Mitochondrial DNA integrity and metabolome profile are preserved in the human induced pluripotent stem cell reference line KOLF2.1J.

Quality control of human induced pluripotent stem cells (iPSCs) is critical to ensure reproducibility of research. Recently, KOLF2.1J was characterized and published as a male iPSC reference line to study neurological disorders. Emerging evidence suggests potential negative effects of mtDNA mutations, but its integrity was not analyzed in the original publication. To assess mtDNA integrity, we conducted a targeted mtDNA analysis followed by untargeted metabolomics analysis. We found that KOLF2.1J mtDNA integrity was intact at the time of publication and is still preserved in the commercially distributed cell line. In addition, the basal KOLF2.1J metabolome profile was similar to that of the two commercially available iPSC lines IMR90 and iPSC12, but clearly distinct from an in-house-generated ERCC6R683X/R683X iPSC line modeling Cockayne syndrome. Conclusively, we validate KOLF2.1J as a reference iPSC line, and encourage scientists to conduct mtDNA analysis and unbiased metabolomics whenever feasible.

Toward Unified Flavor Quantitation in Cocoa-Based Products.

Because food flavor is perceived through a combination of odor and taste, an analytical method that covers both dimensions would be very beneficial for mapping the consistent product quality over the entirety of a manufacturing process. Such a method, so-called "unified flavor quantitation", has been successfully applied to several different food products in recent years. The simultaneous detection of aroma and taste compounds by means of ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) enables the analysis and quantification of an enormously large number of compounds in a single run. To evaluate the limits of this method, chocolate, a high-fat, complex matrix, was selected. In 38 distinct commercial chocolate samples, 20 flavor-active acids, aldehydes, and sugars were analyzed after a simple, rapid extraction step followed by derivatization with 3-nitrophenylhydrazine using a single UHPLC-MS/MS method. The results obtained highlight the great potential of the "unified flavor quantitation" approach and demonstrate the possibility of high-throughput quantitation of key aroma- and taste-active molecules in a single assay.

High-Throughput Analysis of Underivatized Amino Acids and Acylcarnitines in Infant Serum: A Micromethod Based on Stable Isotope Dilution Targeted HILIC-ESI-MS/MS.

Amino acids and acylcarnitines are important biomarkers of the body's energy state and can be used as diagnostic markers of certain inborn errors of metabolism. Few multianalyte methods for high-throughput analysis in serum exist for these compounds, but micromethods suitable for use in young children and infants are lacking. Therefore, we developed a quantitative high-throughput multianalyte hydrophilic interaction liquid chromatography-tandem mass spectrometry method preceded by a derivatization-free sample preparation using minimum amounts of serum (25 µL). Isotopically labeled standards were utilized for quantification. Forty amino acids and amino acid derivatives and 22 acylcarnitines were detected by applying a multiple reaction monitoring mode within a 20 min run. The method was comprehensively validated, comprising linearity, accuracy, (intraday/interday) precision, and quantitation limits, of which the latter ranged from 0.25 to 50 nM for acylcarnitines and from 0.005 to 1 µM for amino acids and their derivatives. Application of the method to 145 serum samples of three- to four-month-old healthy infants showed excellent reproducibility for multiday analyses and enabled simultaneous amino acid and acylcarnitine profiling in this age group.

Toward High-Throughput Analysis of Aroma Compounds Using Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry: Screening of Key Food Odorants in Various Foods.

Recent studies show the immense capacities of the unified quantitation of aroma and taste compounds using liquid chromatography-mass spectrometry (LC-MS). The goal of this study was to highlight the broad application of this unified method. Thus, a stable isotope dilution analysis quantification method of the most important key food odorants in various food categories by LC-MS was developed. Using the well-known derivatization agent 3-nitrophenylhydrazine for carbonyl derivatization and a newly developed approach for alcohol and thiol derivatization, a method for the quantitation of 20 key food odorants was established. Intraday precision was determined to be ≤26%, and interday precision was between 24 and 31%. Limits of quantitation were determined between 0.014 and 283 µg/kg. The work shows that a wide array of aroma compounds can be analyzed accurately by LC-MS.

Sweetener System Intervention Shifted Neutrophils from Homeostasis to Priming.

BACKGROUND: Non-nutritive sweeteners (NNS) are part of personalized nutrition strategies supporting healthy glycemic control. In contrast, the consumption of non-nutritive sweeteners has been related to person-specific and microbiome-dependent glycemic impairments. Reports on the effects of NNS on our highly individual cellular immune system are sparse. The recent identification of taste receptor expression in a variety of immune cells, however, suggested their immune-modulatory relevance. METHODS: We studied the influence of a beverage-typical NNS system on the transcriptional profiling of sweetener-cognate taste receptors, selected cytokines and their receptors, and on Ca2+ signaling in isolated blood neutrophils. We determined plasma concentrations of saccharin, acesulfame-K, and cyclamate by HPLC-MS/MS, upon ingestion of a soft drink-typical sweetener surrogate. In an open-labeled, randomized intervention study, we determined pre- versus post-intervention transcript levels by RT-qPCR of sweetener-cognate taste receptors and immune factors. RESULTS: Here we show that the consumption of a food-typical sweetener system modulated the gene expression of cognate taste receptors and induced the transcriptional regulation signatures of early homeostasis- and late receptor/signaling- and inflammation-related genes in blood neutrophils, shifting their transcriptional profile from homeostasis to priming. Notably, sweeteners at postprandial plasma concentrations facilitated fMLF (N-formyl-Met-Leu-Phe)-induced Ca2+ signaling. CONCLUSIONS: Our results support the notion of sweeteners priming neutrophils to higher alertness towards their adequate stimuli.

Human Gingival Fibroblasts as a Novel Cell Model Describing the Association between Bitter Taste Thresholds and Interleukin-6 Release.

Human gingival fibroblast cells (HGF-1 cells) present an important cell model to investigate the gingiva's response to inflammatory stimuli such as lipopolysaccharides from Porphyromonas gingivalis (Pg-LPS). Recently, we demonstrated trans-resveratrol to repress the Pg-LPS evoked release of the pro-inflammatory cytokine interleukin-6 (IL-6) via involvement of bitter taste sensing receptor TAS2R50 in HGF-1 cells. Since HGF-1 cells express most of the known 25 TAS2Rs, we hypothesized an association between a compound's bitter taste threshold and its repressing effect on the Pg-LPS evoked IL-6 release by HGF-1 cells. To verify our hypothesis, 11 compounds were selected from the chemical bitter space and subjected to the HGF-1 cell assay, spanning a concentration range between 0.1 µM and 50 mM. In the first set of experiments, the specific role of TAS2R50 was excluded by results from structurally diverse TAS2R agonists and antagonists and by means of a molecular docking approach. In the second set of experiments, the HGF-1 cell response was used to establish a linear association between a compound's effective concentration to repress the Pg-LPS evoked IL-6 release by 25% and its bitter taste threshold concentration published in the literature. The Pearson correlation coefficient revealed for this linear association was R2 = 0.60 (p < 0.01), exceeding respective data for the test compounds from a well-established native cell model, the HGT-1 cells, with R2 = 0.153 (p = 0.263). In conclusion, we provide a predictive model for bitter tasting compounds with a potential to act as anti-inflammatory substances.

Ketogenic diets composed of long-chain and medium-chain fatty acids induce cardiac fibrosis in mice.

PURPOSE: Heart diseases are the leading cause of death worldwide. Metabolic interventions via ketogenic diets (KDs) have been used for decades to treat epilepsy, and more recently, also diabetes and obesity, as common comorbidities of heart diseases. However, recent reports linked KDs, based on long-chain triglycerides (LCTs), to cardiac fibrosis and a reduction of heart function in rodents. As intervention using medium-chain triglycerides (MCTs) was recently shown to be beneficial in murine cardiac reperfusion injury, the question arises as to what extent the fatty acid (FA)-composition in a KD alters molecular markers of FA-oxidation (FAO) and modulates cardiac fibrotic outcome. METHODS: The effects of LCT-KD as well as an LCT/MCT mix (8:1 ketogenic ratio) on cardiac tissue integrity and the plasma metabolome were assessed in adult male C57/BL6NRJ mice after eight weeks on the respective diet. RESULTS: Both KDs resulted in increased amount of collagen fibers and cardiac tissue was immunologically indistinguishable between groups. MCT supplementation resulted in i) profound changes in plasma metabolome, ii) reduced hydroxymethylglutaryl-CoA synthase upregulation, and mitofusin 2 downregulation, iii) abrogation of LCT-induced mitochondrial enlargement, and iv) enhanced FAO profile. Contrary to literature, mitochondrial biogenesis was unaffected by KDs. We propose that the observed tissue remodeling is caused by the accumulation of 4-hydroxy-2-nonenal protein adducts, despite an inconspicuous nuclear factor (erythroid-derived 2)-like 2 pathway. CONCLUSION: We conclude that regardless of the generally favorable effects of MCTs, they cannot inhibit 4-hydroxy-2-nonenal adduct formation and fibrotic tissue formation in this setting. Furthermore, we support the burgeoning concern about the effect of KDs on the cardiac safety profile.

Modeling the Orthosteric Binding Site of the G Protein-Coupled Odorant Receptor OR5K1.

With approximately 400 encoding genes in humans, odorant receptors (ORs) are the largest subfamily of class A G protein-coupled receptors (GPCRs). Despite its high relevance and representation, the odorant-GPCRome is structurally poorly characterized: no experimental structures are available, and the low sequence identity of ORs to experimentally solved GPCRs is a significant challenge for their modeling. Moreover, the receptive range of most ORs is unknown. The odorant receptor OR5K1 was recently and comprehensively characterized in terms of cognate agonists. Here, we report two additional agonists and functional data of the most potent compound on two mutants, L1043.32 and L2556.51. Experimental data was used to guide the investigation of the binding modes of OR5K1 ligands into the orthosteric binding site using structural information from AI-driven modeling, as recently released in the AlphaFold Protein Structure Database, and from homology modeling. Induced-fit docking simulations were used to sample the binding site conformational space for ensemble docking. Mutagenesis data guided side chain residue sampling and model selection. We obtained models that could better rationalize the different activity of active (agonist) versus inactive molecules with respect to starting models and also capture differences in activity related to minor structural differences. Therefore, we provide a model refinement protocol that can be applied to model the orthosteric binding site of ORs as well as that of GPCRs with low sequence identity to available templates.

Evaluation of spray-dried eggs as a micronutrient-rich nutritional supplement.

Regular consumption of hen eggs can help to prevent deficiencies of essential nutrients, such as essential amino acids, vitamin A and E or trace elements zinc and selenium, for vulnerable populations. This study focused on assessing the nutritional value of spray-dried eggs, favored by their manufacturability, storability and ease of addition to (complementary) foods. Using a wide range of analytical techniques, we recorded and compared the nutrient profiles of commercially produced pasteurized whole eggs and their respective powder samples spray-dried at 160°C. Important nutrients that were not significantly affected by spray-drying include total fat content, several amino acids, α- and δ-tocopherol, lutein, zeaxanthin, essential trace elements and cobalamin. The most notable mean losses were found for unsaturated fatty acids, e.g., linoleic (by -38.7%, from 4.11 ± 0.45 to 2.52 ± 0.75 g/100 g DM) and linolenic acid (by -60.8%, from 0.76 ± 0.05 to 0.30 ± 0.04 g/100 g DM). Despite recording significant retinol losses in two out of three batches, the overall low reduction of -14% recommend spray-dried eggs as a valuable source of vitamin A. A daily intake of spray-dried egg powder corresponding to one medium sized egg meets dietary reference values for children, e.g., by 100% for vitamin E, by 24% for retinol, by 61% for selenium and by 22% for zinc. In conclusion, even though a dry weight comparison favors supplementation with pasteurized whole eggs, our results demonstrate a high potential for spray-dried eggs as nutritional supplement. However, the spray-drying process should be optimized toward higher retentions of unsaturated fatty acids and retinol.

A Comparison of the Impact of Restrictive Diets on the Gastrointestinal Tract of Mice.

The rate of gut inflammatory diseases is growing in modern society. Previously, we showed that caloric restriction (CR) shapes gut microbiota composition and diminishes the expression of inflammatory factors along the gastrointestinal (GI) tract. The current project aimed to assess whether prominent dietary restrictive approaches, including intermittent fasting (IF), fasting-mimicking diet (FMD), and ketogenic diet (KD) have a similar effect as CR. We sought to verify which of the restrictive dietary approaches is the most potent and if the molecular pathways responsible for the impact of the diets overlap. We characterized the impact of the diets in the context of several dietary restriction-related parameters, including immune status in the GI tract; microbiota and its metabolites; bile acids (BAs); gut morphology; as well as autophagy-, mitochondria-, and energy restriction-related parameters. The effects of the various diets are very similar, particularly between CR, IF, and FMD. The occurrence of a 50 kDa truncated form of occludin, the composition of the microbiota, and BAs distinguished KD from the other diets. Based on the results, we were able to provide a comprehensive picture of the impact of restrictive diets on the gut, indicating that restrictive protocols aimed at improving gut health may be interchangeable.

Discovery and Identification of Tastants and Taste-Modulating N-Acyl Amino Acid Derivatives in Traditional Korean Fermented Dish Kimchi Using a Sensomics Approach.

Sensory-guided fractionation by means of ultrafiltration and gel permeation chromatography followed by high-performance liquid chromatography, synthesis, liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantitation, and taste re-engineering experiments revealed taste-active and taste-enhancing compounds contributing to the umami, mouthful and complex taste profile of the fermented Korean dish, kimchi. Besides basic taste-active compounds, in particular, various N-acylated amino acids deriving from succinic acid and lactic acid imparted taste-modulating properties in food matrices. Taste threshold concentrations were determined to evaluate intrinsic and modulating effects. Quantitation of N-acylated amino acids in kimchi following synthesis revealed the presence of numerous derivatives showing taste-active properties. Sensory evaluation including recombination and partial addition experiments highlighted that both the N-lactoyl- and the N-succinoyl amino acid derivatives contribute to increasing the fullness, volume, and complexity of food matrices, whereas the latter directly contributes to the overall taste of kimchi in natural concentrations.

Sensoproteomic Discovery of Taste-Modulating Peptides and Taste Re-engineering of Soy Sauce.

Soy sauce, one of the most common Asian fermented foods, exhibits a distinctive savory taste profile. In the present study, targeted quantitation of literature-known taste compounds, calculation of dose-over-threshold factors, and taste re-engineering experiments enabled the identification of 34 key tastants. Following the sensoproteomics approach, 14 umami-, kokumi-, and salt-enhancing peptides were identified for the first time, with intrinsic taste threshold concentrations in the range of 166-939 µmol/L and taste-modulating threshold concentrations ranging from 42 to 420 µmol/L. The lowest taste-modulating threshold concentrations were found for the leucyl peptide LDYY with an umami- and salt-enhancing threshold of 42 µmol/L. Addition of the 14 newly identified peptides to the taste recombinate (aRecDipeptides) increased the overall taste intensity and mouthfulness of the recombinate, and comparison with the authentic soy sauce confirmed the identification of all key tastants. Finally, these data as well as the quantitative profiling of several (non)-fermented foods highlight the importance of fermentation with respect to taste formation. On the basis of this knowledge, microorganisms with specific digestion patterns may be used to tailor the taste profile and especially the salt taste sensation of soy sauces.

High-Throughput Flavor Analysis and Mapping of Flavor Alterations Induced by Different Genotypes of Mentha by Means of UHPLC-MS/MS.

The demand for mint is increasing from year to year, and it is more important than ever to secure a sustainable and robust supply of such an important plant. The USDA mint core collection provides the basis for many researches worldwide regarding, e.g., sequencing, cytology, and disease resistances. A recently developed toolbox enables here for the first time the analysis of such a complex collection in terms of the aroma compound composition and the mapping of flavor alterations depending on taxonomy, environmental conditions, and growing stages by means of comprehensive liquid chromatography tandem mass spectrometry. Therefore, in this study, not only the aroma compound composition of 153 genotypes was characterized but it was also demonstrated that the composition varies depending on taxonomy and changes during the growth of the plant. Furthermore, it could be shown that greenhouse conditions have an enormous influence on the concentrations of aroma compounds.

Classification Model for the Second Extracellular Loop of Class A GPCRs.

The extracellular loop 2 (ECL2) is the longest and the most diverse loop among class A G protein-coupled receptors (GPCRs). It connects the transmembrane (TM) helices 4 and 5 and contains a highly conserved cysteine through which it is bridged with TM3. In this paper, experimental ECL2 structures were analyzed based on their sequences, shapes, and intramolecular contacts. To take into account the flexibility, we incorporated into our analyses information from the molecular dynamics trajectories available on the GPCRmd website. Despite the high sequence variability, shapes of the analyzed structures, defined by the backbone volume overlaps, can be clustered into seven main groups. Conformational differences within the clusters can be then identified by intramolecular interactions with other GPCR structural domains. Overall, our work provides a reorganization of the structural information of the ECL2 of class A GPCR subfamilies, highlighting differences and similarities on sequence and conformation levels.

Overlapping activation pattern of bitter taste receptors affect sensory adaptation and food perception.

The composition of menus and the sequence of foodstuffs consumed during a meal underlies elaborate rules. However, the molecular foundations for the observed taste- and pleasure-raising effects of complex menus are obscure. The molecular identification and characterization of taste receptors can help to gain insight into the complex interrelationships of food items and beverages during meals. In our study, we quantified important bitter compounds in chicory and chicory-based surrogate coffee and used them to identify responsive bitter taste receptors. The two receptors, TAS2R43 and TAS2R46, are exquisitely sensitive to lactucin, lactucopicrin, and 11ß,13-dihydrolactucin. Sensory testing demonstrated a profound influence of the sequence of consumption of chicory, surrogate coffee, and roasted coffee on the perceived bitterness by human volunteers. These findings pave the way for a molecular understanding of some of the mixture effects underlying empirical meal compositions.

Identification and Quantitation of Taste-Active Compounds in Dried Scallops by Combined Application of the Sensomics and a Quantitative NMR Approach.

Application of the sensomics concept on dried scallops, a Japanese specialty produced from the adductor muscle of scallops, revealed after activity-guided fractionation with subsequent (comparative) taste dilution analyses besides nucleotides, amino acids, organic acids, and inorganic ions, the presence of taste-modulating quaternary ammonium compounds and opines in highly taste-active fractions. In order to recreate the taste of dried scallops, two independent quantitation approaches were applied and compared. The first approach used multiple targeted UHPLC-MS/MS and high-performance ion chromatography methods. Besides already established quantitation methods for basic taste compounds, a new HILIC-UHPLC-MS/MSMRM method for the quantitation of chromatographically challenging opines, using synthesized stable isotope-labeled standards, was developed. Furthermore, a qHNMR approach was applied, enabling a direct identification and quantitation of organic taste compounds in a food extract without prior fractionation using a reference 1H NMR database. Both methods yielded similar quantitative results of taste-active compounds in dried scallop extracts and subsequent taste recombination experiments based on these data were able to recreate the taste of dried scallops.

Targeted LC-MS/MS Profiling of Bile Acids in Various Animal Tissues.

Bile acids are being increasingly investigated in humans and laboratory animals as markers for various diseases in addition to their important functions, such as promoting the emulsification in fat digestion and preventing gallstone formation. In humans and animals, primary bile acids are formed from cholesterol in the liver, converted in the intestine into various secondary bile acids by the intestinal microbiota and reabsorbed in the terminal ileum, and partially returned to the liver. A universal high-throughput workflow, including a simple workup, was applied as a tool for bile acid analysis in animal studies. The complex bile acid profiles in various tissues, organs, and body fluids from different animals were mapped using a newly developed comprehensive liquid chromatography-tandem mass spectrometry method. The method can also be used in screening food to obtain information about the nutritional content of bile acids. This could be relevant to investigations on various animal diseases and on the bioavailability of bile acids that pass through the gastric tract.

Systematic Evaluation of Liquid Chromatography (LC) Column Combinations for Application in Two-Dimensional LC Metabolomic Studies.

In comparison to proteomics, the application of two-dimensional liquid chromatography (2D LC) in the field of metabolomics is still premature. One reason might be the elevated chemical complexity and the associated challenge of selecting proper separation conditions in each dimension. As orthogonality of dimensions is a major issue, the present study aimed for the identification of successful stationary phase combinations. To determine the degree of orthogonality, first, six different metrics, namely, Pearson's correlation coefficient (1 - |R|), the nearest-neighbor distances (H̅NND), the "asterisk equations" (AO), and surface coverage by bins (SCG), convex hulls (SCCH), and α-convex hulls (SCαH), were critically assessed by 15 artificial 2D data sets, and a systematic parameter optimization of α-convex hulls was conducted. SGG, SCαH with α = 0.1, and H̅NND generated valid results with sensitivity toward space utilization and data distribution and, therefore, were applied to pairs of experimental retention time sets obtained for >350 metabolites, selected to represent the chemical space of human urine. Normalized retention data were obtained for 23 chromatographic setups, comprising reversed-phase (RP), hydrophilic interaction liquid chromatography (HILIC), and mixed-mode separation systems with an ion exchange (IEX) contribution. As expected, no single LC setting provided separation of all considered analytes, but while conventional RP×HILIC combinations appeared rather complementary than orthogonal, the incorporation of IEX properties into the RP dimension substantially increased the 2D potential. Eventually, one of the most promising column combinations was implemented for an offline 2D LC time-of-flight mass spectrometry analysis of a lyophilized urine sample. Targeted screening resulted in a total of 164 detected metabolites and confirmed the outstanding coverage of the 2D retention space.

Assessing the extent and timing of chemosensory impairments during COVID-19 pandemic.

Chemosensory impairments have been established as a specific indicator of COVID-19. They affect most patients and may persist long past the resolution of respiratory symptoms, representing an unprecedented medical challenge. Since the SARS-CoV-2 pandemic started, we now know much more about smell, taste, and chemesthesis loss associated with COVID-19. However, the temporal dynamics and characteristics of recovery are still unknown. Here, capitalizing on data from the Global Consortium for Chemosensory Research (GCCR) crowdsourced survey, we assessed chemosensory abilities after the resolution of respiratory symptoms in participants diagnosed with COVID-19 during the first wave of the pandemic in Italy. This analysis led to the identification of two patterns of chemosensory recovery, partial and substantial, which were found to be associated with differential age, degrees of chemosensory loss, and regional patterns. Uncovering the self-reported phenomenology of recovery from smell, taste, and chemesthetic disorders is the first, yet essential step, to provide healthcare professionals with the tools to take purposeful and targeted action to address chemosensory disorders and their severe discomfort.

Astringent Gallic Acid in Red Wine Regulates Mechanisms of Gastric Acid Secretion via Activation of Bitter Taste Sensing Receptor TAS2R4.

Red wine is rich in phenolic compounds, which chiefly determine its characteristic taste. One of its major phenolic acid constituents for which an astringency, yet no clear contribution to bitter taste has been reported, is gallic acid (GA). In previous studies, we have demonstrated bitter-tasting constituents to regulate cellular proton secretion (PS) as a key mechanism of gastric acid secretion via activation of bitter taste sensing receptors (TAS2Rs). Here, we hypothesized a contributing role of GA to the red wine-stimulated effect on PS in human gastric tumor cells (HGT-1 cells). Sensory analyses revealed that 10 µM GA as the lowest concentration tested more bitter than tap water, with increasing bitter ratings up to 1000 µM. In HGT-1 cells, the concentration of 10 µM GA evoked the most pronounced effect on PS secretion, either when added to cells as in-water solution or when spiked to a red wine matrix. GA-spiking of Zweigelt and Blaufränkisch red wine samples up to a concentration of 10 µM resulted in an equally stimulated PS, whereas the non-GA-spiked wine samples demonstrated contrary effects on PS, indicating a functional role of GA on PS. Involvement of TAS2R4 in the GA-induced PS was verified by means of an HGT-1 homozygote CRISPR-Cas9 TAS2R4 knockout approach. Moreover, gene expression analyses revealed GA to increase TAS2R4. These results demonstrate a functional role of TAS2R4 in GA-evoked PS as a key mechanism of gastric acid secretion aiding digestion. Moreover, our data provide mechanistic insights, which will help to produce stomach-friendly red wines.