2-oxoglutarate Research Articles

Alpha-ketoglutaric acid attenuates oxidative stress and modulates mitochondrial dynamics and autophagy of spleen in a piglet model of lipopolysaccharide-induced sepsis

Alpha-ketoglutaric acid (2-ketoglutaric acid or 2-oxoglutaric acid, AKG), a crucial intermediate in the tricarboxylic acid cycle, is pivotal in animal antioxidative process. The purpose of this study was to investigate whether AKG has the efficacy to mitigate spleen oxidative stress in lipopolysaccharide (LPS)-induced sepsis piglets through the modulation of mitochondrial dynamics and autophagy. Utilizing a 2 × 2 factorial design, the study encompassed 24 piglets subjected to varying diets (basal or 1% AKG) and immune stimulations (saline or LPS) over 21 days. Subsequently, they were injected intraperitoneally with either LPS or saline solution. The results showed that LPS decreased antioxidant capacity, whereas AKG supplementation increased antioxidant activities compared to control group. LPS elevated mitochondrial fission factor, mitochondrial elongation factor 1, mitochondrial elongation factor 2, dynamin-related protein 1, voltage-dependent anion channel 1, and fission 1 mRNA abundance, but reduced mRNA abundance of mitofusin 1, mitofusin 2, and optic atrophy 1 compared to controls. LPS elevated mRNA abundance of autophagy related protein 5, autophagy related protein 7, P62, Beclin1, and interleukin-1β mRNA abundance compared to controls. However, AKG supplementation mitigated these effects induced by LPS. Additionally, AKG intake was associated with lower protein expressions of microtubule-associated protein light chain 3, Parkin, and PTEN-induced putative kinase 1 compared to LPS-challenged piglets. These results suggested that AKG could alleviate spleen oxidative stress caused by LPS by regulating mitochondrial dynamics and autophagy.

Overexpression of the DHA1 family, ChlH and ChlK, leads to enhanced dicarboxylic acids production in koji fungi, Aspergillus luchuensis mut. kawachii and Aspergillus oryzae

The white koji fungus Aspergillus luchuensis mut. kawachii secretes substantial amounts of citric acid through the expression of the citric acid exporter CexA, a member of the DHA1 family. In this study, we aimed to characterize 11 CexA homologs (Chl proteins) encoded in the genome of A.luchuensis mut. kawachii to identify novel transporters useful for organic acid production. We constructed overexpression strains of chl genes using a cexA disruptant of the A.luchuensis mut. kawachii as the host strain, which prevented excessive secretion of citric acid into the culture supernatant. Subsequently, we evaluated the effects of overexpression of chl on producing organic acids by analyzing the culture supernatant. All overexpression strains did not exhibit significant citric acid accumulation in the culture supernatant, indicating that Chl proteins are not responsible for citric acid export. Furthermore, the ChlH overexpression strain displayed an accumulation of 2-oxoglutaric and fumaric acids in the culture supernatant, while the ChlK overexpression strain exhibited the accumulation of 2-oxoglutaric, malic and succinic acids. Notably, the ChlH and ChlK overexpression led to a substantial increase in the production of 2-oxoglutaric acid, reaching approximately 25mM and 50mM, respectively. Furthermore, ChlH and ChlK overexpression also significantly increased the secretory production of dicarboxylic acids, including 2-oxoglutaric acid, in the yellow koji fungus, Aspergillus oryzae. Our study demonstrates that overexpression of DHA1 family gene results in enhanced secretion of organic acids in koji fungi of the genus Aspergillus.

Addition of α-ketoglutaric acid (AKG) reduces deamination in Chinese perch (Siniperca chuatsi) fed with fermented soybean meal as a substitute for fishmeal.

To alleviate amino acid imbalances in fermented soybean meal as a replacement for fishmeal feeds, this study evaluated the effects of adding lysine (Lys), methionine (Met), and α-ketoglutaric acid (AKG) to fermented soybean meals for Chinese perch. Chinese perch (34 ± 3g) were fed five diets for 66days (fishmeal as the protein source of the basal diet [FM]; fermented soybean meal as a substitute for 30% fishmeal in the soybean meal diet [FSM]; addition of crystalline Lys and Met [AA]; addition of α-ketoglutaric acid [AKG]; and simultaneous addition of crystalline Lys, Met, and AKG [BA] to the soybean meal diet). At the end of the feeding trial, the FSM group had the highest feeding rate and the lowest weight gain rate among all the groups. The FM group had the highest protein retention and the lowest feed efficiency among the groups. The mRNA transcription level of genes related to the AMP-activating protein (AMPK) signaling pathway and amino acid response (AAR) signaling pathway (lkb1, atf4, and gcn2) were highest in the AA group (P < 0.05) but lower in the AKG and BA groups. In the AKG group, the mRNA transcription level of the gluconeogenesis pathway-related gene (pepck and g6pase) was significantly higher than that in the other four groups, but the mRNA transcription level of genes related to amino acid catabolism (gdh and ampd) was lower. Among all the groups, the FSM group had the lowest mRNA transcription level of genes associated with the mammalian target of rapamycin (mTOR) signaling pathway (mtor and s6k). These findings imply that the feeding rate of Chinese perch in the fermented soybean meal group was the highest, but the protein retention was the lowest, while the addition of Lys, Met, and AKG improved protein retention. In conclusion, the addition of AKG to fermented soybean meal as a fishmeal substitute reduced amino acid deamination, enhanced gluconeogenesis, and increased protein deposition, which contributed to the growth of Chinese perch, alleviated amino acid imbalances, and improved the feed utilization of Chinese perch.

Glutaminolysis of CD4+ T Cells: A Potential Therapeutic Target in Viral Diseases.

CD4+ T cells play a critical role in the pathogenesis of viral diseases, which are activated by the internal metabolic pathways encountering with viral antigens. Glutaminolysis converts glutamine into tricarboxylicacid (TCA) circulating metabolites by α-ketoglutaric acid, which is essential for the proliferation and differentiation of CD4+ T cells and plays a central role in providing the energy and structural components needed for viral replication after the virus hijacks the host cell. Changes in glutaminolysis in CD4+ T cells are accompanied by changes in the viral status of the host cell due to competition for glutamine between immune cells and host cells. More recently, attempts have been made to treat tumours, autoimmune diseases, and viral diseases by altering the breakdown of glutamine in T cells. In this review, we will discuss the current knowledge of glutaminolysis in the CD4+ T cell subsets from viral diseases, not only increasing our understanding of immunometabolism but also providing a new perspective for therapeutic target in viral diseases.

Immobilization of Thermoplasma acidophilum Glucose Dehydrogenase and Isocitrate Dehydrogenase Through Enzyme-Inorganic Hybrid Nanocrystal Formation

The development of green catalysts, specifically biocatalysts, is crucial for building a sustainable society. To enhance the versatility of biocatalysts, the immobilization of enzymes plays a vital role as it improves their recyclability and robustness. As target enzymes to immobilize, glucose dehydrogenases and carboxylases are particularly important among various kinds of enzymes due to their involvement in two significant reactions: regeneration of the reduced form of coenzyme required for various reactions, and carboxylation reactions utilizing CO2 as a substrate, respectively. In this study, we immobilized Thermoplasma acidophilum glucose dehydrogenase (TaGDH) and T. acidophilum isocitrate dehydrogenase (TaIDH) using a previously reported method involving the formation of enzyme-inorganic hybrid nanocrystals, in the course of our continuing study focusing on carboxylation catalyzed by the free form of TaGDH and TaIDH. Subsequently, we investigated the properties of the resulting immobilized enzymes. Our results indicate the successful immobilization of TaGDH and TaIDH through the formation of hybrid nanocrystals utilizing Mn2+. The immobilization process enhanced TaIDH activity, up to 211%, while TaGDH retained 71% of its original activity. Notably, the immobilized TaGDH exhibited higher activity at temperatures exceeding 87 °C than the free TaGDH. Moreover, these immobilized enzymes could be recycled. Finally, we successfully utilized the immobilized enzymes for the carboxylation of 2-ketoglutaric acid under 1 MPa CO2. In conclusion, this study represents the first immobilization of TaGDH and TaIDH using the hybrid nanocrystal forming method. Furthermore, we achieved significant activity enhancement of TaIDH through immobilization and demonstrated the recyclability of the immobilized enzymes.

LC-MS/MS platform-based serum untargeted screening reveals the diagnostic biomarker panel and molecular mechanism of breast cancer

BackgroundBreast cancer (BC), the most common form of malignant cancer affecting women worldwide, was characterized by heterogeneous metabolic disorder and lack of effective biomarkers for diagnosis. The purpose of this study is to search for reliable metabolite biomarkers of BC as well as triple-negative breast cancer (TNBC) using serum metabolomics approach. MethodsIn this study, an untargeted metabolomics technique based on ultra-high performance liquid chromatography combined with mass spectrometry (UHPLC-MS) was utilized to investigate the differences in serum metabolic profile between the BC group (n = 53) and non-BC group (n = 57), as well as between TNBC patients (n = 23) and non-TNBC subjects (n = 30). The multivariate data analysis, determination of the fold change and the Mann-Whitney U test were used to screen out the differential metabolites. Additionally, machine learning methods including receiver operating curve analysis and logistic regression analysis were conducted to establish diagnostic biomarker panels. ResultsThere were 36 metabolites found to be significantly different between BC and non-BC groups, and 12 metabolites discovered to be significantly different between TNBC and non-TNBC patients. Results also showed that four metabolites, including N-acetyl-D-tryptophan, 2-arachidonoylglycerol, pipecolic acid and oxoglutaric acid, were considered as vital biomarkers for the diagnosis of BC and non-BC with an area under the curve (AUC) of 0.995. Another two-metabolite panel of N-acetyl-D-tryptophan and 2-arachidonoylglycerol was discovered to discriminate TNBC from non-TNBC and produced an AUC of 0.965. ConclusionThis study demonstrated that serum metabolomics can be used to identify BC specifically and identified promising serum metabolic markers for TNBC diagnosis.

Hybrid nutraceutical of 2-ketoglutaric acid in improving inflammatory bowel disease: Role of prebiotics and TAK1 inhibitor

The main cause of inflammatory bowel disease (IBD) is abnormal intestinal permeability due to the disruption of the tight junction of the intestinal barrier through a pathogen-mediated inflammatory mechanism and an imbalance of the gut microbiota. This study aimed to evaluate whether 2-ketoglutaric acid alleviated permeability dysfunction with tight junction localization, activated the transforming growth factor beta-activated kinase 1 (TAK1) inflammation pathway, and regulated the homeostasis of the intestinal microbiome in vitro and in vivo IBD model. Our findings revealed that 2-ketoglutaric acid significantly suppressed abnormal intestinal permeability, delocalization of tight junction proteins from the intestinal cell, expression of inflammatory cytokines, such as TNF-α, both in vitro and in vivo. 2-Ketoglutaric acid was found to directly bind to TAK1 and inhibit the TNF receptor-associated factor 6 (TRAF6)–TAK1 interaction, which is related to the activation of nuclear factor kappa B (NF-κB) pathways, thereby regulating the expression of mitogen-activated protein kinase. Dietary 2-ketoglutaric acid also alleviated gut microbiota dysbiosis and IBD symptoms, as demonstrated by improvements in the intestine length and the abundance of Ligilactobacillus, Coriobacteriaceae_UCG_002, and Ruminococcaceae_unclassified in mice with colitis. This study indicated that 2-ketoglutaric acid binds to TAK1 for activity inhibition which is related to the NF-κB pathway and alleviates abnormal permeability by regulating tight junction localization and gut microbiome homeostasis. Therefore, 2-ketoglutaric acid is an effective nutraceutical agent and prebiotic for the treatment of IBD.

Biochemical characterization and differential expression of genes associated with apple tolerance to Penicillium expansum

The so-called blue mold is one of the most important fungal pathologies causing major pome fruits postharvest losses. To understand apple defense mechanisms, great interest has been put on aspects such as changes in fruit tissue gene expression or biochemical composition during infections. In the present study, the expression patterns of 15 genes and isoforms previously reported to be involved in the defense response of apple against Penicillium expansum were analyzed. Thus, 28 apple accessions from the germplasm bank of the Experimental Station of Aula Dei (EEAD-CSIC) (NE Spain) were wounded and artificially inoculated with P. expansum. At 14 days post inoculation (dpi), lesion diameter and lesion extension were measured, ranking apple accessions according to their susceptibility/tolerance. In addition, several biochemical components were assessed in healthy fruits at the moment of inoculation (0 dpi) and after two weeks of incubation (14 dpi). Sugar and organic acid concentrations, total flavonoids content and ascorbic acid levels were measured. Enzyme activities, including phenylalanine ammonia-lyase (PAL), peroxidase (POX), and polyphenol oxidase (PPO), were also assessed. In summary, some parameters exhibited significant negative correlations with susceptibility, such as PPO enzymatic activity at 0 dpi and total sugars at harvest. Furthermore, positive correlations were identified, including total sugars at 0 dpi and 14 dpi, α-ketoglutaric acid at 14 dpi, total organic acids, and quinic acid at harvest. Notably, the variation in some biochemical components during infection, such as PPO activity, significantly correlated with ‘lesion diameter’. Concerning gene expression, 10 accessions were studied. The PAL1 and the allergen Mal d 1.03 were found to be induced by the inoculation with P. expansum. Furthermore, LDOX and Mal d 1.03 were differentially expressed between tolerant and susceptible cultivars at 14 dpi. In addition, their expression level was positively correlated with tolerance, making them potential biomarkers for future selection programs.

Optimization of conditions to achieve a high content of alizarin and purpurin in the adventitious roots of Rubia cordifolia L.

Rubia cordifolia L. (Indian madder) is a valuable medicinal plant known for its natural dye content, notably alizarin and purpurin, with applications in textiles and cosmetics. This study aims to optimize the concentrations of auxin, salicylic acid, elicitors, and precursors to maximize the production of alizarin and purpurin in adventitious roots. Various concentrations and combinations of auxins were systematically tested to explore their effects on the yield of adventitious root formation and dye compounds. Nodal explants cultivated in MS media supplemented with IAA + IBA + NAA (1.5 + 0.5 + 0.75 mg/L) at a pH of 5.7 exhibited the highest adventitious root production. Post-establishment of root cultures, the impact of salicylic acid, L-phenylalanine, elicitors (yeast extract, pectin, and Xylan), and the precursor α-ketoglutaric acid on the production of anthraquinones (AQ) was evaluated. High-performance liquid Chromatography (HPLC) analysis revealed enriched alizarin, purpurin, and total AQ content. The maximum amount of AQ (64.65 mg/g), alizarin (17.59 mg/g), and purpurin (19.61 mg/g) was observed when cultured on 30 mg/L of α-ketoglutaric acid, compared to other elicitors and precursors. This optimized condition provides valuable insights for the sustainable cultivation and utilization of R. cordifolia as a prominent source of natural dyes. It offers practical implications for the dye industry and its production.

Biological characteristics and functions of a novel glutamate dehydrogenase from Trichinella spiralis.

Glutamate dehydrogenase (GDH) plays an important role in the metabolism of organisms. Its high abundance in mitochondria in particular highlights its core role in cellular physiological processes. GDH catalyzes the mutual conversion between L-glutamic acid and α-ketoglutaric acids. At the same time, this transformation is accompanied by the oxidation-reduction of NAD(H) or NADP(H). This process not only helps to link amino acid metabolism with sugar metabolism, but also helps maintain the balance of intracellular pH and nitrogen homeostasis. In this study, a novel Trichinella spiralis glutamate dehydrogenase (TsGDH) was cloned, expressed and identified. The results revealed that TsGDH was expressed at various stages of development of the nematode T. spiralis, with higher expression levels in the adult worm stage, and was mainly localized in the cuticle, muscular layer, stichosome and female intrauterine embryos. After RNAi treatment, larval natural TsGDH enzyme activity was obviously reduced, and metabolism, molting, growth and reproduction were also significantly inhibited. The results indicate that TsGDH plays an important role in the development and survival of T. spiralis, and it may be a potential molecular target of anti-Trichinella vaccines and drugs.

Preliminary screening of biomarkers and drug candidates in a mouse model of β-thalassemia based on quasi-targeted metabolomics.

β-thalassemia (β-TH) is a hereditary hemolytic anemia that results in deficient hemoglobin (Hb) synthesis. It is characterized by ineffective erythropoiesis, anemia, splenomegaly, and systemic iron overload. Exploration new potential biomarkers and drug candidates is important to facilitate the prevention and treatment of β-TH. We applied quasi-targeted metabolomics between wild type (Wt) and heterozygous β-TH mice (Th3/+), a model of non-transfusion-dependent β-TH intermedia, in plasma and peripheral blood (PB) cells. Futher data was deeply mined by Kyoto Encyclopedia of Genomes (KEGG) and machine algorithms methods. Using KEGG enrichment analysis, we found that taurine and hypotaurine metabolism disorders in plasma and alanine, aspartate and glutamate metabolism disorders in PB cells. After systematically anatomize the metabolites by machine algorithms, we confirmed that alpha-muricholic acidUP and N-acetyl-DL-phenylalanineUP in plasma and Dl-3-hydroxynorvalineUP, O-acetyl-L-serineUP, H-abu-OHUP, S-(Methyl) glutathioneUP, sepiapterinDOWN, and imidazoleacetic acidDOWN in PB cells play key roles in predicting the occurrence of β-TH. Furthermore, Sepiapterin, Imidazoleacetic acid, Methyl alpha-D-glucopyranoside and alpha-ketoglutaric acid have a good binding capacity to hemoglobin E through molecular docking and are considered to be potential drug candidates for β-TH. Those results may help in identify useful molecular targets in the diagnosis and treatment of β-TH and lays a strong foundation for further research.

Comparative metabolomics of MCF-7 and MCF-7/TAMR identifies potential metabolic pathways in tamoxifen resistant breast cancer cells.

Breast cancer is the most common cancer and the leading cause of cancer-related death among women. An Estrogen Receptor (ER) antagonist called tamoxifen is used as an adjuvant therapy for ER-positive breast cancers. Approximately 40% of patients develop tamoxifen resistance (TAMR) while receiving treatment. Cancer cells can rewire their metabolism to develop resistant phenotypes, and their metabolic state determines how receptive they are to chemotherapy. Metabolite extraction from human MCF-7 and MCF-7/TAMR cells was done using the methanol-methanol-water extraction method. After treating the dried samples with methoxamine hydrochloride in pyridine, the samples were derivatized with 2,2,2-Trifluoro-N-methyl-N-(trimethylsilyl)-acetamide, and Chlorotrimethylsilane (MSTFA + 1% TMCS). The Gas chromatography/mass spectrometry (GC-MS) raw data were processed using MSdial and Metaboanalyst for analysis. Univariate analysis revealed that 35 metabolites were elevated in TAMR cells whereas 25 metabolites were downregulated. N-acetyl-D-glucosamine, lysine, uracil, tyrosine, alanine, and o-phosphoserine were upregulated in TAMR cells, while hydroxyproline, glutamine, N-acetyl-L-aspartic acid, threonic acid, pyroglutamic acid, glutamine, o-phosphoethanolamine, oxoglutaric acid, and myoinositol were found to be downregulated. Multivariate analysis revealed a distinct separation between the two cell lines, as evidenced by their metabolite levels. The enriched pathways of deregulated metabolites included valine, leucine, and isoleucine degradation, Citric Acid Cycle, Warburg effect, Malate-Aspartate shuttle, glucose-alanine cycle, propanoate metabolism, and Phospholipid biosynthesis. This study revealed dysregulation of various metabolic processes in TAMR cells, which may be crucial in elucidating the molecular basis of the mechanisms underlying acquired tamoxifen resistance.

Capillary Zone Electrophoresis with Diode-Array Detection as a Tool to Monitor Variations in the Concentration of Organic Acids from the Tricarboxylic Acid Cycle in Human Urine

The tricarboxylic acid (TCA) cycle is responsible for the generation of energy in the human body and may inform about the energy status in different physiological and pathological conditions. It was developed a method to quantify the TCA cycle intermediates (α-ketoglutaric, citric, fumaric, lactic, malic, and succinic acids) by capillary zone electrophoresis with diode array detector (CZE-DAD) in indirect mode. The background electrolyte consisted of 24 mmol L–1 2,6-pyridine carboxylic acid, 76 mmol L–1 β-alanine, and 4 mmol L–1 hexadecyltrimethylammonium hydroxide (pH 4). The method was validated and presented a good linearity (R2 &gt; 0.9922) and adequate limits of detection (0.24-1.84 mg L–1) and quantification (0.74-5.57 mg L–1). The method was successfully applied to samples of individuals with obesity before being submitted to bariatric surgery and one year after the surgery. The citric, lactic, and malic acids were detected in the analyzed samples. To evaluate the changes in concentrations of the TCA intermediates, principal component analysis was carried out, and there was no significant difference between the two sets of samples. Altogether, the data obtained after applying this new method suggest that CZE-DAD could be used to detect and quantify the TCA cycle metabolites in human urine.

Glutamine Metabolism Promotes Renal Fibrosis through Regulation of Mitochondrial Energy Generation and Mitochondrial Fission.

Fibroblast activation and proliferation is an essential phase in the progression of renal fibrosis. Despite the recognized significance of glutamine metabolism in cellular growth and proliferation, its precise pathophysiological relevance in renal fibrosis remains uncertain. Therefore, this study aims to investigate the involvement of glutamine metabolism in fibroblast activation and its possible mechanism. Our findings highlight the importance of glutamine metabolism in fibroblast activation and reveal that patients with severe fibrosis exhibit elevated serum glutamine levels and increased expression of kidney glutamine synthetase. Furthermore, the deprivation of glutamine metabolism in vitro and in vivo could inhibit fibroblast activation, thereby ameliorating renal fibrosis. It was also detected that glutamine metabolism is crucial for maintaining mitochondrial function and morphology. These effects may partially depend on the metabolic intermediate α-ketoglutaric acid. Moreover, glutamine deprivation led to upregulated mitochondrial fission in fibroblasts and the activation of the mammalian target of rapamycin / mitochondrial fission process 1 / dynamin-related protein 1 pathway. Thus, these results provide compelling evidence that the modulation of glutamine metabolism initiates the regulation of mitochondrial function, thereby facilitating the progression of renal fibrosis. Consequently, targeting glutamine metabolism emerges as a novel and promising avenue for therapeutic intervention and prevention of renal fibrosis.

Metabolomics studies in common multifactorial eye disorders: a review of biomarker discovery for age-related macular degeneration, glaucoma, diabetic retinopathy and myopia.

Multifactorial Eye disorders are a significant public health concern and have a huge impact on quality of life. The pathophysiological mechanisms underlying these eye disorders were not completely understood since functional and low-throughput biological tests were used. By identifying biomarkers linked to eye disorders, metabolomics enables early identification, tracking of the course of the disease, and personalized treatment. The electronic databases of PubMed, Scopus, PsycINFO, and Web of Science were searched for research related to Age-Related macular degeneration (AMD), glaucoma, myopia, and diabetic retinopathy (DR). The search was conducted in August 2023. The number of cases and controls, the study's design, the analytical methods used, and the results of the metabolomics analysis were all extracted. Using the QUADOMICS tool, the quality of the studies included was evaluated, and metabolic pathways were examined for distinct metabolic profiles. We used MetaboAnalyst 5.0 to undertake pathway analysis of differential metabolites. Metabolomics studies included in this review consisted of 36 human studies (5 Age-related macular degeneration, 10 Glaucoma, 13 Diabetic retinopathy, and 8 Myopia). The most networked metabolites in AMD include glycine and adenosine monophosphate, while methionine, lysine, alanine, glyoxylic acid, and cysteine were identified in glaucoma. Furthermore, in myopia, glycerol, glutamic acid, pyruvic acid, glycine, cysteine, and oxoglutaric acid constituted significant metabolites, while glycerol, glutamic acid, lysine, citric acid, alanine, and serotonin are highly networked metabolites in cases of diabetic retinopathy. The common top metabolic pathways significantly enriched and associated with AMD, glaucoma, DR, and myopia were arginine and proline metabolism, methionine metabolism, glycine and serine metabolism, urea cycle metabolism, and purine metabolism. This review recapitulates potential metabolic biomarkers, networks and pathways in AMD, glaucoma, DR, and myopia, providing new clues to elucidate disease mechanisms and therapeutic targets. The emergence of advanced metabolomics techniques has significantly enhanced the capability of metabolic profiling and provides novel perspectives on the metabolism and underlying pathogenesis of these multifactorial eye conditions. The advancement of metabolomics is anticipated to foster a deeper comprehension of disease etiology, facilitate the identification of novel therapeutic targets, and usher in an era of personalized medicine in eye research.

Aluminum Supplementation Mediates the Changes in Tea Plant Growth and Metabolism in Response to Calcium Stress.

Tea plants are more sensitive to variations in calcium concentration compared to other plants, whereas a moderate aluminum concentration facilitates the growth and development of tea plants. Aluminum and calcium show a competitive interaction with respect to the uptake of elements, consequently exerting physiological effects on plants. To further explore these interactions, in this study, we used the solution culture method to treat tea plants with two calcium concentrations (0.8 mM and 5.6 mM) and three aluminum concentrations (0 mM, 0.4 mM, and 1 mM). We then determined the influence of the combined treatments on root growth and quality compound accumulation in the tissues by a combination of phenotype, gene expression, and metabolite analyses. Moderate aluminum supplementation (0.4 mM) alleviated the inhibition of root growth caused by high calcium stress. High calcium stress significantly inhibited the accumulation of most amino acids (e.g., Glutamic acid, Citulline, and Arginine) and organic acids (e.g., a-ketoglutaric acid) in the roots, stems, and leaves, whereas aluminum deficiency significantly increased most amino acids in the roots and leaves (except Serine, Alanine, and Phenylalanine in the roots and Ser in the leaves), with a more than two-fold increase in Arg and Lysine. High calcium stress also induced the accumulation of secondary metabolites such as epigallocatechin gallate and procyanidin in the roots, whereas aluminum supplementation significantly reduced the contents of flavonol glycosides such as quercetin, rutin, myricitrin, and kaempferitrin, as well as caffeine, regardless of calcium concentration. Aluminum supplementation reversed some of the changes in the contents of leaf metabolites induced by calcium stress (e.g., 4-dihydroquercetin, apigenin C-pentoside, phenethylamine, and caffeine). Overall, calcium stress caused severe growth inhibition and metabolic disorders in tea plants, which could be reversed by aluminum supplementation, particularly in maintaining the root tips and the accumulation of secondary metabolites. These results provide a theoretical basis for improving calcium-aluminum nutrient management to promote tea plant growth and quality.

Revealing the phosphate-solubilizing characteristics and mechanisms of the plant growth-promoting bacterium Agrobacterium deltaense C1.

This study explores the phosphate (Pi)-solubilizing characteristics and mechanisms of a novel phosphate-solubilizing bacterium, Agrobacterium deltaense C1 (C1 hereafter). The growth-promoting effects of C1 were investigated by gnotobiotic experiments, and the Pi-solubilizing mechanism was revealed by extracellular metabolomics, liquid chromatography analysis, and reverse transcription quantitative polymerase chain reaction. Results showed that C1 significantly increased Arabidopsis biomass and total phosphorus (P) content under P deficiency. Under Ca3(PO4)2 condition, the presence of C1 resulted in a significant and negative correlation between available P content and medium pH changes, implying that Pi dissolution occurs through acid release. Metabolomics revealed C1's ability to release 99 organic acids, with gluconic acid (GA), citric acid, and α-ketoglutaric acid contributing 64.86%, 9.58%, and 0.94%, respectively, to Pi solubilization. These acids were significantly induced by P deficiency. Moreover, C1's Pi solubilization may remain significant even in the presence of available P, as evidenced by substantial pH reduction and high gcd gene expression. Additionally, C1 produced over 10 plant growth-promoting substances. C1 dissolves Pi primarily by releasing GA, which enhances plant growth under P deficiency. Notably, its Pi solubilization effect is not significantly limited by available Pi.

Abstract B040: NCI 10129: A Phase 2 Study of the PARP inhibitor Olaparib (AZD2281) in IDH1 and IDH2 mutant advanced solid tumors

Abstract Introduction: Cholangiocarcinoma (CCA) is a highly aggressive malignancy with mutations in Isocitrate dehydrogenase (IDH) identified in approximately 15% of patients. Neomorphic mutations in IDH 1/2 result in accumulation of the oncometabolite 2-hydroxyglutarate (2G), which has been implicated in tumor progression via inhibitory effects on alpha ketoglutarate. Moreover, the mutant IDH dependent accumulation of 2HG results in homologous recombination deficiency (HRD) and renders these cells exquisitely sensitive to Poly (ADP-Ribose) polymerase (PARP) inhibitors. NCI 10129 evaluated the efficacy of olaparib for IDH mutated solid tumors, and here we present the CCA cohort. Methods: NCI 10129 is a 3 arm, open label phase 2 clinical trial performed in the NCI National Clinical Trials Network evaluating olaparib 300 mg twice daily for IDH mutated solid tumors refractory to standard treatment. Patients with CCA were enrolled in 2 cohorts 1) IDHi pre-treated CCA and 2) IDHi untreated CCA. The primary endpoint was overall response rate (ORR) and secondary endpoints includes progression-free survival (PFS), and overall survival (OS). An exploratory analysis measured the impact of circulating 2HG levels, circulating tumor DNA, and tumor genomics on patient outcomes. Results: Thirty patients with CCA enrolled on NCI10129 (15 each IDHi pre-treated and IDHi treatment naïve), 18 patients received ≥ 2 prior systemic treatments, and 23 patients had IDH1 mutations and 7 patients IDH2 mutations. There were no objective responses seen in the first 15 CCA patients in either cohort and enrollment was closed. The median PFS was 2.4 months 95% CI (1.9 - 6.5) and median OS 12.9 months 95% CI (6.3 – not reached). The IDHi pre-treated cohort had improved median OS compared to IDHi treatment naïve (15.3 vs 7.3, P = 0.04). There were 8/30 (27%) patients with clinical benefit (CB) as defined by PFS &amp;gt; 6 months. Patients with CB had lower baseline mean D-2HG levels compared to those without CB (1.4 umol/L vs 5.9 umol/L, p = 0.02). Whole exome sequencing revealed the study population to be enriched for HRD by sum scores and HRD mutational signatures without alternative HRD mutations other than IDH1/2. No improvement in survival was noted based on HRD status. RNA sequencing Gene Set Enrichment Analysis revealed that cell cycle and interferon alpha pathways were activated in patients with CB (p &amp;lt; 0.01). Olaparib was tolerable with the majority of adverse events grade 1-2. Conclusion: Olaparib did not result in objective responses in IDH mutated CCA. However, a subgroup of patients did demonstrate prolonged PFS comparable to historical controls for refractory CCA as well that seen with targeted agents such as IDHi. Exploratory analysis revealed this subgroup to be enriched for lower baseline 2HG levels. Serial ctDNA analysis is ongoing and will be presented. Given the CB subgroup and OS data, additional clinical trials leveraging the HRD properties of IDH mutations is warranted. Future studies may benefit from DNA damage combination therapies thereby enhancing potency. Citation Format: Michael Cecchini, Mary Jo Pilat, Nataliya Uboha, Nilofer S Azad, May Cho, Elizabeth J Davis, Jordi Rodon Ahnert, Gabriel Tinoco, Geoff I Shapiro, Simon Khagi, Benjamin Powers, Roman Groisberg, Jan Drappatz, Li Chen, Biswajit Das, Xun Bao, Jing Li, Abhijit Patel, Monica Niger, Deborah Doroshow, Diane Durecki, Scott Boerner, Ranjit Bindra, Percy Ivy, Yu Shyr, Patricia LoRusso. NCI 10129: A Phase 2 Study of the PARP inhibitor Olaparib (AZD2281) in IDH1 and IDH2 mutant advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B040.

Imbalanced metabolism induced NH4+ accumulation and its effect on the central metabolism of Methylomonas sp. ZR1.

Nitrogen and carbon are the two most essential nutrient elements, and their metabolism is tightly coupled in single carbon metabolic microorganisms. However, the nitrogen metabolism and the nitrogen/carbon (N/C) metabolic balance in single-carbon metabolism is poorly studied. In this study, the nitrogen metabolism pattern of the fast growing methanotrophs Methylomonas sp. ZR1 grown in methane and methanol was studied. Effect study of different nitrogen sources on the cell growth of ZR1 indicates that nitrate salts are the best nitrogen source supporting the growth of ZR1 using methane and methanol as carbon source. However, its metabolic intermediate ammonium was found to accumulate with high N/C ratio in the medium and consequently inhibit the growth of ZR1. Studies of carbon and nitrogen metabolic kinetic under different N/C ratio conditions indicate that the accumulation of NH4+ is caused by the imbalanced nitrogen and carbon metabolism in ZR1. Feeding carbon skeleton α-ketoglutaric acid could effectively relieve the inhibition effect of NH4+ on the growth of ZR1, which further confirms this assumption. qPCR analysis of the expression level of the central metabolic key enzyme gene indicates that the nitrogen metabolic intermediate ammonium has strong regulation effect on the central nitrogen and carbon metabolism in ZR1. qPCR-combined genomic analysis confirms that a third ammonium assimilation pathway glycine synthesis system is operated in ZR1 to balance the nitrogen and carbon metabolism. Based on the qPCR result, it was also found that ZR1 employs two strategies to relieve ammonium stress in the presence of ammonium: assimilating excess ammonium or cutting off the nitrogen reduction reactions according to the available C1 substrate. Validating the connections between single-carbon and nitrogen metabolism and studying the accumulation and assimilation mechanism of ammonium will contribute to understand how nitrogen regulates cellular growth in single-carbon metabolic microorganisms.

Polyelectrolyte Complex Hydrogels from Controlled Kneading and Annealing-Induced Tightly Wound and Highly Entangled Natural Polysaccharides.

Hydrogels usually are fabricated by using monomers or preexisting polymers in precursor solutions. Here, a polyelectrolyte complex biohydrogel (Bio-PEC hydrogel) made from a precursor dough, by kneading, annealing, and crosslinking the dough of two oppositely charged polysaccharides, cationic chitosan quaternary ammonium salt (HACC) and anionic sodium hyaluronate (HA), photoinitiator (α-ketoglutaric acid), crosslinker glycidyl methacrylate (GMA), and water of very small quantity is reported. Controlled kneading and annealing homogenized the dough with respect to transforming randomly distributed, individual polymer chains into tightly wound double-stranded structures, which, upon UV irradiation, covalently sparsely crosslinked into a highly entangled network and subsequently, upon fully swollen in water, results in Bio-PEC hydrogel, HACC/HA, exhibiting near-perfect elasticity, high tensile strength, and high swelling resistance. Via the same kneading and annealing, tetracarboxyphenylporphyrin iron (Fe-TCPP) metal nanoclusters are incorporated into HACC/HA to obtain photocatalytic, antibacterial, and biocompatible Bio-PEC hydrogel composite, Fe-TCPP@HACC/HA. Using SD rat models, the efficacy of Fe-TCPP@HACC/HA in inhibiting Escherichia coli (E. coli) growth in vitro and the ability to promote wound healing and scar-free skin regeneration in vivo, or its high potential as a wound dressing material for biomedical applications are demonstrated.