Carnosine (β-alanyl-l-histidine) and anserine (β-alanyl-3-methyl-l-histidine) are abundant peptides in the nervous system and skeletal muscle of many vertebrates. Many in vitro and in vivo studies demonstrated that exogenously added carnosine can improve muscle contraction, has antioxidant activity, and can quench various reactive aldehydes. Some of these functions likely contribute to the proposed anti-aging activity of carnosine. However, the physiological role of carnosine and related histidine-containing dipeptides (HCDs) is not clear. In this study, we generated a mouse line deficient in carnosine synthase (Carns1). HCDs were undetectable in the primary olfactory system and skeletal muscle of Carns1-deficient mice. Skeletal muscle contraction in these mice, however, was unaltered, and there was no evidence for reduced pH-buffering capacity in the skeletal muscle. Olfactory tests did not reveal any deterioration in 8-month-old mice lacking carnosine. In contrast, aging (18–24-month-old) Carns1-deficient mice exhibited olfactory sensitivity impairments that correlated with an age-dependent reduction in the number of olfactory receptor neurons. Whereas we found no evidence for elevated levels of lipoxidation and glycation end products in the primary olfactory system, protein carbonylation was increased in the olfactory bulb of aged Carns1-deficient mice. Taken together, these results suggest that carnosine in the olfactory system is not essential for information processing in the olfactory signaling pathway but does have a role in the long-term protection of olfactory receptor neurons, possibly through its antioxidant activity.

Clostridium difficile is an anaerobic and spore-forming bacterium responsible for 15–25% of postantibiotic diarrhea and 95% of pseudomembranous colitis. Peptidoglycan is a crucial element of the bacterial cell wall that is exposed to the host, making it an important target for the innate immune system. The C. difficile peptidoglycan is largely N-deacetylated on its glucosamine (93% of muropeptides) through the activity of enzymes known as N-deacetylases, and this N-deacetylation modulates host–pathogen interactions, such as resistance to the bacteriolytic activity of lysozyme, virulence, and host innate immune responses. C. difficile genome analysis showed that 12 genes potentially encode N-deacetylases; however, which of these N-deacetylases are involved in peptidoglycan N-deacetylation remains unknown. Here, we report the enzymes responsible for peptidoglycan N-deacetylation and their respective regulation. Through peptidoglycan analysis of several mutants, we found that the N-deacetylases PdaV and PgdA act in synergy. Together they are responsible for the high level of peptidoglycan N-deacetylation in C. difficile and the consequent resistance to lysozyme. We also characterized a third enzyme, PgdB, as a glucosamine N-deacetylase. However, its impact on N-deacetylation and lysozyme resistance is limited, and its physiological role remains to be dissected. Finally, given the influence of peptidoglycan N-deacetylation on host defense against pathogens, we investigated the virulence and colonization ability of the mutants. Unlike what has been shown in other pathogenic bacteria, a lack of N-deacetylation in C. difficile is not linked to a decrease in virulence.

Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca2+ entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because O-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 O-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 O-GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 WT (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells. Using these cells, we examined the possible O-GlcNAcylation sites of STIM1 to determine whether the sites were O-GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser621 and Thr626 were O-GlcNAcylated and that Thr626 was O-GlcNAcylated in the steady state but Ser621 was not. The SOCE activity in STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells was lower than that in STIM1-KO-WT-HEK cells because of reduced phosphorylation at Ser621. Treatment with the O-GlcNAcase inhibitor Thiamet G or O-GlcNAc transferase (OGT) transfection, which increases O-GlcNAcylation, reduced SOCE activity, whereas treatment with the OGT inhibitor ST045849 or siOGT transfection, which decreases O-GlcNAcylation, also reduced SOCE activity. Decrease in SOCE activity due to increase and decrease in O-GlcNAcylation was attributable to reduced phosphorylation at Ser621. These data suggest that both decrease in O-GlcNAcylation at Thr626 and increase in O-GlcNAcylation at Ser621 in STIM1 lead to impairment of SOCE activity through decrease in Ser621 phosphorylation. Targeting STIM1 O-GlcNAcylation could provide a promising treatment option for the related diseases, such as neurodegenerative diseases.

Infiltration of peripheral immune cells after blood-brain barrier dysfunction causes severe inflammation after a stroke. Although the endothelial glycocalyx, a network of membrane-bound glycoproteins and proteoglycans that covers the lumen of endothelial cells, functions as a barrier to circulating cells, the relationship between stroke severity and glycocalyx dysfunction remains unclear. In this study, glycosaminoglycans, a component of the endothelial glycocalyx, were studied in the context of ischemic stroke using a photochemically induced thrombosis mouse model. Decreased levels of heparan sulfate and chondroitin sulfate and increased activity of hyaluronidase 1 and heparanase (HPSE) were observed in ischemic brain tissues. HPSE expression in cerebral vessels increased after stroke onset and infarct volume greatly decreased after co-administration of N-acetylcysteine + glycosaminoglycan oligosaccharides as compared with N-acetylcysteine administration alone. These results suggest that the endothelial glycocalyx was injured after the onset of stroke. Interestingly, scission activity of proHPSE produced by immortalized endothelial cells and HEK293 cells transfected with hHPSE1 cDNA were activated by acrolein (ACR) exposure. We identified the ACR-modified amino acid residues of proHPSE using nano LC–MS/MS, suggesting that ACR modification of Lys139 (6-kDa linker), Lys107, and Lys161, located in the immediate vicinity of the 6-kDa linker, at least in part is attributed to the activation of proHPSE. Because proHPSE, but not HPSE, localizes outside cells by binding with heparan sulfate proteoglycans, ACR-modified proHPSE represents a promising target to protect the endothelial glycocalyx.

α-Linked galactose is a common carbohydrate motif in nature that is processed by a variety of glycoside hydrolases from different families. Terminal Galα1–3Gal motifs are found as a defining feature of different blood group and tissue antigens, as well as the building block of the marine algal galactan λ-carrageenan. The blood group B antigen and linear α-Gal epitope can be processed by glycoside hydrolases in family GH110, whereas the presence of genes encoding GH110 enzymes in polysaccharide utilization loci from marine bacteria suggests a role in processing λ-carrageenan. However, the structure–function relationships underpinning the α-1,3-galactosidase activity within family GH110 remain unknown. Here we focus on a GH110 enzyme (PdGH110B) from the carrageenolytic marine bacterium Pseudoalteromonas distincta U2A. We showed that the enzyme was active on Galα1–3Gal but not the blood group B antigen. X-ray crystal structures in complex with galactose and unhydrolyzed Galα1–3Gal revealed the parallel β-helix fold of the enzyme and the structural basis of its inverting catalytic mechanism. Moreover, an examination of the active site reveals likely adaptations that allow accommodation of fucose in blood group B active GH110 enzymes or, in the case of PdGH110, accommodation of the sulfate groups found on λ-carrageenan. Overall, this work provides insight into the first member of a predominantly marine clade of GH110 enzymes while also illuminating the structural basis of α-1,3-galactoside processing by the family as a whole.

Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α2-macroglobulin–like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity–conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1's thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1's protease inhibition at pH 5, indicating that A2ML1's hydroxyl reactivity is not an adaption to its acidic epidermal environment.

The bacterial enhancer-binding protein (bEBP) FlrC, controls motility and colonization of Vibrio cholerae by regulating the transcription of class-III flagellar genes in σ54-dependent manner. However, the mechanism by which FlrC regulates transcription is not fully elucidated. Although, most bEBPs require nucleotides to stimulate the oligomerization necessary for function, our previous study showed that the central domain of FlrC (FlrCC) forms heptamer in a nucleotide-independent manner. Furthermore, heptameric FlrCC binds ATP in “cis-mediated” style without any contribution from sensor I motif 285REDXXYR291 of the trans protomer. This atypical ATP binding raises the question of whether heptamerization of FlrC is solely required for transcription regulation, or if it is also critical for ATPase activity. ATPase assays and size exclusion chromatography of the trans-variants FlrCC-Y290A and FlrCC-R291A showed destabilization of heptameric assembly with concomitant abrogation of ATPase activity. Crystal structures showed that in the cis-variant FlrCC-R349A drastic shift of Walker A encroached ATP-binding site, whereas the site remained occupied by ADP in FlrCC-Y290A. We postulated that FlrCC heptamerizes through concentration-dependent cooperativity for maximal ATPase activity and upon heptamerization, packing of trans-acting Tyr290 against cis-acting Arg349 compels Arg349 to maintain proper conformation of Walker A. Finally, a Trp quenching study revealed binding of cyclic-di-GMP with FlrCC. Excess cyclic-di-GMP repressed ATPase activity of FlrCC through destabilization of heptameric assembly, especially at low concentration of protein. Systematic phylogenetic analysis allowed us to propose similar regulatory mechanisms for FlrCs of several Vibrio species and a set of monotrichous Gram-negative bacteria.

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol-sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81–cholesterol association but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol-unbound) or closed (cholesterol-bound) conformation. The open mutant of CD81 exhibited reduced HCV receptor activity, whereas the closed mutant enhanced activity. These data are consistent with cholesterol sensing switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81–partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry, and CD81's function as a molecular scaffold; these insights are relevant to CD81's varied roles in both health and disease.

Chorismate mutase (CM), an essential enzyme at the branch-point of the shikimate pathway, is required for the biosynthesis of phenylalanine and tyrosine in bacteria, archaea, plants, and fungi. MtCM, the CM from Mycobacterium tuberculosis, has less than 1% of the catalytic efficiency of a typical natural CM and requires complex formation with 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase for high activity. To explore the full potential of MtCM for catalyzing its native reaction, we applied diverse iterative cycles of mutagenesis and selection, thereby raising kcat/Km 270-fold to 5 × 105 m−1s−1, which is even higher than for the complex. Moreover, the evolutionarily optimized autonomous MtCM, which had 11 of its 90 amino acids exchanged, was stabilized compared with its progenitor, as indicated by a 9 °C increase in melting temperature. The 1.5 Å crystal structure of the top-evolved MtCM variant reveals the molecular underpinnings of this activity boost. Some acquired residues (e.g. Pro52 and Asp55) are conserved in naturally efficient CMs, but most of them lie beyond the active site. Our evolutionary trajectories reached a plateau at the level of the best natural enzymes, suggesting that we have exhausted the potential of MtCM. Taken together, these findings show that the scaffold of MtCM, which naturally evolved for mediocrity to enable inter-enzyme allosteric regulation of the shikimate pathway, is inherently capable of high activity.

Identification of antibody-binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome, which is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort, is frequently involved in this cross-reactivity, but no antibody-binding epitopes have been determined so far. To reveal human IgE-binding regions of Art v 3, we produced three murine high-affinity mAbs, which showed 70–90% coverage of the allergenic epitopes from mugwort pollen–allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free 15N-labeled antigen whose 1H-15N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4 as well as a novel Art v 3–specific epitope at the C terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP allergy and facilitates design of therapeutics.

Sirtuin 6, SIRT6, is critical for both glucose and lipid homeostasis and is involved in maintaining genomic stability under conditions of oxidative DNA damage such as those observed in age-related diseases. There is an intense search for modulators of SIRT6 activity, however, not many specific activators have been reported. Long acyl-chain fatty acids have been shown to increase the weak in vitro deacetylase activity of SIRT6 but this effect is modest at best. Herein we report that electrophilic nitro-fatty acids (nitro-oleic acid and nitro-conjugated linoleic acid) potently activate SIRT6. Binding of the nitro-fatty acid to the hydrophobic crevice of the SIRT6 active site exerted a moderate activation (2-fold at 20 μm), similar to that previously reported for non-nitrated fatty acids. However, covalent Michael adduct formation with Cys-18, a residue present at the N terminus of SIRT6 but absent from other isoforms, induced a conformational change that resulted in a much stronger activation (40-fold at 20 μm). Molecular modeling of the resulting Michael adduct suggested stabilization of the co-substrate and acyl-binding loops as a possible additional mechanism of SIRT6 activation by the nitro-fatty acid. Importantly, treatment of cells with nitro-oleic acid promoted H3K9 deacetylation, whereas oleic acid had no effect. Altogether, our results show that nitrated fatty acids can be considered a valuable tool for specific SIRT6 activation, and that SIRT6 should be considered as a molecular target for in vivo actions of these anti-inflammatory nitro-lipids.

Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs −1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical −1 and −2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1β (nsp1β) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1β and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate −1 and −2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1β and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1β:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1β within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1β and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1β and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism.

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

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Viral infection is one environmental factor that may contribute to the initiation of pancreatic β-cell destruction during the development of autoimmune diabetes. Picornaviruses, such as encephalomyocarditis virus (EMCV), induce a pro-inflammatory response in islets leading to local production of cytokines, such as IL-1, by resident islet leukocytes. Furthermore, IL-1 is known to stimulate β-cell expression of iNOS and production of the free radical nitric oxide. The purpose of this study was to determine whether nitric oxide contributes to the β-cell response to viral infection. We show that nitric oxide protects β-cells against virally mediated lysis by limiting EMCV replication. This protection requires low micromolar, or iNOS-derived, levels of nitric oxide. At these concentrations nitric oxide inhibits the Krebs enzyme aconitase and complex IV of the electron transport chain. Like nitric oxide, pharmacological inhibition of mitochondrial oxidative metabolism attenuates EMCV-mediated β-cell lysis by inhibiting viral replication. These findings provide novel evidence that cytokine signaling in β-cells functions to limit viral replication and subsequent β-cell lysis by attenuating mitochondrial oxidative metabolism in a nitric oxide–dependent manner.

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division, the machinery responsible for PG synthesis localizes mid-cell, at the septum, under the control of a multiprotein complex called the divisome. In Escherichia coli, septal PG synthesis and cell constriction rely on the accumulation of FtsN at the division site. Interestingly, a short sequence of FtsN (Leu75–Gln93, known as EFtsN) was shown to be essential and sufficient for its functioning in vivo, but what exactly this sequence is doing remained unknown. Here, we show that EFtsN binds specifically to the major PG synthase PBP1b and is sufficient to stimulate its biosynthetic glycosyltransferase (GTase) activity. We also report the crystal structure of PBP1b in complex with EFtsN, which demonstrates that EFtsN binds at the junction between the GTase and UB2H domains of PBP1b. Interestingly, mutations to two residues (R141A/R397A) within the EFtsN-binding pocket reduced the activation of PBP1b by FtsN but not by the lipoprotein LpoB. This mutant was unable to rescue the ΔponB-ponAts strain, which lacks PBP1b and has a thermosensitive PBP1a, at nonpermissive temperature and induced a mild cell-chaining phenotype and cell lysis. Altogether, the results show that EFtsN interacts with PBP1b and that this interaction plays a role in the activation of its GTase activity by FtsN, which may contribute to the overall septal PG synthesis and regulation during cell division.

Poly(ADP-ribose) polymerase (PARP) superfamily members covalently link either a single ADP-ribose (ADPR) or a chain of ADPR units to proteins using NAD as the source of ADPR. Although the well-known poly(ADP-ribosylating) (PARylating) PARPs primarily function in the DNA damage response, many noncanonical mono(ADP-ribosylating) (MARylating) PARPs are associated with cellular antiviral responses. We recently demonstrated robust up-regulation of several PARPs following infection with murine hepatitis virus (MHV), a model coronavirus. Here we show that SARS-CoV-2 infection strikingly up-regulates MARylating PARPs and induces the expression of genes encoding enzymes for salvage NAD synthesis from nicotinamide (NAM) and nicotinamide riboside (NR), while down-regulating other NAD biosynthetic pathways. We show that overexpression of PARP10 is sufficient to depress cellular NAD and that the activities of the transcriptionally induced enzymes PARP7, PARP10, PARP12 and PARP14 are limited by cellular NAD and can be enhanced by pharmacological activation of NAD synthesis. We further demonstrate that infection with MHV induces a severe attack on host cell NAD+ and NADP+. Finally, we show that NAMPT activation, NAM, and NR dramatically decrease the replication of an MHV that is sensitive to PARP activity. These data suggest that the antiviral activities of noncanonical PARP isozyme activities are limited by the availability of NAD and that nutritional and pharmacological interventions to enhance NAD levels may boost innate immunity to coronaviruses.

COP27: Navigating a difficult road to Sharm El-Sheikh Expert comment NCapeling 6 July 2022

Against a backdrop of rising urgency, COP27 in Egypt will bring all aspects of climate action into the spotlight – but especially the role of the host country.

As COP26 drew to a close in Glasgow, Egyptian officials announced their priorities for COP27, emphasizing climate finance and climate adaptation – a new approach given previous COPs mainly focused on mitigation, reducing emissions to limit climate damage.

This was followed by the COP27 presidency outlining its vision at MENA Climate Week 2022 to achieve ‘substantive and equal progress’ on all aspects of the negotiations, and Egypt emphasizing its intention to focus on implementing existing carbon reduction targets rather than pushing for further carbon cuts.

Egypt argues it is hosting COP27 on behalf of African nations and that, while it is promoting the interests of the developing world, it will be an impartial arbiter. However it is also useful to consider its priorities from the Egyptian government’s perspective.

Agenda drivers

Egypt has long prioritized climate finance and adaptation because it remains in need of technical and financial support to adapt, especially in agriculture and tourism.

It plans to expand its access to climate funding and investment, an area in which Egypt has been relatively successful as it currently receives 27 per cent of all multilateral climate finance in the MENA region and has issued the region’s first sovereign green bonds.

With public debt currently 94 per cent of GDP, Egyptian officials have also called for debt relief for Egypt and other developing countries.

Egypt’s Climate Change Strategy reflects this approach, aiming to enhance Egypt’s rank on the Climate Change Performance Index in order to ‘attract more investments and acquire more climate funding’.

Limiting the mitigation scope and the focus on finance also echoes Egypt’s own reluctance to make carbon reduction commitments. The Egyptian nationally determined contribution (NDC) – its 2030 pledge under the Paris Agreement – does not include any quantifiable emission reduction targets.

Egypt is one of only a few countries which failed to submit an updated NDC in 2021 and its upcoming update will not include an economy-wide carbon reduction target.

Egypt has also never published a long-term strategy and has no decarbonization plans despite independent estimates it should cut rising emissions by one-quarter by 2030, and by two-thirds by 2050 to be aligned with the Paris Agreement. This partly explains why observers rate Egypt’s climate action as highly insufficient.

Furthermore, Egypt’s championing of ‘moving from pledges to implementation’ without having quantifiable carbon reduction pledges of its own effectively exempts it from both pledging and implementation.

As a developing country, Egypt’s negotiating position is supported by UNFCCC provisions which recognize differentiated responsibilities and respective capabilities of nations.

Its proposal to focus COP27 on the implementation of climate action and finance pledges is important in consolidating progress. But not pushing for more emission reductions at this critical moment risks derailing global decarbonization momentum and undermining global climate action.

According to optimistic estimates, if current climate pledges were implemented the world would still remain on track for 2°C of warming by the end of the century, with far worse impacts than if warming was curbed at 1.5°C.

Under a 2°C scenario, 37 per cent of the global population could regularly be exposed to extreme heat waves compared to 14 per cent in a 1.5°C warmer world, with developing countries expected to be worst-affected.

A 2°C trajectory also runs the risk of tipping points such as the melting of ice sheets in Antarctica and Greenland, triggering runaway climate change. Time to change the warming trajectory is running out as the latest IPCC assessment warns the window of opportunity is now ‘brief and rapidly closing’, and the UN Secretary General recently called for faster carbon cuts by the end of 2022 to avoid a ‘climate catastrophe’.

A different energy transition

Egypt opted not to join any of the voluntary sectoral coalitions at COP26 on reducing methane, clean energy transition, transition to zero-emissions vehicles, or moving beyond oil and gas.

This position is explained by its growing role as an exporter and advocate for fossil gas in the energy transition. Egypt is the second-largest producer of natural gas in Africa and is emerging as a fossil gas hub for the eastern Mediterranean, which is shaping its domestic energy policy.

Its 59GW electricity generation capacity is almost double the peak demand and is dominated by gas-powered electricity generation, which currently represents 42 per cent of all Africa’s gas generation.

Egypt’s climate policy is also shaped by fossil gas, and its national Climate Change Strategy encourages the expansion of gas use by promoting a transition to compressed natural gas for vehicles, the expansion of its domestic natural gas network – despite having universal access to electricity – and shifting to a gas-fuelled shipping sector.

Egypt also voiced support for other African countries to extract and deploy fossil gas and oil resources, making it one of the protagonists of the ‘great fossil gas pushback’. These advocates defend the right of developing countries to deploy fossil gas as a ‘transition fuel’ and champion its necessity to solve energy poverty.

But their position is not shared by all African and developing countries, and is rejected by some civil society groups, who argue it risks locking in greenhouse gases and local emissions for decades as well as delaying future development of low carbon energy systems.

Egypt’s huge spare generation capacity has contributed to a slowdown in renewable energy projects over the past two years. With renewables representing just 6GW, Egypt is expected to miss its renewable energy target for 2022, set at 20 per cent of generating capacity.

Engaging Egypt better

But these positions are more malleable than they seem, and Egypt is open to dialogue – not just on refining the COP27 agenda but also on reviewing its own climate priorities and leveraging its energy sector for a more ambitious transition.

Review: Decolonization and its discontents The World Today mhiggins.drupal 1 August 2022

Jenna Marshall on a lively if flawed argument to find a way forward in a debate that has descended ‘into acrimony’.

Against Decolonization: Taking African Agency Seriously
Olufemi Taiwo, Hurst, £14.99

Decolonization was once heralded as a moment of potential and possibility for the formerly imperial world to chart a new way forward no longer tied to the apparatus of empire.

It marked a period of transition to nation states – and an expansion of a rights-based international community that would dispense with the morally bankrupt regime of racism, dispossession and subjugation that characterized colonialism.  
 

The descent of the decolonization debate

Half a century later, the debate surrounding decolonization has descended into acrimony. Some factions cast the issue of decolonization as a sustained attack on British and western culture writ large; other disparate voices coalesce around related social justice issues such as inequality, climate change and education. 

Decolonization has become ubiquitous in the public domain – and its ubiquity is the problem. In Against Decolonization, Olufemi Taiwo renounces a concept he conceives as having been emptied of serious study and analytical purchase; one incapable of addressing the complexities of modern global politics and more importantly, Africa’s place in it. 

Although the book attacks the inexhaustible ways in which the ‘trope of decolonization’ has been deployed, Taiwo is less bothered by the purported failures of scholars he deems ‘decolonizers’ and focuses his attention instead on the political landscape of African countries, past and present. 

Central to the book’s argument are two distinct scholarly strands on decolonization. The first, legal focus, centres on the political and economic forces of state-building. The second essays an ‘ideology’ of decolonization that rests on ‘forcing an ex-colony to forswear on pain of being forever under the yoke of colonization any and every cultural, political, intellectual, social and linguistic artefact, idea, process, institution and practice that retains even the slightest whiff of the colonial past’. 

Whether one should characterize the latter as simply a field of study or a potential set of policy arrangements remains unclear – yet what is certain is that it is too nebulous, too elastic, too open-ended to offer any substantive model or mechanism to understand postcolonial Africa. 
 

Correcting Eurocentric narcissism

 The decolonization research agenda in Africa came to prominence during the Cold War period of national liberation struggles.

The intellectual project that followed centred on dismantling Eurocentrism as colonial subjugation through its promotion of the West as the crucible of legitimate and scientific knowledge. Since then, scholars have argued that decolonization itself has become compromised by its enduringly Eurocentric gaze at the expense of the agency of African thinkers, creatives and statesmen and women. 

Taiwo seeks to correct this narcissism – and the omissions left in its wake – by introducing lesser-known Africans and pan-African scholars and cultural figures. These voices, he hopes, will illuminate issues often ignored by ‘decolonizers’ and spark a ‘renewed interest in an appreciation of the many different ways in which African thinkers have responded to the colonial experience’.
 

The decolonization of language

Taiwo challenges the decolonization of language as an oversold promise.

Romanticizing an imagined, pristine African pre-colonial past, he says, ultimately leads to nativism and atavism. As a case in point, Taiwo highlights bureaucratic instances of ‘language policy planning’ to deploy African languages in places such as Niger, Mali, Cameroon, Senegal and Nigeria that were hindered by multilingualism, education and high rates of illiteracy.

The author goes on to confront the abstract language of decolonization, which, he says, allows western scholars to unproblematically engage with the concept without any serious consideration of their own complicity in upholding systems of exclusion. It entrenches their own institutional power within the academy, amplifies their perspectives at the expense of others, and limits the possibilities for understanding the problems of world politics with deleterious effects on policy.

It is a serious claim, but there are issues to be addressed: Taiwo assumes there is coherence among scholars of decolonization, which is not the case. 

When Taiwo approaches how to foster political systems that cater to the needs of their citizens he dismantles the binary of ‘West as modern’ v ‘Africa as traditional’. Chieftaincies as traditional African governance, he points out, were the product of colonial anthropology, not Africans themselves. From the Fanti Confederation of 1871 to the Egba United Government in what is now Nigeria, Taiwo demonstrates that there has been a sustained tradition towards demands for democratic values. 

As he concedes an intellectual neglect of African philosophers that underlies the design and operations of Africa’s political institutions, Taiwo’s initial dismissal of the significance of cultural decolonization deserves another look. How should political and economic drives toward self-determination be advanced in the absence of knowledge shaped and mediated through African lived experiences?

In this respect, the tale of two decolonizations – the legal alongside the ideological – suggest an unhelpful, if not false, binary.
 

The problem of reconciling modernity and colonialism

What resonates throughout the book is the idea that Europe cannot profess to hold an exclusive intellectual claim to modernity. Its universal aspirations are open to all of humanity, allowing those who have historically been marginalized to be worldmakers. 

The idea that modernity and colonialism are irreconcilable is problematic. For instance, Taiwo argues that the curtailment of capitalism in Africa by restricting the growth of the middle classes while limiting competition between African capitalists and the metropole is the consequence of colonialism.

Yet the celebrated cultural anthropologist Sidney Mintz established an understanding that the matter for debate is not whether non-westerners were part of the modern system, but how and to what degree they were included and able to actualise its ideals. 

The emerging capitalist world economy needed non-western lands and labour, and so they were conscripted to this end. Recent abolitionist and black radical scholarship has built on this argument to maintain that capitalism not only requires inequality for it to function but that race – as a mechanism for producing ‘difference’ – enshrines it.

Acknowledging the unacknowledged

In the end, Taiwo communicates a palpable frustration at the state of academic discourses on contemporary Africa under the guise of emancipatory and radical scholarship. He offers an alternative approach whereby African students might rid themselves of a faddism that is ‘at best unsatisfactory’ and at worst produces ‘confusion, obscurantism, if not outright distortion and falsification’.

Yet out of this irritation, Taiwo’s greatest contribution in Against Decolonization might be to urge an acknowledgement of how and to what degree decolonization has become subdued and its political possibilities curtailed. He ultimately urges us to reconsider the purpose of the decolonization academic movement as an ethics to abide by rather than a social theory of the postcolonial world. 

This requires those ‘decolonizers’ on whose careers the term is built to adopt greater intellectual humility – to resist the posture of the anarchist radical scholar armed for the ‘good fight’. Instead, they should apply a scholarly curiosity to genuinely engage with those already on the battlefield, so to speak; those ‘doing the work’ in practice, but who have been unacknowledged until now.

Review: the rise of Africa’s superwomen The World Today mhiggins.drupal 1 August 2022

From foster care in England to colonialism’s legacy in Zimbabwe, this set of essays on race, feminism and identity is searingly honest, says Masiyaleti Mbewe.

Black and Female
Tsitsi Dangarembga, Faber, £9.99

The 1988 novel Nervous Conditions by the Zimbabwean author Tsitsi Dangarembga is considered one of Africa’s finest literary exports. It won the Commonwealth Writers’ Prize and alongside The Book of Not (2006) and This Mournable Body (2018), shortlisted for the 2020 Booker Prize, forms a trilogy of semi-autobiographical novels that grapple with the gendered colonial oppression of young black girls and women from Southern Rhodesia through to Zimbabwe. 

In Black and Female, Dangarembga continues the interrogation of these intersections in an unflinchingly honest and personal, if occasionally dense, collection of essays. Along the way, she examines the sheer magnitude of colonialism’s effects on African people, and how they ripple through her early childhood in England and her formative years as a writer, filmmaker and feminist activist in post-independence Zimbabwe.

‘Writing While Black and Female’

In 1961 Dangarembga’s parents relocated from Southern Rhodesia to the UK. While they worked and studied in London, they put their two-year-old daughter, her older brother and, later, her younger sister into private foster care in Dover, Kent (as many Africans did – a fact that was new to me). The first essay, ‘Writing While Black and Female’, takes a painful look at the four years she spent with her foster parents, Mummy-Gran and Daddy Henry. 

Blackness, she learned in those years, was a consequence of her non-whiteness. So Dangarembga writes of the momentary elation she felt when a stranger addressed her as a ‘lovely little piccaninny’, giving her ‘a category I could wield against the void of no longer being’. To cope with this sort of racialization and her abandonment, the young Dangarembga turned to disassociation and self-harm. 

As she writes: ‘Blackness is a condition imposed on me, rather than being an experienced identity.’ Instead of ‘black’ people, therefore, Dangarembga prefers the term ‘highly melanated people’. It is a resonant phrase, highlighting the inherently ridiculous nature of racism.

Dangarembga’s ‘Africanness’ shifts into focus upon her return to Rhodesia in 1965. At first, other children refused to play with her and her siblings, calling them ‘varungu’ (white people). As she describes it: ‘The dance of my identities … became frenetic’. 

In ‘Black, Female and the Superwoman Black Feminist’, the second essay, Dangarembga is adamant about the urgent necessity of a black feminist practice that is centred on action to provide real, material change. Along the way, she makes a distinction – a slightly uneasy one to my mind – between the patriarchy that western colonization imposed, based on private ownership, and the patriarchy of pre-colonial African society with its foundations in kinship that devolved power to an extent.

‘Hence women could and did become rulers and warriors, and royal spirit mediums called mhondoro,’ Dangarembga writes approvingly. She is making a nuanced point; but the idea of a more accommodating sort of patriarchy jars a little nonetheless.

While independence may have arrived for Zimbabwe more than 40 years ago, Dangarembga argues strongly that the subjugation of women and feminists at the hands of the ruling Zanu-PF government continues as an extension of colonial rule. Indeed, beyond Zimbabwe, black feminists remain ‘a small, often embattled group’ across Africa, believes Dangarembga. 

As a young black feminist who is part of this ‘small, embattled group’, I should say we have been able to foster large communities digitally and otherwise to work around the hostility we are often faced with. Despite internet shutdowns and restrictions, we resist – an act Dangarembga encourages.  

Resistance, she says, starts with establishing community despite these difficulties. At the nucleus of Dangarembga’s argument is the ‘superwoman’ of the essay’s title, the African woman who doesn’t require external factors to be inspired to action but who continuously draws on what Dangarembga calls ‘internal agency’ that derives from ‘an unrelenting fight for survival and dignity’.

Pointedly, she criticizes global feminism’s greater focus on optics than activism in the practical sense. One only has to observe the performative allyship and ‘Instagram activism’ rampant on the internet today to see her point.
 

The complexities of decolonization

In the final essay, ‘Decolonization as Revolutionary Imagining’, Dangarembga turns her gaze upon the ‘highly stratified’ European societies that outsourced their violent inequality to their empires and ‘the work of decolonization’. However, decolonial discourse is complex, and it is here that the writing occasionally gets bogged down. Fewer recommendations with more elaboration perhaps would have helped.

She herself acknowledges the difficulties of decolonization. Centuries of the Enlightenment and its logic of ‘racism, slavery, genocide and colonization’ are hard to uproot ‘whatever one’s melanin concentration’, she writes.

Nevertheless, Dangarembga concludes with the radical determination to dismantle that is evident throughout this searing yet hopeful collection: ‘The trajectory of current and future generations depends on that uprooting.’

A new vision for African agency in sustainable development Expert comment LJefferson 18 October 2022

Change is necessary not only in global economic structures and attitudes – but in African governance too.

The conventional notion that Africa is mostly a consumer of norms and practices designed by the Global North has been repeatedly challenged and is increasingly being debunked. Increased African agency in international affairs is today a well-established and documented reality. But Africa’s influence still does not match the scale of the challenges that it faces on its pathway to sustainable development. 

Pushing for African agency in sustainable development also warrants a critical assessment of how ‘sustainable development’ should be defined, and how it can be achieved in terms of actual poverty reduction and real improvement in the lives of local poor Africans. Sustainable development has been a political catchphrase for over 30 years – but a genuine transition towards sustainability has yet to begin.

African agency today

Historically, there have been structural limitations on the agency of African stakeholders to shape development pathways. Chief among them, donor-recipient power dynamics have persistently promoted dependency and sustained institutional corruption. Many African countries are also challenged by economic incentives and infrastructure that have favoured the market demands and supply chains of former colonial powers, which largely remain reliant on natural resource extraction, and are marked by limited investment in value-addition activities and technology development.

Today, however, African agency is being exerted in a more assertive fashion. The African Union (AU), individual African states, civil society, the private sector and eminent and ordinary persons are all displaying Africa’s agency in steering global sustainable development priorities, namely by proposing their own development agenda, The African Union Agenda 2063, adopted in 2013. This was followed by the UN Sustainable Development Goals (Agenda 2030), which in many ways mirror Agenda 2063 – a clear demonstration of the influence of Africa in the global arena. 

Agenda 2063 is a strategic roadmap for Africa ‘to build an integrated, prosperous and peaceful Africa, driven and managed by its own citizens and development goals representing a dynamic force in the international arena’. Prepared following a broad-based participatory consultation, it advocates for inclusion and empowerment and provides an excellent vision for African countries and African people. The SDGs address several of the key shortcomings of their predecessor – the MDGs – and incorporate a broader and more transformative agenda that more adequately reflects the complex challenges of the 21st century and the need for structural reforms in the global economy and governance norms.

In international forums on sustainable development, African countries are increasingly using their collective voice to change the discourse on how development can and should be done. For instance, by championing innovative solutions for carbon markets, African policy leaders are enabling access to climate finance for development while preserving Africa’s natural wealth.

In the post-COVID era, championing investments in and leadership of Africa’s global health architecture demonstrates a desire that in the next pandemic, Africa CDC, AMA and continental manufacturers will play leading roles in determining Africa’s public health strategy and implementation.

In trade, building on the groundwork led by the regional economic commissions, the AfCFTA will catalyse and scale regional integration, trade and cooperation, leading to promising new modes of supply chain and self-sufficiency.

Encouraging signs, but persistent shortcomings

Encouraging signs that African agency is gaining momentum cannot disguise the fact that Africa has yet to move from rhetoric to implementation in the realm of sustainable development. Continental visions often fail to go beyond declarations of intent, and have only limited influence on governance systems or national structural transformation, and African states remain vulnerable to economic shocks emanating from the global system.

Change will require governance systems that are coordinated, transparent, efficient, and inclusive, as well as tools, processes, and means (material, technical, and human) for successful implementation. There is an urgent need for a new governance paradigm in Africa and internationally, dealing with long-term social change.

Notably, African agency should not be only seen as emanating from government, but also as being exerted by independent civil society organizations and committed ordinary individuals. Effective agency needs to be multi-faceted and multi-actor, and depends on the inclusiveness of African governments and their willingness to work with civil society in their strategic engagements with external partners.

Both Agenda 2063 and the SDGs hold the potential for transformation, but implementation will depend on continued advocacy to hold authorities to account and change the dominant discourse, logic and rules of engagement at global, regional, national, and local levels. There is a need for a dynamic new model of African ‘development’.

Time for a new vision

Africa’s economic landscape is changing rapidly, with new regional and local value chains, and integrated regional economic corridors to link countries, minimize the burden of high-cost production and logistics, and boost real incomes and international competitiveness. But Africa continues to face structural challenges, including the need for large investment projects – at a time when Africa remains a net exporter of capital.

Donors and development partners must reflect upon their prior modes of engagement and commit to genuine and equitable relationships with African states. Such partnerships must reflect mutual respect of ideas and accountability, and commit to making space in international forums and multi-lateral arrangements for African people and countries to realize their own visions for progress.

But African leaders and actors must also recognize that with the advent of resources and agency comes responsibility for results and outcomes, notably the need to improve governance. Gaps in accountability, widespread corruption, and lack of successful implementation and sustainability of projects must be addressed.

Africa-Japan relations and evolving multilateralism 23 November 2022 — 9:00AM TO 10:30AM Anonymous (not verified) 17 November 2022 Online

This panel discussion reflects on the outcomes of TICAD 8 in 2022 and looks forward to TICAD 9 in 2025.

The eighth edition of the Tokyo International Conference on African Development (TICAD), held in Tunisia from 27–28 August 2022, marked the second time that Japan’s now-triennial summit was hosted in an African country, after TICAD 6 was held in Kenya in 2016.

The summit was attended by 48 representatives of African countries and at least 20 heads of state and government and included a pledge by the Japanese government to commit $30 billion in public and private finance to Africa over the next three years.

In reaffirming the three pillars of TICAD 8 – revolving around the economy, societal resilience, and peace and stability – the newly adopted Tunis Declaration (28 August 2022) also outlined some of the key projects underpinning Japan’s pledge, including a $4 billion fund for a Green Growth Initiative with Africa (GGA).

2023 will mark 30 years since the inception of TICAD in 1993 and ten years since the African Union (AU)’s adoption of its flagship Agenda 2063, on which the Tunis Declaration placed distinct emphasis.

This panel discussion reflects on the outcomes of TICAD 8 in 2022 and looks forward to TICAD 9 in 2025, exploring wider developments in summitry, Africa-Asia relations, and modes of multilateralism.

Questions explored include:

• How has international summitry evolved over the past three decades since the inception of TICAD in 1993, which represented the first periodic high-level summit engagement with Africa by a ‘non-traditional’ partner?
• Looking ahead to TICAD 9 in 2025, what are the priorities for enforcing the stated tenets of TICAD – ‘African ownership, international partnership, inclusivity and openness’ – in cooperation efforts?
• What lessons can be drawn from TICAD’s co-partnership approach (with the African Union Commission and others) – particularly given increasing calls for AU membership of the G20 and Prime Minister Kishida’s pledge at TICAD 8 to support a permanent African UNSC seat during its non-permanent membership in 2023–24? Beyond membership, what are the priorities for furthering agency?
• How are Africa-Asia relations evolving and diverging? How are Japan and other Asian countries perceived by different African countries?

This event is the third in the Chatham House – Japan House London webinar series (2022-2023). The series is held in partnership with Japan House London. You can watch previous webinars from the series here.

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On beyond “Detect and Respond” and “Secure by Design”

Tricia Rowlison
Dec 1, 2020; 19:2090-2103
Research

Yi Liu
Dec 1, 2020; 19:1968-1985
Research

David Durán
Nov 19, 2020; 0:RA120.002276v1-mcp.RA120.002276
Research

Matthias Schittmayer
Dec 1, 2020; 19:2104-2114
Research

We have observed overexpression of PACS-1, a cytosolic sorting protein in primary cervical tumors. Absence of exonic mutations and overexpression at the RNA level suggested a transcriptional and/or posttranscriptional regulation. University of California Santa Cruz genome browser analysis of PACS-1 micro RNAs (miR), revealed two 8-base target sequences at the 3' terminus for hsa-miR-34a and hsa-miR-449a. Quantitative RT-PCR and Northern blotting studies showed reduced or loss of expression of the two microRNAs in cervical cancer cell lines and primary tumors, indicating dysregulation of these two microRNAs in cervical cancer. Loss of PACS-1 with siRNA or exogenous expression of hsa-miR-34a or hsa-miR-449a in HeLa and SiHa cervical cancer cell lines resulted in DNA damage response, S-phase cell cycle arrest, and reduction in cell growth. Furthermore, the siRNA studies showed that loss of PACS-1 expression was accompanied by increased nuclear γH2AX expression, Lys382-p53 acetylation, and genomic instability. PACS-1 re-expression through LNA-hsa-anti-miR-34a or -449a or through PACS-1 cDNA transfection led to the reversal of DNA damage response and restoration of cell growth. Release of cells post 24-h serum starvation showed PACS-1 nuclear localization at G1-S phase of the cell cycle. Our results therefore indicate that the loss of hsa-miR-34a and hsa-miR-449a expression in cervical cancer leads to overexpression of PACS-1 and suppression of DNA damage response, resulting in the development of chemo-resistant tumors.

Kinases are critical components of intracellular signaling pathways and have been extensively investigated with regard to their roles in cancer. p21-activated kinase-1 (PAK1) is a serine/threonine kinase that has been previously implicated in numerous biological processes, such as cell migration, cell cycle progression, cell motility, invasion, and angiogenesis, in glioma and other cancers. However, the signaling network linked to PAK1 is not fully defined. We previously reported a large-scale yeast genetic interaction screen using toxicity as a readout to identify candidate PAK1 genetic interactions. En masse transformation of the PAK1 gene into 4,653 homozygous diploid Saccharomyces cerevisiae yeast deletion mutants identified ∼400 candidates that suppressed yeast toxicity. Here we selected 19 candidate PAK1 genetic interactions that had human orthologs and were expressed in glioma for further examination in mammalian cells, brain slice cultures, and orthotopic glioma models. RNAi and pharmacological inhibition of potential PAK1 interactors confirmed that DPP4, KIF11, mTOR, PKM2, SGPP1, TTK, and YWHAE regulate PAK1-induced cell migration and revealed the importance of genes related to the mitotic spindle, proteolysis, autophagy, and metabolism in PAK1-mediated glioma cell migration, drug resistance, and proliferation. AKT1 was further identified as a downstream mediator of the PAK1-TTK genetic interaction. Taken together, these data provide a global view of PAK1-mediated signal transduction pathways and point to potential new drug targets for glioma therapy.

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.

Mitochondrial DNA (mtDNA) encodes proteins and RNAs that support the functions of mitochondria and thereby numerous physiological processes. Mutations of mtDNA can cause mitochondrial diseases and are implicated in aging. The mtDNA within cells is organized into nucleoids within the mitochondrial matrix, but how mtDNA nucleoids are formed and regulated within cells remains incompletely resolved. Visualization of mtDNA within cells is a powerful means by which mechanistic insight can be gained. Manipulation of the amount and sequence of mtDNA within cells is important experimentally and for developing therapeutic interventions to treat mitochondrial disease. This review details recent developments and opportunities for improvements in the experimental tools and techniques that can be used to visualize, quantify, and manipulate the properties of mtDNA within cells.

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.

Mutations in the GUCY2D gene coding for the dimeric human retinal membrane guanylyl cyclase (RetGC) isozyme RetGC1 cause various forms of blindness, ranging from rod dysfunction to rod and cone degeneration. We tested how the mutations causing recessive congenital stationary night blindness (CSNB), recessive Leber's congenital amaurosis (LCA1), and dominant cone–rod dystrophy-6 (CORD6) affected RetGC1 activity and regulation by RetGC-activating proteins (GCAPs) and retinal degeneration-3 protein (RD3). CSNB mutations R666W, R761W, and L911F, as well as LCA1 mutations R768W and G982VfsX39, disabled RetGC1 activation by human GCAP1, -2, and -3. The R666W and R761W substitutions compromised binding of GCAP1 with RetGC1 in HEK293 cells. In contrast, G982VfsX39 and L911F RetGC1 retained the ability to bind GCAP1 in cyto but failed to effectively bind RD3. R768W RetGC1 did not bind either GCAP1 or RD3. The co-expression of GUCY2D allelic combinations linked to CSNB did not restore RetGC1 activity in vitro. The CORD6 mutation R838S in the RetGC1 dimerization domain strongly dominated the Ca2+ sensitivity of cyclase regulation by GCAP1 in RetGC1 heterodimer produced by co-expression of WT and the R838S subunits. It required higher Ca2+ concentrations to decelerate GCAP-activated RetGC1 heterodimer—6-fold higher than WT and 2-fold higher than the Ser838-harboring homodimer. The heterodimer was also more resistant than homodimers to inhibition by RD3. The observed biochemical changes can explain the dominant CORD6 blindness and recessive LCA1 blindness, both of which affect rods and cones, but they cannot explain the selective loss of rod function in recessive CSNB.

Type I and III interferons induce expression of the “myxovirus resistance proteins” MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms cytoplasmic structures, whereas murine Mx1 forms nuclear bodies. Whereas both HuMxA and MuMx1 are antiviral toward influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral toward vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles (“biomolecular condensates”). In the present study, we investigated whether nuclear murine Mx1 structures might also represent phase-separated biomolecular condensates. The transient expression of murine GFP-Mx1 in human Huh7 hepatoma, human Mich-2H6 melanoma, and murine NIH 3T3 cells led to the appearance of Mx1 nuclear bodies. These GFP-MuMx1 nuclear bodies were rapidly disassembled by exposing cells to 1,6-hexanediol (5%, w/v), or to hypotonic buffer (40–50 mosm), consistent with properties of membraneless phase-separated condensates. Fluorescence recovery after photobleaching (FRAP) assays revealed that the GFP-MuMx1 nuclear bodies upon photobleaching showed a slow partial recovery (mobile fraction: ∼18%) suggestive of a gel-like consistency. Surprisingly, expression of GFP-MuMx1 in Huh7 cells also led to the appearance of GFP-MuMx1 in 20–30% of transfected cells in a novel cytoplasmic giantin-based intermediate filament meshwork and in cytoplasmic bodies. Remarkably, Huh7 cells with cytoplasmic murine GFP-MuMx1 filaments, but not those with only nuclear bodies, showed antiviral activity toward VSV. Thus, GFP-MuMx1 nuclear bodies comprised phase-separated condensates. Unexpectedly, GFP-MuMx1 in Huh7 cells also associated with cytoplasmic giantin-based intermediate filaments, and such cells showed antiviral activity toward VSV.