduction of CYPs. There are a few known inactivators of CYP2D6 including two confirmed protein adductors – SCH66712 and EMTPP, and some heme modifiers including paroxetine and MDMA. To understand the potential similarities and differences between these classes of inactivators, we have used molecular modeling to analyze types of interactions that might lead to inactivation. From our modeling experiments we predicted Thr309 of CYP2D6 to be a likely nucleophilic target of the electrophiles formed from the protein inactivators. Isolation of the CYP2D6‐SCH66712 inhibitory complex allowed us to confirm Thr309 as a putative electrophilic target. Two other potential target residues were also identified. We have mutated these residues to Ala to test for interaction with the different classes of inactivators and will present data on the altered kinetics of interaction of mutant CYP2D6 with known mechanism‐based inactivators. (Support: NIH 1R15‐GM086767–01 & ‐01S1)
Covalent Modification of CYP2D6 Following Inactivation by SCH66712
Authors: Hicks Michael J. , Livezey Mara R. , Furge Laura Lowe
Published: June 2021

SCH66712 is a known potent mechanism‐based inactivator of CYP2D6. It contains a piperazine group and a substituted imidazole ring; the latter is believed to be the specific reactive element required for CYP2D6 inactivation. Recent modeling studies in our lab have suggested that Thr309 could be a potential site for electrophilic inactivation. Using enzyme digestion techniques coupled to LC/MS, labeled peptides from the CYP2D6‐SCH66712 inhibitory complex were identified, including a peptide containing Thr309. Mutants of CYP2D6 at Thr309 and other identified residues have been prepared, expressed, and purified to test for altered inhibition profiles to better understand both the mechanism of CYP2D6 inactivation and the role of potential substrate access and egress channels in the enzyme. (Support: NIH 1R15‐GM086767–01 & ‐01S1
Visualizing the Effect of Iron on the Localization of Intracellular Ferritin in Mosquito Cells
Authors: Conley Zachary Ryan , Geiser Dawn , Winzerling Joy
Published: June 2021

Ferritin is a 24‐subunit molecule, made up of heavy chain (HCH) and light chain (LCH) homologues that stores and controls the release of dietary iron in mammalian, plant, and insect systems. Dietary iron taken in a blood meal is stored inside ferritin, and previous work in mosquitoes demonstrates an important function for dietary iron in the process of egg development. One approach to limit the spread of arthropod‐borne diseases, such as yellow fever, West Nile virus and malaria, is to reduce the number of potential vectors by interfering with this process in mosquito reproduction and thereby to decrease transmission rates. The localization of ferritin subunits inside CCL‐125 (Aedes aegypti) and 4a3b (Anopheles gambiae) mosquito cells under various iron treatment conditions is important to further elucidate the regulation of iron metabolism in these important disease vectors and to observe the dynamics of intracellular ferritin subunits following iron administration. Deconvolution microscopy was used to capture 3D fluorescent images of treated mosquito cells to visualize the LCH ferritin subunit in multiple focal planes as a follow‐up to previous detection of the HCH subunit. Fluorescent probes were used to illuminate cell organelles (i.e. Golgi apparatus, lysosomes, endoplasmic reticulum, nuclei), while secondary probes for ferritin subunits demonstrated abundance and co‐localization within organelles. These images will help to develop a model for the biochemical regulation of ferritin, and perhaps to advance novel hypotheses for the crucial role of iron in mosquito reproduction. The Agricultural Experiment Station and the College of Agriculture and Life Sciences supported this research.
Hypoglycemia increases glycolytic metabolism in the rat ventromedial hypothalamus (VMH)
Authors: Mun Jonathan , Cooper Andrew , Cavaretta John , Lee Beverly J.
Published: June 2021

The VMH is a critical brain‐area involved in energy sensing and responsible for initiating the counter regulatory response (CRR) during hypoglycemia. We hypothesize local metabolic changes following hypoglycemia contribute to hypoglycemia‐associated autonomic failure (HAAF), an impairment of the CRR. VMH and CA1 hippocampus (HPC) were collected from rats (n=30) 6 hours following a single episode of insulin‐induced hypoglycemia (IIH) (acute hypoglycemia; AH), 24 h following the third of 3 recurrent episodes of IIH (recurrent hypoglycemia; RH), or saline‐treated euglycemia (control; SC). Tissue samples were randomly pooled before methanol extraction and analysis by GC/MS and LC/MS/MS. 180 metabolites were identified and quantified. In the VMH lactate, tricarboxylic acid (TCA) cycle intermediates (citrate, cis‐aconitate, and malate), and phospholipid metabolism products (glycerol and glycerol‐3‐phosphate) were increased (p≤0.05) 6 h following AH, but were not significantly different 24 h after RH. These changes were not observed in HPC following IIH. RNA sequencing analysis showed a 0.9, 0.2, and 0.1 fold‐increase in glycerol‐3‐phosphate dehydrogenase, GLUT1, and 2‐oxoglutarate dehydrogenase gene expression, respectively (p≤0.01), in the VMH 24 h after RH. It is unclear at this time whether the hypoglycemia‐induced changes in carbohydrate and lipid metabolism in the VMH contribute to HAAF. Supported by DK082609
Small‐molecule modulators of Nkx6.1 expression in pancreatic progenitor cells during directed differentiation of human embryonic stem cell to insulin producing beta cells
Authors: Peterson Quinn , Melton Douglas
Published: June 2021

Type 1 diabetes is characterized by destruction of the insulin‐producing pancreatic beta cells, resulting in dysregulation of blood glucose levels. As such, the identification of small‐molecules capable of efficiently inducing the differentiation of human embryonic stem cells (hESC) to insulin producing beta cells may provide a route to cell transplantation therapies for these patients. In 2012, several protocols were reported to generate pancreatic precursor cells capable of further differentiation into beta cells when transplanted into mice. However, these pancreatic precursors do not generate functional beta cells in vitro. One possible explanation of this effect is the absence of the critical pancreatic progenitor transcription factor, Nkx6.1, in over 60% of the hESC derived pancreatic progenitor cells in vitro. Thus incomplete induction of Nkx6.1 may represent a barrier to beta cell production in vitro. To this end, we sought to uniformly induce Nkx6.1 expression in pancreatic progenitors. Herein, we report the development of a combinatorial screening platform for the identification of small‐molecules that can induce Nkx6.1 expression in pancreatic precursor cells, and the elucidation of pathways driving this aspect of beta cell differentiation.
Ultraviolet B radiation enhances glycation of human serum albumin and the formation of CML in vitro
Authors: Liu Weixi , Cohenford Menashi A , Frost Leslie , Dain Joel A
Published: June 2021

Non‐enzymatic glycation of human serum albumin (HSA) is a major contributor to the pathology of diabetes. Exposure to ultraviolet B (UVB) irradiation is known to perturb protein structure and generate reactive oxygen species (ROS) which might accelerate protein glycation. The objective of this study was to evaluate the effect of UVB irradiation on the glycation level of HSA by D‐glucose and the formation of carboxymethyl lysine (CML), an Advanced Glycation Endproducts (AGEs) used as a biomarker of oxidatively damaged tissues. Compared to the untreated HSA, UVB exposed HSA showed a more rapid increase in fluorescence intensity over time, as well as enhanced peak areas of AGEs in the HPLC elution profile. Circular dichroism spectrum revealed a more extended loss in protein secondary structure when glycation had taken place under exposure to UVB. More D‐glucose adducts were found on UVB treated HSA, generating a higher mass shift of protein in MALDI profile. Compared to samples in the dark, reactions exposure to UVB produced about double amount of CML within 60 hours of incubation. In conclusion, UVB radiation accelerates the glycation level of HSA and promotes the formation of AGEs, which may be stimulated by the generation of ROS by UV radiation. This study warrants further investigation as there have been few reports on the correlation between the UV radiation of proteins and their enhanced glycation by a reducing sugar.
The Dysregulation of AMPK Suppresses Phosphorylation of Sterol Regulatory Element Binding Protein and Increases Its Activity in the Development of Atherosclerosis in Pig and Human Diabetes.
Authors: Zang Mengwei , Li Yu , Xu Shanqin , Jiang Bingbing , Cohen Richard A
Published: June 2021

Our recent studies discover that AMPK phosphorylates and inactivates the lipogenic factor SREBP as well as ameliorates hepatic steatosis and hyperlipidemia in diabetic mice (Li Y, et al, Cell Metabolism, 2011). However, whether AMPK is linked to SREBP in atherosclerosis remains unknown. By using a well‐established atherosclerotic swine model of diabetes, which develops human‐like atherosclerotic plaques, we show that vascular AMPK activity is suppressed, which in turn reduces Ser‐372 phosphorylation of SREBP‐1. The proteolytic processing of SREBP‐1 and‐2 and expression of their target genes involved in de novo lipogenic processes (ACC1, FAS, SCD1 and HMGCS) are increased. Similarly, AMPK deficiency in cells decreases SREBP‐1 phosphorylation at Ser372 and increases FAS. Immunostaining analysis reveals an elevation in SREBP‐1 localized predominantly in lipid‐laden macrophages and VCAM‐1‐positive vascular cells in fatty streaks, cholesterol crystals, and fibrous caps with necrotic lipid cores. Aberrant SREBP‐1 contributes to increased inflammatory markers in aortic atherosclerosis in pigs. The pro‐atherogenic effect of SREBP‐1 is also supported by co‐localization of SREBP‐1 and VCAM‐1 in atherosclerotic lesions in humans. These studies uncover a previously unrecognized association of dysregulated AMPK‐SREBP signaling in vascular inflammation and atherosclerosis in diabetes (NIH DK076942).
Effects of Oleate and Inflammatory Cytokines on Dermal Fibroblasts in Type 1 Diabetics
Authors: Jones Albert Richard , Deeney Jude T , Corkey Barbara
Published: June 2021

Background

Dermal fibroblasts are crucial to wound healing which can be impaired in type 1 diabetics. We set out to determine if exposure to inflammatory cytokines and oleate changed the metabolism and ROS production of fibroblasts.

Methods

Cells were acutely exposed to 25 or 50 μM of oleate and chronically exposed to 100 μM of oleate. Cells were exposed to 10 ng/mL of TNF‐ α. Oxygen Consumption was measured using the SeaHorse XF24 Flux Analyzer, ROS was measured using DCF dye. Lipid accumulation was measured using Nile Red staining. β‐oxidation was measured using radioactive oleate.

Results

The data shows type 1 diabetics sequester more lipid droplets than controls. As a result, less fatty acid is available for β‐oxidation and the stimulation of respiration, which explains the decreases in oxygen consumption, proton leak, β‐oxidation and ROS generation in diabetic fibroblasts. We believe that diabetics express an inflammatory phenotype that reduces β‐oxidation in the cell causing a decrease in oxygen consumption and ROS production.

Future Research

We plan to measure the mRNA and protein expression of key components of β‐oxidation and inflammation in fibroblasts.

Funded by NIH
High fat feeding enhances the ability of mesenchymal stem cell therapy to modulate mitochondria in the infarcted heart
Authors: Hughey Curtis , Ma Lianli , James Freyja D , Bracy Deanna P , Wasserman David H , Rottman Jeffrey N , Shearer Jane
Published: June 2021

OBJECTIVE

Evaluate the effect of mesenchymal stem cell (MSC) therapy on cardiac mitochondria following a myocardial infarction (MI).

METHODS

C57BL6 mice were high fat‐fed (HFF) for eight weeks and separated into three groups: HFF‐SHAM, HFF‐MI+PBS mice given a MI followed by intramyocardial injection of saline and HFF‐MI+MSC mice receiving MSCs. HFF groups were age‐matched with chow‐fed mice.

RESULTS

Mitochondrial function was evaluated via high‐resolution respirometry in permeabilized, peri‐infarct fibers 5 weeks post‐MI. In chow‐fed mice, maximal oxygen flux (155.30 vs 122.63 vs 135.61 pmol/s/mgww/CS; SHAM vs MI+PBS vs MI+MSC; n=9–10) and oxidative phosphorylation (OXPHOS) efficiency (RCR = 3.17 vs 2.34 vs 2.84) was preserved by MSC therapy. Also, MSC treatment prevented a decline in mitochondrial content as indicated by citrate synthase activity (1.28 vs 0.90 vs 1.34 mmol/min/mg pro). In HFF mice, the MSC treatment maintained mitochondrial content (1.21 vs 0.99 vs 1.24) and OXPHOS efficiency (RCR = 2.18 vs 1.88 vs 2.23). Also, HFF‐MI+MSC hearts exhibited an exaggerated decline in futile oxygen flux (61.39 vs 50.69 vs 38.85; n=8–9). Immunoblots show the HFF‐MI+MSC hearts have lower mitochondrial complex I and II, UCP3 and TFAM protein levels (n=6).

CONCLUSIONS

MSC therapy minimizes mitochondrial dysfunction post‐MI and HFF enhances the therapeutic potential of MSCs. Funded by CIHR and Killam Trusts.
Association of Genetic Polymorphism of Peroxisome Proliferator‐Activated Receptor‐Gamma Gene and Polycystic Ovary Syndrome
Authors: Hemimi Neveen Salah Eldine , Shaafie Ishtiyaq Ahmad , Alshawa Haya Husni A
Published: June 2021

Background

Peroxisome proliferative‐ activated receptor gamma (PPARs) is a nuclear receptor which controls transcription of genes involved in free fatty acid uptake and lipogenesis, and is essential for differention and survival of adipocytes.

Aim

To investigate the association of Pro12Ala polymorphism in PPAR gamma gene with polycystic ovary syndrome (PCOS) and its related metabolic disorders.

Material and Method

The study consisted of 50 women diagnosed with PCOS and 96 healthy women as a control group. Individuals were genotyped for Pro12Ala genetic polymorphism of PPAR gamma by using polymerase chain reaction‐ restriction fragment length polymorphism. Statistical analysis was done by SPSS program.

Results

Frequency of Pro12/Pro12 and Pro12/Ala12 genotypes were 86% and 14% in PCOS group compared to 77.1% and 22.9% in control group. The carriers of Pro12/Pro12 genotype exhibited a significant greater risk for PCOS compared to carriers of Pro12/Ala12 genotype (OR= 1.8, 95%CI= 0.7–4.6). Furthermore they were at higher risk of different metabolic disorders such as obesity, insulin resistance and diabetes mellitus.

Conclusion

Pro12/Pro12 genotype is a risk factor for PCOS and its related metabolic disorders.

Source of Fund: Dubai Medical College for girls.
Retinoid X receptor alpha (RXRα) interaction with sugars
Authors: Soman Frances L , de Jesus Piva Camila , Hostetler Heather A.
Published: June 2021

Nuclear receptors, peroxisome proliferator‐activated receptor alpha (PPARα) and liver X receptor alpha (LXRα), with their heterodimeric partner, RXRα, regulate transcriptional activity of lipid and glucose metabolism. It has been shown that glucose binds with high affinity to PPARα, and that transcriptional activity induced by the PPARα‐RXRα heterodimer increases in the presence of glucose. LXRα has also been shown to bind glucose, resulting in altered activity. As heterodimerization with RXRα is required for activation, we examined the ability of RXRα to interact with glucose and other sugars using fluorescent ligand binding assays and circular dichroism. While preliminary data with a GST‐tagged human RXRα protein suggested strong interactions with sugars, recent data with a tag‐free protein demonstrates significant conformational differences between these proteins. Although glucose did not significantly alter RXRα secondary structure, some sugars and sweeteners did. Such interactions may affect RXRα's role in heterodimerization and gene regulation. This research was supported by USPHS NIH grant DK77573.
Crystal structure and in vitro analysis of human IQGAP1 Calponin homology domain: implication for its interactions with Ca2+ bound Calmodulin and F‐actin
Authors: Liu Jing , Kurella Vinodh , LeCour Louis F. , Worthylake David Kent
Published: June 2021

IQGAP1 is a 190kDa multi‐domain scaffold protein involved in cell signaling, cytoskeleton reorganization, cell‐cell adhesion via its dynamic interactions with various binding partners. A single copy Type‐3 Calponin homology domain locates at its amino terminus (residues 46–160). Here we have determined the crystal structure of human IQGAP1 residues 1–191 (CHD). In the crystal, two CHD molecules form a “head‐to‐head” homodimer. At high concentration in solution, the CHD can also form a dimer. Isothermal titration calorimetry (ITC) and F‐actin co‐sedimentation assays show that the CHD can bind to Ca2+/CaM and F‐actin, respectively. Ca2+/CaM does not affect the interaction between the CHD and F‐actin. The Calmodulin Target Database identified residues 91–103 of the CHD as a potential CaM binding site. Site‐directed mutagenesis and ITC indicate that 92LKKI95 is critical for Ca2+/CaM binding. Structure‐based sequence alignment predicts an F‐actin binding site locating in the dimer interface. A disulfide bond (K161C) was introduced to stabilize the dimer and mask potential F‐actin‐binding residues. Reduced F‐actin binding was observed upon dimer formation. Our results suggest that residues 92LKKI95 contribute to Ca2+/CaM binding, and that F‐actin binding utilizes residues located at the dimer interface. Our results also indicate the self‐association of full‐length IQGAP1 may be a parallel homodimer.
Characterization of a novel interaction between BLIP‐II and Staphylococcus aureus PBP2a
Authors: Adamski Carolyn J , Chow Dar‐Chone , Brown Nicholas , Palzkill Timothy
Published: June 2021

The prediction and manipulation of protein‐protein interactions remains a difficult task. Model systems such as the β‐lactamase inhibitory protein II (BLIP‐II) and β‐lactamases have been used to investigate the principles of protein‐protein interactions. Previous studies focused on the determinants of binding affinity and specificity between BLIP‐II and class A β‐lactamases. However, interactions between BLIP‐II and other proteins have yet to be explored. In this study, we characterized the novel interaction between BLIP‐II and penicillin binding protein 2a (PBP2a) from methicillin resistant Staphylococcus aureus. Mutagenesis and surface plasmon resonance experiments were used to determine how specificity is achieved between BLIP‐II and its binding partners. The results suggest that an outer ring of residues on the BLIP‐II interface plays a critical role in binding PBP2a while an inner ring of residues on the binding surface is primarily responsible for the binding of β‐lactamases. Changes in BLIP‐II binding affinity for PBP2a and β‐lactamases were found to be largely due to changes in kon and koff, respectively. In summary, the results of the study indicate BLIP‐II binds PBP2a in addition to β‐lactamases and provide insights into how BLIP‐II binds a wide range of target proteins.

Research support provided by grants R01 A132956 and R21 A1092063 to T.P.
Monitoring the Structural Dynamics of LRRK2 using Split‐ Luciferase Protein‐Fragment‐Assisted Complementation
Authors: Nardozzi Jonathan , Baker Olivia , Wong Salinda , LaVoie Matthew J.
Published: June 2021

Missense mutation of the PARK8 gene, which encodes the protein Leucine‐Rich Repeat Kinase 2 (LRRK2), is the most common genetic cause of Parkinson's Disease (PD), yet the physiological role of this protein is largely unknown. LRRK2 dimerization has been linked to its activity and localization in the cell, as dimers have higher kinase activity and are preferentially membrane‐associated. In this study we utilized a Split‐Luciferase Protein‐ Fragment‐Assisted Complementation assay (PCA) to assess dimerization of LRRK2. PCA has numerous advantages over other techniques used to study protein‐protein interactions, as the luciferase reporter displays specificity, reversibility, and signal intensity. We have developed a quantitative bioluminescent assay of LRRK2 dimerization as a means to understanding the regulators of LRRK2 structure and function. Numerous split‐luciferase fragment pairs were explored from both the Renilla and Firefly luciferase reporters, and one fragment pair was optimized for investigating LRRK2 dimerization. Interestingly, we observed that fusion and orientation of luciferase fragments to their respective LRRK2 monomers were important factors for reporter efficiency. Efforts to characterize small molecule regulators of LRRK2 dimerization are ongoing.

NIH grant #NS072604
Sensitization of NCI‐H460 Cells to Anticancer Drugs with Aminoglycosides
Authors: Cuccarese Michael
Published: June 2021

Aminoglycosides, characterized by their toxic interaction with the bacterial ribosome, are also known for their effects on eukaryotic cells. While aminoglycosides are routinely used in the clinic as antibiotic agents, they are currently being evaluated therapeutically for their ability to rescue protein synthesis that is suppressed by nonsense‐mutation (e.g., in the case of cystic fibrosis). In this current study we aim to determine if the aminoglycoside read‐though effect on eukaryotic cells can be leveraged for use in cancer chemotherapy.

Herein we describe our efforts to find the aminoglycoside read‐though effect on non‐small cell lung carcinoma cells (NCI‐H460) and it application to cancer cell chemotherapy. Specifically, these studies involve the search for sensitization effects of sub‐toxic doses of known read‐though inducing aminoglycosides in cancer cell cytotoxicity assays with known anticancer agents. For NCI‐H460 cancer cells, a dose dependent sensitization effect was found for known read‐though inducing aminoglycosides for some, but not all anticancer drugs. Studies aimed at the mode of action for sensitization due to aminoglycoside exposure is ongoing.