Lysine lactylation (Kla) is a recently discovered histone mark derived from metabolic lactate. The NAD+ -dependent deacetylase SIRT3, which can also catalyze removal of the lactyl moiety from lysine, is expressed at low levels in hepatocellular carcinoma (HCC) and has been suggested to be an HCC tumor suppressor. Here we report that SIRT3 can delactylate non-histone proteins and suppress HCC development. Using SILAC-based quantitative proteomics, we identify cyclin E2 (CCNE2) as one of the lactylated substrates of SIRT3 in HCC cells. Furthermore, our crystallographic study elucidates the mechanism of CCNE2 K348la delactylation by SIRT3. Our results further suggest that lactylated CCNE2 promotes HCC cell growth, while SIRT3 activation by Honokiol induces HCC cell apoptosis and prevents HCC outgrowth in vivo by regulating Kla levels of CCNE2. Together, our results establish a physiological function of SIRT3 as a delactylase that is important for suppressing HCC, and our structural data could be useful for the future design of activators.

Do we paint too rosy a picture of life as an academic scientist when advising our students?

Upon ex vivo culture, hematopoietic stem cells (HSCs) quickly lose potential and differentiate into progenitors. The identification of culture conditions that maintain the potential of HSCs ex vivo is therefore of high clinical interest. Here, we demonstrate that the potential of murine and human HSCs is maintained when cultivated for 2 days ex vivo at a pH of 6.9, in contrast to cultivation at the commonly used pH of 7.4. When cultivated at a pH of 6.9, HSCs remain smaller, less metabolically active, less proliferative and show enhanced reconstitution ability upon transplantation compared to HSC cultivated at pH 7.4. HSCs kept at pH 6.9 show an attenuated polyamine pathway. Pharmacological inhibition of the polyamine pathway in HSCs cultivated at pH 7.4 with DFMO mimics phenotypes and potential of HSCs cultivated at pH 6.9. Ex vivo exposure to a pH of 6.9 is therefore a positive regulator of HSC function by reducing polyamines. These findings might improve HSC short-term cultivation protocols for transplantation and gene therapy interventions.

HIV-1 uses inositol hexakisphosphate (IP6) to build a metastable capsid capable of delivering its genome into the host nucleus. Here, we show that viruses that are unable to package IP6 lack capsid protection and are detected by innate immunity, resulting in the activation of an antiviral state that inhibits infection. Disrupting IP6 enrichment results in defective capsids that trigger cytokine and chemokine responses during infection of both primary macrophages and T-cell lines. Restoring IP6 enrichment with a single mutation rescues the ability of HIV-1 to infect cells without being detected. Using a combination of capsid mutants and CRISPR-derived knockout cell lines for RNA and DNA sensors, we show that immune sensing is dependent upon the cGAS-STING axis and independent of capsid detection. Sensing requires the synthesis of viral DNA and is prevented by reverse transcriptase inhibitors or reverse transcriptase active-site mutation. These results demonstrate that IP6 is required to build capsids that can successfully transit the cell and avoid host innate immune sensing.

The cellular integrated stress response (ISR) is a central signaling pathway that tunes translation initiation in response to a wide range of cellular insults to promote cell survival. The critical node of this regulation involves the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) by stress kinases. In this issue of EMBO reports, Wu et al (2023) report FAM69C as a novel eIF2α kinase that promotes ISR activation and stress granule (SG) assembly in microglia in response to oxidative stress. This work proposes a protective role for FAM69C and SGs in limiting damaging inflammatory responses commonly associated with neurodegenerative diseases.

Shade avoidance syndrome (SAS) commonly occurs in plants experiencing vegetative shade, triggering a series of morphological and physiological changes for the plants to reach more light. A number of positive regulators, such as PHYTOCHROME-INTERACTING 7 (PIF7), and negative regulators, such as PHYTOCHROMES, are known to ensure appropriate SAS. Here, we identify 211 shade-regulated long non-coding RNAs (lncRNAs) in Arabidopsis. We further characterize PUAR (PHYA UTR Antisense RNA), a lncRNA produced from the intron of the 5' UTR of the PHYTOCHROME A (PHYA) locus. PUAR is induced by shade and promotes shade-induced hypocotyl elongation. PUAR physically associates with PIF7 and represses the shade-mediated induction of PHYA by blocking the binding of PIF7 to the 5' UTR of PHYA. Our findings highlight a role for lncRNAs in SAS and provide insight into the mechanism of PUAR in regulating PHYA gene expression and SAS.

The growth factor Neuregulin-1 (NRG-1) regulates myocardial growth and is currently under clinical investigation as a treatment for heart failure. Here, we demonstrate in several in vitro and in vivo models that STAT5b mediates NRG-1/EBBB4-stimulated cardiomyocyte growth. Genetic and chemical disruption of the NRG-1/ERBB4 pathway reduces STAT5b activation and transcription of STAT5b target genes Igf1, Myc, and Cdkn1a in murine cardiomyocytes. Loss of Stat5b also ablates NRG-1-induced cardiomyocyte hypertrophy. Dynamin-2 is shown to control the cell surface localization of ERBB4 and chemical inhibition of Dynamin-2 downregulates STAT5b activation and cardiomyocyte hypertrophy. In zebrafish embryos, Stat5 is activated during NRG-1-induced hyperplastic myocardial growth, and chemical inhibition of the Nrg-1/Erbb4 pathway or Dynamin-2 leads to loss of myocardial growth and Stat5 activation. Moreover, CRISPR/Cas9-mediated knockdown of stat5b results in reduced myocardial growth and cardiac function. Finally, the NRG-1/ERBB4/STAT5b signaling pathway is differentially regulated at mRNA and protein levels in the myocardium of patients with pathological cardiac hypertrophy as compared to control human subjects, consistent with a role of the NRG-1/ERBB4/STAT5b pathway in myocardial growth.

Mitochondria play central roles in cellular energy production and metabolism. Most proteins required to carry out these functions are synthesized in the cytosol and imported into mitochondria. A growing number of metabolic disorders arising from mitochondrial dysfunction can be traced to errors in mitochondrial protein import. The mechanisms underlying the import of precursor proteins are commonly studied using radioactively labeled precursor proteins imported into purified mitochondria. Here, we establish a fluorescence-based import assay to analyze protein import into mitochondria. We show that fluorescently labeled precursors enable import analysis with similar sensitivity to those using radioactive precursors, yet they provide the advantage of quantifying import with picomole resolution. We adapted the import assay to a 96-well plate format allowing for fast analysis in a screening-compatible format. Moreover, we show that fluorescently labeled precursors can be used to monitor the assembly of the F1 F0 ATP synthase in purified mitochondria. Thus, we provide a sensitive fluorescence-based import assay that enables quantitative and fast import analysis.

As stem cells divide, they acquire mutations that can be passed on to daughter cells. To mitigate potentially deleterious outcomes, cells activate the DNA damage response (DDR) network, which governs several cellular outcomes following DNA damage, including repairing DNA or undergoing apoptosis. At the helm of the DDR are three PI3-like kinases including Ataxia-Telangiectasia Mutated (ATM). We report here that knockdown of ATM in planarian flatworms enables stem cells to withstand lethal doses of radiation which would otherwise induce cell death. In this context, stem cells circumvent apoptosis, replicate their DNA, and recover function using homologous recombination-mediated DNA repair. Despite radiation exposure, atm knockdown animals survive long-term and regenerate new tissues. These effects occur independently of ATM's canonical downstream effector p53. Together, our results demonstrate that in planarians, ATM promotes radiation-induced apoptosis. This acute, ATM-dependent apoptosis is a key determinant of long-term animal survival. Our results suggest that inhibition of ATM in these organisms could, therefore, potentially favor cell survival after radiation without obvious effects on stem cell behavior.

Synaptic vesicle (SV) fusion with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. The effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Using spray-mixing plunge-freezing cryo-electron tomography we observe events following synaptic stimulation at nanometer resolution in near-native samples. Our data suggest that during the stage that immediately follows stimulation, termed early fusion, PM and SV membrane curvature changes to establish a point contact. The next stage-late fusion-shows fusion pore opening and SV collapse. During early fusion, proximal tethered SVs form additional tethers with the PM and increase the inter-SV connector number. In the late-fusion stage, PM-proximal SVs lose their interconnections, allowing them to move toward the PM. Two SNAP-25 mutations, one arresting and one disinhibiting spontaneous release, cause connector loss. The disinhibiting mutation causes loss of membrane-proximal multiple-tethered SVs. Overall, tether formation and connector dissolution are triggered by stimulation and respond to spontaneous fusion rate manipulation. These morphological observations likely correspond to SV transition from one functional pool to another.

Vesicular transport is a means of communication. While cells can communicate with each other via secretion of extracellular vesicles, less is known regarding organelle-to organelle communication, particularly in the case of mitochondria. Mitochondria are responsible for the production of energy and for essential metabolic pathways in the cell, as well as fundamental processes such as apoptosis and aging. Here, we show that functional mitochondria isolated from Saccharomyces cerevisiae release vesicles, independent of the fission machinery. We isolate these mitochondrial-derived vesicles (MDVs) and find that they are relatively uniform in size, of about 100 nm, and carry selective protein cargo enriched for ATP synthase subunits. Remarkably, we further find that these MDVs harbor a functional ATP synthase complex. We demonstrate that these vesicles have a membrane potential, produce ATP, and seem to fuse with naive mitochondria. Our findings reveal a possible delivery mechanism of ATP-producing vesicles, which can potentially regenerate ATP-deficient mitochondria and may participate in organelle-to-organelle communication.

Regulatory T (T reg) cells developing in the thymus are essential to maintain tolerance and prevent fatal autoimmunity in mice and humans. Expression of the T reg lineage-defining transcription factor FoxP3 is critically dependent upon T cell receptor (TCR) and interleukin-2 (IL-2) signaling. Here, we report that ten-eleven translocation (Tet) enzymes, which are DNA demethylases, are required early during double-positive (DP) thymic T cell differentiation and prior to the upregulation of FoxP3 in CD4 single-positive (SP) thymocytes, to promote Treg differentiation. We show that Tet3 selectively controls the development of CD25- FoxP3lo CD4SP Treg cell precursors in the thymus and is critical for TCR-dependent IL-2 production, which drive chromatin remodeling at the FoxP3 locus as well as other Treg-effector gene loci in an autocrine/paracrine manner. Together, our results demonstrate a novel role for DNA demethylation in regulating the TCR response and promoting Treg cell differentiation. These findings highlight a novel epigenetic pathway to promote the generation of endogenous Treg cells for mitigation of autoimmune responses.

Microspherule protein 1 (Mcrs1) is a component of the nonspecific lethal (NSL) complex and the chromatin remodeling INO80 complex, which participates in transcriptional regulation during mitosis. Here, we investigate the roles of Mcrs1 during female meiosis in mice. We demonstrate that Mcrs1 is a novel regulator of the meiotic G2/M transition and spindle assembly in mouse oocytes. Mcrs1 is present in the nucleus and associates with spindle poles and chromosomes of oocytes during meiosis I. Depletion of Mcrs1 alters HDAC2-mediated H4K16ac, H3K4me2, and H3K9me2 levels in nonsurrounded nucleolus (NSN)-type oocytes, and reduces CDK1 activity and cyclin B1 accumulation, leading to G2/M transition delay. Furthermore, Mcrs1 depletion results in abnormal spindle assembly due to reduced Aurora kinase (Aurka and Aurkc) and Kif2A activities, suggesting that Mcrs1 also plays a transcription-independent role in regulation of metaphase I oocytes. Taken together, our results demonstrate that the transcription factor Mcrs1 has important roles in cell cycle regulation and spindle assembly in mouse oocyte meiosis.

Stress granules are dynamic cytoplasmic ribonucleoprotein granules that assemble in response to cellular stress. Aberrant formation of stress granules has been linked to neurodegenerative diseases. However, the molecular mechanisms underlying the initiation of stress granules remain elusive. Here we report that the brain-enriched protein kinase FAM69C promotes stress granule assembly through phosphorylation of eukaryotic translation initiation factor 2 (eIF2α). FAM69C physically interacts with eIF2α and functions as a stress-specific kinase for eIF2α, leading to stress-induced protein translation arrest and stress granule assembly. Primary microglia derived from Fam69c knockout mice exhibit aberrant stress granule assembly in response to oxidative stress and ATP. Defective stress granule assembly in microglia correlates with the formation of ASC specks and NLRP3 inflammasome activation, whereas induction of stress granule precludes inflammasome formation. Consistently, increased NLRP3 levels, caspase-1 cleavage and Il18 expression corroborate microglia-associated neuroinflammation in aged Fam69c knockout mice. Our study demonstrates that FAM69C is critical for stress granule assembly and suggests its role in the regulation of microglia function.

Throughout the SARS-CoV-2 pandemic, limited diagnostic capacities prevented sentinel testing, demonstrating the need for novel testing infrastructures. Here, we describe the setup of a cost-effective platform that can be employed in a high-throughput manner, which allows surveillance testing as an acute pandemic control and preparedness tool, exemplified by SARS-CoV-2 diagnostics in an academic environment. The strategy involves self-sampling based on gargling saline, pseudonymized sample handling, automated RNA extraction, and viral RNA detection using a semiquantitative multiplexed colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay with an analytical sensitivity comparable with RT-qPCR. We provide standard operating procedures and an integrated software solution for all workflows, including sample logistics, analysis by colorimetry or sequencing, and communication of results. We evaluated factors affecting the viral load and the stability of gargling samples as well as the diagnostic sensitivity of the RT-LAMP assay. In parallel, we estimated the economic costs of setting up and running the test station. We performed > 35,000 tests, with an average turnover time of < 6 h from sample arrival to result announcement. Altogether, our work provides a blueprint for fast, sensitive, scalable, cost- and labor-efficient RT-LAMP diagnostics, which is independent of potentially limiting clinical diagnostics supply chains.

Multisubunit Tethering Complexes (MTCs) are a set of conserved protein complexes that tether vesicles at the acceptor membrane. Interactions with other components of the trafficking machinery regulate MTCs through mechanisms that are partially understood. Here, we systematically investigate the interactome that regulates MTCs. We report that P4-ATPases, a family of lipid flippases, interact with MTCs that participate in the anterograde and retrograde transport at the Golgi, such as TRAPPIII. We use the P4-ATPase Drs2 as a paradigm to investigate the mechanism and biological relevance of this interplay during transport of Atg9 vesicles. Binding of Trs85, the sole-specific subunit of TRAPPIII, to the N-terminal tail of Drs2 stabilizes TRAPPIII on membranes loaded with Atg9 and is required for Atg9 delivery during selective autophagy, a role that is independent of P4-ATPase canonical functions. This mechanism requires a conserved I(S/R)TTK motif that also mediates the interaction of the P4-ATPases Dnf1 and Dnf2 with MTCs, suggesting a broader role of P4-ATPases in MTC regulation.

The Sin3 transcriptional regulator homolog A (Sin3A) is the core member of a multiprotein chromatin-modifying complex. Its inactivation at the CD4/CD8 double-negative stage halts further thymocyte development. Among various functions, Sin3A regulates STAT3 transcriptional activity, central to the differentiation of Th17 cells active in inflammatory disorders and opportunistic infections. To further investigate the consequences of conditional Sin3A inactivation in more mature precursors and post-thymic T cell, we have generated CD4-Cre and CD4-CreERT2 Sin3AF/F mice. Sin3A inactivation in vivo hinders both thymocyte development and peripheral T-cell survival. In vitro, in Th17 skewing conditions, Sin3A-deficient cells proliferate and acquire memory markers and yet fail to properly upregulate Il17a, Il23r, and Il22. Instead, IL-2+ and FOXP3+ are mostly enriched for, and their inhibition partially rescues IL-17A+ T cells. Notably, Sin3A deletion also causes an enrichment of genes implicated in the mTORC1 signaling pathway, overt STAT3 activation, and aberrant cytoplasmic RORγt accumulation. Thus, together our data unveil a previously unappreciated role for Sin3A in shaping critical signaling events central to the acquisition of immunoregulatory T-cell phenotypes.

ADAR1 -mediated A-to-I RNA editing is a self-/non-self-discrimination mechanism for cellular double-stranded RNAs. ADAR mutations are one cause of Aicardi-Goutières Syndrome, an inherited paediatric encephalopathy, classed as a "Type I interferonopathy." The most common ADAR1 mutation is a proline 193 alanine (p.P193A) mutation, mapping to the ADAR1p150 isoform-specific Zα domain. Here, we report the development of an independent murine P195A knock-in mouse, homologous to human P193A. The Adar1P195A/P195A mice are largely normal and the mutation is well tolerated. When the P195A mutation is compounded with an Adar1 null allele (Adar1P195A/- ), approximately half the animals are runted with a shortened lifespan while the remaining Adar1P195A/- animals are normal, contrasting with previous reports. The phenotype of the Adar1P195A/- animals is both associated with the parental genotype and partly non-genetic/environmental. Complementation with an editing-deficient ADAR1 (Adar1P195A/E861A ), or the loss of MDA5, rescues phenotypes in the Adar1P195A/- mice.

In the arthropod gut, commensal microbiota maintain the immune deficiency (Imd)/Relish pathway for expression of antimicrobial peptides, whereas pathogenic bacteria induce dual oxidase 2 (Duox2) for production of extracellular microbicidal reactive oxygen species (ROS). The Imd/Relish pathway and the Duox2/ROS system are regarded as independent systems. Here, we report that these two systems are bridged by the tumor necrosis factor (TNF) ortholog PcEiger in the red swamp crayfish Procambarus clarkii. PcEiger expression is induced by commensal bacteria or the Imd/Relish pathway. PcEiger knockdown alters bacterial abundance and community composition due to variations in the oxidative status of the intestine. PcEiger induces Duox2 expression and ROS production by regulating the activity of the transcription factor Atf2. Moreover, PcEiger mediates regulation of the Duox2/ROS system by commensal bacteria and the Imd/Relish pathway. Our findings suggest that the Imd/Relish pathway regulates the Duox2/ROS system via PcEiger in P. clarkii, and they provide insights into the crosstalk between these two important mechanisms for arthropod intestinal immunity.

The observation that soil-dwelling fungi seem to exchange information via electrical impulses has raised new interest about their interactions with plants and their ecological significance.

Climate change is having dramatic effects on forest health and growth - tree genomics provides tools for understanding and mitigating these effects.

Plant-based industries generate huge amounts of lignin waste that could be converted to useful bioproducts. Efforts to recycle lignin include GM plants, microbial cell factories and "lignin-first" approaches.

Due to its low cost and easy handling, nanopore sequencing has great potential to become a consumer product. The widespread use of DNA sequencers would, however, create new social, legal, and ethical challenges.