Amelioration of hepatic steatosis by dietary essential amino acid-induced ubiquitination.

Nonalcoholic fatty liver disease (NAFLD) is a global health concern with no approved drugs. High-protein dietary intervention is currently the most effective treatment. However, its underlying mechanism is unknown. Here, using Drosophila oenocytes, the specialized hepatocyte-like cells, we find that dietary essential amino acids ameliorate hepatic steatosis by inducing polyubiquitination of Plin2, a lipid droplet-stabilizing protein. Leucine and isoleucine, two branched-chain essential amino acids, strongly bind to and activate the E3 ubiquitin ligase Ubr1, targeting Plin2 for degradation. We further show that the amino acid-induced Ubr1 activity is necessary to prevent steatosis in mouse livers and cultured human hepatocytes, providing molecular insight into the anti-NAFLD effects of dietary protein/amino acids. Importantly, split-intein-mediated trans-splicing expression of constitutively active UBR2, an Ubr1 family member, significantly ameliorates obesity-induced and high fat diet-induced hepatic steatosis in mice. Together, our results highlight activation of Ubr1 family proteins as a promising strategy in NAFLD treatment.

Integration of light and temperature sensing by liquid-liquid phase separation of phytochrome B.

Light and temperature in plants are perceived by a common receptor, phytochrome B (phyB). How phyB distinguishes these signals remains elusive. Here, we report that phyB spontaneously undergoes phase separation to assemble liquid-like droplets. This capacity is driven by its C terminus through self-association, whereas the intrinsically disordered N-terminal extension (NTE) functions as a biophysical modulator of phase separation. Light exposure triggers a conformational change to subsequently alter phyB condensate assembly, while temperature sensation is directly mediated by the NTE to modulate the phase behavior of phyB droplets. Multiple signaling components are selectively incorporated into phyB droplets to form concentrated microreactors, allowing switch-like control of phyB signaling activity through phase transitions. Therefore, light and temperature cues are separately read out by phyB via allosteric changes and spontaneous phase separation, respectively. We provide a conceptual framework showing how the distinct but highly correlated physical signals are interpreted and sorted by one receptor.

A swapped genetic code prevents viral infections and gene transfer.

Engineering the genetic code of an organism has been proposed to provide a firewall from natural ecosystems by preventing viral infections and gene transfer1-6. However, numerous viruses and mobile genetic elements encode parts of the translational apparatus7-9, potentially rendering a genetic-code-based firewall ineffective. Here we show that such mobile transfer RNAs (tRNAs) enable gene transfer and allow viral replication in Escherichia coli despite the genome-wide removal of 3 of the 64 codons and the previously essential cognate tRNA and release factor genes. We then establish a genetic firewall by discovering viral tRNAs that provide exceptionally efficient codon reassignment allowing us to develop cells bearing an amino acid-swapped genetic code that reassigns two of the six serine codons to leucine during translation. This amino acid-swapped genetic code renders cells resistant to viral infections by mistranslating viral proteomes and prevents the escape of synthetic genetic information by engineered reliance on serine codons to produce leucine-requiring proteins. As these cells may have a selective advantage over wild organisms due to virus resistance, we also repurpose a third codon to biocontain this virus-resistant host through dependence on an amino acid not found in nature10. Our results may provide the basis for a general strategy to make any organism safely resistant to all natural viruses and prevent genetic information flow into and out of genetically modified organisms.

C. difficile intoxicates neurons and pericytes to drive neurogenic inflammation.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections1,2. The exaggerated colonic inflammation caused by C. difficile toxins such as toxin B (TcdB) damages tissues and promotes C. difficile colonization3-6, but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI.

A zebrafish embryo culture system defines factors that promote vertebrate myogenesis across species.

Ex vivo expansion of satellite cells and directed differentiation of pluripotent cells to mature skeletal muscle have proved difficult challenges for regenerative biology. Using a zebrafish embryo culture system with reporters of early and late skeletal muscle differentiation, we examined the influence of 2,400 chemicals on myogenesis and identified six that expanded muscle progenitors, including three GSK3ß inhibitors, two calpain inhibitors, and one adenylyl cyclase activator, forskolin. Forskolin also enhanced proliferation of mouse satellite cells in culture and maintained their ability to engraft muscle in vivo. A combination of bFGF, forskolin, and the GSK3ß inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs) and produced engraftable myogenic progenitors that contributed to muscle repair in vivo. In summary, these studies reveal functionally conserved pathways regulating myogenesis across species and identify chemical compounds that expand mouse satellite cells and differentiate human iPSCs into engraftable muscle.

Mitochondrial base editor induces substantial nuclear off-target mutations.

DddA-derived cytosine base editors (DdCBEs)-which are fusions of split DddA halves and transcription activator-like effector (TALE) array proteins from bacteria-enable targeted C•G-to-T•A conversions in mitochondrial DNA1. However, their genome-wide specificity is poorly understood. Here we show that the mitochondrial base editor induces extensive off-target editing in the nuclear genome. Genome-wide, unbiased analysis of its editome reveals hundreds of off-target sites that are TALE array sequence (TAS)-dependent or TAS-independent. TAS-dependent off-target sites in the nuclear DNA are often specified by only one of the two TALE repeats, challenging the principle that DdCBEs are guided by paired TALE proteins positioned in close proximity. TAS-independent off-target sites on nuclear DNA are frequently shared among DdCBEs with distinct TALE arrays. Notably, they co-localize strongly with binding sites for the transcription factor CTCF and are enriched in topologically associating domain boundaries. We engineered DdCBE to alleviate such off-target effects. Collectively, our results have implications for the use of DdCBEs in basic research and therapeutic applications, and suggest the need to thoroughly define and evaluate the off-target effects of base-editing tools.

ABO genotype alters the gut microbiota by regulating GalNAc levels in pigs.

The composition of the intestinal microbiome varies considerably between individuals and is correlated with health1. Understanding the extent to which, and how, host genetics contributes to this variation is essential yet has proved to be difficult, as few associations have been replicated, particularly in humans2. Here we study the effect of host genotype on the composition of the intestinal microbiota in a large mosaic pig population. We show that, under conditions of exacerbated genetic diversity and environmental uniformity, microbiota composition and the abundance of specific taxa are heritable. We map a quantitative trait locus affecting the abundance of Erysipelotrichaceae species and show that it is caused by a 2.3 kb deletion in the gene encoding N-acetyl-galactosaminyl-transferase that underpins the ABO blood group in humans. We show that this deletion is a ≥3.5-million-year-old trans-species polymorphism under balancing selection. We demonstrate that it decreases the concentrations of N-acetyl-galactosamine in the gut, and thereby reduces the abundance of Erysipelotrichaceae that can import and catabolize N-acetyl-galactosamine. Our results provide very strong evidence for an effect of the host genotype on the abundance of specific bacteria in the intestine combined with insights into the molecular mechanisms that underpin this association. Our data pave the way towards identifying the same effect in rural human populations.

Memory B cell repertoire from triple vaccinees against diverse SARS-CoV-2 variants.

Omicron (B.1.1.529), the most heavily mutated SARS-CoV-2 variant so far, is highly resistant to neutralizing antibodies, raising concerns about the effectiveness of antibody therapies and vaccines1,2. Here we examined whether sera from individuals who received two or three doses of inactivated SARS-CoV-2 vaccine could neutralize authentic Omicron. The seroconversion rates of neutralizing antibodies were 3.3% (2 out of 60) and 95% (57 out of 60) for individuals who had received 2 and 3 doses of vaccine, respectively. For recipients of three vaccine doses, the geometric mean neutralization antibody titre for Omicron was 16.5-fold lower than for the ancestral virus (254). We isolated 323 human monoclonal antibodies derived from memory B cells in triple vaccinees, half of which recognized the receptor-binding domain, and showed that a subset (24 out of 163) potently neutralized all SARS-CoV-2 variants of concern, including Omicron. Therapeutic treatments with representative broadly neutralizing monoclonal antibodies were highly protective against infection of mice with SARS-CoV-2 Beta (B.1.351) and Omicron. Atomic structures of the Omicron spike protein in complex with three classes of antibodies that were active against all five variants of concern defined the binding and neutralizing determinants and revealed a key antibody escape site, G446S, that confers greater resistance to a class of antibodies that bind on the right shoulder of the receptor-binding domain by altering local conformation at the binding interface. Our results rationalize the use of three-dose immunization regimens and suggest that the fundamental epitopes revealed by these broadly ultrapotent antibodies are rational targets for a universal sarbecovirus vaccine.

Single-cell dissection of cervical cancer reveals key subsets of the tumor immune microenvironment.

The tumor microenvironment (TME) directly determines patients' outcomes and therapeutic efficiencies. An in-depth understanding of the TME is required to improve the prognosis of patients with cervical cancer (CC). This study conducted single-cell RNA and TCR sequencing of six-paired tumors and adjacent normal tissues to map the CC immune landscape. T and NK cells were highly enriched in the tumor area and transitioned from cytotoxic to exhaustion phenotypes. Our analyses suggest that cytotoxic large-clone T cells are critical effectors in the antitumor response. This study also revealed tumor-specific germinal center B cells associated with tertiary lymphoid structures. A high-germinal center B cell proportion in patients with CC is predictive of improved clinical outcomes and is associated with elevated hormonal immune responses. We depicted an immune-excluded stromal landscape and established a joint model of tumor and stromal cells to predict CC patients' prognosis. The study revealed tumor ecosystem subsets linked to antitumor response or prognosis in the TME and provides information for future combinational immunotherapy.

The MBD-ACD DNA methylation reader complex recruits MICRORCHIDIA6 to regulate ribosomal RNA gene expression in Arabidopsis.

DNA methylation is an important epigenetic mark implicated in selective rRNA gene expression, but the DNA methylation readers and effectors remain largely unknown. Here, we report a protein complex that reads DNA methylation to regulate variant-specific 45S ribosomal RNA (rRNA) gene expression in Arabidopsis (Arabidopsis thaliana). The complex, consisting of METHYL-CpG-BINDING DOMAIN PROTEIN5 (MBD5), MBD6, ALPHA-CRYSTALLIN DOMAIN PROTEIN15.5 (ACD15.5), and ACD21.4, directly binds to 45S rDNA. While MBD5 and MBD6 function redundantly, ACD15.5 and ACD21.4 are indispensable for variant-specific rRNA gene expression. These 4 proteins undergo phase separation in vitro and in vivo and are interdependent for their phase separation. The α-crystallin domain of ACD15.5 and ACD21.4, which is essential for their function, enables phase separation of the complex, likely by mediating multivalent protein interactions. The effector MICRORCHIDIA6 directly interacts with ACD15.5 and ACD21.4, but not with MBD5 and MBD6, and is recruited to 45S rDNA by the MBD-ACD complex to regulate variant-specific 45S rRNA expression. Our study reveals a pathway in Arabidopsis through which certain 45S rRNA gene variants are silenced, while others are activated.

Stuxnet fine-tunes Notch dose during development using a functional Polycomb response element.

Evolutionarily conserved Notch signaling is highly sensitive to changes in Notch receptor dose caused by intrinsic and environmental fluctuations. It is well known that epigenetic regulation responds dynamically to genetic, cellular and environmental stresses. However, it is unclear whether the Notch receptor dose is directly regulated at the epigenetic level. Here, by studying the role of the upstream epigenetic regulator Stuxnet (Stx) in Drosophila developmental signaling, we find that Stx promotes Notch receptor mRNA expression by counteracting the activity of Polycomb repressive complex 1 (PRC1). In addition, we provide evidence that Notch is a direct PRC1 target by identifying and validating in vivo the only bona fide Polycomb response element (PRE) among the seven Polycomb group (PcG)-binding sites revealed by DamID-seq and ChIP-seq analysis. Importantly, in situ deletion of this PRE results in increased Notch expression and phenotypes resembling Notch hyperactivation in cell fate specification. These results not only underscore the importance of epigenetic regulation in fine-tuning the Notch activity dose, but also the need to assess the physiological significance of omics-based PcG binding in development.

Comparative analysis of nascent RNA sequencing methods and their applications in studies of cotranscriptional splicing dynamics.

High-throughput detection of nascent RNA is critical for studies of transcription and much more challenging than that of mRNA. Recently, several massively parallel nascent RNA sequencing methods were established in eukaryotic cells. Here, we systematically compared 3 classes of methods on the same pure or crude nuclei preparations: GRO-seq for sequence nuclear run-on RNAs, pNET-seq for sequence RNA polymerase II-associated RNAs, and CB RNA-seq for sequence chromatin-bound (CB) RNAs in Arabidopsis (Arabidopsis thaliana). To improve the resolution of CB RNAs, 3'CB RNA-seq was established to sequence the 3' ends of CB RNAs. In addition, we modified pNET-seq to establish the Chromatin Native Elongation Transcript sequencing (ChrNET) method using chromatin as the starting material for RNA immunoprecipitation. Reproducibility, sensitivity and accuracy in detecting nascent transcripts, experimental procedures, and costs were analyzed, which revealed the strengths and weaknesses of each method. We found that pNET and GRO methods best detected active RNA polymerase II. CB RNA-seq is a simple and cost-effective alternative for nascent RNA studies, due to its high correlation with pNET-seq and GRO-seq. Compared with pNET, ChrNET has higher specificity for nascent RNA capture and lower sequencing cost. 3'CB is sensitive to transcription-coupled splicing. Using these methods, we identified 1,404 unknown transcripts, 4,482 unannotated splicing events, and 60 potential recursive splicing events. This comprehensive comparison of different nascent/chromatin RNA sequencing methods highlights the strengths of each method and serves as a guide for researchers aiming to select a method that best meets their study goals.

Oxysterol Sensing Through GPR183 Triggers Endothelial Senescence in Hypertension.

BACKGROUND: Despite endothelial dysfunction being an initial step in the development of hypertension and associated cardiovascular/renal injuries, effective therapeutic strategies to prevent endothelial dysfunction are still lacking. GPR183 (G protein-coupled receptor 183), a recently identified G protein-coupled receptor for oxysterols and hydroxylated metabolites of cholesterol, has pleiotropic roles in lipid metabolism and immune responses. However, the role of GPR183 in the regulation of endothelial function remains unknown. METHODS: Endothelial-specific GPR183 knockout mice were generated and used to examine the role of GPR183 in endothelial senescence by establishing 2 independent hypertension models: desoxycorticosterone acetate/salt-induced and Ang II (angiotensin II)-induced hypertensive mice. Echocardiography, transmission electron microscopy, blood pressure measurement, vasorelaxation response experiments, flow cytometry analysis, and chromatin immunoprecipitation analysis were performed in this study. RESULTS: Endothelial GPR183 was significantly induced in hypertensive mice, which was further confirmed in renal biopsies from subjects with hypertensive nephropathy. Endothelial-specific deficiency of GPR183 markedly alleviated cardiovascular and renal injuries in hypertensive mice. Moreover, we found that GPR183 regulated endothelial senescence in both hypertensive mice and aged mice. Mechanistically, GPR183 disrupted circadian signaling by inhibiting PER1 (period 1) expression, thereby facilitating endothelial senescence and dysfunction through the cAMP/PKA (protein kinase A)/CREB (cAMP-response element binding protein) signaling pathway. Importantly, pharmacological inhibition of the oxysterol-GPR183 axis by NIBR189 or clotrimazole ameliorated endothelial senescence and cardiovascular/renal injuries in hypertensive mice. CONCLUSIONS: This study discovers a previously unrecognized role of GPR183 in promoting endothelial senescence. Pharmacological targeting of GPR183 may be an innovative therapeutic strategy for hypertension and its associated complications.

XIAP-mediated degradation of IFT88 disrupts HSC cilia to stimulate HSC activation and liver fibrosis.

Activation of hepatic stellate cells (HSCs) plays a critical role in liver fibrosis. However, the molecular basis for HSC activation remains poorly understood. Herein, we demonstrate that primary cilia are present on quiescent HSCs but exhibit a significant loss upon HSC activation which correlates with decreased levels of the ciliary protein intraflagellar transport 88 (IFT88). Ift88-knockout mice are more susceptible to chronic carbon tetrachloride-induced liver fibrosis. Mechanistic studies show that the X-linked inhibitor of apoptosis (XIAP) functions as an E3 ubiquitin ligase for IFT88. Transforming growth factor-ß (TGF-ß), a profibrotic factor, enhances XIAP-mediated ubiquitination of IFT88, promoting its proteasomal degradation. Blocking XIAP-mediated IFT88 degradation ablates TGF-ß-induced HSC activation and liver fibrosis. These findings reveal a previously unrecognized role for ciliary homeostasis in regulating HSC activation and identify the XIAP-IFT88 axis as a potential therapeutic target for liver fibrosis.

Gut stem cell necroptosis by genome instability triggers bowel inflammation.

The aetiology of inflammatory bowel disease (IBD) is a multifactorial interplay between heredity and environment1,2. Here we report that deficiency in SETDB1, a histone methyltransferase that mediates the trimethylation of histone H3 at lysine 9, participates in the pathogenesis of IBD. We found that levels of SETDB1 are decreased in patients with IBD, and that mice with reduced SETDB1 in intestinal stem cells developed spontaneous terminal ileitis and colitis. SETDB1 safeguards genome stability3, and the loss of SETDB1 in intestinal stem cells released repression of endogenous retroviruses (retrovirus-like elements with long repeats that, in humans, comprise approximately 8% of the genome). Excessive viral mimicry generated by motivated endogenous retroviruses triggered Z-DNA-binding protein 1 (ZBP1)-dependent necroptosis, which irreversibly disrupted homeostasis of the epithelial barrier and promoted bowel inflammation. Genome instability, reactive endogenous retroviruses, upregulation of ZBP1 and necroptosis were all seen in patients with IBD. Pharmaceutical inhibition of RIP3 showed a curative effect in SETDB1-deficient mice, which suggests that targeting necroptosis of intestinal stem cells may represent an approach for the treatment of severe IBD.

Accelerated discovery of CO2 electrocatalysts using active machine learning.

The rapid increase in global energy demand and the need to replace carbon dioxide (CO2)-emitting fossil fuels with renewable sources have driven interest in chemical storage of intermittent solar and wind energy1,2. Particularly attractive is the electrochemical reduction of CO2 to chemical feedstocks, which uses both CO2 and renewable energy3-8. Copper has been the predominant electrocatalyst for this reaction when aiming for more valuable multi-carbon products9-16, and process improvements have been particularly notable when targeting ethylene. However, the energy efficiency and productivity (current density) achieved so far still fall below the values required to produce ethylene at cost-competitive prices. Here we describe Cu-Al electrocatalysts, identified using density functional theory calculations in combination with active machine learning, that efficiently reduce CO2 to ethylene with the highest Faradaic efficiency reported so far. This Faradaic efficiency of over 80 per cent (compared to about 66 per cent for pure Cu) is achieved at a current density of 400 milliamperes per square centimetre (at 1.5 volts versus a reversible hydrogen electrode) and a cathodic-side (half-cell) ethylene power conversion efficiency of 55 ± 2 per cent at 150 milliamperes per square centimetre. We perform computational studies that suggest that the Cu-Al alloys provide multiple sites and surface orientations with near-optimal CO binding for both efficient and selective CO2 reduction17. Furthermore, in situ X-ray absorption measurements reveal that Cu and Al enable a favourable Cu coordination environment that enhances C-C dimerization. These findings illustrate the value of computation and machine learning in guiding the experimental exploration of multi-metallic systems that go beyond the limitations of conventional single-metal electrocatalysts.

Promoting C-F bond activation via proton donor for CF4 decomposition.

Tetrafluoromethane (CF4), the simplest perfluorocarbons, is a permanently potent greenhouse gas due to its powerful infrared radiation adsorption capacity. The highly symmetric and robust C-F bond structure makes its activation a great challenge. Herein, we presented an innovated approach that efficiently activates C-F bond utilizing protonated sulfate (-HSO4) modified Al2O3@ZrO2 (S-Al2O3@ZrO2) catalyst, resulting in highly efficient CF4 decomposition. By combining in situ infrared spectroscopy tests and density function theory simulations, we demonstrate that the introduced -HSO4 proton donor has a stronger interaction on the C-F bond than the hydroxyl (-OH) proton donor, which can effectively stretch the C-F bond for its activation. Consequently, the obtained S-Al2O3@ZrO2 catalyst achieved a stable 100% CF4 decomposition at a record low temperature of 580 °C with a turnover frequency value of ~8.3 times higher than the Al2O3@ZrO2 catalyst without -HSO4 modification, outperforming the previously reported results. This work paves a new way for achieving efficient C-F bond activation to decompose CF4 at a low temperature.

Spatial transcriptomics reveals light-induced chlorenchyma cells involved in promoting shoot regeneration in tomato callus.

Callus is a reprogrammed cell mass involved in plant regeneration and gene transformation in crop engineering. Pluripotent callus cells develop into fertile shoots through shoot regeneration. The molecular basis of the shoot regeneration process in crop callus remains largely elusive. This study pioneers the exploration of the spatial transcriptome of tomato callus during shoot regeneration. The findings reveal the presence of highly heterogeneous cell populations within the callus, including epidermis, vascular tissue, shoot primordia, inner callus, and outgrowth shoots. By characterizing the spatially resolved molecular features of shoot primordia and surrounding cells, specific factors essential for shoot primordia formation are identified. Notably, chlorenchyma cells, enriched in photosynthesis-related processes, play a crucial role in promoting shoot primordia formation and subsequent shoot regeneration. Light is shown to promote shoot regeneration by inducing chlorenchyma cell development and coordinating sugar signaling. These findings significantly advance our understanding of the cellular and molecular aspects of shoot regeneration in tomato callus and demonstrate the immense potential of spatial transcriptomics in plant biology.

Application of stem cells in engineered vascular graft and vascularized organs.

Recent studies report that stem cell therapies have been applied successfully to patients, This has increased anticipations that this regeneration strategy could be a potential method to treat a wide range of intractable diseases some day. Stem cells offer new prospects for the treatment of incurable diseases and for tissue regeneration and repairation because of their unique biological properties. Angiogenesis a key process in tissue regeneration and repairation. Vascularization of organs is one of the main challenges hindering the clinical application of engineered tissues. Efficient production of engineered vascular grafts and vascularized organs is of critical importance for regenerative medicine. In this review, we focus on the types of stem cells that are widely used in tissue engineering and regeneration, as well as their application of these stem cells in the construction of tissue-engineered vascular grafts and vascularization of tissue-engineered organs.

Loss of the ceramide synthase HYL-2 from Caenorhabditis elegans impairs stress responses and alters sphingolipid composition.

Sphingolipids, essential membrane components and signaling molecules in cells, have ceramides at the core of their metabolic pathways. Initially termed as "longevity assurance genes", the encoding genes of ceramide synthases are closely associated with individual aging and stress responses, although the mechanisms remain unclear. This study aims to explore the alterations and underlying mechanisms of three ceramide synthases, HYL-1, HYL-2, and LAGR-1, in the aging and stress responses of Caenorhabditis elegans. Our results showed the knockdown of HYL-1 extends the lifespan and enhance stress resistance in worms, whereas the loss of HYL-2 function significantly impairs tolerances to heat, oxidation, and ultraviolet stress. Stress intolerance induced by HYL-2 deficiency may result from intracellular mitochondrial dysfunction, accumulation of reactive oxygen species, and abnormal nuclear translocation of DAF-16 under stress conditions. Loss of HYL-2 led to a significant reduction of predominant ceramides (d17:1/C20∼C23) as well as corresponding complex sphingolipids. Furthermore, the N-acyl chain length composition of sphingolipids underwent dramatic modifications, characterized by a decrease in C22 sphingolipids and an increase in C24 sphingolipids. Extra d18:1-ceramides resulted in diminished stress resilience in wild-type worms, while supplementation of d18:1/C16 ceramide to HYL-2-deficient worms marginally improved stress tolerance to heat and oxidation. These findings indicate the importance of appropriate ceramide content and composition in maintaining subcellular homeostasis and nuclear-cytoplasmic signal transduction during healthy aging and stress responses.