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Norway Report
Quality of Democracy
SGI experts on Norway
Norway SGI 2022
Kåre Hagen
Director, The Centre for Welfare and Labour Research, Oslo Metropolitan University
Kåre Hagen is director at The Centre for Welfare and Labour Research at Oslo Metropolitan University. He is a political scientist, and his fields of research are comparative welfare state policies, public sector reform and implications of European integration on EU member states. He has had positions at the Department for Political Science at the University of Oslo and at the Norwegian School of Management (BI). Hagen if frequently used by Norwegian Governments to prepare reports on public sector reform.
Tobias Bach
Prof., Department of Political Science, University of Oslo
Tobias Bach is Professor at the Department of Political Science, where he leads the research group Policy, Bureaucracy, and Organization. He is also Senior Researcher at ARENA Centre for European Studies. He obtained his PhD at the University of Potsdam (2013). He previously worked at the Hertie School, the University of Hannover, and the University of Potsdam. He was a visiting researcher at KU Leuven and the University of Bergen. His research addresses the structure and organization of government and executive politics in an internationally comparative perspective.
Detlef Jahn (Coordinator)
Professor, University of Greifswald
Detlef Jahn has been professor of comparative politics at the University of Greifswald since 1999 and was a research professor at Nottingham Trent University from 1996 to 1999. He studied political science, sociology and history at the universities of Duisburg, Bielefeld and Edinburgh and holds a PhD (1991) from the European University Institute (Florence). He has been a guest professor at several universities in the United States (Irvine, Los Angeles, New York, Las Vegas), Sweden (Göteborg, Södertörn), Australia (NAU), and New Zealand (Lincoln). He is a member of the international advisory board at the Centre of Excellence on Democracy Research of the Åbo Akedemi University. Currently he is a permanent fellow at the KFG at the Freie Universität Berlin. His research interests include the study of institutions, party preferences and social and environmental policy studies. He has created a comprehensive database for analysis of the political process in modern democracies that is updated regularly and can be accessed at: http://comparativepolitics.uni-greifswald.de.
With strong outcomes in nearly every category, Norway falls into the top group worldwide (rank 3) with regard to the quality of democracy. Its score on this measure has declined by 0.2 points relative to 2014.
The country's electoral processes are free and fair. Political party financing is mostly public, with strong reporting requirements. Political advertising is extensively regulated to ensure voters are aware of funders. The media are independent, showing considerable plurality of opinion, but digital media and particularly social media platforms are drawing audiences and advertising away from traditional media.
Civil rights and political liberties receive strong protections, and gender-equality provisions are robust, but the labor market remains strongly segregated by gender and occupation. Some labor market discrimination against immigrants persists, despite being illegal.
The legal system is transparent, predictable and respected. Corruption is rare, with considerable social stigma attached.
Electoral Processes
How fair are procedures for registering candidates and parties?
Legal regulations provide for a fair registration procedure for all elections; candidates and parties are not discriminated against.
A few restrictions on election procedures discriminate against a small number of candidates and parties.
Some unreasonable restrictions on election procedures exist that discriminate against many candidates and parties.
Discriminating registration procedures for elections are widespread and prevent a large number of potential candidates or parties from participating.
Candidacy Procedures
Procedures for registering candidates and political parties are considered fair and have not been contested or subject to public debate. The formal requirements for starting a new party is the registration of a unique name and the support of 5,000 persons with the right to vote. Parties nominate candidates for elections. After being elected, a candidate may change their party membership without losing their position.
To what extent do candidates and parties have fair access to the media and other means of communication?
All candidates and parties have equal opportunities of access to the media and other means of communication. All major media outlets provide a fair and balanced coverage of the range of different political positions.
Candidates and parties have largely equal opportunities of access to the media and other means of communication. The major media outlets provide a fair and balanced coverage of different political positions.
Candidates and parties often do not have equal opportunities of access to the media and other means of communication. While the major media outlets represent a partisan political bias, the media system as a whole provides fair coverage of different political positions.
Candidates and parties lack equal opportunities of access to the media and other means of communications. The major media outlets are biased in favor of certain political groups or views and discriminate against others.
Media Access
Candidates and parties are free to purchase political advertising in print publications and in social media. Advertisements from political parties are not allowed on television or radio. This ban has been subject to some controversy, with the populist Progress Party advocating a removal of the restriction. The other political parties are opposed to changing the law. Political advertising during election campaigns is extensively regulated to ensure that voters are aware of the sources behind such advertising.
Television and radio broadcasters, both public and private, organize many electoral debates, to which all major parties (those with a vote share larger than 3% in the previous election) have fair access. There is no government interference in choosing the themes of the debates. In general, however, representatives of the larger parties are interviewed more often and participate in more debates than do small-party candidates.
To what extent do all citizens have the opportunity to exercise their right of participation in national elections?
All adult citizens can participate in national elections. All eligible voters are registered if they wish to be. There are no discriminations observable in the exercise of the right to vote. There are no disincentives to voting.
The procedures for the registration of voters and voting are for the most part effective, impartial and nondiscriminatory. Citizens can appeal to courts if they feel being discriminated. Disincentives to voting generally do not constitute genuine obstacles.
While the procedures for the registration of voters and voting are de jure non-discriminatory, isolated cases of discrimination occur in practice. For some citizens, disincentives to voting constitute significant obstacles.
The procedures for the registration of voters or voting have systemic discriminatory effects. De facto, a substantial number of adult citizens are excluded from national elections.
Voting and Registration Rights
All Norwegian citizens who are 18 years old or older have the right to vote in parliamentary elections. In local elections, individuals without Norwegian citizenship aged 18 years or older who have resided in Norway for at least three years have the right to vote. The three-year rule does not apply to Nordic citizens with a permanent residence in Norway. There is no requirement of prior registration. Each eligible citizen receives a voting card sent by mail. It is possible to vote before the election through mail-in-ballot or at specific locations, including at Norwegian embassies abroad. There has been no allegation from any political party that the electoral process is not inclusive. Election turnout is high, and discrimination is rarely reported. Young voters "learn" voting behavior in schools by participating in a school vote prior to reaching the age of voting eligibility. Some municipalities have experimented with a voting age of 16 in local elections.
To what extent is private and public party financing and electoral campaign financing transparent, effectively monitored and in case of infringement of rules subject to proportionate and dissuasive sanction?
The state enforces that donations to political parties are made public and provides for independent monitoring to that respect. Effective measures to prevent evasion are effectively in place and infringements subject to effective, proportionate and dissuasive sanctions.
The state enforces that donations to political parties are made public and provides for independent monitoring. Although infringements are subject to proportionate sanctions, some, although few, loopholes and options for circumvention still exist.
The state provides that donations to political parties shall be published. Party financing is subject to some degree of independent monitoring but monitoring either proves regularly ineffective or proportionate sanctions in case of infringement do not follow.
The rules for party and campaign financing do not effectively enforce the obligation to make the donations public. Party and campaign financing is neither monitored independently nor, in case of infringements, subject to proportionate sanctions.
Party Financing
Political parties in Norway are funded primarily by public grants that are proportional to the number of votes received by a party during the last held parliamentary election. On average, parties receive about three-quarters of their revenues through state subsidies (ranging from 60% to 80%). Membership fees are an insignificant source of party finances. Parties also receive private donations. For example, the Labor Party receives funds from particular trade unions, while the Conservative Party receives donations from individuals and business organizations.
Since 1998, political parties have been obliged to publish an overview of the source of their revenues, and detailed reports have been required since 2005. Thus, all party organizations, central and local, are today obliged to submit detailed income reports, with full information on the source of income, on an annual basis. Information on contributions of NOK 35,000 or more must be provided separately, with the identity of the donor included. In election years, contributions of NOK 10,000 or more must be reported. Income reports are submitted to the Central Bureau of Statistics and are published in detail.
Do citizens have the opportunity to take binding political decisions when they want to do so?
Citizens have the effective opportunity to actively propose and take binding decisions on issues of importance to them through popular initiatives and referendums. The set of eligible issues is extensive, and includes national, regional, and local issues.
Citizens have the effective opportunity to take binding decisions on issues of importance to them through either popular initiatives or referendums. The set of eligible issues covers at least two levels of government.
Citizens have the effective opportunity to vote on issues of importance to them through a legally binding measure. The set of eligible issues is limited to one level of government.
Citizens have no effective opportunity to vote on issues of importance to them through a legally binding measure.
Popular Decision-Making
Government decision-making is inclusive in that organized interests have access to and are incorporated into regular processes of planning and implementation. The system makes no provision for direct citizen participation in the form of legally binding public votes or citizen referendum initiatives. Referendums have been held, but only on unique occasions, the last one was held in 1994 on EU membership. Formally, referendums are strictly consultative, though they are treated as binding in practice. Referendums have been more frequently held at the local level, on issues like alcohol restrictions, the primary language of instruction at schools, and on mergers with/separation from other municipalities. Local governments must formally take the initiative in order for a local referendum to be held.
To what extent are the media independent from government?
Public and private media are independent from government influence; their independence is institutionally protected and fully respected by the incumbent government.
The incumbent government largely respects the independence of media. However, there are occasional attempts to exert influence.
The incumbent government seeks to ensure its political objectives indirectly by influencing the personnel policies, organizational framework or financial resources of public media, and/or the licensing regime/market access for private media.
Major media outlets are frequently influenced by the incumbent government promoting its partisan political objectives. To ensure pro-government media reporting, governmental actors exert direct political pressure and violate existing rules of media regulation or change them to benefit their interests.
Media market and media consumption behaviors are rapidly changing in a country featuring the widespread use of digital media. The dominant TV and radio channel is the state-owned Norwegian Broadcasting Cooperation (NRK). It is a public service channel, financed by public grants set by the parliament. One national, commercially financed private TV channel receives financial compensation for producing a broad range of content, a condition that is anchored in an agreement with the government. In addition, several broadcasters operate from other countries. Aside from commercials for tobacco, alcohol and gambling, and commercials directed at children, which are banned, there are no restrictions on content.
The state-owned broadcaster (NRK) is organized in a way that ensures considerable autonomy. It is independent in all aspects of editorial policy, and the government does not intervene in the organization's daily operations or editorial decisions. The head of NRK reports to a board of directors. Board members are appointed by the government. A separate institution called the Broadcasting Council (Kringkastingsrådet) plays an oversight role, monitoring, debating and expressing views about the management and activities of the state-funded broadcast media. It can also provide advice on administrative and economic issues. The issues debated by the council can originate with the chairman of the state broadcasting organization or from the public (often in the form of criticism and complaints). The opinions expressed by the Kringkastingsrådet carry substantial weight, and recommendations from this council are usually implemented. Eight council members are appointed by the parliament, and an additional six by the government.
Newspapers are all privately owned. The freedom of the press is explicitly guaranteed in the constitution; the article addressing press freedoms was amended and strengthened with a constitutional amendment in 2004. In Norway there is a historical tradition of two or more local newspapers, often representing different political views. In order to maintain this pluralism, the state provides financial support for the smaller newspapers through unconditional grants.
All TV channels and media outlets have developed digital platforms. Increased numbers of digital publications and other changes in the media world have burdened many of the media houses. Some major media houses have experimented with new combinations of marketing and journalism that might challenge consumers' faith in the independence of journalism. New technology is rapidly changing the media landscape, drawing audiences away from TV and newspapers to digital media platforms. Social media platforms such as Facebook and Google increasingly draw advertisement revenues away from traditional media in Norway and elsewhere. In addition, the media landscape is becoming more diversified and national media increasingly competes with international digital news sources.
To what extent are the media characterized by an ownership structure that ensures a pluralism of opinions?
Diversified ownership structures characterize both the electronic and print media market, providing a well-balanced pluralism of opinions. Effective anti-monopoly policies and impartial, open public media guarantee a pluralism of opinions.
Diversified ownership structures prevail in the electronic and print media market. Public media compensate for deficiencies or biases in private media reporting by representing a wider range of opinions.
Oligopolistic ownership structures characterize either the electronic or the print media market. Important opinions are represented but there are no or only weak institutional guarantees against the predominance of certain opinions.
Oligopolistic ownership structures characterize both the electronic and the print media market. Few companies dominate the media, most programs are biased, and there is evidence that certain opinions are not published or are marginalized.
Media Pluralism
In terms of audience ratings, the state-owned Norwegian Broadcasting Corporation (NRK) dominates in TV, radio and, increasingly, digital platforms. There is a great number of private, commercial TV and radio channels, many of them based in, and sending from, other countries. A special body called the Norwegian Media Authority (Medietilsynet) is responsible for monitoring and regulating the market, although foreign actors cannot be controlled.
The stated goal of government regulation of the broadcast-media market is to guarantee that quality remains high and that coverage is national. Cable TV is essentially unregulated beyond the effect of general laws (e.g., bans on commercial for alcohol, tobacco, gambling and political statements).
Newspapers operate independently and express a plurality of views. As elsewhere in the world, newspaper circulation is on the decline, as is print advertising. As a result, many newspapers are under financial strain and have in recent years been forced to cut back on editorial staff. Web-based news outlets are replacing print newspapers and are accounting for a steadily growing market share of media advertising. In the last few years, local newspapers in particular have come under increasing strain resulting from reductions in advertising income and subscription rates.
The concentration of ownership has to date not been perceived as a threat to media plurality. However, private ownership is becoming increasingly oligopolistic across print and broadcast media. The distributors of digital signals have also used their power to change marketplace dynamics. Since digital distribution is becoming increasingly important, the structure of ownership in this channel has a larger negative implication for media plurality. Although there is a tradition of nonintervention by owners in editorial matters, the print media as has at critical junctures become politically biased. The media landscape as a whole, as well as the general public debate, demonstrates a noticeable and sometimes-narrow political correctness. Broadband internet is widely used and accessible across the entire country.
Increasingly, international companies such as Facebook and Google, have gained a huge share of the advertising market, and this has triggered a renewed debate about the role of monopolies in media.
To what extent can citizens obtain official information?
Legal regulations guarantee free and easy access to official information, contain few, reasonable restrictions, and there are effective mechanisms of appeal and oversight enabling citizens to access information.
Access to official information is regulated by law. Most restrictions are justified, but access is sometimes complicated by bureaucratic procedures. Existing appeal and oversight mechanisms permit citizens to enforce their right of access.
Access to official information is partially regulated by law, but complicated by bureaucratic procedures and some poorly justified restrictions. Existing appeal and oversight mechanisms are often ineffective.
Access to official information is not regulated by law; there are many restrictions of access, bureaucratic procedures and no or ineffective mechanisms of enforcement.
Access to Government Information
Freedom of information legislation gives every person right of access to official documents held by any public authority. Official documents are defined as information that is recorded and can be listened to, displayed or transferred, and which is either created, dispatched or received by an authority.
All records are indexed at the time of creation or receipt. Some ministries make these electronic indexes available on the internet or through e-mail. Requests can be made in any form (even anonymously) and must be responded to without undue delay, generally (according to Ministry of Justice guidelines) within three days.
Documents can be withheld if they are made secret by another law or if they refer to issues of national security, national defense or international relations, financial management, the minutes of the State Council, appointments or security measures in the civil service, regulatory or oversight measures, test answers, annual fiscal budgets or long-term budgets, or photographs of persons entered in a personal data register. If access is denied, individuals can appeal to a higher authority and then to the parliament's ombudsman for public administration, or to a court. The ombudsman's decisions are not binding but are generally followed. There have been very few court cases dealing with this issue.
The 1998 Security Act sets rules on the classification of information. It creates four levels of classification and mandates that information cannot be classified for more than 30 years. The Act on Defense Secrets prohibits the disclosure of military secrets by government officials, as well as the collection (in the form of sketches, photographs or notes) and disclosure of secrets by others, including journalists. Articles 90 and 91 of the criminal code criminalize the disclosure of secrets, and provide for imprisonment of up to 10 years for violations of these provisions.
Civil Rights and Political Liberties
To what extent does the state respect and protect civil rights and how effectively are citizens protected by courts against infringements of their rights?
All state institutions respect and effectively protect civil rights. Citizens are effectively protected by courts against infringements of their rights. Infringements present an extreme exception.
The state respects and protects rights, with few infringements. Courts provide protection.
Despite formal protection, frequent infringements of civil rights occur and court protection often proves ineffective.
State institutions respect civil rights only formally, and civil rights are frequently violated. Court protection is not effective.
State institutions respect and protect civil rights. Personal liberties are well-protected against abuse by state and non-state actors. People cannot be detained without a formal charge for more than 24 hours. A court decision is needed if a suspect is to be held in prison during an investigation, a question given more serious consideration in Norway than in many other countries.
Access to the courts is free and easy, and the judiciary system is generally regarded by the general public as being fair and efficient. However, if one loses a case brought to the court, one risks having to pay the full cost of the proceedings. This may deter citizens from bringing cases to court, in addition to the prohibitive fees that lawyers may charge. For those with an extremely low income there is a state program for funding costs, and most labor union memberships (which are widespread) include an insurance against high expenses.
To what extent does the state concede and protect political liberties?
All state institutions concede and effectively protect political liberties.
All state institutions for the most part concede and protect political liberties. There are only few infringements.
State institutions concede political liberties but infringements occur regularly in practice.
Political liberties are unsatisfactory codified and frequently violated.
Political Liberties
Political liberties are protected by the constitution and the law. The right to free expression was strengthened through a constitutional amendment in 2004. Limitations to freedom of speech (such as regarding hate speech/discrimination) are regulated by law. All citizens may comment on legislative proposals in hearing procedures. In 2014, the Sámi minority was granted explicit rights to their own language and cultural expressions. Norway has ratified all international conventions on human and civil rights. The European Convention on Human Rights is incorporated into national law. The right to free worship and other religious activities are ensured. The historical tradition of a privileged, state-owned Lutheran church was ended in 2017, and now, all religious communities are treated equally. Political liberties are respected by state institutions.
How effectively does the state protect against different forms of discrimination?
State institutions effectively protect against and actively prevent discrimination. Cases of discrimination are extremely rare.
State anti-discrimination protections are moderately successful. Few cases of discrimination are observed.
State anti-discrimination efforts show limited success. Many cases of discrimination can be observed.
The state does not offer effective protection against discrimination. Discrimination is widespread in the public sector and in society.
Equality of opportunity and equality before the law are firmly established in Norway. There is an ombudsperson for civil rights and one for the right to non-discrimination based on gender, disability, ethnicity and sexual orientation. As of 2021, Norway also has an ombudsperson tasked with protecting the elderly from ageism. The Sámi minority living in the north have a limited right to self-rule, though there still are some unsettled issues over the use of natural resources in this area.
Men and women are nearly at par in terms of education levels. Women's labor-force participation rate is comparatively high among OECD countries. Women earn on average 87.5% of what men do. However, once specifics such as hours worked, occupation, education and seniority are taken into consideration, it is difficult to verify significant differences between the earnings of men and women. This finding does not per se imply that there is no gender discrimination whatsoever in the labor market (e.g., men may be more readily hired for high-paying occupations). In 2017, several instances of gender-based discrimination were disclosed as a result of the #metoo campaign. That being said, affirmative action in favor of women has been used extensively in the labor market, particularly within the public sector. Even so, the labor market remains by international comparison strongly segregated by gender and occupation.
Some discrimination against non-Western immigrants seems to persist. In some areas of the economy, immigrants find it comparatively difficult to find work and are generally paid lower wages. Unemployment rates are also substantially higher among immigrant populations than that seen among native Norwegians. Although discrimination against immigrants (including in the labor market) is illegal, it occurs in some areas of Norwegian society, though very few discrimination cases are prosecuted.
To what extent do government and administration act on the basis of and in accordance with legal provisions to provide legal certainty?
Government and administration act predictably, on the basis of and in accordance with legal provisions. Legal regulations are consistent and transparent, ensuring legal certainty.
Government and administration rarely make unpredictable decisions. Legal regulations are consistent, but leave a large scope of discretion to the government or administration.
Government and administration sometimes make unpredictable decisions that go beyond given legal bases or do not conform to existing legal regulations. Some legal regulations are inconsistent and contradictory.
Government and administration often make unpredictable decisions that lack a legal basis or ignore existing legal regulations. Legal regulations are inconsistent, full of loopholes and contradict each other.
Legal Certainty
Norway's government and administration act predictably and in accordance with the law. Norway has a sound and transparent legal system. Corruption within the legal system is a rather marginal problem. The state bureaucracy is regarded as both efficient and reliable. Norwegian citizens generally trust their institutions. In principle, the Supreme Court may test the constitutional legality of a government decision, though it has not done so for many years.
To what extent do independent courts control whether government and administration act in conformity with the law?
Independent courts effectively review executive action and ensure that the government and administration act in conformity with the law.
Independent courts usually manage to control whether the government and administration act in conformity with the law.
Courts are independent, but often fail to ensure legal compliance.
Courts are biased for or against the incumbent government and lack effective control.
Norway's court system provides for the review of actions by the executive. The legal system is grounded in the principles of the so-called Scandinavian civil-law system. There is no general codification of private or public law, as in civil-law countries. Rather, there are comprehensive statutes codifying central aspects of the criminal law and the administration of justice, among other things.
Norwegian courts do not attach the same weight to judicial precedents as does the judiciary in common-law countries. Court procedure is relatively informal and simple, and there is a strong lay influence in the judicial assessment of criminal cases.
At the top of the judicial hierarchy is the Supreme Court, which is followed by the High Court. The majority of criminal matters are settled summarily in the district courts. A Court of Impeachment is available to hear charges brought against government ministers, members of parliament and Supreme Court judges, although it is very rarely used. The courts are independent of any influence exerted by the executive. Professional standards and the quality of internal organization are high. The selection of judges is rarely disputed and is not seen as involving political issues.
To what extent does the process of appointing (supreme or constitutional court) justices guarantee the independence of the judiciary?
Justices are appointed in a cooperative appointment process with special majority requirements.
Justices are exclusively appointed by different bodies with special majority requirements or in a cooperative selection process without special majority requirements.
Justices are exclusively appointed by different bodies without special majority requirements.
All judges are appointed exclusively by a single body irrespective of other institutions.
Appointment of Justices
All judges are formally appointed by a government decision that is made based on a recommendation issued by an autonomous body, the Instillingsrådet. This body is composed of three judges, one lawyer, a legal expert from the public sector and two members who are not from the legal profession. The government almost always follows the recommendations. Supreme Court justices are not considered to be in any way political and their tenure security is guaranteed in the constitution. There is a firm tradition of autonomy in the Supreme Court. The appointment of judges attracts limited attention and rarely leads to public debate.
To what extent are public officeholders prevented from abusing their position for private interests?
Legal, political and public integrity mechanisms effectively prevent public officeholders from abusing their positions.
Most integrity mechanisms function effectively and provide disincentives for public officeholders willing to abuse their positions.
Some integrity mechanisms function, but do not effectively prevent public officeholders from abusing their positions.
Public officeholders can exploit their offices for private gain as they see fit without fear of legal consequences or adverse publicity.
There are few very few instances of corruption in Norway. The cases that have surfaced in recent years have been at the municipal level and are related to public procurement. As a rule, corrupt officeholders are prosecuted under established laws. There is a great social stigma against corruption, even in its minor manifestations. During the last decade, some incidences of corruption related to investments and overseas Norwegian business activities have been revealed.


Only two months into 2018 and it's already been quite a year. When the 2017 Tax Cuts and Jobs Act was signed into law at the end of December, we knew we had to share our insights on what the new tax provisions mean for solar investments.
With that now behind us, we can focus on our year-in-review post. Let's take a look at what SDC Energy accomplished with your help in 2017.
As you take your tax planning forward you can feel good about realizing the 30% tax benefit that comes from investing in solar projects. Combined, our investors' tax offset from the Federal Tax Investment Credit (ITC) was to the tune of $1.3 million last year. Not too shabby.
And that's not counting the $3.7 million of income write-offs our investors gained by putting their money into one of the smartest tax investment – keeping more dollars in their pocket.
Help us spread the word. Share this post using #SDCEnergy and let others know how effective investing in solar can be to lower taxes.
Some of the projects funded in 2017 are already generating revenue so you know first-hand that solar routinely yields great returns. Last year our investors' solar installations generated over $400,000 of revenue with an expected $4,000,000 over the next decade.
Financing solar doesn't just lower your taxes; it protects you from volatile markets with a reliable cash flow that guarantees income over the life of your investment. This doesn't seem so important when the market is riding high, but as the Dow plunged 1,175 on Feb. 5 – the biggest one-day point decline in history – "steady solar" sure looked more attractive.
By investing in SDC Energy solar projects you can double your impact with socially-minded solar financing. Together, we made quite a difference in 2017.
You brought much-needed financing to 12 churches, schools, and other non-profits last year, helping these cash-strapped community-builders cut operating costs and achieve sustainability goals with clean solar energy.
Solar does great things for your portfolio while offsetting dirty fossil fuels and keeping the air cleaner for generations to come.
Combined, your 2017 solar investments will remove over 1,000 tons of harmful pollutants from the atmosphere during their first year of operation. That's like planting 1,000 acres of forest, or taking 225 cars off the road, or avoiding burning 1 million pounds of coal – or planting 1,000 acres of forest — or taking 225 cars off the road, each year for the 20-plus years your solar investment produces energy.
That's plenty of good both for your pocketbook and the environment.
We are proud to have such committed investors and want to thank you for your trust in SDC Energy, both last year and into the future. Let's create more positive change together so that you can cut your tax bill while supporting communities and the planet.
Help us promote all of these amazing benefits. Share your thoughts on what your solar investment means to you and we'll use your comments to reach new investors.
Send us an email or share this post on LinkedIn or Facebook with the hashtag #SDCEnergy.


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*Restore ministries is reposting this blog because we failed to include all of the content.
You can read the original post at www.infoforfamilies.com.
Also, there is a part 2 to this which will be posted so be watching for that as well.


text:
Meeting abstract
The role of probiotics in nosocomial infections
Gian Vincenzo Zuccotti1 and
Fabio Meneghin1
Italian Journal of Pediatrics201440 (Suppl 2) :A16
© Zuccotti and Meneghin; licensee BioMed Central Ltd. 2014
Nosocomial Infection
Nosocomial Pneumonia
Acute Gastroenteritis
Probiotic Supplementation
Nosocomial infections are among the leading causes of mortality and morbidity especially in neonatal intensive care unit (NICU) [1]. The intestinal microbiota of the gut is nowadays considered to play an important functional role in the host's health through nutritional, physiological and immunological processes. For these reasons, probiotics may exert actions of prevention and therapy of infectious diseases.
The mechanisms of action of probiotics are strain specific but can be summarized mainly in three areas: changes of gut ecology, modulation of gut mucosal barrier and regulation of the immune response through interaction with gut-associated immune system [2]. Several studies regarding the supplementation of probiotics in nosocomial infections have been conducted mainly in adult population. Among pediatric studies major findings have been observed in treatment of acute gastroenteritis, primarily caused by Rotavirus [3, 4], and in the prevention of antibiotic associated diarrhea (AAD) [5]. Supplementation with probiotics has proven useful even in the treatment of Clostridium difficile disease (CDD), the most common pathogen involved in AAD [6]. Data from meta-analysis and cochrane review on the prevention of necrotizing enterocolitis (NEC) show an overall benefit of probiotic supplementation [7]. The limitations of the above cited studies are mainly related to heterogeneity in terms of strain, dosage and duration of treatment and the lack of studies on extremely low birth weight preterm infants. Data on nosocomial pneumonia and ventilator-associated pneumonia in neonatal and pediatric age is scanty. In a large randomized, double-blind placebo controlled study, Hojsak et al demonstrated that supplementation with Lactobacillus GG significantly decreased the risk of nosocomial respiratory tract infections [8]. On the other hand, the data from adult studies have been conflicting, with a tendency towards the demonstration of probiotic efficacy in reducing the incidence of ventilator-associated pneumonia [9]. Meticillin-resistant Staphylococcus aureus is a multidrug-resistant nosocomial pathogen; a recent review of literature [10] showed that many probiotic strains inhibit MRSA growth in vitro. Furthermore, this review describes that there is little published clinical data on the use of probiotics in prophylaxis or treatment of MRSA-mediated infections.
Due to the significant heterogeneity between the studies in literature it is not possible to draw consistent conclusions on extensive use of probiotics in prevention and treatment of nosocomial infections, except for acute gastroenteritis, AAD, CDD and NEC.
Department of Pediatrics, University of Milan, Luigi Sacco Hospital, Milan, 20157, Italy
Polin RA, Denson S, Brady MT: Epidemiology and Diagnosis of Health Care–Associated Infections in the NICU. Pediatrics. 2012, 129: e1104-e1109.View ArticlePubMedGoogle Scholar
Kotzampassi K, Giamarellos-Bourboulis EJ: Probiotics for infectious diseases: more drugs, less dietary supplementation. International Journal of Antimicrobial Agents. 2012, 40: 288-296. 10.1016/j.ijantimicag.2012.06.006.View ArticlePubMedGoogle Scholar
Allen SJ, Martinez EG, Gregorio GV: Probiotics for treating acute infectious diarrhoea (Review). Cochrane Database of Systematic Reviews. 2010, CD003048-10.1002/14651858.CD003048.pub3. 11Google Scholar
Guarino A, Ashkenazi S, Gendrel D, Vecchio AL, Shamir R, Szajewska H: European Society for Pediatric Gastroenterology, Hepatology, and Nutrition/European Society for Pediatric Infectious Diseases Evidence-Based Guidelines for the Management of Acute Gastroenteritis in Children in Europe: Update 2014. JPGN. 2014, 59: 132-152.PubMedGoogle Scholar
McFarland LV: Meta-Analysis of Probiotics for the Prevention of Antibiotic Associated Diarrhea and the Treatment of Clostridium difficile Disease Meta-Analysis of Probiotics for the Prevention of Antibiotic Associated Diarrhea and the Treatment of Clostridium difficile Disease. Am J Gastroenterol. 2006, 101: 812-822. 10.1111/j.1572-0241.2006.00465.x.View ArticlePubMedGoogle Scholar
Goldenberg JZ, Ma SSY, Saxton JD, Martzen MR, Vandvik PO, Thorlund K, Guyatt GH, Johnston BC: Probiotics for the prevention of Clostridium difficile associated diarrhea in adults and children. Cochrane Database of Systematic Reviews. 2013, CD006095-10.1002/14651858.CD006095.pub3. 5Google Scholar
AlFaleh K, Anabrees J, Bassler D: Probiotics for prevention of necrotizing enterocolitis in preterm infants . Cochrane Database of Systematic Reviews. 2011, CD005496-10.1002/14651858.CD005496.pub3. 3Google Scholar
Hojsak I, Abdovic S, Szajewska H, Milosevic M, Krznaric Z, Kolacek S: Lactobacillus GG in the Prevention of Nosocomial Gastrointestinal and Respiratory Tract Infections. Pediatrics. 2010, 125: e1171-e1177. 10.1542/peds.2009-2568.View ArticlePubMedGoogle Scholar
Siempos II, Ntaidou TK, Falagas ME: Impact of the administration of probiotics on the incidence of ventilator-associated pneumonia: A meta-analysis of randomized controlled trials. Crit Care Med. 2010, 38: 954-962. 10.1097/CCM.0b013e3181c8fe4b.View ArticlePubMedGoogle Scholar
Sikorska H, Smoragiewicz W: Role ofprobioticsinthepreventionandtreatmentof meticillin-resistant Staphylococcus aureus infections. International Journal of Antimicrobial Agents. 2013, 42: 475-481. 10.1016/j.ijantimicag.2013.08.003.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

redpajama_set_name: RedPajamaCommonCrawl


Nike's jumbo-branded 1990s hoops classic the Air More Uptempo is back again, this time donning a Chicago Bulls colorway in white/red.
The Air More Uptempo's 2016 return has been very successful so far, with the first black/white release even moving the needle on the resell market. Thankfully, that shoe's come back in waves, helping eliminate the need to pay over retail.
Also on the way for this model is a white/gum colorway set to release later this year.
Readers can find the white/red pair now at Kicks USA.


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Israeli Blogger's Arrest Causes Serious Concern says Karabakh
in Artsakh, International, Latest, Top Stories
Alexander Lapshin, Israeli blogger who was detained in Minsk, Belarus by the order of Azerbaijani authorities for visiting Nagorno-Karabakh (Photo: Alexander Lapshin)
STEPANAKERT (ArmRadio)—"The launching of a criminal case against famous blogger Alexander Lapshin and his detention cause serious concern," press service of the Nagorno-Karabakh Republic (Artsakh) Foreign Ministry said during an interview with News.am.
Lapshin, who holds Russian and Israeli citizenship, was arrested in Belarus last Wednesday at the request of the Azerbaijani government over his visits to Nagorno-Karabakh.
"Azerbaijan, where harassment and political persecution against civil activists and journalists on fabricated pretexts have long become a tradition, tries to spread its "standards" of human rights beyond the borders of the country," said the Artsakh Foreign Ministry.
"Such actions are not only an obvious violation of fundamental human rights, namely, the freedom of movement and freedom of speech, but also a policy of intimidation, which should be adequately assessed by the international community," the Ministry said.
Lapshin was arrested in Belarus last Wednesday at the request of the Azerbaijani government over his visits to Nagorno-Karabakh, Haaretz reports.
Lapshin wrote several critical posts against Azerbaijan and its president, Ilham Aliyev, in recent months. His family is afraid that if he's extradited to Azerbaijan, his life will be in danger.
Lapshin, 40, writes a blog in which he records his travels throughout the world, and writes about the various countries he has visited.
On December 13, he and his wife arrived in the Belarus capital, Minsk. A day later, shortly before midnight, Lapshin wrote on his Facebook page: "At this moment I was arrested by the police in Minsk at the request of Azerbaijan. I'm at the Piersamajskaja station in Minsk."
The Azeri government claims that in April 2011 and October 2012, Lapshin visited Nagorno-Karabakh and wrote about the visits in his blog. After the last visit, Lapshin's name was placed on the Baku government's blacklist, and he in effect became a wanted man.
Last June, Lapshin entered Azerbaijan itself, using a Ukrainian passport. Because his name in Ukrainian is slightly different from the name listed in his Russian or Israeli passport, he was not arrested upon entering the country. After a few days traveling around the country, he left without any problem.
In subsequent blog posts, Lapshin criticized the poverty in certain parts of the capital, Baku; the dictatorial rule of Aliyev; and the issue of Nagorno-Karabakh.
Israel on Sunday asked Belarus to not hand Lapshin over to Azerbaijan, the Times of Israel reported.
Life Sentence Upheld for Murderer of Gyumri Family


text:
Cellular Physiology & Biochemistry
International Journal of Experimental Cellular Physiology, Biochemistry and Pharmacology
https://doi.org/10.33594/000000474
Corresponding Author: Ghada Bouguerra
Laboratoire d'Hématologie Moléculaire et Cellulaire, Institut Pasteur de Tunis, 13 Place Pasteur, PO BOX no74, 1002, Belvédère, Tunis (Tunisia)
Tel. +216 71 84 66 59, Fax +216 71 79 18 33, E-Mail ghada.bouguerra@yahoo.fr
Enhanced Eryptosis in Glucose-6-Phosphate Dehydrogenase Deficiency
Ghada Bouguerraa,b Khaoula Talbib,c Nawel Trabelsib,c Dorra Chaouachib Imen Boudrigab Salem Abbèsa,b Samia Menifa,b
aUniversité de Tunis El Manar, Faculté de Médecine de Tunis, Tunis, Tunisie, bUniversité de Tunis El Manar, Laboratoire d'Hématologie Moléculaire et Cellulaire, Institut Pasteur de Tunis, Tunis, Tunisie, cUniversité de Tunis El Manar, Faculté des Sciences de Tunis, Tunis, Tunisie
Eryptosis • Phosphatidylserine exposure • Anemia • G6PD deficiency • Oxidative stress
Background/Aims: Defects in the Glucose-6-Phosphate Dehydrogenase (G6PD) enzyme enhance cellular oxidative damage, thus impairing erythrocytes and radically shortening their lifespan. We aimed to study programmed erythrocyte cell death in G6PD-deficient patients, describe the molecular genetics basis of G6PD and investigate phenotype-genotype correlations. Methods: We explored eryptosis using the annexin V-binding assay, taken as an indicator of PS exposure at the erythrocyte surface. We assessed reactive oxygen species (ROS) production, intracellular calcium concentrations and ceramide formation at the cell surface. Prior to and following treatments, cells were analyzed by flow cytometry. Finally, we explored G6PD gene mutations through PCR-Sanger sequencing. Results: Before stimulation, PS-exposing erythrocytes were significantly higher in G6PD-deficient patients than in healthy volunteers. This was paralleled by a significant increase in reactive oxygen species production, suggesting that oxidative stress is the main trigger of PS exposure in G6PD-deficient erythrocytes. Five previously described mutations were detected in our patients. Two genotypes correlated with a significantly higher percentage of PS-exposing cells. Conclusion: Our study uncovers a novel effect detected in G6PD-deficient erythrocytes which is cell membrane scrambling with PS translocation to the erythrocyte surface. Our findings shed a light on the mechanisms of premature erythrocyte clearance in G6PD deficiency.
Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is the most common hereditary enzymopathy worldwide [1]. Due to the disease's genotypic heterogeneity [2], clinical manifestations vary from mild to severe syndromes such as neonatal jaundice (NNJ) [3], favism [4], congenital non-spherocytic hemolytic anemia (CNSHA) [5] and drug or infection-induced hemolytic anemia (DIHA and IIHA respectively) [6]. Predominantly, the pathophysiology in G6PD deficiency is characterized by episodes of hemolysis which are triggered either by the exposure to oxidative stress through infection [7], the ingestion of certain foods such as fava beans [8] or oxidative treatments such as the antimalarial Primaquine [9].
As G6PD catalyzes the first step in the pentose phosphate pathway (PPP), the enzyme not only controls the generation of the only reducing power in erythrocytes in the form of reduced nicotinamide adenine dinucleotide phosphate (NADPH) [10] but also sustains a constant supply of the reduced form of the antioxidant enzyme Glutathione (GSH) [11]. GSH plays a crucial role in maintaining membrane integrity as well as maintaining hemoglobin (Hb) at its functional state by neutralizing reactive oxygen species (ROS) such as free radicals, hydrogen peroxide, and other oxidative species produced as a result of regular oxygen metabolism in erythrocytes [12]. Consequently, a decrease in G6PD levels jeopardizes the erythrocyte's antioxidant machinery, which in turn, enhances cellular oxidative damage [13],
thus impairing erythrocytes [14].
A housekeeping gene, located on the X chromosome, encodes the G6PD enzyme [15]. So far, more than 200 genetic variants were described, in association with the aforementioned wide range of clinical manifestations [16]. In regions such as Tunisia, where the deficiency is endemic [17], predominant variants include the African variant A- (G6PD:c.376A>G, G6PD:c.202G>A) [18, 19], classified as a type III deficiency by the World Health Organization and associated with both CNSHA and DIHA, and the Mediterranean variant B- (G6PD:c.563C>T), classified as a type II deficiency and associated with favism and NNJ [17]. Evidence suggests that both variants are associated with a premature cellular breakdown through severe shortening of erythrocyte half-life from 60 to 22 days [20].
The premature cellular breakdown of erythrocytes could be a consequence of either hemolysis or eryptosis [21]. Eryptosis is a process of erythrocyte programmed cell death, which is characterized by biochemical and morphological stigmas mimicking the apoptosis of nucleated cells [22]. Biochemical hallmarks of eryptosis include calcium (Ca2+) entry in the cell with an increase of cytosolic Ca2+ activity ([Ca2+]i) [23], alteration of intracellular ion homeostasis [24], activation of cysteine proteases such as µ-calpain [25] and caspase 3 [26] as well as the generation of free radicals paralleled by the inactivation of antioxidant enzymes [27]. During this cellular death process, morphological changes include cell shrinkage [28], membrane blebbing, and exposure of the phospholipid phosphatidylserine (PS) at the outer leaflet [29]. Under physiological conditions, PS is retained within the inner membrane leaflet through the ATP-dependent aminophospholipid translocase (APLT), thus ensuring an asymmetrical phospholipid distribution through the membrane [30]. PS-exposing erythrocytes are recognized by macrophages belonging to the endothelial system through their vitronectin receptors, then rapidly and efficiently cleared from the circulation [31, 32].
Although an enhanced sensitivity to eryptosis was, previously reported in G6PD deficiency [33], there is as yet no consistent data on PS exposure, nor a consensus on the underlying mechanisms of premature programmed erythrocyte cell death. Furthermore, there were no thorough examinations of the relationship between the nature of the molecular defects and the enhanced eryptosis in G6PD.
In this study, we explore eryptosis in G6PD-deficient patients through the quantification of PS exposure at the cell surface. We further examine the signaling pathways orchestrating the enhanced eryptosis in this pathological context. Finally, we study the G6PD gene in order to investigate the potential correlation between G6PD genetic variants and enhanced eryptosis in G6PD-deficient patients.
Study Subjects
The study involved 19 subjects diagnosed with G6PD deficiency. Patients were chosen based on age (older than a year), transfusion status (no history of transfusion), and the absence of concomitant hematological disorders such as sickle cell anemia, thalassemia, and PK deficiency. The control group included 21 healthy volunteers. All patients and healthy volunteers gave their informed written consent per the recommendations of the ethical committee of the Pasteur Institute of Tunis and the declaration of Helsinki.
Hematological explorations
Blood samples were collected in EDTA-containing tubes. Complete blood counts (CBC) were obtained through a Pentra 60C+ analyzer (ABX Diagnostics, Montpelier, France).
Hemoglobin fractions analysis was carried out through capillary electrophoresis using a Capillarys 2 FLEX Piercing analyzer (Sebia, Evry, France).
G6PD activity was measured through quantitative spectrophotometric analysis of the rate of NADPH production from NADP as previously described [34]. G6PD activity was considered normal when ranging between 5.26 IU/g of Hb and 7.94 IU/g of Hb.
Flow cytometry experimental procedures were performed as previously described by Jemaà et. al [35]. Data acquisition was carried out on 50,000 cells for each sample. For all experiments, fluorescence was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm in a logarithmic scale using the flow cytometer FACS Canto II (BD Biosciences, France). Forward scatter (FSC) and Side Scatter (SSC) were determined on a linear scale. An arbitrary threshold was set at 5000 in order to exclude cellular debris.
Pretreatment of Samples. Blood samples were centrifuged at 500xg for 20 minutes and the leucocytes and platelets containing supernatant was discarded. Erythrocytes were resuspended in Ringer solution (pH 7.4) containing 125 mM NaCl, 5 mM KCl,1 mM MgSO4, 32 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 mM glucose and 1 mM CaCl2 at 0.4% hematocrit. All incubations were performed at 37°C.
Where indicated, oxidative stress was induced by the addition of 0.3 mM tetra-butyl-hydroxyperoxide (TBHP, Sigma Aldrich, Germany) to Ringer solution, followed by the incubation of samples for 1 hour at 37°C. Similarly, opening of Ca2+ channels was induced by the addition of 1 μM of the calcium ionophore Ionomycin (Sigma Aldrich, Germany) in dimethyl sulfoxide (DMSO) to Ringer solution, followed by the incubation of samples for 2 hours at 37°C.
Annexin-V binding assay. Annexin-V binding was determined in freshly-drawn blood. For this purpose, a suspension of 150 µl from each sample was centrifuged at 1600 rpm for 3 min. Cells were subsequently stained with annexin-V-FITC (1:200 dilution in Ringer solution containing 5 mM CaCl2; BD Biosciences, France) and incubated at 37°C for 15 min under protection from light.
A marker was placed in order to separate annexin-V-binding cells from control cells.
Assessment of intracellular Ca2+. Intracellular Calcium was determined through Fluo3/AM assay. Cells were washed in Ringer solution containing 5 mM CaCl2, pelleted and then loaded with 5 μM Fluo3/AM (Sigma Aldrich, Germany). Pellets were incubated at 37°C for 30 min then resuspended in Ringer solution.
Ceramide formation at the cell surface. Cells were stained for 1 hour at 37°C with 1 µg/ml monoclonal anti-ceramide antibody (clone MID 15B4, Sigma Aldrich, Germany) in PBS containing 0.1 % bovine serum albumin BSA at a dilution of 1:10. The samples were washed twice with PBS-BSA. Subsequently, the cells were stained for 30 min with polyclonal fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG and IgM antibody (BD Biosciences, Belgium) diluted 1:50 in PBS-BSA. Unbound secondary antibody was removed by washing twice with PBS-BSA.
Reactive Oxygen Species quantification. Oxidative stress was determined using 2',7'-dichlorodihydrofluorescein diacetate (DCFDA). After incubation, a 150 µl suspension was washed in Ringer solution and then stained with DCFDA (Thermo Fisher, France) in Ringer solution containing DCFDA at a final concentration of
10 µM. Erythrocytes were incubated at 37°C for 30 min in the dark and then washed three times in Ringer solution.
PCR-Sanger sequencing
Genomic DNA was extracted from peripheral blood using the standard phenol-chloroform method [36]. Mutations spanning the G6PD gene were determined by polymerase chain reaction (PCR) followed by Sanger Sequencing. A screening of the entire coding sequence of the gene (exons 2 to13) was carried out using the primer pairs listed in Table 1. PCR was carried-out in a 25μl mixture containing 50 μg of genomic DNA; 10X PCR buffer; 10 mM dNTPs; 20 μM of each primer; 25 mM MgCl2;
5 IU Taq polymerase and ultrapure H2O. Reaction conditions were as follows: 95˚C for 5 min, followed by 35 cycles of denaturation at 95˚C for 30 sec, annealing at 58°C for 30 Sec (exon 13), at 60°C for 45 sec (exon2, 6-7 and 8), at 62°C for 30 sec (exon 10-12), at 65°C for 30 sec (exon 3-4, 5 and 9), and an extension at 72˚C for 45 sec. The final extension was performed at 72˚C for 7 min.
Primer sequences used in the molecular exploration of the G6PD gene
Data are expressed as arithmetic means ± Standard Deviation (SD). In order to determine statistical significance between groups, Mann-Whitney test, or ANOVA test with Tukey's test as post-test were performed as appropriate using GraphPad Prism version 5.00 (GraphPad Software, USA). For all experiments, n denotes the number of individuals and the threshold of significance was set at p<0.05.
Hematological data
Except for two individuals who had microcytosis, most patients showed normal hematological indices, as shown in Table 2.
Hematological data of healthy volunteers and G6PD-deficient patients. ***p<0.001 indicates a significate difference in the G6PD-deficient group compared to healthy volunteers
Erythrocytes, entering eryptosis, were identified through the annexin V-binding assay, which directly reflected the percentage of phosphatidylserine exposure at the cell surface. As depicted in Fig. 1 (A-B), the percentage of annexin V-binding erythrocytes was significantly higher in cells taken from G6PD-deficient patients (1.38 ± 1.08) than in cells drawn from healthy controls (0.64 ± 0.29).
To determine erythrocytes' cell volume, forward scatter was measured. Similarly, we assessed cell density through the study of side scatter (Fig. 2 (A-D)). Despite the slight decrease noted in cells from G6PD-deficient patients (55,142 ± 10,273), there was no significant difference in forward scatter compared to erythrocytes from the control group (66,875 ± 25,853). However, a marked and significant decrease in SSC was noted in cells from G6PD-deficient patients scatters (14,342 ± 7493), compared to the control group (29,085 ± 19,891).
Because calcium is the main actor of significant morphological and biochemical changes during eryptosis, we investigated the intracellular calcium content in G6PD-
deficient erythrocytes through a Fluo3/am assay. Fluo3/am-dependent fluorescence was measured in erythrocytes before and following the treatment of fresh erythrocytes
with the calcium ionophore ionomycin. As shown in Fig. 3 (A-C), Fluo3/AM-dependent fluorescence was lower in G6PD-deficient cells (180.2 ± 74.02) than in cells from healthy individuals (210.9 ± 124.1). This decrease in fluorescence only reaches a statistical difference following the treatment with ionomycin (1 µM) in Ringer solution for 2 hours at 37°C. Indeed, we detected a significantly lower Fluo3/AM-dependent fluorescence in patients (391.1 ± 183.7) compared to that measured in controls (615.7 ± 383).
We designed a third set of experiments to determine the intracellular content in reactive oxygen species as well as the involvement of extracellular oxidative stress in enhancing eryptosis in G6PD deficiency. DCFDA-dependent fluorescence, taken as a measure of intracellular ROS content, was measured before and after exposure to the oxidant TBHP. Fig. 4 (A-C) shows that there seems to be no significant difference in DCFDA fluorescence between cells taken from G6PD-deficient patients (419.3 ± 165.4) and cells taken from healthy individuals (379.9 ± 131). However, following the exposure to 0.3 mM TBHP for 1 hour, a significantly higher DCFDA fluorescence was recorded in cells from deficient patients than in cells from controls (1784 ± 476.7 and 1396 ± 268.5 respectively).
Finally, ceramide formation at the cell surface was quantified using FITC-labeled antibodies. As shown in Fig. 5 (A-B), ceramide abundance was slightly lower in G6PD-deficient cells than in control cells (respectively 127.5 ± 39.78 and 131.8 ± 37.88), a difference that did not reach statistical difference.
Fig. 1. Phosphatidylserine exposure in G6PD-deficient patients. A. Arithmetic means ± SEM (n = 21 - 19) of the percentage of Annexin V-binding erythrocytes in freshly drawn blood from healthy individuals (controls, black circles) and G6PD-deficient patients (black triangles). *** (p<0.001) indicates significant difference from healthy volunteers. (Mann-Whitney test). B. Original histograms of Annexin-V-binding of erythrocytes taken from healthy subjects (grey area) and G6PD-deficient patients (black line).
Fig. 2. Forward and Side scatter in G6PD-deficient patients. A, B. Arithmetic means ± SEM (n = 21 - 19) of erythrocyte Forward scatter and Side scatter in freshly drawn blood from healthy individuals (white) and G6PD-deficient patients (black). *(p<0.05) indicates significant difference from healthy volunteers. (Mann-Whitney test). C, D. Original histograms of Forward scatter and side scatter of erythrocytes taken from healthy subjects (grey area) and G6PD-deficient patients (black line).
Fig. 3. Intracellular Ca2+ activity in G6PD-deficient erythrocytes. A. Arithmetic means ± SEM (n = 21 - 19) of Fluo3-AM fluorescence in freshly drawn blood from healthy individuals (white) and G6PD-deficient patients (black) prior to and following treatment with ionomycin (1µM). *(p<0.05) indicates significant difference from healthy volunteers (Mann-Whitney test). B, C. Original histograms of Fluo3-AM fluorescence of erythrocytes taken from healthy subjects (grey area) and G6PD-deficient patients (black line) prior to and following treatment with ionomycin (1µM).
Fig. 4. ROS activity in G6PD-deficient erythrocytes. A. Arithmetic means ± SEM (n = 21 - 19) of DCFDA-dependent fluorescence in freshly drawn blood from healthy individuals (white) and G6PD-deficient patients (black) prior to and following treatment with 0.3 mM TBHP. **(p<0.01) indicates significant difference from healthy volunteers. (Mann-Whitney test). B, C. Original histograms of DCFDA-dependent fluorescence of erythrocytes taken from healthy subjects (grey area) and G6PD-deficient patients (black line) prior to and following treatment with 0.3 mM TBHP.
Fig. 5. Ceramide quantification at the cell surface of G6PD-deficient erythrocytes. Arithmetic means ± SEM (n = 21 - 19) of anti-ceramide dependent fluorescence in freshly drawn blood from healthy individuals (white) and G6PD-deficient patients (black). (Mann-Whitney test).
Molecular Findings
DNA sequencing of the G6PD gene revealed a total of five previously-described mutations, accounting for the enzymatic deficiency in our subjects. The most frequently-encountered mutation, G6PD: c.202G>A, associated with the African variant A-, was found in eleven patients (0.53), including eight hemizygous males as well as in three heterozygous females. The second mutation accounting for the African variant G6PD: c.376A>G, was found in a total of eight patients (0.33), five of whom were hemizygous males, and three were heterozygous females.
The G6PD: c.563C>T mutation, associated with the Mediterranean variant, was only found in two males in the hemizygous form (0.13).
Furthermore, DNA sequencing revealed the presence of two rare mutations. The first was a substitution in exon 11, G6PD: c.1311C>T, which was only found in the heterozygous form in three females. The second was an intronic mutation, G6PD: c.1365-13T>C, encountered in one heterozygous female, one homozygous female, and three hemizygous males. Allele frequencies, calculated only for male patients, are summarized in Table 3.
No mutations spanning the entire coding sequence of the G6PD gene were detected in three deficient patients, all-male.
Allele frequencies of G6PD mutations in G6PD-deficient male patients
Phenotype-Genotype correlations
Due to lionization, female patients present with heterogeneous erythrocytes. To further explore the impact of G6PD genetic variants on eryptosis, we excluded female patients from further analysis. We separated male G6PD-deficient patients into four groups according to their genotypes. The first group encompassed seven patients who presented with the African variant A-. The second included three patients presenting with the African variant as well as the c.1311 C>T mutation. The third group included only two patients who presented with the Mediterranean variant B-. Finally, the fourth group included four patients presenting with the intronic mutation c.1365-13T>C.
Compared to erythrocytes taken from healthy individuals, cells taken from patients belonging to the first and the fourth group presented a significantly higher percentage of annexin-V binding cells (Fig. 6). Furthermore, cells taken from the fourth group showed the highest percentage of annexin-V binding cells, compared to all groups, and seem to present significantly higher annexin-V binding cells when compared to the A- group.
Fig. 6. Phosphatidylserine exposure according to genotypes in G6PD-deficient patients. Arithmetic means ± SEM of the percentage of Annexin V-binding erythrocytes in freshly drawn blood from healthy individuals (white), G6PD-deficient patients presenting with the A- variant (vertical white stripes), the A- variant associated to the c.1311 C>T mutation (horizontal white stripes), the B- variant (black) and the intronic mutation c.1365-13T>C (slanted white lines). ***(p<0.001) and **(p<0.01) indicate significant difference from healthy volunteers. #(p<0.05) indicates significant difference from the A- group. ANOVA test.
Our study demonstrates enhanced eryptosis in G6PD-deficient erythrocytes through significant cell membrane scrambling in non-stimulated cells. Using the annexin V-binding assay, we established that non-stimulated erythrocytes significantly expose PS at the membrane surface, thus leading to membrane scrambling.
Flow cytometry data shows that the loss of membrane asymmetry in G6PD-deficient erythrocytes is paralleled by a significant increase in cell density, which could be explained by the exit of osmotically obliged water following the activation of K+ channels (the Gardos effect). The latter phenomenon is usually paralleled by a significant decrease in forward scatter, reflecting cell shrinkage during eryptosis. However, we found that although the decrease in cell size was notable in samples retrieved from G6PD-deficient patients, it did not reach statistical significance.
This result could be due to the absence of a prominent intracellular calcium concentration in unstimulated cells, which would activate the Ca2+-sensitive K+ channels. Furthermore, our data show that calcium levels were significantly lower in G6PD-deficient erythrocytes than in cells taken from healthy volunteers.
So far, the role of calcium in eryptosis signaling, in the context of G6PD deficiency, remains greatly debated. While a study of forty (40) G6PD-deficient patients by Feng et., al [37] confirms the absence of a significant role of calcium in eryptosis signaling, even following stimulation, a recent study by Hertz et al. [38], shows significantly increased intracellular calcium concentrations in fresh G6PD deficient erythrocytes.
Other mechanisms involved in eryptosis signaling include oxidative stress. Whether intracellular or extracellular, oxidative stress participates in erythrocyte death in G6PD deficiency. Due to the instability of the active form of the G6PD enzyme, reactive oxygen species escape the diminished antioxidant defenses and thrive in this defective milieu [39]. Through DCFDA staining, we quantified ROS production in stimulated and unstimulated cells. Although increased compared to the control group, ROS production in erythrocytes from G6PD-deficient patients did not reach statistical significance. Yet, when exposed to an oxidant (0.3 mM TBHP for 1 hour), ROS production significantly increases in erythrocytes taken from G6PD deficient patients.
This phenomenon could be due to the episodic nature of erythrocyte death in G6PD deficiency. Indeed, none of the patients suffered from a chronic hemolytic syndrome. They were referred to our laboratory based on the physician's recommendations, mainly following the ingestion of fava beans. By the time erythrocyte cell death was assessed, the hemolytic episode had already passed, which would explain the baseline levels ROS in non-stimulated erythrocytes.
Our results suggest that ROS seem to be the chief mediators of enhanced eryptosis in G6PD-deficient erythrocytes. In this case, the primary signaling pathway triggered by oxidative stress would bypass calcium signaling. The increase in intracellular calcium concentration is not always sufficient on its own to disrupt the phospholipid asymmetry regulation machinery such as the aminophospholipid translocase (APLT), the membrane scramblases, and floppases. Consequently, PS externalization does not automatically correlate with the increase in cytosolic calcium concentration [40].
Along those lines, the study by Mandal et al., describes a calcium-independent-Fas-mediated signaling pathway involving caspases in erythrocytes. The pathway is orchestrated by Caspase 8, which is activated following the exposure of erythrocytes to oxidative stress [26]. In particular, Mandal and al.'s results suggest that the correlation between the activation of caspases and the externalization of PS could be due either to the inhibition of the activity of flippase through the proteolytic cleavage of the membrane transporter by caspase-3 or indirectly by affecting other regulators of floppases [41].
Further investigations into the mechanisms of eryptosis led us to the quantification of ceramide abundance at the cell surface. Using FITC-labeled anti-ceramide antibodies, we showed that ceramide tended to be lower in cells from patients than in controls, a difference that did not reach statistical significance. Thus, it is highly likely that ceramide is involved in eryptosis signaling in G6PD deficiency.
Finally, we investigated the potential correlation between G6PD genetic variants and enhanced eryptosis in deficient male patients. Patients presenting with the A- variant showed a significant percentage of Annexin-binding cells compared to the control group. From a structural point of view, in vitro folding of the A- variant was shown to produce partially folded polypeptides that do not undergo the dimerization needed for the enzymatic activity of G6PD [42]. The latter decreases to about 5 to 10% of normal activity which would explain the enhanced eryptosis in A- cells.
Erythrocytes from the group of G6PD-deficient patients carrying the c.1365-13T>C intronic mutation presented with the highest percentage of PS-exposure, a rather surprising result. Indeed, c.1365-13T>C is a non-synonymous single nucleotide variant that does not seem to have any deleterious consequences on the G6PD protein. However, Sirdah et al., previously described a possible association for this variant, in conjunction with the 3′UTR c.*+357A>G (rs1050757) mutation, with G6PD deficiency [43]. Therefore, further molecular explorations of the 3'UTR region of the G6PD gene need to be performed on our study subjects. Similarly, further investigations of the 3'UTR and the promoter regions need to be completed on the three G6PD-deficient patients who did not present any defects in the explored G6PD gene sequences.
In conclusion, enhanced eryptosis effectively contributes to anemia in G6PD deficiency. Furthermore, the genetic variants associated with the enzyme deficiency seem to contribute to the degree of enhanced eryptosis. Our findings shed a light on the mechanisms of premature erythrocyte clearance in G6PD deficiency and suggest a potential use of the annexin V-binding assay in the biological monitoring of G6PD-deficient patients.
B.G. designed the project, carried out the experiments and wrote the manuscript. T.K. assisted with in vitro cell experiments and data collection. T.N. participated in flow cytometry data analysis and critical discussion of the study. C.D. and B.I. assisted in the collection of samples and G6PD enzymatic activity measurements. A.S. supervised in vitro experiments and manuscript preparation. M.S. supervised and assisted in critical discussions of the study.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
All patients and healthy volunteers gave their informed written consent per the recommendations of the ethical committee of the Pasteur Institute of Tunis and the declaration of Helsinki.
The authors declare that no conflicts of interest exist.
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Ženská čtyřhra Tallinn Open 2022 probíhala ve druhé polovině září 2022. Do deblové soutěže tallinnského tenisového turnaje hraného na tvrdém halovém povrchu nastoupilo šestnáct dvojic. Jednalo se o úvodní ročník historicky prvního estonského turnaje hraného na okruhu WTA Tour.

Vítězem se stal třetí nasazený pár ukrajinských dvojčat Ljudmily a Nadiji Kičenokových, která ve finále zdolala americko-německé turnajové jedničky Nicole Melicharovou-Martinezovou a Lauru Siegemundovou. Po dvousetovém průběhu 7–5 a 4–6 rozhodl až supertiebreak poměrem míčů [10–7]. Šampionky si do žebříčku WTA připsaly 280 bodů.  Na okruhu WTA Tour Ukrajinky vybojovaly osmé individuální tituly ze čtyřhry a čtvrtou společnou trofej.

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Tallinn Open
WTA Tour 2022

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