Use of Integrated Metabolic Maps as a Framework for Teaching Biochemical Pathways in the Pre-clinical Medical Curriculum.

Introduction: The Larner College of Medicine has steadily transitioned to primarily active learning-based instruction. Although evaluations praise session formats, students often highlight difficulties in synthesizing preparatory materials to integrate biochemical pathways. A student/faculty collaboration led to the development of interactive metabolic maps that illustrate pathways and link to a broader framework of metabolism. Methods: A review of the session materials identified relevant biochemical pathways, and for each pathway, we created a fillable visual diagram to highlight the interactions between all substrates, enzymes, and cofactors. Implementation of the metabolic maps began for first-year medical students in fall 2022. Evaluation data included standard student session evaluations (Likert scale and qualitative comments) and a survey specific to the metabolic maps. Results: After implementing the maps, student ratings of biochemistry/metabolism session materials significantly improved (3.2 ± 1.04 to 4.3 ± 0.87, p < 0.001), and students made positive comments about their effectiveness. Most students (77.8%) used the metabolic maps to aid in studying biochemistry content for exams and found the metabolic maps important for integrating information about metabolic pathways. The median performance on metabolism-specific questions was higher, although not statistically significant (69.23 to 77.28, ns). Discussion: The implementation of integrated metabolic maps improved student satisfaction of biochemistry/metabolism session materials. Limitations include confounding factors related to student population differences and other simultaneous curriculum changes. Implementing interactive visual aids to integrate metabolism pathways and concepts is applicable to any medical curriculum, and other longitudinal topics may benefit from this type of curricular framework. Supplementary Information: The online version contains supplementary material available at 10.1007/s40670-024-02073-1.

Syntaxin-6 delays prion protein fibril formation and prolongs presence of toxic aggregation intermediates.

Prions replicate via the autocatalytic conversion of cellular prion protein (PrPC) into fibrillar assemblies of misfolded PrP. While this process has been extensively studied in vivo and in vitro, non-physiological reaction conditions of fibril formation in vitro have precluded the identification and mechanistic analysis of cellular proteins, which may alter PrP self-assembly and prion replication. Here, we have developed a fibril formation assay for recombinant murine and human PrP (23-231) under near-native conditions (NAA) to study the effect of cellular proteins, which may be risk factors or potential therapeutic targets in prion disease. Genetic screening suggests that variants that increase syntaxin-6 expression in the brain (gene: STX6) are risk factors for sporadic Creutzfeldt-Jakob disease (CJD). Analysis of the protein in NAA revealed, counterintuitively, that syntaxin-6 is a potent inhibitor of PrP fibril formation. It significantly delayed the lag phase of fibril formation at highly sub-stoichiometric molar ratios. However, when assessing toxicity of different aggregation time points to primary neurons, syntaxin-6 prolonged the presence of neurotoxic PrP species. Electron microscopy and super-resolution fluorescence microscopy revealed that, instead of highly ordered fibrils, in the presence of syntaxin-6 PrP formed less-ordered aggregates containing syntaxin-6. These data strongly suggest that the protein can directly alter the initial phase of PrP self-assembly and, uniquely, can act as an 'anti-chaperone', which promotes toxic aggregation intermediates by inhibiting fibril formation.

Protocol for analyzing TRAIL- and Fas-induced signaling complexes by immunoprecipitation from human cells.

Engagement of TRAIL or Fas death receptors can trigger the assembly of cytoplasmic caspase-8/FADD/RIPK1 (FADDosome) signaling complexes that promote nuclear factor κB (NF-κB) activation. Here, we present a protocol for immunoprecipitation of TRAIL- or Fas-induced FADDosomes from human cell lines. We describe steps for stimulating human cells with TRAIL or Fas ligand, followed by preparation of membrane death receptor-associated, as well as cytoplasmic FADDosome, signaling complexes. This protocol has application in the analysis of death receptor-induced signaling complex formation. For complete details on the use and execution of this protocol, please refer to Davidovich et al.1.

Impact of protein and small molecule interactions on kinase conformations.

Protein kinases act as central molecular switches in the control of cellular functions. Alterations in the regulation and function of protein kinases may provoke diseases including cancer. In this study we investigate the conformational states of such disease-associated kinases using the high sensitivity of the kinase conformation (KinCon) reporter system. We first track BRAF kinase activity conformational changes upon melanoma drug binding. Second, we also use the KinCon reporter technology to examine the impact of regulatory protein interactions on LKB1 kinase tumor suppressor functions. Third, we explore the conformational dynamics of RIP kinases in response to TNF pathway activation and small molecule interactions. Finally, we show that CDK4/6 interactions with regulatory proteins alter conformations which remain unaffected in the presence of clinically applied inhibitors. Apart from its predictive value, the KinCon technology helps to identify cellular factors that impact drug efficacies. The understanding of the structural dynamics of full-length protein kinases when interacting with small molecule inhibitors or regulatory proteins is crucial for designing more effective therapeutic strategies.

Corosolic acid attenuates cardiac ischemia/reperfusion injury through the PHB2/PINK1/parkin/mitophagy pathway.

Despite advances in treatment, myocardial infarction remains the leading cause of heart failure and death worldwide, and the restoration of coronary blood flow can also cause heart damage. In this study, we found that corosolic acid (CA), also known as plant insulin, significantly protects the heart from ischemia-reperfusion (I/R) injury. In addition, CA can inhibit oxidative stress and improve mitochondrial structure and function in cardiomyocytes. Subsequently, our study demonstrated that CA improved the expression of the mitophagy-related proteins Prohibitin 2 (PHB2), PTEN-induced putative kinase protein-1 (PINK1), and Parkin. Meanwhile, through molecular docking, we found an excellent binding between CA and PHB2 protein. Finally, the knockdown of PHB2 eliminated the protective effect of CA on hypoxia-reoxygenation in cardiomyocytes. Taken together, our study reveals that CA increases mitophagy in cardiomyocytes via the PHB2/PINK1/Parkin signaling pathway, inhibits oxidative stress response, and maintains mitochondrial function, thereby improving cardiac function after I/R.

Review of AlphaFold 3: Transformative Advances in Drug Design and Therapeutics.

Google DeepMind Technologies Limited (London, United Kingdom) recently released its new version of the biomolecular structure predictor artificial intelligence (AI) model named AlphaFold 3. Superior in accuracy and more powerful than its predecessor AlphaFold 2, this innovation has astonished the world with its capacity and speed. It takes humans years to determine the structure of various proteins and how the shape works with the receptors but AlphaFold 3 predicts the same structure in seconds. The version's utility is unimaginable in the field of drug discoveries, vaccines, enzymatic processes, and determining the rate and effect of different biological processes. AlphaFold 3 uses similar machine learning and deep learning models such as Gemini (Google DeepMind Technologies Limited). AlphaFold 3 has already established itself as a turning point in the field of computational biochemistry and drug development along with receptor modulation and biomolecular development. With the help of AlphaFold 3 and models similar to this, researchers will gain unparalleled insights into the structural dynamics of proteins and their interactions, opening up new avenues for scientists and doctors to exploit for the benefit of the patient. The integration of AI models like AlphaFold 3, bolstered by rigorous validation against high-standard research publications, is set to catalyze further innovations and offer a glimpse into the future of biomedicine.

Deciphering the actin structure-dependent preferential cooperative binding of cofilin.

The mechanism underlying the preferential and cooperative binding of cofilin and the expansion of clusters toward the pointed-end side of actin filaments remains poorly understood. To address this, we conducted a principal component analysis based on available filamentous actin (F-actin) and C-actin (cofilins were excluded from cofilactin) structures and compared to monomeric G-actin. The results strongly suggest that C-actin, rather than F-ADP-actin, represented the favourable structure for binding preference of cofilin. High-speed atomic force microscopy explored that the shortened bare half helix adjacent to the cofilin clusters on the pointed end side included fewer actin protomers than normal helices. The mean axial distance (MAD) between two adjacent actin protomers along the same long-pitch strand within shortened bare half helices was longer (5.0-6.3 nm) than the MAD within typical helices (4.3-5.6 nm). The inhibition of torsional motion during helical twisting, achieved through stronger attachment to the lipid membrane, led to more pronounced inhibition of cofilin binding and cluster formation than the presence of inorganic phosphate (Pi) in solution. F-ADP-actin exhibited more naturally supertwisted half helices than F-ADP.Pi-actin, explaining how Pi inhibits cofilin binding to F-actin with variable helical twists. We propose that protomers within the shorter bare helical twists, either influenced by thermal fluctuation or induced allosterically by cofilin clusters, exhibit characteristics of C-actin-like structures with an elongated MAD, leading to preferential and cooperative binding of cofilin.

Nanodisc assembly from bacterial total lipid extracts.

Nanodiscs are discoidal lipoproteins that have often been used as vehicles to study membrane proteins in their native configuration. Nanodiscs have been primarily made from synthetic lipids. However, nanodiscs also offer a format by which native lipids can be studied in their natural configuration. Here, we present a method to synthesize nanodiscs from bacterial total lipid extracts using the biothreat agent, Yersinia pestis, as a proof-of-concept. The creation of nanoparticles entirely composed of bacterial lipids supports membrane characterization and vaccine antigen discovery without the inherent safety concerns associated with live bacterial cells of this Tier 1 select agent pathogen.

Protocol for the production and reconstitution of VDAC1 for functional assays.

The voltage-dependent anion channel (VDAC) is an abundant and multifunctional outer mitochondrial membrane protein, playing key roles in neurodegeneration, apoptosis, and mitochondrial membrane biogenesis. Here, we present a protocol to produce and reconstitute high yields of detergent-solubilized VDAC, expressed as inclusion bodies in E. coli. We describe steps for purification by affinity chromatography and refolding in lauryldimethylamine-N-oxide (LDAO). We then detail procedures for reconstituting VDAC into membrane vesicles to assay its channel and phospholipid scramblase activity via fluorescence-based assays. For complete details on the use and execution of this protocol, please refer to Bergdoll et al.,1 Queralt-Martín et al., 2 and Jahn et al.3.

Protocol for assaying irreversible inhibitors of thioredoxin reductase 1.

Selenoprotein thioredoxin reductase 1 (TXNRD1) is a promising therapeutic target, with several inhibitors reported to inhibit TXNRD1 activity. These inhibitors have the potential for applications such as anti-tumor medications. Here, we present a protocol for assessing irreversible inhibitors of TXNRD1. We describe four assays covering cellular TXNRD activity measurement, recombinant enzyme-based activity determination, differential scanning fluorimetry (DSF), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. This protocol will facilitate the screening and development of potential small-molecule inhibitors of TXNRD1.

Lipidome and metabolome profiling of longissimus lumborum beef with different ultimate pH and postmortem aging.

The objective of this exploratory study was to assess the changes on lipidome and metabolome profiling of Longissimus lumborum bull muscle with different ultimate pH (pHu) and aging periods. The bull muscles classified as normal, intermediate, or high pHu were collected from a Brazilian commercial slaughterhouse, cut into steaks, individually vacuum-packaged, and aged for 3 days (3-d) or 21 days (21-d) at 2 °C. Muscle extracts were analyzed for the profiles of both lipids, by mass spectrometry (via direct flow-injection), and metabolites, by nuclear magnetic resonance, with downstream multivariate data analysis. As major results, pairwise comparisons identified C12:0 and C14:0 acylcarnitines as potential biomarkers of the intermediate pHu-muscle, which are related to lipid catabolism for alternative energy metabolism and indicate less protein breakage postmortem. Interestingly, the concentration of arginine at early postmortem aging (3-d) may influence the previously reported improved tenderness in normal and high pHu-muscles. Moreover, upregulation of fumarate, formate, and acetate with increased pHu muscle at 21-d aging indicate more intense tricarboxylic acid cycle, amino acid degradation, and pyruvate oxidation by reactive oxygen species, respectively. These three compounds (fumarate, formate, and acetate) discriminated statistically the muscle with high pHu at 21-d aging. The normal pHu-muscle showed higher concentrations of glycogenolysis and glycolysis metabolites, including glucose, mannose, and pyruvate. Hence, our results enhance knowledge of postmortem biochemical changes of beef within different pHu groups aged up to 21 days, which is essential to understand the mechanisms underpinning bull meat quality changes.

Insights into the physiology, biochemistry and ecological significance of the red seaweed Tricleocarpa fragilis in the Andaman Sea.

The present study focused on the physiological and biochemical aspects of Tricleocarpa fragilis, red seaweed belonging to the phylum Rhodophyta, along the South Andaman coast, with particular attention given to its symbiotic relationships with associated flora and fauna. The physicochemical parameters of the seawater at the sampling station, such as its temperature, pH, and salinity, were meticulously analyzed to determine the optimal harvesting period for T. fragilis. Seaweeds attach to rocks, dead corals, and shells in shallow areas exposed to moderate wave action because of its habitat preferences. Temporal variations in biomass production were estimated, revealing the highest peak in March, which was correlated with optimal seawater conditions, including a temperature of 34 ± 1.1 °C, a pH of 8 ± 0.1, and a salinity of 32 ± 0.8 psu. GC‒MS analysis revealed n-hexadecanoic acid as the dominant compound among the 36 peaks, with major bioactive compounds identified as fatty acids, diterpenes, phenolic compounds, and hydrocarbons. This research not only enhances our understanding of ecological dynamics but also provides valuable insights into the intricate biochemical processes of T. fragilis. The established antimicrobial potential and characterization of bioactive compounds from T. fragilis lay a foundation for possible applications in the pharmaceutical industry and other industries.

Morphological, Physiological, and Photosynthetic Differences of Tartary Buckwheat Induced by Post-Anthesis Drought.

Tartary buckwheat (Fagopyrum tataricum (L.) Gaertn) is a crop of significant interest due to its nutritional value and resilience to drought conditions. However, drought, particularly following flowering, is a major factor contributing to yield reduction. This research employed two distinct Tartary buckwheat genotypes to investigate the effects of post-anthesis drought on growth and physicochemical characteristics. The study aimed to elucidate the response of Tartary buckwheat to drought stress. The findings indicated that post-anthesis drought adversely impacted the growth, morphology, and biomass accumulation of Tartary buckwheat. Drought stress enhanced the maximum photosynthetic capacity (Fv/Fm) and light protection ability (NPQ) of the 'Xiqiao-2' genotype. In response to drought stress, 'Dingku-1' and 'Xiqiao-2' maintained osmotic balance by accumulating soluble sugars and proline, respectively. Notably, 'Xiqiao-2' exhibited elevated levels of flavonoids and polyphenols in its leaves, which helped mitigate oxidative damage caused by drought. Furthermore, rewatering after a brief drought period significantly improved plant height, stem diameter, and biomass accumulation in 'Dingku-1'. Overall, 'Xiqiao-2' demonstrated greater long-term tolerance to post-anthesis drought, while 'Dingku-1' was less adversely affected by short-term post-anthesis drought.

"CARBGAME" (CARd & Board GAmes in Medical Education) as an innovative gamification tool for learning clinical enzymology in biochemistry for first year medical students.

Gamification is emerging as an active learning innovation in medical education to enhance student engagement and promote life-long learning in a unique and collaborative environment. Clinical enzymology in biochemistry is one of the core topics in the medical curriculum. However, students face challenges in comprehension and retention of information. Hence, CARd & Board GAmes in Medical Education (CARBGAME) was introduced and evaluated for its effectiveness in enhancing learning, application, and retention of knowledge in clinical enzymology via gamification context. This mixed-method study involved 150 first-year undergraduate medical students. Before the game, students completed a pre-test in clinical enzymology. Later they were divided into 25 small groups to compete in the board game designed for enzymology in biochemistry. The students took turns throwing the dice and answering the questions on the game board to continue moving forward. The first team to reach 100 and solve the case-based question was deemed the winner. Following the board game, the students took up the post-test to compare the educational impact of the innovation. Also, the subsequent internal assessment scores were compared with previous batch who were not implemented with this intervention. Then students evaluated the effectiveness of CARBGAME-Clinical Enzymology using a 32-item questionnaire on 5-point Likert scale. The feedback obtained on a 10-point rating scale and for qualitative analysis, students' and faculty perceptions were recorded in small groups. CARBGAME received overwhelmingly positive feedback from both students and faculty. It was perceived well by students for being fun, relevant, consistent, motivating, collaborative, and promoting experiential learning. The game's low-stakes approach, effective feedback, and sense of accomplishment were highly appreciated, making it a valuable tool for education. A significant improvement in knowledge was recorded, from a mean score of 8.37 ± 1.126 on a 20-point scoring scale before the game to 16.53 ± 1.219 after with a p-value of 0.0001. The comparison of the internal assessment scores between the intervention and non-intervention group of students also showed a significant improvement among those implemented with CARBGAME (p < 0.0001). The CARBGAME innovation has achieved the intended outcome of promoting active learning and enhanced performance in clinical enzymology. Highly positive responses from faculty and students also indicate the exigent need to introduce innovative components like games into curricula to achieve student engagement and promote a meaningful learning experience.

Prominin 1 and Tweety Homology 1 both induce extracellular vesicle formation.

Prominin-1 (Prom1) is a five-transmembrane-pass integral membrane protein that associates with curved regions of the plasma membrane. Prom1 interacts with membrane cholesterol and actively remodels the plasma membrane. Membrane bending activity is particularly evident in photoreceptors, where Prom1 loss-of-function mutations cause failure of outer segment homeostasis, leading to cone-rod retinal dystrophy (CRRD). The Tweety Homology (Ttyh) protein family has been proposed to be homologous to Prominin, but it is not known whether Ttyh proteins have an analogous membrane-bending function. Here, we characterize the membrane-bending activity of human Prom1 and Ttyh1 in native bilayer membranes. We find that Prom1 and Ttyh1 both induce formation of extracellular vesicles (EVs) in cultured mammalian cells and that the EVs produced are physically similar. Ttyh1 is more abundant in EV membranes than Prom1 and produces EVs with membranes that are more tubulated than Prom1 EVs. We further show that Prom1 interacts more stably with membrane cholesterol than Ttyh1 and that this may contribute to membrane bending inhibition in Prom1 EVs. Intriguingly, a loss-of-function mutation in Prom1 associated with CRRD induces particularly stable cholesterol binding. These experiments provide mechanistic insight into Prominin function in CRRD and suggest that Prom and Ttyh belong to a single family of functionally related membrane-bending, EV-generating proteins.

Safety evaluation of Indocalamus Iatifolius McClure leaves based on acute toxicity, harmful metals, and 9 organochlorine residue determination.

To investigate the safety of Indocalamu Iatifolius McClur leaves sold in the market, a study was conducted using Indocalamu Iatifolius McClur leaves randomly collected from an online store and a large supermarket. Acute toxicity experiments were performed on mice, and their body weight was monitored for 14 days after administration. After the observation period, blood samples were collected for biochemical analysis, and organ pathology was examined. Then, the content of copper (Cu), lead (Pb), cadmium (Cd), mercury (Hg), arsenic (As), and the residues of nine organochlorine pesticides in Indocalamu Iatifolius McClur leaves were measured according to the National Food Safety Standard (GB/T5009-2003) and the pesticide residue determination methods in the 2020 edition of the Chinese Pharmacopoeia. The results showed that the mice in the Indocalamu Iatifolius McClur leaves (online store) group experienced mortality and severe liver and lung damage. The levels of lead, cadmium, mercury, arsenic, and the nine organochlorine pesticides met the relevant standards and regulations. However, the copper content in the Indocalamu Iatifolius McClur leaves (online store) group was nearly 80 times higher than that in the supermarket group. Mice in the Indocalamu Iatifolius McClur leaves (supermarket) group remained healthy without any abnormalities, and the levels of harmful metals and organochlorine pesticides complied with the standards and regulations. The study suggests the need for regulatory policies and safety standards for the sale of Indocalamu Iatifolius McClur leaves.

Selenium nanoparticle inclusion in broiler diets for enhancing sustainable production and health.

This study aimed to evaluate the effects of dietary supplementation of nanoparticles of Selenium (Nano-Se) on productive performance, nutrient digestibility, carcass criteria, selenium retention, blood biochemistry, and histopathological examination of broiler chicken. A total of 192 1-day-old male broiler chickens (Cobb 500) were randomly assigned to one of four treatment diets, with each diet given to six replicates of eight chicks. The birds were randomly assigned to one of four treatment groups, each of which included Nano-Se at levels of 0, 0.2, 0.3, or 0.4 mg/kg. The feeding experiment lasted 35 days. Nano-Se addition to broiler diets at 0.2 and 0.3 mg/kg enhanced body weight and body weight gain linearly compared to the control diet and 0.4 mg/kg. The apparent digestibility coefficient of ether extracts linearly increased with increasing Nano-Se levels up to 0.4 mg/kg. Increasing Nano-Se decreased serum cholesterol, triglycerides, alanine aminotransaminase, aspartate aminotransaminase, and creatinine in broiler chickens. Also, serum antioxidants showed a significant increase with increasing Nano-Se levels. As Nano-Se levels were supplemented, improvements in cooking loss, water-holding capacity, and antioxidants were observed as compared to the control. Additionally, a noticeable improvement in meat quality was observed regarding the obtained meat characters. It was preferred to use low doses of Nano-Se (0.3 mg/kg), as tissue retention of Se for both meat and liver was more comparable to the control. In conclusion, nutritional supplementation with Nano-Se increased growth performance, nutrient digestibility, selenium retention, meat quality, blood biochemistry, histological indices, and antioxidant activity of broiler chickens. Overall, the best performance of broilers was observed with Nano-Se supplementation at 0.3 mg/kg, highlighting its potential as a novel supplement for broiler diets.

Physcomitrium patens flavodiiron proteins form heterotetrametric complexes.

Flavodiiron proteins (FLVs) catalyze the reduction of oxygen to water by using electrons from Photosystem I (PSI). In several photosynthetic organisms such as cyanobacteria, green algae, mosses and gymnosperms, FLV-dependent electron flow protects PSI from over-reduction and consequent damage especially under fluctuating light conditions. In this work we investigated biochemical and structural properties of FLVA and FLVB from the model moss Physcomitrium patens. The two proteins, expressed and purified from Escherichia coli, bind both iron and flavin cofactors and show NAD(P)H oxidase activity as well as oxygen reductase capacities. Moreover, the co-expression of both FLVA and FLVB, coupled to a tandem affinity purification procedure with two different affinity tags, enabled the isolation of the stable and catalytically active FLVA/B hetero tetrameric protein complex with cooperative nature. The multimeric organization was shown to be stabilized by inter-subunit disulfide bonds. This investigation provides valuable new information on the biochemical properties of FLVs, with new insights into their in vivo activity.

Early hypophosphataemia and refeeding syndrome in extremely low birthweight babies and outcomes to 2 years of age: secondary cohort analysis from the ProVIDe trial.

OBJECTIVE: To investigate in extremely low birthweight (ELBW; <1000 g) babies the associations between refeeding syndrome (serum phosphate <1.4 mmol·L-1 and serum total calcium>2.8 mmol·L-1) and hypophosphataemia in the first week and death or neurodisability at 2 years' corrected age (CA). DESIGN: Secondary cohort analysis of the ProVIDe trial participants with serum biochemistry within 7 days of birth. At 2 years' CA, neurodisability was assessed by Bayley Scales of Infant Development Edition III and neurological examination. Associations between neurodisability and other variables were analysed using t-tests and logistic regression adjusted for sex and smallness-for-gestational age. SETTING: Six tertiary neonatal intensive care units (NICUs) in New Zealand. PARTICIPANTS: 352 ELBW babies born between 29 April 2014 and 30 October 2018. MAIN OUTCOME MEASURE: Death or neurodisability at 2 years' CA. RESULTS: Fifty-nine babies died, two after discharge from the NICU. Of the 336 babies who survived to 2 years' CA, 277 had neurodevelopmental assessment and 107 (39%) had a neurodisability. Death or neurodisability was more likely in babies who had refeeding syndrome (aOR 1.96 (95% CI 1.09 to 3.53), p=0.02) and in babies who had hypophosphataemia (aOR 1.74 (95% CI 1.09 to 2.79), p=0.02). Hypophosphataemia was associated with increased risk of death (aOR 2.07 (95% CI 1.09 to 3.95), p=0.03)) and severe hypophosphataemia (<0.9 mmol·L-1) with increased risk of death (aOR 2.67 (95% CI 1.41 to 5.00), p=0.002) and neurodisability (aOR 2.31 (95% CI 1.22 to 4.35), p=0.01). CONCLUSIONS: In ELBW babies, refeeding syndrome and hypophosphataemia in the first week are associated with death or neurodisability. Until optimal phosphate requirements are determined through further research, monitoring for hypophosphataemia and mitigation strategies are indicated. TRIAL REGISTRATION NUMBER: ACTRN12612001084875.

A high-resolution 13C NMR approach for profiling fatty acid unsaturation in lipid extracts and in live C. elegans.

Unsaturated fatty acids (UFA) play a crucial role in central cellular processes in animals, including membrane function, development, and disease. Disruptions in UFA homeostasis can contribute to the onset of metabolic, cardiovascular, and neurodegenerative disorders. Consequently, there is a high demand for analytical techniques to study lipid compositions in live cells and multicellular organisms. Conventional analysis of UFA compositions in cells, tissues and organisms involves solvent extraction procedures coupled with analytical techniques such as gas chromatography,mass spectrometry (MS) and/or nuclear magnetic resonance (NMR) spectroscopy. As a non-destructive and non-targeted technique, NMR spectroscopy is uniquely capable of characterizing the chemical profiling of living cells and multicellular organisms. Here we use NMR spectroscopy to analyze C. elegans, enabling the determination of their lipid compositions and fatty acid unsaturation levels both in cell-free lipid extracts and in vivo. The NMR spectra of lipid extracts from wild-type and fat-3 mutant C. elegans strains revealed notable differences due to the absence of Δ-6 fatty acid desaturase activity, including the lack of arachidonic and eicosapentaenoic acyl chains. Uniform 13C-isotope labeling and high-resolution 2D solution-state NMR of live worms confirmed these findings, indicating that the signals originated from fast-tumbling lipid molecules within lipid droplets. Overall, this strategy permits the analysis of lipid storage in intact worms and has enough resolution and sensitivity to identify differences between wild type and mutant animals with impaired fatty acid desaturation. Our results establish methodological benchmarks for future investigations of fatty acid regulation in live C. elegans using NMR.