Isotope Labeling Research Articles

Robust approach for production of the human oncology target Aurora kinase B in complex with its binding partner INCENP

Protein kinases are key players in many eukaryotic signal transduction cascades and are as a result often linked to human disease. In humans, the mitotic protein kinase family of Aurora kinases consist of three members: Aurora A, B and C. All three members are involved in cell division with proposed implications in various human cancers. The human Aurora kinase B has in particular proven challenging to study with structural biology approaches, and this is mainly due to difficulties in producing the large quantities of active enzyme required for such studies. Here, we present a novel and E. coli-based production system that allows for production of milligram quantities of well-folded and active human Aurora B in complex with its binding partner INCENP. The complex is produced as a continuous polypeptide chain and the resulting fusion protein is cleaved with TEV protease to generate a stable and native heterodimer of the Aurora B:INCENP complex. The activity, stability and degree of phosphorylation of the protein complex was quantified by using a coupled ATPase assay, 31P NMR spectroscopy and mass spectrometry. The developed production system enables isotope labeling and we here report the first 1H–15N-HSQC of the human Aurora B:INCENP complex. Our developed production strategy paves the way for future structural and functional studies of Aurora B and can as such assist the development of novel anticancer drugs targeting this important mitotic protein kinase.

Unexpected asymmetric distribution of cholesterol and phospholipids in equilibrium model membranes

Lipid compositional asymmetry across the leaflets of the plasma membrane is a ubiquitous feature in eukaryotic cells. How this asymmetry is maintained is thought to be primarily controlled by active transport of lipids between leaflets. This strategy is facilitated by the fact that long tail phospholipids and sphingolipids diffuse through the lipid bilayer slowly – taking many hours or days. However, a lipid like cholesterol – which is the most abundant lipid in the plasma membrane of animal cells – has been harder to pin-point in terms of its favored side. In the present work we show that when a saturated lipid is added to a mix of the unsaturated lipid palmitoyl-oleoyl-phosphatidylcholine (POPC) and cholesterol, both cholesterol and the long tail phospholipids organize asymmetrically across the membrane’s leaflets naturally. In these extruded unilamellar vesicles, most cholesterol as well as the saturated lipid – dipalmitoylphosphatidylcholine (DPPC) or sphingomyelin (SM) – segregated to the inner leaflet while POPC preferentially localized in the outer leaflet. This asymmetric arrangement generated a slight phospholipid number imbalance favoring the outer leaflet and thus opposite to where cholesterol and the saturated lipids preferentially partitioned. These results were obtained using Magic Angle Spinning (MAS) NMR in combination with Small Angle Neutron Scattering (SANS) using isotope labeling to differentiate lipid species. We suggest that sidedness in membranes can be driven by thermodynamic processes. In addition, our MAS NMR results show that the lower bound for cholesterol’s flip-flop half-time at 45°C is 10ms, which is at least two orders of magnitude slower than current MD simulations predict. This result stands in stark contrast to previous work that suggested that cholesterol’s flip-flop half-time at 37°C has an upper bound of 10ms.

Enhanced carbon use efficiency and warming resistance of soil microorganisms under organic amendment

The frequency and intensity of extreme weather events, including rapid temperature fluctuations, are increasing because of climate change. Long-term fertilization practices have been observed to alter microbial physiology and community structure, thereby affecting soil carbon sequestration. However, the effects of warming on the carbon sequestration potential of soil microbes adapted to long-term fertilization remain poorly understood. In this study, we utilized 18O isotope labeling to assess microbial carbon use efficiency (CUE) and employed stable isotope probing (SIP) with 18O-H2O to identify growing taxa in response to temperature changes (5–35 °C). Organic amendment with manure or straw residue significantly increased microbial CUE by 86–181 % compared to unfertilized soils. The microorganisms inhabiting organic amended soils displayed greater resistance of microbial CUE to high temperatures (25–35 °C) compared to those inhabiting soils fertilized only with minerals. Microbial growth patterns determined by the classification of taxa into incorporators or non-incorporators based on 18O incorporation into DNA exhibited limited phylogenetic conservation in response to temperature changes. Microbial clusters were identified by grouping taxa with similar growth patterns across different temperatures. Organic amendments enriched microbial clusters associated with increased CUE, whereas clusters in unfertilized or mineral-only fertilized soils were linked to decreased CUE. Specifically, shifts in the composition of growing bacteria were correlated with enhanced microbial CUE, whereas modifications in the composition of growing fungi were associated with diminished CUE. Notably, the responses of microbial CUE to temperature fluctuations were primarily driven by changes in the bacterial composition. Overall, our findings demonstrate that organic amendments enhance soil microbial CUE and promote the enrichment of specific microbial clusters that are better equipped to cope with temperature changes. This study establishes a theoretical foundation for manipulating soil microbes to enhance carbon sequestration under global climate scenarios.

Integrative phosphoproteomic analysis reveal hemostatic-endothelial signaling interplay

BackgroundThe vascular endothelial cell (EC) monolayer plays a crucial part in maintaining hemostasis. An extensive array of G protein-coupled receptors (GPCRs) allows ECs to dynamically act on key hemostatic stimuli such as thrombin and histamine. The impact of these individual stimuli on EC signal transduction has been the subject of various studies, but insight into discordant and concordant EC signaling between different GPCRs remain limited. ObjectivesTo elucidate histamine and protease activated receptor (PAR1-4) signaling cascades in endothelial cells, discern overlapping and diverging regulation between these stimuli and their effect on the EC monolayer. MethodsWe employed stable isotope labelling by amino acids in cell culture (SILAC) mass spectrometry-based phosphoproteomics on in vitro cultured BOECs, stimulated with histamine and different protease activated receptor peptides (PAR1-4). We investigated key phosphosites through immuno(fluorescence)- staining and determined effects on barrier function through trans endothelial resistance assays. ResultsEC histamine activation initiated an extensive (kinase-) signaling network (among which, MAPK3, STAT3 and CTNND1). PAR1 and PAR2 receptors induced highly similar signaling cascades, wheras PAR3 and PAR4 induced minimal phospho-regulation. Integration of all applied stimuli indicated uniquely activated proteins between both stimuli, as well as a general overlapping activation of cell-junction and actin cytoskeletal proteins. ConclusionWe provide an integrative phosphoproteomic analysis of histamine and PAR agonists in the endothelium that highlights the endothelial response programs that are at the bases of regulating hemostasis.

Natural Abundance 195Pt-13C Correlation NMR Spectroscopy on Surfaces Enabled by Fast MAS Dynamic Nuclear Polarization

Surface organometallic chemistry has developed as an effective strategy for the rational design and synthesis of well-defined, single-site Pt-based heterogeneous catalysts. Given its high sensitivity to changes in electronic structure, 195Pt solid-state NMR spectroscopy offers a unique approach to investigate the chemical structure and local environment of Pt surface sites, providing invaluable insights for establishing structure-activity relationships. However, this approach is typically hindered by severe sensitivity issues, due to the low loading of Pt sites and the often-encountered large 195Pt chemical shift anisotropies. To overcome this limitation, 195Pt NMR signature of surface metal centers can be indirectly detected through protons. Indirect detection on 13C spins, has also been demonstrated to be feasible by combining isotopic labeling with dynamic nuclear polarization (DNP). Here, we extend this methodology to a supported Pt complex at natural abundance. The material was prepared by grafting (COD)PtMeOSi(OtBu)3 (COD = 1,5-cyclooctadiene, Me = methyl and tBu = tert-butyl) onto partially dehydroxylated silica. DNP enhanced two-dimensional through-bond 13C{195Pt} heteronuclear correlation experiments were successfully implemented at fast magic angle spinning. They enabled the detection of the 0.37% NMR-responsive surface species, thereby showcasing the remarkable sensitivity of this approach and its broad applicability. Key bonding information was obtained by measuring the correlated 13C and 195Pt isotopic chemical shifts as well as 1J(13C-195Pt) coupling constants, confirming directly the coordination structure of the surface Pt sites.

Soil carbon and nitrogen cycling at the atmosphere-soil interface: Quantifying the responses of biocrust-soil interactions to global change.

In drylands, where water scarcity limits vascular plant growth, much of the primary production occurs at the soil surface. This is where complex macro- and microbial communities, in an intricate bond with soil particles, form biological soil crusts (biocrusts). Despite their critical role in regulating C and N cycling in dryland ecosystems, there is limited understanding of the fate of biologically fixed C and N from biocrusts into the mineral soil, or how climate change will affect C and N fluxes between the atmosphere, biocrusts, and subsurface soils. To address these gaps, we subjected biocrust-soil systems to experimental warming and drought under controlled laboratory conditions, monitored CO2 fluxes, and applied dual isotopic labeling pulses (13CO2 and 15N2). This allowed detailed quantification of elemental pathways into specific organic matter (OM) pools and microbial biomass via density fractionation and phospholipid fatty acid analyses. While biocrusts modulated CO2 fluxes regardless of the temperature regime, drought severely limited their photosynthetic C uptake to the extent that the systems no longer sustained net C uptake. Furthermore, the effect of biocrusts extended into the underlying 1 cm of mineral soil, where C and N accumulated as mineral-associated OM (MAOM<63μm). This was strongly associated with increased relative dominance of fungi, suggesting that fungal hyphae facilitate the downward C and N translocation and subsequent MAOM formation. Most strikingly, however, these pathways were disrupted in systems exposed to warming, where no effects of biocrusts on the elemental composition of the underlying soil nor on MAOM were determined. This was further associated with reduced net biological N fixation under combined warming and drought, highlighting how changing climatic conditions diminish some of the most fundamental ecosystem functions of biocrusts, with detrimental repercussions for C and N cycling and the persistence of soil organic matter pools in dryland ecosystems.

Transformation of Biochar from Plant Biomass in Gray Forest Soil: Evaluation by Isotopic Labeling Method

Pyrolysis is considered to be one of promising methods for processing agricultural waste and for producing fertilizers. The efficiency of the resulting biochar as a fertilizer has been proven, but the preferential way of decomposition of organic substances in it—biotic or abiotic—is still open to argument. The ways of transformation of biochar obtained from corn (a plant of the C4 type of photosynthesis with an increased 13C content) were assessed in this work, using the solid-phase CP/MAS 13C NMR spectroscopy. Biochar was placed into the top layer of a monolith of gray forest soil, and the precipitation regime characteristic of Central Russia was simulated for 90 days. The peak at 129 ppm typical for aromatic compounds increased during the experiment in the obtained NMR spectra of soil samples with biochar in the upper soil layer, but not in other layers. This testifies that biochar particles do not migrate down the soil profile during one season. The intensity of cumulative microbial respiration in the presence of biochar increases from 85.0 g CO2 kg–1 in the control sample to 201.4 g CO2 kg–1 in the sample with biochar (the topsoil). According to the NMR spectra of the salt formed during mineralization of carbon dioxide released from the soil, it contains labeled carbon: there is a peak at 169 ppm characteristic of carbonates. The cumulative volume of CO2 released from the soil with biochar is 1.9 times greater as compared to the control soil. The addition of microorganisms-decomposers caused an additional increase in the CO2 volume: 2.4 times relative to the control, which indicates the role of microorganisms in the destruction of soil organic matter and of biochar. However, based on the stability of the total carbon content in the soil, it can be concluded that only a small proportion of biochar components is susceptible to biotic decomposition.

LuCl3/B(C6F5)3 Cocatalyzed Reductive Deoxygenation of Ketones, Aldehydes, Alcohols, and Ethers to Alkanes with Pinacolborane.

This report describes the LuCl3/B(C6F5)3 cocatalyzed reductive deoxygenation of 67 ketones, aldehydes, alcohols, and ethers to alkanes under mild conditions. The strategy tolerates reactive amino, hydroxyl, nitro, halogen, vinyl, and ester functional groups, and the results demonstrate rare chemoselective deoxygenation of α,β-unsaturated ketones. Isotopic labeling experiments, control experiments, and derivatization studies are used to elucidate the reaction mechanism.

Surfactant Phospholipid Kinetics in Ventilated Children after Therapeutic Surfactant Supplementation

Acute lung Injury leads to alterations in surfactant lipid composition and metabolism. Although several mechanisms contribute to dysregulated surfactant metabolism, studies investigating in vivo surfactant metabolism are limited. The aim of this study is to characterise surfactant phospholipid composition and flux utilising a stable isotope labelling technique in mechanically ventilated paediatric patients. Paediatric patients (&lt;16 years of age) received 3.6 mg/kg intravenous methyl-D9-choline chloride followed by the endotracheal instillation of 100 mg/kg of exogenous surfactant after 24 h. Bronchioalveolar fluid samples were taken at baseline and 12, 24, 36, 48, 72 and 96 h after methyl-D9-choline infusion. Nine participants (median age of 48 days) were recruited. The primary phosphatidylcholine (PC) composition consisted of PC16:0/16:0 or DPPC (32.0 ± 4.5%). Surfactant supplementation resulted in a 30% increase in DPPC. Methyl-D9 PC enrichment was detected after 12 h and differed significantly between patients, suggesting variability in surfactant synthesis/secretion by the CDP-choline pathway. Peak enrichment was achieved (0.94 ± 0.15% of total PC) at 24 h after methyl-D9-choline infusion. There was a trend towards reduced enrichment with the duration of mechanical ventilation prior to study recruitment; however, this was not statistically significant (p = 0.19). In this study, we demonstrated the fractional molecular composition and turnover of surfactant phospholipids, which was highly variable between patients.

Topological Synthesis of 2D High-Entropy Multimetallic (Oxy)hydroxide for Enhanced Lattice Oxygen Oxidation Mechanism.

Owing to sluggish reaction kinetics and high potential, oxygen evolution reaction (OER) electrocatalysts face a trade-off between activity and stability. Herein, an innovative topological strategy is presented for preparing 2D multimetallic (oxy)hydroxide, including ternary CoFeZn, quaternary CoFeMnZn, and high-entropy CoFeMnCuZn. The key to the synthesis lies in using Ca-rich brownmillerite oxide as a precursor, which possesses inherent structural flexibility enabling tailored elemental adjustments and topologically transforms from a point-shared structure of metal-oxygen octahedrons into an edge-shared structure under alkaline conditions. The presence of Zn in the catalysts causes a shift in the center of the O2p band toward the Fermi level, resulting in more Co4+ species, which drive holes into oxygen ligands to promote intramolecular oxygen coupling. The triggered lattice oxidation mechanism is identified by detecting peroxo-like (O2 2-) negative species using tetramethylammonium chemical probe, along with 18O isotope labeling experiments. As a result, the catalyst demonstrates an overpotential of 267mV at 10 mA cm-2, ranking it among the top-performing non-Ni-based catalysts. Importantly, the catalysts also show high Fe-leaching resistance during OER compared to conventional NiFe and CoFe hydroxides/(oxy)hydroxides. The assembled zinc-air battery enables stable operation for over 225 h at a low charging voltage of 1.93V.

Disruption of millipede-gut microbiota in E. pulchripes and G. connexa highlights the limited role of litter fermentation and the importance of litter-associated microbes for nutrition.

Millipedes are thought to depend on their gut microbiome for processing plant-litter-cellulose through fermentation, similar to many other arthropods. However, this hypothesis lacks sufficient evidence. To investigate this, we used inhibitors to disrupt the gut microbiota of juvenile Epibolus pulchripes (tropical, CH4-emitting) and Glomeris connexa (temperate, non-CH4-emitting) and isotopic labelling. Feeding the millipedes sterile or antibiotics-treated litter reduced faecal production and microbial load without major impacts on survival or weight. Bacterial diversity remained similar, with Bacteroidota dominant in E. pulchripes and Pseudomonadota in G. connexa. Sodium-2-bromoethanesulfonate treatment halted CH4 emissions in E. pulchripes, but it resumed after returning to normal feeding. Employing 13C-labeled leaf litter and RNA-SIP revealed a slow and gradual prokaryote labelling, indicating a significant density shift only by day 21. Surprisingly, labelling of the fungal biomass was somewhat quicker. Our findings suggest that fermentation by the gut microbiota is likely not essential for the millipede's nutrition.

Metabolic preferences of astrocytes: Functional metabolic mapping reveals butyrate outcompetes acetate.

Disruptions to the gut-brain-axis have been linked to neurodegenerative disorders. Of these disruptions, reductions in the levels of short-chain fatty acids (SCFAs), like butyrate, have been observed in mouse models of Alzheimer's disease (AD). Butyrate supplementation in mice has shown promise in reducing neuroinflammation, amyloid-β accumulation, and enhancing memory. However, the underlying mechanisms remain unclear. To address this, we investigated the impact of butyrate on energy metabolism in mouse brain slices, primary cultures of astrocytes and neurons and in-vivo by dynamic isotope labelling with [U-13C]butyrate and [1,2-13C]acetate to map metabolism via mass spectrometry. Metabolic competition assays in cerebral cortical slices revealed no competition between butyrate and the ketone body, β-hydroxybutyrate, but competition with acetate. Astrocytes favoured butyrate metabolism compared to neurons, suggesting that the astrocytic compartment is the primary site of butyrate metabolism. In-vivo metabolism investigated in the 5xFAD mouse, an AD pathology model, showed no difference in 13C-labelling of TCA cycle metabolites between wild-type and 5xFAD brains, but butyrate metabolism remained elevated compared to acetate in both groups, indicating sustained uptake and metabolism in 5xFAD mice. Overall, these findings highlight the role of astrocytes in butyrate metabolism and the potential use of butyrate as an alternative brain fuel source.

Carbon isotopic labelling of carboxylic acids enabled by organic photoredox-catalysed cyanation

Carbon isotopic labelling of carboxylic acids enabled by organic photoredox-catalysed cyanation

Heterocyclic Surgery for Isotopic Labeling

Heterocyclic Surgery for Isotopic Labeling

Single-step conversion of methane-steam to methanol on single-atom Cu1/γ-Al2O3 catalyst prepared via electrostatic anchoring

Direct conversion of methane to methanol in heterogeneous catalysis is of paramount importance but remains a challenging issue in improving catalytic activity and selectivity. Here, we report that Cu single-atom sites anchored on γ-Al2O3 oxides (Cu1/γ-Al2O3) using surface electrostatic adsorption, are highly active (methanol yield of ∼47 mmol/molCu/h) and stable in continuous conversion of methane-steam to methanol at 200 °C, comparable to most of the state-of-the-art Cu-zeolite catalysts. The combined DFT calculation and isotope labeling experiments reveal the methane-to-methanol reaction pathway on Cu single-atom sites and methanol formation is the rate limiting step. The further theoretical study indicates that single Cu atom as active site can preferentially activate methane C-H bond instead of methanol and has larger advantage for highly-selective methanol yield compared with [Cu(II)-OH]+ and [CuOCu]2+ active sites. Our molecular-level findings on Cu1/γ-Al2O3 single-atom catalyst pave the way to prepare better non-zeolite catalysts for methane to methanol.

Age-induced changes in skeletal muscle mitochondrial DNA synthesis, quantity, and quality in genetically unique rats

AbstractMitochondrial genomic integrity is a key element of physiological processes and health. Changes in the half-life of the mitochondrial genome are implicated in the generation and accumulation of age-induced mitochondrial DNA (mtDNA) mutations, which are implicated in skeletal muscle aging and sarcopenia. There are conflicting data on the half-life of mtDNA, and there is limited information on how aging affects half-life in skeletal muscle. We hypothesized that skeletal muscle mtDNA synthesis rates would decrease with age in both female and male rats concomitant with changes in mtDNA integrity reflected in mtDNA copy number and mutation frequency. We measured mitochondrial genome half-life using stable isotope labeling over a period of 14 days and assessed mtDNA copy number and deletion mutation frequency using digital PCR in the quadriceps muscle of 9-month-old and 26-month-old male and female OKC-HET rats. We found a significant age-related increase in mtDNA half-life, from 132 days at 9 months to 216 days at 26 months of age in OKC-HET quadriceps. Concomitant with the increase in mtDNA half-life, we found an age-related increase in mtDNA deletion mutation frequency in both male and female rats. Notably, 26-month-old female rats had a lower mutation frequency than male rats, and there were no changes in mtDNA copy number with sex, age, or mitochondrial genotype. These data reveal several key findings: (1) mtDNA turnover in rat skeletal muscle decreases with age, (2) mtDNA half-lives in skeletal muscle are approximately an order of magnitude longer than what is reported for other tissues, and (3) muscle mtDNA turnover differs significantly from the turnover of other mitochondrial macromolecules including components of the mitochondrial nucleoid. These findings provide insight into the factors driving age-induced mtDNA mutation accumulation, which contribute to losses of mitochondrial genomic integrity and may play a role in skeletal muscle dysfunction.

CDS2 expression regulates de novo phosphatidic acid synthesis.

CDS enzymes (CDS1 and 2 in mammals) convert phosphatidic acid (PA) to CDP-DG, an essential intermediate in the de novo synthesis of PI. Genetic deletion of CDS2 in primary mouse macrophages resulted in only modest changes in the steady-state levels of major phospholipid species, including PI, but substantial increases in several species of PA, CDP-DG, DG and TG. Stable isotope labelling experiments employing both 13C6- and 13C6D7-glucose revealed loss of CDS2 resulted in a minimal reduction in the rate of de novo PI synthesis but a substantial increase in the rate of de novo PA synthesis from G3P, derived from DHAP via glycolysis. This increased synthesis of PA provides a potential explanation for normal basal PI synthesis in the face of reduced CDS capacity (via increased provision of substrate to CDS1) and increased synthesis of DG and TG (via increased provision of substrate to LIPINs). However, under conditions of sustained GPCR-stimulation of PLC, CDS2-deficient macrophages were unable to maintain enhanced rates of PI synthesis via the 'PI cycle', leading to a substantial loss of PI. CDS2-deficient macrophages also exhibited significant defects in calcium homeostasis which were unrelated to the activation of PLC and thus probably an indirect effect of increased basal PA. These experiments reveal that an important homeostatic response in mammalian cells to a reduction in CDS capacity is increased de novo synthesis of PA, likely related to maintaining normal levels of PI, and provides a new interpretation of previous work describing pleiotropic effects of CDS2 deletion on lipid metabolism/signalling.

Unlocking Lattice Oxygen on Selenide-Derived NiCoOOH for Amine Electrooxidation and Efficient Hydrogen Production.

In pursuit of advancing the electrooxidation of amines, which is typically encumbered by the inertness of C(sp3)-H/N(sp3)-H bonds, our study introduces a high-performance electrocatalyst that significantly enhances the production efficiency of vital chemicals and fuels. We propose a novel electrocatalytic strategy employing a uniquely designed (NixCo1-x)Se2-R electrocatalyst, which is activated through Se-O exchange and electron orbital spin manipulation. This catalyst efficiently generates M4+ species, thus enabling the activation of lattice oxygen and streamlining the electrooxidation of amines. Empirical evidence from isotope labeling, molecular probes, and computational analyses indicates that the electrocatalyst fosters the formation of energetically favorable peroxy radical intermediates, which substantially expedite the reaction kinetics. The refined electrocatalyst achieves an exceptional current density of 20 mA cm-2 at a potential of 1.315 V, with selectivity surpassing 99% for propionitrile, while demonstrating remarkable stability over 560 h. This work emphasizes the criticality of deciphering the fundamental mechanisms of amine electrooxidation and charts a more sustainable pathway for the nitrile and hydrogen production, marking a substantial advancement in the field of electrocatalysis.

Spliceosomal vulnerability of MYCN-amplified neuroblastoma is contingent on PRMT5-mediated regulation of epitranscriptomic and metabolomic pathways

Approximately 50 % of poor prognosis neuroblastomas arise due to MYCN over-expression. We previously demonstrated that MYCN and PRMT5 proteins interact and PRMT5 knockdown led to apoptosis of MYCN-amplified (MNA) neuroblastoma. Here we evaluate the highly selective first-in-class PRMT5 inhibitor GSK3203591 and its in vivo analogue GSK3326593 as targeted therapeutics for MNA neuroblastoma. Cell-line analyses show MYCN-dependent growth inhibition and apoptosis, with approximately 200-fold greater sensitivity of MNA neuroblastoma lines. RNA sequencing of three MNA neuroblastoma lines treated with GSK3203591 reveal deregulated MYCN transcriptional programmes and altered mRNA splicing, converging on key regulatory pathways such as DNA damage response, epitranscriptomics and cellular metabolism. Stable isotope labelling experiments in the same cell lines demonstrate that glutamine metabolism is impeded following GSK3203591 treatment, linking with disruption of the MLX/Mondo nutrient sensors via intron retention of MLX mRNA. Interestingly, glutaminase (GLS) protein decreases after GSK3203591 treatment despite unchanged transcript levels. We demonstrate that the RNA methyltransferase METTL3 and cognate reader YTHDF3 proteins are lowered following their mRNAs undergoing GSK3203591-induced splicing alterations, indicating epitranscriptomic regulation of GLS; accordingly, we observe decreases of GLS mRNA m6A methylation following GSK3203591 treatment, and decreased GLS protein following YTHDF3 knockdown. In vivo efficacy of GSK3326593 is confirmed by increased survival of Th-MYCN mice, with drug treatment triggering splicing events and protein decreases consistent with in vitro data. Together our study demonstrates the PRMT5-dependent spliceosomal vulnerability of MNA neuroblastoma and identifies the epitranscriptome and glutamine metabolism as critical determinants of this sensitivity.

NQuant Enables Precise Quantitative N-Glycomics.

N-glycosylation is a highly heterogeneous post-translational modification that modulates protein function. Defects in N-glycosylation are directly linked to various human diseases. Despite the importance of quantifying N-glycans with high precision, existing glycoinformatics tools are limited. Here, we developed nQuant, a glycoinformatics tool that enables label-free and isotopic labeling quantification of N-glycomics data obtained via LC-MS/MS, ensuring a low false quantitation rate. Using the label-free quantification module, we profiled the N-glycans released from purified glycoproteins and HEK293 cells as well as the dynamic changes of N-glycosylation during mouse corpus callosum development. Through the isotopic labeling quantification module, we revealed the dynamic changes of N-glycans in acute promyelocytic leukemia cells after all-trans retinoic acid treatment. Taken together, we demonstrate that nQuant enables fast and precise quantitative N-glycomics.