By Eireann Van Natta Dr. Anthony Fauci was granted $15 million in taxpayer-funded security as a private citizen, according to a new report. An agreement between the U.S. Marshals Service (USMS) and the U.S. Department of Health and Human Services (HHS) provided Fauci protection from Jan. 4, 2023 to Sept. 20, 2024, according to FOIA documents […]

The post Fauci Received 15 Million Dollars In Taxpayer Funds For Private Security Detail, Report Says appeared first on Liberty Unyielding.

A new drug that slows the pace of Alzheimer's disease is too expensive for too little benefit to be used on the NHS, the watchdog says.

velocityconf: Help your dev + ops teams be cross-functional and more successful. http://t.co/1mqGK3zh0U Free webcast 5/22 w/ @lnxchk

When my job announced we were returning to the office, I was able to quit and rely on my emergency fund while job searching. I still wish I had saved more.

Tech accelerator Startupbootcamp, British-East African business tycoon Ashish Thakkar’s Mara Group and Blend Financial Services are planning a $250 million (R4.4 billion) fund to invest in new African technology companies.

The University of KwaZulu-Natal will share R2 million in research funding from the UK government to improve 3D-printing techniques for rocket engine components.

Plan a trip to Rome, and you’ll wish you were there for weeks! From the Colosseum to the Vatican Museums, there’s a lot in the Eternal City to check off your bucket list.  But if you can tear yourself away from Rome’s top attractions, there are plenty of excellent day trips from Rome. Rome is […]

The post 18 Fun Day Trips from Rome appeared first on Adventurous Kate.

There’s so much more to Pisa than the Leaning Tower. There are so many cool things to do in Pisa — this is a big, bustling city with a mind and attitude of its own! So many travelers swoop in on a day trip from Florence to take photos with the Leaning Tower of Pisa. And […]

The post 16 Fun Things to Do in Pisa, Italy appeared first on Adventurous Kate.

Funding Ukraine’s recovery 15 October 2024 — 2:00PM TO 3:30PM Anonymous (not verified) 24 September 2024 Online

The expert panel explores the role of the private sector in financing recovery projects.

Despite the ongoing war, recovery and reconstruction is under way in Ukraine. The question of how to push and finance the pipeline of the recovery projects remains one of the key challenges for the Ukrainian government. Most importantly, there is an outstanding question about how foreign investors can participate in the process.

Energy, together with access to finance and uninterrupted export routes is one of the key pillars of Ukraine’s wartime economy. In the coming months, Ukraine faces an ultimate test on its capacity to implement energy projects to restore energy generation. It is a test case for how effectively Kyiv raises public and private funds and delivers results to mitigate destruction as the war continues.

This expert panel discusses key questions including:

• What funding streams and mechanisms are currently in place?
• What does the new Ukraine Investment Framework offer?
• How successful is the energy sector in generating necessary funding?
• What role for private-public partnerships? Do we have examples?

This event is organised in partnership with Ukrainian project ‘The Recovery Spending Watchdog’ financed by the EU. The project is a joint effort of the Centre for Economic Strategy, the Institute for Economic Research and Policy Consulting, and the NGO ‘Technologies for Progress’.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane anion channel that plays a key role in controlling transepithelial fluid movement. Excessive activation results in intestinal fluid loss during secretory diarrheas, whereas CFTR mutations underlie cystic fibrosis (CF). Anion permeability depends both on how well CFTR channels work (permeation/gating) and on how many are present at the membrane. Recently, treatments with two drug classes targeting CFTR—one boosting ion-channel function (potentiators) and the other increasing plasma membrane density (correctors)—have provided significant health benefits to CF patients. Here, we present an image-based fluorescence assay that can rapidly and simultaneously estimate both CFTR ion-channel function and the protein's proximity to the membrane. We monitor F508del-CFTR, the most common CF-causing variant, and confirm rescue by low temperature, CFTR-targeting drugs and second-site revertant mutation R1070W. In addition, we characterize a panel of 62 CF-causing mutations. Our measurements correlate well with published data (electrophysiology and biochemistry), further confirming validity of the assay. Finally, we profile effects of acute treatment with approved potentiator drug VX-770 on the rare-mutation panel. Mapping the potentiation profile on CFTR structures raises mechanistic hypotheses on drug action, suggesting that VX-770 might allow an open-channel conformation with an alternative arrangement of domain interfaces. The assay is a valuable tool for investigation of CFTR molecular mechanisms, allowing accurate inferences on gating/permeation. In addition, by providing a two-dimensional characterization of the CFTR protein, it could better inform development of single-drug and precision therapies addressing the root cause of CF disease.

The inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), which form tetrameric channels, play pivotal roles in regulating the spatiotemporal patterns of intracellular calcium signals. Mutations in IP3Rs have been increasingly associated with many debilitating human diseases such as ataxia, Gillespie syndrome, and generalized anhidrosis. However, how these mutations affect IP3R function, and how the perturbation of as-sociated calcium signals contribute to the pathogenesis and severity of these diseases remains largely uncharacterized. Moreover, many of these diseases occur as the result of autosomal dominant inheritance, suggesting that WT and mutant subunits associate in heterotetrameric channels. How the in-corporation of different numbers of mutant subunits within the tetrameric channels affects its activities and results in different disease phenotypes is also unclear. In this report, we investigated representative disease-associated missense mutations to determine their effects on IP3R channel activity. Additionally, we designed concatenated IP3R constructs to create tetrameric channels with a predefined subunit composition to explore the functionality of heteromeric channels. Using calcium imaging techniques to assess IP3R channel function, we observed that all the mutations studied resulted in severely attenuated Ca2+ release when expressed as homotetramers. However, some heterotetramers retained varied degrees of function dependent on the composition of the tetramer. Our findings suggest that the effect of mutations depends on the location of the mutation in the IP3R structure, as well as on the stoichiometry of mutant subunits assembled within the tetrameric channel. These studies provide insight into the pathogenesis and penetrance of these devastating human diseases.

IQGAP1 is a key scaffold protein that regulates numerous cellular processes and signaling pathways. Analogous to many other cellular proteins, IQGAP1 undergoes post-translational modifications, including phosphorylation. Nevertheless, very little is known about the specific sites of phosphorylation or the effects on IQGAP1 function. Here, using several approaches, including MS, site-directed mutagenesis, siRNA-mediated gene silencing, and chemical inhibitors, we identified the specific tyrosine residues that are phosphorylated on IQGAP1 and evaluated the effect on function. Tyr-172, Tyr-654, Tyr-855, and Tyr-1510 were phosphorylated on IQGAP1 when phosphotyrosine phosphatase activity was inhibited in cells. IQGAP1 was phosphorylated exclusively on Tyr-1510 under conditions with enhanced MET or c-Src signaling, including in human lung cancer cell lines. This phosphorylation was significantly reduced by chemical inhibitors of MET or c-Src or by siRNA-mediated knockdown of MET. To investigate the biological sequelae of phosphorylation, we generated a nonphosphorylatable IQGAP1 construct by replacing Tyr-1510 with alanine. The ability of hepatocyte growth factor, the ligand for MET, to promote AKT activation and cell migration was significantly greater when IQGAP1-null cells were reconstituted with IQGAP1 Y1510A than when cells were reconstituted with WT IQGAP1. Collectively, our data suggest that phosphorylation of Tyr-1510 of IQGAP1 alters cell function. Because increased MET signaling is implicated in the development and progression of several types of carcinoma, IQGAP1 may be a potential therapeutic target in selected malignancies.

Ilka Wittig
Jul 1, 2007; 6:1215-1225
Research

Ulrich Rothbauer
Feb 1, 2008; 7:282-289
Research

Candida albicans and Aspergillus fumigatus are dangerous fungal pathogens with high morbidity and mortality, particularly in immunocompromised patients. Innate immune-mediated programmed cell death (pyroptosis, apoptosis, necroptosis) is an integral part of host defense against pathogens. Inflammasomes, which are canonically formed upstream of pyroptosis, have been characterized as key mediators of fungal sensing and drivers of proinflammatory responses. However, the specific cell death pathways and key upstream sensors activated in the context of Candida and Aspergillus infections are unknown. Here, we report that C. albicans and A. fumigatus infection induced inflammatory programmed cell death in the form of pyroptosis, apoptosis, and necroptosis (PANoptosis). Further, we identified the innate immune sensor Z-DNA binding protein 1 (ZBP1) as the apical sensor of fungal infection responsible for activating the inflammasome/pyroptosis, apoptosis, and necroptosis. The Zα2 domain of ZBP1 was required to promote this inflammasome activation and PANoptosis. Overall, our results demonstrate that C. albicans and A. fumigatus induce PANoptosis and that ZBP1 plays a vital role in inflammasome activation and PANoptosis in response to fungal pathogens.

Harris and Trump's shared goal masks a fundamental AI policy divide Expert comment rgold.drupal 3 November 2024

Both presidential candidates will pursue US tech dominance but differ on the means to achieve it.

There remain some differences between the US presidential candidates’ positions on the governance of artificial intelligence and other emerging technology, notably around competition. 

But under either future administration, US decision-making looks set to be heavily influenced by growing securitization, the aims of the US technology industry and broader competition with China. 

‘Safe’ AI development 

Vice President Kamala Harris attended the inaugural Global AI Safety summit in the UK in 2023, where she announced President Joe Biden’s Executive Order on AI. This significant move by the federal government sought to define national and cyber security guidelines for AI developers and outline privacy and transparency guarantees. It also committed the federal government to a review of the National Security implications of this emerging technology, which was published last week.

In her remarks at the summit, Harris was clear that her definitions of safety extended beyond catastrophic risk mitigation to societal and personal harm. She noted the corrosive effects of some algorithmic decision-making and disinformation on democracy, appealing for AI that is developed ‘in the service of the public interest’.

A number of initiatives developed during the Biden administration have attempted to steer emerging technology as it takes root in society. This includes the establishment of the US AI Safety Institute, various schemes on global AI governance and investment in Public AI projects like the National AI Research Resource (NAIRR).

The US public is largely supportive: polling by the AI Policy Institute (AIPI) points to a majority preference for safety standards governing the US effort to lead in AI, rather than pedal-to-the-metal development. Investment in public options on AI and the infrastructure required to develop and sustain it is a bold direction that the incoming administration should consider expanding.

Biden’s Executive Order on AI has come under fire by the Trump campaign. At a rally in Iowa, Trump explained that he would cancel the order ‘on day one’, echoing a Republican platform that described it as imposing ‘Radical Leftwing ideas’ . 

Trump does, however, have his own track record in technology policy. During his first presidency, his administration passed an Executive Order on AI, stressing that ‘continued American leadership in Artificial Intelligence is of paramount importance to maintaining the economic and national security of the United States’. The tools and institutions announced as part of the order – including AI research investment, national AI research institutes and AI regulatory guidance including on federal use of AI – echo those of the current administration. 

Four years is a long time in AI, however. As the power of this technology is revealed, talk of safety may give way to talk of security. While the candidates may disagree on the role of the federal government in setting standards, there will be close alignment on the central need for US supremacy in national security. Neither administration seems likely to erect barriers to securitization of AI should it emerge as a critical strategic asset.

AI regulation at home 

American industry will remain the pivotal force shaping the US AI ecosystem, particularly as America jostles for position as the maker of the global rules governing AI. A notable aspect of Biden’s AI Executive Order was where it staked responsibility. Reports by AI and Cloud companies on the safety of their tools and infrastructure are sent to the Department of Commerce.

Under Secretary Gina Raimondo, whose star continues to rise, the department has become significantly more engaged with technology companies. In the absence of any other legal authority, AI governance has therefore sat under the purview of a secretary who herself has noted that they are ‘not a regulator’. 

The trend of industry leaders driving the government agenda on AI is replicated in other departments. There was an outcry over the composition of the new Department of Homeland Security advisory panel, the Artificial Intelligence and Security Board, with civil society groups concerned about the preponderance of industry voices: the 22-member panel includes the CEOs of OpenAI, Anthropic, NVIDIA, IBM, AWS, Adobe, Microsoft and Alphabet.

Under a Harris presidency, these trends seem likely to continue. Plus with deadlock in Congress probable, establishing new legal authorities for emerging technology will be difficult. That will likely mean emerging tech governance remains heavily influenced by the Department for Commerce. 

Like Harris, Trump has his allies in industry. While the Biden administration has courted the CEOs of AI companies who have historically spoken out in favour of regulation, Trump’s allies tend towards a more deregulatory agenda. 

Silicon Valley billionaires Elon Musk and Marc Andreessen have backed Trump’s plans to minimize AI regulation, lauding his support for ‘little tech’. They have also backed reported plans for so-called ‘Manhattan Projects’ to develop military technology, stewarded by ‘industry-led’ agencies. Trump’s aversion to strong regulatory institutions may mean an end to Biden’s anti-trust efforts, benefitting the biggest voices in the room, though his VP pick may disagree. JD Vance has somewhat surprisingly come out strongly in defence of the current chair of the Federal Trade Commission, Lina Khan, and her anti-trust efforts targeting US big tech.

Regardless, business interests will likely shape either a Harris or Trump administration’s approach as the US grapples with balancing the ambitions of its industry with an increasingly protectionist stance towards its biggest import market, China.

Competition abroad

China looms large in the imaginations of both campaigns. 

The US has signalled to its allies that American AI standards should replace Chinese standards. Export controls on semiconductors were expanded in September this year, with key voices in the industry – notably the Netherlands, Japan and South Korea – describing the restrictions as ‘economically motivated’ despite nominally being tied to national security by the US. 

Jorge H. Capdevila
Feb 1, 2000; 41:163-181
Reviews

Linda J. Pike
Jul 1, 2006; 47:1597-1598
Report

JP Segrest
Feb 1, 1992; 33:141-166
Reviews

Saverio Cinti
Nov 1, 2005; 46:2347-2355
Research Articles

Expert

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

E. A. Rakhmanov and S. P. Suetin
Trans. Moscow Math. Soc. 83 (), 269-290.
Abstract, references and article information

A. I. Aptekarev and M. L. Yattselev
Trans. Moscow Math. Soc. 83 (), 251-268.
Abstract, references and article information

M. A. Stepanova
Trans. Moscow Math. Soc. 83 (), 1-13.
Abstract, references and article information

Haiyong Wang
Math. Comp. 93 (), 2861-2884.
Abstract, references and article information

Ming-Lun Hsieh and Shunsuke Yamana
Trans. Amer. Math. Soc. 377 (), 5617-5672.
Abstract, references and article information

Bero Roos
Theor. Probability and Math. Statist. 111 (), 137-151.
Abstract, references and article information

Lasse L. Wolf and Hong-Wei Zhang
Proc. Amer. Math. Soc. 152 (), 5445-5453.
Abstract, references and article information

Ferdinand Ihringer
Proc. Amer. Math. Soc. 152 (), 5355-5365.
Abstract, references and article information

Sudip Ranjan Bhuia
Proc. Amer. Math. Soc. 152 (), 5249-5263.
Abstract, references and article information

Lawford Hatcher
Proc. Amer. Math. Soc. 152 (), 5191-5205.
Abstract, references and article information

R. Gibara and D. Kinzebulatov
St. Petersburg Math. J. 35 (), 445-460.
Abstract, references and article information

Mohammed Abouzaid and Nathaniel Bottman
Bull. Amer. Math. Soc. 61 (), 525-608.
Abstract, references and article information

As I walked into the embalming room at Jones Funeral Home and Supplies in Kingston on Sunday, I immediately felt the weight of the room. The air was thick with the scent of various chemicals used in the trade permeating the space. Tools such as...

In a touching display of compassion and solidarity, a group of St Thomas residents has come together to organise the funeral of a a well-known and beloved man with intellectual challenges. For years, Donovan Sinclair was a familiar face in the...

Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell–mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell–mediated anti-tumor immunity, but many patients do not respond and a significant proportion develop inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization, and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. In silico predictions revealed that kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1–triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1–targeting therapeutic approaches.

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.

Aspergillus terreus is an allergenic fungus, in addition to causing infections in both humans and plants. However, the allergens in this fungus are still unknown, limiting the development of diagnostic and therapeutic strategies. We used a proteomic approach to search for allergens, identifying 16 allergens based on two-dimensional immunoblotting with A. terreus susceptible patient sera. We further characterized triose-phosphate isomerase (Asp t 36), one of the dominant IgE (IgE)-reactive proteins. The gene was cloned and expressed in Escherichia coli. Phylogenetic analysis showed Asp t 36 to be highly conserved with close similarity to the triose-phosphate isomerase protein sequence from Dermatophagoides farinae, an allergenic dust mite. We identified four immunodominant epitopes using synthetic peptides, and mapped them on a homology-based model of the tertiary structure of Asp t 36. Among these, two were found to create a continuous surface patch on the 3D structure, rendering it an IgE-binding hotspot. Biophysical analysis indicated that Asp t 36 shows similar secondary structure content and temperature sensitivity with other reported triose-phosphate isomerase allergens. In vivo studies using a murine model displayed that the recombinant Asp t 36 was able to stimulate airway inflammation, as demonstrated by an influx of eosinophils, goblet cell hyperplasia, elevated serum Igs, and induction of Th2 cytokines. Collectively, our results reveal the immunogenic property of Asp t 36, a major allergen from A. terreus, and define a new fungal allergen more broadly. This allergen could serve as a potent candidate for investigating component resolved diagnosis and immunotherapy.

The recent structural elucidation of ex vivo Drosophila Orb2 fibrils revealed a novel amyloid formed by interdigitated Gln and His residue side chains belonging to the prion-like domain. However, atomic-level details on the conformational transitions associated with memory consolidation remain unknown. Here, we have characterized the nascent conformation and dynamics of the prion-like domain (PLD) of Orb2A using a nonconventional liquid-state NMR spectroscopy strategy based on 13C detection to afford an essentially complete set of 13Cα, 13Cβ, 1Hα, and backbone 13CO and 15N assignments. At pH 4, where His residues are protonated, the PLD is disordered and flexible, except for a partially populated α-helix spanning residues 55–60, and binds RNA oligos, but not divalent cations. At pH 7, in contrast, His residues are predominantly neutral, and the Q/H segments adopt minor populations of helical structure, show decreased mobility and start to self-associate. At pH 7, the His residues do not bind RNA or Ca2+, but do bind Zn2+, which promotes further association. These findings represent a remarkable case of structural plasticity, based on which an updated model for Orb2A functional amyloidogenesis is suggested.

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.

Mingkun Yang
Nov 24, 2020; 0:RA120.002144v1-mcp.RA120.002144
Research

Gle1 is a conserved, essential regulator of DEAD-box RNA helicases, with critical roles defined in mRNA export, translation initiation, translation termination, and stress granule formation. Mechanisms that specify which, where, and when DDXs are targeted by Gle1 are critical to understand. In addition to roles for stress-induced phosphorylation and inositol hexakisphosphate binding in specifying Gle1 function, Gle1 oligomerizes via its N-terminal domain in a phosphorylation-dependent manner. However, a thorough analysis of the role for Gle1 self-association is lacking. Here, we find that Gle1 self-association is driven by two distinct regions: a coiled-coil domain and a novel 10-amino acid aggregation-prone region, both of which are necessary for proper Gle1 oligomerization. By exogenous expression in HeLa cells, we tested the function of a series of mutations that impact the oligomerization domains of the Gle1A and Gle1B isoforms. Gle1 oligomerization is necessary for many, but not all aspects of Gle1A and Gle1B function, and the requirements for each interaction domain differ. Whereas the coiled-coil domain and aggregation-prone region additively contribute to competent mRNA export and stress granule formation, both self-association domains are independently required for regulation of translation under cellular stress. In contrast, Gle1 self-association is dispensable for phosphorylation and nonstressed translation initiation. Collectively, we reveal self-association functions as an additional mode of Gle1 regulation to ensure proper mRNA export and translation. This work also provides further insight into the mechanisms underlying human gle1 disease mutants found in prenatally lethal forms of arthrogryposis.

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.

Neurodegeneration in Parkinson's disease (PD) can be recapitulated in animals by administration of α-synuclein preformed fibrils (PFFs) into the brain. However, the mechanism by which these PFFs induce toxicity is unknown. Iron is implicated in PD pathophysiology, so we investigated whether α-synuclein PFFs induce ferroptosis, an iron-dependent cell death pathway. A range of ferroptosis inhibitors were added to a striatal neuron-derived cell line (STHdhQ7/7 cells), a dopaminergic neuron–derived cell line (SN4741 cells), and WT primary cortical neurons, all of which had been intoxicated with α-synuclein PFFs. Viability was not recovered by these inhibitors except for liproxstatin-1, a best-in-class ferroptosis inhibitor, when used at high doses. High-dose liproxstatin-1 visibly enlarged the area of a cell that contained acidic vesicles and elevated the expression of several proteins associated with the autophagy-lysosomal pathway similarly to the known lysosomal inhibitors, chloroquine and bafilomycin A1. Consistent with high-dose liproxstatin-1 protecting via a lysosomal mechanism, we further de-monstrated that loss of viability induced by α-synuclein PFFs was attenuated by chloroquine and bafilomycin A1 as well as the lysosomal cysteine protease inhibitors, leupeptin, E-64D, and Ca-074-Me, but not other autophagy or lysosomal enzyme inhibitors. We confirmed using immunofluorescence microscopy that heparin prevented uptake of α-synuclein PFFs into cells but that chloroquine did not stop α-synuclein uptake into lysosomes despite impairing lysosomal function and inhibiting α-synuclein toxicity. Together, these data suggested that α-synuclein PFFs are toxic in functional lysosomes in vitro. Therapeutic strategies that prevent α-synuclein fibril uptake into lysosomes may be of benefit in PD.

Akt3 regulates mitochondrial content in endothelial cells through the inhibition of PGC-1α nuclear localization and is also required for angiogenesis. However, whether there is a direct link between mitochondrial function and angiogenesis is unknown. Here we show that Akt3 depletion in primary endothelial cells results in decreased uncoupled oxygen consumption, increased fission, decreased membrane potential, and increased expression of the mitochondria-specific protein chaperones, HSP60 and HSP10, suggesting that Akt3 is required for mitochondrial homeostasis. Direct inhibition of mitochondrial homeostasis by the model oxidant paraquat results in decreased angiogenesis, showing a direct link between angiogenesis and mitochondrial function. Next, in exploring functional links to PGC-1α, the master regulator of mitochondrial biogenesis, we searched for compounds that induce this process. We found that, sildenafil, a phosphodiesterase 5 inhibitor, induced mitochondrial biogenesis as measured by increased uncoupled oxygen consumption, mitochondrial DNA content, and voltage-dependent anion channel protein expression. Sildenafil rescued the effects on mitochondria by Akt3 depletion or pharmacological inhibition and promoted angiogenesis, further supporting that mitochondrial homeostasis is required for angiogenesis. Sildenafil also induces the expression of PGC-1 family member PRC and can compensate for PGC-1α activity during mitochondrial stress by an Akt3-independent mechanism. The induction of PRC by sildenafil depends upon cAMP and the transcription factor CREB. Thus, PRC can functionally substitute during Akt3 depletion for absent PGC-1α activity to restore mitochondrial homeostasis and promote angiogenesis. These findings show that mitochondrial homeostasis as controlled by the PGC family of transcriptional activators is required for angiogenic responses.