Stop codon read-through (SCR) is a process of continuation of translation beyond a stop codon. This phenomenon, which occurs only in certain mRNAs under specific conditions, leads to a longer isoform with properties different from that of the canonical isoform. MTCH2, which encodes a mitochondrial protein that regulates mitochondrial metabolism, was selected as a potential read-through candidate based on evolutionary conservation observed in the proximal region of its 3' UTR. Here, we demonstrate translational read-through across two evolutionarily conserved, in-frame stop codons of MTCH2 using luminescence- and fluorescence-based assays, and by analyzing ribosome-profiling and mass spectrometry (MS) data. This phenomenon generates two isoforms, MTCH2x and MTCH2xx (single- and double-SCR products, respectively), in addition to the canonical isoform MTCH2, from the same mRNA. Our experiments revealed that a cis-acting 12-nucleotide sequence in the proximal 3' UTR of MTCH2 is the necessary signal for SCR. Functional characterization showed that MTCH2 and MTCH2x were localized to mitochondria with a long t1/2 (>36 h). However, MTCH2xx was found predominantly in the cytoplasm. This mislocalization and its unique C terminus led to increased degradation, as shown by greatly reduced t1/2 (<1 h). MTCH2 read-through–deficient cells, generated using CRISPR-Cas9, showed increased MTCH2 expression and, consistent with this, decreased mitochondrial membrane potential. Thus, double-SCR of MTCH2 regulates its own expression levels contributing toward the maintenance of normal mitochondrial membrane potential.

Expert

Expert

A. V. Domrina
Trans. Moscow Math. Soc. 83 (), 201-215.
Abstract, references and article information

A. P. Starovoitov and N. V. Ryabchenko
Trans. Moscow Math. Soc. 83 (), 15-31.
Abstract, references and article information

Gurmeet K. Bakshi, Jyoti Garg and Gabriela Olteanu
Math. Comp. 93 (), 3027-3058.
Abstract, references and article information

Buyang Li, Zongze Yang and Zhi Zhou
Math. Comp. 93 (), 2557-2586.
Abstract, references and article information

Yinji Li, Zhiwei Wang and Xiangyu Zhou
Trans. Amer. Math. Soc. 377 (), 5947-5992.
Abstract, references and article information

Ning Jiang, Yi-Long Luo and Shaojun Tang
Trans. Amer. Math. Soc. 377 (), 5323-5359.
Abstract, references and article information

John D. Berman
Proc. Amer. Math. Soc. 152 (), 5055-5066.
Abstract, references and article information

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.

Laura F Fielden
Nov 11, 2020; 0:RA120.002370v1-mcp.RA120.002370
Research

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.

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.

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.

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

Junhwan Kim
Dec 1, 2020; 61:1707-1719
Research Articles

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

Babunageswararao Kanuri
Nov 17, 2020; 0:jlr.RA120001101v1-jlr.RA120001101
Research Articles

When viewing a SAS Visual Analytics report, you might intermittently see an error that includes content similar to the following:

When you use SAS/ACCESS Interface to PostgreSQL to query a Yellowbrick database, the SAS OBS= option is not generating a limit clause on the query that is passed to the database. Click the

Smith-Lemli-Opitz Syndrome (SLOS) is a developmental disorder (OMIM #270400) caused by autosomal recessive mutations in the Dhcr7 gene, which encodes the enzyme 3β-hydroxysterol-7 reductase. SLOS patients present clinically with dysmorphology and neurological, behavioral and cognitive defects, with characteristically elevated levels of 7-dehydrocholesterol (7-DHC) in all bodily tissues and fluids. Previous mouse models of SLOS have been hampered by postnatal lethality when Dhcr7 is knocked out globally, while a hypomorphic mouse model showed improvement in the biochemical phenotype with ageing, and did not manifest most other characteristic features of SLOS. We report the generation of a conditional knockout of Dhcr7 (Dhcr7flx/flx), validated by generating a mouse with a liver-specific deletion (Dhcr7L-KO). Phenotypic characterization of liver-specific knockout mice revealed no significant changes in viability, fertility, growth curves, liver architecture, hepatic triglyceride secretion, or parameters of systemic glucose homeostasis. Furthermore, qPCR and RNA-Seq analyses of livers revealed no perturbations in pathways responsible for cholesterol synthesis, either in male or female Dhcr7L-KO mice, suggesting hepatic disruption of post-squalene cholesterol synthesis leads to minimal impact on sterol metabolism in the liver. This validated conditional Dhcr7 knockout model may now allow us to systematically explore the pathophysiology of SLOS, by allowing for temporal, cell and tissue-specific loss of DHCR7.

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

Phospholipids, including ether phospholipids, are composed of numerous isomeric and isobaric species that have the same backbone and acyl chains. This structural resemblance results in similar fragmentation patterns by collision-induced dissociation of phospholipids regardless of class, yielding complicated MS/MS spectra when isobaric species are analyzed together. Furthermore, the presence of isobaric species can lead to misassignment of species when made solely based on their molecular weights. In this study, we used normal-phase HPLC for ESI-MS/MS analysis of phospholipids from bovine heart mitochondria. Class separation by HPLC eliminates chances for misidentification of isobaric species from different classes of phospholipids. Chromatography yields simple MS/MS spectra without interference from isobaric species, allowing clear identification of peaks corresponding to fragmented ions containing monoacylglycerol backbone derived from losing one acyl chain. Using these fragmented ions, we characterized individual and isomeric species in each class of mitochondrial phospholipids, including unusual species, such as PS, containing an ether linkage and species containing odd-numbered acyl chains in cardiolipin, PS, PI, and PG. We also characterized monolysocardiolipin and dilysocardiolipin, the least abundant but nevertheless important mitochondrial phospholipids. The results clearly show the power of HPLC-MS/MS for identification and characterization of phospholipids, including minor species.

Modulation of the host cell is integral to the survival and replication of microbial pathogens. Several intracellular bacterial pathogens deliver bacterial proteins, termed ‘effector proteins’ into the host cell during infection by sophisticated protein translocation systems, which manipulate cellular processes and functions. The functional contribution of individual effectors is poorly characterised, particularly in intracellular bacterial pathogens with large effector protein repertoires. Technical caveats have limited the capacity to study these proteins during a native infection, with many effector proteins having only been demonstrated to be translocated during over-expression of tagged versions. Here we developed a novel strategy to examine effector proteins in the context of infection. We coupled a broad, unbiased proteomics-based screen with organelle purification to study the host-pathogen interactions occurring between the host cell mitochondrion and the Gram-negative, Q fever pathogen Coxiella burnetii. We identify 4 novel mitochondrially-targeted C. burnetii effector proteins, renamed Mitochondrial Coxiella effector protein (Mce) B to E. Examination of the subcellular localisation of ectopically expressed proteins confirmed their mitochondrial localisation, demonstrating the robustness of our approach. Subsequent biochemical analysis and affinity enrichment proteomics of one of these effector proteins, MceC, revealed the protein localises to the inner membrane and can interact with components of the mitochondrial quality control machinery. Our study adapts high-sensitivity proteomics to study intracellular host-pathogen interactions, providing a robust strategy to examine the sub-cellular localisation of effector proteins during native infection. This approach could be applied to a range of pathogens and host cell compartments to provide a rich map of effector dynamics throughout infection.

Shohei Ohtani remains a must-watch player for Angels fans in 2019, and his recovery from Tommy John surgery will be worth monitoring throughout the season as he prepares to pitch again in '20.

An employment judge has cleared the way for Farah Jameel, a former chair of the BMA’s General Practitioners Committee for England (GPCE), to go ahead with claims of discrimination and unfair dismissal against the association over her removal from the post during maternity leave.Jameel, who was elected the first female chair in November 2021, was put on temporary suspension in 2022 after complaints by BMA staff. The BMA told her in August 2023 that her contract was being terminated.The contract described her as a contractor providing consultancy services rather than an employee. But in a preliminary ruling the employment judge Natasha Joffe has held that Jameel was in reality an office holder and an employee, opening the way for her claims to proceed to a full hearing by an employment tribunal.The GPCE passed a vote of no confidence in Jameel in July 2023, as a means of electing a new...

Limited expectations as Scholz heads to Kyiv and Moscow Expert comment NCapeling 14 February 2022

After heavy criticism for an initially timid response, the approach of Germany’s new government to the Ukraine crisis is improving as its position on Russia evolves.

Olaf Scholz is a famously quiet and cautious communicator and, as concerns mounted in Washington and London about the possibility of a Russian invasion of Ukraine, the new German chancellor remained silent.

This time his silence reflected the fact that his government was unprepared for its first major foreign policy test, with marked divisions between the SPD and the Greens on how to manage a rapidly deteriorating relationship with Russia.

When he did speak, Scholz caused dismay in several NATO capitals by defending the controversial Nord Stream 2 pipeline. He called it ‘a purely private economic project’ even though the company building the pipeline is a subsidiary of Russia’s state-owned Gazprom.

The absence of crisis management from Berlin was a stark reminder of the gap left by Angela Merkel. Her knowledge of Russia and Ukraine, and her personal experience of dealing with Vladimir Putin, were key assets in 2014 in developing a unified western response to Russia’s annexation of Crimea and its destabilization of south-eastern Ukraine.

Germany has coordinated with its allies

Scholz has prepared the ground carefully for his visits to Kyiv and Moscow, extensively coordinating with NATO allies, such as France and Poland – as part of the Weimar Triangle – and the three Baltic states. His inaugural visit to Washington helped align positions on US-EU efforts to develop an economic deterrent against further Russian intervention in Ukraine.

Scholz has also repeated Merkel’s language from 2014 to signal to Moscow that Germany and its partners are ‘united and determined’ to stand their ground. And he has spoken of Germany’s continuing double strategy of ‘clear announcements’ to Moscow of the price it would pay for further aggression accompanied by a readiness to use all possible channels of dialogue to preserve peace.

The continuation of Germany’s established policy will not surprise Moscow. But under the surface Germany’s discourse on Russia is evolving as the political class internalizes the fact that the current Russian leadership is openly threatening to use military force to impose its will on Ukraine and re-configure Europe’s security arrangements.

Despite deep concerns about Germany’s dependence on Russian gas – more than half of the gas it consumes comes from Russia – there is broad acceptance that Nord Stream 2 cannot go ahead if Russia invades Ukraine. But typically, Scholz has not yet said as much publicly, limiting himself to stating ‘all options are on the table.’

Robert Habeck, Germany’s vice-chancellor as well as economic affairs and climate action minister, says the ‘geopolitical situation’ requires Germany to diversify its gas supplies and infrastructure for importing gas. For years, a strong pro-Russian gas lobby has dominated the thinking of the Economics Ministry and one consequence is the complete absence of facilities to import liquefied natural gas.

The German debate on Russia is shifting

A public debate has also started about weapons deliveries to Ukraine, even though this runs counter to deeply embedded pacifist tendencies in German society. Some MPs, former diplomats, and experts have begun to make the case for Germany to supply weapons to Ukraine so it can exercise its right to self-defence and deter military attack.

Christoph Heusgen, a former long-standing foreign policy adviser in the Federal Chancellery, says Berlin’s decision not to deliver arms to Ukraine to avoid endangering the Minsk peace process requires revision now Moscow has abandoned the path of negotiation.

Despite its mantra that there is no alternative to the Minsk Agreements, Berlin now admits these currently offer no prospect of resolving the war in Donbas because Russia is not prepared to implement them properly.

Other taboos are also being broken inside Germany. Heavyweight left-of-centre Die Zeit published the first results of its investigation into the SPD’s links with Russia and their place in Russia’s channels of influence in Germany. And even the SPD’s Sigmar Gabriel, an outspoken champion of Nord Stream 2 during his time as a government minister, has raised the question of Germany terminating energy imports from Russia if there is a drastic deterioration of the security situation.

SANTA CLARA, Calif., Oct. 25, 2024 — ServiceNow has announced that enterprise software industry veteran Amit Zavery will join the company as president, chief product officer (CPO), and chief operating officer […]

The post Google Cloud and Oracle Veteran Amit Zavery to Spearhead Product and Engineering at ServiceNow appeared first on HPCwire.

Mike Gesicki and Jonnu Smith are among UPI senior sports writer Alex Butler's four must-start fantasy football tight ends for Week 10.

Chicago Bears coach Matt Eberflus has fired offensive coordinator Shane Waldron, the team said Tuesday. The dismissal came less than a day after Eberflus committed to quarterback Caleb Williams, but promised "changes."

Instead of its usual quantum summit this year, IBM will hold its first IBM Quantum Developer Conference which the company is calling, “an exclusive, first-of-its-kind.” It’s planned as an in-person […]

The post IBM Quantum Summit Evolves into Developer Conference appeared first on HPCwire.

Nvidia will discuss how it uses the RISC-V architecture at the RISC-V Summit from October 22 to 24. The GPU maker has used the RISC-V CPU architecture in its GPU […]

The post Nvidia, Google to Speak About RISC-V Use at Annual Summit appeared first on HPCwire.

Nov. 8, 2024 — This month, over 100 academics, researchers, industry experts, and national lab staff gathered at Berkeley Lab’s Artificial Intelligence Science Summit (BLASS) to explore AI’s potential to drive […]

The post Berkeley Lab AI Science Summit Fuels Collaboration appeared first on HPCwire.

Cameron Crowe, the writer and director behind "We Bought a Zoo," "Almost Famous" and "Jerry Maguire," has a new baby girl with girlfriend Anais Smith.

The Mediterranean island nation of Cyprus signed the U.S.-led Artemis Accords on Wednesday, becoming the 46th signatory to the agreement that establishes principles for the safe exploration of space.

The state decided that the Smarter Balanced Assessment Consortium offers it a chance to share assessment goals with neighboring states.

The U.S. Department of Education's office for civil rights said that some students were placed in self-contained special education settings without an individualized justification for doing so.

About $8 million will be eligible for compensatory education to students who were affected by an illegal state-imposed age cap on special education enrollment.

A bill prohibiting anti-Semitism in Florida's public schools and universities is going to Republican Gov. Ron DeSantis.