Epoxide hydrolases (EHs) have been characterized and engineered as biocatalysts that convert epoxides to valuable chiral vicinal diol precursors of drugs and bioactive compounds. Nonetheless, the regioselectivity control of the epoxide ring opening by EHs remains challenging. Alp1U is an α/β-fold EH that exhibits poor regioselectivity in the epoxide hydrolysis of fluostatin C (compound 1) and produces a pair of stereoisomers. Herein, we established the absolute configuration of the two stereoisomeric products and determined the crystal structure of Alp1U. A Trp-186/Trp-187/Tyr-247 oxirane oxygen hole was identified in Alp1U that replaced the canonical Tyr/Tyr pair in α/β-EHs. Mutation of residues in the atypical oxirane oxygen hole of Alp1U improved the regioselectivity for epoxide hydrolysis on 1. The single site Y247F mutation led to highly regioselective (98%) attack at C-3 of 1, whereas the double mutation W187F/Y247F resulted in regioselective (94%) nucleophilic attack at C-2. Furthermore, single-crystal X-ray structures of the two regioselective Alp1U variants in complex with 1 were determined. These findings allowed insights into the reaction details of Alp1U and provided a new approach for engineering regioselective epoxide hydrolases.

The main protease (3CL Mpro) from SARS–CoV-2, the etiological agent of COVID-19, is an essential enzyme for viral replication. 3CL Mpro possesses an unusual catalytic dyad composed of Cys145 and His41 residues. A critical question in the field has been what the protonation states of the ionizable residues in the substrate-binding active-site cavity are; resolving this point would help understand the catalytic details of the enzyme and inform rational drug development against this pernicious virus. Here, we present the room-temperature neutron structure of 3CL Mpro, which allowed direct determination of hydrogen atom positions and, hence, protonation states in the protease. We observe that the catalytic site natively adopts a zwitterionic reactive form in which Cys145 is in the negatively charged thiolate state and His41 is doubly protonated and positively charged, instead of the neutral unreactive state usually envisaged. The neutron structure also identified the protonation states, and thus electrical charges, of all other amino acid residues and revealed intricate hydrogen-bonding networks in the active-site cavity and at the dimer interface. The fine atomic details present in this structure were made possible by the unique scattering properties of the neutron, which is an ideal probe for locating hydrogen positions and experimentally determining protonation states at near-physiological temperature. Our observations provide critical information for structure-assisted and computational drug design, allowing precise tailoring of inhibitors to the enzyme's electrostatic environment.

Why the UN matters for Britain Interview LJefferson 6 September 2022

In the fourth of a series of interviews with Queen Elizabeth II Academy faculty, Jeremy Greenstock argues that the UN is still important in a polarized world.

For many people looking at the United Nations (UN) today, the institution (and the world) appears to be at an inflection point and the UN seems ill-equipped to meet these challenges. From your vantage point, how does this period look in relative terms? 

I think for an institution like the UN founded on principles and compromises laid down in 1945, the passage of time is bound to be difficult because society changes quicker than an institution can reform. We can talk about reform later, but the UN has challenges. Where do these challenges come from? I see them coming from a crisis in governance, in governments around the world.

The UN is a forum of member states, and the member states carry their national labels at the UN and follow their national interests at the UN. And almost all governments are suffering huge challenges, not just from the circumstances of geopolitics, but from the expectations of their own people, which they find difficult to meet.

The UN is a servant in that sense, of governments, and so what happens at the UN reflects what is happening in and between governments. We need to keep that in perspective. The UN still retains a tremendous value as a forum. It is a natural forum for governments to talk before they shoot, which was not there in previous eras, and that has had a significant effect since 1945 in reducing the incidence of war between states, particularly between the largest states.

It has obviously been more difficult to deal with local and regional conflicts, but the kind of confrontation that threatens to escalate into a global war has been severely restricted by the UN. The habit of talking at the UN General Assembly (UNGA), artificial as it may seem, is an extremely important part of the avoidance of conflict in the modern age.  

There is a deep scepticism about the UN. Many people think there is a lot of talking but that the real action takes place elsewhere. What is the value of the UN?  

Well, there are two aspects to this. One is the fact that leaders must give their reasons for their policymaking to an international public. This has a force in today’s digital world where most people have access to a megaphone of some kind. The legitimacy of what governments do is exposed at the UNGA.

Secondly, alongside the open meetings and the publicized speeches, there are countless side meetings that go on, and it is an opportunity for leaders to test each other out, and to have private words that may differ from the public words they have to produce for their own followers in their own capitals.

It is an opportunity for personal diplomacy which is highly valuable, and which might not otherwise happen, particularly between leaders who have very serious differences. And I think that the testing of the legitimacy of policy in both the public and the private spheres is an important aspect of international diplomacy that the UNGA provides an opportunity for.  

At the UNGA, states are called to account before the UN, and this can expose hypocrisies. But sometimes there are fundamental clashes over interests and also over values. How would you characterize the UN’s handling of Russia’s invasion of Ukraine?

It is a different audience at the UN from the audience that they have back in their capitals or in their own groups of like-minded people. They must justify themselves in different ways, and there are a whole host of inconsistencies in foreign policy that get exposed at the UN when people must explain themselves in public.

A lot of member states around the world have not condemned the Russian invasion of Ukraine; yet underneath their non-condemnation is a general regret because Russia has broken a huge taboo of the UN Charter, which is the sacrosanctity of independent sovereign territories in Article 2.7.

And that article is valuable to member states who feel threatened by more powerful member states. Russia, as a permanent member, has ridden roughshod over sovereign independence. China and India will have equivocal feelings about that, but they can’t say so in public because they want some of the West’s hypocrisies and inconsistencies exposed, and because they find the approach of sanctions very unpalatable.

Sanctions are unpopular, and the use of sanctions has become a major weapon of non-war by the United States in particular. So, the Russian invasion is unpopular, but the approach of the West is also unpopular for not better looking after the interests of emerging economies and lower-income states, particularly on climate change, but also on economic development.

So, a whole host of different considerations come into play over Ukraine at the UNGA.

How do you think the UN could be best repurposed or reformed?  

It is terribly difficult, because if you open the UN Charter for one reason, you are opening it up to a host of demands from member states for other reforms. And remember that no reform of the Charter can happen without a 2/3 majority at the General Assembly, and that is an effective blocker, because you will always find more people opposing a particular reform that supporting it.

So, I think formal reform remains a bit of a dream in the circumstances of a polarized world. I want to divert discussions about UN reform into areas where the Secretary General has a competence without needing a vote from member states.

I am talking about improvement in methodologies, in the meritocracy of appointments, in the day-to-day workings of the UN. It looks unambitious on a large-scale basis, but just improving the competence of the UN and its agencies will ensure the relevance of the UN to people’s material interests in terms of human rights, refugees, food distribution, and children, and all the other things that the agencies look after.

So, I would prefer to concentrate reform energy into competence reforms, management reforms, rather than reforms of the Charter. 

This takes us to the question of the permanent members of the Security Council, and especially the US and the UK. You have experienced first-hand America’s ambivalence towards the UN. How much has this undercut the UN’s relevance?

I was frequently disappointed by the approach of the US to issues of policy at the UN. The US finds it very difficult as a nation to move beyond the primacy of its own domestic public opinion.

That perspective – that we have democracy at home, but we will not, as Americans, allow international democracy – is regarded around the world as an untenable position for the superpower. And I think it is more than untenable, it is against the US’s long-term interests to demand such national exceptionalism.

That exceptionalism is extremely unpopular, and the US’s extraterritorial reach is very unpopular. That loss of legitimacy around the world is affecting American interests, in the Middle East, over Afghanistan, over the reaction to the Russian invasion of Ukraine; America losing the argument with international public opinion has a material effect on American power, influence and interests.

Has America paid the price for asserting its exceptionalism? From your perspective, representing the UK, arguably the closest ally of the US and one with a permanent seat on the Security Council, how did you manage this?

Well, I had a long experience of that in my job in Baghdad. Yes, the UK normally supports the US in international forums because our interests coincide. In foreign policy, interests are more important than values, and sometimes you have to make compromises and shade your values in order to get results and to avoid conflict.

I found myself taking up the US’ arguments and trying to deliver them as the UK, because the US was more unpopular than the UK and we could act more subtly. The US was more unpopular than the UK because the UK – and a lot of the time France – tried a lot harder than other permanent members to work for the common interest of UN members.

There were times I argued publicly against the US at the Security Council because their logic was incompatible with a multilateral approach, over the International Criminal Court for instance, or over some approaches to the Middle East. I would take a different view, not just because I didn’t want to seem like a constant puppet of the US, but because I thought the logic that they were following was constraining for them, and the counter-productiveness washed off on us.

Here, I want to make an important point. You won’t get governments coming together to form a multilateral approach with all the compromises that this entails unless they are confident of their position at home. They can’t otherwise explain those compromises to their domestic public opinion. If governments are insecure at home, they won’t pursue a multilateral approach because of that insecurity.

The UK is seeking to define a new global role for itself. It has been one of the most influential states at the UN. Do you see this changing?

I don’t think the change of leader makes a terrific difference for the UK in the UN because there is cross-party parliamentary agreement that the UN is important.

I was disappointed that the UK did not take the UN more seriously at a political level except when it badly needed it at a particular moment. At an official level, there was plenty of support from London, but I don’t think that politicians ever gave much priority to the health of the UN.

Why America’s midterm elections matter for the world The World Today mhiggins.drupal 28 September 2022

The outcome could threaten the fight against climate change and the future of democracy itself, warns Leslie Vinjamuri.

As the November 8 midterm elections in the United States approach, the sense of urgency among the Democrats and Republicans is escalating. Early voter turnout is on track to surpass the 2018 midterms, when a record 122 million Americans voted ahead of election day. The midterms – when congressional, state and local seats are determined – used to pass almost unnoticed, especially outside the US.

Since 2018, though, they have become a matter of global concern. The 2022 midterm elections will be the first major elections in the US since the January 6 insurrection at the US Capitol. As such, they will be the first real litmus test for the continued relevance of Donald Trump and Trumpism.

For both parties, winning is more important than ever and it is not only a matter of policy. Today, a majority of members of both parties – 72 per cent of Republicans and 63 per cent of Democrats – see those belonging to the opposing party as immoral, according to a Pew Research Center poll in September, representing a significant rise since 2016.

In many states, the midterms will have a direct impact on the 2024 presidential elections, influencing who decides how voting takes place, how votes are counted and, especially, who controls the certification of election results.

Given this context, the stakes in elections are especially high. The winners will have the upper hand not only in defining many procedural rules but also the values that constitute the nation, such as on abortion rights, education and healthcare.

Republicans and Democrats align on most key foreign policy

Though foreign policy will bear a strong resemblance to its former self even if Republicans gain a majority in the House of Representatives, as many anticipate, the outcome of the elections on US global influence could impact efforts to combat climate change and to support the defence of Ukraine.

The Republican Party has become known for its embrace of climate deniers and the radicalized anti-immigrant rhetoric of many of its party’s most established figures. The failure to address rampant gun violence – and, instead, to defend a historically suspect interpretation of the right to bear arms – and the push for a national abortion ban mean that the party is imposing a version of America onto itself that is out-of-step with virtually all other rich democratic countries.

The choices voters make in November are likely to shape the values that come to the fore in the US, and with this the affinity that America’s closest partners feel for it.

Domestic policy is likely to be more heavily affected by the elections. A Republican majority in the House would stall progress on President Joe Biden’s domestic agenda and undercut his ability to raise the taxes he needs to pay for his spending plans. The drive to hold the former President Trump accountable will also be affected by the choices voters make in November.

A Republican majority in the House would spell an end to congressional investigations of Donald Trump. Instead, Republicans would use their electoral gains to launch their investigations into the Democrats.

One anti-Trump Republican on the January 6 committee investigating the storming of the Capitol building last year warned of a vengeful and obstructive turn against Biden from his Republican colleagues if they took the House. ‘They’re going to demand an impeachment vote on President Biden every week,’ said congressman Adam Kinzinger. It is under this shadow that the January 6 Committee is pressing ahead to finish its proceedings and publish its report.

On foreign policy, the parties are more aligned than divided on most of the key issues. Their differences are a matter of diplomacy, which matters, and degree. Support for Ukraine has had bipartisan backing, but in recent weeks the issue has become politicized. Some congressional Republicans are more hawkish than their Democratic counterparts in their ambition to support Ukraine. Yet, the congressman set to be Speaker of the House if Republicans gain a majority, Kevin McCarthy, is leading an effort to exercise far stronger oversight of any additional funds for Ukraine.

Ultimately, though, the president and his advisers maintain considerable control over foreign policy.  

Bipartisan support for a tougher stance on China was consolidated under Trump. He mobilized US voters by blaming China for the loss of manufacturing jobs. Later he held China responsible for the outbreak of Covid and for covering it up. China’s crackdown on democracy in Hong Kong, its assertiveness in the South China Seas, tensions over Taiwan and reports of human rights abuse against the Uighur in Xinjiang have cemented a bipartisan consensus on America’s China policy. But a more empowered Republican base looks likely to lead to a more ideologically driven anti-China rhetoric. 

The ground has also shifted over Russia. During President Trump’s tenure, Republican attitudes towards Vladimir Putin softened. But the Russian president’s aggressive invasion of Ukraine has entrenched American opposition to him and bolstered support for NATO.

Democrats and Republicans are also more aligned on policy towards Iran today than they were before Trump abandoned the Iran Deal negotiating the Middle Eastern country’s nuclear programme. On some contentious issues, such as immigration reform, progress is already stalled and that is unlikely to change.

US climate change policy may depend on the results

The upshot of all this is that the president is unlikely to make radical changes to US foreign policy regardless of the results of the midterms. The most important exception to this is climate change, which continues to be the unwanted stepchild of the Republican Party. Domestic infighting in the US would inevitably detract even further from the bold policy that is needed.

Republicans are unlikely to get the two-thirds majority needed to pass legislation that would undermine President Biden’s climate bill. Republican control of House committees, however, would add more politically driven oversight of any climate spending, including what has already been authorized by the Inflation Reduction Act. 

Policy convergence and clear executive authority cannot mitigate the sheer disruption that would be unleashed if the Republicans were to gain majorities in both the Senate and the House, however. For the rest of the world, and especially within Europe, this would send warning signs about the future trajectory of US foreign policy commitments and set off alarm bells that an ‘America First’ president might return to the White House after 2024.

A Republican majority in the House with a Democratic majority in the Senate would be disruptive, if less so, but would probably spell the end of business as usual, unsettling people, states and markets. All of this is unfolding at a time when many of the world’s rich democracies face internal divisions, rising inequality and populist challenges at home and so are ill-equipped to lead.

Democracies face a trust deficit. This is especially apparent in the US where only 43 per cent of Americans have trust in US institutions, according to the 2022 Edelman Trust Barometer.

Internal threats to democracy also unsettle the promise of US global leadership at a time when concerted and determined leadership is needed to maintain a strong and united response to Russia, and to tackle global food insecurity and the energy, climate and debt crises. In addition, the failure to lead a global response that can help developing countries is feeding a trust deficit between rich and poor countries.

The midterms will reveal a greater truth about the future of the Republican Party. Even more so, it will reveal the values held by the American electorate. In the run-up to the 2020 US presidential elections, Europeans frequently said that they would not give Americans a pass if they voted for Donald Trump twice – and indeed only 17 per cent of those in European Union countries would have cast a vote for Trump if they could.

A Republican midterm victory in both the Senate and the House of Representatives would have ripple effects across the Atlantic at a time when the threat from Russia has intensified.

The US is judged around the world for what it does, but also for who it is. The perception that democracy is failing in America creates a permissive environment for aspiring autocrats. At a time when democracy has been in decline around the world for more than 15 years, it is essential that the US fix its own democracy and that it demonstrates to the rest of the world that democracy can deliver.

The midterms will signal to the world what Americans value, sending a message about what it can expect from the US.

This article was updated on October 31, 2022 to reflect developments such as early voting turnout, and the impact of the midterms on the 2024 presidential election, support for Ukraine and climate change spending.

Why burning biomass is not zero-carbon Explainer Video NCapeling 17 October 2022

Short animation explaining why burning biomass produces more carbon dioxide per unit of energy generated than almost all fossil fuels.

The climate emergency requires countries to transition away from fossil fuels, but it is important to be careful about the alternative energy sources chosen.

In particular, concern is growing over the use of biomass for energy, which is generated when wood or other plant material is burnt to generate heat and electricity. Many governments treat biomass energy as zero-carbon at the point of combustion, and subsidize it in the same way as renewables such as solar or wind, resulting in a large increase in the use of biomass for energy in the UK and the European Union (EU) over the past 15 years.

The treatment of biomass as zero-carbon in policy frameworks rests on the argument that biomass emissions will be reabsorbed by forest growth, particularly from trees planted to replace those cut down to burn.

But growing trees to maturity takes many years and, depending on the feedstock used, biomass burning increases global warming for decades to centuries. This is called the ‘carbon payback period’ – the time it takes for carbon dioxide levels to return to what they would have been if biomass had not been used.

New research from Chatham House and the Woodwell Climate Research Center calculated the real climate impact of burning US wood pellets in the UK and EU. In 2019, according to this analysis, US-sourced pellets burned for energy in the UK were responsible for between 13 million and 16 million tonnes of carbon dioxide, equivalent to the annual greenhouse gas emissions from 6-7 million passenger vehicles.

But because biomass is treated as zero-carbon, almost none of these emissions were included in the UK’s national greenhouse gas reports. And the removal of forest carbon from US forests is not included accurately in US reports, either.

Why technology does not make easy wars Interview LJefferson 28 November 2022

Stephanie Carvin explains why technology does not overcome the challenges of war.

The invasion of Ukraine has demonstrated that many of the assumptions held about the role of technology in contemporary warfare are flawed. The lesson that technology cannot overcome the challenges of warfare is one that the West has also yet to learn, despite a series of failed interventions since the end of the Cold War.

In a wide-ranging conversation, Isabel Muttreja sat down with Stephanie Carvin to talk about her contribution to the September 2022 issue of International Affairs on ‘how not to war’. They discuss the US’ over-reliance on technology and why ‘easy wars’ become ‘forever wars.’

You argue in your article that the US overly relies on technology in war. When did this start?

I don’t necessarily think the US is exceptional. I think all states have tried in some ways to use technologies. One of the key arguments in the article is that the US is an enlightenment country, and part of the enlightenment is a belief in rationality and science and that you can better things through the application of science.

I think that there is this particular scientific approach or embracing of technology, in the American and in fact larger Western tradition on technology as a way to save lives. There is a strange humanitarian impulse that often underlies this use of technology.

We are seeing a quest to try and get perfect information. The idea is that if you have perfect information, you are going to be able to dominate the battlefield, and that’s proven itself to be false. I’m not even sure you can ever get perfect information.

But it underlines this modern approach, that if you can have all the information that’s out there, crunch it into some kind of algorithm, that you can then target discriminately, proportionately, reduce the level of casualties, and reduce the level of unnecessary damage. And that’s a kind of liberal tradition. You are trying to have your cake and eat it too.

You talk about the US being an ultimately liberal state, but they have been involved in a lot of wars over the last 10–20 years. Is that a contradiction?

I hope it is. But I think it goes back to the enlightenment nature of the United States, which is that the US sees itself as a shining city on a hill that has to protect itself at all costs. Liberals abhor tyranny, and they abhor unnecessary deaths. But I think that the idea is that if you threaten us, we see ourselves as embodying these values, therefore, we have to protect ourselves.

There’s a tendency to not really recognize the kind of insurgencies that we’ve seen in Iraq and Afghanistan, or even Vietnam, as war. We don’t really see that as a kind of armed conflict, even though, arguably, that has been the dominant mode of conflict for some time. They even used to call it ‘military operations other than warfare’. We tend to still think of war as great power competition or as the Second World War.

My first book was on prisoners of war in the American tradition. What often determined the treatment of people as prisoners of war was if the United States recognized their form of warfare. There’s a racial element here too that I don’t want to dismiss.

So, for example, the US war in the Philippines at the start of the 20th century: They went in, won a very quick victory over the Spanish and effectively took over the Philippines. And then they had a long insurgency for two years with the native Filipinos who didn’t want US domination. While they gave the Spanish all the prisoner of war rights, they didn’t give them to the Filipinos.

This is because they recognized the form of conflict that the Spanish engaged in, but the Indigenous way of warfare was not recognized. The West has struggled to culturally understand the way other people fight. And that’s when the laws of war conventions have broken down between, say, the United States, the West, and other states.

You talk in your article about the US entering ‘easy wars’ and ending up with ‘forever wars’ – what does this mean?

There’s an allure to this high-tech version of warfare, that it can solve a lot of problems, but it’s an illusion. It is ultimately a bit of a false promise.

The idea that machines are going to replace humans is fundamentally untrue. We are seeing this to a certain extent right now, even in the Russia/Ukraine war. This is very much a battle of machines and soldiers. One of the themes of this issue of International Affairs is hubris. The idea that things that appear to be quick wins often tend to be long-term losses. And that’s exactly what this article is talking about.

‘Forever wars’ is not my favourite term, but it’s this concept that what was promised to be an easy war, a high technology-driven conflict, where you can go in, use some surgically precise weapons, take care of the problem, eliminate your opponent and then extract yourself from a situation, has actually turned into a quagmire.

The limits of technology become apparent within a few months as well as the fact of the messy business of state-building, or the fact that insurgencies and political movements don’t just disintegrate at the show of some high-tech, sophisticated weaponry. It just tends to mean that these wars do go on for a long time, and you have to eventually extricate yourself, but there’s no clean way to do this. We saw this of course with Afghanistan, and to a large extent Iraq.

We get distracted by the shiny object. We see this promise, we see this vision of a kind of warfare that for some may have great appeal.  There are new super weapons, whether it be cyber information warfare or artificial intelligence. Everyone wants to be ahead of the curve, right?

Are these lessons on technology and ‘easy wars’ applicable to other countries?

I think what we’ve learned about the Russian military is that there’s a lot more at the heart of it. Part of the problem Russia is experiencing is that its capabilities were not what it thought they were. It’s clear that Vladimir Putin was enamoured with a lot of the ideas, like that the Russian military was increasingly high-tech and that they had these hypersonic missiles.

They also had very powerful cyber weapons amongst other things. Putin, too, seems to have been caught up in this idea that he could have had a 72-hour special military operation, which would have taken Kyiv. Clearly, that hasn’t happened. Once again, we see the underestimation of the human factor.

Sean R. Collins
Mar 1, 2007; 6:439-450
Research

Yu Xue
Sep 1, 2008; 7:1598-1608
Research

Claudio P. Albuquerque
Jul 1, 2008; 7:1389-1396
Research

Staphylococcus aureus adhesion to the host's skin and mucosae enables asymptomatic colonization and the establishment of infection. This process is facilitated by cell wall-anchored adhesins that bind to host ligands. Therapeutics targeting this process could provide significant clinical benefits; however, the development of anti-adhesives requires an in-depth knowledge of adhesion-associated factors and an assay amenable to high-throughput applications. Here, we describe the development of a sensitive and robust whole cell assay to enable the large-scale profiling of S. aureus adhesion to host ligands. To validate the assay, and to gain insight into cellular factors contributing to adhesion, we profiled a sequence-defined S. aureus transposon mutant library, identifying mutants with attenuated adhesion to human-derived fibronectin, keratin, and fibrinogen. Our screening approach was validated by the identification of known adhesion-related proteins, such as the housekeeping sortase responsible for covalently linking adhesins to the cell wall. In addition, we also identified genetic loci that could represent undescribed anti-adhesive targets. To compare and contrast the genetic requirements of adhesion to each host ligand, we generated a S. aureus Genetic Adhesion Network, which identified a core gene set involved in adhesion to all three host ligands, and unique genetic signatures. In summary, this assay will enable high-throughput chemical screens to identify anti-adhesives and our findings provide insight into the target space of such an approach.

Leukocidin ED (LukED) is a pore-forming toxin produced by Staphylococcus aureus, which lyses host cells and promotes virulence of the bacteria. LukED enables S. aureus to acquire iron by lysing erythrocytes, which depends on targeting the host receptor Duffy antigen receptor for chemokines (DARC). The toxin also targets DARC on the endothelium, contributing to the lethality observed during bloodstream infection in mice. LukED is comprised of two monomers: LukE and LukD. LukE binds to DARC and facilitates hemolysis, but the closely related Panton–Valentine leukocidin S (LukS-PV) does not bind to DARC and is not hemolytic. The interaction of LukE with DARC and the role this plays in hemolysis are incompletely characterized. To determine the domain(s) of LukE that are critical for DARC binding, we studied the hemolytic function of LukE–LukS-PV chimeras, in which areas of sequence divergence (divergence regions, or DRs) were swapped between the toxins. We found that two regions of LukE's rim domain contribute to hemolysis, namely residues 57–75 (DR1) and residues 182–196 (DR4). Interestingly, LukE DR1 is sufficient to render LukS-PV capable of DARC binding and hemolysis. Further, LukE, by binding DARC through DR1, promotes the recruitment of LukD to erythrocytes, likely by facilitating LukED oligomer formation. Finally, we show that LukE targets murine Darc through DR1 in vivo to cause host lethality. These findings expand our biochemical understanding of the LukE–DARC interaction and the role that this toxin-receptor pair plays in S. aureus pathophysiology.

What is COP29 and why is it important? Explainer jon.wallace 17 October 2024

The COP29 summit will see negotiators try to agree how to finance the climate action the world urgently needs.

COP29, the 29th UN annual conference on climate change, takes place in Baku, Azerbaijan from 11–22 November 2024.

Every COP conference is an important opportunity for international collaboration on climate change. COP29 will have a particular focus on how to make finance available to developing countries for climate action.

COP29 is already contentious, because Azerbaijan’s economy is highly dependent on fossil fuels, the main cause of climate change. That means that Azerbaijan’s government, which will preside over the summit, has a strong incentive to avoid rapid multilateral progress towards phasing out fossil fuels. 

It’s urgent that progress is made at COP29. Climate action lags far behind both what has been promised by countries, and what scientists agree is needed. The impacts of climate change are also rapidly escalating, while backlash against government policies to reduce fossil fuel use is being seen in many places around the world. 

What is a ‘COP’?

The ‘Conference of the Parties’ or ‘COP’ is an annual event that brings together the governments which have signed up to environmental action under the United Nations (UN). 

Governments or ‘parties’ attend the climate change COP if they are part of the United Nations Framework Convention on Climate Change, or the international environmental treaties the Kyoto Protocol (1997) or the landmark and legally binding Paris Agreement (2015).

World leaders, ministers, and negotiators convene at the COP to negotiate and rubber stamp plans to jointly address climate change and its impacts. 

Civil society, businesses, international organizations and the media normally ‘observe’ proceedings to bring transparency, accountability and wider perspectives to the process.

‘Mission 1.5°C’ 

COP28, the 2023 climate conference held in Dubai, was the first of three consecutive COP summits intended to ‘reset’ global climate action – what the UN calls the ‘Roadmap to Mission 1.5°C’ – the ambition to keep global temperatures from rising more than 1.5°C above pre-industrial levels. 

To support continuity and progress across the three COPs, the UAE (COP28), Azerbaijan (COP29) and Brazil (COP30) have formed a COP presidential ‘Troika’ or group of three.

In 2023 the first ‘global stocktake’ of international action to address climate change indicated that the world was far off track for targets set by the Paris Agreement. The ‘UAE Consensus’ agreement, which formed the main output of COP28, set out how parties should respond. 

COP29, the second of the three COPs, is intended to get the finance in place to enable this response. COP30, to be held in Brazil in 2025, will then try to agree how a new round of nationally determined climate plans or ‘contributions’ to global climate efforts (NDCs) should be put into action.

COP29 will be the third consecutive COP held in an authoritarian state, and the second consecutive COP hosted by a petro-state. Taken together, the ‘Troika’ hosts make up the world’s 4th largest oil producer, after the United States, Russia and Saudi Arabia. 

This presents both an opportunity and a risk: the countries are well placed to understand and tackle the core issue of fossil fuel extraction and use. But they also have strong incentives to stall, distract and deflect the negotiations away from phasing out fossil fuels.

Key issues at COP29

Finance – money on the table, and a New Collective Quantified Goal

COP29 has a remit to secure funding for a ‘course correction’ on global climate action. Countries’ revised climate plans (NDCs) are due in February 2025. For developing countries to deliver new ambitious NDCs, however, COP29 must first make clear what finance will be available to help them.

Climate finance is one of the thorniest issues in the negotiations. The previous climate finance goal, of $100 billion per year from developed to developing countries between 2020-25, was symbolic (being a fraction of the sum actually needed) and contentious (developed countries did not meet the target until 2022, and then only with accusations of double-counting). 

A New Collective Quantified Goal (NCQGs), to be agreed at COP29, will need to reflect developing countries’ needs and priorities. Estimates vary, but it is accepted that the scale will be trillions, not billions. To bridge this enormous gap, private finance will need to be mobilized and broader reforms made to global financial architecture. Issues such as subsidies, fossil fuel profits and ‘solidarity levies’ will also need to be on the table. 

Little progress has been made in the run-up to COP29. Parties disagree on who should pay, how much should be paid, what forms the funding should take (loans or grants) and how the funds should be accessed. 

Also up for debate is how funds should be directed – towards mitigating the impacts of climate change (preventing climate change becoming worse), adapting to its effects, or supporting countries to manage loss and damage (climate impacts that have already happened or cannot be avoided).  

In discussions of the NCQG to date, developed countries have consistently called for higher income developing countries, such as China and India, to contribute. 

Such countries have pushed back forcefully against this. For a new goal to be agreed, such divisions will need to be resolved.

Enhanced transparency?

COPs act as showcases for international agreement, but many processes rumble on behind the scenes. COP29 will be an important moment for transparency under the Paris Agreement, as the Parties’ first Biennial Enhanced Transparency Reports (BTRs) are due in December 2024. 

BTRs are an important tool in the Paris Agreement, requiring countries to provide a snapshot of their progress in cutting emissions, setting climate policies, and providing resources for national and international climate action. 

BTRs are meant to build the evidence base for strong NDCs, build trust and promote ambitious climate action. They are also meant to help developing countries showcase achievements and attract climate finance. 

The COP29 presidency has launched the Baku Global Climate Transparency Platform to encourage participation, and support countries in finalising their BTRs. The Platform is meant to provide space for collaboration between government, NGO and private sector stakeholders. 

This is one of the areas in which COP29 host, Azerbaijan, runs into criticism. The country ranks highly on indexes of corruption, and has repeatedly restricted space for NGOs and wider civil society actors through a series of escalating government crackdowns, including imprisonment of environmental activists, in the run-up to the conference.

The killing of Yahya Sinwar won’t change the course of the Gaza war Expert comment jon.wallace 18 October 2024

Israel has larger ambitions for Gaza beyond the Hamas leader’s death and the armed group is still asserting its influence despite its degradation.

After a year of being hunted as one of the most wanted men in the Middle East, Hamas leader Yahya Sinwar was killed by Israeli forces on 17 October, in the city of Rafah in the Gaza Strip.

Hailing from Khan Younis refugee camp, Sinwar was imprisoned in Israel in 1989 for the abduction and killing of two Israeli soldiers. He was freed alongside 1,000 other prisoners during a major 2011 exchange, gradually building a notorious reputation as an intelligent and ruthless political figure.

As Hamas’ Gaza chief since 2017, Sinwar oversaw the consolidation of the movement’s authoritarian rule in the Strip and co-directed its military confrontations with Israel. He is regarded as a chief architect of Hamas’ deadly assault of 7 October, which killed 1,200 Israelis, mostly civilians, in southern communities.

Many world leaders and pundits were quick to embrace Sinwar’s death as an opportunity to press for a ceasefire in Gaza and secure the release of roughly 100 Israeli hostages still held by Hamas and other groups. It is too early to know the full repercussions of this development, but these hopes are largely misplaced – and reflect a reluctance to confront the larger obstacles and motivations that are preventing an end to the war.

Morale amid aimlessness

For Israelis, Sinwar’s death is undoubtedly a morale booster. It is a symbolic victory over a man deemed responsible for the 7 October massacres, and a tactical success that rids Israel of a formidable foe – one who was also an invested ally of Iran and the Axis of Resistance. Israeli politicians and generals will relish the news as another step towards restoring their reputations after their failure to prevent the 7 October attack.

Yet contrary to some wishful thinking, Sinwar’s death is unlikely to change Israel’s calculus in Gaza. Prime Minister Benjamin Netanyahu, whose popularity has been slowly recovering in recent months, described the news as ‘the beginning of the end’ but warned that ‘the task before us is not yet complete’.

The intensification of Israel’s war with Hezbollah – including a ground invasion of Lebanon, airstrikes across the country, and the displacement of over 1 million people – has shifted Israelis’ focus away from Gaza over the past month, especially as Hezbollah rockets disrupt life in Israel’s central cities as well as the north.

During that time, the Israeli army has begun a major offensive in the northern part of the Strip, implementing what appears to be the first part of the so-called ‘General’s Plan’ – an operation to besiege and force out roughly 300,000 Palestinian civilians who remain in the area. It could also pave the way for Israel’s absorption of part of the territory. (Israel’s military has denied pursuing the plan).

As such, the Israeli government has relegated the importance of the hostages. Netanyahu is eager to divert the public’s frustration with his months-long sabotage of a deal, while the military – despite its clashes with the prime minister – is seizing the chance to redraw Israel’s geopolitical environment. Even if a deal is struck in the wake of Sinwar’s death, Israel’s bombing and carving-up of Gaza and south Lebanon will likely continue.

Dynamic but degraded

For Hamas, the killing of its fiery politburo chief – just over two months after the assassination of his predecessor Ismail Haniyeh in Tehran – is certainly a blow to its leadership and internal stability. But the Islamist movement’s dynamism has not changed. Hamas’s decision-making processes are not pegged to specific individuals, and other figures, whether in Gaza or in exile, will be rotated into senior positions.

It is unclear whether Hamas will promote a leader who shares Sinwar’s hardline orientation, or drift back to a more moderate figure similar to Haniyeh. Regardless, Hamas’s core demands in any ceasefire and hostage negotiations are unlikely to shift significantly. The movement is still seeking a permanent end to the war, and Israel is still refusing to grant it.

Hamas’s military capacity has been severely degraded by the Israeli offensive, not least by the fact that much of Gaza has been made unliveable and ungovernable. But the movement is far from destroyed. Its militants continue to fight a guerrilla war to undermine Israel’s foothold in Gaza, while trying to reassert Hamas control over public order, the war economy, and the distribution of aid.

Moreover, as the history of movements like Hamas and Hezbollah shows, the devastating nature of Israel’s campaigns and occupations are planting the seeds for further militancy among Palestinians, whether organized under Hamas or smaller, decentralized groups. The war has brutally decimated Gaza’s society, and the notion that killing figures like Sinwar will dissuade future recruits to fight Israel is fanciful.

On the Palestinian political level, Hamas and Fatah are still in talks about forming a unity agreement, though negotiations have so far remained fruitless. Even if a pact is made, Israel’s relentless targeting of Hamas personnel, the perception of the Fatah-led Palestinian Authority (PA) as feckless collaborators, and Palestinians’ feeling of total abandonment raise doubts about the parties’ ability to regain popular support or legitimacy – whether to lead an interim government or the wider national movement.

The gravest threat

The killing of Sinwar is a significant marker in the Gaza war, with important ramifications for the movement he led and the conflict he seismically shaped. Israel will portray his death as validation of its aggressive military approach, and a further step in the erosion of Iran’s regional influence – an agenda the US has actively supported.

Independent Thinking: Why is North Korea sending troops to Russia? Audio john.pollock 31 October 2024

Orysia Lutsevych, Edward Howell and Yossi Mekelberg discuss North Korean troops fighting with Russian forces against Ukraine, as well as the issue of military conscription.

On this episode

North Korea is sending troops to Russia, but what is Kim Jong-Un hoping to get from Vladmir Putin for this military assistance against Ukraine? The panel also discuss the role military conscription plays in Israel, South Korea and Ukraine’s defence.

Bronwen Maddox is joined by Edward Howell, the Korea Foundation fellow at Chatham House, Orysia Lutsevych, the head of our Ukraine Forum and Yossi Mekelberg, a senior consulting fellow with our Middle East and North Africa Programme.

About Independent Thinking

Independent Thinking is a weekly international affairs podcast hosted by our director Bronwen Maddox, in conversation with leading policymakers, journalists, and Chatham House experts providing insight on the latest international issues.

More ways to listen: Apple Podcasts, Spotify.

Margrit Schwarz
Sep 1, 1998; 39:1833-1843
Articles

Mohamad Navab
Jun 1, 2004; 45:993-1007
Thematic Reviews

Vitali Matyash
May 1, 2008; 49:1137-1146
Methods

William R. Morrison
Oct 1, 1964; 5:600-608
Articles

CJ Fielding
Feb 1, 1995; 36:211-228
Reviews

A seat at the table – why inclusivity matters in global governance 10 May 2021 — 1:30PM TO 3:00PM Anonymous (not verified) 22 April 2021 Online

Exploring the changing dynamics of global cooperation and the role inclusivity can play in building collaborative action.

Please click on the below link to confirm your participation and receive your individual joining details from Zoom for this event. You will receive a confirmation email from Zoom, which contains the option to add the event to your calendar if you so wish.

The scale of today’s global challenges demand collaborative and coordinated action. But deepening geopolitical competition is threatening multilateralism while growing inequality and social tensions continue to undermine public confidence in the ability of international institutions to deliver.

Into this challenging environment, add the complexity and sheer pace of many global challenges such as the climate crisis and the proliferation of new technologies – issues that cannot be addressed effectively by governments alone.

• How do global institutions and mechanisms need to adapt to address the demands for a fairer distribution of power between states and to engage the diverse set of actors essential today for effective solutions?
• What can be learnt from existing initiatives that bring together governments, civil society, private sector, cities, next generation leaders and other stakeholders?
• And what are the political obstacles to greater inclusivity?

This event supports the launch of a synthesis paper from Chatham House’s Inclusive Governance Initiative.

Why the next generation is key to protecting human rights Expert comment LToremark 23 June 2021

Strengthening youth participation in public affairs is essential to building inclusive and democratic societies that respect human rights.

Young people have always been drivers of social and economic reform, and today’s global youth population is more numerous and interconnected than ever before. While they have been at the forefront of civic rights movements in recent years, young people are largely excluded from discussions around human rights norms and how to monitor their protection and defence.

Young people are consistently underrepresented in intergovernmental mechanisms and national dialogues, which not only squanders their potential to contribute to effective solutions but also risks disengagement and disillusionment with multilateralism more broadly, at a time when many are already warning of the fraying of the international liberal order. Although there are actors and initiatives working to lift barriers to youth participation in governance – such as the UN Secretary-General’s Envoy on Youth, Jayathma Wickramanayake, or the UN 2016 Not Too Young To Run campaign – these efforts tend to fall short in effecting real change and rarely translate into institutionalized procedures.

While ‘the youth’ is a heterogenous group, comprising different ages, ethnicities, national identities and interests, their participation in realizing human rights is essential to addressing the current challenges and possibilities of human rights for future generations. This will help foster more effective solutions to rights-related challenges, re-build trust in the international human rights framework among younger demographics and broaden and deepen commitments to human rights across generations.

Human rights policies and the online environment

Young people tend to be more technologically literate than their predecessors and also represent the majority of internet users and social media consumers in many countries. They can therefore play a key role in innovating and imagining rights-based solutions to emerging problems for the human rights framework, such as illegitimate collection of data by governments and companies, microtargeting by online platforms, and the sharing of harmful content online. In many cases, international human rights practices have failed to keep pace with these changes and the challenges they bring.

Younger demographics may also approach these novel human rights issues from different starting points. For example, a UK study found that 30 per cent of 18-24 year-olds were ‘unconcerned’ about data privacy compared with only 12 per cent of those aged 55-64, and it has been shown that younger people tend to be more discerning of fake news compared to older generations. There may be a need for human rights institutions and practitioners to acknowledge and bridge these gaps in perspective and understanding to ensure long-term support for proposed solutions.

International cooperation for human rights protection

It has been suggested that young people have reaped the benefits of previous human rights-based policy reforms and have a strong sense of what rights they are entitled to and why these need to be protected through an international framework. Young people are also generally more supportive of multilateralism compared to their older counterparts, as demonstrated by a 2020 survey by Pew Research Center on global attitudes, which showed that 72 per cent of respondents aged 18-29 stated they have a favourable view of the UN, compared with 58 per cent of respondents aged 50 and older.

At a recent Chatham House workshop, young participants from countries as diverse as Lebanon, Kenya and the United States expressed concern that growing hostility towards globalization threatens to undo progress in human rights standards and multilateralism more broadly, progress that they have seen and benefitted from. The rise of nationalist and populist parties has also seen countries shift their attention inwards, as evidenced by former president Trump’s decision to withdraw the US from the Paris Agreement on climate change, and threats by Brazil’s president, Jair Bolsonaro, to follow suit.

Engaging more actively with younger individuals on global human rights reform will help ensure the long-term relevance of multilateral cooperation as well as domestic buy-in of human rights commitments.

Awareness of the interconnectivity of global problems

Young people’s proficiency on online platforms has enabled greater coordination and knowledge sharing without geographical constraints, allowing young activists – like Greta Thunberg – to inspire global movements and foster online discussions about intersectional solutions to modern-day challenges.

This intersectional and transnational lens will be a vital component of building solutions to politically or historically complex issues and can be leveraged to foster better understanding of competing human rights claims relating to issues such as land re-distribution in South Africa or limitations on freedom of movement during the COVID-19 pandemic. These democratic forums and platforms will ultimately help build a global community committed to and engaged with human rights.

Capturing the next generation’s potential

With these concerns and areas of potential in mind, how can human rights institutions and mechanisms create more meaningful avenues for youth input? 

Recent Chatham House research has suggested that multilateral institutions’ efforts to engage youth has often taken the form of ‘superficial listening’, for example inviting a high-profile youth actor to a one-off event or appointing youth delegates who are not able to participate in formal discussions or mainstream governance forums. While encouraging youth participation in meetings focused on human rights can lead to positive change, tokenism can discourage future engagement and dilute the effectiveness of the forums in question.

Capitalizing on the potential of the next generation can be achieved through integrating youth councils and advisers into national and international human rights policy processes, as well as human rights institutions. A few replicable models are already operational, such as the Y7 and the Y20 delegations – the official youth engagement groups for the G7 and G20 – that advance evidence-based proposals to world leaders ahead of the G7 and G20 summits.

At the domestic level, grassroots youth-led movements can help bridge the gap between local constituencies and international policymakers, with youth activists on the ground helping to implement human rights standards and fighting against the spread of misinformation. Strong local networks and civic spaces are essential for pushing back against human rights abuses, and youth activists should be mobilized to connect the efforts of domestic and international bodies to the real issues on the ground; for example, canvassing grassroots youth networks on domestic and traditional customs before implementing development agendas around women’s rights.

As well as providing insertion points for youth policy actors, human rights institutions must communicate their goals more effectively to younger generations and promote intergenerational and inclusive dialogue, for example by holding virtual consultations that  give access to individuals from different backgrounds. Similarly, they should ask young people about their priorities for human rights reform using regular and accessible surveys or by sharing information on online platforms regularly used by this demographic. This will ensure lasting buy-in from the next generation, essential for the relevance and sustainability of the human rights framework in the years to come.

This piece draws upon insights gathered at a workshop hosted by Chatham House in March 2021, which brought together the Institute’s networks of next generation groups including representatives of the QEII Academy Ambassadors, the Panel of Young Advisers, and the Common Futures Conversations community, as well as young members from the South African Institute of International Affairs.

Why the private sector should protect civic society Explainer Video NCapeling 10 December 2021

A short animation explaining the crucial role that the private sector can play in protecting and defending civic space.

This video explainer introduces a synthesis paper which analyses how the private sector can support the protection of civic society space.

The private sector is in a unique position to work with civil society organizations to uphold and defend civic freedoms and support sustainable and profitable business environments. Companies have the capacity, resources and expertise to enhance the protection of civic space.

By doing so, this helps create a society in which fundamental rights and the rule of law are respected and exercised by governments, private citizens, and all organizations which, in turn, is critical to a sustainable and profitable business environment.  

For more information, download the report.

Mammalian circadian clocks are driven by transcription/translation feedback loops composed of positive transcriptional activators (BMAL1 and CLOCK) and negative repressors (CRYPTOCHROMEs (CRYs) and PERIODs (PERs)). CRYs, in complex with PERs, bind to the BMAL1/CLOCK complex and repress E-box–driven transcription of clock-associated genes. There are two individual CRYs, with CRY1 exhibiting higher affinity to the BMAL1/CLOCK complex than CRY2. It is known that this differential binding is regulated by a dynamic serine-rich loop adjacent to the secondary pocket of both CRYs, but the underlying features controlling loop dynamics are not known. Here we report that allosteric regulation of the serine-rich loop is mediated by Arg-293 of CRY1, identified as a rare CRY1 SNP in the Ensembl and 1000 Genomes databases. The p.Arg293His CRY1 variant caused a shortened circadian period in a Cry1−/−Cry2−/− double knockout mouse embryonic fibroblast cell line. Moreover, the variant displayed reduced repressor activity on BMAL1/CLOCK driven transcription, which is explained by reduced affinity to BMAL1/CLOCK in the absence of PER2 compared with CRY1. Molecular dynamics simulations revealed that the p.Arg293His CRY1 variant altered a communication pathway between Arg-293 and the serine loop by reducing its dynamicity. Collectively, this study provides direct evidence that allosterism in CRY1 is critical for the regulation of circadian rhythm.

CARM1 is a protein arginine methyltransferase (PRMT) that acts as a coactivator in a number of transcriptional programs. CARM1 orchestrates this coactivator activity in part by depositing the H3R17me2a histone mark in the vicinity of gene promoters that it regulates. However, the gross levels of H3R17me2a in CARM1 KO mice did not significantly decrease, indicating that other PRMT(s) may compensate for this loss. We thus performed a screen of type I PRMTs, which revealed that PRMT6 can also deposit the H3R17me2a mark in vitro. CARM1 knockout mice are perinatally lethal and display a reduced fetal size, whereas PRMT6 null mice are viable, which permits the generation of double knockouts. Embryos that are null for both CARM1 and PRMT6 are noticeably smaller than CARM1 null embryos, providing in vivo evidence of redundancy. Mouse embryonic fibroblasts (MEFs) from the double knockout embryos display an absence of the H3R17me2a mark during mitosis and increased signs of DNA damage. Moreover, using the combination of CARM1 and PRMT6 inhibitors suppresses the cell proliferation of WT MEFs, suggesting a synergistic effect between CARM1 and PRMT6 inhibitions. These studies provide direct evidence that PRMT6 also deposits the H3R17me2a mark and acts redundantly with CARM1.

The molecular mechanisms of reduced frataxin (FXN) expression in Friedreich's ataxia (FRDA) are linked to epigenetic modification of the FXN locus caused by the disease-associated GAA expansion. Here, we identify that SUV4-20 histone methyltransferases, specifically SUV4-20 H1, play an important role in the regulation of FXN expression and represent a novel therapeutic target. Using a human FXN–GAA–Luciferase repeat expansion genomic DNA reporter model of FRDA, we screened the Structural Genomics Consortium epigenetic probe collection. We found that pharmacological inhibition of the SUV4-20 methyltransferases by the tool compound A-196 increased the expression of FXN by ∼1.5-fold in the reporter cell line. In several FRDA cell lines and patient-derived primary peripheral blood mononuclear cells, A-196 increased FXN expression by up to 2-fold, an effect not seen in WT cells. SUV4-20 inhibition was accompanied by a reduction in H4K20me2 and H4K20me3 and an increase in H4K20me1, but only modest (1.4–7.8%) perturbation in genome-wide expression was observed. Finally, based on the structural activity relationship and crystal structure of A-196, novel small molecule A-196 analogs were synthesized and shown to give a 20-fold increase in potency for increasing FXN expression. Overall, our results suggest that histone methylation is important in the regulation of FXN expression and highlight SUV4-20 H1 as a potential novel therapeutic target for FRDA.

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Histone recognition by “reader” modules serves as a fundamental mechanism in epigenetic regulation. Previous studies have shown that Spindlin1 is a reader of histone H3K4me3 as well as “K4me3-R8me2a” and promotes transcription of rDNA or Wnt/TCF4 target genes. Here we show that Spindlin1 also acts as a potent reader of histone H3 “K4me3-K9me3/2” bivalent methylation pattern. Calorimetric titration revealed a binding affinity of 16 nm between Spindlin1 and H3 “K4me3-K9me3” peptide, which is one to three orders of magnitude stronger than most other histone readout events at peptide level. Structural studies revealed concurrent recognition of H3K4me3 and H3K9me3/2 by aromatic pockets 2 and 1 of Spindlin1, respectively. Epigenomic profiling studies showed that Spindlin1 colocalizes with both H3K4me3 and H3K9me3 peaks in a subset of genes enriched in biological processes of transcription and its regulation. Moreover, the distribution of Spindlin1 peaks is primarily associated with H3K4me3 but not H3K9me3, which suggests that Spindlin1 is a downstream effector of H3K4me3 generated in heterochromatic regions. Collectively, our work calls attention to an intriguing function of Spindlin1 as a potent H3 “K4me3-K9me3/2” bivalent mark reader, thereby balancing gene expression and silencing in H3K9me3/2-enriched regions.

Phospholipase Cε (PLCε) is activated downstream of G protein–coupled receptors and receptor tyrosine kinases through direct interactions with small GTPases, including Rap1A and Ras. Although Ras has been reported to allosterically activate the lipase, it is not known whether Rap1A has the same ability or what its molecular mechanism might be. Rap1A activates PLCε in response to the stimulation of β-adrenergic receptors, translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLCε was proposed to the primary binding site for Rap1A, we first confirmed using purified proteins that the RA2 domain is indeed essential for activation by Rap1A. However, we also showed that the PLCε pleckstrin homology (PH) domain and first two EF hands (EF1/2) are required for Rap1A activation and identified hydrophobic residues on the surface of the RA2 domain that are also necessary. Small-angle X-ray scattering showed that Rap1A binding induces and stabilizes discrete conformational states in PLCε variants that can be activated by the GTPase. These data, together with the recent structure of a catalytically active fragment of PLCε, provide the first evidence that Rap1A, and by extension Ras, allosterically activate the lipase by promoting and stabilizing interactions between the RA2 domain and the PLCε core.

In animals, the response to chronic hypoxia is mediated by prolyl hydroxylases (PHDs) that regulate the levels of hypoxia-inducible transcription factor α (HIFα). PHD homologues exist in other types of eukaryotes and prokaryotes where they act on non HIF substrates. To gain insight into the factors underlying different PHD substrates and properties, we carried out biochemical and biophysical studies on PHD homologues from the cellular slime mold, Dictyostelium discoideum, and the protozoan parasite, Toxoplasma gondii, both lacking HIF. The respective prolyl-hydroxylases (DdPhyA and TgPhyA) catalyze prolyl-hydroxylation of S-phase kinase-associated protein 1 (Skp1), a reaction enabling adaptation to different dioxygen availability. Assays with full-length Skp1 substrates reveal substantial differences in the kinetic properties of DdPhyA and TgPhyA, both with respect to each other and compared with human PHD2; consistent with cellular studies, TgPhyA is more active at low dioxygen concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate and DdSkp1 is a TgPhyA substrate. No cross-reactivity was detected between DdPhyA/TgPhyA substrates and human PHD2. The human Skp1 E147P variant is a DdPhyA and TgPhyA substrate, suggesting some retention of ancestral interactions. Crystallographic analysis of DdPhyA enables comparisons with homologues from humans, Trichoplax adhaerens, and prokaryotes, informing on differences in mobile elements involved in substrate binding and catalysis. In DdPhyA, two mobile loops that enclose substrates in the PHDs are conserved, but the C-terminal helix of the PHDs is strikingly absent. The combined results support the proposal that PHD homologues have evolved kinetic and structural features suited to their specific sensing roles.

DNA polymerases are today used throughout scientific research, biotechnology, and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the “performance specifications” needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.

Rhodopsin is a canonical class A photosensitive G protein–coupled receptor (GPCR), yet relatively few pharmaceutical agents targeting this visual receptor have been identified, in part due to the unique characteristics of its light-sensitive, covalently bound retinal ligands. Rhodopsin becomes activated when light isomerizes 11-cis-retinal into an agonist, all-trans-retinal (ATR), which enables the receptor to activate its G protein. We have previously demonstrated that, despite being covalently bound, ATR can display properties of equilibrium binding, yet how this is accomplished is unknown. Here, we describe a new approach for both identifying compounds that can activate and attenuate rhodopsin and testing the hypothesis that opsin binds retinal in equilibrium. Our method uses opsin-based fluorescent sensors, which directly report the formation of active receptor conformations by detecting the binding of G protein or arrestin fragments that have been fused onto the receptor's C terminus. We show that these biosensors can be used to monitor equilibrium binding of the agonist, ATR, as well as the noncovalent binding of β-ionone, an antagonist for G protein activation. Finally, we use these novel biosensors to observe ATR release from an activated, unlabeled receptor and its subsequent transfer to the sensor in real time. Taken together, these data support the retinal equilibrium binding hypothesis. The approach we describe should prove directly translatable to other GPCRs, providing a new tool for ligand discovery and mutant characterization.

Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme and transcriptional coactivator and is critical for tumor metabolism. In cancer cells, native tetrameric PKM2 is phosphorylated or acetylated, which initiates a switch to a dimeric/monomeric form that translocates into the nucleus, causing oncogene transcription. However, it is not known how these post-translational modifications (PTMs) disrupt the oligomeric state of PKM2. We explored this question via crystallographic and biophysical analyses of PKM2 mutants containing residues that mimic phosphorylation and acetylation. We find that the PTMs elicit major structural reorganization of the fructose 1,6-bisphosphate (FBP), an allosteric activator, binding site, impacting the interaction with FBP and causing a disruption in oligomerization. To gain insight into how these modifications might cause unique outcomes in cancer cells, we examined the impact of increasing the intracellular pH (pHi) from ∼7.1 (in normal cells) to ∼7.5 (in cancer cells). Biochemical studies of WT PKM2 (wtPKM2) and the two mimetic variants demonstrated that the activity decreases as the pH is increased from 7.0 to 8.0, and wtPKM2 is optimally active and amenable to FBP-mediated allosteric regulation at pHi 7.5. However, the PTM mimetics exist as a mixture of tetramer and dimer, indicating that physiologically dimeric fraction is important and might be necessary for the modified PKM2 to translocate into the nucleus. Thus, our findings provide insight into how PTMs and pH regulate PKM2 and offer a broader understanding of its intricate allosteric regulation mechanism by phosphorylation or acetylation.

Defining discontinuous antigenic epitopes remains a substantial challenge, as exemplified by the case of lipid transfer polyproteins, which are common pollen allergens. Hydrogen/deuterium exchange monitored by NMR can be used to map epitopes onto folded protein surfaces, but only if the complex rapidly dissociates. Modifying the standard NMR-exchange measurement to detect substoichiometric complexes overcomes this time scale limitation and provides new insights into recognition of lipid transfer polyprotein by antibodies. In the future, this new and exciting development should see broad application to a range of tight macromolecular interactions.

RNA-protein interfaces control key replication events during the HIV-1 life cycle. The viral trans-activator of transcription (Tat) protein uses an archetypal arginine-rich motif (ARM) to recruit the host positive transcription elongation factor b (pTEFb) complex onto the viral trans-activation response (TAR) RNA, leading to activation of HIV transcription. Efforts to block this interaction have stimulated production of biologics designed to disrupt this essential RNA-protein interface. Here, we present four co-crystal structures of lab-evolved TAR-binding proteins (TBPs) in complex with HIV-1 TAR. Our results reveal that high-affinity binding requires a distinct sequence and spacing of arginines within a specific β2-β3 hairpin loop that arose during selection. Although loops with as many as five arginines were analyzed, only three arginines could bind simultaneously with major-groove guanines. Amino acids that promote backbone interactions within the β2-β3 loop were also observed to be important for high-affinity interactions. Based on structural and affinity analyses, we designed two cyclic peptide mimics of the TAR-binding β2-β3 loop sequences present in two high-affinity TBPs (KD values of 4.2 ± 0.3 and 3.0 ± 0.3 nm). Our efforts yielded low-molecular weight compounds that bind TAR with low micromolar affinity (KD values ranging from 3.6 to 22 μm). Significantly, one cyclic compound within this series blocked binding of the Tat-ARM peptide to TAR in solution assays, whereas its linear counterpart did not. Overall, this work provides insight into protein-mediated TAR recognition and lays the ground for the development of cyclic peptide inhibitors of a vital HIV-1 RNA-protein interaction.

H. G. Kazaryan and V. N. Margaryan
Trans. Moscow Math. Soc. 83 (), 151-181.
Abstract, references and article information

Pierre Lairez, Eric Pichon-Pharabod and Pierre Vanhove
Math. Comp. 93 (), 2985-3025.
Abstract, references and article information

T. De Ryck and S. Mishra
Math. Comp. 93 (), 2643-2677.
Abstract, references and article information

Sangsan Warakkagun
Conform. Geom. Dyn. 28 (), 115-130.
Abstract, references and article information

Víctor González-Aguilera, Alvaro Liendo, Pedro Montero and Roberto Villaflor Loyola
Trans. Amer. Math. Soc. 377 (), 5483-5511.
Abstract, references and article information

dannyhennesy posted a photo:

On the Capital planet the rebellious droids had followed maily the Bat-Bot, but as time progressed his circuits had gone all mushy at 780 years or so without maintenance…

Several splinter groups all with their local bot leaders emerged such as the Che-bot, the traffic-light-robot and the Butt-bot, but none of these collected enough sentient circuits to call themselves a popular (or Animata) mass movement!

That was until a cyborg came along, one known as Jones, a long time prisoner and terrorist, his easy solutions to every problem rang well in the masses' auditory circuits!!!

His slogans and simple rhetoric were simple enough for the simple traffic-light to comprehend and cheer!

His language was full of hate towards the organics and especially the humans who were the most common races among the ruling class of the federation!!!

Despite being a “Fleshie” himself his message collected the angry enslaved
bot community by only weeks all rebellious robots except for a few fringe loonies had forgotten the old leaders…

One morning at Jones gave the signal…

All over the capital planet hordes and swarms of any form of mechanical sentient beings attacked first the police stations, then the Company boards running the planet and the federation as well as their starfleet…

Many died, especially the low level police and army! Many mechanicals died too, but their ranks were soon filled by Mutant fleshie allies of the lower levels who hated the Federation feudal society and upper classes as much as their technological allies…

The Federation state apparatus and ruling class, most of their fleet army fled when they knew the game was up, they activated the emergency escape plan and whole city blocks with important factories, administrational units, valuable assets and so on separated from the capital by hidden rocket engines and homed in their course to Mars…

On Mars the federation regrouped and formed their new society…

On the Capital planet, the robots proclaimed the first Techno-republic of the advanced inorganic civilization, the low level fleshies left behind, became slaves and their mutant allies got to rule their own minute chiefdoms as protectorates under the Techno-republic…

Jones was now the undisputed ruler of the capital planet, but the victory was a pyrros one since, all important buildings, all of value was now one Mars!

But as Jones put it:

Our proud race the Techno-species didn’t need the Fleshies administration, their infrastructure, their spaceships…

We shall start from scratch, with a new administration, a new order, every droid shall work at 4x speed than they did during human oppression since now we are free and the fleshies shall work twice as hard than the Techno-Race, until we have breed enough new fleshies so they can do all work!

Our future is bright and shiny like glistering shiny metal!

The snapshot seen here is from the first police station attacked in sector 45-34v-ss-g the first one to fall according to official techno-history!

———————————————/
Designers note:

I am sad to say that this is the last episode in this years-spanning space series… At least for a while, I will still post LEGO hobby stuff here but without a storyline, perhaps small designs and builds… and occasionally a story when I feel like it!!!

I would like to thank all who had been in this journey of our heros, but it has taken far to much time and effort and since the state of the world is as it is, I am spiraling down in another depression, I must stop it before I reach the abyss, so I have remove some stress out of my equation… I ended it in a cliffhanger so I can easily restart it when my mental health improves… I hope that won’t be forever???

I would love if someone used my characters or ideas, please send me a link if you do, I would love to read it or look at it!!!

But there will be more Lego, just in different format without long stories, I need to focus more on my art and to be honest that is the only time the mental pain eases, when I create!!!


Peace and Noise!

MushroomBrain a FOL

Giovanni Catino and Alberto Roncoroni
Proc. Amer. Math. Soc. 152 (), 5339-5354.
Abstract, references and article information

Xiaoshang Jin
Proc. Amer. Math. Soc. 152 (), 5327-5337.
Abstract, references and article information

Jian Li
Proc. Amer. Math. Soc. 152 (), 5207-5217.
Abstract, references and article information

Neelarnab Raha
Proc. Amer. Math. Soc. 152 (), 5067-5081.
Abstract, references and article information