Metabolic Branch Research Articles

Proteomics and Lysine Acetylation Modification Reveal the Responses of Pakchoi (Brassica rapa L. ssp. chinensis) to Oxybenzone Stress.

The broad-spectrum UV filter oxybenzone is toxic to plants at environmentally relevant concentrations. Lysine acetylation (LysAc) is one of the essential post-translational modifications (PTMs) in plant signaling responses. The goal of this study was to uncover the LysAc regulatory mechanism in response to toxic exposures to oxybenzone as a first step in elucidating xenobiotic acclimatory reactions by using the model Brassica rapa L. ssp. chinensis. A total of 6124 sites on 2497 proteins were acetylated, 63 proteins were differentially abundant, and 162 proteins were differentially acetylated under oxybenzone treatment. Bioinformatics analysis showed that a large number of antioxidant proteins were significantly acetylated under oxybenzone treatment, implying that LysAc alleviated the adverse effects of reactive oxygen species (ROS) by inducing antioxidant systems and stress-related proteins; the significant changes in acetylation modification of enzymes involved in different branches of carbon metabolism in plants under oxybenzone treatment mean that plants can change the direction of carbon flow allocation by regulating the activities of carbon metabolism-related enzymes. Our results profile the protein LysAc under oxybenzone treatment and propose an adaptive mechanism at the post-translational level of vascular plants in response to pollutants, providing a dataset reference for future related research.

Efficient Synthesis of Limonene in Saccharomyces cerevisiae Using Combinatorial Metabolic Engineering Strategies

Limonene is a volatile monoterpene compound that is widely used in food additives, pharmaceutical products, fragrances, and toiletries. We herein attempted to perform efficient biosynthesis of limonene in Saccharomyces cerevisiae using systematic metabolic engineering strategies. First, we conducted de novo synthesis of limonene in S. cerevisiae and achieved a titer of 46.96 mg/L. Next, by dynamic inhibition of the competitive bypass of key metabolic branches regulated by ERG20 and optimization of the copy number of tLimS, a greater proportion of the metabolic flow was directed toward limonene synthesis, achieving a titer of 640.87 mg/L. Subsequently, we enhanced the acetyl-CoA and NADPH supply, which increased the limonene titer to 1097.43 mg/L. Then, we reconstructed the limonene synthesis pathway in the mitochondria. Dual regulation of cytoplasmic and mitochondrial metabolism further increased the limonene titer to 1586 mg/L. After optimization of the process of fed-batch fermentation, the limonene titer reached 2.63 g/L, the highest ever reported in S. cerevisiae.

D-Glucosamine induces circadian phase delay by promoting BMAL1 degradation through AMPK/mTOR pathway

Circadian rhythms are closely linked to the metabolic network through circadian feedback regulation. The hexosamine biosynthetic pathway (HBP) is a branch of glucose metabolism that affects circadian rhythms through the O-linked N-acetylglucosamine modification (O-GlcNAcylation) of clock proteins. Here, we found out that, among the downstream metabolites regulated by d-glucosamine (GlcN) in HBP salvage pathway, only GlcN is able to induce circadian phase delay both in vitro and in vivo. Mechanistic studies indicated that the phase-shift induced by GlcN is independent of O-GlcNAcylation. Instead, GlcN selectively up-regulates p-AMPK activity, leading to the inhibition of mTOR signaling pathway, and thus down-regulation of p-BMAL1 both in human cell line and mouse tissues. Moreover, GlcN promoted BMAL1 degradation via proteasome pathway. These findings reveal a novel molecular mechanism of GlcN in regulating clock phase and suggest the therapeutic potential of GlcN as new use for an old drug in the future treatment of shift work and circadian misalignment.

Abstract 2047: Flux estimation analysis systematically characterizes the metabolic shifts of the central metabolism pathway in human cancer

Abstract Background: Glucose and glutamine are major carbon and energy sources that promote the fast proliferation of cancer cells. The Warburg effect, characterized by shifted flux ratio between aerobic and anaerobic respirations, is considered as a common metabolic reprogramming mechanism in cancer. The further discovery of glutaminolysis pathway elucidated the role of glutamate in providing energy and carbon source in cancer metabolism. Variations in the branches of the central metabolism of glucose and glutamate have also been observed. However, most of these observations were made on cell line or mouse systems, which cannot mimic the dysbalanced nutrient, redox, pH and oxygen levels in a real tumor microenvironment (TME), which may heavily shift in a TME and determines the metabolic phenotypes of a cancer. Analysis: In this study, we conducted a systematic evaluation of the metabolic reprogramming and characteristics via a computational analysis by using pan-cancer transcriptomics data of 11 cancer subtypes and 9 matched adjacent normal tissue types. We focused on the flux distribution and variations of the central energy metabolism and its key branches. We first reconstructed the central metabolism pathway in a subcellular resolution by including glycolytic pathway, production of lactate, TCA cycle, nucleic acids synthesis, glutaminolysis, glutaminate, glutamine and glutathione metabolism, and other amino acids synthesis. We applied our in-house developed scFEA method on TCGA pan-cancer transcriptomics data. Results: Our analysis confirms the increased influx in glucose uptake and glycolysis and decreased upper part of TCA cycle, i.e., Warburg effect in almost all the analyzed cancer types. However, increased lactate production and the second half of TCA cycle were only seen in certain cancer types. More interestingly, we did not see cancer tissues have highly shifted glutaminolysis compared to their adjacent normal tissues. A systems biology model of metabolic shifts through cancer and tissue types is further developed and analyzed. We observed that (1) normal tissues have distinct metabolic phenotypes, (2) cancer types have drastically different metabolic shifts compared to their adjacent normal controls, and (3) the different shifts happened to tissue specific metabolic phenotypes result in a converged metabolic phenotype through cancer types and cancer progression. This study strongly suggests the possibility to have a unified framework for studies of cancer-inducing stressors, adaptive metabolic reprogramming, and cancerous behaviors. Citation Format: Alex Lu, Haiqi Zhu, Kevin Hu, Grace Yang, Shaoyang Huang, Pengtao Dang, Sha Cao, Chi Zhang. Flux estimation analysis systematically characterizes the metabolic shifts of the central metabolism pathway in human cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2047.

Function of ALA Content in Porphyrin Metabolism Regulation of Ananas comosus var. bracteatus.

Chlorophyll and heme are essential molecules for photosynthesis and respiration, which are competing branches of the porphyrin metabolism pathway. Chlorophyll and heme balance regulation is very important for the growth and development of plants. The chimeric leaves of Ananas comosus var. bracteatus were composed of central photosynthetic tissue (PT) and marginal albino tissue (AT), which were ideal materials for the study of porphyrin metabolism mechanisms. In this study, the regulatory function of ALA content on porphyrin metabolism (chlorophyll and heme balance) was revealed by comparing PT and AT, 5-Aminolevulinic Acid (ALA) exogenous supply, and interference of hemA expression. The AT remained similar in porphyrin metabolism flow level to the PT by keeping an equal ALA content in both tissues, which was very important for the normal growth of the chimeric leaves. As the chlorophyll biosynthesis in AT was significantly inhibited, the porphyrin metabolism flow was directed more toward the heme branch. Both tissues had similar Mg2+ contents; however, Fe2+ content was significantly increased in the AT. The chlorophyll biosynthesis inhibition in the white tissue was not due to a lack of Mg2+ and ALA. A 1.5-fold increase in ALA content inhibited chlorophyll biosynthesis while promoting heme biosynthesis and hemA expression. The doubling of ALA content boosted chlorophyll biosynthesis while decreasing hemA expression and heme content. HemA expression interference resulted in a higher ALA content and a lower chlorophyll content, while the heme content remained at a relatively low and stable level. Conclusively, a certain amount of ALA was important for the stability of porphyrin metabolism and the normal growth of plants. The ALA content appears to be able to regulate chlorophyll and heme content by bidirectionally regulating porphyrin metabolism branch direction.

In the battle of the disease: a transcriptomic analysis of European foulbrood-diseased larvae of the Western honey bee (Apis mellifera)

BackgroundEuropean foulbrood is a significant bacterial brood disease of Apis sp. and can cause severe and devastating damages in beekeeping operations. Nevertheless, the epidemiology of its causative agent Melissococcus plutonius has been begun to uncover but the underlying mechanisms of infection and cause of disease still is not well understood. Here, we sought to provide insight into the infection mechanism of EFB employing RNAseq in in vitro reared Apis mellifera larvae of two developmental stages to trace transcriptional changes in the course of the disease, including Paenibacillus alvei secondary infected individuals.ResultsIn consideration of the progressing development of the larva, we show that infected individuals incur a shift in metabolic and structural protein-encoding genes, which are involved in metabolism of crucial compounds including all branches of macronutrient metabolism, transport protein genes and most strikingly chitin and cuticle associated genes. These changes underpin the frequently observed developmental retardation in EFB disease. Further, sets of expressed genes markedly differ in different stages of infection with almost no overlap. In an earlier stage of infection, a group of regulators of the melanization response cascade and complement component-like genes, predominantly C-type lectin genes, are up-regulated while a differential expression of immune effector genes is completely missing. In contrast, late-stage infected larvae up-regulated the expression of antimicrobial peptides, lysozymes and prominent bacteria-binding haemocyte receptor genes compared to controls. While we clearly show a significant effect of infection on expressed genes, these changes may partly result from a shift in expression timing due to developmental alterations of infection. A secondary infection with P. alvei elicits a specific response with most of the M. plutonius associated differential immune effector gene expression missing and several immune pathway genes even down-regulated.ConclusionWe conclude that with progressing infection diseased individuals undergo a systemic response with a change of metabolism and their activated immune defence repertoire. Moreover, larvae are capable of adjusting their response to a secondary invasion in late stage infections.

Evolutionary engineering of Fusarium fujikuroi for enhanced production of gibberellic acid

Fusarium fujikuroi is the microorganism that used for industrial production of gibberellic acids (GAs), a commercially very important plant hormone. A stable, high-yielding F. fujikuroi strain is essential for efficient bio-production. Although, there is a quick development in molecular tools and metabolic engineering research for this microbe in the past decade, the current industrially applying strains are mostly obtained from numerous rounds of mutagenesis and screening, while the industry is still dedicating in strain improvement work based mostly on this method. However, after over half a century’s effort, the yield is still maintained at a low level today. We designed an efficient strategy for strain improvement by amphotericin B resistance prescreening and evolutionary engineering. A superior strain was obtained with over 25% increased yield after testing merely 640 isolates. The superior strain was later confirmed to be genetically very stable for GA production. Minimum inhibitory concentration (MIC) assay verified its increased resistance to amphotericin B, whose target ergosterol shares a same precursor with GAs. Decreased ergosterol accumulation indicated the increased GA3 synthesis accompanied with attenuation of metabolic branch flux. In the end, we found the increased GA3 titer of the superior strain also accompanied with faster growing biomass.

The phosphorylation of AMPKβ1 is critical for increasing autophagy and maintaining mitochondrial homeostasis in response to fatty acids

Fatty acids are vital for the survival of eukaryotes, but when present in excess can have deleterious consequences. The AMP-activated protein kinase (AMPK) is an important regulator of multiple branches of metabolism. Studies in purified enzyme preparations and cultured cells have shown that AMPK is allosterically activated by small molecules as well as fatty acyl-CoAs through a mechanism involving Ser108 within the regulatory AMPK β1 isoform. However, the in vivo physiological significance of this residue has not been evaluated. In the current study, we generated mice with a targeted germline knock-in (KI) mutation of AMPKβ1 Ser108 to Ala (S108A-KI), which renders the site phospho-deficient. S108A-KI mice had reduced AMPK activity (50 to 75%) in the liver but not in the skeletal muscle. On a chow diet, S108A-KI mice had impairments in exogenous lipid-induced fatty acid oxidation. Studies in mice fed a high-fat diet found that S108A-KI mice had a tendency for greater glucose intolerance and elevated liver triglycerides. Consistent with increased liver triglycerides, livers of S108A-KI mice had reductions in mitochondrial content and respiration that were accompanied by enlarged mitochondria, suggestive of impairments in mitophagy. Subsequent studies in primary hepatocytes found that S108A-KI mice had reductions in palmitate- stimulated Cpt1a and Ppargc1a mRNA, ULK1 phosphorylation and autophagic/mitophagic flux. These data demonstrate an important physiological role of AMPKβ1 Ser108 phosphorylation in promoting fatty acid oxidation, mitochondrial biogenesis and autophagy under conditions of high lipid availability. As both ketogenic diets and intermittent fasting increase circulating free fatty acid levels, AMPK activity, mitochondrial biogenesis, and mitophagy, these data suggest a potential unifying mechanism which may be important in mediating these effects.

Cellular Stress-Induced Metabolites in Escherichia coli.

Escherichia coli isolates commonly inhabit the human microbiota, yet the majority of E. coli's small-molecule repertoire remains uncharacterized. We previously employed erythromycin-induced translational stress to facilitate the characterization of autoinducer-3 (AI-3) and structurally related pyrazinones derived from "abortive" tRNA synthetase reactions in pathogenic, commensal, and probiotic E. coli isolates. In this study, we explored the "missing" tryptophan-derived pyrazinone reaction and characterized two other families of metabolites that were similarly upregulated under erythromycin stress. Strikingly, the abortive tryptophanyl-tRNA synthetase reaction leads to a tetracyclic indole alkaloid metabolite (1) rather than a pyrazinone. Furthermore, erythromycin induced two naphthoquinone-functionalized metabolites (MK-hCys, 2; and MK-Cys, 3) and four lumazines (7-10). Using genetic and metabolite analyses coupled with biomimetic synthesis, we provide support that the naphthoquinones are derived from 4-dihydroxy-2-naphthoic acid (DHNA), an intermediate in the menaquinone biosynthetic pathway, and the amino acids homocysteine and cysteine. In contrast, the lumazines are dependent on a flavin intermediate and α-ketoacids from the aminotransferases AspC and TyrB. We show that one of the lumazine members (9), an indole-functionalized analogue, possesses antioxidant properties, modulates the anti-inflammatory fate of isolated TH17 cells, and serves as an aryl-hydrocarbon receptor (AhR) agonist. These three systems described here serve to illustrate that new metabolic branches could be more commonly derived from well-established primary metabolic pathways.

The appropriate expression and coordination of glycolate oxidase and catalase are vital to the successful construction of the photorespiratory metabolic pathway.

Photorespiration has emerged as a hotspot in the evolution of photosynthesis owing to the energy loss during the process. To ensure the physiological functions of photorespiration such as light protection, H2O2 signaling, and stress resistance, separate the photorespiration glycolic acid flow, and minimize photorespiration loss, a balance must be maintained during the construction of photorespiratory metabolic branch. In this study, glycolate oxidase (GLO) and catalase (CAT) were introduced into potato (Solanum tuberosum) chloroplasts through the expression of fusion protein. Through the examination of phenotypic characteristics, photosynthesis, anatomical structure, and enzyme activity, the efficiency of the photorespiration pathway was demonstrated. The results showed that certain transgenic lines plants had shorter plant height and deformed leaves and tubers in addition to the favorable photosynthetic phenotypes of thicker leaves and larger and denser mesophyll cells. By Diaminobenzidine (DAB) staining analysis of the leaves, the intermediate H2O2 could not be decomposed in time to cause biomass decline and malformation, and the excessive glycolate shunt formed by the overexpression of the fusion protein affected other important physiological activities. Hence, the appropriate and coordinated expression of glycolate oxidase and catalase is essential for the establishment of photorespiration pathways in chloroplasts.

NKT cells adopt a glutamine-addicted phenotype to regulate their homeostasis and function

Natural killer T (NKT) cells operate distinctly different metabolic programming from CD4 Tcells, including a strict requirement for glutamine to regulate cell homeostasis. However, the underlying mechanisms remain unknown. Here, we report that at a steady state, NKT cells have higher glutamine levels than CD4 Tcells and that NKT cells increase glutaminolysis on activation. Activated NKT cells use glutamine to fuel the tricarboxylic acid cycle and glutathione synthesis. In addition, glutamine-derived nitrogen enables protein glycosylation via the hexosamine biosynthesis pathway (HBP). Each of these branches of glutamine metabolism seems to be critical for NKT cell homeostasis and mitochondrial functions. Glutaminolysis and HBP differentially regulate interleukin-4 (IL-4) and interferon γ (IFNγ) production. Glutamine metabolism appears to be controlled by AMP-activated protein kinase (AMPK)-mammalian target of rapamycin complex 1 (mTORC1) signaling. These findings highlight a distinct metabolic requirement of NKT cells compared with CD4 Tcells, which may have therapeutic implications in the treatment of certain nutrient-restricted diseases.

Spatial engineering of E. coli with addressable phase-separated RNAs

Biochemical processes often require spatial regulation and specific microenvironments. The general lack of organelles in bacteria limits the potential of bioengineering complex intracellular reactions. Here, we demonstrate synthetic membraneless organelles in Escherichia coli termed transcriptionally engineered addressable RNA solvent droplets (TEARS). TEARS are assembled from RNA-binding protein recruiting domains fused to poly-CAG repeats that spontaneously drive liquid-liquid phase separation from the bulk cytoplasm. Targeting TEARS with fluorescent proteins revealed multilayered structures with composition and reaction robustness governed by non-equilibrium dynamics. We show that TEARS provide organelle-like bioprocess isolation for sequestering biochemical pathways, controlling metabolic branch points, buffering mRNA translation rates, and scaffolding protein-protein interactions. We anticipate TEARS to be a simple and versatile tool for spatially controlling E.coli biochemistry. Particularly, the modular design of TEARS enables applications without expression fine-tuning, simplifying the design-build-test cycle of bioengineering.

Tartary buckwheat FtF3'H1 as a metabolic branch switch to increase anthocyanin content in transgenic plant.

Tartary buckwheat (TB) is a pseudocereal rich in flavonoids, mainly including flavonols and anthocyanins. The flavonoid 3′-hydroxylase (F3′H) is a key enzyme in flavonoid biosynthesis and is encoded by two copies in TB genome. However, its biological function and effects on flavonol and anthocyanin synthesis in TB have not been well validated yet. In this study, we cloned the full-length FtF3′H1 gene highly expressed in all tissues (compared with FtF3′H2) according to TB flowering transcriptome data. The corresponding FtF3′H1 protein contains 534 amino acids with the molecular properties of the typical plant F3′H and belongs to the CYP75B family. During the flowering stage, the FtF3′H1 expression was highest in flowers, and its expression pattern showed a significant and positive correlation with the total flavonoids (R2 > 0.95). The overexpression of FtF3′H1 in Arabidopsis thaliana, Nicotiana tabacum and TB hairy roots resulted in a significant increase in anthocyanin contents (p < 0.05) but a decrease in rutin (p < 0.05). The average anthocyanin contents were 2.94 mg/g (fresh weight, FW) in A. thaliana (about 135% increase), 1.18 mg/g (FW) in tobacco (about 17% increase), and 1.56 mg/g (FW) TB hairy roots (about 44% increase), and the rutin contents were dropped to about 53.85, 14.99, 46.31%, respectively. However, the expression of genes involved in anthocyanin (DFRs and ANSs) and flavonol (FLSs) synthesis pathways were significantly upregulated (p < 0.05). In particular, the expression level of DFR, a key enzyme that enters the anthocyanin branch, was upregulated thousand-fold in A. thaliana and in N. tabacum. These results might be attributed to FtF3′H1 protein with a higher substrate preference for anthocyanin synthesis substrates. Altogether, we identified the basic biochemical activity of FtF3′H1 in vivo and investigated its involvement in anthocyanin and flavonol metabolism in plant.

New insight into the sucrose biosynthesis pathway from genome-wide identification, gene expression analysis, and subcellular localization in hexaploid wheat (Triticum aestivum L.)

Sucrose, the main synthesized product and transported form of photoassimilates, moves from leaves to support plant growth and storage substance biosynthesis occurring in the heterotrophic sink organs. Enhancing sucrose biosynthesis efficiency is a top priority for crop yield breeding programs. However, the molecular mechanism of sucrose biosynthesis is still elusive, especially in wheat. We performed transcriptome sequencing, subcellular localization, and bioinformatics analysis to identify key isoforms and metabolic branches associated with sucrose biosynthesis in wheat. Our analysis identified 45 genes from 13 families that exhibited high expression in young leaves with an evident diurnal change. The carbon flux from photoassimilates to sucrose was divided into two pathways. In the cytoplasm, assimilates initiating at phosphotrioses (TPs) exported by TaTPT1 from chloroplasts flowed along the TP-Sucrose branch formed by TaALD6, TaFBP5, TacPGI, TacPGM, TaUGP1, TaSPS5, and TaSPP1. Intermediates either from the Calvin cycle or TP-Sucrose branch were converted into ADPGlc to synthesize the simple starch, which was transiently degraded by a series of enzymes, including TaBAM4 and TaSEX4 in the chloroplast. Similar to the starch-biosynthesis branch in endosperms, the TP-Sucrose branch is the most prominent in leaves because each reaction can be catalyzed by at least one highly expressed isoform with expected cytosolic localization. The key isoforms and major branches highlighted in the wheat sucrose biosynthesis pathway expand our molecular understanding of crop sucrose biosynthesis and provide clues to increase wheat yield by enhancing the sucrose synthesis efficiency of leaves.

Transcriptional Regulatory Mechanism of Differential Metabolite Formation in Root and Stem of Ephedra sinica.

Ephedra sinica, a well-known Chinese medicinal plant, is characterized as having the opposite medicinal effect among its root and stem. However, there is a lack of understanding to differentiate the active components present in the root and stem of E. sinica, as well as the molecular mechanisms underlying the formation of the differential compounds, which has significantly hampered the further development and utilization of E. sinica resource. In this study, forty-five differential metabolic markers are affiliated to alkaloids, flavonoids, terpenoids, and organic acids between root and stem of E. sinica, and sixty genes of key enzymes are involved in their biosynthesis distributed in metabolic pathway branches such as phenylalanine metabolism, flavonoid biosynthesis and phenylpropane biosynthesis, based on combination non-targeted metabolome with transcriptome technologies. The finding revealed that the expression activity changes of these enzyme genes had a direct impact on the distinction of differential metabolic markers in the root and stem of E. sinica. This study will help to understand the molecular mechanism of the differentiation and biosynthesis of the primary active metabolites in the root and stem of E. sinica, providing a theoretical foundation for its quality control and promotion in cultivation.

Anti-Inflammatory Properties of Metformin During Cultivation of Primary Rat Astrocytes in a Medium with High Glucose Concentration.

Investigation of the relationship between inflammation and energy metabolism is important for understanding biology of chronic noncommunicable diseases. Use of metformin, a drug for treatment of diabetes, is considered as a promising direction for treatment of neurodegenerative diseases and other neuropathologies with an inflammatory component. Astrocytes play an important role in the regulation of energy metabolism and neuroinflammation; therefore, we studied the effect of metformin on the cellular responses of primary rat astrocytes cultured in a medium with high glucose concentration (22.5 mM, 48-h incubation). Lipopolysaccharide (LPS) was used to stimulate inflammation. The effects of metformin were assessed by monitoring changes in the expression of proinflammatory cytokines and synthesis of oxylipins, assayed with ultra-high-performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS). Changes at the intracellular level were assessed by analyzing phosphorylation of ERK kinase and transcription factor STAT3, as well as enzymes mediating oxylipin synthesis, cyclooxygenase 1 and 2 (COX). It was found that, independent on glucose concentration, metformin reduced the LPS-stimulated release of cytokines IL-1β and IL-6, decreased activity of the transcription factor STAT3, ERK kinase, synthesis of the derivatives of the cyclooxygenase branch of metabolism of oxylipins and anandamide, and did not affect formation of ROS. The study of energy phenotype of the cells showed that metformin activated glycolysis and inhibited mitochondrial respiration and oxidative phosphorylation, independent on LPS stimulation and cell cultivation at high glucose concentration. Thus, it has been shown that metformin exhibits anti-inflammatory effects, and its effect on the synthesis of cytokines, prostaglandins, and other lipid mediators could determine beneficial effects of metformin in models of neuropathology.

Measuring Vitamin D3 Metabolic Status, Comparison between Vitamin D Deficient and Sufficient Individuals

The main branch of vitamin D3 metabolism involves several hydroxylation reactions to obtain mono-, di- and trihydroxylated metabolites, including the circulating and active forms—25(OH)D3 and 1,25(OH)2D3, respectively. However, most clinical trials strictly target the determination of 25(OH)D3 to offer a view of the metabolic status of vitamin D3. Due to the growing interest in expanding this restricted view, we have developed a method for measuring vitamin D3 metabolism by determination of vitamin D3, 25(OH)D3, 24,25(OH)2D3, 1,25(OH)2D3 and 1,24,25(OH)3D3 in human plasma. The method was based on SPE–LC–MS/MS with a large volume injection of human plasma (240 µL). Detection of di- and trihydroxymetabolites, found at the picogram per milliliter level, was attained by the combined action of high preconcentration and clean-up effects. The method allows obtaining information about ratios such as the known vitamin D metabolite ratio (24,25(OH)2D3/25(OH)D3), which can provide complementary views of vitamin D3 metabolic status. The method was applied to a cohort of obese patients and a reference cohort of healthy volunteers to find metabolic correlations between target analytes as well as differences as a function of vitamin D levels within and between cohorts.

Immunonutritional Targeting of Cancer via Eicosanoids

Cancer causes widespread mortality via a systemic pro‐inflammatory and dysregulated immune response. Immunotherapy is the front‐line treatment in many cancers with a response rate of approximately 10–30%, although it remains unclear why the response rate is so diverse. Dietary intervention provides an opportunity to optimize the host immune response in cancer patients, however, mechanisms of immunonutritional regulation in cancer are poorly understood. Key components of immune surveillance include the action of cytotoxic T‐lymphocyte‐associated protein‐4 (CTLA4), as certain cancers can utilize CTLA4 to evade immune surveillance. Thus, several monoclonal antibodies targeting CTLA4 are FDA approved. Epidemiological and experimental studies provide evidence that omega‐3 polyunsaturated fatty acids (PUFAs) are beneficial for reducing the risk of cancer, whereas omega‐6 PUFAs can stimulate cancer. Cytochrome P450 epoxygenases represent one branch of PUFA metabolism, generating biologically active omega‐3 epoxy fatty acids (omega‐3 epoxides). Cytochrome P450 epoxygenases convert the omega‐6 PUFA arachidonic acid into epoxyeicosatrienoic acids, which suppress inflammation. Because the half‐life of fatty acid epoxides is rapid, drugs that stabilize their levels by inhibiting soluble epoxide hydrolase (sEHi) are in clinical trials for hyperinflammatory diseases. sEHi may synergize with omega‐3 fatty acids in reducing inflammation and cancer. Since immune checkpoint inhibitors (e.g. CTLA4) can augment cytotoxic T cell responses, we hypothesized that omega‐3 fatty acid supplementation in conjunction with sEHi would enhance immunotherapy in various cancer types. Mice were fed with standard diet, high omega‐6 diet, or high omega‐3 diet for 10 days prior to tumor inoculation and for the duration of the studies. One week following tumor injection, mice were randomized into treatment groups: control, sEHi TPPU (5 mg/kg/day via drinking water), immunotherapy (anti‐CTLA4; 200 µg initial dose, 100 µg Q3D), and TPPU + anti‐CTLA4. Here, we demonstrate in poorly immune responsive tumors (Lewis lung carcinoma (LLC) and B16F10 melanoma) that oral administration of TPPU enhances immunotherapy in mice on a high omega‐3 fatty acid diet to transform “cold” unresponsive tumors into “hot” responsive tumors. Indeed, the combination of TPPU and anti‐CTLA4 resulted in synergistic antitumor activity in mice on the high omega‐3 diet, with 52‐67% inhibition in LLC and B16F10 melanoma compared to control. Anti‐CTLA4 inhibited LLC primary tumor growth in mice on an omega‐3 fatty acid diet. In marked contrast, immune checkpoint blockade (anti‐CTLA4) accelerated LLC growth in mice on an omega‐6 fatty acid diet by 225% compared to control after 15 days of treatment. sEHis alone or in combination with an increased omega‐3 PUFA/omega‐6 PUFA dietary ratio may be a promising new approach to enhance immune checkpoint blockade in cancer. Together, these findings identify omega‐3 epoxides as important regulators of immunotherapy and sEH as a druggable target in cancer.

Inhibition of Anaplerosis Prevented Mitochondrial Dysfunction and Vascular Remodeling in Pulmonary Hypertension

IntroductionPulmonary hypertension (PH) is a deadly disease of the lung vasculature. Recent studies reported mitochondrial dysfunction as a significant contributor to PH pathobiology, the prominent type of dysfunction is still unclear. Importantly, partial mitochondrial functioning is necessary to provide the metabolic intermediates for the proliferative growth of vascular cells in PH. Recently, we reported that in PH, the mitochondrial anabolic branch of metabolism ‐ anaplerosis plays a critical role in the upregulation of the tricarboxylic acid cycle in pulmonary vascular cells with mitochondrial dysfunction.HypothesisWe hypothesize that the inhibition of both anaplerosis targets ‐ pyruvate carboxylase (PC) and glutaminolysis ‐ glutamate dehydrogenase 1 (GLUD1) in the early stages of PH could effectively attenuate mitochondrial dysfunction and vascular remodeling.MethodsPH was induced in female Sprague Dawley rats by subcutaneous injection of sugen (50 mg/kg)/hypoxia (10% O2). The treatment groups received sugen/hypoxia (Su/Hx) and two‐weeks intraperitoneal injection of inhibitors (PC inhibitor, phenylacetic acid (PAA), 20 mg/kg/every other day and R162, an inhibitor of GLUD1 (30mg/kg/daily).ResultsHemodynamic analysis of five weeks of Su/Hx rats showed a progressive PH phenotype with increased right ventricular (RV) systolic pressure (111.08 ± 6.87 mmHg). Inhibiting anaplerosis at early stages of PH (2 weeks Su/Hx), decreased the RV hypertrophy (0.5 ± 0.02 to 0.42 ± 0.02, p&lt;0.05), RV systolic pressure (71.88 ± 3.59 mmHg to 54.14 ± 2.48 mmHg, p&lt;0.05), and Max dP/dt (3863.89 ± 180.27 mmHg/s to 2890.63 ± 154.52 mmHg/s, p&lt;0.01). Su/Hx group showed an increased glycolytic flux with hexokinase‐I upregulation (p&lt;0.05), leading to an upregulation of anaplerosis. This was followed by the impairment in mitochondrial function with a reduction of pyruvate dehydrogenase (PDH) expression (p&lt;0.001) and activation of mitochondrial glycerol‐3‐phosphate‐dehydrogenase (GPD2) (p&lt;0.001), an important mediator of the glycerophosphate shuttle. The increased glycolysis also fluxed into the pentose phosphate pathway (PPP) through myo‐inositol oxygenase (MIOX) (p&lt;0.001), a novel source of reactive oxygen species (ROS) generation in PH. These metabolic alterations lead to the upregulation of proliferative signaling pathways Akt (p&lt;0.01), STAT3 (p&lt;0.001), and P38 (p&lt;0.01). Importantly, the inhibition of the anaplerotic pathways by combination (PAA+R162) treatment in the Su/Hx PH model significantly resolved the activation of PPP, restored mitochondrial metabolism, and prevented the proliferative signaling leading to lung vascular remodeling.ConclusionThe results suggest that inhibition of the anaplerotic pathway effectively attenuated mitochondrial dysfunction and vascular remodeling in Su/HX animals. Therefore, our findings indicate that simultaneous targeting of both PC and glutamine‐mediated anaplerosis is a promising therapeutic target for the resolution of vascular remodeling in PH.

Design of stable and self-regulated microbial consortia for chemical synthesis

Microbial coculture engineering has emerged as a promising strategy for biomanufacturing. Stability and self-regulation pose a significant challenge for the generation of intrinsically robust cocultures for large-scale applications. Here, we introduce the use of multi-metabolite cross-feeding (MMCF) to establish a close correlation between the strains and the design rules for selecting the appropriate metabolic branches. This leads to an intrinicially stable two-strain coculture where the population composition and the product titer are insensitive to the initial inoculation ratios. With an intermediate-responsive biosensor, the population of the microbial coculture is autonomously balanced to minimize intermediate accumulation. This static-dynamic strategy is extendable to three-strain cocultures, as demonstrated with de novo biosynthesis of silybin/isosilybin. This strategy is generally applicable, paving the way to the industrial application of microbial cocultures.