Comparison of untargeted gas chromatography-mass spectrometry analysis algorithms with implications to the interpretation and putative identification of volatile aroma compositions.

The aroma profiling process requires the identification of the volatile compounds in a sample or its headspace. Typically, the identification of compounds relies on automated feature finding and matching algorithms to (putatively) identify and report compounds based on retention index and mass spectra matching against a compound library. We investigated the use of five different workflows and proposed three metrics (target accuracy A, identification percentage I, uniqueness U) to quantify their impact on generated aroma profiles of a mixture of fragrance standards and a commercial grade essential oil. All workflows accurately identified target compounds (100% in standards, >90% in samples) and reported similar compound identities for major GC-MS features, but beyond that could differ by up to 40-50%. Despite the variances, different workflows did not report conflicting compound identities. Aroma compositions primarily contained unreported or extra (putatively) identified compounds due to variations in mass spectral elucidations within the various workflows. Considering these differences, we show how the proposed metrics, I and U, could be modified to help the analyst interpret and evaluate reported volatile aroma compositions of unknown materials.

Co-culture of Kluyveromyces marxianus and Meyerozyma guilliermondii with In Situ Product Recovery of 2-Phenylethanol.

Production of 2-phenylethanol (2-PE) via Kluyveromyces marxianus is well-established. However, co-culture with other microbes in combination with in situ product recovery (ISPR) yields improved selectivity and volumetric productivity. Fermentation ofK. marxianus (MUCL 53775) with direct inclusion of absorptive polymer Hytrel3548 achieved ISPR, but accumulation of the byproduct phenylethyl acetate (PEA) was strongly favored. Co-culture of K. marxianus (MUCL 53775) with Meyerozyma guilliermondii (MUCL 28072) with ISPR limited PEA production, thereby improving the 2-PE selectivity from 13 to 90%, compared to a pure culture of K. marxianus (MUCL 53775) under similar conditions. This improved the volumetric productivity by 85% compared to 2-PE ISPR with a pure culture of K. marxianus. This is the first report of co-culture in a two-phase fermentation for 2-PE bioproduction and demonstrates that interactions between co-culture and ISPR techniques can modulate bioproduction between 2-PE and byproduct PEA, and this technique will be explored for other strain combinations and for other high-value molecules of interest.

Aromatic Yeasts: Interactions and Implications in Coffee Fermentation Aroma Profiles.

Nontraditional yeasts prevalent in tropical agricultural fermentations such as coffee and cocoa are known to contribute to aroma profiles, yet the functional roles and interactions between the associated microbial consortia in a farm fermentation are unclear. Here, boiled green bean extract (GBE) from green coffee beans was developed as a rich screening medium to deconstruct the microbial consortia and their interactions during the fermentation of dried green coffee beans. When cultivated in coculture with S. cerevisiae on GBE, strain-specific groupings with distinct volatile organic profiles were observed for nontraditional yeasts (e.g., Hanseniaspora spp., Pichia kudriavzevii). Further changes are evident when constructed consortia composed of nontraditional yeast, S. cerevisiae, and Lactococcus lactis var. cremoris were cultured in GBE, and a comparison with abiotically acidified GBE suggests that pH plays a major role in the influence of lactic acid bacteria (LAB) on fermentation aromas. This approach represents a tool for the development of starter culture formulations to create different flavor profiles in coffee fermentation.

Profiling of Aroma-Active Compounds in Ylang-Ylang Essential Oils by Aroma Extract Dilution Analysis (AEDA) and Chemometric Methods.

The aroma-active compounds in the extra, first, and third grades of ylang-ylang essential oils (YYEO) from Comoros and Madagascar were identified by gas chromatography-mass spectrometry with olfactometry (GC-MS/O) using an aroma extract dilution analysis (AEDA) technique. In the previous study, the authors investigated differences in volatile compound profiles between YYEO of different grades and regions using GC coupled with a flame ionization detector (FID) and GC-MS. This study follows up with identification of the aroma-active compounds present in YYEO of various grades from both origins and to profile the aroma of those oils. For the first time, principal component analysis (PCA) on AEDA logarithmic flavor dilution (LFD) data was performed, in comparison with the corresponding PCA on GC-FID-MS data. Based on AEDA data, 21 aroma-active compounds were found across all samples and grades of YYEO, with 8 common ones previously identified by GC-FID. Linalool had the highest odor activity and is the major component of YYEO, followed by geraniol, although the latter only appeared as a much smaller peak in the chromatogram. Other trace compounds such as eugenol and vanillin were also found to be significant to the aroma of YYEO. Using PCA on resulting LFD data, YYEO from Comoros were found to have spicier odor qualities as compared to those from Madagascar. The main contributors that determine the difference in a spicy aroma profile of Comoros and Madagascar oils are vanillin, methyl eugenol, and trans-cinnamyl acetate.

Methionine is a metabolic dependency of tumor-initiating cells.

Understanding cellular metabolism holds immense potential for developing new classes of therapeutics that target metabolic pathways in cancer. Metabolic pathways are altered in bulk neoplastic cells in comparison to normal tissues. However, carcinoma cells within tumors are heterogeneous, and tumor-initiating cells (TICs) are important therapeutic targets that have remained metabolically uncharacterized. To understand their metabolic alterations, we performed metabolomics and metabolite tracing analyses, which revealed that TICs have highly elevated methionine cycle activity and transmethylation rates that are driven by MAT2A. High methionine cycle activity causes methionine consumption to far outstrip its regeneration, leading to addiction to exogenous methionine. Pharmacological inhibition of the methionine cycle, even transiently, is sufficient to cripple the tumor-initiating capability of these cells. Methionine cycle flux specifically influences the epigenetic state of cancer cells and drives tumor initiation. Methionine cycle enzymes are also enriched in other tumor types, and MAT2A expression impinges upon the sensitivity of certain cancer cells to therapeutic inhibition.

Publisher Correction: Methionine is a metabolic dependency of tumor-initiating cells.

In the version of this article originally published, there is an error in Fig. 5a. Originally, 'MAT2A' appeared between 'Methionine' and 'Homocysteine'. 'MAT2A' should have been 'MTR'. The error has been corrected in the PDF and HTML versions of this article.

MMSET I acts as an oncoprotein and regulates GLO1 expression in t(4;14) multiple myeloma cells.

Multiple myeloma (MM) is characterized by recurrent chromosomal translocations. T(4;14) MM overexpresses multiple myeloma SET domain-containing protein (MMSET). MMSET has three major isoforms: the full-length form MMSET II and the short isoforms REIIBP and MMSET I. Here we show that the short isoform MMSET I is an oncoprotein that promoted cell survival and tumorigenesis in vitro and in vivo. Gene expression array analysis indicated that MMSET I increased glyoxalase I (GLO1) expression. Chromatin immunoprecipitation (ChIP) coupled with qPCR indicated that MMSET I bound upstream of the GLO1 transcription start site. Ectopic overexpression of MMSET I or its mutants showed MMSET I depended on its C terminus to regulate GLO1 expression. GLO1 knockdown (KD) induced apoptosis and reduced colony formation. MMSET I or GLO1 KD reduced the levels of anti-apoptosis factors such as MCL1 and BCL2. Ectopic overexpression of GLO1 resulted in the significant rescue of KMS11 cells from MMSET I KD-induced apoptosis and glycolysis inhibition. This suggested that GLO1 may be of functional importance target downstream of MMSET I. Cumulatively, our study suggests that MMSET I is an oncoprotein and potential therapeutic target for t(4;14) MM.

Excessive interferon-α signaling in autoimmunity alters glycosphingolipid processing in B cells.

Excessive interferon-α (IFN-α) production by innate immune cells is a hallmark of autoimmune diseases. What other cell type secretes IFN-α and how IFN-α affects immune cell metabolism and homeostasis in autoimmunity are largely unclear. Here, we report that autoimmune B cells, arising from two different B cell-specific genetic lesions in mice, secrete IFN-α. In addition, IFN-α, found in abundance in autoimmunity, elicited profound changes in the B cell lipidome, increasing their expression of glycosphingolipids (GSLs) and leading to their CD1d-mediated depletion of iNKT cells in vitro and in vivo. IFN-α receptor blockade could reverse the loss of iNKT cells. Excessive stimulation of B cells with IFN-α altered the expression of enzymes that catalyze critical steps in GSL processing, increasing the expressions of glucosylceramide synthase (GCS) and globotrihexosylceramide synthase (Gb3S) but decreasing that of α-galactosidase A (α-galA). Inhibiting GCS or restoring α-galA expression prevented iNKT depletion by IFN-α-activated B cells. Taken together, our work indicated that excessive IFN-α perturbs GSL metabolism in B cells which in turn adversely affects iNKT homeostasis.

Lipidomic Profiling of Lung Pleural Effusion Identifies Unique Metabotype for EGFR Mutants in Non-Small Cell Lung Cancer.

Cytology and histology forms the cornerstone for the diagnosis of non-small cell lung cancer (NSCLC) but obtaining sufficient tumour cells or tissue biopsies for these tests remains a challenge. We investigate the lipidome of lung pleural effusion (PE) for unique metabolic signatures to discriminate benign versus malignant PE and EGFR versus non-EGFR malignant subgroups to identify novel diagnostic markers that is independent of tumour cell availability. Using liquid chromatography mass spectrometry, we profiled the lipidomes of the PE of 30 benign and 41 malignant cases with or without EGFR mutation. Unsupervised principal component analysis revealed distinctive differences between the lipidomes of benign and malignant PE as well as between EGFR mutants and non-EGFR mutants. Docosapentaenoic acid and Docosahexaenoic acid gave superior sensitivity and specificity for detecting NSCLC when used singly. Additionally, several 20- and 22- carbon polyunsaturated fatty acids and phospholipid species were significantly elevated in the EGFR mutants compared to non-EGFR mutants. A 7-lipid panel showed great promise in the stratification of EGFR from non-EGFR malignant PE. Our data revealed novel lipid candidate markers in the non-cellular fraction of PE that holds potential to aid the diagnosis of benign, EGFR mutation positive and negative NSCLC.

Aberrant presentation of self-lipids by autoimmune B cells depletes peripheral iNKT cells.

Invariant natural killer T (iNKT) cells provide cognate help via CD1d to lipid antigen-presenting B cells for antibody production, but whether B cells reciprocally regulate iNKT cells remains largely unexplored. Here, we found peripheral, but not thymic, iNKT cells to be numerically reduced in autoimmune mice lacking Fas specifically in B cells. The residual iNKT cells were antigenically overstimulated, had altered cytokine production, and manifested enhanced proliferation and apoptosis. B cell-specific ablation of CD1d ameliorated these iNKT defects, suggesting that inappropriate presentation of CD1d-restricted self-lipids by autoimmune B cell-depleted peripheral iNKT cells. CD1d(+) autoimmune B cells have reduced α-galactosidase A expression and, as revealed by lipidomic profiling, altered lipidome with aberrant accumulation of certain self-lipids and reduction of others. These findings unveil a critical link between autoimmunity, B cell lipidome, and the maintenance of peripheral iNKT cells and highlight an essential homeostatic function of B cells beyond antibody production.