Pectins are a major dietary nutrient source for the human gut microbiota. The prominent gut microbe Bacteroides thetaiotaomicron was recently shown to encode the founding member (BT1017) of a new family of pectin methylesterases essential for the metabolism of the complex pectin rhamnogalacturonan-II (RG-II). However, biochemical and structural knowledge of this family is lacking. Here, we showed that BT1017 is critical for the metabolism of an RG-II–derived oligosaccharide ΔBT1017oligoB generated by a BT1017 deletion mutant (ΔBT1017) during growth on carbohydrate extract from apple juice. Structural analyses of ΔBT1017oligoB using a combination of enzymatic, mass spectrometric, and NMR approaches revealed that it is a bimethylated nonaoligosaccharide (GlcA-β1,4-(2-O-Me-Xyl-α1,3)-Fuc-α1,4-(GalA-β1,3)-Rha-α1,3-Api-β1,2-(Araf-α1,3)-(GalA-α1,4)-GalA) containing components of the RG-II backbone and its side chains. We showed that the catalytic module of BT1017 adopts an α/β-hydrolase fold, consisting of a central twisted 10-stranded β-sheet sandwiched by several α-helices. This constitutes a new fold for pectin methylesterases, which are predominantly right-handed β-helical proteins. Bioinformatic analyses revealed that the family is dominated by sequences from prominent genera of the human gut microbiota, including Bacteroides and Prevotella. Our re-sults not only highlight the critical role played by this family of enzymes in pectin metabolism but also provide new insights into the molecular basis of the adaptation of B. thetaiotaomicron to the human gut.

Pyrroline-5-carboxylate reductase 1 (PYCR1) catalyzes the biosynthetic half-reaction of the proline cycle by reducing Δ1-pyrroline-5-carboxylate (P5C) to proline through the oxidation of NAD(P)H. Many cancers alter their proline metabolism by up-regulating the proline cycle and proline biosynthesis, and knockdowns of PYCR1 lead to decreased cell proliferation. Thus, evidence is growing for PYCR1 as a potential cancer therapy target. Inhibitors of cancer targets are useful as chemical probes for studying cancer mechanisms and starting compounds for drug discovery; however, there is a notable lack of validated inhibitors for PYCR1. To fill this gap, we performed a small-scale focused screen of proline analogs using X-ray crystallography. Five inhibitors of human PYCR1 were discovered: l-tetrahydro-2-furoic acid, cyclopentanecarboxylate, l-thiazolidine-4-carboxylate, l-thiazolidine-2-carboxylate, and N-formyl l-proline (NFLP). The most potent inhibitor was NFLP, which had a competitive (with P5C) inhibition constant of 100 μm. The structure of PYCR1 complexed with NFLP shows that inhibitor binding is accompanied by conformational changes in the active site, including the translation of an α-helix by 1 Å. These changes are unique to NFLP and enable additional hydrogen bonds with the enzyme. NFLP was also shown to phenocopy the PYCR1 knockdown in MCF10A H-RASV12 breast cancer cells by inhibiting de novo proline biosynthesis and impairing spheroidal growth. In summary, we generated the first validated chemical probe of PYCR1 and demonstrated proof-of-concept for screening proline analogs to discover inhibitors of the proline cycle.

Environmental factors, such as viral infection, are proposed to play a role in the initiation of autoimmune diabetes. In response to encephalomyocarditis virus (EMCV) infection, resident islet macrophages release the pro-inflammatory cytokine IL-1β, to levels that are sufficient to stimulate inducible nitric oxide synthase (iNOS) expression and production of micromolar levels of the free radical nitric oxide in neighboring β-cells. We have recently shown that nitric oxide inhibits EMCV replication and EMCV-mediated β-cell lysis and that this protection is associated with an inhibition of mitochondrial oxidative metabolism. Here we show that the protective actions of nitric oxide against EMCV infection are selective for β-cells and associated with the metabolic coupling of glycolysis and mitochondrial oxidation that is necessary for insulin secretion. Inhibitors of mitochondrial respiration attenuate EMCV replication in β-cells, and this inhibition is associated with a decrease in ATP levels. In mouse embryonic fibroblasts (MEFs), inhibition of mitochondrial metabolism does not modify EMCV replication or decrease ATP levels. Like most cell types, MEFs have the capacity to uncouple the glycolytic utilization of glucose from mitochondrial respiration, allowing for the maintenance of ATP levels under conditions of impaired mitochondrial respiration. It is only when MEFs are forced to use mitochondrial oxidative metabolism for ATP generation that mitochondrial inhibitors attenuate viral replication. In a β-cell selective manner, these findings indicate that nitric oxide targets the same metabolic pathways necessary for glucose stimulated insulin secretion for protection from viral lysis.

Towards just transition in Africa: Green financing for urban energy solutions and job creation 9 June 2022 — 7:30AM TO 11:00AM Anonymous (not verified) 18 May 2022 Nairobi and online

This event explores the major openings and potential impediments to the development of a just transition policy in Africa.

Global climate policies towards a ‘just transition’ under the Paris Agreement should also align with and support African states’ national sustainable development priorities. In particular, the need for decent and fair job creation and the establishment of sufficient, resilient and sustainable power supply, accessible to all, and efficient energy use.

Achieving green growth requires innovative and more accessible financing models, especially as wealthy nations’ financial pledges have fallen short. Ahead of the ‘African COP27’ set to take place in Egypt in November 2022, there is a need for transformational strategic thinking and context-specific action from African governments, civil society, businesses and financiers in their green financing demands and national implementation plans.

Sustainable urban energy solutions represent a critical zone of opportunity for the development of new and more reliable green finance pathways. Africa’s rapidly expanding cities present a significant economic opportunity and source of growth. However, urban centres are also where income and energy inequalities are at their starkest. The acceleration of sustainable energy generation and use could have a transformative impact on SMEs and livelihoods across value chains.

At this event, participants will discuss the major openings and potential impediments to the development of a credible ‘just transition’ policy in Africa towards net zero goals, with a particular focus on establishing and enhancing links between green financing innovation, employment creation, sustainable power supply and generation, and sustainable energy usage and consumption in an urban environment.

This event is held in partnership with the Pan African Climate Justice Alliance (PACJA). It is part of a series on Towards Just Transition: Connecting Green Financing and Sustainable Job Creation in Africa, supported by the Chatham House Sustainability Accelerator.

This event will be held in English and French with simultaneous interpretation.

Will Africans’ calls for better democracy be met? The World Today mhiggins.drupal 29 July 2022

Voters want the continent’s ageing leaders to step aside to usher in a new age of political engagement and robust democracy, say the experts of Afrobarometer.

Across Africa, recent years have been marked by both encouraging democratic highs and troubling anti-democratic lows. Notable advances from last year include the Gambia’s successful presidential election, a ruling-party transition in Zambia and the first democratic transfer of power in Niger. In the lead up to this, add Malawi’s retake of its flawed presidential election in 2020 and an earlier succession of oustings of long-serving autocrats in Sudan, Zimbabwe and the Gambia. 

Contrast these gains, though, with setbacks elsewhere, including increasing restrictions on opposition parties in Benin, Senegal and Tanzania; the use of violence and intimidation during elections in Côte d’Ivoire and Uganda; and military coups, with the latest in Burkina Faso this year and last year in Chad, Mali, Sudan and Guinea.

These contradictory developments join dire warnings from experts that democracy is losing ground on the continent. But what can we learn about the state of democracy on the continent from Africans themselves?

Afrobarometer, a pan-African, non-partisan research network, has been surveying people about their views on democracy, governance and quality of life for more than 20 years. After interviewing nearly 50,000 citizens across 34 countries during Afrobarometer Round 8, which spans 2019-2021, we find that despite the efforts of some leaders to undermine democratic norms, Africans remain committed to democracy and democratic institutions.

They believe that the military should stay out of politics, that political parties should freely compete for power, that elections are an imperfect but essential tool for choosing their leaders, and that it is time for the old men who cling to power to step aside.

But their political reality falls short of these expectations. The perception of widespread and worsening corruption is particularly corrosive, leaving people increasingly dissatisfied with political systems that are yet to deliver on their aspirations to live in societies that are democratically and accountably governed. And although citizens find myriad ways to voice their concerns, they feel that their governments are not listening.

Simply put, Africans want more democratic and accountable governance than they think they are getting.

Africans’ democratic aspirations

Over the past decade, democracy watchers have been alarmed by declining trends in Africa. Concerns have been exacerbated in the past two years as some governments have taken advantage of the Covid pandemic to limit freedoms, restrict fair campaigning or postpone elections. Activists fear that supposedly temporary rollbacks in hard-won governance reforms could become permanent.

But for the most part, African citizens remain committed to democracy and democratic institutions. Across 30 countries that Afrobarometer has surveyed consistently since Round 5 (2011–2013), most indicators are strong and quite steady.

For example, seven in 10 Africans say that ‘democracy is preferable to any other kind of government’. While this is down modestly from 73 per cent a decade ago, more specific indicators seem to affirm popular commitment to democracy. Large and steady majorities consistently reject authoritarian alternatives, including one-person or ‘strongman’ rule (82 per cent), one-party rule (77 per cent) and military rule (75 per cent), which is clearly rejected even in many of the countries rocked by recent military coups.

Africans also express strong support for a limit to presidential terms, a feature of democratic governance that researchers and activists argue nurtures political participation, demonstrates that change via the ballot box is possible, and reduces the risk of personality cults, authoritarianism, corruption and coups. Across 34 countries, an average of 76 per cent favour limiting their presidents to two terms, including a majority (54 per cent) who ‘strongly’ support this rule. Term limits enjoy majority support in every surveyed country. 

The public’s democratic commitment is undergirded by strong and in some cases growing support for core democratic institutions. Support for multiparty competition and parliamentary oversight of leaders remains steady, while expectations that governments should be accountable to the courts have increased significantly over the past decade.

In addition, growing numbers of people say it is more important to have a government that is accountable to the people rather than one that just ‘gets things done’, an especially strong indicator of deepening commitment to democratic norms among citizens. 

Trouble at the polling booth

Elections remain a central, though controversial, institution of democracy for Africans. They have served as the foundation for real change, as in Zambia last year. But in other cases, such as Uganda’s January 2021 poll, they have been marred by violence and human rights abuses, as well as the weaponization of Covid to justify restrictions on campaigning.

The public is also sceptical about the capacity of elections to bring about real change: fully 50 per cent say they do not think elections are effective in enabling voters ‘to remove from office leaders who do not do what the people want’.

At the same time, large majorities report positively on their country’s election environment. Asked about their most recent election, at least eight in 10 say they did not observe intimidation (87 per cent) or interference (81 per cent) by security forces and did not fear violence (80 per cent).

We must keep in mind that these encouraging averages can obscure deep problems in some countries. For example, while only 3 per cent of Namibians say votes are ‘often’ not counted fairly, between a quarter and one-third cite inaccurate counts as a frequent problem in Zimbabwe, Sudan and Gabon. 

In addition, confidence in the fairness of the media environment is drastically lower, on average just 36 per cent.

But perhaps most importantly, almost nine in 10 Africans (87 per cent) say they are free to vote as they choose, including sizeable majorities in every surveyed country. And a solid majority of 63 per cent rate their most recent election as completely or mostly free and fair. 

All of this may help to explain still-strong support for competitive elections as the best system for selecting leaders. A robust three-quarters confirm their commitment to elections, though this has fallen slightly over the past decade, probably reflecting disillusionment with electoral processes that are too often torn by violence and produce contested results. 

A growing number of people may also be recognizing that elections, especially poor-quality ones, are not enough to guarantee democracy and better governance, and that a healthy democracy must include such other features as government accountability, respect for the rule of law, responsiveness and citizen participation.

The ‘democratic disappointment’ gap

To what extent does political reality align with Africans’ democratic aspirations? Our findings suggest that it is falling well short of expectations.

While a slim majority has steadily reported that their country is a ‘full democracy’ or one ‘with minor problems’ over the past decade, satisfaction, however, has dropped to 43 per cent in that time. 

What explains this growing dissatisfaction? Other indicators of democratic supply offer some clues. While ratings of election quality have held steady, favourable public assessments of presidential accountability to parliament and to the courts have both declined.

The rising scourge of corruption

But one of the most significant driving factors may be burgeoning corruption, a trend that appears to parallel declining democratic satisfaction. On average across 34 countries, around six in 10 say both that corruption in their country increased over the past year, and that their government is doing a poor job of controlling it.

These perceptions matter. Over time, when perceptions of corruption rise or fall, levels of dissatisfaction with democracy tend to follow suit. 

In South Africa, dissatisfaction with democracy grew steadily alongside scandals involving President Jacob Zuma, and has continued to rise under his successor, Cyril Ramaphosa, whose office has been tainted by ‘Farmgate’ and a major Covid-relief scandal. The ‘Fishrot’ scandal in Namibia has had similar consequences.

Are governments listening?

African citizens are raising their voices, calling on their governments to fulfil their democratic aspirations. Since April 2017, the Carnegie Endowment for International Peace has recorded more than 70 episodes in 35 African countries of protests focused on issues ranging from demands for democracy in eSwatini to resisting police brutality, presidential third-term attempts and Covid restrictions. 

Citizen participation and government responsiveness are cornerstones of democracy. But are governments listening?

Voting is the most obvious and popular way for citizens to express themselves, and Africans take advantage of this opportunity. Two-thirds said they voted in their most recent national election. But elections occur only occasionally, and they force individuals to compress a wide array of views into very few choices. How do Africans find their voice during the long intervals between elections?

Many invest in personal efforts to act as agents of change. In fact, nearly half say they joined with others to raise an issue at least once in the past year, and a third contacted a political leader. A quarter report they acted with others to request government action. Less common but still important modes of engagement include asking for help from or lodging a complaint with government, contacting the media, and joining a demonstration.

These robust levels of citizen engagement suggest that people feel they can make a difference. Unfortunately, decision-makers aren’t always receptive or responsive to citizen voices. Less than a quarter of people think local government officials listen to them – and even fewer think their members of parliament do. 

What is more troubling is that fully two-thirds say they are at risk of retaliation or some form of negative consequences if they take action by reporting incidents of corruption. 

Lack of government responsiveness and respect for popular voices may have direct implications for both citizen engagement and citizen satisfaction. For example, we find that people are more likely to contact leaders or take other actions to solve problems if they believe that government officials respect and listen to them; that they will get a response if they raise an issue; and if they do not need to fear retaliation. 

Similarly, when we compare country averages for government responsiveness to the percentage of citizens who are satisfied with democracy, we again find positive associations. 

When governments are responsive, citizens are more likely to engage in addressing community needs and to be satisfied with their political system and optimistic about the future. Respectful and responsive governance has the potential to spur citizen action to solve critical development challenges – and may be the cure for what ails democracy.

Towards democracy in Sudan: Reflections on the transitional period 20 September 2022 — 2:00PM TO 4:00PM Anonymous (not verified) 31 August 2022 Online

In this webinar, panellists discuss the key outcomes of the evaluation workshop and reflect on how the experience of the transition to date should inform the realization of Sudan’s democratic aspirations going forward.

The military coup on 25 October 2021 halted Sudan’s transition to democracy and prospects for sustainable peace. Since then, members of the former regime have regained political influence, with many reinstated to senior positions.

The coup has provoked a strong reaction from the country’s pro-democracy movement and youth-led resistance committees who have led continuous peaceful protests demanding civilian democratic transition, despite a brutal crackdown by state security forces.

Those supportive of the coup have blamed the Forces of Freedom and Change (FFC), a wide coalition of pro-democracy political and civilian groups, for the mistakes of the transitional government.

However, the country’s political, security and economic situation has sharply deteriorated since the military’s action, and the progress achieved by the transitional government has been reversed, leading to an accelerating economic crisis, increased food insecurity and political instability.

The FFC, which played a key role in appointing the transitional government, has acknowledged its errors. In July 2022, it held a workshop in Khartoum, which was broadcast on social media, to evaluate its performance and identify lessons learned, in discussion with civil society actors and activists.

In this webinar, leading pro-democracy movement figures and independent experts discuss the key outcomes of the evaluation workshop and reflect on how the experience of the transition to date should inform the realization of Sudan’s democratic aspirations going forward.

This event is part of a Chatham House Africa programme project on supporting Sudan’s civilian-led democratic transition.

Towards just transition in Africa: Continental coordination on green financing and job creation 6 October 2022 — 7:00AM TO 3:30PM Anonymous (not verified) 8 September 2022 Addis Ababa and online

At this hybrid conference in Addis Ababa, speakers take stock of preparations ahead of the ‘African COP27’ in November and discuss the key priorities for streamlining continental cooperation on policy approaches to just transition.

At this hybrid conference in Addis Ababa, speakers will take stock of policy efforts and preparations ahead of the ‘African COP27’ in November and discuss the key priorities for streamlining continental cooperation on policy approaches to just transition.

Global climate policies towards a ‘just transition’ under the Paris Agreement should align with and support African states’ national sustainable development priorities – in particular, the need for decent and fair job creation, as well as resilient and sustainable land, environment and ecosystem management policies.

They must also be cognizant of African nations’ urgent requirements for sustainable and accessible energy to underpin economic development. Achieving green growth requires innovative and more accessible financing models, especially as wealthy nations’ financial pledges have fallen short. It also requires clarity and cooperation to unlock investment in both renewable and transitional energy.

African countries face collective climate and employment-related challenges. However, policymaking often remains regionally siloed according to differing political, energy sector and ecological realities. There is a need for transformational strategic thinking and context-specific action from African governments, civil society, businesses and financiers, in their green financing demands and national implementation plans.

At this hybrid conference in Addis Ababa, speakers will take stock of policy efforts and preparations ahead of the ‘African COP27’ in November and discuss the key priorities for streamlining continental cooperation on policy approaches to just transition, job creation and green financing.

This event is the third in a series on Towards just transition: Connecting green financing and sustainable job creation in Africa, supported by the Chatham House Sustainability Accelerator.

A critical juncture for Sudan’s democratic transition Expert comment LJefferson 28 March 2023

International pressure is essential to reach an agreement that establishes a credible civilian government.

The signing of the Framework Agreement (FA) on 5 December 2022 between Sudan’s military leaders and its leading pro-democracy parties is a major step to reversing the damage done by the disastrous military coup in October 2021.

The FA removes any formal role for the military in Sudan’s politics. A civilian head of state and prime minister will select the cabinet and chair the Defence and Security Council. The armed forces will be prohibited from non-military business activities and security sector reform will lead to a unified, professional and non-partisan national army. Elections are due to take place at the end of a two-year transitional period. 

Signatories included General Abdel Fatah Al Burhan, chair of the Sovereign Council and head of the Sudanese Armed Forces (SAF), General Mohamed Hamdan Dagalo (known as Hemedti), his deputy and Commander of the paramilitary Rapid Support Forces (RSF) and more than 40 civilian entities, including the Forces of Freedom and Change- Central Council (FFC-CC), a few other political parties, former armed movements, civil society organizations and professional associations. However, the agreement has faced criticism from the street for not being sufficiently radical, has been overshadowed at times by heightened tension between the two military leaders, and has seen sabotage attempts by supporters of the Bashir regime.

Building consensus on the Framework Agreement

The agreement meets most of the demands of the anti-coup camp, at least on paper. Yet doubts persist as to whether the military are genuine about handing over power, particularly among the neighbourhood-based resistance committees – the heart of the youth-led mobilization that forced the military to recognize the failure of their power grab. Peaceful protests against the coup have seen 125 killed and over 8,000 injured by government security forces. Many want to see Burhan and Hemedti held accountable.

Recognizing the need to expand popular support, FFC-CC leaders have been reaching out to other pro-democracy forces to build a united civilian front. They report increased buy-in from some resistance committees in the last few months, recognizing that street protests alone were not sufficient to overthrow the coup, and that engagement with the military is necessary to find a way out of the impasse.

The FA offers the only currently available path to embedding civilian politics in Sudan and has received active diplomatic support from UNITAMS, AU and IGAD (who form the Tripartite Mechanism), the Troika of the US, UK and Norway, alongside the EU, as well as Saudi Arabia and the UAE (who are members of ‘the Quad’ with the US and UK).

Broader public participation has also been developed through a series of conferences, facilitated by the Tripartite Mechanism, on five contentious issues – dismantling the old regime, the Juba Peace Agreement, Eastern Sudan, Transitional Justice and Security Sector Reform. Recommendations will be incorporated in a final political agreement. 

The political process has been overshadowed by increasingly visible tension between Burhan and Hemedti, seen in parallel foreign visits, conflicting public statements, and a heavy military presence in Khartoum. But concerns that SAF and the RSF were heading towards confrontation appear to have been assuaged thanks to international pressure and preliminary agreements reached between military and civilian signatories of the FA on security sector reform and integration. In a significant breakthrough, both sides have now agreed to draft the final agreement and transitional constitution, with the aim of forming a civilian government by 11 April.  

Potential spoilers and interests from Sudan’s regions

Progress has been made, but significant challenges remain, notably from supporters of the former Bashir regime in ‘the deep state’ and from Sudan’s historically marginalized peripheries. Old regime elements have been intensifying social media campaigns to derail the agreement and drive a wedge between the SAF and RSF, and have been accused of deliberately inciting instability in the peripheries to undermine the democratic transition. 

The Popular Defence Forces, established by the National Islamic Front in the 1990s, have been reactivated under different names in several parts of the country and there are reports of mobilization and recruitment of armed militias in Darfur. The recent public appearance of Ali Karti, the Secretary-General of the Islamic Movement, who has close relations with Islamists in SAF, has also caused renewed concern.

Two Darfuri armed movement leaders who signed the October 2020 Juba Peace Agreement (JPA) and are members of the current military-led government, have not signed the FA, allegedly due to concerns about their representation in the next government.

Despite intensive efforts to bring them on board, there is continuing disagreement over the inclusion of other members of ‘the Democratic Bloc’, a political alliance backed by Sudan’s influential neighbour Egypt, which is reportedly angry at being excluded from the Quad. The FFC-CC say that the door is open for the two Darfuri leaders and some other political parties, but they will not allow the agreement to be ‘diluted’ with political forces who intend to torpedo the transition, including by imposing a weak prime minister.

Both Burhan and Hemedti have courted support from the regions. Burhan used the 2020 SAF takeover of Al Fashaga in the contested eastern border region with Ethiopia to boost his national standing and secure backing from local tribal leaders; while Hemedti has sought to position himself as a champion of the peripheries, particularly in his Darfur heartlands, while simultaneously advancing his business interests.

Competition between them in building domestic powerbases, as well as alliances with neighbouring states, risks reigniting tensions, particularly given deep grievances and contrasting ambitions between and among Sudan’s diverse regional leaders and communities.

Visual Abstract

Felicia Grasso
Dec 1, 2020; 19:1986-1996
Research

Eugen Netz
Dec 1, 2020; 19:2157-2167
Technological Innovation and Resources

Peter Lasch
Dec 1, 2020; 19:2125-2138
Technological Innovation and Resources

Colin T. McDowell
Nov 24, 2020; 0:RA120.002256v1-mcp.RA120.002256
Research

Adult progenitor cell populations typically exist in a quiescent state within a controlled niche environment. However, various stresses or forms of damage can disrupt this state, which often leads to dysfunction and aging. We built a glucocorticoid (GC)-induced liver damage model of mice, found that GC stress induced liver damage, leading to consequences for progenitor cells expansion. However, the mechanisms by which niche factors cause progenitor cells proliferation are largely unknown. We demonstrate that, within the liver progenitor cells niche, Galectin-3 (Gal-3) is responsible for driving a subset of progenitor cells to break quiescence. We show that GC stress causes aging of the niche, which induces the up-regulation of Gal-3. The increased Gal-3 population increasingly interacts with the progenitor cell marker CD133, which triggers focal adhesion kinase (FAK)/AMP-activated kinase (AMPK) signaling. This results in the loss of quiescence and leads to the eventual stemness exhaustion of progenitor cells. Conversely, blocking Gal-3 with the inhibitor TD139 prevents the loss of stemness and improves liver function. These experiments identify a stress-dependent change in progenitor cell niche that directly influence liver progenitor cell quiescence and function.

The Wnt/β-catenin pathway is one of the major pathways that regulates embryonic development, adult homeostasis, and stem cell self-renewal. In this pathway, transcription factors T-cell factor and lymphoid enhancer factor (TCF/LEF) serve as a key switch to repress or activate Wnt target gene transcription by recruiting repressor molecules or interacting with the β-catenin effector, respectively. It has become evident that the protein stability of the TCF/LEF family members may play a critical role in controlling the activity of the Wnt/β-catenin signaling pathway. However, factors that regulate the stability of TCF/LEFs remain largely unknown. Here, we report that pVHL binding protein 1 (VBP1) regulates the Wnt/β-catenin signaling pathway by controlling the stability of TCF/LEFs. Surprisingly, we found that either overexpression or knockdown of VBP1 decreased Wnt/β-catenin signaling activity in both cultured cells and zebrafish embryos. Mechanistically, VBP1 directly binds to all four TCF/LEF family members and von Hippel-Lindau tumor-suppressor protein (pVHL). Either overexpression or knockdown of VBP1 increases the association between TCF/LEFs and pVHL and then decreases the protein levels of TCF/LEFs via proteasomal degradation. Together, our results provide mechanistic insights into the roles of VBP1 in controlling TCF/LEFs protein stability and regulating Wnt/β-catenin signaling pathway activity.

Mitochondrial dysfunction is associated with a variety of human diseases including neurodegeneration, diabetes, nonalcohol fatty liver disease (NAFLD), and cancer, but its underlying causes are incompletely understood. Using the human hepatic cell line HepG2 as a model, we show here that endoplasmic reticulum-associated degradation (ERAD), an ER protein quality control process, is critically required for mitochondrial function in mammalian cells. Pharmacological inhibition or genetic ablation of key proteins involved in ERAD increased cell death under both basal conditions and in response to proinflammatory cytokines, a situation frequently found in NAFLD. Decreased viability of ERAD-deficient HepG2 cells was traced to impaired mitochondrial functions including reduced ATP production, enhanced reactive oxygen species (ROS) accumulation, and increased mitochondrial outer membrane permeability. Transcriptome profiling revealed widespread down-regulation of genes underpinning mitochondrial functions, and up-regulation of genes associated with tumor growth and aggression. These results highlight a critical role for ERAD in maintaining mitochondrial functional and structural integrity and raise the possibility of improving cellular and organismal mitochondrial function via enhancing cellular ERAD capacity.

Large regions in tumor tissues, particularly pancreatic cancer, are hypoxic and nutrient-deprived because of unregulated cell growth and insufficient vascular supply. Certain cancer cells, such as those inside a tumor, can tolerate these severe conditions and survive for prolonged periods. We hypothesized that small molecular agents, which can preferentially reduce cancer cell survival under nutrient-deprived conditions, could function as anticancer drugs. In this study, we constructed a high-throughput screening system to identify such small molecules and screened chemical libraries and microbial culture extracts. We were able to determine that some small molecular compounds, such as penicillic acid, papyracillic acid, and auranofin, exhibit preferential cytotoxicity to human pancreatic cancer cells under nutrient-deprived compared with nutrient-sufficient conditions. Further analysis revealed that these compounds target to redox systems such as GSH and thioredoxin and induce accumulation of reactive oxygen species in nutrient-deprived cancer cells, potentially contributing to apoptosis under nutrient-deprived conditions. Nutrient-deficient cancer cells are often deficient in GSH; thus, they are susceptible to redox system inhibitors. Targeting redox systems might be an attractive therapeutic strategy under nutrient-deprived conditions of the tumor microenvironment.

Elevated levels of fasting insulin release and insufficient glucose-stimulated insulin secretion (GSIS) are hallmarks of diabetes. Studies have established cross-talk between integrin signaling and insulin activity, but more details of how integrin-dependent signaling impacts the pathophysiology of diabetes are needed. Here, we dissected integrin-dependent signaling pathways involved in the regulation of insulin secretion in β-cells and studied their link to the still debated autocrine regulation of insulin secretion by insulin/insulin-like growth factor (IGF) 2–AKT signaling. We observed for the first time a cooperation between different AKT isoforms and focal adhesion kinase (FAK)–dependent adhesion signaling, which either controlled GSIS or prevented insulin secretion under fasting conditions. Indeed, β-cells form integrin-containing adhesions, which provide anchorage to the pancreatic extracellular matrix and are the origin of intracellular signaling via FAK and paxillin. Under low-glucose conditions, β-cells adopt a starved adhesion phenotype consisting of actin stress fibers and large peripheral focal adhesion. In contrast, glucose stimulation induces cell spreading, actin remodeling, and point-like adhesions that contain phospho-FAK and phosphopaxillin, located in small protrusions. Rat primary β-cells and mouse insulinomas showed an adhesion remodeling during GSIS resulting from autocrine insulin/IGF2 and AKT1 signaling. However, under starving conditions, the maintenance of stress fibers and the large adhesion phenotype required autocrine IGF2-IGF1 receptor signaling mediated by AKT2 and elevated FAK-kinase activity and ROCK-RhoA levels but low levels of paxillin phosphorylation. This starved adhesion phenotype prevented excessive insulin granule release to maintain low insulin secretion during fasting. Thus, deregulation of the IGF2 and adhesion-mediated signaling may explain dysfunctions observed in diabetes.

In Alzheimer's disease (AD), tau, a microtubule-associated protein (MAP), becomes hyperphosphorylated, aggregates, and accumulates in the somato-dendritic compartment of neurons. In parallel to its intracellular accumulation in AD, tau is also released in the extracellular space, as revealed by its increased presence in cerebrospinal fluid (CSF). Consistent with this, recent studies, including ours, have reported that neurons secrete tau, and several therapeutic strategies aim to prevent the intracellular tau accumulation. Previously, we reported that late endosomes were implicated in tau secretion. Here, we explore the possibility of preventing intracellular tau accumulation by increasing tau secretion. Using neuronal models, we investigated whether overexpression of the vesicle-associated membrane protein 8 (VAMP8), an R-SNARE found on late endosomes, could increase tau secretion. The overexpression of VAMP8 significantly increased tau secretion, decreasing its intracellular levels in the neuroblastoma (N2a) cell line. Increased tau secretion by VAMP8 was also observed in murine hippocampal slices. The intracellular reduction of tau by VAMP8 overexpression correlated to a decrease of acetylated tubulin induced by tau overexpression in N2a cells. VAMP8 staining was preferentially found on late endosomes in N2a cells. Using total internal reflection fluorescence (TIRF) microscopy, the fusion of VAMP8-positive vesicles with the plasma membrane was correlated to the depletion of tau in the cytoplasm. Finally, overexpression of VAMP8 reduced the intracellular accumulation of tau mutants linked to frontotemporal dementia with parkinsonism and α-synuclein by increasing their secretion. Collectively, the present data indicate that VAMP8 could be used to increase tau and α-synuclein clearance to prevent their intracellular accumulation.

Acute lung injury (ALI), is a rapidly progressing heterogenous pulmonary disorder that possesses a high risk of mortality. Accumulating evidence has implicated the activation of the p65 subunit of NF-κB [NF-κB(p65)] activation in the pathological process of ALI. microRNAs (miRNAs), a group of small RNA molecules, have emerged as major governors due to their post-transcriptional regulation of gene expression in a wide array of pathological processes, including ALI. The dysregulation of miRNAs and NF-κB activation has been implicated in human diseases. In the current study, we set out to decipher the convergence of miR-99b and p65 NF-κB activation in ALI pathology. We measured the release of pro-inflammatory cytokines (IL-1β, IL-6, and TNFα) in bronchoalveolar lavage fluid using ELISA. MH-S cells were cultured and their viability were detected with cell counting kit 8 (CCK8) assays. The results showed that miR-99b was up-regulated, while PRDM1 was down-regulated in a lipopolysaccharide (LPS)-induced murine model of ALI. Mechanistic investigations showed that NF-κB(p65) was enriched at the miR-99b promoter region, and further promoted its transcriptional activity. Furthermore, miR-99b targeted PRDM1 by binding to its 3'UTR, causing its down-regulation. This in-creased lung injury, as evidenced by increased wet/dry ratio of mouse lung, myeloperoxidase activity and pro-inflammatory cytokine secretion, and enhanced infiltration of inflammatory cells in lung tissues. Together, our findings indicate that NF-κB(p65) promotion of miR-99b can aggravate ALI in mice by down-regulating the expression of PRDM1.

RAS genes are the most commonly mutated in human cancers and play critical roles in tumor initiation, progression, and drug resistance. Identification of targets that block RAS signaling is pivotal to develop therapies for RAS-related cancer. As RAS translocation to the plasma membrane (PM) is essential for its effective signal transduction, we devised a high-content screening assay to search for genes regulating KRAS membrane association. We found that the tyrosine phosphatase PTPN2 regulates the plasma membrane localization of KRAS. Knockdown of PTPN2 reduced the proliferation and promoted apoptosis in KRAS-dependent cancer cells, but not in KRAS-independent cells. Mechanistically, PTPN2 negatively regulates tyrosine phosphorylation of KRAS, which, in turn, affects the activation KRAS and its downstream signaling. Consistently, analysis of the TCGA database demonstrates that high expression of PTPN2 is significantly associated with poor prognosis of patients with KRAS-mutant pancreatic adenocarcinoma. These results indicate that PTPN2 is a key regulator of KRAS and may serve as a new target for therapy of KRAS-driven cancer.

Postoperative recurrence from microscopic residual disease must be prevented to cure intractable cancers, including pancreatic cancer. Key to this goal is the elimination of cancer stem cells (CSCs) endowed with tumor-initiating capacity and drug resistance. However, current therapeutic strategies capable of accomplishing this are insufficient. Using in vitro models of CSCs and in vivo models of tumor initiation in which CSCs give rise to xenograft tumors, we show that dexamethasone induces expression of MKP-1, a MAPK phosphatase, via glucocorticoid receptor activation, thereby inactivating JNK, which is required for self-renewal and tumor initiation by pancreatic CSCs as well as for their expression of survivin, an anti-apoptotic protein implicated in multidrug resistance. We also demonstrate that systemic administration of clinically relevant doses of dexamethasone together with gemcitabine prevents tumor formation by CSCs in a pancreatic cancer xenograft model. Our study thus provides preclinical evidence for the efficacy of dexamethasone as an adjuvant therapy to prevent postoperative recurrence in patients with pancreatic cancer.

Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence. Previously, using an unbiased screen, we identified ALK-1 as a high-capacity receptor for low-density lipoprotein (LDL) in endothelial cells that mediates its transcytosis in a nondegradative manner. Here we examine the crosstalk between BMP-9 and LDL and how it influences their interactions with ALK-1. Treatment of endothelial cells with BMP-9 triggers the extensive endocytosis of ALK-1, and it is mediated by caveolin-1 (CAV-1) and dynamin-2 (DNM2) but not clathrin heavy chain. Knockdown of CAV-1 reduces BMP-9–mediated internalization of ALK-1, BMP-9–dependent signaling and gene expression. Similarly, treatment of endothelial cells with LDL reduces BMP-9–induced SMAD1/5 phosphorylation and gene expression and silencing of CAV-1 and DNM2 diminishes LDL-mediated ALK-1 internalization. Interestingly, BMP-9–mediated ALK-1 internalization strongly re-duces LDL transcytosis to levels seen with ALK-1 deficiency. Thus, BMP-9 levels can control cell surface levels of ALK-1, via CAV-1, to regulate both BMP-9 signaling and LDL transcytosis.

The HIV-1 protein Gag assembles at the plasma membrane and drives virion budding, assisted by the cellular endosomal complex required for transport (ESCRT) proteins. Two ESCRT proteins, TSG101 and ALIX, bind to the Gag C-terminal p6 peptide. TSG101 binding is important for efficient HIV-1 release, but how ESCRTs contribute to the budding process and how their activity is coordinated with Gag assembly is poorly understood. Yeast, allowing genetic manipulation that is not easily available in human cells, has been used to characterize the cellular ESCRT function. Previous work reported Gag budding from yeast spheroplasts, but Gag release was ESCRT-independent. We developed a yeast model for ESCRT-dependent Gag release. We combined yeast genetics and Gag mutational analysis with Gag-ESCRT binding studies and the characterization of Gag-plasma membrane binding and Gag release. With our system, we identified a previously unknown interaction between ESCRT proteins and the Gag N-terminal protein region. Mutations in the Gag-plasma membrane–binding matrix domain that reduced Gag-ESCRT binding increased Gag-plasma membrane binding and Gag release. ESCRT knockout mutants showed that the release enhancement was an ESCRT-dependent effect. Similarly, matrix mutation enhanced Gag release from human HEK293 cells. Release enhancement partly depended on ALIX binding to p6, although binding site mutation did not impair WT Gag release. Accordingly, the relative affinity for matrix compared with p6 in GST-pulldown experiments was higher for ALIX than for TSG101. We suggest that a transient matrix-ESCRT interaction is replaced when Gag binds to the plasma membrane. This step may activate ESCRT proteins and thereby coordinate ESCRT function with virion assembly.

Priyanka Tripathi
Dec 30, 2020; 0:jlr.RA120001190v1-jlr.RA120001190
Research Articles

Oktawia Nilsson
Nov 17, 2020; 0:jlr.RA120000920v1-jlr.RA120000920
Research Articles

Jenny E. Kanter
Dec 8, 2020; 0:jlr.ILR120001217v1-jlr.ILR120001217
Images in Lipid Research

In SAS Customer Intelligence Studio, you can choose to create a new calculated variable in the Edit Value dialog box when you populate a treatment custom detail. Following creation of the new calculated

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Certain tables that are created in SAS Scalable Performance Data (SPD) Server might not be displayed correctly by SAS Federation Server Manager. Tables that have Latin5 characters in column names encounter this

Functions of the gut microbiome have a growing number of implications for host metabolic health, with diet being one of the most significant influences on microbiome composition. Compelling links between diet and the gut microbiome suggest key roles for various macronutrients, including lipids, yet how individual classes of dietary lipids interact with the microbiome remains largely unknown. Sphingolipids are bioactive components of most foods and are also produced by prominent gut microbes. This makes sphingolipids intriguing candidates for shaping diet–microbiome interactions. Here, we used a click chemistry–based approach to track the incorporation of bioorthogonal dietary omega-alkynyl sphinganine (sphinganine alkyne [SAA]) into the murine gut microbial community (Bioorthogonal labeling). We identified microbial and SAA-specific metabolic products through fluorescence-based sorting of SAA-containing microbes (Sort), 16S rRNA gene sequencing to identify the sphingolipid-interacting microbes (Seq), and comparative metabolomics to identify products of SAA assimilation by the microbiome (Spec). Together, this approach, termed Bioorthogonal labeling-Sort-Seq-Spec (BOSSS), revealed that SAA assimilation is nearly exclusively performed by gut Bacteroides, indicating that sphingolipid-producing bacteria play a major role in processing dietary sphinganine. Comparative metabolomics of cecal microbiota from SAA-treated mice revealed conversion of SAA to a suite of dihydroceramides, consistent with metabolic activities of Bacteroides and Bifidobacterium. Additionally, other sphingolipid-interacting microbes were identified with a focus on an uncharacterized ability of Bacteroides and Bifidobacterium to metabolize dietary sphingolipids. We conclude that BOSSS provides a platform to study the flux of virtually any alkyne-labeled metabolite in diet–microbiome interactions.

The cornea is densely innervated, mainly by sensory nerves of the ophthalmic branch of the trigeminal ganglia (TG). These nerves  are important to maintain corneal homeostasis, and nerve damage can lead to a decrease in wound healing, an increase in corneal ulceration and dry eye disease (DED), and neuropathic pain. Pathologies, such as diabetes, aging, viral and bacterial infection, as well as  prolonged use of contact lenses and surgeries to correct vision can produce nerve damage. There are no effective therapies to alleviate DED (a multifunctional disease) and several clinical trials using -3 supplementation show unclear and sometimes negative results. Using animal models of corneal nerve damage, we show that treating corneas with pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA) increases nerve regeneration, wound healing, and tear secretion. The mechanism involves the activation of a calcium-independent phospholipase A2 (iPLA2) that releases the incorporated DHA from phospholipids and enhances the synthesis of docosanoids neuroprotectin D1 (NPD1) and a new resolvin stereoisomer  RvD6i. NPD1 stimulates the synthesis of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and of semaphorin 7A (Sema7A).  RvD6i treatment of injured corneas modulates gene expression in the TG resulting in enhanced neurogenesis; decreased neuropathic pain and increased sensitivity. Taken together, these results represent a promising therapeutic option to re-establish the homeostasis of the cornea.

Sphingolipids have emerged as bioactive lipids involved in the regulation of many physiological and pathological processes. In the retina, they have been established to participate in numerous processes, such as neuronal survival and death, proliferation and migration of neuronal and vascular cells, inflammation, and neovascularization. Dysregulation of sphingolipids is, therefore, crucial in the onset and progression of retinal diseases. This review examines the involvement of sphingolipids in retinal physiology and diseases. Ceramide (Cer) emerges as a common mediator of inflammation and death of neuronal and retinal pigment epithelium cells in animal models of retinopathies such as glaucoma, age-related macular degeneration (AMD), and retinitis pigmentosa. Sphingosine-1-phosphate (S1P) has opposite roles, preventing photoreceptor and ganglion cell degeneration but also promoting inflammation, fibrosis, and neovascularization in AMD, glaucoma, and pro-fibrotic disorders. Alterations in Cer, S1P, and ceramide-1-phosphate may also contribute to uveitis. Notably, use of inhibitors that either prevent Cer increase or modulate S1P signaling, such as Myriocin, desipramine, and Fingolimod (FTY720), preserves neuronal viability and retinal function. These findings underscore the relevance of alterations in the sphingolipid metabolic network in the etiology of multiple retinopathies and highlight the potential of modulating their metabolism for the design of novel therapeutic approaches.

Roux-en-Y gastric bypass (RYGB) is one of the most commonly performed weight-loss procedures, but how severe obesity and RYGB affects circulating HDL-associated microRNAs (miRNAs) remains unclear. Here, we aim to investigate how HDL-associated miRNAs are regulated in severe obesity and how weight loss after RYGB surgery affects HDL-miRNAs. Plasma HDL were isolated from patients with severe obesity (n=53) before, 6 and 12 months after RYGB by immunoprecipitation using goat anti-human apoA-I microbeads. HDL were also isolated from 18 healthy participants. miRNAs were extracted from isolated HDL and levels of miR-24, miR-126, miR-222 and miR-223 were determined by TaqMan miRNA assays. We found that HDL-associated miR-126, miR-222 and miR-223 levels, but not miR-24 levels, were significantly higher in patients with severe obesity when compared with healthy controls. There were significant increases in HDL-associated miR-24, miR-222 and miR-223 at 12 months after RYGB. Additionally, cholesterol efflux capacity and paraoxonase (PON1) activity were increased and intracellular adhesion molecule-1 (ICAM-1) levels decreased. The increases in HDL-associated miR-24 and miR-223 were positively correlated with increase in cholesterol efflux capacity (r=0.326, P=0.027 and r=0.349, P=0.017 respectively). An inverse correlation was observed between HDL-associated miR-223 and ICAM-1 at baseline. Together, these findings show that HDL-associated miRNAs are differentially regulated in healthy versus patients with severe obesity and are altered after RYGB. These findings provide insights into how miRNAs are regulated in obesity before and after weight reduction, and may lead to the development of novel treatment strategies for obesity and related metabolic disorders.

Apolipoprotein A-I (ApoA-I) of high-density lipoprotein (HDL) is essential for the transportation of cholesterol between peripheral tissues and the liver. However, specific mutations in Apolipoprotein A-I (ApoA-I) of high-density lipoprotein (HDL) are responsible for a late-onset systemic amyloidosis, the pathological accumulation of protein fibrils in tissues and organs. Carriers of these mutations do not exhibit increased cardiovascular disease risk despite displaying reduced levels of ApoA-I/ HDL-cholesterol. To explain this paradox, we show that the HDL particle profile of patients carrying either L75P or L174S ApoA-I amyloidogenic variants a higher relative abundance of the 8.4 nm vs 9.6 nm particles, and that serum from patients, as well as reconstituted 8.4 and 9.6 nm HDL particles (rHDL), possess increased capacity to catalyze cholesterol efflux from macrophages. Synchrotron radiation circular dichroism and hydrogen-deuterium exchange revealed that the variants in 8.4 nm rHDL have altered secondary structure composition and display a more flexible binding to lipids compared to their native counterpart. The reduced HDL-cholesterol levels of patients carrying ApoA-I amyloidogenic variants are thus balanced by higher proportion of small, dense HDL particles and better cholesterol efflux due to altered, region-specific protein structure dynamics.

Microtubules are polymers composed of αβ-tubulin subunits that provide structure to cells and play a crucial role in in the development and function of neuronal processes and cilia, microtubule-driven extensions of the plasma membrane that have sensory (primary cilia) or motor (motile cilia) functions. To stabilize microtubules in neuronal processes and cilia, α tubulin is modified by the posttranslational addition of an acetyl group, or acetylation. We discovered that acetylated tubulin in microtubules interacts with the membrane sphingolipid, ceramide. However, the molecular mechanism and function of this interaction are not understood. Here, we show that in human iPS cell-derived neurons, ceramide stabilizes microtubules, which indicates a similar function in cilia. Using proximity ligation assays, we detected complex formation of ceramide with acetylated tubulin in C. reinhardtii flagella and cilia of human embryonic kidney (HEK293T) cells, primary cultured mouse astrocytes, and ependymal cells. Using incorporation of palmitic azide and click chemistry-mediated addition of fluorophores, we show that a portion of acetylated tubulin is S-palmitoylated. S-palmitoylated acetylated tubulin is colocalized with ceramide-rich platforms (CRPs) in the ciliary membrane, and it is coimmunoprecipitated with Arl13b, a GTPase that mediates transport of proteins into cilia. Inhibition of S-palmitoylation with 2-bromo palmitic acid or inhibition of ceramide biosynthesis with fumonisin B1 reduces formation of the Arl13b-acetylated tubulin complex and its transport into cilia, concurrent with impairment of ciliogenesis. Together, these data show, for the first time, that CRPs mediate membrane anchoring and interaction of S-palmitoylated proteins that are critical for cilium formation, stabilization, and function. 

Atherosclerosis is characterized by the pathological accumulation of cholesterol-laden macrophages in the arterial wall. Atherosclerosis is also the main underlying cause of CVDs, and its development is largely driven by elevated plasma cholesterol. Strong epidemiological data find an inverse association between plasma β-carotene with atherosclerosis, and we recently showed that β-carotene oxygenase 1 (BCO1) activity, responsible for β-carotene cleavage to vitamin A, is associated with reduced plasma cholesterol in humans and mice. In this study, we explore whether intact β-carotene or vitamin A affects atherosclerosis progression in the atheroprone LDLR-deficient mice. Compared with control-fed Ldlr^–/– mice, β-carotene-supplemented mice showed reduced atherosclerotic lesion size at the level of the aortic root and reduced plasma cholesterol levels. These changes were absent in Ldlr^–/–/Bco1^–/– mice despite accumulating β-carotene in plasma and atherosclerotic lesions. We discarded the implication of myeloid BCO1 in the development of atherosclerosis by performing bone marrow transplant experiments. Lipid production assays found that retinoic acid, the active form of vitamin A, reduced the secretion of newly synthetized triglyceride and cholesteryl ester in cell culture and mice. Overall, our findings provide insights into the role of BCO1 activity and vitamin A in atherosclerosis progression through the regulation of hepatic lipid metabolism.

N-acylethanolamines (NAEs) are endogenous lipid-signaling molecules derived from fatty acids that regulate numerous biological functions, including in the brain. Interestingly, NAEs are elevated in the absence of fatty acid amide hydrolase (FAAH) and following CO₂-induced ischemia/hypercapnia, suggesting a neuroprotective response. Tetracosahexaenoic acid (THA) is a product and precursor to DHA; however, the NAE product, tetracosahexaenoylethanolamide (THEA), has never been reported. Presently, THEA was chemically synthesized as an authentic standard to confirm THEA presence in biological tissues. Whole brains were collected and analyzed for unesterified THA, total THA, and THEA in wild-type and FAAH-KO mice that were euthanized by either head-focused microwave fixation, CO₂ + microwave, or CO₂ only. PPAR activity by transient transfection assay and ex vivo neuronal output in medium spiny neurons (MSNs) of the nucleus accumbens by patch clamp electrophysiology were determined following THEA exposure. THEA in the wild-type mice was nearly doubled (P < 0.05) following ischemia/hypercapnia (CO₂ euthanization) and up to 12 times higher (P < 0.001) in the FAAH-KO compared with wild-type. THEA did not increase (P > 0.05) transcriptional activity of PPARs relative to control, but 100 nM of THEA increased (P < 0.001) neuronal output in MSNs of the nucleus accumbens. Here were identify a novel NAE, THEA, in the brain that is elevated upon ischemia/hypercapnia and by KO of the FAAH enzyme. While THEA did not activate PPAR, it augmented the excitability of MSNs in the nucleus accumbens. Overall, our results suggest that THEA is a novel NAE that is produced in the brain upon ischemia/hypercapnia and regulates neuronal excitation.

Bile acids, which are synthesized from cholesterol by the liver, are chemically transformed along the intestinal tract by the gut microbiota, and the products of these transformations signal through host receptors, affecting overall host health. These transformations include bile acid deconjugation, oxidation, and 7α-dehydroxylation. An understanding of the biogeography of bile acid transformations in the gut is critical because deconjugation is a prerequisite for 7α-dehydroxylation and because most gut microorganisms harbor bile acid transformation capacity. Here, we used a coupled metabolomic and metaproteomic approach to probe in vivo activity of the gut microbial community in a gnotobiotic mouse model. Results revealed the involvement of Clostridium scindens in 7α-dehydroxylation, of the genera Muribaculum and Bacteroides in deconjugation, and of six additional organisms in oxidation (the genera Clostridium, Muribaculum, Bacteroides, Bifidobacterium, Acutalibacter, and Akkermansia). Furthermore, the bile acid profile in mice with a more complex microbiota, a dysbiosed microbiota, or no microbiota was considered. For instance, conventional mice harbor a large diversity of bile acids, but treatment with an antibiotic such as clindamycin results in the complete inhibition of 7α-dehydroxylation, underscoring the strong inhibition of organisms that are capable of carrying out this process by this compound. Finally, a comparison of the hepatic bile acid pool size as a function of microbiota revealed that a reduced microbiota affects host signaling but not necessarily bile acid synthesis. In this study, bile acid transformations were mapped to the associated active microorganisms, offering a systematic characterization of the relationship between microbiota and bile acid composition.

Oxylipins are potent lipid mediators involved in a variety of physiological processes. Their profiling has the potential to provide a wealth of information regarding human health and disease and is a promising technology for translation into clinical applications. However, results generated by independent groups are rarely comparable, which increases the need for the implementation of internationally agreed upon protocols. We performed an interlaboratory comparison for the MS-based quantitative analysis of total oxylipins. Five independent laboratories assessed the technical variability and comparability of 133 oxylipins using a harmonized and standardized protocol, common biological materials (i.e., seven quality control plasmas), standard calibration series, and analytical methods. The quantitative analysis was based on a standard calibration series with isotopically labeled internal standards. Using the standardized protocol, the technical variance was within ±15% for 73% of oxylipins; however, most epoxy fatty acids were identified as critical analytes due to high variabilities in concentrations. The comparability of concentrations determined by the laboratories was examined using consensus value estimates and unsupervised/supervised multivariate analysis (i.e., principal component analysis and partial least squares discriminant analysis). Interlaboratory variability was limited and did not interfere with our ability to distinguish the different plasmas. Moreover, all laboratories were able to identify similar differences between plasmas. In summary, we show that by using a standardized protocol for sample preparation, low technical variability can be achieved. Harmonization of all oxylipin extraction and analysis steps led to reliable, reproducible, and comparable oxylipin concentrations in independent laboratories, allowing the generation of biologically meaningful oxylipin patterns.

Some cases of chylomicronemia are caused by autoantibodies against glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1), an endothelial cell protein that shuttles LPL to the capillary lumen. GPIHBP1 autoantibodies prevent binding and transport of LPL by GPIHBP1, thereby disrupting the lipolytic processing of triglyceride-rich lipoproteins. Here, we review the "GPIHBP1 autoantibody syndrome" and summarize clinical and laboratory findings in 22 patients. All patients had GPIHBP1 autoantibodies and chylomicronemia, but we did not find a correlation between triglyceride levels and autoantibody levels. Many of the patients had a history of pancreatitis, and most had clinical and/or serological evidence of autoimmune disease. IgA autoantibodies were present in all patients, and IgG4 autoantibodies were present in 19 of 22 patients. Patients with GPIHBP1 autoantibodies had low plasma LPL levels, consistent with impaired delivery of LPL into capillaries. Plasma levels of GPIHBP1, measured with a monoclonal antibody–based ELISA, were very low in 17 patients, reflecting the inability of the ELISA to detect GPIHBP1 in the presence of autoantibodies (immunoassay interference). However, GPIHBP1 levels were very high in five patients, indicating little capacity of their autoantibodies to interfere with the ELISA. Recently, several GPIHBP1 autoantibody syndrome patients were treated successfully with rituximab, resulting in the disappearance of GPIHBP1 autoantibodies and normalization of both plasma triglyceride and LPL levels. The GPIHBP1 autoantibody syndrome should be considered in any patient with newly acquired and unexplained chylomicronemia.

The adipocyte-derived hormone leptin increases trafficking of KATP and Kv2.1 channels to the pancreatic β-cell surface, resulting in membrane hyperpolarization and suppression of insulin secretion. We have previously shown that this effect of leptin is mediated by the NMDA subtype of glutamate receptors (NMDARs). It does so by potentiating NMDAR activity, thus enhancing Ca2+ influx and the ensuing downstream signaling events that drive channel trafficking to the cell surface. However, the molecular mechanism by which leptin potentiates NMDARs in β-cells remains unknown. Here, we report that leptin augments NMDAR function via Src kinase–mediated phosphorylation of the GluN2A subunit. Leptin-induced membrane hyperpolarization diminished upon pharmacological inhibition of GluN2A but not GluN2B, indicating involvement of GluN2A-containing NMDARs. GluN2A harbors tyrosine residues that, when phosphorylated by Src family kinases, potentiate NMDAR activity. We found that leptin increases phosphorylation of Tyr-418 in Src, an indicator of kinase activation. Pharmacological inhibition of Src or overexpression of a kinase-dead Src mutant prevented the effect of leptin, whereas a Src kinase activator peptide mimicked it. Using mutant GluN2A overexpression, we show that Tyr-1292 and Tyr-1387 but not Tyr-1325 are responsible for the effect of leptin. Importantly, β-cells from db/db mice, a type 2 diabetes mouse model lacking functional leptin receptors, or from obese diabetic human donors failed to respond to leptin but hyperpolarized in response to NMDA. Our study reveals a signaling pathway wherein leptin modulates NMDARs via Src to regulate β-cell excitability and suggests NMDARs as a potential target to overcome leptin resistance.

SLC19A2 and SLC19A3, also known as thiamine transporters (THTR) 1 and 2, respectively, transport the positively charged thiamine (vitamin B1) into cells to enable its efficient utilization. SLC19A2 and SLC19A3 are also known to transport structurally unrelated cationic drugs, such as metformin, but whether this charge selectivity extends to other molecules, such as pyridoxine (vitamin B6), is unknown. We tested this possibility using Madin-Darby canine kidney II (MDCKII) cells and human embryonic kidney 293 (HEK293) cells for transfection experiments, and also using Caco-2 cells as human intestinal epithelial model cells. The stable expression of SLC19A2 and SLC19A3 in MDCKII cells (as well as their transient expression in HEK293 cells) led to a significant induction in pyridoxine uptake at pH 5.5 compared with control cells. The induced uptake was pH-dependent, favoring acidic conditions over neutral to basic conditions, and protonophore-sensitive. It was saturable as a function of pyridoxine concentration, with an apparent Km of 37.8 and 18.5 μm, for SLC19A2 and SLC19A3, respectively, and inhibited by the pyridoxine analogs pyridoxal and pyridoxamine as well as thiamine. We also found that silencing the endogenous SLC19A3, but not SLC19A2, of Caco-2 cells with gene-specific siRNAs lead to a significant reduction in carrier-mediated pyridoxine uptake. These results show that SLC19A2 and SLC19A3 are capable of recognizing/transporting pyridoxine, favoring acidic conditions for operation, and suggest a possible role for these transporters in pyridoxine transport mainly in tissues with an acidic environment like the small intestine, which has an acidic surface microclimate.

In-depth coverage of proteomic analysis could enhance our understanding to the mechanism of the protein functions. Unfortunately, many highly hydrophobic proteins and low-abundance proteins, which play critical roles in signaling networks, are easily lost during sample preparation, mainly attributed to the fact that very few extractants can simultaneously satisfy the requirements on strong solubilizing ability to membrane proteins and good enzyme compatibility. Thus, it is urgent to screen out ideal extractant from the huge compound libraries in a fast and effective way. Herein, by investigating the interior mechanism of extractants on the membrane proteins solubilization and trypsin compatibility, a molecular dynamics simulation system was established as complement to the experimental procedure to narrow down the scope of candidates for proteomics analysis. The simulation data shows that the van der Waals interaction between cation group of ionic liquid and membrane protein is the dominant factor in determining protein solubilization. In combination with the experimental data, 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) is on the shortlist for the suitable candidates from comprehensive aspects. Inspired by the advantages of C12Im-Cl, an ionic liquid-based filter-aided sample preparation (i-FASP) method was developed. Using this strategy, over 3,300 proteins were confidently identified from 10³ HeLa cells (~100 ng proteins) in a single run, an improvement of 53% over the conventional FASP method. Then the i-FASP method was further successfully applied to the label-free relative quantitation of human liver cancer and para-carcinoma tissues with obviously improved accuracy, reproducibility and coverage than the commonly used urea-based FASP method. The above results demonstrated that the i-FASP method could be performed as a versatile tool for the in-depth coverage proteomic analysis of biological samples.

Ion mobility separates molecules in the gas-phase based on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a TOF mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking MS (XL-MS). Cross-linking reagents covalently link amino acids in proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent – termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500 Ų and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50-70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides.