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Metabolic phospholipid labeling of intact bacteria enables a fluorescence assay that detects compromised outer membranes

Inga Nilsson
Mar 10, 2020; 0:jlr.RA120000654v1-jlr.RA120000654
Research Articles




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The fatty acids from LPL-mediated processing of triglyceride-rich lipoproteins are taken up rapidly by cardiomyocytes

Haibo Jiang
Apr 2, 2020; 0:jlr.ILR120000783v1-jlr.ILR120000783
Images in Lipid Research




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Skin barrier lipid enzyme activity in Netherton patients is associated with protease activity and ceramide abnormalities

Jeroen van Smeden
Apr 7, 2020; 0:jlr.RA120000639v1-jlr.RA120000639
Research Articles




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Dispersed lipid droplets: an intermediate site for lipid transport and metabolism in primary human adipocytes.

Björn Morén
Apr 15, 2020; 0:jlr.ILR120000808v1-jlr.ILR120000808
Images in Lipid Research




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GPIHBP1, a partner protein for lipoprotein lipase, is expressed only in capillary endothelial cells

Xia Meng
May 1, 2020; 61:591-591
Images in Lipid Research




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Lipid-tuned Zinc Transport Activity of Human ZnT8 Protein Correlates with Risk for Type-2 Diabetes [Molecular Bases of Disease]

Zinc is a critical element for insulin storage in the secretory granules of pancreatic beta cells. The islet-specific zinc transporter ZnT8 mediates granular sequestration of zinc ions. A genetic variant of human ZnT8 arising from a single nonsynonymous nucleotide change contributes to increased susceptibility to type-2 diabetes (T2D), but it remains unclear how the high risk variant (Arg-325), which is also a higher frequency (>50%) allele, is correlated with zinc transport activity. Here, we compared the activity of Arg-325 with that of a low risk ZnT8 variant (Trp-325). The Arg-325 variant was found to be more active than the Trp-325 form following induced expression in HEK293 cells. We further examined the functional consequences of changing lipid conditions to mimic the impact of lipid remodeling on ZnT8 activity during insulin granule biogenesis. Purified ZnT8 variants in proteoliposomes exhibited more than 4-fold functional tunability by the anionic phospholipids, lysophosphatidylcholine and cholesterol. Over a broad range of permissive lipid compositions, the Arg-325 variant consistently exhibited accelerated zinc transport kinetics versus the Trp-form. In agreement with the human genetic finding that rare loss-of-function mutations in ZnT8 are associated with reduced T2D risk, our results suggested that the common high risk Arg-325 variant is hyperactive, and thus may be targeted for inhibition to reduce T2D risk in the general populations.




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Episode 14 - The Internet of Cleaning Products in the Sky (IoCPitS): games, nVidia, EE vs Three

In this week's fiery episode host Matt Egan talks war and gaming with Macworld UK's David Price, the greatest graphics ever with Christopher Minasians of PC Advisor and Macworld UK (14:21), and the best networks in the UK with fellow PCA and MWUK stalwart Henry Burrell (25:23). Expect songs and laughter, and tech ever after.  


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Episode 15 - The Internet of ill Advised Man Buns (IoIAMB) Google I/O and terrible film adaptations

Host Matt Egan is joined by producer Chris to talk about the raft of announcements coming out of the Google I/O developer conference this week, including Android N, Google assistant and Allo. Techworld.com editor Charlotte Jee joins in to discuss driverless cars (17:00) following the Queen's speech this week. Finally, acting editor at Macworld.co.uk David Price talks about terrible film adaptations of video games, from Angry Birds to World of Warcraft (29:00). Don't forget to like, share and tell your friends!  


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Episode 45 - The Internet of Social Responsibility (IoSR)

Host Henry Burrell leads the team into a tangle of nitty gritty tech topics in the penultimate pod of the year. Macworld UK Acting Editor David Price tells us why Apple and Nintendo very nearly scored a home run with Super Mario Run, but just missed out. The team, led by Charlotte Jee, Editor of Techworld then tackle Amazon's latest endeavour to solve first world problems (provided you live in Cambridge) (13 mins). Tamlin Magee, Online Editor at Techworld then leads us onto the sticky topic of defining extreme terror online and which big companies have a responsibility to police the Internet (27 mins).  


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Episode 50 - The Internet of Fiftieth Episode Special (IoFES) Anything goes, tbh

Momentous scenes as the pod celebrates its Golden Jubilee. Matt Egan is joined by some very special guests to congratulate the gang on the milestone while indulging in some points based tech quizzing. Join Chris Martin, Charlotte Jee, Dom Preston and Henry Burrell to recap questions from pods gone by with a new twist, and have a long hard think about what tech will be 50 more episodes from now. The prize? A small bronze jug. Obviously. NB. May not actually be 50 minutes, sorry.  


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Episode 51 - The Internet of Sick Burns (IoSB) Snapchat IPO, Android Wear 2.0 and Apple in India

Techworld Editor Charlotte Jee conducts the tech orchestra this week, trying to get a tune out of this cold, creaking week. Online Editor at Computerworld UK Scott Carey lets us know all about Snapchat's upcoming IPO, billions of dollars, and the gang comes to the realisation they'll perhaps never understand what it's for. Then Senior Staff Writer at PC Advisor Henry Burrell recaps on Google and LG's launch of Android Wear 2.0 and ask - frankly - is it a big deal? Finally Acting Editor at Macworld UK David Price discusses Apple's rumoured plans to move manufacturing to India, which inevitably moves us on to Tech Trumps.  


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Episode 52 - The Internet of Nostalgia (IoN) Nokia 3310, drone taxis and Apple on the telly

Matt Egan takes us where no other pod has dared gone before (September 2000) and asks Digital Arts Editor Neil Bennett if anyone cares that Nokia is rereleasing the famous 3310. Is it cool to rock one now? Then Techworld Audience Development Editor Christina Mercer lets us know that the future is already here with self driving drone taxis. Would you hitch a lift round Dubai on a massive quadcopter? Finally Deputy Editor at Macworld David Price ruminates on Apple's forecast foray into the world of snackable media content - will it challenge Netflix or try to buy it?  


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Episode 64 - The Internet of Hype (IoH) Apple Special, again!

June means WWDC, and boy did Apple deliver this year. HomePod! iPad! Software! It's all here. Join the Macworld massive David Price, Ashleigh Macro, Dom Preston and Henry Burrell as they rifle through 40 minutes of on-the-nose critiques of Apple's latest announcements. With the new iMac Pro, iPad 10.5in and HomePod, the company introduced hardware for the first time since 2013, while the introduction of iOS 11 sets us up for the iPhone 8 in September. To the orchard!  


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Episode 100 - The Internet of Interval Timers (IoIT) Bumper 100 topic special!

Here it is folks, the insane 100th episode we have been planned for weeks (ahem). What better way to spend just over 50 minutes than with 100 tech topics covering over two years, one taken from each of the last 99 episodes of the pod plus the bonus round at the end.


Henry Burrell leads Tamlin Magee, Charlotte Jee, Scott Carey, Karen Khan and Christina Mercer down the tech rabbit hole in a game show-style quick fire test of our tech brains. In this game, everyone is a winner. Naw.


Thanks a lot for listening - here's to the next 100.

 

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Episode 101 - The Internet of Chucking Tech in the Bin (IoCTitB) Room 101 special

Episode 101 is a second special in a row as we take tech to trial and consign our least favourite digital surplus to Room 101.


The Orwellian nightmare is compered by Henry Burrell as he, David Price, Tamlin Magee and Scott Carey each pick two things to banish forever. Cue a a bumper edition where we throw people, buzzwords, fads and entire companies down the trash chute.


May contain arguments.

 

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Episode 102 - The Internet of Tennis Max (IoTM) Apple event special, new iPhones and Apple Watch

A quick-fire reaction Apple special, recorded the morning after new iPhones and an Apple Watch were announced at an event in California.


Henry Burrell joins David Price, Ashleigh Macro and Scott Carey to discuss which products we'll be buying, avoiding and coveting over the Christmas period.


Is the new iPhone named terribly? Is it an upgrade at all? What wasn't announced? And does Scott give the new Apple Watch any praise at all? It's all here.

 

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Episode 103 - The Internet of Dystopia (IoD) Skirting the line between fact and fiction

It's a pop culture episode this week as we do a deep dive into dystopian fiction. Scott Carey is your host as he chats to Dominic Preston and Tamlin Magee about the best novels, films, TV shows and video games set in dystopian universes.


We talk about how the world is closer to fiction than ever before, and how this impacts the way we think about fictional dystopias, and end on a positive note to discuss our favourite utopian fiction too.

 

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Episode 112 - The Internet of Controversial Dildos (IoCD) CES roundup ft. sex toys and smart wood

This week our host Scott Carey catches up with Sean Bradley and Dominic Preston fresh off the back of the International Consumer Electronics Show (CES) in Las Vegas.


Dom explains why he was surprised, both in a good and bad way, by the latest foldable screen technology and Sean talks about what is going on in the gaming laptop space, as Alienware looks to soften its image.


Then Dom talks about a piece of connected wood and the gang break down the big controversy from the show floor regarding a certain innovative dildo.


We are also announcing the sad news that the UK Tech Weekly Podcast will be going on an indefinite hiatus while we reassess our podcasting output. We would like to thank anyone that has taken the time to listen to us for these one hundred and twelve (112!) episodes and rest assured we will be back in some guise soon.

 

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Metabolic phospholipid labeling of intact bacteria enables a fluorescence assay that detects compromised outer membranes [Research Articles]

Gram-negative bacteria possess an asymmetric outer membrane (OM) composed primarily of lipopolysaccharides (LPS) on the outer leaflet and phospholipids (PLs) on the inner leaflet. Loss of this asymmetry due to mutations in the lipopolysaccharide (LPS) biosynthesis or transport pathways causes externalization of PLs to the outer leaflet of the OM and leads to OM permeability defects. Here, we employed metabolic labeling to detect a compromised OM in intact bacteria. Phosphatidylcholine synthase (Pcs) expression in Escherichia coli allowed for incorporation of exogenous propargylcholine (PCho) into phosphatidyl(propargyl)choline (PPC) and for incorporation of exogenous 1-azidoethyl-choline (AECho) into phosphatidyl(azidoethyl)choline (AEPC) as confirmed by LC-MS analyses. A fluorescent copper-free click reagent poorly labeled AEPC in intact wild-type cells, but readily labeled AEPC from lysed cells. Fluorescence microscopy and flow cytometry analyses confirmed the absence of significant AEPC labeling from intact wild-type E. coli strains, and revealed significant AEPC labeling in an E. coli LPS transport mutant (lptD4213) and an LPS biosynthesis mutant (E. coli lpxC101). Our results suggest that metabolic PL labeling with AECho is a promising tool to detect a compromised bacterial OM, reveal aberrant PL externalization, and identify or characterize novel cell-active inhibitors of LPS biosynthesis or transport.




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The fatty acids from LPL-mediated processing of triglyceride-rich lipoproteins are taken up rapidly by cardiomyocytes [Images in Lipid Research]




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Roles of endogenous ether lipids and associated PUFA in the regulation of ion channels and their relevance for disease [Research Articles]

Ether lipids (ELs) are lipids characterized by the presence of either an ether linkage (alkyl lipids) or a vinyl ether linkage (i.e. plasmalogens [Pls]) at the sn1 position of the glycerol backbone and they are enriched in PUFAs at the sn2 position. In this review, we highlight that ELs have various biological functions, act as a reservoir for second messengers (such as PUFAs), and have roles in many diseases. Some of the biological effects of ELs may be associated with their ability to regulate ion channels that control excitation-contraction/secretion/mobility coupling and therefore cell physiology. These channels are embedded in lipid membranes, and lipids can regulate their activities directly or indirectly as second messengers or by incorporating into membranes. Interestingly, ELs and EL-derived PUFAs have been reported to play a key role in several pathologies, including neurological disorders, cardiovascular diseases, and cancers. Investigations leading to a better understanding of their mechanisms of action in pathologies have opened a new field in cancer research. In summary, newly identified lipid regulators of ion channels, such as ELs and PUFAs, may represent valuable targets to improve disease diagnosis and advance the development of new therapeutic strategies for managing a range of diseases and conditions.




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Skin barrier lipid enzyme activity in Netherton patients is associated with protease activity and ceramide abnormalities [Research Articles]

Individuals with Netherton syndrome (NTS) have increased serine protease activity, which strongly impacts the barrier function of the skin epidermis and leads to skin inflammation. Here, we investigated how serine protease activity in NTS correlates with changes in the stratum corneum ceramides, which are crucial components of the skin barrier. We examined two key enzymes involved in epidermal ceramide biosynthesis, glucocerebrosidase (GBA) and acid-sphingomyelinase (ASM). We compared in situ expression levels and activities of GBA and ASM between NTS patients and controls and correlated the expression and activities with i) stratum corneum ceramide profiles, ii) in situ serine protease activity, and iii) clinical presentation of patients. Using activity-based probe labeling, we visualized and localized active, epidermal GBA, and a newly developed in situ zymography method enabled us to visualize and localize active ASM. Reduction in active GBA in NTS patients coincided with increased ASM activity, particularly in areas with increased serine protease activity. NTS patients with scaly erythroderma exhibited more pronounced anomalies in GBA and ASM activities than patients with ichthyosis linearis circumflexa. They also displayed a stronger increase in stratum corneum ceramides processed via ASM. We conclude that changes in the localization of active GBA and ASM correlate with i) altered stratum corneum ceramide composition in NTS patients, ii) local serine protease activity, and iii) the clinical manifestation of NTS. 




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Circulating oxidized LDL increased in patients with acute myocardial infarction is accompanied by heavily modified HDL. [Research Articles]

Oxidized low-density lipoprotein (oxLDL) is a known risk factor for atherogenesis. This study aimed to reveal structural features of oxLDL present in human circulation related to atherosclerosis. When LDL was fractionated on an anion-exchange column, in vivo-oxLDL, detected by the anti-oxidized phosphatidylcholine (oxPC) monoclonal antibody, was recovered in flow-through and electronegative LDL (LDL(-)) fractions. The amount of the electronegative in vivo-oxLDL, namely oxLDL in LDL(-) fraction, present in patients with acute myocardial infarction (AMI) was three-fold higher than that observed in healthy subjects. Surprisingly, LDL(-) fraction contained apoA1 in addition to apoB, and HDL-sized particles were observed with transmission electron microscopy. In LDL(-) fractions, acrolein adducts were identified at all lysine residues in apoA1, with only a small number of acrolein-modified residues were identified in apoB. The amount of oxPC adducts of apoB was higher in LDL(-) than in L1 fraction as determined using western blotting. The electronegative in vivo-oxLDL was immunologically purified from the LDL(-) fraction with an anti-oxPC monoclonal antibody. Majority of PC species was not oxidized, whereas oxPC and lysoPC did not accumulate. Here, we propose that there are two types of in vivo-oxLDL in human circulating plasma and the electronegative in vivo-oxLDL accompanies oxidized HDL.




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LDL subclass lipidomics in atherogenic dyslipidemia:Effect of statin therapy on bioactive lipids and dense LDL [Patient-Oriented and Epidemiological Research]

Atherogenic LDL particles are physicochemically and metabolically heterogeneous. Can bioactive lipid cargo differentiate LDL subclasses, and thus potential atherogenicity?  What is the effect of statin treatment? Obese, hypertriglyceridemic, hypercholesterolemic males (n=12; Lp(a) <10 mg/dL) received pitavastatin calcium (4mg/day) for 180 days in a single-phase, unblinded study. The lipidomic profiles (23 lipid classes) of five LDL subclasses fractionated from baseline and post-statin plasmas were determined by LC-MS. At baseline and on statin treatment, very small dense LDL (LDL5) was preferentially enriched (up to 3-fold) in specific lysophospholipids (lysophosphatidylcholine (LPC); lysophosphatidylinositol (LPI); lyso-platelet activating factor (LPC(O)); 9,0.2 and 0.14 mol/mol apoB respectively; all p<0.001 versus LDL1-4), suggesting  elevated inflammatory potential per particle. In contrast, lysophosphatidylethanolamine was uniformly distributed among LDL subclasses. Statin treatment markedly reduced absolute plasma concentrations of all LDL subclasses (up to 33.5%), including LPC, LPI and LPC(O) contents (up to -52%), consistent with reduction in cardiovascular risk. Despite such reductions, lipotoxic ceramide load per particle in LDL1-5 (1.5 - 3 mol/mol apoB; 3 - 7 mmol/mol phosphatidylcholine) was either conserved or elevated. Bioactive lipids may constitute biomarkers for the cardiometabolic risk associated with specific LDL subclasses in atherogenic dyslipidemia at baseline, and with residual risk on statin therapy.




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Dispersed lipid droplets: an intermediate site for lipid transport and metabolism in primary human adipocytes. [Images in Lipid Research]




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Phosphatidylinositol Metabolism, Phospholipases, Lipidomics, and Cancer:In Memoriam of Michael J. O. Wakelam (1955-2020) [Tribute]




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Sphingolipids distribution at mitochondria-associated membranes (MAM) upon induction of apoptosis. [Research Articles]

The levels and composition of sphingolipids and related metabolites are altered in aging and common disorders such as diabetes and cancers, as well as in neurodegenerative, cardiovascular, and respiratory diseases. Changes in sphingolipids have been implicated as being an essential step in mitochondria-driven cell death. However, little is known about the precise sphingolipid composition and modulation in mitochondria or related organelles. Here, we used LC–MS/MS to analyze the presence of key components of the ceramide metabolic pathway in vivo and in vitro in purified endoplasmic reticulum (ER), mitochondria-associated membranes (MAM), and mitochondria. Specifically, we analyzed the sphingolipids in the three pathways that generate ceramide: sphinganine in the de novo ceramide pathway, sphingomyelin in the breakdown pathway, and sphingosine in the salvage pathway. We observed sphingolipid profiles in mouse liver, mouse brain, and a human glioma cell line (U251). We analyzed the quantitative and qualitative changes of these sphingolipids during staurosporine (STS)-induced apoptosis in U251 cells. Ceramide, especially C16-ceramide, levels increased during early apoptosis possibly through a conversion from mitochondrial sphinganine and sphingomyelin, but sphingosine and lactosyl- and glucosyl-ceramide levels were unaffected. We also found that ceramide generation is enhanced in mitochondria when sphingomyelin levels are decreased in the MAM. This decrease was associated with an increase in acid sphingomyelinase (ASM) activity in MAM. We conclude that meaningful sphingolipid modifications occur in MAM, the mitochondria, and ER during the early phases of apoptosis.




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2-Chlorofatty acids are biomarkers of sepsis mortality and mediators of barrier dysfunction in rats [Research Articles]

Sepsis is defined as the systemic, dysregulated host immune response to an infection that leads to injury to host organ systems, and, often, death. Complex interactions between pathogens and their hosts elicit microcirculatory dysfunction. Neutrophil myeloperoxidase (MPO) is critical for combating pathogens, but MPO-derived hypochlorous acid (HOCl) can react with host molecular species as well. Plasmalogens are targeted by HOCl, leading to the production of 2-chlorofatty acids (2-CLFAs). 2-CLFAs are associated with human sepsis mortality, decrease in vitroendothelial barrier function, and activate human neutrophil extracellular trap formation. Here, we sought to examine 2-CLFAs in an in vivorat sepsis model. Intraperitoneal cecal slurry sepsis with clinically relevant rescue therapies led to ~73% mortality and evidence of microcirculatory dysfunction. Plasma concentrations of 2-CLFAs assessed 8h after sepsis induction were lower in rats that survived sepsis than in non-survivors. 2-CLFA levels were elevated in kidney, liver, spleen, lung, colon and ileum in septic animals. In vivo, exogenous 2-CLFA treatments increased kidney permeability, and in in vitroexperiments 2-CLFA also increased epithelial surface expression of vascular cell adhesion molecule 1 and decreased epithelial barrier function. Collectively, these studies support a role of free 2-CLFAs as biomarkers of sepsis mortality, potentially mediated, in part, by 2-CLFA-elicited endothelial and epithelial barrier dysfunction.




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Russia’s Uncertain Regime Transformation

11 March 2020

Professor Nikolai Petrov

Senior Research Fellow, Russia and Eurasia Programme, Chatham House

Dr Ben Noble

Lecturer in Russian Politics, University College London; Senior Research Fellow, HSE, Moscow
Despite the drama, Vladimir Putin’s announcement endorsing a constitutional change allowing him to remain president from 2024 does surprisingly little to change the status quo.

2020-03-11-Putin-Constitution.jpg

Russian President Vladimir Putin addresses lawmakers debating on the second reading of the constitutional reform bill during a session of the State Duma, Russia's lower house of parliament March 10, 2020. Photo by ALEXANDER NEMENOV/AFP via Getty Images.

With Putin’s current term as head of state due to run out in 2024, the question everybody has been asking is what he will do to remain in power. The Russian president’s recent speech, made in person in the State Duma during the second reading of his own constitutional reform bill, has been interpreted by many as a clear answer. Summaries such as “Putin forever” and “perpetual Putin” abound. But the reality is not so clear.

Putin has not committed to standing for re-election in 2024, never mind staying in power until 2036, when two additional six-year terms from 2024 would run out. What he has done is provide the constitutional grounds to retain power as president. It creates a highly credible option without committing him to it.

And the uncertainty matters. Because as long as members of the elite are unsure whether Putin will take up the option to remain president, they are kept in check.

Broader constitutional reform

With the flurry of interest around Putin’s announcement, we should not lose sight of his moves to further strengthen the presidency. As part of the broader constitutional reform package, Russia’s existing “super-presidency” will gain additional powers, such as the authority to fire top-tier judges and to block legislation when the legislature has overridden a presidential veto (in other words, a “super-veto”).

The proposals also put the autonomy of local self-government at risk, with Moscow and regional executives gaining the constitutional power to hire and fire officials who are not even technically part of the state. And the president now has a formalised role as “general leader” of the government. Putin is creating the “Great Presidency”.

However, the majority of constitutional changes do not relate to the presidency – they have different purposes. Firstly, to revitalise support for the regime which took a hit following unpopular pension reforms in 2018. Secondly, to distract or appease those worried by Putin remaining in a strengthened presidency. And perhaps most significantly, to boost turnout in the nationwide vote on reforms.

This desire to re-energise popular support becomes apparent as the changes – some of which will have to be inserted rather awkwardly into the constitution’s structure – focus on three elements aimed squarely at improving the regime’s appeal: increased material support from the state for citizens, including indexing state pensions; an emphasis on “traditional values”, including a declaration that marriage can only be a union between a man and a woman; and increased Russian sovereignty, including a “nationalisation” of the elite, with a constitutional ban on top-level officials having bank accounts abroad. 

Constitutional reform is, moreover, the most visible part of a broader political transformation already underway, including a major propaganda drive. Putin has promised a significant increase in resources for its “maternity capital” programme, putting more money in the pockets of young Russian families.

And he has instructed Prime Minister Mikhail Mishustin’s government to focus on delivering his “national projects” – goals aimed at improving Russians’ lives across a range of areas, from infrastructure to education and healthcare.

Taking advantage of several imminent historical milestones is also on the cards. It has been reported Putin will sign the constitutional reform bill on March 18 – the anniversary of Russia’s annexation of Crimea. And May 9 is the 75th anniversary of the end of the Great Patriotic War (the Russian term for the Second World War), with foreign dignitaries invited to attend events in Moscow.

Putin has also been filling the airwaves with a high-production-values series called “20 Questions for Vladimir Putin”, as well as holding public meetings with citizens in provinces such as Cherepovets and Ivanovo. There is a clear aim to demonstrate the president is not only still in control, but also concerned with the well-being of everyday Russians.

With parliamentary elections scheduled for September 2021 the Kremlin knows that, to maintain its control of a super-majority of seats in the State Duma, its ratings-raising drive has to work – even if it does always have the option of using manifestly authoritarian methods for realising desired election results. A proposal to call early State Duma elections was made during the second reading of Putin’s reform bill, but was quickly withdrawn after Putin spoke out against the idea.

Russia’s complex architecture of “power”

Throughout this transformation, maintaining control of the elite – particularly of the siloviki – is key for Putin. A reshuffling and removal of senior officials in the Procuracy has seen Yury Chaika replaced as general prosecutor by Ivan Krasnov, previously a deputy chair of the Investigative Committee, which is widely seen as a rival structure in Russia’s complex architecture of “power” bodies.

When considered alongside the constitutional changes giving the president broader powers in appointing regional prosecutors, this is textbook “divide and rule”. Power balancing is also on display with the Security Council, as the job description for Dmitry Medvedev’s new role as its deputy chair could provide fertile ground for clashes with the body’s secretary, Nikolai Patrushev.

Pitting rival patronal networks against each other means Putin can keep rivals in check within the broader structure of the “Great Presidency”, while staying firmly in control himself.

The prospect of Putin remaining president is unlikely to be popular. According to data from independent Russian polling agency the Levada Centre, only 27 per cent of Russians want Putin to stay in the post after 2024. This figure could, of course, change in either direction as the prospect becomes more real for Russians. But if Putin’s announcement galvanises mass opposition, the authorities may well use responses to the COVID-19 outbreak to keep protesters at bay – something already on display in Moscow.

What this all means for Russia is that, despite the drama, considerable uncertainty remains following Putin’s announcement. What we can say for certain, however, is that it dashes hopes of serious political change any time soon.




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In a COVID-19 World, Russia Sticks to International Distancing

29 March 2020

Mathieu Boulègue

Research Fellow, Russia and Eurasia Programme
While a global response is needed against the coronavirus crisis, Russia does not see it as in its interests to contribute – and in fact the Kremlin is using the crisis to further destabilise the world.

2020-03-29-Coronavirus-Russia-Moscow

Young woman wearing a face mask in front of St. Basil's Cathedral, Moscow. Photo by ALEXANDER NEMENOV/AFP via Getty Images.

Persistent internet rumours claiming the coronavirus outbreak originated from a secret American pharmaceutical company with the aim of destroying China from within were quickly discredited. Pop culture fans recognised the supposed activities of the Umbrella Corporation as being from the famous Japanese video games series Resident Evil.

However, although fake news, it can likely be attributed to Russian trolls conducting this and other similar activities online, especially when considered within the wider context of how the Russian regime is using this worldwide crisis to further destabilize the West and test its resolve.

Russian trolls never sleep

Russia’s COVID-19 related actions first and foremost take the form of a vast information warfare campaign, with media outlets simultaneously downplaying the threat of the pandemic - ‘it is less dangerous than seasonal flu’ - while stoking fear about what is happening elsewhere in Europe.

For the domestic audience in Russia, some media are reporting the pandemic marks the collapse of the Western world and liberalism altogether, calling it a form of collective punishment. Other point out how fast liberal democracies have curbed individual and entrepreneurial freedoms in order to slow down the viral outbreak, and seek to diminish the credibility of the Western response to the crisis.

Exploiting the coronavirus crisis in this way is a new low in Russia’s wider political warfare campaign to undermine global governance overall, as these activities are detrimental to people's very safety. For example, in Ukraine, it is thought a Russian-engineered disinformation operation may have caused the outburst of violence in the city of Novi Sanzhary following the arrival of evacuees from China.

In the military realm, fake news has been targeting the US-led multinational exercise DEFENDER-Europe 2020. The Russian leadership criticized the exercise as an offensive ‘anti-Russian scenario’ but then used accompanying propaganda that it could actively facilitate the spread of COVID-19 across Europe because of the arrival and movement of large numbers of troops.

The large-scale drills were planned to involve 18 participating nations and should have taken place across ten European countries from April to May 2020. But the exercise has now been scaled down – as has the Russian disinformation targeting it.

And while the world is pre-occupied with managing COVID-19, Moscow is able to grow bolder in its provocations. Recent air incursions were reported into Irish controlled airspace as well as over the North Sea. Although this practice is - unfortunately - routine as part of Russian constant military sabre-rattling, it does increase the risk of tactical errors and miscalculation.

Self-isolation, Kremlin style

Meanwhile, just when a global response is needed to fight the pandemic, Moscow’s response has been, at best, self-serving. On March 22, Russian military reportedly started sending medical equipment and supplies to Italy. While the nature and the scope of this assistance can be doubted, it still represents a charm offensive for Russia to be brought back in from the cold in Europe - since successive Italian leaderships have been accommodating to the Kremlin. And sending virologists to Italy might also be a useful learning curve for Russia’s regime.

But within Russia itself, Vladimir Putin does have to face the problem that, on top of all the projected social and healthcare costs, the coronavirus is also having negative political consequences. On March 25, the ‘popular vote’ - a mock referendum designed to rubber-stamp Putin’s recent constitutional changes - was pushed back. And the Ministry of Communications has been forced to postpone a major exercise aimed at ensuring the ‘stable and safe operation of Runet’ - namely eliminating vulnerabilities in the Russian ‘sovereign’ internet to potential external threats.

Certainly it would be naive to believe Moscow will put self-interest to one side during this pandemic. ‘International distancing’ is not new for the Kremlin, and Russia has been practising self-isolation since at least 2008 through its own actions, most notably in Georgia and Ukraine.

Its self-perception as a ‘besieged fortress’ is being reinforced by this crisis and Russia will, at the very least, likely come out of the crisis feeling vindicated in its view that internationalism is dying or already dead.

With the health systems of many countries under massive strain, and societal resilience being tested by social distancing, the Kremlin continues to probe for weaknesses, and is also carefully watching other countries’ responses to the crisis in terms of adaptation and mobilization of resources.

COVID-19 provides a major intelligence-gathering opportunity for Moscow to learn how well others can implement wartime-like planning in peacetime. In a rapidly changing world, Russia is still Russia.




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COVID-19 and the Iranian Shadows of War

8 April 2020

Dr Sanam Vakil

Deputy Director and Senior Research Fellow, Middle East and North Africa Programme
Coronavirus has plunged Iran into the country’s biggest crisis since its war with Iraq. More than 30 years later, the lingering effects of the war are shaping Iran’s reaction to the pandemic.

2020-04-08-Iran-COVID-Tehran

Spraying disinfectant at Tajrish bazaar in Tehran, Iran, during the coronavirus pandemic in March 2020. Photo by Majid Saeedi/Getty Images.

In the face of the COVID-19 pandemic, many global leaders have invoked war analogies – from the Pearl Harbor attack to the collective spirit on display during the Second World War – to highlight past lessons learned or rally their populations.

For Iran’s leadership, more recent war analogies hold resonance and help explain the ideological and political conundrum limiting an effective COVID-19 response. While the Islamic Republic has weathered a multitude of challenges, COVID-19 is putting unprecedented strain on Iran’s already fragile, heavily-sanctioned economy and further exposing domestic political fissures amid ongoing international tensions.

Iran has been identified as the regional epicentre of the pandemic with a steadily rising number of deaths, including several of the country’s political and military elite. Yet the Iranian government has not evoked the collective memory of the war as an opportunity for national resistance and mobilization.

Sluggish and poorly managed

This is unsurprising, because thus far the Iranian government’s response to COVID-19 has been sluggish and poorly managed. After an initial slow response, Iran then attempted to downplay the impact of the virus, covering up the number of cases and deaths and blaming the United States, before implementing a poorly coordinated action plan marred by government infighting.

For the Iranian leadership, the Iran-Iraq war has been the single most influential and defining period – it has impacted its political ideology, domestic and security policies and international relations. More than half a million Iranians died, and a paranoid worldview and sense of isolation was cemented among many elite leaders such as Supreme Leader Ali Khamenei.

The war created a valiant culture of leadership from Qassem Soleimani to presidents Mahmoud Ahmadinejad and Hashemi Rafsanjani and, over time, has resulted in the development of Iran’s asymmetrical defense capabilities.

The war enabled a dark purge of political opponents and the gradual birth of Iran’s reformist faction, all while the ethos of sacrifice and martyrdom was linked to the collective notions of resistance.

These would be carried forward in other resistance campaigns both regional and economic. Most defining was Ayatollah Khomeini’s infamous 1988 decision to ‘drink the poisoned chalice’ and end the long war. Three decades later, Iran continues to contend with those outcomes.

To acknowledge that the COVID-19 crisis could have equally profound consequences would add further pressure to the Islamic Republic at a time of incomparable vulnerability. Even before this crisis, the Iranian government linked sanctions to economic warfare, making future negotiations conditional on sanctions relief.

Iranian hardliners used the opportunity to promote Iran’s subsistence-based resistance economy designed to insulate Iran’s economy from external shocks such as sanctions. While both groups recognize the economic urgency, their contending strategies help explain the muddled government response and the ongoing ideological competition between the political elites.

Rouhani has argued that a full lockdown of the Iranian economy is impossible because it is already under significant strain from sanctions - the Iranian economy experienced a 9.5% contraction in 2019 and is expected to worsen in the coming year.

That said, through Iran’s New Year holidays the government did take action to slow the spread of the virus, discouraging travel and shutting schools, pilgrimage sites and cancelling Friday prayers. Finally, on 4 April, after receiving permission from Khamenei to do so, Rouhani withdrew $1 billion from Iran’s National Development Fund and is distributing the money through loans and credits to 23 million households.

Aid from a number of Iran’s parastatal agencies was also announced. Conversely, in his annual New Year’s speech the supreme leader securitized the crisis by laying blame on the United States for spreading the virus as a form of biological terrorism. Iran’s army chief of staff Major General Bagheri was tasked with building hospitals and the Islamic Revolution Guard Corps given authority to clear the streets.

The recent expulsion of Médecins Sans Frontières from Iran highlights the mix of paranoia and resistance culture still on display. US sanctions on Iran have significantly weakened Iran’s economy and limited Tehran’s ability to purchase much needed medical supplies and equipment. Unable to access its foreign reserves due to sanctions, the Rouhani government has applied for a $5 billion loan from the IMF.

European countries alongside a number of US members of congress have appealed to the Trump administration to ease sanctions on humanitarian grounds. While Washington continues to pursue its steadfast approach, referring to Iran’s campaign as a ‘sanctions relief scam’, Germany, France, and the UK have offered $5 million in aid and launched INSTEX – a trading mechanism designed to circumvent sanctions to allow non-sanctioned humanitarian trade.

The impact of coronavirus on Iranian society remains to be seen. But the impact of sanctions has placed heavy economic and psychological burden on the people. Feeling abandoned by the Iranian state and the United States could produce a mix of contradictory nationalistic and independent impulses.

The social contract – already fragile amid protests and government repression – reveals declining trust. Without national mobilization and calls for unity reminiscent of the war period, Iranians have stepped in, highlighting the continued resilience of civil society. Support for the medical establishment has been celebrated throughout the country and on social media. Charities, the private sector - through one initiative known as Campaign Nafas (Breathe) - and diaspora groups have initiated fundraising drives and assistance measures.

Iran’s relations with the international community, and specifically the United States, remain an unresolved consequence of the war. The 2015 Iranian nuclear agreement was the closest Tehran and Washington came to resolving decades of tensions, containment and sanctions.

COVID-19 has further heightened the trajectory of tensions between Tehran and Washington suggesting that any new deal, while necessary, is not on the cards. Tit-for-tat military exchanges have been on the rise in Iraq and Yemen while American and Iranian leaders issue threats and warnings of potential escalation.

Abdullah Nasseri, an advisor to Iran’s reformists, recently stated that in order to manage the coronavirus crisis, the Iranian government needed to make a decision akin to the 1988 United Nations resolution 598 that ended war hostilities. Ayatollah Khomeini famously commented on that ceasefire, stating: ‘Happy are those who have departed through martyrdom. Unhappy am I that I still survive.… Taking this decision is more deadly than drinking from a poisoned chalice. I submitted myself to Allah's will and took this drink for His satisfaction’. 

While a similar compromise today might appear deadly to the political establishment, it is clear that a paradigm shift away from the shadows of Iran’s last war is urgently needed to manage the challenges stemming from COVID-19.




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Beware Russian and Chinese Positioning for After the Pandemic

9 April 2020

Keir Giles

Senior Consulting Fellow, Russia and Eurasia Programme
Authoritarian regimes can use the COVID-19 crisis to improve their international standing, taking advantage of others’ distraction. Their aims are different, but their methods have much in common.

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An airlifter of the Russian Aerospace Forces prepares to fly to Serbia carrying equipment and professionals during the COVID-19 crisis. Photo by Russian Defence MinistryTASS via Getty Images.

Both Russia and China have mounted combined charm offensives and disinformation campaigns on the back of the pandemic. Shipments of ‘aid’ – reportedly of questionable utility and quality - have gone hand in hand with a concerted effort to deflect any blame from China for the early spread, and an ongoing drive by Russia to undermine states’ confidence and have sanctions lifted.

These concurrent operations have very different objectives, as Russia seeks to subvert international order while China is continuing its bid to demonstrate global leadership - but in both cases, they are seeking long-term gains by exploiting the inattention and distraction of their targets.

Both seek to present themselves as globally responsible stakeholders, but for divergent reasons – especially China which needs the rest of the world to recover and return to stability to ensure its own economic recovery. But despite this, the two campaigns appear superficially similar.

Fertile ground for disinformation

One reason lies in the unique nature of the current crisis. Unlike political issues that are local or regional in nature, COVID-19 affects everybody worldwide. The perceived lack of reliable information about the virus provides fertile ground for information and disinformation campaigns, especially feeding on fear, uncertainty and doubt. But Russia in particular would not be succeeding in its objectives without mis-steps and inattention by Western governments.

Confused reporting on Russia sending medical supplies to the United States showed Moscow taking advantage of a US administration in apparent disarray. Claims Russia was sending ’humanitarian aid’ were only belatedly countered by the US State Department pointing out it had been paid for. Meanwhile the earlier arrival of Russian military equipment in Italy also scored a propaganda victory for Russia, facilitated by curious passivity by the Italian government.

In both cases Russia also achieved secondary objectives. With the United States, Russia scored bonus points by shipping equipment produced by a subsidiary of a company under US sanctions. In the case of Italy, Russian state media made good use of misleading or heavily edited video clips to give the impression of widespread Italian acclaim for Russian aid, combined with disdain for the efforts of the EU.

Beijing’s external information campaigns have sought to deflect or defuse criticism of its early mishandling and misinformation on coronavirus and counter accusations of secrecy and falsifying data while also pursuing an opportunity to exercise soft power. For Moscow, current efforts boost a long-standing and intensive campaign to induce the lifting of sanctions, demonstrating if nothing else that sanctions are indeed an effective measure. Official and unofficial lobbying has intensified in numerous capital cities, and will inevitably find supporters.

But both the aid and the information campaigns are seriously flawed. While appropriate and useful aid for countries that are struggling should of course be welcomed, both Russian and Chinese equipment delivered to Europe has repeatedly been found to be inappropriate or defective

Russian photographs of cardboard boxes stacked loose and unsecured in a transport aircraft bound for the United States sparked alarm and disbelief among military and aviation experts - and there has still been no US statement on what exactly was purchased, and whether it was found to be fit for purpose when it arrived.

Reporting from Italy that the Russian equipment delivered there was ‘80% useless’ has not been contradicted by the Italian authorities. In fact, although the Italian sources criticizing Russia remain anonymous it is striking that - President Trump aside - no government has publicly endorsed materials and assistance received from Russia as actually being useful and helpful.

Even in Serbia, with its traditionally close ties with Russia, the only information forthcoming on the activities of the Russian Nuclear, Biological and Chemical Protection Troops and their equipment that arrived on April 3 was from Russian press releases.

Both countries’ strategic communications efforts are similarly fallible. China’s notoriously heavy-handed approach to its critics is of only limited use in the face of such a severe and immediate threat. One suggestion that the virus originated in the US – an early response to US criticism – has already been walked back by the Chinese diplomat who made it.

And Russia continues to be capable of spectacularly misjudging its targets. When investigative journalists looked more closely at the nature of the assistance to Italy, Russia’s official response was rage and personal threats, laying bare the real nature of the campaign and immediately alienating many of those whom Moscow had sought to win over.

Errors and deficiencies such as these provide opportunities to mitigate the worst side-effects of the campaigns. And actions by individuals can also mitigate much of the impact. The most effective disinformation plays on deeply emotional issues and triggers visceral rather than rational reactions.

Advocates of ’informational distancing’ as well as social distancing suggest a tactical pause to assess information calmly, instead of reacting or spreading it further unthinkingly. This approach would bolster not only calm dispassionate assessment of the real impact of Russian and Chinese actions, but also counter spreading of misinformation on the pandemic as a whole - especially when key sources of disinformation are national leaders seeking to politicize or profit from the crisis.

Limitations of Russian and Chinese altruism must be stated clearly and frankly to fill gaps in public understanding. Where help is genuine, it should of course be welcomed: but if it is the case that assistance received from Moscow or Beijing is not appropriate, not useful, or not fit for purpose, this should be acknowledged publicly.

Even without central direction or coordination with other Russian strategic communications efforts, the self-perpetuating Russian disinformation ecosystem continues to push narratives designed to undermine confidence in institutions and their ability to deal with the crisis. This too must continue to be monitored closely and countered where it matters.

In all cases, miscalculations by Russia or China that expose the true intent of their campaigns – no matter how different their objectives might be - should be watched for closely and highlighted where they occur.

Despite the enormity of the present emergency it is not a time for any government to relax its vigilance over longer-term threats. States must not lose sight of manoeuvres seeking to exploit weakness and distraction. If Russia and China emerge from the current crisis with enhanced authority and unjustifiably restored reputations, this will make it still harder to resist their respective challenges to the current rules-based international order in the future.




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Belarusians Left Facing COVID-19 Alone

16 April 2020

Ryhor Astapenia

Robert Bosch Stiftung Academy Fellow, Russia and Eurasia Programme

Anaïs Marin

Associate Fellow, Russia and Eurasia Programme
The way the epidemic is being mismanaged creates a risk of political destabilisation and leaves the country exposed to external influence.

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Playing accordion in front of dummy football fans in Brest, Belarus as the country's championship continues despite the COVID-19 outbreak. Photo by SERGEI GAPON/AFP via Getty Images.

Since the World Health Organisation (WHO) declared COVID-19 a pandemic, few countries have chosen to ignore social distancing recommendations. But, even among those states which have, the Belarusian official response to its epidemic remains unique.

President Aliaksandr Lukashenka’s statements that vodka, sauna and tractors are protecting Belarusians from coronavirus attracted amused attention in international media. Lukashenka also described other societies’ response to COVID-19 as ‘a massive psychosis’.

Although Lukashenka is notorious for his awkward style of public communication, the fact that Belarus is refusing to impose comprehensive confinement measures is of concern. Belarusians continue to work, play football and socialise.

Lukashenka, himself playing ice hockey in front of state cameras, claims it is the best way to stay healthy. Belarusian authorities clearly appear to be in denial – and this could have dire humanitarian consequences.

From denial to half measures

Belarus actually has one of the largest numbers of hospital beds in the world per 1,000 of the population. But in the absence of quarantine measures its health system, already crippled by corruption and embezzlement, is likely to be overwhelmed.

Patients being treated for pneumonia in hospitals have suggested medical staff are uninformed and inadequately equipped. It is claimed doctors are not reporting COVID-19 as the suspected cause of death, either through a lack of testing or for fear of reprisals.

Observers believe the real mortality rate is already well above official figures (40 deaths as of 16 April). Based on an Imperial College London model, between 15,000 and 32,000 people could die under the current mild confinement regime – and such a high death toll would hugely impact the country’s political stability. Citing personal data protection, the Ministry of Health has imposed a total news blackout; the only cluster officially acknowledged so far is the city of Vitsebsk.

Although specific Belarusian cities and some individuals started changing their approach – by extending school vacations or cancelling weddings – such measures remain half-hearted.

Clearly a major reason for such an apparently irresponsible reaction is that Belarus cannot afford a massive lockdown that would freeze its already underdeveloped economy and drive it deeper into recession. Unlike many other nations, Belarus lacks budgetary resources for a sizable stimulus package. But a delayed response might backfire on the economy.

Economic recession has been forecast to amount to at least 10% of GDP. For Lukashenka, who openly challenged conventional wisdom regarding the need for quarantine and isolation, such an economic downturn would harm his confidence rating in the eyes of Belarusian voters, mindful of the state’s mismanagement of the crisis. And it could create doubt within the ruling elite itself, with Lukashenka seeking re-election for a sixth mandate in late August.

Against this backdrop, a radicalization of the opposition-minded part of society is also to be expected, with greater reliance on social networks in the face of official secrecy and disinformation. The expected response of the regime is then likely to be pre-emptive repression. Evidence is emerging that law enforcement agencies have already stepped up judicial and paralegal harassment of dissenters, notably independent journalists and bloggers.

Russia’s initial reluctance to address the coronavirus crisis may also have influenced Belarus. Lukashenka and his administration often react to public health challenges by the Soviet rulebook, reminiscent of the Soviet authorities’ mismanagement of the Chernobyl disaster in 1986.

Russia has unilaterally closed its borders with Belarus and, as bilateral relations continue to deteriorate, this casts further doubt on the viability of the Union State of Belarus and Russia. Pro-Russian media forecast Moscow will be unwilling to alleviate the expected socio-economic crisis, as it continues to reject Minsk’s demands regarding subsidised oil deliveries. Yet the Kremlin might use the crisis as an opportunity to resume its integrationist pressure on Belarus.

China, with which Belarus engaged in a seemingly privileged strategic partnership in the 2010s, was actually the first country to dispatch humanitarian aid to beef up Belarusian capacity to fight the virus.

But Minsk should not expect Beijing to rescue its economy and, unless it commits to more internal reforms, Belarus is not likely to receive much from the EU either. The regime has already applied to the IMF for emergency financial support, but conditions are attached and, even if successful, the funds would amount to no more than $900m.

The government’s decision to take only half measures so far is rooted in the hope COVID-19 is not as bad as foreign experts fear. But, unless the leadership acknowledges the public health crisis and mitigates its economic impact, COVID-19 will accelerate Belarus’s slide back into international self-isolation. If combined with a humanitarian crisis, this will put the Belarusian regime under considerable stress.

This crisis does risk a new ‘Chernobyl moment’ for the authorities, but the population could react more vocally this time. As volunteers self-organise to fight the epidemic, it might become more difficult for the authorities to say that it is efficient in running the country. But the bottom line is Belarus desperately needs money. Whoever steps up to support Belarus financially will also be able to heavily influence its politics.




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Nigeria’s Political Leaders Need to Win Trust to Tackle COVID-19

23 April 2020

Elizabeth Donnelly

Deputy Director, Africa Programme

Idayat Hassan

Director, Centre for Democracy and Development
COVID-19 will require Nigeria's government to rely on already stretched communities and informal institutions. But there is a yawning gap in trust and accountability between citizens and the state in Nigeria – the crisis will force the state to attempt to bridge this divide.

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News stand in Lagos, Nigeria on April 12, 2020. Photo by PIUS UTOMI EKPEI/AFP via Getty Images.

Nigeria is better placed than many to respond to the arrival of the coronavirus disease. In 2014, it successfully contained a deadly Ebola virus outbreak and the country’s current score on the Epidemic Preparedness Index (38.9 per cent) is higher than the African and global averages.

But the outbreak is compounding Nigeria’s numerous pre-existing crises. It was already grappling with a Lassa fever outbreak that has claimed more than one hundred lives in 2020, the aftermath of recession, and conflict and insecurity within its borders.

Effective leadership to build confidence will be vital. However, President Muhammadu Buhari has made few appearances, delivering his first speech on Nigeria’s response more than one month after the country’s first recorded case. And the indefinite suspension of meetings of the Federal Executive Council has raised questions on the efficacy of the response.

Extended lockdown imposed

The recent loss of President Buhari’s steadfast chief of staff Abba Kyari as a result of contracting COVID-19 is a further significant setback for the presidency. But the administration has established a presidential task force to develop a national strategy and an extended lockdown has been imposed on the most affected states  Lagos, Ogun and the Federal Capital Territory of Abuja. The country has also closed national borders and is expanding testing capacity to 1,500 per day.

However, when Nigeria’s first case was recorded on February 27 it was state governments that initially took action  shutting schools, closing state borders and imposing lockdowns. Going forwards, the 36 state governments will have a key role to play although their governance capacity and commitment varies widely.

The federal government has released $2.7 million to support the Nigeria Centre for Disease Control (NCDC), and promised an additional $18 million  but this falls well short of the estimated $330 million needed to tackle the coronavirus disease in Nigeria. The government is looking to its private sector to help make up the difference. The country’s finances are under severe pressure with Nigerian crude oil  the main source of government revenue and foreign exchange reserves  selling for as low as $12 or $13 a barrel (with production costs of around $22 per barrel), and a debt servicing to revenue ratio of more than 50 per cent even before the oil price crash.

Facing its second recession in four years, with -3.4 per cent GDP growth forecast by the IMF, the country has little economic resilience. Nigeria will not be able to sustain restrictions on its 81.15 million-strong workforce, 83.2 per cent of which operate in the informal sector. One area at particular risk is food security, as the pandemic is disrupting farming, supply chains and trade. By building on past benefit programmes, the federal government is providing cash and distributing food to vulnerable households, but this important effort is being hampered by poor communication, inefficiencies and a lack of transparency  longstanding challenges in many aspects of public service delivery in Nigeria.

In the absence of a reliable social safety net, Nigerians trust and rely on their families, communities and the informal economy to see them through difficult times. It is these informal mechanisms that lend Nigeria its oft-referenced resilience, which has enabled society to function and continue while a largely disconnected political class has focused on self-enrichment.

It is through these traditional channels that the government will need to deliver information, support, testing and treatment. But without high levels of trust, the administration may find it difficult to do so. Many Nigerians initially considered the pandemic a hoax, some describing it as a ‘rich man’s disease’, while others see it as another conspiracy by politicians to loot the treasury.

Lockdown measures have also heightened tensions across the country. Some citizens are rebelling and in one instance burned down a police station in response to the closure of mosques in Katsina state. Marking a further breakdown in the relationship between the population and its leaders, the Nigerian National Human Rights Commission (NHRC) recently reported security services enforcing the lockdown have extrajudicially killed 18 people, while, so far, COVID-19 has killed 25 people in Nigeria.

Mitigating the spread and worst consequences of the virus will depend on the state rebuilding trust with its citizens through effective communication and action. It is particularly important that the community mechanisms of support are protected as they come under growing pressure as communities become increasingly affected by the virus.

The stark choice facing most Nigerians  between risking starvation and risking contagion  means a sustained lockdown is not a tenable option. People will choose to go to work. This will especially be the case as people grow weary of measures imposed upon them by a state that the vast majority of the population believe does not serve or care for them.

Having largely ignored the needs of Nigeria’s citizens for decades, the political class face an uphill battle in building trust with the population. Earning this trust is not only crucial for the struggle against COVID-19 but also for Nigeria’s longer-term progress and system of political governance.




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Fighting COVID-19 the Ukrainian Way

28 April 2020

Orysia Lutsevych

Research Fellow and Manager, Ukraine Forum, Russia and Eurasia Programme
Coronavirus has exposed vulnerabilities in Ukraine but also activated private sector and citizen engagement in delivering help. This could accelerate social change if a smart response is adopted and political reforms follow.

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Girls wearing face masks at the monument to Chernobyl victims in Slavutich during a memorial ceremony amid the COVID-19 pandemic. Photo by SERGEI SUPINSKY/AFP via Getty Images.

Ukrainians are accustomed to crisis. As COVID-19 spread, forest fires were raging in the Chernobyl exclusion zone, turning Kyiv into the most polluted city in the world. The fighting in Donbas continued, claiming the lives of more Ukrainian soldiers, bringing the total to more than 4,000 — and, on top of that, President Zelenskyy overhauled his government. So Ukraine is fighting three battles at the same time — war with Russia, the struggle against its own ineffective system, and now COVID-19.

Every crisis is a reality check — the coronavirus provoked and exposed the strategic vulnerabilities and deep-rooted features of Ukraine’s system of governance. Three trends have come to the fore. First, the inefficiency and paralysis of many state agencies, particularly the lack of coordination between them and the prevalence of vested interests. Second, the reliance of the country’s leaders on large financial-industrial groups (FIGs) to compensate for weak institutional capacity. Third, a strong societal and private sector mobilization to fill the gaps in the dilapidated public health system.

State agencies are rigid and ineffective. Despite the modern Prozorro digital public procurement system, and the government’s allocation of $2.5 million from the early days of the epidemic, the Ministry of Health blocked COVID-related purchases for over a month. This was a tactic by — now ex-minister — Yemets to pressure the state medical procurement agency into appointing a protégé of his as one of its deputy heads.

Lowest testing rate in Europe

Similarly, in some regions, notably Odesa, procurement stalled and orders went to politically connected businesses at higher-than-market prices. Lack of tests and laboratory equipment means Ukraine has administered only 72,000 tests within a population of 42 million to date — the lowest rate in Europe.

Doctors were given orders to ensure they only test patients in hospitals with COVID-19 symptoms and only those arriving from Asia, while ignoring the fact that millions of Ukrainian labour migrants were in Europe. Indeed, the first confirmed case was imported from Italy.

Ukrainian government and public health officials lack information to take informed decisions. There is no accurate electronic database of registered deaths and reporting is lagging behind events. Information on testing availability in the regions is missing.

Thirteen days after the first case of the virus was recorded, Zelenskyy exhorted business tycoons to come to the rescue. Taking a populist tone, Zelenskyy said ‘Ukraine has been feeding you for a long time and it is time that you helped the country’. The tycoons divided the regions among themselves to deliver relief efforts according to the location of their enterprises.

It is believed FIGs have donated around $25 million to procure testing kits, ventilators, personal protective equipment (PPE) and disinfectants. This may sound impressive, but many of those same tycoons actually owe millions to the state, some even billions, and cause serious problems by perpetuating the current rent-seeking system, where public resources benefit those groups resulting in serious social losses.

Reliance on these groups makes Zelenskyy a hostage to their favour in any potential reform efforts. It is a dangerous solution, as these tycoons often obstruct Ukraine’s economic development.

An alternative — and more transformative — trend of public-private partnerships is emerging in some regions. Across Ukraine, hundreds of small- and medium-sized enterprises (SMEs) have led efforts to deliver PPE, support the vulnerable with food supplies, and to procure ventilators for key hospitals.

They have mobilised hundreds of volunteers to deliver assistance and partnered with local non-profits. Fundraising initiatives have begun in Lviv, Odesa, Kyiv and Poltava with donations and expenditure has been posted online for transparency. Companies have repurposed to produce PPE kits and medical equipment. The efforts unfolded quickly and, in some cases, in smooth collaboration with municipal and regional authorities.

Ukraine cannot afford to ‘waste’ this crisis, which could help accelerate healthcare reform, decentralization, modernize governance, and boost citizen empowerment. But for this to happen, the country has to deploy a ‘smart response’.

Such ‘smart response’ means applying a resilience framework that nurtures the agility of the system of governance, ensures a diversity of actors in decision-making, supporting both self-regulation and better coordination. Rather than reaching out to tycoons, Zelenskyy should enter a coalition with true agents of change — SME leaders, volunteers, and mayors who have mobilized effective grassroots action. These actors demand a level playing field with accountable governance and effective state institutions.

Civic COVID-19 response hubs and local authorities should be joined in a network that spans the regions, and connected with the national agencies designing pandemic responses. For a national strategy to be effective, central headquarters should draw information from local communities and manage a ‘team of teams’ in a decentralised fashion.

Ensuring effective public service delivery without compromising integrity and keeping the risk of corruption low should also be a priority of political reform, with volunteers and the private sector ensuring civic oversight of both regional and national funding.

Civic engagement such as this can be transformative as it defies the Soviet legacy of paternalism and expands the belief among citizens that society can work for them. By assisting the relief effort, citizens are gaining valuable insights into quality of public services and participate in holding them to account.

Citizens are also developing a better understanding of the purpose of having effective armed forces, police, border guards and modern hospitals. They are coming to understand the value of taxpayer money and witnessing how corruption erodes institutions.

This survival mobilization — if properly harnessed by the state — could drive transformative change and make Ukraine more resilient, not just against present crises, but future ones too.




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Algeria’s Perfect Storm: COVID-19 and Its Fallout

6 May 2020

Adel Hamaizia

Associate Fellow, Middle East and North Africa Programme

Yahia H. Zoubir

Senior Professor of International Studies, KEDGE Business School; Visiting Fellow, Brookings Doha Center
Coronavirus is a godsend for Algeria’s government to introduce restrictive measures beyond those needed to contain COVID-19. But its new leaders are missing a chance to gain legitimacy, which will offset the socio-economic fallout of the drop in oil prices.

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Algerian volunteers prepare personal protection equipment (PPE) to help combat the coronavirus epidemic in the capital Algiers. Photo by RYAD KRAMDI/AFP via Getty Images.

Although protests successfully ended Abdelaziz Bouteflika’s 20-year sultanistic rule a little over one year ago, demands have been continuing to dismantle the system, get rid of the old personnel, and institute democracy.

The controversial election in December of Abdelmadjid Tebboune — who has inherited a disastrous situation — has not tempered the determination of the Hirak protest movement. As a former minister and prime minister under Bouteflika, the new president has won little legitimacy, and protests have continued.

Now COVID-19 is worsening already dire economic conditions, such as a sharp drop in oil prices. By the beginning of May, statistics showed 10% of confirmed cases have ended in fatality, the highest percentage in the region.

Maintaining an authoritarian style

Hirak had already called for the suspension of the marches — mobilising online instead — before the government’s measures, which include curfews and lockdowns, demonstrating a high sense of duty. But instead of appeasing Hirak’s demands, the government has maintained the authoritarian style of its predecessors.

Tebboune released more than 5,000 prisoners on March 31 but kept prisoners of conscience and leaders of the hirak imprisoned, then subsequently imprisoned journalists and activists. It even passed a controversial penal law, that also covers fake news, and may be used to justify actions against journalists.

The regime wishes to see an end to the Hirak, and rejects accusations of totalitarianism by insisting freedom and a democratic climate exist in Algeria.

Tebboune’s actions contradict his praise for the ‘blessed’ hirak and his promises of instituting the rule of law. In proclaiming the measures, the government has shown disappointing leadership, acting in an authoritarian fashion.

Tebboune also declared proudly that Algeria was fully prepared to fight the coronavirus epidemic, an optimistic claim given the country has only 400 intensive care unit (ICU) beds, or one per 100,000 people. Despite hundreds of billions of hydrocarbon dollars accumulating during the Bouteflika-era, Algeria’s health system ranks 173 out of 195 countries.

Algerians often refer to hospitals as ‘mouroirs’, meaning ‘places for the dying’. Not only has the state failed to build modern hospitals but basic hygienic conditions are lacking, and government officials prefer being treated overseas. A 2014 project to build five university hospitals was abandoned, leaving the health sector in deplorable shape.

Before Chinese assistance arrived, the glaring lack of equipment to protect caregivers and care for the sick was evident. Prime Minister Abdelaziz Djerad admitted the health system required a ‘total overhaul’. The president recently stated Algeria’s doctors are among the 'best in the world' but didn't address why almost 15,000 Algerian doctors practice in France.

Strict containment measures are in sync with most countries but implementation is challenging when most people live in overcrowded urban dwellings (the average household consists of 5.9 members).

Water shortages in many areas makes good hygiene and decontamination impossible, while schools and universities find online teaching difficult when many students do not possess laptops or internet connections. And only 20% of Algerians have debit cards in a cash-dominated economy because of low trust in the public-dominated banking sector, making online shopping capability low.

An already declining macroeconomic situation is worsening due to COVID-19. The IMF revised its 2020 estimates for Algeria, forecasting a catastrophic contraction of -5.2% in a country where hydrocarbons account for 93% of export revenues and 60% of its budget.

Foreign currency reserves are now an estimated $55 billion (expected to fall to $44billion by the end of 2020), down from $200 billion in 2014, and Algerian crude has recently traded close to production costs, with the fiscal breakeven oil price at $157.

In line with its historic aversion to external borrowing, Tebboune recently ruled out seeking financial support from the ‘IMF or other foreign banks’, as he argued such borrowing undermines sovereign foreign policy because - when indebted - ‘we cannot talk about either Palestine or Western Sahara’, two causes dear to Algeria. ‘Friendly countries’ - most likely a reference to China - are said to have offered to grant loans which have been declined for now.

The government is forecasted to face a 20% budget shortfall this year, but Algeria’s fiscal response to COVID-19 is actually the largest among the regional hydrocarbon exporters at an estimated 8% of GDP, compared to an average of 3.2%. However, the government revised downwards its 2020 public spending by 50% (a second cut in a month, from an initial 30% reduction), halting state projects and slashing its $41 billion import bill by 25% while expanding agricultural production. National oil company SONATRACH will also cut planned investment by half to $7 billion but plans have been revealed to develop other natural resources including gold, uranium and phosphates.

But recent growth rates are insufficient to create jobs for those entering the labour market. Despite government attempts to support a rather anaemic ‘formal’ private sector, estimates are 700,000 jobs could be lost due to potential bankruptcies from reduced activity and a loss of markets abroad.

Facing potential social unrest and the quasi-preservation of a tired social contract, the government has committed to upholding public sector wages - including for 50% of the civil servants told to stay home - protecting sacrosanct, unsustainable subsidies, and increasing health expenditure to strengthen the capacity to combat COVID-19.

A supplementary finance law will include various measures that support businesses and the economic fallout. However, while the government is to be commended for its efforts to aid businesses, supporting large swathes of the population is challenging as approximately 50% of the workforce operate in the informal economy.

Weak administrative capacity and insufficient data to implement cash transfers makes the planned ‘solidarity allowance’ of 10,000 dinars ($80) for Ramadan difficult to allocate to those who most need it (especially those in the informal sector). Families, communities, and religious organisations continue to be a social safety net.

So COVID-19 has not created new problems, it has merely magnified and exacerbated the numerous inequalities and failures of the Bouteflika regime to sufficiently invest in human security (economic, food, health environmental, personal, community, and political). Typically, whenever oil prices and related earnings dwindle, the political system promises to reform and diversify the economy. Tebboune is repeating this same old tune.

There are positive elements, such as the government’s realization it must initiate genuine reforms. And local enterprises have been successfully producing artificial respirators, surgical masks, and other materials. Algerians, including the Hirak, are showing great social solidarity.

But the government must capitalize on these positive actions by introducing real change. Because, if not, Hirak will certainly be back in force once the crisis is over, and operating in an environment of worsening socioeconomic problems. The medicine of the past will not work.




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Oxidative stress-mediated regulation of proteasome complexes [Other]

Oxidative stress has been implicated in aging and many human diseases, notably neurodegenerative disorders and various cancers. The reactive oxygen species that are generated by aerobic metabolism and environmental stressors can chemically modify proteins and alter their biological functions. Cells possess protein repair pathways to rescue oxidized proteins and restore their functions. If these repair processes fail, oxidized proteins may become cytotoxic. Cell homeostasis and viability are therefore dependent on the removal of oxidatively damaged proteins. Numerous studies have demonstrated that the proteasome plays a pivotal role in the selective recognition and degradation of oxidized proteins. Despite extensive research, oxidative stress-triggered regulation of proteasome complexes remains poorly defined. Better understanding of molecular mechanisms underlying proteasome function in response to oxidative stress will provide a basis for developing new strategies aimed at improving cell viability and recovery as well as attenuating oxidation-induced cytotoxicity associated with aging and disease. Here we highlight recent advances in the understanding of proteasome structure and function during oxidative stress and describe how cells cope with oxidative stress through proteasome-dependent degradation pathways.




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The ProteoRed MIAPE web toolkit: A user-friendly framework to connect and share proteomics standards [Technology]

The development of the HUPO-PSI's (Proteomics Standards Initiative) standard data formats and MIAPE (Minimum Information About a Proteomics Experiment) guidelines should improve proteomics data sharing within the scientific community. Proteomics journals have encouraged the use of these standards and guidelines to improve the quality of experimental reporting and ease the evaluation and publication of manuscripts. However, there is an evident lack of bioinformatics tools specifically designed to create and edit standard file formats and reports, or embed them within proteomics workflows. In this article, we describe a new web-based software suite (The ProteoRed MIAPE web toolkit) that performs several complementary roles related to proteomic data standards. Firstly, it can verify the reports fulfill the minimum information requirements of the corresponding MIAPE modules, highlighting inconsistencies or missing information. Secondly, the toolkit can convert several XML-based data standards directly into human readable MIAPE reports stored within the ProteoRed MIAPE repository. Finally, it can also perform the reverse operation, allowing users to export from MIAPE reports into XML files for computational processing, data sharing or public database submission. The toolkit is thus the first application capable of automatically linking the PSI's MIAPE modules with the corresponding XML data exchange standards, enabling bidirectional conversions. This toolkit is freely available at http://www.proteored.org/MIAPE/.




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Bayesian Proteoform Modeling Improves Protein Quantification of Global Proteomic Measurements [Technology]

As the capability of mass spectrometry-based proteomics has matured, tens of thousands of peptides can be measured simultaneously, which has the benefit of offering a systems view of protein expression. However, a major challenge is that with an increase in throughput, protein quantification estimation from the native measured peptides has become a computational task. A limitation to existing computationally-driven protein quantification methods is that most ignore protein variation, such as alternate splicing of the RNA transcript and post-translational modifications or other possible proteoforms, which will affect a significant fraction of the proteome. The consequence of this assumption is that statistical inference at the protein level, and consequently downstream analyses, such as network and pathway modeling, have only limited power for biomarker discovery. Here, we describe a Bayesian model (BP-Quant) that uses statistically derived peptides signatures to identify peptides that are outside the dominant pattern, or the existence of multiple over-expressed patterns to improve relative protein abundance estimates. It is a research-driven approach that utilizes the objectives of the experiment, defined in the context of a standard statistical hypothesis, to identify a set of peptides exhibiting similar statistical behavior relating to a protein. This approach infers that changes in relative protein abundance can be used as a surrogate for changes in function, without necessarily taking into account the effect of differential post-translational modifications, processing, or splicing in altering protein function. We verify the approach using a dilution study from mouse plasma samples and demonstrate that BP-Quant achieves similar accuracy as the current state-of-the-art methods at proteoform identification with significantly better specificity. BP-Quant is available as a MatLab ® and R packages at https://github.com/PNNL-Comp-Mass-Spec/BP-Quant.




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The Proteomics of Networks and Pathways: A Movie is Worth a Thousand Pictures [Editorial]

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WITHDRAWN: Heralds of parallel MS: Data-independent acquisition surpassing sequential identification of data dependent acquisition in proteomics [Research]

This article has been withdrawn by the authors. This article did not comply with the editorial guidelines of MCP. Specifically, single peptide based protein identifications of 9-19% were included in the analysis and discussed in the results and conclusions. We wish to withdraw this article and resubmit a clarified, corrected manuscript for review.




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DEqMS: a method for accurate variance estimation in differential protein expression analysis [Technological Innovation and Resources]

Quantitative proteomics by mass spectrometry is widely used in biomarker research and basic biology research for investigation of phenotype level cellular events. Despite the wide application, the methodology for statistical analysis of differentially expressed proteins has not been unified. Various methods such as t-test, linear model and mixed effect models are used to define changes in proteomics experiments. However, none of these methods consider the specific structure of MS-data. Choices between methods, often originally developed for other types of data, are based on compromises between features such as statistical power, general applicability and user friendliness. Furthermore, whether to include proteins identified with one peptide in statistical analysis of differential protein expression varies between studies. Here we present DEqMS, a robust statistical method developed specifically for differential protein expression analysis in mass spectrometry data. In all datasets investigated there is a clear dependence of variance on the number of PSMs or peptides used for protein quantification. DEqMS takes this feature into account when assessing differential protein expression. This allows for a more accurate data-dependent estimation of protein variance and inclusion of single peptide identifications without increasing false discoveries. The method was tested in several datasets including E.coli proteome spike-in data, using both label-free and TMT-labelled quantification. In comparison to previous statistical methods used in quantitative proteomics, DEqMS showed consistently better accuracy in detecting altered protein levels compared to other statistical methods in both label-free and labelled quantitative proteomics data. DEqMS is available as an R package in Bioconductor.




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Immediate adaptation analysis implicates BCL6 as an EGFR-TKI combination therapy target in NSCLC [Research]

Drug resistance is a major obstacle to curative cancer therapies, and increased understanding of the molecular events contributing to resistance would enable better prediction of therapy response, as well as contribute to new targets for combination therapy. Here we have analyzed the early molecular response to epidermal growth factor receptor (EGFR) inhibition using RNA sequencing data covering 13 486 genes and mass spectrometry data covering 10 138 proteins. This analysis revealed a massive response to EGFR inhibition already within the first 24 hours, including significant regulation of hundreds of genes known to control downstream signaling, such as transcription factors, kinases, phosphatases and ubiquitin E3-ligases. Importantly, this response included upregulation of key genes in multiple oncogenic signaling pathways that promote proliferation and survival, such as ERBB3, FGFR2, JAK3 and BCL6, indicating an early adaptive response to EGFR inhibition. Using a library of more than 500 approved and experimental compounds in a combination therapy screen, we could show that several kinase inhibitors with targets including JAK3 and FGFR2 increased the response to EGFR inhibitors. Further, we investigated the functional impact of BCL6 upregulation in response to EGFR inhibition using siRNA-based silencing of BCL6. Proteomics profiling revealed that BCL6 inhibited transcription of multiple target genes including p53, resulting in reduced apoptosis which implicates BCL6 upregulation as a new EGFR inhibitor treatment escape mechanism. Finally, we demonstrate that combined treatment targeting both EGFR and BCL6 act synergistically in killing lung cancer cells. In conclusion, or data indicates that multiple different adaptive mechanisms may act in concert to blunt the cellular impact of EGFR inhibition, and we suggest BCL6 as a potential target for EGFR inhibitor-based combination therapy.




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Characterization of signaling pathways associated with pancreatic {beta}-cell adaptive flexibility in compensation of obesity-linked diabetes in db/db mice [Research]

The onset of obesity-linked type 2 diabetes (T2D) is marked by an eventual failure in pancreatic β-cell function and mass that is no longer able to compensate for the inherent insulin resistance and increased metabolic load intrinsic to obesity. However, in a commonly used model of T2D, the db/db mouse, β-cells have an inbuilt adaptive flexibility enabling them to effectively adjust insulin production rates relative to the metabolic demand. Pancreatic β-cells from these animals have markedly reduced intracellular insulin stores, yet high rates of (pro)insulin secretion, together with a substantial increase in proinsulin biosynthesis highlighted by expanded rough endoplasmic reticulum and Golgi apparatus. However, when the metabolic overload and/or hyperglycemia is normalized, β-cells from db/db mice quickly restore their insulin stores and normalize secretory function. This demonstrates the β-cell’s adaptive flexibility and indicates that therapeutic approaches applied to encourage β-cell rest are capable of restoring endogenous β-cell function. However, mechanisms that regulate β-cell adaptive flexibility are essentially unknown. To gain deeper mechanistic insight into the molecular events underlying β-cell adaptive flexibility in db/db β-cells, we conducted a combined proteomic and post-translational modification specific proteomic (PTMomics) approach on islets from db/db mice and wild-type controls (WT) with or without prior exposure to normal glucose levels. We identified differential modifications of proteins involved in redox homeostasis, protein refolding, K48-linked deubiquitination, mRNA/protein export, focal adhesion, ERK1/2 signaling, and renin-angiotensin-aldosterone signaling, as well as sialyltransferase activity, associated with β-cell adaptive flexibility. These proteins are all related to proinsulin biosynthesis and processing, maturation of insulin secretory granules, and vesicular trafficking—core pathways involved in the adaptation of insulin production to meet metabolic demand. Collectively, this study outlines a novel and comprehensive global PTMome signaling map that highlights important molecular mechanisms related to the adaptive flexibility of β-cell function, providing improved insight into disease pathogenesis of T2D.




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Modulation of natural HLA-B*27:05 ligandome by ankylosing spondylitis-associated endoplasmic reticulum aminopeptidase 2 (ERAP2) [Research]

The human leucocyte antigen (HLA)-B*27:05 allele and the endoplasmic reticulum-resident aminopeptidases are strongly associated with ankylosing spondylitis (AS), a chronic inflammatory spondyloarthropathy. This study examined the effect of endoplasmic reticulum aminopeptidase 2 (ERAP2) in the generation of the natural HLA-B*27:05 ligandome in live cells. Complexes of HLA-B*27:05-bound peptide pools were isolated from human ERAP2-edited cell clones and the peptides were identified using high throughput mass spectrometry analyses. The relative abundance of thousand ligands was established by quantitative tandem mass spectrometry and bioinformatics analysis. The residue frequencies at different peptide position, identified in presence or absence of ERAP2, determined structural features of ligands and their interactions with specific pockets of antigen binding site of HLA-B*27:05 molecule. Sequence alignment of ligands identified with species of bacteria associated with HLA-B*27-dependent reactive arthritis was performed. In the absence of ERAP2, peptides with N-terminal basic residues, and minority canonical P2 residues are enriched in the natural ligandome. Further, alterations of residue frequencies and hydrophobicity profile at P3, P7, and P positions were detected. In addition, several ERAP2-dependent cellular peptides were highly similar to protein sequences of arthritogenic bacteria, including one human HLA-B*27:05 ligand fully conserved in a protein from Campylobacter jejuni. These findings highlight the pathogenic role of this aminopeptidase in the triggering of AS autoimmune disease.




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Quantitative proteomics of human heart samples collected in vivo reveal the remodeled protein landscape of dilated left atrium without atrial fibrillation [Research]

Genetic and genomic research has greatly advanced our understanding of heart disease. Yet, comprehensive, in-depth, quantitative maps of protein expression in hearts of living humans are still lacking. Using samples obtained during valve replacement surgery in patients with mitral valve prolapse (MVP), we set out to define inter-chamber differences, the intersect of proteomic data with genetic or genomic datasets, and the impact of left atrial dilation on the proteome of patients with no history of atrial fibrillation (AF).  We collected biopsies from right atria (RA), left atria (LA) and left ventricle (LV) of seven male patients with mitral valve regurgitation with dilated LA but no history of AF. Biopsy samples were analyzed by high-resolution mass spectrometry (MS), where peptides were pre-fractionated by reverse phase high-pressure liquid chromatography prior to MS measurement on a Q-Exactive-HF Orbitrap instrument. We identified 7,314 proteins based on 130,728 peptides. Results were confirmed in an independent set of biopsies collected from three additional individuals. Comparative analysis against data from post-mortem samples showed enhanced quantitative power and confidence level in samples collected from living hearts. Our analysis, combined with data from genome wide association studies suggested candidate gene associations to MVP, identified higher abundance in ventricle for proteins associated with cardiomyopathies and revealed the dilated LA proteome, demonstrating differential representation of molecules previously associated with AF, in non-AF hearts. This is the largest dataset of cardiac protein expression from human samples collected in vivo. It provides a comprehensive resource that allows insight into molecular fingerprints of MVP and facilitates novel inferences between genomic data and disease mechanisms. We propose that over-representation of proteins in ventricle is consequent not to redundancy but to functional need, and conclude that changes in abundance of proteins known to associate with AF are not sufficient for arrhythmogenesis.




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Proteaphagy in mammalian cells can function independent of ATG5/ATG7 [Research]

The degradation of intra- and extracellular proteins is essential in all cell types and mediated by two systems, the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway. This study investigates the changes in autophagosomal and lysosomal proteomes upon inhibition of proteasomes by bortezomib (BTZ) or MG132. We find an increased abundance of more than 50 proteins in lysosomes of cells in which the proteasome is inhibited. Among those are dihydrofolate reductase (DHFR), ß-Catenin and 3-hydroxy-3-methylglutaryl-coenzym-A (HMGCoA)-reductase. Since these proteins are known to be degraded by the proteasome they seem to be compensatorily delivered to the autophagosomal pathway when the proteasome is inactivated. Surprisingly, most of the proteins which show increased amounts in the lysosomes of BTZ or MG132 treated cells are proteasomal subunits. Thus an inactivated, non-functional proteasome is delivered to the autophagic pathway. Native gel electrophoresis shows that the proteasome reaches the lysosome intact and not disassembled. Adaptor proteins, which target proteasomes to autophagy, have been described in Arabidopsis, Saccharomyces and upon starvation in mammalians. However, in cell lines deficient of these proteins or their mammalian orthologues, respectively, the transfer of proteasomes to the lysosome is not impaired. Obviously, these proteins do not play a role as autophagy adaptor proteins in mammalian cells. We can also show that chaperone-mediated autophagy (CMA) does not participate in the proteasome delivery to the lysosomes. In autophagy-related (ATG)-5 and ATG7 deficient cells the delivery of inactivated proteasomes to the autophagic pathway was only partially blocked, indicating the existence of at least two different pathways by which inactivated proteasomes can be delivered to the lysosome in mammalian cells.




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HIGD2A is required for assembly of the COX3 module of human mitochondrial complex IV [Research]

Assembly factors play a critical role in the biogenesis of mitochondrial respiratory chain complexes I-IV where they assist in the membrane insertion of subunits, attachment of co-factors, and stabilization of assembly intermediates. The major fraction of complexes I, III and IV are present together in large molecular structures known as respiratory chain supercomplexes. A number of assembly factors have been proposed as required for supercomplex assembly, including the hypoxia inducible gene 1 domain family member HIGD2A. Using gene-edited human cell lines and extensive steady state, translation and affinity enrichment proteomics techniques we show that loss of HIGD2A leads to defects in the de novo biogenesis of mtDNA-encoded COX3, subsequent accumulation of complex IV intermediates and turnover of COX3 partner proteins. Deletion of HIGD2A also leads to defective complex IV activity. The impact of HIGD2A loss on complex IV was not altered by growth under hypoxic conditions, consistent with its role being in basal complex IV assembly. While in the absence of HIGD2A we show that mitochondria do contain an altered supercomplex assembly, we demonstrate it to harbor a crippled complex IV lacking COX3. Our results redefine HIGD2A as a classical assembly factor required for building the COX3 module of complex IV.




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Flow-induced reorganization of laminin-integrin networks within the endothelial basement membrane uncovered by proteomics [Research]

The vessel wall is continuously exposed to hemodynamic forces generated by blood flow. Endothelial mechanosensors perceive and translate mechanical signals via cellular signaling pathways into biological processes that control endothelial development, phenotype and function. To assess the hemodynamic effects on the endothelium on a system-wide level, we applied a quantitative mass spectrometry approach combined with cell surface chemical footprinting. SILAC-labeled endothelial cells were subjected to flow-induced shear stress for 0, 24 or 48 hours, followed by chemical labeling of surface proteins using a non-membrane permeable biotin label, and analysis of the whole proteome and the cell surface proteome by LC-MS/MS analysis. These studies revealed that of the >5000 quantified proteins 104 were altered, which were highly enriched for extracellular matrix proteins and proteins involved in cell-matrix adhesion. Cell surface proteomics indicated that LAMA4 was proteolytically processed upon flow-exposure, which corresponded to the decreased LAMA4 mass observed on immunoblot. Immunofluorescence microscopy studies highlighted that the endothelial basement membrane was drastically remodeled upon flow exposure. We observed a network-like pattern of LAMA4 and LAMA5, which corresponded to the localization of laminin-adhesion molecules ITGA6 and ITGB4. Furthermore, the adaptation to flow-exposure did not affect the inflammatory response to tumor necrosis factor α, indicating that inflammation and flow trigger fundamentally distinct endothelial signaling pathways with limited reciprocity and synergy. Taken together, this study uncovers the blood flow-induced remodeling of the basement membrane and stresses the importance of the subendothelial basement membrane in vascular homeostasis.




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A cross-linking mass spectrometry approach defines protein interactions in yeast mitochondria [Research]

Protein cross-linking and the analysis of cross-linked peptides by mass spectrometry is currently receiving much attention. Not only is this approach applied to isolated complexes to provide information about spatial arrangements of proteins but it is also increasingly applied to entire cells and their organelles. As in quantitative proteomics, the application of isotopic labelling further makes it possible to monitor quantitative changes in the protein-protein interactions between different states of a system. Here, we cross-linked mitochondria from Saccharomyces cerevisiae grown on either glycerol- or glucose-containing medium to monitor protein-protein interactions under non-fermentative and fermentative conditions. We investigated qualitatively the protein-protein interactions of the 400 most abundant proteins applying stringent data-filtering criteria, i.e. a minimum of two cross-linked peptide spectrum matches and a cut-off in the spectrum scoring of the used search engine. The cross-linker BS3 proved to be equally suited for connecting proteins in all compartments of mitochondria when compared with its water-insoluble but membrane-permeable derivative DSS. We also applied quantitative cross-linking to mitochondria of both the growth conditions using stable-isotope labelled BS3. Significant differences of cross-linked proteins under glycerol and glucose conditions were detected, however, mainly due to the different copy numbers of these proteins in mitochondria under both the conditions. Results obtained from the glycerol condition indicate that the internal NADH:ubiquinone oxidoreductase Ndi1 is part of an electron transport chain supercomplex. We have also detected several hitherto uncharacterized proteins and identified their interaction partners. Among those, Min8 was found to be associated with cytochrome c oxidase. BN-PAGE analyses of min8 mitochondria suggest that Min8 promotes the incorporation of Cox12 into cytochrome c oxidase.