Optimization of skin sampling based on tape stripping for lipidome analysis by nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry.

In this study, skin sampling by tape stripping for lipid analysis was optimized by examining the lipid profiles of the stratum corneum (SC), focusing on the composition and levels of ceramides (Cer), diacylglycerols (DG), and triacylglycerols (TG), using nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry. Significant variations in the number and composition of the identified lipids, particularly Cer and neutral lipid species, were observed across different skin locations, including the forearm, forehead, cheek, and neck. Analysis of the layer-to-layer lipid profiles of the seven consecutive layers revealed a gradual decrease in DG and TG levels from the outermost to the innermost layers, with certain Cer subclasses showing increases in the second to fourth layers and subsequent decreases. Comparative analysis of lipid profiles from adjacent spots demonstrated statistical consistency and persistent differences between spots. Pooling layers were evaluated as an alternative method for representing SC layers, and their efficiencies were assessed by varying the number of pooled layers. We found that pooling five consecutive layers was effective in terms of the number and levels of identified lipids. Additionally, investigations into the matrix effect and extraction efficiency upon pooling layers indicated that pooling up to five layers did not significantly affect ionization suppression or reduce extraction recovery.

Toxoplasma acyl-CoA synthetase TgACS3 is crucial to channel host fatty acids in lipid droplets and for parasite propagation.

Apicomplexa comprise important pathogenic parasitic protists that heavily depend on lipid acquisition to survive within their human host cells. Lipid synthesis relies on the incorporation of an essential combination of fatty acids (FAs) either generated by a metabolically adaptable de novo synthesis in the parasite or by scavenging from the host cell. The incorporation of FAs into membrane lipids depends on their obligate metabolic activation by specific enzyme groups, acyl-CoA synthetases (ACSs). Each ACS has its own specificity, so they can fulfill specific metabolic functions. Whilst such functionalities have been well studied in other eukaryotic models, their roles and importance in Apicomplexa is currently very limited, especially for Toxoplasma gondii. Here, we report the identification of 7 putative ACSs encoded by the genome of T. gondii (TgACS), which localize to different sub-cellular compartments of the parasite, suggesting exclusive functions. We show that the perinuclear/cytoplasmic TgACS3 regulates replication and growth of Toxoplasma tachyzoites. Conditional disruption of TgACS3 shows that the enzyme is required for parasite propagation and survival, especially under high host nutrient content. Lipidomic analysis of parasites lacking TgACS3 reveals its role in the activation of host-derived FAs that are used for i) parasite membrane phospholipid and ii) storage triacylglycerol (TAG) syntheses, allowing proper membrane biogenesis of parasite progenies. Altogether, our results reveal the role of TgACS3 as the bulk FA activator for membrane biogenesis allowing intracellular division and survival in T. gondii tachyzoites, further pointing at the importance of ACS and FA metabolism for the parasite.

Hepatocyte-Specific Yap1 Knockout Maintained the Liver Homeostasis of Lipid Metabolism in Mice.

Introduction: Yes-associated protein 1 (YAP1) is a crucial molecule in the Hippo pathway. The impact of hepatocyte-specific Yap1 knockout (Yap1 LKO) on hepatic lipid droplets (LD) and pePLIN2 in metabolic fatty liver has not been reported. This study aims to explore whether Yap1 LKO could offer a protective effect in a liver injury model. Methods: Three-week-old Yap1 LKO and Yap1 Flox mice were given aristolochic acid I (AAI) combined carbon tetrachloride (CCL4) establish liver injury model. Eight-week-old Yap1 LKO and Yap1 Flox mice were fed with a high-fat diet for 18 weeks to establish obesity-related liver injury model. Further biochemical, histomorphological, immunohistochemical, and lipidomic analyses were performed on serum and liver tissues of these mice to elucidate the effects of hepatocyte-specific Yap1 knockout on hepatic lipid metabolism. Results: Yap1 LKO reduced triglyceride (TG) content and PLIN2 expression level in the liver during the intervention of AAI combined CCl4. Moreover, Yap1 LKO improved lipid metabolism homeostasis in the liver by increasing the beneficial lipid molecules and reducing the harmful lipid molecules through lipidomics. Finally, Yap1 LKO reduced TG content in the serum and liver, hepatic vacuolar degeneration, and hepatic PLIN2 expression level in mice fed with a high-fat diet (HFD). Conclusion: Yap1 LKO is protective in regulating liver and blood TG when induced with toxic substances AAI combined CCl4 and a high-fat diet.

The hepatokine FGF21 stopped lipogenesis and reduced testosterone production in mLTC-1 Leydig Cell Line.

Beyond their link to metabolic issues like type 2 diabetes, factors like lifestyle, environment, and excess weight may also influence fertility. Fibroblast growth factor 21 (FGF21), a liver-derived hormone linked to energy balance, has recently emerged as a potential player in female mammalian reproduction. In male, only two studies have described potential effects of FGF21 on fertility. A recent study has described a negative correlation observed in obese patients presenting a low testosterone level associated with elevated FGF21 plasma levels. To investigate the role of FGF21 in steroidogenesis, we have studied the involvement of FGF21 in lipid and steroid activity by Leydig cells. Leydig cell model expressed all FGF21 receptors and ß-Klotho cofactor as determined by RT-qPCR and by western-blot. Cultured mLTC-1 Leydig cell line exposed to increasing FGF21 concentration induced phosphorylation (Ser 473) of Akt and modified the CREB factor activity, suggesting the functionality of the FGF21 pathway. FGF21 consequences on mLTC-1 Leydig cells are inhibition of the lipid synthesis, leading to a reduction in the content of lipid droplets. The drop in lipid synthesis is associated with a reduction in the amount of lipids (mainly PUFA, cholesterol esterified, and triglycerides) as measured by lipidomic approach. The main consequence is to reduce the quantity of cholesterol, the steroid precursor, in mLTC-1 Leydig cells and is associated with a low production in testosterone. The decrease in androgens was also associated with a reduction in the steroid enzyme genes expression, which are under the control of CREB activity, and present a lower activity due to low cAMP intracellular levels. In vivo, steroid production was lowering after FGF21 administration in adult male mice associated to a decrease in progressive motility and velocity of sperm. In addition, these experimental data are reinforced by a data mining analysis focused on "gonad" terms in 1,319,905 article references showing the link already described between FGF21 with the fatty acids pathways, cholesterol storage, and steroid production. In conclusion, we demonstrated that Leydig cells in the testes present a functional FGF21 pathway, which regulates lipid metabolism and steroid function. In mLTC-1 Leydig cells, FGF21 reduced cholesterol, PUFA content, and testosterone production. Finally, this work highlighted that the hepatokine FGF21 could have a negative impact on androgen synthesis and testicular activity.

Spatially lipidomic characterization of patient-derived organoids by whole-mount autofocusing SMALDI mass spectrometry imaging.

BACKGROUND: Patient-derived organoids (PDOs) are multi-cellular cultures with specific three-dimensional (3D) structures. Tumor organoids (TOs) offer a personalized perspective for assessing treatment response. However, the presence of normal organoid (NO) residuals poses a potential threat to their utility for personalized medicine. There is a crucial need for an effective platform capable of distinguishing between TO and NO in cancer organoid cultures. RESULTS: We introduced a whole-mount (WM) preparation protocol for in-situ visualization of the lipidomic distribution of organoids. To assess the efficacy of this method, nine breast cancer organoids (BCOs) and six normal breast organoids (NBOs) were analyzed. Poly-l-lysine (PLL) coated slides, equipped with 12 well chambers, were utilized as a carrier for the high-throughput analysis of PDOs. Optimizing the fixation time to 30 min, preserved the integrity of organoids and the fidelity of lipid compounds. The PDOs derived from the same organoid lines exhibited similar lipidomic profiles. BCOs and NBOs were obviously distinguished based on their lipidomic signatures detected by WM autofocusing (AF) scanning microprobe matrix-assisted laser desorption/ionization (SMALDI) mass spectrometry imaging (MSI). SIGNIFICANCE: A whole-mount (WM) preparation protocol was developed to visualize lipidomic distributions of the organoids' surface. Using poly-l-lysine coated slides for high-throughput analysis, the method preserved organoid integrity and distinguished breast cancer organoids (BCOs) from normal breast organoids (NBOs) based on their unique lipidomic profiles using autofocusing scanning microprobe matrix-assisted laser desorption/ionization (SMALDI) mass spectrometry imaging.

The role of ATP citrate lyase in myelin formation and maintenance.

Formation of myelin by Schwann cells is tightly coupled to peripheral nervous system development and is important for neuronal function and long-term maintenance. Perturbation of myelin causes a number of specific disorders that are among the most prevalent diseases affecting the nervous system. Schwann cells synthesize myelin lipids de novo rather than relying on uptake of circulating lipids, yet one unresolved matter is how acetyl CoA, a central metabolite in lipid formation is generated during myelin formation and maintenance. Recent studies have shown that glucose-derived acetyl CoA itself is not required for myelination. However, the importance of mitochondrially-derived acetyl CoA has never been tested for myelination in vivo. Therefore, we have developed a Schwann cell-specific knockout of the ATP citrate lyase (Acly) gene to determine the importance of mitochondrial metabolism to supply acetyl CoA in nerve development. Intriguingly, the ACLY pathway is important for myelin maintenance rather than myelin formation. In addition, ACLY is required to maintain expression of a myelin-associated gene program and to inhibit activation of the latent Schwann cell injury program.

Rapid Characterization of Phospholipids from Biological Matrix Enabled by Indium Tin Oxide (ITO) Coated Slide Assisted Enrichment MALDI Mass Spectrometry.

Lipidomic analysis of human serum is essential to monitor the individual's health status. Herein, we develop a facile strategy for rapid characterization of phospholipids in human serum via indium tin oxide (ITO) coated glass slide solid phase extraction MALDI mass spectrometry (ITO-SPE-MALDI-MS). Phospholipid species are retained on ITO slide via solid phase extraction owing to the unique property of the ITO material; the measurement of phospholipid species from 1 µl human serum within 2 min is achievable. A comparison of ITO-SPE strategy with conventional extraction methods was further carried out using liquid chromatography-mass spectrometry (LC-MS) and ion-mobility mass spectrometry (IM-MS), resulting in a comparable enrichment performance for the phospholipid analysis. Furthermore, rapid lipidomic profiling of serum samples from human colorectal cancer patients and cell lines was demonstrated. Our results indicate that ITO-SPE-MALDI-MS provides a higher throughput strategy for the analysis of phospholipid species in complex biological mixtures, showcasing its potential for applications in the analysis of clinical biofluids.

Integrative metagenomic and lipidomic analyses reveal alterations in children with obesity and after lifestyle intervention.

Background: Despite emerging evidence linking alterations in gut microbiota to childhood obesity, the metabolic mechanisms linking gut microbiota to the lipid profile during childhood obesity and weight loss remain poorly understood. Methodology: In this study, children with obesity were treated with lifestyle weight loss therapy. Metagenomics association studies and serum untargeted lipidomics analyses were performed in children with obesity and healthy controls before and after weight loss. Main findings: We identified alterations in gut microbiota associated with childhood obesity, as well as variations in circulating metabolite concentrations. Children with obesity showed significant decreases in the levels of s-Rothia_kristinae and s-Enterobacter_roggenkampii, alongsige elevated levels of s-Clostridiales_bacterium_Marseille-P5551. Following weight loss, the levels of s-Streptococcus_infantarius and s-Leuconostoc_citreum increased by factors of 3.354 and 1.505, respectively, in comparison to their pre-weight loss levels. Correlation analyses indicated a significant positive relationship between ChE(2:0) levels and both with s-Lachnospiraceae_bacterium_TF09-5 and fasting glucose levels. CoQ8 levels were significantly negatively correlated with s-Rothia_kristinae and HOMA-IR. Conclusion: We linked altered gut microbiota and serum lipid levels in children with obesity to clinical indicators, indicating a potential impact on glucose metabolism via lipids. This study contributes to understanding the mechanistic relationship between altered gut microbiota and childhood obesity and weight loss, suggesting gut microbiome as a promising target for intervention. Clinical trial registration: https://www.chictr.org.cn/showproj.html?proj=178971, ChiCTR2300072179.

Investigation of Human Milk as a Biological System in a Multicenter Mother-Infant Cohort: Protocol Design and Cohort Profile of the Phoenix Study.

Breastfeeding and human milk are the gold standard for infant feeding. Studying human milk with a systems biology approach in a large longitudinal cohort is needed to understand its complexity and health implications. The Phoenix study is a multicenter cohort study focusing on the interactions of maternal characteristics, human milk composition, infant feeding practices, and health outcomes of Chinese mothers and infants. A total of 779 mother-infant dyads were recruited from November 2021 to September 2022, and 769 mother-infant dyads were enrolled in the study. Scheduled home visits took place at 1, 4, 6, and 12 months postpartum, and 696 dyads (90.5% participants) completed the 12-month visit. At each visit, maternal and infant anthropometry was assessed. Questionnaires were administered to collect longitudinal information on maternal characteristics and lifestyle, infant feeding, and health. Digital diaries were used to record maternal dietary intake, infant feeding, and stool character. Human milk, maternal feces, infant feces, and infant saliva were collected. An external pharmaceutical-level quality assurance approach was implied to ensure the trial quality. Multi-omics techniques (including glycomics, lipidomics, proteomics, and microbiomics) and machine learning algorithms were integrated into the sample and data analysis. The protocol design of the Phoenix study provides a framework for prospective cohort studies of mother-infant dyads and will provide insights into the complex dynamics of human milk and its interplay with maternal and infant health outcomes in the Chinese population.

Lipid biomarkers in colorectal cancer, with particular emphasis on exosomes - current status and future inferences.

INTRODUCTION: Colorectal cancer (CRC) is one of the most deadly cancers on a global scale. Diagnosis of CRC is challenging and it is often detected at a late stage. Identification of relevant biomarkers could lead to the development of effective diagnostic methods for CRC. AREAS COVERED: We reviewed the literature on lipid (including exosomal) biomarkers that have the potential to become common, minimally invasive and effective diagnostic tools for CRC. We showed that differences in lipid levels (single compounds and entire panels) make it possible to classify patients into diseased or healthy groups, determine the stage of CRC, as well as accompanying inflammation and immune reactions associated with tumorigenesis. We also discussed exosomes which are important components of the tumor microenvironment that influence tumor progression and for which only a small number of studies were conducted so far in this area. EXPERT OPINION: A rapid development in the field of lipid-based biomarkers, including exosomal lipid biomarkers, is expected as growing evidence shows their potential application and good accuracy. However, one of the major issues that needs to be addressed within this topic is to translate findings into a noninvasive and versatile diagnostic test robustly validated in clinical conditions.

Lipidomics combined with random forest machine learning algorithms to reveal freshness markers for duck eggs during storage in different rearing systems.

The differences in lipids in duck eggs between the 2 rearing systems during storage have not been fully studied. Herein, we propose untargeted lipidomics combined with a random forest (RF) algorithm to identify potential marker lipids based on ultra-performance liquid chromatography‒mass spectrometry (UPLPC-MS/MS). A total of 106 and 16 differential lipids (DL) were screened in egg yolk and white, respectively. In yolk, metabolic pathway analysis of DLs revealed that glycerophospholipid metabolism and sphingolipid metabolism were the key metabolic pathways in the traditional free-range system (TFS) during storage, glycosylphosphatidylinositol-anchored biosynthesis and glyceride metabolism were the key pathways in the floor-rearing system (FRS). In egg white, the key pathway in both systems is the biosynthesis of unsaturated fatty acids. Combined with RF algorithm, 12 marker lipids were screened during storage. Therefore, this study elucidates the changes in lipids in duck eggs during storage in 2 rearing systems and provides new ideas for screening marker lipids during storage. This approach is highly important for evaluating the quality of egg and egg products and provides guidance for duck egg production.

Lipoprotein Lipidomics as a Frontier in Non-Alcoholic Fatty Liver Disease Biomarker Discovery.

Non-alcoholic fatty liver disease (NAFLD) is a progressive liver disease characterized by the build-up of fat in the liver of individuals in the absence of alcohol consumption. This condition has become a burden in modern societies aggravated by the lack of appropriate predictive biomarkers (other than liver biopsy). To better understand this disease and to find appropriate biomarkers, a new technology has emerged in the last two decades with the ability to explore the unmapped role of lipids in this disease: lipidomics. This technology, based on the combination of chromatography and mass spectrometry, has been extensively used to explore the lipid metabolism of NAFLD. In this review, we aim to summarize the knowledge gained through lipidomics assays exploring tissues, plasma, and lipoproteins from individuals with NAFLD. Our goal is to identify common features and active pathways that could facilitate the finding of a reliable biomarker from this field. The most frequent observation was a variable decrease (1-9%) in polyunsaturated fatty acids in phospholipids and non-esterified fatty acids in NAFLD patients, both in plasma and liver. Additionally, a reduction in phosphatidylcholines is a common feature in the liver. Due to the scarcity of studies, further research is needed to properly detect lipoprotein, plasma, and tissue lipid signatures of NAFLD etiologies, and NAFLD subtypes, and to define the relevance of this technology in disease management strategies in the push toward personalized medicine.

Lipid profile alterations and biomarker identification in type 1 diabetes mellitus patients under glycemic control.

BACKGROUND: Type 1 diabetes mellitus (T1DM) is well-known to trigger a disruption of lipid metabolism. This study aimed to compare lipid profile changes in T1DM patients after achieving glucose control and explore the underlying mechanisms. In addition, we seek to identify novel lipid biomarkers associated with T1DM under conditions of glycemic control. METHODS: A total of 27 adults with T1DM (age: 34.3 ± 11.2 yrs) who had maintained glucose control for over a year, and 24 healthy controls (age: 35.1 + 5.56 yrs) were recruited. Clinical characteristics of all participants were analyzed and plasma samples were collected for untargeted lipidomic analysis using mass spectrometry. RESULTS: We identified 594 lipid species from 13 major classes. Differential analysis of plasma lipid profiles revealed a general decline in lipid levels in T1DM patients with controlled glycemic levels, including a notable decrease in triglycerides (TAGs) and diglycerides (DAGs). Moreover, these T1DM patients exhibited lower levels of six phosphatidylcholines (PCs) and three phosphatidylethanolamines (PEs). Random forest analysis determined DAG(14:0/20:0) and PC(18:0/20:3) to be the most prominent plasma markers of T1DM under glycemic control (AUC = 0.966). CONCLUSIONS: The levels of all metabolites from the 13 lipid classes were changed in T1DM patients under glycemic control, with TAGs, DAGs, PCs, PEs, and FFAs demonstrating the most significant decrease. This research identified DAG(14:0/20:0) and PC(18:0/20:3) as effective plasma biomarkers in T1DM patients with controled glycemic levels.

Ethanol Extract of Propolis Attenuates Liver Lipid Metabolism Disorder in High-Fat Diet-Fed SAMP8 Mice.

SCOPE: The prevalence of high-fat diet (HFD) consumption is increasing among middle-aged and older adults, which accelerates the aging process of this population and is more likely to induce lipid metabolism disorders. But the alleviation of ethanolic extract of propolis (EEP) on lipid metabolism disorders during aging remains unclear. METHODS AND RESULTS: This study assesseed the impact of EEP intervention (200 mg kg-1 bw) on aging and lipid metabolism disorders in HFD-fed senescence accelerate mouse prone 8 (SAMP8) mice. Findings indicate that EEP ameliorates hair luster degradation and weight gain, reduces systemic inflammation and metabolism levels, enhances hepatic antioxidant enzyme activities, and improves the hepatic expression of senescence-associated secretory phenotype and aging-related genes in HFD-fed SAMP8 mice. Histological staining demonstrates that EEP improves hepatic lipid deposition and inflammatory cell infiltration. Transcriptomic and lipidomic analysis reveal that EEP promotes fatty acid ß-oxidation by activating PPAR pathway, resulting in reduced hepatic lipid deposition, and attenuates bile acid (BA) accumulation by improving BA metabolism, which were ensured through qPCR validation of key genes and immunoblot validation of key proteins. CONCLUSIONS : EEP can regulate lipid metabolic dysregulation during aging accompanied by an HFD, potentially delaying the onset and progression of age-related diseases. This provides new approach for supporting healthy aging.

Metabolomic and lipidomic profiling of the spinal cord in type 2 diabetes mellitus rats with painful neuropathy.

In this paper we investigated lipid and metabolite changes in diabetic neuropathy, using untargeted lipidomics and metabolomics analyses of the spinal cords from streptozotocin-treated diabetic rats.170 metabolites and 45 lipids were dysregulated in the painful diabetic neuropathy (PDN) phase. Pathway enrichment analysis revealed perturbations in starch and sucrose, tryptophan, pyrimidine, cysteine and methionine, thiamine, tyrosine, and nucleotides. The disturbance of tyrosine, tryptophan, methionine, triacylglycerol, and phosphatidylethanolamine metabolism indicated that pathological mechanisms in the PDN involved energy metabolism, oxidative stress, and neural reparative regeneration. These revelations offered potential biomarkers for PDN and enriched the comprehension of the complex molecular mechanisms characterizing PDN, establishing a solid foundation for subsequent inquiries into neural convalescence and recovery after PDN.

Oxidative stress and plasma ceramides in broiler chickens.

The selection for rapid growth in chickens has rendered meat-type (broiler) chickens susceptible to develop metabolic syndrome and thus inflammation. The sphingolipid ceramide has been linked as a marker of oxidative stress in mammals, however, the relationship between sphingolipid ceramide supply and oxidative stress in broiler chickens has not been investigated. Therefore, we employed a lipidomic approach to investigate the changes in circulating sphingolipid ceramides in context of allopurinol-induced oxidative stress in birds. Day zero hatched chicks (n = 60) were equally divided into six groups; an unsupplemented control, an allopurinol group (25 mg/kg body weight), a conjugated linoleic acid (CLA) group where half of the oil used in the control diet was substituted for a CLA oil mixture, a CLA and an allopurinol group utilizing the same dose of CLA and allopurinol, a berberine (BRB) group consisting of berberine supplementation (200 mg/kg feed), and a BRB and allopurinol group, utilizing the same dose of BRB and allopurinol. Conjugated linoleic acid and berberine were utilized to potentially enhance antioxidant activity and suppress the oxidative stress induced by allopurinol treatment. Body weight, plasma uric acid, nonesterified fatty acids (NEFA) and sphingolipid ceramides were quantified. Allopurinol induced an inflammatory state as measured by a significant reduction in plasma uric acid - an antioxidant in birds as well as a metabolic waste product. Results showed that both total and saturated sphingolipid ceramides declined (p < 0.05) with age in unsupplemented chicks, although plasma ceramides C16:0 and 18:0 increased in concentration over the study period. Simple total and saturated sphingolipid ceremide's were further decreased (p < 0.05) with allopurinol supplementation, however, this may be an indirect consequence of inducing an inflammatory state. Neither CLA or BRB were able to significantly attenuate the decline. The administration of allopurinol specifically targets the liver which in birds, is the primary organ for fatty acids synthesis. For this reason, sphingolipid ceramide production might have been unwittingly affected by the addition of allopurinol.

Detailed Protocol for Solid-Phase Extraction for Lipidomic Analysis.

Developing robust analytical techniques is a vital phase to facilitate understanding the roles and impacts of various omic profilings in cellular functions. The comprehensive analysis of various biological molecules within a biological system requires a precise sample preparation technique. Solid-Phase Extraction (SPE) has proven to be an indispensable method in lipidomic analysis, providing an uncomplicated and user-friendly technique for extraction and purification of lipid components from complex biological matrices. Of all the factors influencing the reliability and success of SPE, column or adsorbent materials, flow rate, and storage conditions are paramount in terms of their significance. In this chapter, we will discuss in detail the SPE steps for lipidomic analysis in biofluid samples (serum and plasma) and muscle tissues.

Lipidomic Risk Score to Enhance Cardiovascular Risk Stratification for Primary Prevention.

BACKGROUND: Accurate risk stratification is vital for primary prevention of cardiovascular disease (CVD). However, traditional tools such as the Framingham Risk Score (FRS) may underperform within the diverse intermediate-risk group, which includes individuals requiring distinct management strategies. OBJECTIVES: This study aimed to develop a lipidomic-enhanced risk score (LRS), specifically targeting risk prediction and reclassification within the intermediate group, benchmarked against the FRS. METHODS: The LRS was developed via a machine learning workflow using ridge regression on the Australian Diabetes, Obesity, and Lifestyle Study (AusDiab; n = 10,339). It was externally validated with the Busselton Health Study (n = 4,492), and its predictive utility for coronary artery calcium scoring (CACS)-based outcomes was independently validated in the BioHEART cohort (n = 994). RESULTS: LRS significantly improved discrimination metrics for the intermediate-risk group in both AusDiab and Busselton Health Study cohorts (all P < 0.001), increasing the area under the curve for CVD events by 0.114 (95% CI: 0.1123-0.1157) and 0.077 (95% CI: 0.0755-0.0785), with a net reclassification improvement of 0.36 (95% CI: 0.21-0.51) and 0.33 (95% CI: 0.15-0.49), respectively. For CACS-based outcomes in BioHEART, LRS achieved a significant area under the curve improvement of 0.02 over the FRS (0.76 vs 0.74; P < 1.0 × 10-5). A simplified, clinically applicable version of LRS was also created that had comparable performance to the original LRS. CONCLUSIONS: LRS, augmenting the FRS, presents potential to improve intermediate-risk stratification and to predict atherosclerotic markers using a simple blood test, suitable for clinical application. This could facilitate the triage of individuals for noninvasive imaging such as CACS, fostering precision medicine in CVD prevention and management.

PhosphoLipidome Alteration Induced by Clostridioides difficile Toxin B in Enteric Glial Cells.

Clostridioides difficile (C. difficile) is responsible for a spectrum of nosocomial/antibiotic-associated gastrointestinal diseases that are increasing in global incidence and mortality rates. The C. difficile pathogenesis is due to toxin A and B (TcdA/TcdB), both causing cytopathic and cytotoxic effects and inflammation. Recently, we demonstrated that TcdB induces cytopathic and cytotoxic (apoptosis and necrosis) effects in enteric glial cells (EGCs) in a dose/time-dependent manner and described the underlying signaling. Despite the role played by lipids in host processes activated by pathogens, to counter infection and/or induce cell death, to date no studies have investigated lipid changes induced by TcdB/TcdA. Here, we evaluated the modification of lipid composition in our in vitro model of TcdB infection. Apoptosis, cell cycle, cell viability, and lipidomic profiles were evaluated in EGCs treated for 24 h with two concentrations of TcdB (0.1 ng/mL; 10 ng/mL). In EGCs treated with the highest concentration of TcdB, not only an increased content of total lipids was observed, but also lipidome changes, allowing the separation of TcdB-treated cells and controls into different clusters. The statistical analyses also allowed us to ascertain which lipid classes and lipid molecular species determine the clusterization. Changes in lipid species containing inositol as polar head and plasmalogen phosphatidylethanolamine emerged as key indicators of altered lipid metabolism in TcdB-treated EGCs. These results not only provide a picture of the phospholipid profile changes but also give information regarding the lipid metabolism pathways altered by TcdB, and this might represent an important step for developing strategies against C. difficile infection.