An in silico MS/MS library for automatic annotation of novel FAHFA lipids.

BackgroundA new lipid class named 'fatty acid esters of hydroxyl fatty acids' (FAHFA) was recently discovered in mammalian adipose tissue and in blood plasma and some FAHFAs were found to be associated with type 2 diabetes. To facilitate the automatic annotation of FAHFAs in biological specimens, a tandem mass spectra (MS/MS) library is needed. Due to the limitation of the commercial available standard compounds, we proposed building an in silico MS/MS library to extend the coverage of molecules.ResultsWe developed a computer-generated library with 3267 tandem mass spectra (MS/MS) for 1089 FAHFA species. FAHFA spectra were generated based on authentic standards with negative mode electrospray ionization and 10, 20, and 40 V collision induced dissociation at 4 spectra/s as used in in ultra-high performance liquid chromatography-QTOF mass spectrometry studies. However, positional information of the hydroxyl group is only obtained either at lower QTOF spectra acquisition rates of 1 spectrum/s or at the MS(3) level in ion trap instruments. Therefore, an additional set of 4290 fragment-rich MS/MS spectra was created to enable distinguishing positional FAHFA isomers. The library was generated based on ion fragmentations and ion intensities of FAHFA external reference standards, developing a heuristic model for fragmentation rules and extending these rules to large swaths of computer-generated structures of FAHFAs with varying chain lengths, degrees of unsaturation and hydroxyl group positions. Subsequently, we validated the new in silico library by discovering several new FAHFA species in egg yolk, showing that this library enables high-throughput screening of FAHFA lipids in various biological matrices.ConclusionsThe developed library and templates are freely available for commercial or noncommercial use at http://fiehnlab.ucdavis.edu/staff/yanma/fahfa-lipid-library. This in silico MS/MS library allows users to annotate FAHFAs from accurate mass tandem mass spectra in an easy and fast manner with NIST MS Search or PepSearch software. The developing template is provided for advanced users to modify the parameters and export customized libraries according to their instrument features. Graphical abstractExample of experimental and in silico MS/MS spectra for FAHFA lipids

Integrating Multiple Analytical Datasets to Compare Metabolite Profiles of Mouse Colonic-Cecal Contents and Feces.

The pattern of metabolites produced by the gut microbiome comprises a phenotype indicative of the means by which that microbiome affects the gut. We characterized that phenotype in mice by conducting metabolomic analyses of the colonic-cecal contents, comparing that to the metabolite patterns of feces in order to determine the suitability of fecal specimens as proxies for assessing the metabolic impact of the gut microbiome. We detected a total of 270 low molecular weight metabolites in colonic-cecal contents and feces by gas chromatograph, time-of-flight mass spectrometry (GC-TOF) and ultra-high performance liquid chromatography, quadrapole time-of-flight mass spectrometry (UPLC-Q-TOF). Of that number, 251 (93%) were present in both types of specimen, representing almost all known biochemical pathways related to the amino acid, carbohydrate, energy, lipid, membrane transport, nucleotide, genetic information processing, and cancer-related metabolism. A total of 115 metabolites differed significantly in relative abundance between both colonic-cecal contents and feces. These data comprise the first characterization of relationships among metabolites present in the colonic-cecal contents and feces in a healthy mouse model, and shows that feces can be a useful proxy for assessing the pattern of metabolites to which the colonic mucosum is exposed

Umbilical cord blood metabolomics reveal distinct signatures of dyslipidemia prior to bronchopulmonary dysplasia and pulmonary hypertension

Pulmonary hypertension (PH) is a common consequence of bronchopulmonary dysplasia (BPD) and remains a primary contributor to increased morbidity and mortality among preterm infants. Unfortunately, at the present time, there are no reliable early predictive markers for BPD-associated PH. Considering its health consequences, understanding in utero perturbations that lead to the development of BPD and BPD-associated PH and identifying early predictive markers is of utmost importance. As part of the discovery phase, we applied a multiplatform metabolomics approach consisting of untargeted and targeted methodologies to screen for metabolic perturbations in umbilical cord blood (UCB) plasma from preterm infants that did ( n = 21; cases) or did not ( n = 21; controls) develop subsequent PH. A total of 1,656 features were detected, of which 407 were annotated by metabolite structures. PH-associated metabolic perturbations were characterized by reductions in major choline-containing phospholipids, such as phosphatidylcholines and sphingomyelins, indicating altered lipid metabolism. The reduction in UCB abundances of major choline-containing phospholipids was confirmed in an independent validation cohort consisting of UCB plasmas from 10 cases and 10 controls matched for gestational age and BPD status. Subanalyses in the discovery cohort indicated that elevations in the oxylipins PGE1, PGE2, PGF2a, 9- and 13-HOTE, 9- and 13-HODE, and 9- and 13-KODE were positively associated with BPD presence and severity. This expansive evaluation of cord blood plasma identifies compounds reflecting dyslipidemia and suggests altered metabolite provision associated with metabolic immaturity that differentiate subjects, both by BPD severity and PH development

Validation of a Blood-Based Protein Biomarker Panel for a Risk Assessment of Lethal Lung Cancer in the Physicians\u27 Health Study

This study aimed to assess a four-marker protein panel (4MP)\u27s performance, including the precursor form of surfactant protein B, cancer antigen 125, carcinoembryonic antigen, and cytokeratin-19, for predicting lung cancer in a cohort enriched with never- and ever-smokers. Blinded pre-diagnostic plasma samples collected within 2 years prior to a lung cancer diagnosis from 25 cases and 100 sex-, age-, and smoking-matched controls were obtained from the Physicians\u27 Health Study (PHS). The 4MP yielded AUC performance estimates of 0.76 (95% CI: 0.61-0.92) and 0.69 (95% CI: 0.56-0.82) for predicting lung cancer within one year and within two years of diagnosis, respectively. When stratifying into ever-smokers and never-smokers, the 4MP had respective AUCs of 0.77 (95% CI: 0.63-0.92) and 0.72 (95% CI: 0.17-1.00) for a 1-year risk of lung cancer. The AUCs of the 4MP for predicting metastatic lung cancer within one year and two years of the blood draw were 0.95 (95% CI: 0.87-1.00) and 0.78 (95% CI: 0.62-0.94), respectively. Our findings indicate that a blood-based biomarker panel may be useful in identifying ever- and never-smokers at high risk of a diagnosis of lung cancer within one-to-two years

A Metabolite-Based Liquid Biopsy for Detection of Ovarian Cancer

Serial CA125 and second line transvaginal ultrasound (TVS) screening in the UKCTOCS indicated a shift towards detection of earlier stage ovarian cancer (OvCa), but did not yield a significant mortality reduction. There remains a need to establish additional biomarkers that can complement CA125 for even earlier and at a larger proportion of new cases. Using a cohort of plasma samples from 219 OvCa cases (59 stage I/II and 160 stage III/IV) and 409 female controls and a novel Sensitivity Maximization At A Given Specificity (SMAGS) method, we developed a blood-based metabolite-based test consisting of 7 metabolites together with CA125 for detection of OvCa. At a 98.5% specificity cutpoint, the metabolite test achieved sensitivity of 86.2% for detection of early-stage OvCa and was able to capture 64% of the cases with low CA125 levels (\u3c 35 units/mL). In an independent test consisting of 65 early-stage OvCa cases and 141 female controls, the metabolite panel achieved sensitivity of 73.8% at a 91.4% specificity and captured 13 (44.8%) out of 29 early-stage cases with CA125 levels \u3c 35 units/mL. The metabolite test has utility for ovarian cancer screening, capable of improving upon CA125 for detection of early-stage disease

Spatial Transcriptomics of Intraductal Papillary Mucinous Neoplasms of the Pancreas Identifies NKX6-2 as a Driver of Gastric Differentiation and Indolent Biological Potential

Intraductal Papillary Mucinous Neoplasms (IPMNs) of the pancreas are bona fide precursor lesions of pancreatic ductal adenocarcinoma (PDAC). The most common subtype of IPMNs harbor a gastric foveolar-type epithelium, and these low-grade mucinous neoplasms are harbingers of IPMNs with high-grade dysplasia and cancer. The molecular underpinning of gastric differentiation in IPMNs is unknown, although identifying drivers of this indolent phenotype might enable opportunities for intercepting progression to high-grade IPMN and cancer. We conducted spatial transcriptomics on a cohort of IPMNs, followed by orthogonal and cross species validation studies, which established the transcription factor NKX6-2 as a key determinant of gastric cell identity in low-grade IPMNs. Loss of NKX6-2 expression is a consistent feature of IPMN progression, while re-expression of NKX6-2 in murine IPMN lines recapitulates the aforementioned gastric transcriptional program and glandular morphology. Our study identifies NKX6-2 as a previously unknown transcription factor driving indolent gastric differentiation in IPMN pathogenesis

Low-density lipoprotein electronegativity and risk of death after acute coronary syndromes: A case-cohort analysis.

BACKGROUND AND AIMS Low-density lipoprotein (LDL)-cholesterol (LDL-C) promotes atherosclerotic cardiovascular disease (ASCVD), with changes in LDL electronegativity modulating its pro-atherogenic/pro-thrombotic effects. Whether such alterations associate with adverse outcomes in patients with acute coronary syndromes (ACS), a patient population at particularly high cardiovascular risk, remains unknown. METHODS This is a case-cohort study using data from a subset of 2619 ACS patients prospectively recruited at four university hospitals in Switzerland. Isolated LDL was chromatographically separated into LDL particles with increasing electronegativity (L1-L5), with the L1-L5 ratio serving as a proxy of overall LDL electronegativity. Untargeted lipidomics revealed lipid species enriched in L1 (least) vs. L5 (most electronegative subfraction). Patients were followed at 30 days and 1 year. The mortality endpoint was reviewed by an independent clinical endpoint adjudication committee. Multivariable-adjusted hazard ratios (aHR) were calculated using weighted Cox regression models. RESULTS Changes in LDL electronegativity were associated with all-cause mortality at 30 days (aHR, 2.13, 95% CI, 1.07-4.23 per 1 SD increment in L1/L5; p=.03) and 1 year (1.84, 1.03-3.29; p=.04), with a notable association with cardiovascular mortality (2.29; 1.21-4.35; p=.01; and 1.88; 1.08-3.28; p=.03). LDL electronegativity superseded several risk factors for the prediction of 1-year death, including LDL-C, and conferred improved discrimination when added to the updated GRACE score (area under the receiver operating characteristic curve 0.74 vs. 0.79, p=.03). Top 10 lipid species enriched in L1 vs. L5 were: cholesterol ester (CE) (18:2), CE (20:4), free fatty acid (FA) (20:4), phosphatidyl-choline (PC) (36:3), PC (34:2), PC (38:5), PC (36:4), PC (34:1), triacylglycerol (TG) (54:3), and PC (38:6) (all p < .001), with CE (18:2), CE (20:4), PC (36:3), PC (34:2), PC (38:5), PC (36:4), TG (54:3), and PC (38:6) independently associating with fatal events during 1-year of follow-up (all p < .05). CONCLUSIONS Reductions in LDL electronegativity are linked to alterations of the LDL lipidome, associate with all-cause and cardiovascular mortality beyond established risk factors, and represent a novel risk factor for adverse outcomes in patients with ACS. These associations warrant further validation in independent cohorts

Exercise Training Reduces the Inflammatory Response and Promotes Intestinal Mucosa-Associated Immunity in Lynch Syndrome

PURPOSE: Lynch syndrome (LS) is a hereditary condition with a high lifetime risk of colorectal and endometrial cancers. Exercise is a non-pharmacologic intervention to reduce cancer risk, though its impact on patients with LS has not been prospectively studied. Here, we evaluated the impact of a 12-month aerobic exercise cycling intervention in the biology of the immune system in LS carriers. PATIENTS AND METHODS: To address this, we enrolled 21 patients with LS onto a non-randomized, sequential intervention assignation, clinical trial to assess the effect of a 12-month exercise program that included cycling classes 3 times weekly for 45 minutes versus usual care with a one-time exercise counseling session as control. We analyzed the effects of exercise on cardiorespiratory fitness, circulating, and colorectal-tissue biomarkers using metabolomics, gene expression by bulk mRNA sequencing, and spatial transcriptomics by NanoString GeoMx. RESULTS: We observed a significant increase in oxygen consumption (VO2peak) as a primary outcome of the exercise and a decrease in inflammatory markers (prostaglandin E) in colon and blood as the secondary outcomes in the exercise versus usual care group. Gene expression profiling and spatial transcriptomics on available colon biopsies revealed an increase in the colonic mucosa levels of natural killer and CD8+ T cells in the exercise group that were further confirmed by IHC studies. CONCLUSIONS: Together these data have important implications for cancer interception in LS, and document for the first-time biological effects of exercise in the immune system of a target organ in patients at-risk for cancer

Modulating a Prebiotic Food Source Influences Inflammation and Immune-Regulating Gut Microbes and Metabolites: Insights from the BE GONE Trial

BACKGROUND: Accessible prebiotic foods hold strong potential to jointly target gut health and metabolic health in high-risk patients. The BE GONE trial targeted the gut microbiota of obese surveillance patients with a history of colorectal neoplasia through a straightforward bean intervention. METHODS: This low-risk, non-invasive dietary intervention trial was conducted at MD Anderson Cancer Center (Houston, TX, USA). Following a 4-week equilibration, patients were randomized to continue their usual diet without beans (control) or to add a daily cup of study beans to their usual diet (intervention) with immediate crossover at 8-weeks. Stool and fasting blood were collected every 4 weeks to assess the primary outcome of intra and inter-individual changes in the gut microbiome and in circulating markers and metabolites within 8 weeks. This study was registered on ClinicalTrials.gov as NCT02843425, recruitment is complete and long-term follow-up continues. FINDINGS: Of the 55 patients randomized by intervention sequence, 87% completed the 16-week trial, demonstrating an increase on-intervention in diversity [n = 48; linear mixed effect and 95% CI for inverse Simpson index: 0.16 (0.02, 0.30); p = 0.02] and shifts in multiple bacteria indicative of prebiotic efficacy, including increased Faecalibacterium, Eubacterium and Bifidobacterium (all p \u3c 0.05). The circulating metabolome showed parallel shifts in nutrient and microbiome-derived metabolites, including increased pipecolic acid and decreased indole (all p \u3c 0.002) that regressed upon returning to the usual diet. No significant changes were observed in circulating lipoproteins within 8 weeks; however, proteomic biomarkers of intestinal and systemic inflammatory response, fibroblast-growth factor-19 increased, and interleukin-10 receptor-α decreased (p = 0.01). INTERPRETATION: These findings underscore the prebiotic and potential therapeutic role of beans to enhance the gut microbiome and to regulate host markers associated with metabolic obesity and colorectal cancer, while further emphasizing the need for consistent and sustainable dietary adjustments in high-risk patients. FUNDING: This study was funded by the American Cancer Society

Blood-based Proteomic Signatures Associated With MEN1-related Duodenopancreatic Neuroendocrine Tumor Progression

PURPOSE: Patients with multiple endocrine neoplasia type 1 (MEN1) are predisposed to develop duodenopancreatic neuroendocrine tumors (dpNETs), and metastatic dpNET is the primary cause of disease-related mortality. Presently, there is a paucity of prognostic factors that can reliably identify patients with MEN1-related dpNETS who are at high risk of distant metastasis. In the current study, we aimed to establish novel circulating molecular protein signatures associated with disease progression. EXPERIMENTAL DESIGN: Mass spectrometry-based proteomic profiling was conducted on plasmas procured through an international collaboration between MD Anderson Cancer Center, the National Institutes of Health, and the University Medical Center Utrecht from a cohort of 56 patients with MEN1 [14 with distant metastasis dpNETs (cases) and 42 with either indolent dpNETs or no dpNETs (controls)]. Findings were compared to proteomic profiles generated from serially collected plasmas from a mouse model of Men1-pancreatic neuroendocrine tumors (Men1fl/flPdx1-CreTg) and control mice (Men1fl/fl). RESULTS: A total of 187 proteins were found to be elevated in MEN1 patients with distant metastasis compared to controls, including 9 proteins previously associated with pancreatic cancer and other neuronal proteins. Analyses of mouse plasmas revealed 196 proteins enriched for transcriptional targets of oncogenic MYCN, YAP1, POU5F1, and SMAD that were associated with disease progression in Men1fl/flPdx1-CreTg mice. Cross-species intersection revealed 19 proteins positively associated with disease progression in both human patients and in Men1fl/flPdx1-CreTg mice. CONCLUSIONS: Our integrated analyses identified novel circulating protein markers associated with disease progression in MEN1-related dpNET