Walking the Talk: How to Identify Anti-Pluralist Parties.

The recent increase of democratic declines around the world - "the third wave of autocratization" - has sparked a new generation of studies on the topic. Scholars tend to agree that the main threat to contemporary democracy arises from democratically elected rulers who gradually erode democratic norms. Is it possible to identify future autocratizers before they win power in elections? Linz (1978) and Levitsky and Ziblatt (2018) suggest that a lacking commitment to democratic norms reveals would-be autocratizers before they reach office. This article argues that the concept of anti-pluralism rather than populism or extreme ideology captures this. We use a new expert-coded data set on virtually all relevant political parties worldwide from 1970 to 2019 (V-Party) to create a new Anti-Pluralism Index (API) to provide the first systematic empirical test of this argument. We find substantial evidence validating that the API and Linz's litmus-test indicators signal leaders and parties that will derail democracy if and when they come into power.

How democracies prevail: democratic resilience as a two-stage process.

This article introduces a novel conceptualization of democratic resilience - a two-stage process where democracies avoid democratic declines altogether or avert democratic breakdown given that such autocratization is ongoing. Drawing on the Episodes of Regime Transformation (ERT) dataset, we find that democracies have had a high level of resilience to onset of autocratization since 1900. Nevertheless, democratic resilience has become substantially weaker since the end of the Cold War. Fifty-nine episodes of sustained and substantial declines in democratic practices have occurred since 1993, leading to the unprecedented breakdown of 36 democratic regimes. Ominously, we find that once autocratization begins, only one in five democracies manage to avert breakdown. We also analyse which factors are associated with each stage of democratic resilience. The results suggest that democracies are more resilient when strong judicial constraints on the executive are present and democratic institutions were strong in the past. Conversely and adding nuance to the literature, economic development is only associated with resilience to onset of autocratization, not to resilience against breakdown once autocratization has begun.

Data for Politics: Creating an International Research Infrastructure Measuring Democracy.

Questions such as how democratic a country is, how free are its media, or how independent is its judiciary are highly important to researchers and decision makers. We describe a research infrastructure that produces the world's largest dataset on democracy, governance, human rights, and related topics. The dataset is far more resolved and accurate than previous efforts, currently covers 202 political units from 1789 until the present, and is regularly updated each spring. The infrastructure involves an online survey of over 3,000 experts from 180 countries. Survey design and advanced statistical techniques are crucial for assuring data validity. The infrastructure also provides reports and analyses based on the data and easy-to-use tools for exploring and graphing the data.

Explaining the homogeneous diffusion of COVID-19 nonpharmaceutical interventions across heterogeneous countries.

We analyze the adoption of nonpharmaceutical interventions in the Organisation for Economic Co-operation and Development (OECD) countries during the early phase of the coronavirus disease 2019 (COVID-19) pandemic. Given the complexity associated with pandemic decisions, governments are faced with the dilemma of how to act quickly when their core decision-making processes are based on deliberations balancing political considerations. Our findings show that, in times of severe crisis, governments follow the lead of others and base their decisions on what other countries do. Governments in countries with a stronger democratic structure are slower to react in the face of the pandemic but are more sensitive to the influence of other countries. We provide insights for research on international policy diffusion and research on the political consequences of the COVID-19 pandemic.

Institutionalising electoral uncertainty and authoritarian regime survival.

Authoritarian incumbents routinely use democratic emulation as a strategy to extend their tenure in power. Yet, there is also evidence that multiparty competition makes electoral authoritarianism more vulnerable to failure. Proceeding from the assumption that the outcomes of authoritarian electoral openings are inherently uncertain, it is argued in this article that the institutionalisation of elections determines whether electoral authoritarianism promotes stability or vulnerability. By 'institutionalisation', it is meant the ability of authoritarian regimes to reduce uncertainty over outcomes as they regularly hold multiparty elections. Using discrete-time event-history models for competing risks, the effects of sequences of multiparty elections on patterns of regime survival and failure in 262 authoritarian regimes from 1946 to 2010 are assessed, conditioned on their degree of competitiveness. The findings suggest that the institutionalisation of electoral uncertainty enhances authoritarian regime survival. However, for competitive electoral authoritarian regimes this entails substantial risk. The first three elections substantially increase the probability of democratisation, with the danger subsequently diminishing. This suggests that convoking multiparty competition is a risky game with potentially high rewards for autocrats who manage to institutionalise elections. Yet, only a small number of authoritarian regimes survive as competitive beyond the first few elections, suggesting that truly competitive authoritarianism is hard to institutionalise. The study thus finds that the question of whether elections are dangerous or stabilising for authoritarianism is dependent on differences between the ability of competitive and hegemonic forms of electoral authoritarianism to reduce electoral uncertainty.

Linking democracy and biodiversity conservation: Empirical evidence and research gaps.

Increasing human pressure threatens plant and animal species with extinction worldwide. National political institutions constitute an important arena for biodiversity conservation. Yet, the relationship between how democratic these national institutions are and a country's efforts towards and track-record for biodiversity conservation remains poorly understood. In this review, we outline the theoretical links between democracy and biodiversity conservation and review the empirical literature testing them. While more studies reported a positive than a negative relation between democracy and biodiversity conservation (15 vs. 11), the most common result was a mixed relationship (28), often conditioned on economic factors. The use of different proxies to measure biodiversity, including deforestation, protected areas, threatened species, and fishery statistics emerged as a primary obstacle for synthesis. We suggest overcoming this caveat together with a consistent definition of democratic institutions and a standardized statistical framework as research priorities to improve policies against the global biodiversity loss.

Sequential Requisites Analysis: A New Method for Analyzing Sequential Relationships in Ordinal Data.

OBJECTIVES: This article presents a new method inspired by evolutionary biology for analyzing longer sequences of requisites for the emergence of particular outcome variables across numerous combinations of ordinal variables in social science analysis. METHODS: The approach is a sorting algorithm through repeated pairwise investigations of states in a set of variables and identifying what states in the variables occur before states in all other variables. We illustrate the proposed method by analyzing a set of variables from version 7.1 of the V-Dem data set (Coppedge et al. 2017. Varieties of Democracy (V-Dem) Project; Pemstein et al. 2017. University of Gothenburg, Varieties of Democracy Institute: Working Paper No. 21). With a large set of indicators measured over many years, the method makes it possible to identify and compare long, complex sequences across many variables. RESULTS: This affords an opportunity, for example, to disentangle the sequential requisites of failing and successful sequences in democratization, or if requisites are different during different time periods. CONCLUSIONS: For policy purposes, this is instrumental: Which components of democracy occur earlier and which later? Which components of democracy are therefore the ideal targets for democracy promotion at different stages?

Women's Political Empowerment: A New Global Index, 1900-2012.

The political empowerment of women is a societal process crucial to development and progress. The V-Dem women's political empowerment index (WPEI) provides information about women's civil liberties, civil society participation, and political participation globally. Spanning from 1900 to 2012, three dimensions of empowerment, and over 170 countries, it is among the most comprehensive measures of women's empowerment available. This paper presents a conceptualization of women's political empowerment and provides an overview of the construction of the index and operationalization of its three sub-dimensions: Women's civil liberties, civil society participation, and political participation. Compared to other indices measuring women's empowerment, such as the GDI, the GEM, the GII, and the CIRI data on human rights, the V-Dem index allows more precise measurement and is superior in temporal scope and coverage of countries of the Global South. The paper demonstrates the benefits of this new index and its sub-dimensions through several empirical illustrations.

Women's rights in democratic transitions: A global sequence analysis, 1900-2012.

What determines countries' successful transition to democracy? This article explores the impact of granting civil rights in authoritarian regimes and especially the gendered aspect of this process. It argues that both men's and women's liberal rights are essential conditions for democratisation to take place: providing both women and men rights reduces an inequality that affects half of the population, thus increasing the costs of repression and enabling the formation of women's organising - historically important to spark protests in initial phases of democratisation. This argument is tested empirically using data that cover 173 countries over the years 1900-2012 and contain more nuanced measures than commonly used. Through novel sequence analysis methods, the results suggest that in order to gain electoral democracy a country first needs to furnish civil liberties to both women and men.

SCARA Involvement in the Uptake of Nanoparticles Formed by Cell-Penetrating Peptides.

The investigation of uptake mechanisms for cell-penetrating peptides (CPPs) is and has been an ongoing project for as long as the peptides have been known, a time period that now spans over two decades. The ultimate answer is yet to be revealed and the current understanding is that no "one" mechanism will ever be found. The reason for this is that the uptake mechanism seems to be dependent on a multitude of factors that include which CPP, what cells are used, whether or not there is cargo and what the cargo is. CPPs are capable of delivering a variety of bio-macromolecules that are by themselves unable to enter into cells. Our group has reported on many different peptides in recent years, many aimed at delivering various oligonucleotide-based cargoes. These peptides have utilized the inherent positive charge of the peptides and some rationally designed modifications to non-covalently complex oligonucleotides and bring them into cells. In this chapter, we present a brief overview of the current proposals for the uptake mechanisms of CPPs and describe methods for detecting and evaluating the role of scavenger receptor class A receptors in the uptake of non-covalent cell-penetrating peptide:oligonucleotide complexes.

A convergent uptake route for peptide- and polymer-based nucleotide delivery systems.

Cell-penetrating peptides (CPPs) have been used as vehicles to deliver various cargos into cells and are promising as tools to deliver therapeutic biomolecules such as oligonucleotides both in vitro and in vivo. CPPs are positively charged and it is believed that CPPs deliver their cargo in a receptor-independent manner by interacting with the negatively charged plasma membrane and thereby inducing endocytosis. In this study we examine the mechanism of uptake of several different, well known, CPPs that form complexes with oligonucleotides. We show that these CPP:oligonucleotide complexes are negatively charged in transfection-media and their uptake is mediated by class A scavenger receptors (SCARA). These receptors are known to promiscuously bind to, and mediate uptake of poly-anionic macromolecules. Uptake of CPP:oligonucleotide complexes was abolished using pharmacological SCARA inhibitors as well as siRNA-mediated knockdown of SCARA. Additionally, uptake of CPP:oligonucleotide was significantly increased by transiently overexpressing SCARA. Furthermore, SCARA inhibitors also blocked internalization of cationic polymer:oligonucleotide complexes. Our results demonstrate that the previous held belief that CPPs act receptor independently does not hold true for CPP:oligonucleotide complexes, as scavenger receptor class A (SCARA) mediates the uptake of all the examined CPP:oligonucleotide complexes in this study.

PDGF beta targeting in cervical cancer cells suggest a fine-tuning of compensatory signalling pathways to sustain tumourigenic stimulation.

The platelet-derived growth factor (PDGF) signalling pathway has been reported to play an important role in human cancers by modulating autocrine and paracrine processes such as tumour growth, metastasis and angiogenesis. Several clinical trials document the benefits of targeting this pathway; however, in cervical cancer the role of PDGF signalling in still unclear. In this study, we used siRNA against PDGF beta (PDGFBB) to investigate the cellular and molecular mechanisms of PDGFBB signalling in Ca Ski and HeLa cervical cancer cells. Our results show that PDGFBB inhibition in Ca Ski cells led to rapid alterations of the transcriptional pattern of 579 genes, genes that are known to have antagonistic roles in regulating tumour progression. Concomitantly, with the lack of significant effects on cervical cancer cells proliferation, apoptosis, migration or invasion, these findings suggests that cervical cancer cells shift between compensatory signalling pathways to maintain their behaviour. The observed autocrine effects were limited to cervical cancer cells ability to adhere to an endothelial cell (EC) monolayer. However, by inhibiting PDGFBB on cervical cells, we achieved reduced proliferation of ECs in co-culture settings and cellular aggregation in conditioned media. Because of lack of PDGF receptor expression on ECs, we believe that these effects are a result of indirect PDGFBB paracrine signalling mechanisms. Our results shed some light into the understanding of PDGFBB signalling mechanism in cervical cancer cells, which could be further exploited for the development of synergistic anti-tumour and anti-angiogenic therapeutic strategies.

Development of a novel nanoparticle by dual modification with the pluripotential cell-penetrating peptide PepFect6 for cellular uptake, endosomal escape, and decondensation of an siRNA core complex.

Development of novel devices for effective nucleotide release from nanoparticles is required to improve the functionality of nonviral delivery systems, because decondensation of nucleotide/polycation complexes is considered as a key step for cytoplasmic delivery of nucleotides. Previously, PepFect6 (PF6) comprised chloroquine analog moieties and a stearylated cell-penetrating peptide to facilitate endosomal escape and cellular uptake, respectively, was developed as a device for efficient siRNA delivery. As PF6 contains bulky chloroquine analog moieties, the polyplexes are expected to be loose structure, which facilitates decondensation. In the present study, siRNA was electrostatically condensed by PF6, and the PF6/siRNA complexes were coated with lipid membranes. The surface of the nanoparticles encapsulating the PF6/siRNA core (PF6-NP) was modified with PF6 for endosomal escape (PF6/PF6-NP). The RNAi effect of PF6/PF6-NP was compared with those of stearylated cell-penetrating peptide octaarginine (R8)-modified PF6-NP, R8-modified nanoparticles encapsulating the R8/siRNA core (R8-NP) and PF6-modified R8-NP. Nanoparticles encapsulating the PF6 polyplex, especially PF/PF-NP, showed a significant knockdown effect on luciferase activity of B16-F1 cells stably expressing luciferase. siRNA was widely distributed within the cytoplasm after transfection of the nanoparticles encapsulating the PF6 polyplex, while siRNA encapsulated in the R8-presenting nanoparticles was localized within the nuclei. Thus, the siRNA distribution was dependent on the manner of peptide-modification. In conclusion, we have successfully developed PF6/PF6-NP exhibiting a potent RNAi effect resulting from high cellular uptake, efficient endosomal escape and decondensation of the polyplexes based on the multifunctional cell penetrating peptide PF6. PF6 is therefore a useful pluripotential device for siRNA delivery.

Identification of cell-penetrating peptides that are bactericidal to Neisseria meningitidis and prevent inflammatory responses upon infection.

Meningococcal disease is characterized by a fast progression and a high mortality rate. Cell-penetrating peptides (CPPs), developed as vectors for cargo delivery into eukaryotic cells, share structural features with antimicrobial peptides. A screen identified two CPPs, transportan-10 (TP10) and model amphipathic peptide (MAP), with bactericidal action against Neisseria meningitidis. Both peptides were active in human whole blood at micromolar concentrations, while hemolysis remained negligible. Additionally, TP10 exhibited significant antibacterial activity in vivo. Uptake of SYTOX green into live meningococci was observed within minutes after TP10 treatment, suggesting that TP10 may act by membrane permeabilization. Apart from its bactericidal activity, TP10 suppressed inflammatory cytokine release from macrophages infected with N. meningitidis as well as from macrophages stimulated with enterobacterial and meningococcal lipopolysaccharide (LPS). Finally, incubation with TP10 reduced the binding of LPS to macrophages. This novel endotoxin-inhibiting property of TP10, together with its antimicrobial activity in vivo, indicates the possibility to design peptide-based therapies for infectious diseases.

Molecular parameters of siRNA--cell penetrating peptide nanocomplexes for efficient cellular delivery.

Cell-penetrating peptides (CPPs) are versatile tools for the intracellular delivery of various biomolecules, including siRNA. Recently, CPPs were introduced that showed greatly enhanced delivery efficiency. However, the molecular basis of this increased activity is poorly understood. Here, we performed a detailed analysis of the molecular and physicochemical properties of seven different siRNA-CPP nanoparticles. In addition, we determined which complexes are internalized most efficiently into the leukemia cell-line SKNO-1, and subsequently inhibited the expression of a luciferase reporter gene. We demonstrated effective complexation of siRNA for all tested CPPs, and optimal encapsulation of the siRNA was achieved at very similar molar ratios independent of peptide charge. However, CPPs with an extreme high or low overall charge proved to be exceptions, suggesting an optimal range of charge for CPP-siRNA nanoparticle formation based on opposite charge. The most active CPP (PepFect6) displayed high serum resistance but also high sensitivity to decomplexation by polyanionic macromolecules, indicating the necessity for partial decomplexation for efficient uptake. Surprisingly, CPP-siRNA complexes acquired a negative ζ-potential in the presence of serum. These novel insights shed light on the observation that cell association is necessary but not sufficient for activity and motivate new research into the nature of the nanoparticle-cell interaction. Overall, our results provide a comprehensive molecular basis for the further development of peptide-based oligonucleotide transfection agents.

Modeling the endosomal escape of cell-penetrating peptides using a transmembrane pH gradient.

Cell-penetrating peptides (CPPs) can internalize into cells with covalently or non-covalently bound biologically active cargo molecules, which by themselves are not able to pass the cell membrane. Direct penetration and endocytosis are two main pathways suggested for the cellular uptake of CPPs. Cargo molecules which have entered the cell via an endocytotic pathway must be released from the endosome before degradation by enzymatic processes and endosomal acidification. Endosomal entrapment seems to be a major limitation in delivery of these molecules into the cytoplasm. Bacteriorhodopsin (BR) asymmetrically introduced into large unilamellar vesicles (LUVs) was used to induce a pH gradient across the lipid bilayer. By measuring pH outside the LUVs, we observed light-induced proton pumping mediated by BR from the outside to the inside of the LUVs, creating an acidic pH inside the LUVs, similar to the late endosomes in vivo. Here we studied the background mechanism(s) of endosomal escape. 20% negatively charged LUVs were used as model endosomes with incorporated BR into the membrane and fluorescein-labeled CPPs entrapped inside the LUVs, together with a fluorescence quencher. The translocation of different CPPs in the presence of a pH gradient across the membrane was studied. The results show that the light-induced pH gradient induced by BR facilitates vesicle membrane translocation, particularly for the intermediately hydrophobic CPPs, and much less for hydrophilic CPPs. The presence of chloroquine inside the LUVs or addition of pyrenebutyrate outside the LUVs destabilizes the vesicle membrane, resulting in significant changes of the pH gradient across the membrane.

PepFect15, a novel endosomolytic cell-penetrating peptide for oligonucleotide delivery via scavenger receptors.

Gene-regulatory biomolecules such as splice-correcting oligonucleotides and anti-microRNA oligonucleotides are important tools in the struggle to understand and treat genetic disorders caused by defective gene expression or aberrant splicing. However, oligonucleotides generally suffer from low bioavailability, hence requiring efficient and non-toxic delivery vectors to reach their targets. Cell-penetrating peptides constitute a promising category of carrier molecules for intracellular delivery of bioactive cargo. In this study we present a novel cell-penetrating peptide, PepFect15, comprising the previously reported PepFect14 peptide modified with endosomolytic trifluoromethylquinoline moieties to facilitate endosomal escape. Pepfect15 efficiently delivers both splice-correcting oligonucleotides and anti-microRNA oligonucleotides into cells through a non-covalent complexation strategy. To our knowledge this is the first work that describes peptide-mediated anti-microRNA delivery. The peptide and its cargo form stable, negatively charged nanoparticles that are taken up by cells largely through scavenger receptor type A mediated endocytosis.

Intracellular Delivery of Short Interfering RNA in Rat Organ of Corti Using a Cell-penetrating Peptide PepFect6.

RNA interference (RNAi) using short interfering RNA (siRNA) is an attractive therapeutic approach for treatment of dominant-negative mutations. Some rare missense dominant-negative mutations lead to congenital-hearing impairments. A variety of viral vectors have been tested with variable efficacy for modulating gene expression in inner ear. However, there is concern regarding their safety for clinical use. Here, we report a novel cell-penetrating peptide (CPP)-based nonviral approach for delivering siRNA into inner ear tissue using organotypic cultures as model system. PepFect6 (PF6), a variant of stearyl-TP10, was specially designed for improved delivery of siRNA by facilitating endosomal release. We show that PF6 was internalized by all cells without inducing cytotoxicity in cochlear cultures. PF6/siRNA nanoparticles lead to knockdown of target genes, a housekeeping gene and supporting cell-specific connexin 26. Interestingly, application of PF6/connexin 26 siRNA exhibited knockdown of both connexin 26 and 30 mRNA and their absence led to impaired intercellular communication as demonstrated by reduced transfer of calcein among the PF6/connexin 26-siRNA-treated cells. Thus, we conclude that PF6 is an efficient nonviral vector for delivery of siRNA, which can be applied as a tool for the development of siRNA-based therapeutic applications for hearing impairments.Molecular Therapy - Nucleic Acids (2012) 1, e61; doi:10.1038/mtna.2012.50; published online 11 December 2012.

Expanded ataxin-7 cause toxicity by inducing ROS production from NADPH oxidase complexes in a stable inducible Spinocerebellar ataxia type 7 (SCA7) model.

BACKGROUND: Spinocerebellar ataxia type 7 (SCA7) is one of nine inherited neurodegenerative disorders caused by polyglutamine (polyQ) expansions. Common mechanisms of disease pathogenesis suggested for polyQ disorders include aggregation of the polyQ protein and induction of oxidative stress. However, the exact mechanism(s) of toxicity is still unclear. RESULTS: In this study we show that expression of polyQ expanded ATXN7 in a novel stable inducible cell model first results in a concomitant increase in ROS levels and aggregation of the disease protein and later cellular toxicity. The increase in ROS could be completely prevented by inhibition of NADPH oxidase (NOX) complexes suggesting that ATXN7 directly or indirectly causes oxidative stress by increasing superoxide anion production from these complexes. Moreover, we could observe that induction of mutant ATXN7 leads to a decrease in the levels of catalase, a key enzyme in detoxifying hydrogen peroxide produced from dismutation of superoxide anions. This could also contribute to the generation of oxidative stress. Most importantly, we found that treatment with a general anti-oxidant or inhibitors of NOX complexes reduced both the aggregation and toxicity of mutant ATXN7. In contrast, ATXN7 aggregation was aggravated by treatments promoting oxidative stress. CONCLUSION: Our results demonstrates that oxidative stress contributes to ATXN7 aggregation as well as toxicity and show that anti-oxidants or NOX inhibition can ameliorate mutant ATXN7 toxicity.

Scavenger receptor-mediated uptake of cell-penetrating peptide nanocomplexes with oligonucleotides.

Cell-penetrating peptides (CPPs) are short cationic peptides that penetrate cells by interacting with the negatively charged plasma membrane; however, the detailed uptake mechanism is not clear. In contrary to the conventional mode of action of CPPs, we show here that a CPP, PepFect14 (PF14), forms negatively charged nanocomplexes with oligonucleotides and their uptake is mediated by class-A scavenger receptors (SCARAs). Specific inhibitory ligands of SCARAs, such as fucoidin, polyinosinic acid, and dextran sulfate, totally inhibit the activity of PF14-oligonucleotide nanocomplexes in the HeLa pLuc705 splice-correction cell model, while nonspecific, chemically related molecules do not. Furthermore, RNA interference (RNAi) knockdown of SCARA subtypes (SCARA3 and SCARA5) that are expressed in this cell line led to a significant reduction of the activity to <50%. In line with this, immunostaining shows prevalent colocalization of the nanocomplexes with the receptors, and electron microscopy images show no binding or internalization of the nanocomplexes in the presence of the inhibitory ligands. Interestingly, naked oligonucleotides also colocalize with SCARAs when used at high concentrations. These results demonstrate the involvement of SCARA3 and SCARA5 in the uptake of PF14-oligonucleotide nanocomplexes and suggest for the first time that some CPP-based systems function through scavenger receptors, which could yield novel possibilities to understand and improve the transfection by CPPs.