Equivalent carbon number-based targeted odd-chain fatty acyl lipidomics reveals triacylglycerol profiling in clinical colon cancer

Odd-chain FAs (OCFAs) are present in very low level at nearly 1% of total FAs in human plasma, and thus, their functions were usually ignored. Recent epidemiological studies have shown that OCFAs are inversely associated with a variety of disease risks. However, the contribution of OCFAs incorporated into complex lipids remains elusive. Here, we developed a targeted odd-chain fatty acyl-containing lipidomics method based on equivalent carbon number and retention time prediction. The method displayed good reproducibility and robustness as shown by peak width at half height within 0.7 min and coefficient of variation under 20%. A total number of 776 lipid species with odd-chain fatty acyl residues could be detected in the ESI mode of reverse-phase LC-MS, of which 309 lipids were further validated using multiple reaction monitoring transitions. Using this method, we quantified odd-chain fatty acyl-containing lipidome in tissues from 12 colon cancer patients, revealing the remodeling of triacylglycerol. The dynamics of odd-chain fatty acyl lipids were further consolidated by the association with genomic and proteomic features of altered catabolism of branched-chain amino acids and triacylglycerol endogenous synthesis in colon cancer. This lipidomics approach will be applicable for screening of dysregulated odd-chain fatty acyl lipids, which enriches and improves the methods for diagnosis and prognosis evaluation of cancer using lipidomics.     

Targeted lipidomics reveals a novel role for glucosylceramides in glucose response

The addition of excess glucose to the diet drives a coordinated response of lipid metabolism pathways to tune the membrane composition to the altered diet. Here, we have employed targeted lipidomic approaches to quantify the specific changes in the phospholipid and sphingolipid populations that occur in elevated glucose conditions. The lipids within wild-type Caenorhabditis elegans are strikingly stable with no significant changes identified in our global mass spectrometry–based analysis. Previous work has identified ELO-5, an elongase that is critical for the synthesis of monomethyl branched-chain fatty acids (mmBCFAs), as essential for surviving elevated glucose conditions. Therefore, we performed targeted lipidomics on elo-5 RNAi-fed animals and identified several significant changes in these animals in lipid species that contain mmBCFAs as well as in species that do not contain mmBCFAs. Of particular note, we identified a specific glucosylceramide (GlcCer 17:1;O2/22:0;O) that is also significantly upregulated with glucose in wild-type animals. Furthermore, compromising the production of the glucosylceramide pool with elo-3 or cgt-3 RNAi leads to premature death in glucose-fed animals. Taken together, our lipid analysis has expanded the mechanistic understanding of metabolic rewiring with glucose feeding and has identified a new role for the GlcCer 17:1;O2/22:0;O.     

Integration of lipidomics and metabolomics for in-depth understanding of cellular mechanism and disease progression

Mass spectrometry(MS)-based omics technologies are now widely used to profile small molecules in multiple matrices to confer comprehensive snapshots of cellular metabolic phenotypes.The metabolomes of cells,tissues,and organisms comprise a variety of molecules including lipids,amino acids,sugars,organic acids,and so on.Metabolomics mainly focus on the hydrophilic classes,while lipidomics has emerged as an independent omics owing to the complexities of the organismal lipidomes.The potential roles of lipids and small metabolites in disease pathogenesis have been widely investigated in various human diseases,but system-level understanding is largely lacking,which could be partly attributed to the insufficiency in terms of metabolite coverage and quantitation accuracy in current analytical technologies.While scientists are continuously striving to develop high-coverage omics approaches,integration of metabolomics and lipidomics is becoming an emerging approach to mechanistic investigation.Integration of metabolome and lipidome offers a complete atlas of the metabolic landscape,enabling comprehensive network analysis to identify critical metabolic drivers in disease pathology,facilitating the study of interconnection between lipids and other metabolites in disease progression.In this review,we summarize omics-based findings on the roles of lipids and metabolites in the pathogenesis of selected major diseases threatening public health.We also discuss the advantages of integrating lipidomics and metabolomics for in-depth understanding of molecular mechanism in disease pathogenesis.     

The foundations and development of lipidomics

For over a century, the importance of lipid metabolism in biology was recognized but difficult to mechanistically understand due to the lack of sensitive and robust technologies for identification and quantification of lipid molecular species. The enabling technological breakthroughs emerged in the 1980s with the development of soft ionization methods (Electrospray Ionization and Matrix Assisted Laser Desorption/Ionization) that could identify and quantify intact individual lipid molecular species. These soft ionization technologies laid the foundations for what was to be later named the field of lipidomics. Further innovative advances in multistage fragmentation, dramatic improvements in resolution and mass accuracy, and multiplexed sample analysis fueled the early growth of lipidomics through the early 1990s. The field exponentially grew through the use of a variety of strategic approaches, which included direct infusion, chromatographic separation, and charge-switch derivatization, which facilitated access to the low abundance species of the lipidome. In this Thematic Review, we provide a broad perspective of the foundations, enabling advances, and predicted future directions of growth of the lipidomics field.     

Sensitive UHPLC-MS/MS quantification method for 4β- and 4α-hydroxycholesterol in plasma for accurate CYP3A phenotyping

4β-Hydroxycholesterol (4β-OHC) is formed by Cytochrome P450 (CYP)3A and has drawn attention as an endogenous phenotyping probe for CYP3A activity. However, 4β-OHC is also increased by cholesterol autooxidation occurring in vitro due to dysregulated storage and in vivo by oxidative stress or inflammation, independent of CYP3A activity. 4α-hydroxycholesterol (4α-OHC), a stereoisomer of 4β-OHC, is also formed via autooxidation of cholesterol, not by CYP3A, and thus may have clinical potential in reflecting the state of cholesterol autooxidation. In this study, we establish a sensitive method for simultaneous quantification of 4β-OHC and 4α-OHC in human plasma using ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Plasma samples were prepared by saponification, two-step liquid-liquid extraction, and derivatization using picolinic acid. Intense [M+H]+ signals for 4β-OHC and 4α-OHC di-picolinyl esters were monitored using electrospray ionization. The assay fulfilled the requirements of the US Food and Drug Administration guidance for bioanalytical method validation, with a lower limit of quantification of 0.5 ng/ml for both 4β-OHC and 4α-OHC. Apparent recovery rates from human plasma ranged from 88.2% to 101.5% for 4β-OHC, and 91.8% to 114.9% for 4α-OHC. Additionally, matrix effects varied between 86.2% and 117.6% for 4β-OHC and between 89.5% and 116.9% for 4α-OHC. Plasma 4β-OHC and 4α-OHC concentrations in healthy volunteers, stage 3–5 chronic kidney disease (CKD) patients, and stage 5D CKD patients as measured by the validated assay were within the calibration ranges in all samples. We propose this novel quantification method may contribute to accurate evaluation of in vivo CYP3A activity.     

Ex vivo instability of lipids in whole blood: preanalytical recommendations for clinical lipidomics studies

Reliability, robustness, and interlaboratory comparability of quantitative measurements is critical for clinical lipidomics studies. Lipids’ different ex vivo stability in blood bears the risk of misinterpretation of data. Clear recommendations for the process of blood sample collection are required. We studied by UHPLC-high resolution mass spectrometry, as part of the “Preanalytics interest group” of the International Lipidomics Society, the stability of 417 lipid species in EDTA whole blood after exposure to either 4°C, 21°C, or 30°C at six different time points (0.5 h–24 h) to cover common daily routine conditions in clinical settings. In total, >800 samples were analyzed. 325 and 288 robust lipid species resisted 24 h exposure of EDTA whole blood to 21°C or 30°C, respectively. Most significant instabilities were detected for FA, LPE, and LPC. Based on our data, we recommend cooling whole blood at once and permanent. Plasma should be separated within 4 h, unless the focus is solely on robust lipids. Lists are provided to check the ex vivo (in)stability of distinct lipids and potential biomarkers of interest in whole blood. To conclude, our results contribute to the international efforts towards reliable and comparable clinical lipidomics data paving the way to the proper diagnostic application of distinct lipid patterns or lipid profiles in the future.     

Cholesterol metabolism: from lipidomics to immunology

Oxysterols, the oxidized forms of cholesterol or of its precursors, are formed in the first steps of cholesterol metabolism. Oxysterols have interested chemists, biologists, and physicians for many decades, but their exact biological relevance in vivo, other than as intermediates in bile acid biosynthesis, has long been debated. However, in the first quarter of this century, a role for side-chain oxysterols and their C-7 oxidized metabolites has been convincingly established in the immune system. 25-Hydroxycholesterol has been shown to be synthesized by macrophages in response to the activation of Toll-like receptors and to offer protection against microbial pathogens, whereas 7α,25-dihydroxycholesterol has been shown to act as a chemoattractant to lymphocytes expressing the G protein-coupled receptor Epstein-Barr virus-induced gene 2 and to be important in coordinating the action of B cells, T cells, and dendritic cells in secondary lymphoid tissue. There is a growing body of evidence that not only these two oxysterols but also many of their isomers are of importance to the proper function of the immune system. Here, we review recent findings related to the roles of oxysterols in immunology.     

Validation of Diagnostic Coding for Diabetes Mellitus in Hospitalized Patients

ObjectiveSome studies have shown that there is an undercoding of diabetes mellitus among hospitalized patients, which can have adverse clinical and financial implications for health systems. We aimed to validate the discharge diagnostic coding of diabetes mellitus in hospitalized patients using clinical and laboratory-based diagnostic indicators as the reference.MethodsThis was a retrospective cohort study of 83 690 discharges of 48 615 unique adult patients who were hospitalized in an academic medical center over 4.5 years and had at least 4 blood glucose measurements during admission. A missing diabetes code (MDC) was defined using 2 criteria. MDC₁ was defined as the presence of any of the following: blood glucose ≥200 (x2), A1C ≥6.5%, home antihyperglycemic medication, or preadmission code for diabetes, whereas MDC₂ was defined as preadmission diabetes or at least 2 other criteria in MDC₁. Multivariable logistic regression was used to identify factors associated with MDC compared to the present diabetes code.ResultsMDC₁ and MDC₂ were present in 12 186 (14.6%) and 3542 (4.7%) discharges, respectively. Factors associated with both MDC₁ and MDC₂ were medium-dose steroid use [adjusted odds ratio (aOR) 2.11, 2.01], high-dose steroid use (aOR 4.70, 2.50), intermediate medical care service (aOR 1.65, 1.55), infection (aOR 1.21, 1.34), and hepatic disease (aOR 1.93, 1.92).ConclusionIn this retrospective study, MDC ranged from 5% to 15% and was associated with various clinical factors. Further prospective studies are needed to validate these findings, explore the mechanisms behind these associations, and understand the clinical and financial implications.     

Evaluating Use of the Octreotide Acetate Pen Injector in a Summative Human Factors Validation Study

ObjectiveSubcutaneous injections of octreotide acetate require chronic administration by health care providers (HCPs). We aimed to validate the safe and effective use of the octreotide acetate pen injector, its labeling, and instructions for use (IFU) by patients, caregivers, and HCPs and to mitigate use-related risks.MethodsThis summative human factors validation study enrolled adults with neuroendocrine tumors and related diarrhea or flushing, adult caregivers, and HCPs. Before simulated use, participants self-familiarized as desired. Each participant was assigned 1 injection site for administration into an injection pad. The first of 2 unaided injections assessed first use and required priming; the second assessed routine use and dose change. Participants gave subjective feedback after each injection and completed knowledge probes and reading comprehension questions after the second injection.ResultsThe study enrolled 45 participants (15 per group). Forty-two participants completed the first injection successfully by administering the dose correctly. Three participants did not dose successfully; 3 failed to prime the pen, and 1 failed to dial the correct dose. Besides dosing, 2 participants failed to remove the needle after injection. Forty-four participants completed the second injection, but 1 participant failed to dial the correct dose. No other errors were observed. Overall success rates on knowledge probes and reading comprehension questions were 99.1% and 99.6%, respectively. All participants found the IFU easy to follow and understand.ConclusionThe octreotide acetate pen injector, labeling, and IFU enabled intended users to administer subcutaneous octreotide safely and effectively. The residual risks of use are low and acceptable.     

Improving diagnostic strategies for ovarian cancer in Filipino women using ultrasound imaging and a multivariate index assay

ObjectiveTo evaluate the clinical performance and overall utility of imaging and biomarker assays in discriminating between benign and malignant ovarian masses in a Filipino population.MethodsThis is a prospective cohort study among Filipino women undergoing assessment for an ovarian mass in a tertiary center. All included patients underwent a physical examination before level III specialist ultrasonographic and Doppler evaluation, multivariate index assay (MIA2G), and surgery for an adnexal mass. Ovarian tumors were classified as high-risk for malignancy based on the International Ovarian Tumour Analysis (IOTA) – Logistic Regression 2 (LR2) score. The ovarian imaging and biomarker results were correlated with the reference standard: histological findings.ResultsAmong the 379 women with adnexal masses enrolled in this study, 291 were evaluable with ultrasound imaging, biomarker assays, and histopathological results. The risk of malignancy was higher for women classified as high-risk based on IOTA-LR2 (≥10%). The sensitivity, specificity, and diagnostic accuracy for the prediction of malignancy were 81.2%, 81%, and 0.81 (95% CI: 0.77–0.86) for IOTA-LR2; 77.5%, 66.7%, and 0.72 (95% CI: 0.67–0.77) for CA-125; and 91.3%, 41.2%, and 0.66 (95% CI: 0.62–0.71) for MIA2G. A combination of IOTA-LR2 and MIA2G significantly influenced the diagnostic performance and the result. When MIA2G was combined with IOTA-LR2 in parallel, the sensitivity (94.2%) and NPV (87.7%) increased, but the specificity (37.3%) decreased. When combined with IOTA-LR2 in series, there were fewer false positives, which resulted in improved specificity (85%).ConclusionThis study determined the utility of ovarian imaging and a second-generation multivariate index assay in predicting the risk of ovarian malignancy. IOTA-LR2 and MIA2G were useful in classifying patients with a high risk for ovarian malignancy.     

Enhanced targeted DNA methylation of the CMV and endogenous promoters with dCas9-DNMT3A3L entails distinct subsequent histone modification changes in CHO cells

With the emergence of new CRISPR/dCas9 tools that enable site specific modulation of DNA methylation and histone modifications, more detailed investigations of the contribution of epigenetic regulation to the precise phenotype of cells in culture, including recombinant production subclones, is now possible. These also allow a wide range of applications in metabolic engineering once the impact of such epigenetic modifications on the chromatin state is available.In this study, enhanced DNA methylation tools were targeted to a recombinant viral promoter (CMV), an endogenous promoter that is silenced in its native state in CHO cells, but had been reactivated previously (β-galactoside α-2,6-sialyltransferase 1) and an active endogenous promoter (α-1,6-fucosyltransferase), respectively. Comparative ChIP-analysis of histone modifications revealed a general loss of active promoter histone marks and the acquisition of distinct repressive heterochromatin marks after targeted methylation. On the other hand, targeted demethylation resulted in autologous acquisition of active promoter histone marks and loss of repressive heterochromatin marks. These data suggest that DNA methylation directs the removal or deposition of specific histone marks associated with either active, poised or silenced chromatin. Moreover, we show that de novo methylation of the CMV promoter results in reduced transgene expression in CHO cells. Although targeted DNA methylation is not efficient, the transgene is repressed, thus offering an explanation for seemingly conflicting reports about the source of CMV promoter instability in CHO cells.Importantly, modulation of epigenetic marks enables to nudge the cell into a specific gene expression pattern or phenotype, which is stabilized in the cell by autologous addition of further epigenetic marks. Such engineering strategies have the added advantage of being reversible and potentially tunable to not only turn on or off a targeted gene, but also to achieve the setting of a desirable expression level.     

Relationship between hepatic and mitochondrial ceramides: a novel in vivo method to track ceramide synthesis

Ceramides (CERs) are key intermediate sphingolipids implicated in contributing to mitochondrial dysfunction and the development of multiple metabolic conditions. Despite the growing evidence of CER role in disease risk, kinetic methods to measure CER turnover are lacking, particularly using in vivo models. The utility of orally administered ¹³C₃, ¹⁵N l-serine, dissolved in drinking water, was tested to quantify CER 18:1/16:0 synthesis in 10-week-old male and female C57Bl/6 mice. To generate isotopic labeling curves, animals consumed either a control diet or high-fat diet (HFD; n = 24/diet) for 2 weeks and varied in the duration of the consumption of serine-labeled water (0, 1, 2, 4, 7, or 12 days; n = 4 animals/day/diet). Unlabeled and labeled hepatic and mitochondrial CERs were quantified using liquid chromatography tandem MS. Total hepatic CER content did not differ between the two diet groups, whereas total mitochondrial CERs increased with HFD feeding (60%, P < 0.001). Within hepatic and mitochondrial pools, HFD induced greater saturated CER concentrations (P < 0.05) and significantly elevated absolute turnover of 16:0 mitochondrial CER (mitochondria: 59%, P < 0.001 vs. liver: 15%, P = 0.256). The data suggest cellular redistribution of CERs because of the HFD. These data demonstrate that a 2-week HFD alters the turnover and content of mitochondrial CERs. Given the growing data on CERs contributing to hepatic mitochondrial dysfunction and the progression of multiple metabolic diseases, this method may now be used to investigate how CER turnover is altered in these conditions.     

High Sensitivity and Wide Linearity LC-MS/MS Method for Oxylipin Quantification in Multiple Biological Samples

Oxylipins are important biological regulators that have received extensive research attention. Due to the extremely low concentrations, large concentration variations, and high structural similarity of many oxylipins, the quantitative analysis of oxylipins in biological samples is always a great challenge. Here, we developed a liquid chromatography-tandem mass spectrometry-based method with high sensitivity, wide linearity, and acceptable resolution for quantitative profiling of oxylipins in multiple biological samples. A total of 104 oxylipins, some with a high risk of detection crosstalk, were well separated on a 150 mm column over 20 min. The method showed high sensitivity with lower limits of quantitation for 87 oxylipins, reaching 0.05–0.5 pg. Unexpectedly, we found that the linear range for 16, 18, and 17 oxylipins reached 10,000, 20,000, and 40,000 folds, respectively. Due to the high sensitivity, while reducing sample consumption to below half the volume of previous methods, 74, 78, and 59 low-abundance oxylipins, among which some were difficult to detect like lipoxins and resolvins, were well quantified in the tested mouse plasma, mouse liver, and human plasma samples, respectively. Additionally, we determined that analytes with multifarious concentrations of over a 1,000-fold difference could be well quantified simultaneously due to the wide linearity. In conclusion, most likely due to the instrumental advancement, this method effectively improves the quantitative sensitivity and linear range over existing methods, which will facilitate and advance the study of the physiological and pathophysiological functions of oxylipins.     

Overview of commercial treatment planning systems for targeted radionuclide therapy

IntroductionTargeted Radionuclide Therapy (TRT) is a branch of cancer medicine dealing with the therapeutic use of radioisotopes associated with biological vectors accumulating in the tumors/targets, indicated as Molecular Radiotherapy (MRT), or directly injected into the arteries that supply blood to liver tumour vasculature, indicated as Selective RT (SRT). The aim of this work is to offer a panoramic view on the increasing number of commercially-available TRT treatment planning systems (TPSs).Materials and methodsA questionnaire was sent to manufacturers' representatives. Academic software were not considered. Questions were grouped as follows: general information, clinical workflow, calibration procedure, image processing/reconstruction, image registration and segmentation tools, time-activity curve (TAC) fitting and absorbed dose calculation.ResultsAll software reported have CE-marking. TPSs were divided between SRT-dedicated software [4] and MRT [5] dosimetry software. In SRT, since no kinetic process is involved, absorbed dose calculation does not require TAC fitting, and image registration is not fully developed in all TPS. All software requires a radionuclide-specific calibration. In SRT, a relative image calibration can be obtained by scaling the counts to a known activity. Automated VOI contouring and rigid/deformable propagation between different acquisitions time-points is implemented in most TPSs, although DICOM export is rare. Different TAC fits are available depending on the number of time-points. Voxel S-value and Local deposition methods are the most frequent dosimetric approaches; dose-voxel kernel convolution and semi-Monte Carlo method are also available.ConclusionsAvailable TPSs allows performing personalized dosimetry in clinical practice. Individual variations in methodology/algorithms must be considered in the standardisation/harmonization processes.     

Development and Internal Validation of A Prediction Tool To Assist Clinicians Selecting Second-Line Therapy Following Metformin Monotherapy For Type 2 Diabetes

ObjectiveAdults with type 2 diabetes (T2D) face increased risk of many long-term adverse outcomes. While managing patients with T2D, clinicians are challenged to stay informed regarding all new therapies and must consider potential risks and benefits resultant to their use. Metformin (MET) is typically prescribed as first-line therapy, but a second line is often needed, given MET can be insufficient for maintaining long-term glycemic control. Our objective was to develop a predictive decision-making tool to help clinicians use an outcome-based approach to select second-line therapies for patients when MET monotherapy is insufficient for glycemic control.MethodsElectronic health records of 19 277 adults with T2D on MET monotherapy and ≥3 months of either GLP-1RA, DPP-4i, Insulin, SGLT-2i, SFU, or TZD therapy were reviewed at Cleveland Clinic from patient visits occurring between 2005 and 2019. Separate models were developed to predict likelihood of each main outcome measure (stroke, myocardial infarction, worsening hypertension, renal failure, and death). Discrimination and calibration were assessed with bootstrapping.ResultsThe median follow-up time for those without an event was 3.6 years (interquartile range 1.9, 6.3). Model discrimination ability was evaluated by concordance indices (goodness of fit metric with values ranging between 0 and 1: 1 indicates perfect discrimination ability; 0.5 reflects same discrimination ability as chance) demonstrating strong discrimination ability, with concordance index values for outcomes as follows: myocardial infarction (0.786), stroke (0.805), worsening hypertension (0.855), renal failure (0.808), and death (0.827).ConclusionA decision-making tool has been developed that may afford clinicians a more objective and individualized approach to choosing a second-line therapy to control glycemia for persons with T2D.     

Near-infrared mediated polymer-coated carbon nanodots loaded cisplatin for targeted care management of lung cancer therapy

The Cisplatin and its analogues are scarce for the second leading human health problem, cancer. Therefore, we have effectively engineered the PEGlyated carbon nanodots loaded with cisplatin PEG-CDs@Cis-Pt(IV) for targeted lung cancer therapy. The PEG-CDs@Cis-Pt(IV) confirmed by the electroscopic and spectroscopic methods. The in vitro cytotoxicity of the nanoparticles evaluated two lung cancers (A549 and HEL-299), and one non-cancerous cell line (HUVECs) were used in this study by using MTT assay. The nanoparticles are effectively killing cancer cells without affecting the nano-cancerous cells. Further, dual AO-EB fluorescent staining assay established the programmed cell death through cell morphological changes. Further, flow cytometry confirmed the induction of apoptosis by S phase arrest of the cancer cell cycle by the complexes. The results of our investigations also bear witness to CDs@Cis-Pt(IV)+NIR-NPs promising scope and potency care management for precise cancer therapy beyond platinum drugs.     

Key taste components in two wild edible Boletus mushrooms using widely targeted metabolomics

Compounds from wild edible mushrooms has been reported to exert biological activities and contribute to the different flavors of mushrooms in our diet. Wild edible Boletus mushrooms are popular in Southwest China. In this study, we performed ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) based on widely targeted metabolomics analysis to identify key components. A total of 194 metabolites (113 upregulated, 81 downregulated) divided into 11 groups (49 metabolites in group lipids, 34 in amino acids and derivatives, 30 in organic acids, 18 in phenolic acids, 16 in nucleotides and derivatives, 13 in alkaloids, 6 in flavonoids, 3 in lignans and coumarins, 3 in tannins, 2 in terpenoids, and 20 in others) were found among Boletus bainiugan compared with B. subsplendidus. Through clustering analysis, principal component analysis (PCA), and orthogonal signal correction and partial least squares-discriminant analysis (OPLS-DA), different metabolites from fruiting bodies were clearly identified. Significant differences were observed in the metabolites between Boletus bainiugan and B. subsplendidus. These metabolites are involved in important biological functions. Our results provide new insights into important metabolites and theoretical basis for the taste difference of two wild edible Boletus mushrooms.