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Introduction: The lens's metabolic demands are met through a continuous circulation of aqueous humor, encompassing a spectrum of components such as organic and inorganic ions, carbohydrates, glutathione, urea, amino acids, proteins, oxygen, carbon dioxide, and water. Metabolomics is a pivotal tool, offering an initial insight into the complexities of integrated metabolism. In this investigative study, we systematically scrutinize the composition of intraocular fluid in individuals afflicted with cataracts. Methods: The investigation involved a comprehensive analysis of aqueous humor samples from a cohort comprising 192 patients. These individuals were stratified by utilizing the SPONCS classification system, delineating distinct groups characterized by the hardness of cataracts. The analytical approach employed targeted quantitative metabolite analysis using HILIC-based liquid chromatography coupled with high-resolution mass spectrometric detection. The metabolomics data analysis was performed with MetaboAnalyst 5.0. Results: The results of the enrichment analysis have facilitated the inference that the discerned disparities among groups arise from disruptions in taurine and hypotaurine metabolism, variations in tryptophan metabolism, and modifications in mitochondrial beta-oxidation of short-chain saturated fatty acids and pyrimidine metabolism. Conclusion: A decline in taurine concentration precipitates diminished glutathione activity, prompting an elevated requirement for NAD+ and instigating tryptophan metabolism along the kynurenine pathway. Activation of this pathway is additionally prompted by interferon-gamma and UV radiation, leading to the induction of IDO. Concurrently, heightened mitochondrial beta-oxidation signifies a distinctive scenario in translocating fatty acids into the mitochondria, enhancing energy production. [ABSTRACT FROM AUTHOR]

Introduction: Hereditary angioedema (HAE) is a rare, life-threatening autosomal dominant genetic disorder caused by a deficient and/or dysfunctional C1 esterase inhibitor (C1-INH) (type 1 and type 2) leading to recurrent episodes of edema. This study aims to explore HAE patients’ metabolomic profiles and identify novel potential diagnostic biomarkers for HAE. The study also examined distinguishing HAE from idiopathic angioedema (AE). Methods: Blood plasma samples from 10 HAE (types 1/2) patients, 15 patients with idiopathic AE, and 20 healthy controls were collected in Latvia and analyzed using LC-MS based targeted metabolomics workflow. T-test and fold change calculation were used to identify metabolites with significant differences between diseases and control groups. ROC analysis was performed to evaluate metabolite based classification model. Results: A total of 33 metabolites were detected and quantified. The results showed that isovalerylcarnitine, cystine, and hydroxyproline were the most significantly altered metabolites between the disease and control groups. Aspartic acid was identified as a significant metabolite that could differentiate between HAE and idiopathic AE. The mathematical combination of metabolites (hydroxyproline * cystine)/(creatinine * isovalerylcarnitine) was identified as the diagnosis signature for HAE. Furthermore, glycine/asparagine ratio could differentiate between HAE and idiopathic AE. Conclusion: Our study identified isovalerylcarnitine, cystine, and hydroxyproline as potential biomarkers for HAE diagnosis. Identifying new biomarkers may offer enhanced prospects for accurate, timely, and economical diagnosis of HAE, as well as tailored treatment selection for optimal patient care. [ABSTRACT FROM AUTHOR]

The musculoskeletal (MS) system consists of bone, cartilage, tendon, ligament, and skeletal muscle, which forms the basic framework of the human body. This system plays a vital role in appropriate body functions, including movement, the protection of internal organs, support, hematopoiesis, and postural stability. Therefore, it is understandable that the damage or loss of MS tissues significantly reduces the quality of life and limits mobility. Tissue engineering and its applications in the healthcare industry have been rapidly growing over the past few decades. Tissue engineering has made significant contributions toward developing new therapeutic strategies for the treatment of MS defects and relevant disease. Among various biomaterials used for tissue engineering, natural polymers offer superior properties that promote optimal cell interaction and desired biological function. Natural polymers have similarity with the native ECM, including enzymatic degradation, bio-resorb and non-toxic degradation products, ability to conjugate with various agents, and high chemical versatility, biocompatibility, and bioactivity that promote optimal cell interaction and desired biological functions. This review summarizes recent advances in applying natural-based scaffolds for musculoskeletal tissue engineering. [ABSTRACT FROM AUTHOR]

Perturbations of metabolite profiles in human and canine enteropathies have been reported before. However, data in dogs are scarce and inconsistent. Currently, the metabolite profile in Yorkshire Terrier enteropathy (YTE) and the impact of treatment is unknown. The objective of this study was to investigate the plasma metabolome of 13 Yorkshire Terriers with YTE and compare it to 20 healthy Yorkshire Terriers. Furthermore, we studied the impact of treatment on the metabolome. In this prospective observational study, plasma metabolite profiles were analyzed by flow injection analysis-tandem mass spectrometry (FIA-MS/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a targeted metabolomics kit. Metabolite analysis revealed that YTE is accompanied by changes in lipid and bile acid metabolism. YTE was associated with a significant decrease of long-chain fatty acids (octadecenoic acid, eicosadienoic acid, eicosatrienoic acid) and lower levels of long-chain acylcarnitines (tetradecanoylcarnitine, hexadecanoylcarnitine, hexadecenoylcarnitine, octadecenoylcarnitine) compared with healthy controls. Furthermore, taurodeoxycholic acid, a secondary bile acid, was decreased in plasma from YTE patients. These changes might be breed-specific and might be involved in the pathogenesis of YTE. Interestingly, changes in metabolite levels were not recovered after treatment and differed considerably from healthy controls. [ABSTRACT FROM AUTHOR]

The number of mast cells in various organs is elevated manifold in individuals with systemic mastocytosis. Degranulation can lead to life-threatening symptomatology. No data about the alterations of the metabolome and lipidome during an attack have been published. Our aim was to analyze changes in metabolomics and lipidomics during the acute phase of a severe mast cell activation event. A total of 43 metabolites and 11 lipid classes comprising 200 subvariants from multiple plasma samples in duplicate, covering 72 hours of a severe mast cell activation attack with nausea and vomiting, were compared with 2 baseline samples by using quantitative liquid chromatography–mass spectrometry. A strong enterocyte dysfunction reflected in an almost 20-fold reduction in the functional small bowel length was extrapolated from strongly reduced ornithine and citrulline concentrations and was very likely secondary to severe endothelial cell dysfunction with hypoperfusion and extensive vascular leakage. Highly increased histamine and lactate concentrations accompanied the peak in clinical symptoms. Elevated asymmetric and symmetric dimethylarginine levels combined with reduced arginine levels compromised endothelial nitric oxide synthase activity and nitric oxide signaling. Specific and extensive depletion of many lysophosphatidylcholine variants indicates localized autotaxin activation and lysophosphatidic acid release. A strong correlation of clinical parameters with histamine concentrations and symptom reduction after 100-fold elevated plasma diamine oxidase concentrations implies that histamine is the key driver of the acute phase. Rapid elimination of elevated histamine concentrations through use of recombinant human diamine oxidase, supplementation of lysophosphatidylcholine for immunomodulation, inhibition of autotaxin activity, and/or blockade of lysophosphatidic acid receptors might represent new treatment options for life-threatening mast cell activation events. [ABSTRACT FROM AUTHOR]

The companion dog has recently been promoted as powerful translational model of aging. However, while dogs share environments with their human owners and develop many of the same age-related morbidities, little is known about the underlying mechanisms that drive their health and longevity. In addition, dogs have a well described phenotypic pattern in which small dogs live significantly longer than large dogs, such that weight can be used as a crude proxy for longevity. To investigate this pattern, we completed a small lipidomics study on 41 dogs in the Birmingham, Alabama, United States, area to determine individual circulating lipids that were associated with age and body weight. We discovered that sphingomyelins were significantly higher in large, short-lived dogs, independent of age, and triglycerides were higher in older dogs of all sizes. Our results point towards physiological differences that may explain a portion of the variation in longevity seen in companion dogs. [ABSTRACT FROM AUTHOR]

The liver is a central regulator of metabolic homeostasis and serum metabolite levels. Hepatocytes are the functional units of the liver parenchyma and not only responsible for turnover of biomolecules but also act as central immune signaling platforms. Hepatotropic viruses infect liver tissue, resulting in inflammatory responses, tissue damage and hepatitis. Combining well-established in vitro and in vivo model systems with transcriptomic analyses, we show that type I interferon signaling initiates a robust antiviral immune response in hepatocytes. Strikingly, we also identify IFN-I as both, sufficient and necessary, to induce wide-spread metabolic reprogramming in hepatocytes. IFN-I specifically rewired tryptophan metabolism and induced hepatic tryptophan oxidation to kynurenine via Tdo2, correlating with altered concentrations of serum metabolites upon viral infection. Infected Tdo2-deficient animals displayed elevated serum levels of tryptophan and, unexpectedly, also vast increases in the downstream immune-suppressive metabolite kynurenine. Thus, Tdo2-deficiency did not result in altered serum homeostasis of the tryptophan to kynurenine ratio during infection, which seemed to be independent of hepatocyte-intrinsic compensation via the IDO-axis. These data highlight that inflammation-induced reprogramming of systemic tryptophan metabolism is tightly regulated in viral hepatitis. Author summary: Viral hepatitis is responsible for more than one million annual deaths worldwide and may progress to liver cirrhosis and hepatocellular carcinoma. The main metabolic cell type of the liver is the hepatocyte. In viral hepatitis, type I interferon (IFN-I) signaling rewires hepatocyte metabolism and serum metabolites to shape disease pathophysiology–an immune-regulatory circuit that might be therapeutically exploited. Here, we show that hepatocyte-intrinsic antiviral IFN-I signaling is both necessary and sufficient to induce wide-spread metabolic changes in hepatocytes. We identify an IFN-I-mediated induction of the hepatic kynurenine pathway via the rate-limiting and liver-specific enzyme TDO2, which controls serum homeostasis of tryptophan by converting it into kynurenine. Loss of TDO2 triggers a so far unknown compensatory mechanism, resulting in a vast increase of circulating kynurenine independent of hepatocyte intrinsic activity of the related IDO-enzymes. This study provides new insights into how inflammation reprograms metabolism of the liver and the kynurenine pathway during viral hepatitis. [ABSTRACT FROM AUTHOR]

Antiquitin (ATQ) deficiency leads to tissue, plasma, and urinary accumulation of alpha‐aminoadipic semialdehyde (AASA) and its Schiff base delta‐1‐piperideine‐6‐carboxylate (P6C). Although genetic testing of ALDH7A1 is the most definitive diagnostic method, quantifications of pathognomonic metabolites are important for the diagnosis and evaluation of therapeutic and dietary interventions. Current metabolite quantification methods use laborious, technically highly complex, and expensive liquid chromatography‐tandem mass spectro‐metry, which is available only in selected laboratories worldwide. Incubation of ortho‐aminobenzaldehyde (oABA) with P6C leads to the formation of a triple aromatic ring structure with characteristic absorption and fluorescence properties. The mean concentration of P6C in nine urine samples from seven ATQ‐deficient patients under standard treatment protocols was statistically highly significantly different (P <.001) compared to the mean of 74 healthy controls aged between 2 months and 57 years. Using this limited data set the specificity and sensitivity is 100% for all tested age groups using a P6C cut‐off of 2.11 μmol/mmol creatinine, which represents the 99% prediction interval of the P6C concentrations in 17 control urine samples from children below 6 years of age. Plasma P6C concentrations were only elevated in one ATQ subject, possibly because P6C is trapped by pyridoxal‐5‐phosphate (PLP) blocking fusing with oABA. Nevertheless, both urine and plasma samples were amenable to the quantification of exogenous P6C with high response rates. The P6C quantification method using fusion of oABA with P6C is fast, simple, and inexpensive and might be readily implemented into routine clinical diagnostic laboratories for the early diagnosis of neonatal pyridoxine‐dependent epilepsy. [ABSTRACT FROM AUTHOR]

Hyperlipidemia is a risk factor for initiation and progression of diabetic nephropathy but the metabolic pathways altered in the diabetic kidney in a context of hyperlipidemia remain incompletely described. Assuming that changes in urine composition reflect the alteration of renal metabolism and function, we analyzed the urine metabolite composition of diabetic (streptozotocin-treatment) and control (non diabetic) ApoE−/− mice fed a high cholesterol diet using targeted quantitative metabolomics. Urine metabolome was also compared to the plasma metabolome of the same animals. As previously shown, urine albuminuria/urine creatinine ratio (uACR) and glomerular area and plasma lipids (cholesterol, triglycerides) were more elevated in diabetic mice compared to control. After adjustment to urine creatinine, the abundance of 52 urine metabolites was significantly different in diabetic mice compared to control. Among them was a unique metabolite, C14:2-OH (3-hydroxytetradecadienoylcarnitine) that, in diabetic mice, was positively and significantly correlated with uACR, glomerular hypertrophy, blood glucose and plasma lipids. That metabolite was not detected in plasma. C14:2-OH is a long-chain acylcarnitine reminiscent of altered fatty acid beta oxidation. Other acylcarnitines, particularly the short chains C3-OH, C3-DC, C4:1, C5-DC, C5-M-DC, C5-OH that are reminiscent of altered oxidation of branched and aromatic amino acids were also exclusively detected in urine but were only correlated with plasma lipids. Finally, the renal gene expression of several enzymes involved in fatty acid and/or amino acid oxidation was significantly reduced in diabetic mice compared to control. This included the bifunctional enoyl-CoA hydratase/3-hydroxyacyl-CoA (Ehhadh) that might play a central role in C14:2-OH production. This study indicate that the development of diabetes in a context of hyperlipidemia is associated with a reduced capacity of kidney to oxidize fatty acids and amino acids with the consequence of an elevation of urinary acetylcarnitines including C14:2-OH that specifically reflects diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Objective: Our basic understanding of ascending thoracic aortic aneurysm (ATAA) pathogenesis is still very limited, hampering early diagnosis, risk prediction, and development of treatment options. “Omics”-technologies, ideal to reveal tissue alterations from the normal physiological state due to disease have hardly been applied in the field. Using a metabolomic approach, with this study the authors seek to define tissue differences between controls and various forms of ATAAs. Methods: Using a targeted FIA-MS/MS metabolomics approach, we analysed and compared the metabolic profiles of ascending thoracic aortic wall tissue of age-matched controls (n = 8), bicuspid aortic valve-associated aneurysms (BAV-A; n = 9), tricuspid aortic valve-associated aneurysms (TAV-A; n = 14), and tricuspid aortic valve-associated aortic dissections (TAV-Diss; n = 6). Results: With sphingomyelin (SM) (OH) C22:2, SM C18:1, SM C22:1, and SM C24:1 only 4 out of 92 detectable metabolites differed significantly between controls and BAV-A samples. Between controls and TAV-Diss samples only phosphatidylcholine (PC) ae C32:1 differed. Importantly, our analyses revealed a general increase in the amount of total sphingomyelin levels in BAV-A and TAV-Diss samples compared to controls. Conclusions: Significantly increased levels of sphingomyelins in BAV-A and TAV-Diss samples compared to controls may argue for a repression of sphingomyelinase activity and the sphingomyelinase-ceramide pathway, which may result in an inhibition of tissue regeneration; a potential basis for disease initiation and progression. [ABSTRACT FROM AUTHOR]

Introduction Recently, quantitative metabolomics identified a panel of 10 plasma lipids that were highly predictive of conversion to Alzheimer's disease (AD) in cognitively normal older individuals (n = 28, area under the curve [AUC] = 0.92, sensitivity/specificity of 90%/90%). Methods Quantitative targeted metabolomics in serum using an identical method as in the index study. Results We failed to replicate these findings in a substantially larger study from two independent cohorts—the Baltimore Longitudinal Study of Aging ([BLSA], n = 93, AUC = 0.642, sensitivity/specificity of 51.6%/65.7%) and the Age, Gene/Environment Susceptibility-Reykjavik Study ([AGES-RS], n = 100, AUC = 0.395, sensitivity/specificity of 47.0%/36.0%). In analyses applying machine learning methods to all 187 metabolite concentrations assayed, we find a modest signal in the BLSA with distinct metabolites associated with the preclinical and symptomatic stages of AD, whereas the same methods gave poor classification accuracies in the AGES-RS samples. Discussion We believe that ours is the largest blood biomarker study of preclinical AD to date. These findings underscore the importance of large-scale independent validation of index findings from biomarker studies with relatively small sample sizes. [ABSTRACT FROM AUTHOR]

Background: Some yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source. Among them, Pichia pastoris (syn. Komagataella sp.) is frequently used for the production of heterologous proteins and also serves as a model organism for organelle research. Our current knowledge of methylotrophic lifestyle mainly derives from sophisticated biochemical studies which identified many key methanol utilization enzymes such as alcohol oxidase and dihydroxyacetone synthase and their localization to the peroxisomes. C1 assimilation is supposed to involve the pentose phosphate pathway, but details of these reactions are not known to date. Results: In this work we analyzed the regulation patterns of 5,354 genes, 575 proteins, 141 metabolites, and fluxes through 39 reactions of P. pastoris comparing growth on glucose and on a methanol/glycerol mixed medium, respectively. Contrary to previous assumptions, we found that the entire methanol assimilation pathway is localized to peroxisomes rather than employing part of the cytosolic pentose phosphate pathway for xylulose-5-phosphate regeneration. For this purpose, P. pastoris (and presumably also other methylotrophic yeasts) have evolved a duplicated methanol inducible enzyme set targeted to peroxisomes. This compartmentalized cyclic C1 assimilation process termed xylose-monophosphate cycle resembles the principle of the Calvin cycle and uses sedoheptulose- 1,7-bisphosphate as intermediate. The strong induction of alcohol oxidase, dihydroxyacetone synthase, formaldehyde and formate dehydrogenase, and catalase leads to high demand of their cofactors riboflavin, thiamine, nicotinamide, and heme, respectively, which is reflected in strong up-regulation of the respective synthesis pathways on methanol. Methanol-grown cells have a higher protein but lower free amino acid content, which can be attributed to the high drain towards methanol metabolic enzymes and their cofactors. In context with up-regulation of many amino acid biosynthesis genes or proteins, this visualizes an increased flux towards amino acid and protein synthesis which is reflected also in increased levels of transcripts and/or proteins related to ribosome biogenesis and translation. Conclusions: Taken together, our work illustrates how concerted interpretation of multiple levels of systems biology data can contribute to elucidation of yet unknown cellular pathways and revolutionize our understanding of cellular biology. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a severe and chronic neurodegenerative disorder of the brain. The laboratory diagnosis is limited to the analysis of three biomarkers in cerebrospinal fluid (CSF): amyloid-β42 (Aβ42), total tau, and phospho-tau-181 (P-tau-181). However, there is a need to find more biomarkers in CSF that can improve the sensitivity and specificity. The aim of the present study was to analyze endogenous small metabolites (metabolome) in the CSF, which may provide potentially new insights into biochemical processes involved in AD. One hundred CSF samples were dichotomized by normal (n = 50) and pathological decreased Aβ42 and increased tau and P-tau-181 levels (n = 50; correlating to an AD-like pathology). These CSF samples were analyzed using the AbsoluteIDQ® p180 Kit (BIOCRATES Life Sciences), which included 40 acylcarnitines, 21 amino acids, 19 biogenic amines, 15 sphingolipids, and 90 glycerophospholipids. Our data show that two sphingomyelins (SM (d18:1/18:0) and SM (d18:1/18:1)), 5 glycerophospholipids (PC aa C32:0, PC aa C34:1, PC aa C36:1, PC aa C38:4 and PC aa C38:6), and 1 acylcarnitine (C3-DC-M/C5-OH) were significantly altered in the CSF with pathological 'AD-like pathology'. Sphingomyelin SM (d18:1/18:0) proved to be a specific (76%) and sensitive (66%) biomarker with a defined cut-off of 546 nM. Correct diagnoses for 21 out of 32 unknown samples could be achieved using this SM (d18:1/18:0) cut-off value. In conclusion, the sphingolipid SM (d18:1/18:0) is significantly increased in CSF of patients displaying pathological levels of Aβ42, tau, and P-tau-181. [ABSTRACT FROM AUTHOR]

In this work, a fully automated parallel LC column method was established in order to perform orthogonal hydrophilic interaction chromatography (HILIC) and reversed-phase (RPLC) chromatography within one analytical run for targeted quantitative mass spectrometric determination of metabolites from central carbon metabolism. In tins way, the analytical throughput could be significantly improved compared to previously established dual separation work flows involving two separate analytical runs. Two sample aliquots were simultaneously injected onto a dual column setup columns using a ten-port valve, and parallel separations were carried out. Sub 2 μm particle size stationary phases were employed for both separation methods. HILIC and RPLC eluents were combined post column followed by ESI-MS/MS detection. The orthogonal separations were optimized, aiming at an overall separation with 2 retention time segments, while reversed-phase separation was accomplished within 5.5 min; metabolites on the HILIC phase were retained for a minimum time of 6 min. The overall run time was 15 min. The setup was applied to the quantification of 30 primary intercellular metabolites, including amino acids, organic acids, and nucleotides employing internal standardization by a fully 13C-labeled yeast extract. The comparison with HILIC-MS/MS and RPLC-MS/MS in separate analytical runs revealed that an excellent analytical performance was achieved by the parallel LC column method. The experimental repeatability (N - 5) was on average <5% (only for 2 compounds >5%). Moreover, limits of detection for the new approach ranging from 0.002-15 μM were in a good agreement with ones obtained in separate HILIC- MS/MS and RPLC-MS/MS runs (ranging from 0.01-44 μM). [ABSTRACT FROM AUTHOR]

Anion exchange chromatography combined with inductively coupled plasma mass spectrometry was introduced and optimized for the separation and accurate quantification of sugar phosphates in cell extracts. Sugar phosphates have been separated on an anion exchange quaternary ammonium functionalized stationary phase (Dionex CarboPAC PA1) via gradient elution with 160 mM Na2CO3 and 50 mM NaOH. After separation, the sugar phosphates were on-line transferred to an inductively coupled plasma mass spectrometer after having passed an anion self-regenerating suppressor. Detection was performed via phosphorus as PO+ (m/z 47) was generated by the dynamic reaction cell technique using oxygen as a reaction gas. The ionization suppression process in the inductively coupled plasma caused by the high buffer strength of the chromatographic gradient was assessed by post-column flow injection analysis. It could be shown that the anion self-regenerating suppressor led to a significant reduction of signal suppression. With the new methodology excellent relative standard deviations of retention times of sugar phosphates for short- and long-term measurements were achieved. The relative standard deviations of short-term and long-term repeatability of peak areas were below 3.0 and 10.0% respectively. The absolute on-column LODs and LOQs of the optimized method were in the sub- picomole range. The developed method was applied for the purity assessment of commercially available sugar phosphate standards and evaluated for the quantification of sugar phosphates in yeast samples (Pichia pastoris) after extraction with boiling ethanol. Due to the lack of reference materials, the method accuracy was validated by method inter-comparison with an LCxLC-ESI-MS/MS based method, which has been developed in our laboratory. [ABSTRACT FROM AUTHOR]

A mass spectrometric quantitative assay was developed for the analysis of 10 sugar phosphates in the yeast Pichia pastoris. As a novelty, two-dimensional chromatography based on a fully automated heart-cutting LC-LC technique was introduced. Using a ten-port valve, ten fractions of the first chromatographic dimension, i.e. anion exchange chromatography (AEC), were transferred and separated by the orthogonal second dimension, i.e. separation on porous graphitized carbon. The chromatographic separation on the second dimension was optimized for each transferred fraction minimizing the separation time and ensuring complete removal of the salt constituents of the AEC eluents. The latter being crucial for electrospray mass spectrometric detection was confirmed by combining the LC-LC separation with on-line ICP-MS detection. These measurements showed that sodium elution was completed after 0.8 min. Consequently, an analysis time of 1 min per transferred peak was established. In this way, the excellent peak capacity given by ion exchange could be conserved in the second dimension at the same time enabling mass spectrometric detection. Sub-μM limits of detection could be obtained by the new LC-LC-MS/MS methods ranging between 0.03 and 0.19 μM for the investigated compounds (only 3GAP showed a LOD of 1 μM). The method was applied to the quantification of ten sugar phosphates in yeast extracts utilizing internal standardization with a fully labeled 13C yeast extract. Typically, the standard uncertainties for N = 3 replicates assessed by the LC-LC-MS/MS set-up were <5%. [ABSTRACT FROM AUTHOR]

Quantitative metabolic profiling is preceded by dedicated sample preparation protocols. These multistep procedures require detailed optimization and thorough validation. In this work, a uniformly 13 C-labeled ( U13 C) cell extract was used as a tool to evaluate the recoveries and repeatability precisions of the cell extraction and the extract treatment. A homogenous set of biological replicates ( n = 15 samples of Pichia pastoris) was prepared for these fundamental experiments. A range of less than 30 intracellular metabolites, comprising amino acids, nucleotides, and organic acids were measured both in monoisotopic 12 C and U13 C form by LC- MS/ MS employing triple quadrupole MS, reversed phase chromatography, and HILIC. Recoveries of the sample preparation procedure ranging from 60 to 100% and repeatability precisions below 10% were obtained for most of the investigated metabolites using internal standardization approaches. Uncertainty budget calculations revealed that for this complex quantification task, in the optimum case, total combined uncertainty of 12% could be achieved. The optimum case would be represented by metabolites, easy to extract from yeast with high and precise recovery. In other cases the total combined uncertainty was significantly higher. [ABSTRACT FROM AUTHOR]

Abstract: Lunasin is a novel, cancer-preventive, anti-inflammatory and cholesterol-reducing peptide that was originally isolated from soy and later from barley, wheat and rye. We report the first discovery of lunasin in triticale (X Triticosecale Wittmack). Moreover, we report first data of lunasin content in winter rye and wheat genotypes grown in Northern Europe. These data are novel as previously published data on finding of lunasin in cereals were obtained in genotypes grown in Korea. Lunasin content was uncovered using a previously published procedure for isolation from cereals and identified by LC-MS/MS assay. We found that triticale was the most lunasin-rich cereal, with the tested genotypes displaying the following trend in lunasin content: genotype 0002-26 > Dinaro > DSGU 10/94 > 0213-22 > 0317-14 > 0006-31. The greatest lunasin content was 6.46 mg/g in the grain of triticale genotype 0002-26. In comparison, the highest lunasin content in rye variety Dankovske Diament was 1.5 mg/g, and the highest lunasin content in the winter wheat variety Fredis was 0.23 mg/g. We conclude that triticale can play a significant role as functional food, with great potential for the use of triticale products in human and animal diets. [Copyright &y& Elsevier]

Irritable bowel syndrome (IBS) and inflammatory bowel diseases result in a substantial reduction in quality of life and a considerable socioeconomic impact. In IBS, diagnosis and treatment options are limited, but evidence for involvement of the gut microbiome in disease pathophysiology is emerging. Here we analyzed the prevalence of endoscopically visible mucosal biofilms in gastrointestinal disease and associated changes in microbiome composition and metabolism. The presence of mucosal biofilms was assessed in 1426 patients at 2 European university-based endoscopy centers. One-hundred and seventeen patients were selected for in-depth molecular and microscopic analysis using 16S ribosomal RNA gene amplicon-sequencing of colonic biopsies and fecal samples, confocal microscopy with deep learning–based image analysis, scanning electron microscopy, metabolomics, and in vitro biofilm formation assays. Biofilms were present in 57% of patients with IBS and 34% of patients with ulcerative colitis compared with 6% of controls (P <.001). These yellow-green adherent layers of the ileum and right-sided colon were microscopically confirmed to be dense bacterial biofilms. 16S-sequencing links the presence of biofilms to a dysbiotic gut microbiome, including overgrowth of Escherichia coli and Ruminococcus gnavus. R. gnavus isolates cultivated from patient biofilms also formed biofilms in vitro. Metabolomic analysis found an accumulation of bile acids within biofilms that correlated with fecal bile acid excretion, linking this phenotype with a mechanism of diarrhea. The presence of mucosal biofilms is an endoscopic feature in a subgroup of IBS and ulcerative colitis with disrupted bile acid metabolism and bacterial dysbiosis. They provide novel insight into the pathophysiology of IBS and ulcerative colitis, illustrating that biofilm can be seen as a tipping point in the development of dysbiosis and disease. [Display omitted] Bacterial biofilms were observed by colonoscopy as yellow-green membranous layers on the mucosa of the small and large intestinal junction and are specifically prevalent in irritable bowel syndrome and inflammatory bowel disease. [ABSTRACT FROM AUTHOR]

Identifying the changes in endogenous metabolites in response to intrinsic and extrinsic factors has excellent potential to obtain an understanding of cells, biofluids, tissues, or organisms' functions and interactions with the environment. The advantages provided by the metabolomics strategy have promoted studies in bone research fields, including an understanding of bone cell behaviors, diagnosis and prognosis of diseases, and the development of treatment methods such as implanted biomaterials. This review article summarizes the metabolism changes during osteogenesis, osteoclastogenesis, and immunoregulation in hard tissue. The second section of this review is dedicated to describing and discussing metabolite changes in the most relevant bone diseases: osteoporosis, bone injuries, rheumatoid arthritis, and osteosarcoma. We consolidated the most recent finding of the metabolites and metabolite pathways affected by various bone disorders. This collection can serve as a basis for future metabolomics-driven bone research studies to select the most relevant metabolites and metabolic pathways. Additionally, we summarize recent metabolic studies on metabolomics for the development of bone disease treatment including biomaterials for bone engineering. With this article, we aim to provide a comprehensive summary of metabolomics in bone research, which can be helpful for interdisciplinary researchers, including material engineers, biologists, and clinicians. [ABSTRACT FROM AUTHOR]

Objectives: Bone remodelling is a highly dynamic process dependent on the precise coordination of osteoblasts and haematopoietic-cell derived osteoclasts. Changes in core metabolic pathways during osteoclastogenesis, however, are largely unexplored and it is unknown whether and how these processes are involved in bone homeostasis., Methods: We metabolically and transcriptionally profiled cells during osteoclast and osteoblast generation. Individual gene expression was characterised by quantitative PCR and western blot. Osteoblast function was assessed by Alizarin red staining. immunoresponsive gene 1 ( Irg1 ) - deficient mice were used in various inflammatory or non-inflammatory models of bone loss. Tissue gene expression was analysed by RNA in situ hybridisation., Results: We show that during differentiation preosteoclasts rearrange their tricarboxylic acid cycle, a process crucially depending on both glucose and glutamine. This rearrangement is characterised by the induction of Irg1 and production of itaconate, which accumulates intracellularly and extracellularly. While the IRG1-itaconate axis is dispensable for osteoclast generation in vitro and in vivo, we demonstrate that itaconate stimulates osteoblasts by accelerating osteogenic differentiation in both human and murine cells. This enhanced osteogenic differentiation is accompanied by reduced proliferation and altered metabolism. Additionally, supplementation of itaconate increases bone formation by boosting osteoblast activity in mice. Conversely, Irg1- deficient mice exhibit decreased bone mass and have reduced osteoproliferative lesions in experimental arthritis., Conclusion: In summary, we identify itaconate, generated as a result of the metabolic rewiring during osteoclast differentiation, as a previously unrecognised regulator of osteoblasts., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Metformin is widely used for treating type 2 diabetes mellitus (T2D). However, the efficacy of metformin monotherapy is highly variable within the human population. Understanding the potential indirect or synergistic effects of metformin on gut microbiota composition and encoded functions could potentially offer new insights into predicting treatment efficacy and designing more personalized treatments in the future. We combined targeted metabolomics and metagenomic profiling of gut microbiomes in newly diagnosed T2D patients before and after metformin therapy to identify potential pre-treatment biomarkers and functional signatures for metformin efficacy and induced changes in metformin therapy responders. Our sequencing data were largely corroborated by our metabolic profiling and identified that pre-treatment enrichment of gut microbial functions encoding purine degradation and glutamate biosynthesis was associated with good therapy response. Furthermore, we identified changes in glutamine-associated amino acid (arginine, ornithine, putrescine) metabolism that characterize differences in metformin efficacy before and after the therapy. Moreover, metformin Responders' microbiota displayed a shifted balance between bacterial lipidA synthesis and degradation as well as alterations in glutamate-dependent metabolism of N-acetyl-galactosamine and its derivatives (e.g. CMP-pseudaminate) which suggest potential modulation of bacterial cell walls and human gut barrier, thus mediating changes in microbiome composition. Together, our data suggest that glutamine and associated amino acid metabolism as well as purine degradation products may potentially condition metformin activity via its multiple effects on microbiome functional composition and therefore serve as important biomarkers for predicting metformin efficacy.

Rationale: Lung transplantation is the ultimate treatment option for patients with end-stage respiratory diseases but bears the highest mortality rate among all solid organ transplantations due to chronic lung allograft dysfunction (CLAD). The mechanisms leading to CLAD remain elusive due to an insufficient understanding of the complex post-transplant adaptation processes., Objectives: To better understand these lung adaptation processes after transplantation and to investigate their association with future changes in allograft function., Methods: We performed an exploratory cohort study of bronchoalveolar lavage samples from 78 lung recipients and donors. We analysed the alveolar microbiome using 16S rRNA sequencing, the cellular composition using flow cytometry, as well as metabolome and lipidome profiling., Measurements and Main Results: We established distinct temporal dynamics for each of the analysed data sets. Comparing matched donor and recipient samples, we revealed that recipient-specific as well as environmental factors, rather than the donor microbiome, shape the long-term lung microbiome. We further discovered that the abundance of certain bacterial strains correlated with underlying lung diseases even after transplantation. A decline in forced expiratory volume during the first second (FEV 1 ) is a major characteristic of lung allograft dysfunction in transplant recipients. By using a machine learning approach, we could accurately predict future changes in FEV 1 from our multi-omics data, whereby microbial profiles showed a particularly high predictive power., Conclusion: Bronchoalveolar microbiome, cellular composition, metabolome and lipidome show specific temporal dynamics after lung transplantation. The lung microbiome can predict future changes in lung function with high precision., Competing Interests: Conflict of interest: M.L. Watzenböck has nothing to disclose. Conflict of interest: A.-D. Gorki has nothing to disclose. Conflict of interest: F. Quattrone has nothing to disclose. Conflict of interest: R. Gawish has nothing to disclose. Conflict of interest: S. Schwarz has nothing to disclose. Conflict of interest: C. Lambers has nothing to disclose. Conflict of interest: P. Jaksch has nothing to disclose. Conflict of interest: K. Lakovits has nothing to disclose. Conflict of interest: S. Zahalka has nothing to disclose. Conflict of interest: N. Rahimi has nothing to disclose. Conflict of interest: P. Starkl has nothing to disclose. Conflict of interest: D. Symmank has nothing to disclose. Conflict of interest: T. Artner has nothing to disclose. Conflict of interest: C. Pattaroni has nothing to disclose. Conflict of interest: N. Fortelny has nothing to disclose. Conflict of interest: K. Klavins has nothing to disclose. Conflict of interest: F. Frommlet has nothing to disclose. Conflict of interest: B.J. Marsland has nothing to disclose. Conflict of interest: K. Hoetzenecker has nothing to disclose. Conflict of interest: S. Widder reports grants from Austrian Science Fund (Elise Richter V585-B31), during the conduct of the study. Conflict of interest: S. Knapp reports grants from FWF, during the conduct of the study., (Copyright ©The authors 2022. For reproduction rights and permissions contact permissions@ersnet.org.)

The heterogeneity in severity and outcome of COVID-19 cases points out the urgent need for early molecular characterization of patients followed by risk-stratified care. The main objective of this study was to evaluate the fluctuations of serum metabolomic profiles of COVID-19 patients with severe illness during the different disease stages in a longitudinal manner. We demonstrate a distinct metabolomic signature in serum samples of 32 hospitalized patients at the acute phase compared to the recovery period, suggesting the tryptophan (tryptophan, kynurenine, and 3-hydroxy-DL-kynurenine) and arginine (citrulline and ornithine) metabolism as contributing pathways in the immune response to SARS-CoV-2 with a potential link to the clinical severity of the disease. In addition, we suggest that glutamine deprivation may further result in inhibited M2 macrophage polarization as a complementary process, and highlight the contribution of phenylalanine and tyrosine in the molecular mechanisms underlying the severe course of the infection. In conclusion, our results provide several functional metabolic markers for disease progression and severe outcome with potential clinical application. IMPORTANCE Although the host defense mechanisms against SARS-CoV-2 infection are still poorly described, they are of central importance in shaping the course of the disease and the possible outcome. Metabolomic profiling may complement the lacking knowledge of the molecular mechanisms underlying clinical manifestations and pathogenesis of COVID-19. Moreover, early identification of metabolomics-based biomarker signatures is proved to serve as an effective approach for the prediction of disease outcome. Here we provide the list of metabolites describing the severe, acute phase of the infection and bring the evidence of crucial metabolic pathways linked to aggressive immune responses. Finally, we suggest metabolomic phenotyping as a promising method for developing personalized care strategies in COVID-19 patients.

The epidermis is a multi-layered epithelium that consists mainly of keratinocytes which proliferate in its basal layer and then differentiate to form the stratum corneum, the skin's ultimate barrier to the environment. During differentiation keratinocyte function, chemical composition, physical properties, metabolism and secretion are profoundly changed. Extrinsic or intrinsic stressors, like ultraviolet (UV) radiation thus may differently affect the epidermal keratinocytes, depending on differentiation stage. Exposure to UV elicits the DNA damage responses, activation of pathways which detoxify or repair damage or induction of programmed cell death when the damage was irreparable. Recently, rapid diversion of glucose flux into the pentose phosphate pathway (PPP) was discovered as additional mechanism by which cells rapidly generate reduction equivalents and precursors for nucleotides - both being in demand after UV damage. There is however little known about the correlation of such metabolic activity with differentiation state, cell damage and tissue localization of epidermal cells. We developed a method to correlate the activity of G6PD, the first and rate-limiting enzyme of this metabolic UV response, at cellular resolution to cell type, differentiation state, and cell damage in human skin and in organotypic reconstructed epidermis. We thereby could verify rapid activation of G6PD as an immediate UVB response not only in basal but also in differentiating epidermal keratinocytes and found increased activity in cells which initiated DNA damage responses. When keratinocytes had been UVB irradiated before organotypic culture, their distribution within the skin equivalent was abnormal and the G6PD activity was reduced compared to neighboring cells. Finally, we found that the anti-diabetic and potential anti-aging drug metformin strongly induced G6PD activity throughout reconstructed epidermis. Activation of the protective pentose phosphate pathway may be useful to enhance the skin's antioxidant defense systems and DNA damage repair capacity on demand., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Solute carriers (SLCs) are the largest family of transmembrane transporters in humans and are major determinants of cellular metabolism. Several SLCs have been shown to be required for the uptake of chemical compounds into cellular systems, but systematic surveys of transporter-drug relationships in human cells are currently lacking. We performed a series of genetic screens in a haploid human cell line against 60 cytotoxic compounds representative of the chemical space populated by approved drugs. By using an SLC-focused CRISPR-Cas9 library, we identified transporters whose absence induced resistance to the drugs tested. This included dependencies involving the transporters SLC11A2/SLC16A1 for artemisinin derivatives and SLC35A2/SLC38A5 for cisplatin. The functional dependence on SLCs observed for a significant proportion of the screened compounds suggests a widespread role for SLCs in the uptake and cellular activity of cytotoxic drugs and provides an experimentally validated set of SLC-drug associations for a number of clinically relevant compounds.

Patients with active acromegaly (ACRO) exhibit low hepatocellular lipids (HCL), despite pronounced insulin resistance (IR). This contrasts the strong association of IR with nonalcoholic fatty liver disease in the general population. Since low HCL levels in ACRO might be caused by changes in oxidative substrate metabolism, we investigated mitochondrial activity and plasma metabolomics/lipidomics in active ACRO. Fifteen subjects with ACRO and seventeen healthy controls, matched for age, BMI, sex, and body composition, underwent 31P/1H-7-T MR spectroscopy of the liver and skeletal muscle as well as plasma metabolomic profiling and an oral glucose tolerance test. Subjects with ACRO showed significantly lower HCL levels, but the ATP synthesis rate was significantly increased compared with that in controls. Furthermore, a decreased ratio of unsaturated-to-saturated intrahepatocellular fatty acids was found in subjects with ACRO. Within assessed plasma lipids, lipidomics, and metabolomics, decreased carnitine species also indicated increased mitochondrial activity. We therefore concluded that excess of growth hormone (GH) in humans counteracts HCL accumulation by increased hepatic ATP synthesis. This was accompanied by a decreased ratio of unsaturated-to-saturated lipids in hepatocytes and by a metabolomic profile, reflecting the increase in mitochondrial activity. Thus, these findings help to better understanding of GH-regulated antisteatotic pathways and provide a better insight into potentially novel therapeutic targets for treating NAFLD.

Infections induce complex host responses linked to antiviral defense, inflammation, and tissue damage and repair. We hypothesized that the liver, as a central metabolic hub, may orchestrate systemic metabolic changes during infection. We infected mice with chronic lymphocytic choriomeningitis virus (LCMV), performed RNA sequencing and proteomics of liver tissue, and integrated these data with serum metabolomics at different infection phases. Widespread reprogramming of liver metabolism occurred early after infection, correlating with type I interferon (IFN-I) responses. Viral infection induced metabolic alterations of the liver that depended on the interferon alpha/beta receptor (IFNAR1). Hepatocyte-intrinsic IFNAR1 repressed the transcription of metabolic genes, including Otc and Ass1, which encode urea cycle enzymes. This led to decreased arginine and increased ornithine concentrations in the circulation, resulting in suppressed virus-specific CD8 + T cell responses and ameliorated liver pathology. These findings establish IFN-I-induced modulation of hepatic metabolism and the urea cycle as an endogenous mechanism of immunoregulation. VIDEO ABSTRACT., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Destruxins, secondary metabolites of entomopathogenic fungi, exert a wide variety of interesting characteristics ranging from antiviral to anticancer effects. Although their mode of action was evaluated previously, the molecular mechanisms of resistance development are unknown. Hence, we have established destruxin-resistant sublines of HCT116 colon carcinoma cells by selection with the most prevalent derivatives, destruxin (dtx)A, dtxB and dtxE. Various cell biological and molecular techniques were applied to elucidate the regulatory mechanisms underlying these acquired and highly stable destruxin resistance phenotypes. Interestingly, well-known chemoresistance-mediating ABC efflux transporters were not the major players. Instead, in dtxA- and dtxB-resistant cells a hyper-activated mevalonate pathway was uncovered resulting in increased de-novo cholesterol synthesis rates and elevated levels of lanosterol, cholesterol as well as several oxysterol metabolites. Accordingly, inhibition of the mevalonate pathway at two different steps, using either statins or zoledronic acid, significantly reduced acquired but also intrinsic destruxin resistance. Vice versa, cholesterol supplementation protected destruxin-sensitive cells against their cytotoxic activity. Additionally, an increased cell membrane adhesiveness of dtxA-resistant as compared to parental cells was detected by atomic force microscopy. This was paralleled by a dramatically reduced ionophoric capacity of dtxA in resistant cells when cultured in absence but not in presence of statins. Summarizing, our results suggest a reduced ionophoric activity of destruxins due to cholesterol-mediated plasma membrane re-organization as molecular mechanism underlying acquired destruxin resistance in human colon cancer cells. Whether this mechanism might be valid also in other cell types and organisms exposed to destruxins e.g. as bio-insecticides needs to be evaluated., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.