Central Carbon Metabolism Research Articles

Providing insight into the mechanism of action of cationic lipidated oligomers using metabolomics.

The increasing resistance of clinically relevant microbes against current commercially available antimicrobials underpins the urgent need for alternative and novel treatment strategies. Cationic lipidated oligomers (CLOs) are innovative alternatives to antimicrobial peptides and have reported antimicrobial potential. An understanding of their antimicrobial mechanism of action is required to rationally design future treatment strategies for CLOs, either in monotherapy or synergistic combinations. In the present study, metabolomics was used to investigate the potential metabolic pathways involved in the mechanisms of antibacterial activity of one CLO, C12-o-(BG-D)-10, which we have previously shown to be effective against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300. The metabolomes of MRSA ATCC 43300 at 1, 3, and 6 h following treatment with C12-o-(BG-D)-10 (48 µg/mL, i.e., 3× MIC) were compared to those of the untreated controls. Our findings reveal that the studied CLO, C12-o-(BG-D)-10, disorganized the bacterial membrane as the first step toward its antimicrobial effect, as evidenced by marked perturbations in the bacterial membrane lipids and peptidoglycan biosynthesis observed at early time points, i.e., 1 and 3 h. Central carbon metabolism and the biosynthesis of DNA, RNA, and arginine were also vigorously perturbed, mainly at early time points. Moreover, bacterial cells were under osmotic and oxidative stress across all time points, as evident by perturbations of trehalose biosynthesis and pentose phosphate shunt. Overall, this metabolomics study has, for the first time, revealed that the antimicrobial action of C12-o-(BG-D)-10 may potentially stem from the dysregulation of multiple metabolic pathways.IMPORTANCEAntimicrobial resistance poses a significant challenge to healthcare systems worldwide. Novel anti-infective therapeutics are urgently needed to combat drug-resistant microorganisms. Cationic lipidated oligomers (CLOs) show promise as new antibacterial agents against Gram-positive pathogens like methicillin-resistant Staphylococcus aureus (MRSA). Understanding their molecular mechanism(s) of antimicrobial action may help design synergistic CLO treatments along with monotherapy. Here, we describe the first metabolomics study to investigate the killing mechanism(s) of CLOs against MRSA. The results of our study indicate that the CLO, C12-o-(BG-D)-10, had a notable impact on the biosynthesis and organization of the bacterial cell envelope. C12-o-(BG-D)-10 also inhibits arginine, histidine, central carbon metabolism, and trehalose production, adding to its antibacterial characteristics. This work illuminates the unique mechanism of action of C12-o-(BG-D)-10 and opens an avenue to design innovative antibacterial oligomers/polymers for future clinical applications.

Comparative transcriptome analysis reveals the redirection of metabolic flux from cell growth to astaxanthin biosynthesis in Yarrowia lipolytica.

Engineering Yarrowia lipolytica to produce astaxanthin provides a promising route. Here, Y. lipolytica M2 producing a titer of 181 mg/L astaxanthin was isolated by iterative atmospheric and room-temperature plasma mutagenesis and diphenylamine-mediated screening. Interestingly, a negative correlation was observed between cell biomass and astaxanthin production. To reveal the underlying mechanism, RNA-seq analysis of transcriptional changes was performed in high producer M2 and reference strain M1, and a total of 1379 differentially expressed genes were obtained. Data analysis revealed that carbon flux was elevated through lipid metabolism, acetyl-CoA and mevalonate supply, but restrained through central carbon metabolism in strain M2. Moreover, upregulation of other pathways such as ATP-binding cassette transporter and thiamine pyrophosphate possibly provided more cofactors for carotenoid hydroxylase and relieved cell membrane stress caused by astaxanthin insertion. These results suggest that balancing cell growth and astaxanthin production may be important to promote efficient biosynthesis of astaxanthin in Y. lipolytica.

A Point Mutation in Cassette Relieves the Repression Regulation of CcpA Resulting in an Increase in the Degradation of 2,3-Butanediol in Lactococcus lactis

In lactic acid bacteria, the global transcriptional regulator CcpA regulates carbon metabolism by repressing and activating the central carbon metabolism pathway, thus decreasing or increasing the yield of certain metabolites to maximize carbon flow. However, there are no reports on the deregulation of the inhibitory effects of CcpA on the metabolism of secondary metabolites. In this study, we identified a single-base mutant strain of Lactococcus lactis N8-2 that is capable of metabolizing 2,3-butanediol. It has been established that CcpA dissociates from the catabolite responsive element (cre) site due to a mutation, leading to the activation of derepression and expression of the 2,3-butanediol dehydrogenase gene cluster (butB and butA). Transcriptome analysis and quantitative polymerase chain reaction (Q-PCR) results showed significant upregulation of transcription of butB and butA compared to the unmutated strain. Furthermore, micro-scale thermophoresis experiments confirmed that CcpA did not bind to the mutated cre. Furthermore, in a bacterial two-plasmid fluorescent hybridization system, it was similarly confirmed that the dissociation of CcpA from cre eliminated the repressive effect of CcpA on downstream genes. Finally, we investigated the differing catalytic capacities of the 2,3-butanediol dehydrogenase gene cluster in L. lactis N8-1 and L. lactis N8-2 for 2,3-butanediol. This led to increased expression of butB and butA, which were deregulated by CcpA repression. This is the first report on the elimination of the deterrent effect of CcpA in lactic acid bacteria, which changes the direction of enzymatic catalysis and alters the direction of carbon metabolism. This provides new perspectives and strategies for metabolizing 2,3-butanediol using bacteria in synthetic biology.

Transcriptional and Metabolic Response of a Strain of Escherichia coli PTS− to a Perturbation of the Energetic Level by Modification of [ATP]/[ADP] Ratio

The intracellular [ATP]/[ADP] ratio is crucial for Escherichia coli’s cellular functions, impacting transport, phosphorylation, signaling, and stress responses. Overexpression of F1-ATPase genes in E. coli increases glucose consumption, lowers energy levels, and triggers transcriptional responses in central carbon metabolism genes, particularly glycolytic ones, enhancing carbon flux. In this contribution, we report the impact of the perturbation of the energetic level in a PTS− mutant of E. coli by modifying the [ATP]/[ADP] ratio by uncoupling the cytoplasmic activity of the F1 subunit of the ATP synthase. The disruption of [ATP]/[ADP] ratio in the evolved strain of E. coli PB12 (PTS−) was achieved by the expression of the atpAGD operon encoding the soluble portion of ATP synthase F1-ATPase (strain PB12AGD+). The analysis of the physiological and metabolic response of the PTS− strain to the ATP disruption was determined using RT–qPCR of 96 genes involved in glucose and acetate transport, glycolysis and gluconeogenesis, pentose phosphate pathway (PPP), TCA cycle and glyoxylate shunt, several anaplerotic, respiratory chain, and fermentative pathways genes, sigma factors, and global regulators. The apt mutant exhibited reduced growth despite increased glucose transport due to decreased energy levels. It heightened stress response capabilities under glucose-induced energetic starvation, suggesting that the carbon flux from glycolysis is distributed toward the pentose phosphate and the Entner–Duodoroff pathway with the concomitant. Increase acetate transport, production, and utilization in response to the reduction in the [ATP]/[ADP] ratio. Upregulation of several genes encoding the TCA cycle and the glyoxylate shunt as several respiratory genes indicates increased respiratory capabilities, coupled possibly with increased availability of electron donor compounds from the TCA cycle, as this mutant increased respiratory capability by 240% more than in the PB12. The reduction in the intracellular concentration of cAMP in the atp mutant resulted in a reduced number of upregulated genes compared to PB12, suggesting that the mutant remains a robust genetic background despite the severe disruption in its energetic level.

Functional proteomics reveals that Slr0237 is a SigE-regulated glycogen debranching enzyme pivotal for glycogen breakdown.

The group 2 σ factor for RNA polymerase SigE plays important role in regulating central carbon metabolism in cyanobacteria. However, the regulation of SigE for these pathways at a proteome level remains unknown. Using a sigE-deficient strain (ΔsigE) of Synechocystis sp. PCC 6803 and quantitative proteomics, we found that SigE depletion induces differential protein expression for sugar catabolic pathways including glycolysis, oxidative pentose phosphate (OPP) pathway, and glycogen catabolism. Two glycogen debranching enzyme homologues Slr1857 and Slr0237 are found differentially expressed in ΔsigE. Glycogen determination indicated that Δslr0237 accumulated glycogen under photomixotrophic condition but was unable to utilize these reserves in the dark, whereas Δslr1857 accumulates and utilizes glycogen in a similar way as the WT strain does in the same condition. These results suggest that Slr0237 plays the major role as the glycogen debranching enzyme in Synechocystis.

Kidney-tonifying blood-activating decoction delays ventricular remodeling in rats with chronic heart failure by regulating gut microbiota and metabolites and p38 mitogen-activated protein kinase/p65 nuclear factor kappa-B/aquaporin-4 signaling pathway

Ethnopharmacological relevanceMyocardial infarction has likely contributed to the increased prevalence of heart failure(HF).As a result of ventricular remodeling and reduced cardiac function, colonic blood flow decreases, causing mucosal ischemia and hypoxia of the villous structure of the intestinal wall.This damage in gut barrier function increases bowel wall permeability, leading to fluid metabolism disorder,gut microbial dysbiosis, increased gut bacteria translocation into the circulatory system and increased circulating endotoxins, thus promoting a typical inflammatory state.Traditional Chinese Medicine plays a key role in the prevention and treatment of HF.Kidney-tonifying Blood-activating(KTBA) decoction has been proved for clinical treatment of chronic HF.However,the mechanism of KTBA decoction on chronic HF is still unclear. Aims of the studyThe effect of KTBA decoction on gut microbiota and metabolites and p38MAPK/p65NF-κB/AQP4 signaling in rat colon was studied to investigate the mechanism that KTBA decoction delays ventricular remodeling and regulates water metabolism disorder in rats with HF after myocardial infarction based on the theory of "Kidney Storing Essence and Conducting Water". Material and methodsIn vivo,a rat model of HF after myocardial infarction was prepared by ligating the left anterior descending coronary artery combined with exhaustive swimming and starvation.The successful modeling rats were randomly divided into five groups:model group, tolvaptan group(gavaged 1.35mg/(kg•D) tolvaptan),KTBA decoction group(gavaged 15.75g/(kg•D) of KTBA decoction),KTBA decoction combined with SB203580(p38MAPK inhibitor) group(gavaged 15.75g/(kg•D) of KTBA decoction and intraperitoneally injected 1.5mg/(kg•D) of SB203580),and KTBA decoction combined with PDTC(p65NF-kB inhibitor) group(gavaged 15.75g/(kg•D) of KTBA decoction and intraperitoneally injected 120mg/(kg•D) of PDTC).The sham-operation group and model group were gavaged equal volume of normal saline.After 4 weeks of intervention with KTBA decoction,the effect of KTBA decoction on the cardiac structure and function of chronic HF model rats was observed by ultrasonic cardiogram.General state and cardiac index in rats were evaluated.Enzyme linked immunosorbent assay(ELISA) was used to measure N-terminal pro-brain natriuretic peptide (NT-proBNP) concentration in rat serum.Hematoxylin and eosin(H&E) staining,and transmission electron microscope(TEM) were used to observe the morphology and ultrastructure of myocardial and colonic tissue,and myocardial fibrosis was measured by Masson's staining.Cardiac E-cadherin level was detected by Western blot.The mRNA expression and protein expression levels of p38MAPK,I-κBα, p65NF-κB,AQP4,Occludin and ZO-1 in colonic tissue were detected by reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR) and immunohistochemistry. Protein expression of p38MAPK, p-p38MAPK,I-κBα,p-I-κBα,p65NF-κB, p-p65NF-κB,AQP4,Occludin and ZO-1 in rat colon was detected using Western blot.Colonic microbiota and serum metabolites were respectively analyzed by amplicon sequencing and liquid chromatography-mass spectrometry.In vitro, CCD-841CoN cell was placed in the ischemic solution under hypoxic conditions (94%N2,5%CO2,and 1%O2) in a 37 °C incubator to establish an ischemia and hypoxia model.The CCD-841CoN cells were divided into 7 groups, namely blank group and model group with normal rat serum plus control siRNA, tolvaptan group with rat serum containing tolvaptan plus control siRNA, KTBA group with rat serum containing KTBA plus control siRNA, KTBA plus p38MAPK siRNA group, KTBA plus p65NF-κB siRNA group,and KTBA plus AQP4siRNA group.After 24h and 48h of intervention with KTBA decoction,RT-qPCR,immunofluorescence and Western blot was used to detect the mRNA expression and protein expression levels of p38MAPK,I-κBα,p65NF-κB,AQP4, Occludin and ZO-1 in CCD-841CoN cells. ResultsCompared with the model, KTBA decoction improved the general state, decraesed the serum NT-proBNP level,HW/BW ratio, LVIDd and LVIDs, increased E-cadherin level,EF and FS,reduced number of collagen fibers deposited in the myocardial interstitium,and recovered irregular arrangement of myofibril and swollen or vacuolated mitochondria with broken crista in myocardium.Moreover, KTBA decoction inhibited the expression of p38MAPK,I-κBα,and p65NF-κB and upregulated AQP4, Occludin and ZO-1 in colon tissues and CCD-841CoN cells.Additionally,p38siRNA or SB203580, p65siRNA or PDTC, and AQP4siRNA partially weakened the protective effects of KTBA in vitro and vivo.Notably,The LEfSe analysis results showed that there were six gut biomaker bacteria in model group, including Allobaculum, Bacillales,Turicibacter, Turicibacterales,Turicibacteraceae,and Bacilli. Besides, three gut biomaker bacteria containing Deltaproteobacteria, Desulfovibrionaceae,and Desulfovibrionales were enriched by KTBA treatment in chronic HF model.There were five differential metabolites, including L-Leucine,Pelargonic acid, Capsidiol,beta-Carotene,and L- Erythrulose, which can be regulated back in the same changed metabolic routes by the intervention of KTBA.L-Leucine had the positive correlation with Bacillales, Turicibacterales,Turicibacteraceae,and Turicibacter.L-Leucine significantly impacts Protein digestion and absorption, Mineral absorption,and Central carbon metabolism in cancer regulated by KTBA, which is involved in the expression of MAPK and tight junction in intestinal epithelial cells. ConclusionsKTBA decoction manipulates the expression of several key proteins in the p38MAPK/p65NF-κB/AQP4 signaling pathway, modulates gut microbiota and metabolites toward a more favorable profile, improves gut barrier function, delays cardiomyocyte hypertrophy and fibrosis,and improves cardiac function.

Metabolomics in human SGBS cells as new approach method for studying adipogenic effects: Analysis of the effects of DINCH and MINCH on central carbon metabolism

Growing evidence suggests that exposure to certain metabolism-disrupting chemicals (MDCs), such as the phthalate plasticizer DEHP, might promote obesity in humans, contributing to the spread of this global health problem. Due to the restriction on the use of phthalates, there has been a shift to safer declared substitutes, including the plasticizer diisononyl-cyclohexane-1,2-dicarboxylate (DINCH). Notwithstanding, recent studies suggest that the primary metabolite monoisononyl-cyclohexane-1,2-dicarboxylic acid ester (MINCH), induces differentiation of human adipocytes and affects enzyme levels of key metabolic pathways. Given the lack of methods for assessing metabolism-disrupting effects of chemicals on adipose tissue, we used metabolomics to analyze human SGSB cells exposed to DINCH or MINCH. Concentration analysis of DINCH and MINCH revealed that uptake of MINCH in preadipocytes was associated with increased lipid accumulation during adipogenesis. Although we also observed intracellular uptake for DINCH, the solubility of DINCH in cell culture medium was limited, hampering the analysis of possible effects in the μM concentration range. Metabolomics revealed that MINCH induces lipid accumulation similar to peroxisome proliferator activated receptor gamma (PPARG)-agonist rosiglitazone through upregulation of the pyruvate cycle, which was recently identified as a key driver of de novo lipogenesis. Analysis of the metabolome in the presence of the PPARG-inhibitor GW9662 indicated that the effect of MINCH on metabolism was mediated at least partly by a PPARG-independent mechanism. However, all effects of MINCH were only observed at high concentrations of 10 μM, which are three orders of magnitudes higher than the current concentrations of plasticizers in human serum. Overall, the assessment of the effects of DINCH and MINCH on SGBS cells by metabolomics revealed no adipogenic potential at physiologically relevant concentrations. This finding aligns with previous in vivo studies and supports the potential of our method as a New Approach Method (NAM) for the assessment of adipogenic effects of environmental chemicals.

Elevated Nutrient Availability Enhances Chondrocyte Metabolism and Biosynthesis in Tissue-Engineered Cartilage

ObjectiveChondrocytes, which typically rely on anaerobic metabolism, exhibit upregulated biosynthetic activity when subjected to conditions that elicit mixed aerobic-anaerobic metabolism. Previously, we observed that increasing media volume resulted in the transition from anaerobic to mixed aerobic-anaerobic metabolism. Maximal extracellular matrix (ECM) accumulation occurred at this transition as a result of changes in HIF-1α signaling and associated hypoxic gene expression. This study aimed to explore the effect of further increases in media availability on ECM synthesis and chondrocyte metabolism. MethodsPrimary bovine chondrocytes were grown in 3D high-density tissue culture under varying levels of media availability (4 – 16 mL/106 cells). Changes in ECM accumulation and metabolism were determined through biochemical assays and 13C-metabolic flux analysis (13C-MFA). ResultsIncreasing media volumes resulted in higher accumulation of cartilaginous extracellular matrix (collagen and proteoglycans) and cellularity. Extracellular metabolite measurements revealed that elevated media availability led to increased glucose and glutamine metabolism, along with increased anaerobic activity. 13C-MFA utilizing [U-13C] glucose demonstrated that increased media availability significantly impacted central carbon metabolism, upregulating all glucose-related metabolic pathways (glycolysis, lactate fermentation, the TCA cycle, hexosamine biosynthetic pathway, and the malate-aspartate shuttle). Furthermore, 13C-MFA indicated that glutamine was donating carbons to the TCA cycle, and additional studies involving [U-13C] glutamine tracing supported this notion. ConclusionsElevated media availability upregulates extracellular matrix synthesis and leads to significant changes in metabolic phenotype. Glutamine plays an important role in chondrocyte metabolism and increases in glutamine metabolism correlate with increases in extracellular matrix accumulation.

Transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae

Congenital infection caused by vertical transmission of microsporidia N. bombycis can result in severe economic losses in the silkworm-rearing industry. Whole-transcriptome analyses have revealed non-coding RNAs and their regulatory networks in N. bombycis infected embryos and larvae. However, transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae remains unclear. Here, we simultaneously compared the transcriptomes of N. bombycis and its host B. mori embryos of 5-day and larvae of 1-, 5- and 10-day during congenital infection. For the transcriptome of N. bombycis, a comparison of parasite expression patterns between congenital-infected embryos and larva showed most genes related to parasite central carbon metabolism were down-regulated in larvae during infection, whereas the majority of genes involved in parasite proliferation and growth were up-regulated. Interestingly, a large number of distinct or shared differentially expressed genes (DEGs) were revealed by the Venn diagram and heat map, many of them were connected to infection related factors such as Ricin B lectin, spore wall protein, polar tube protein, and polysaccharide deacetylase. For the transcriptome of B. mori infected with N. bombycis, beyond numerous DEGs related to DNA replication and repair, mRNA surveillance pathway, RNA transport, protein biosynthesis, and proteolysis, with the progression of infection, a large number of DEGs related to immune and infection pathways, including phagocytosis, apoptosis, TNF, Toll-like receptor, NF-kappa B, Fc epsilon RI, and some diseases, were successively identified. In contrast, most genes associated with the insulin signaling pathway, 2-oxacarboxylic acid metabolism, amino acid biosynthesis, and lipid metabolisms were up-regulated in larvae compared to those in embryos. Furthermore, dozens of distinct and three shared DEGs that were involved in the epigenetic regulations, such as polycomb, histone-lysine-specific demethylases, and histone-lysine-N-methyltransferases, were identified via the Venn diagram and heat maps. Notably, many DEGs of host and parasite associated with lipid-related metabolisms were verified by RT-qPCR. Taken together, simultaneous transcriptomic analyses of both host and parasite genes lead to a better understanding of changes in the microsporidia proliferation and host responses in embryos and larvae in N. bombycis congenital infection.

Detoxification of fermentation inhibitors using a waste biomass-based adsorbent of AEPA250 to enhance bioethanol production: The adsorption analysis and microbial mechanism

Environmental pollution and energy scarcity have promoted the development of renewable alternative energy of rice straw-based bioethanol. However, the production of main fermentation inhibitors from the pretreatment of rice straw raw material reduces the bioethanol yield. In order to improve bioethanol production, a waste biomass-based adsorbent (AEPA250) was used to adsorb three main inhibitors of ferulic acid, vanillin, and furfural from the rice straw hydrolysate in this study. The adsorption percentages were calculated using the high-performance liquid chromatography method. The results showed that the adsorption percentages of three inhibitors were all above 70% in 10 min, demonstrating the efficient detoxification of AEPA250 on rice straw hydrolysate. The adsorption contributions related to the attribute features of three inhibitors and AEPA250 were evaluated for the first time. The density functional theory calculations and AEPA250 characterizations were used to obtain these attribute parameters. Analysis of the correlation heat map between adsorption efficiencies and attribute features of AEPA250 (or fermentation inhibitors) indicated that AEPA250 possessed the lower activation energy (Ea) and pre-exponential factor (A) when it was prepared, as well as fermentation inhibitors had the higher ionization constant (pKa) and lower electrophilicity index (ω), resulting in the enhancement of bioethanol production. The contribution of pKa was greater than A followed by Ea and ω for adsorption process. Meanwhile, the key metabolic pathways involved in central carbon metabolism were analyzed for AEPA250 detoxification using proteomics method. The results indicated that the up-regulation of SDH3, ACO1, CIT2 and MLS1 (or down-regulation of SCT1) genes contributed to inhibitors detoxification using AEPA250, which effectively provided more nicotinamide adenine dinucleotide (NADH) for the reduction of these inhibitors. This study explores the detoxification mechanisms at the molecular level for boosting bioethanol production in the future, which lays the foundation for the development of efficient adsorbents and resistant engineering strains.

High-throughput profiling of metabolic responses to exogenous nutrients in Synechocystis sp. PCC 6803.

Cyanobacteria fix carbon dioxide and release carbon-containing compounds into the wider ecosystem, yet they are sensitive to small metabolites that may impact their growth and physiology. Several cyanobacteria can grow mixotrophically, but we currently lack a molecular understanding of how specific nutrients may alter the compounds they release, limiting our knowledge of how environmental factors might impact primary producers and the ecosystems they support. In this study, we develop a high-throughput phytoplankton culturing platform and identify how the model cyanobacterium Synechocystis sp. PCC 6803 responds to nutrient supplementation. We assess growth responses to 32 nutrients at two concentrations, identifying 15 that are utilized mixotrophically. Seven nutrient sources significantly enhance growth, while 19 elicit negative growth responses at one or both concentrations. High-throughput exometabolomics indicates that oxidative stress limits Synechocystis' growth but may be alleviated by antioxidant metabolites. Furthermore, glucose and valine induce strong changes in metabolite exudation in a possible effort to correct pathway imbalances or maintain intracellular elemental ratios. This study sheds light on the flexibility and limits of cyanobacterial physiology and metabolism, as well as how primary production and trophic food webs may be modulated by exogenous nutrients.IMPORTANCECyanobacteria capture and release carbon compounds to fuel microbial food webs, yet we lack a comprehensive understanding of how external nutrients modify their behavior and what they produce. We developed a high throughput culturing platform to evaluate how the model cyanobacterium Synechocystis sp. PCC 6803 responds to a broad panel of externally supplied nutrients. We found that growth may be enhanced by metabolites that protect against oxidative stress, and growth and exudate profiles are altered by metabolites that interfere with central carbon metabolism and elemental ratios. This work contributes a holistic perspective of the versatile response of Synechocystis to externally supplied nutrients, which may alter carbon flux into the wider ecosystem.

Revealing the comprehensive impact of organic compounds on the partial nitrification-anammox system during incineration leachate treatment: metabolic hierarchy and adaptation

Organics, as widespread pollutants in high-strength ammonia wastewater, typically exert adverse effects on the performance of partial nitrification-anammox (PNA) systems. However, the in-depth knowledge on how microbial consortia respond to these disturbances remains limited. In this study, we unveiled the evolution of complex organic matter flow and its impact on the metabolic hierarchy and adaptation of microbial consortia, employing multi-omics approaches, i.e., 16S amplicon sequencing, metagenomics, and metabolomics. In a two-stage PNA system sequentially treating synthetic wastewater and incineration leachate over 230 days, partial nitrification stayed stable (nitrite accumulation > 97%) while anammox efficiency dropped (nitrogen removal decreased from 86% to 78%). The phenomenon was revealed to be correlated with the evolution of dissolved organic matter (DOM) and xenobiotic organic compounds (XOCs). In the PN stage, ammonia-oxidizing bacteria (AOB) exhibited excellent adaptability through active metabolic regulation after treating leachate. Numerous heterotrophs proliferated to utilize DOM and XOCs, triggering a “boom” state evident in the glycerophospholipid metabolism. However, in the anammox stage, the competition between carbon fixation and central carbon metabolism within autotrophs and heterotrophs became evident. Increased biosynthesis costs inhibited the central metabolism (specific anammox activity decreased by 66%) and the Wood-Ljungdahl pathway of anammox bacteria (AnAOB) in the presence of recalcitrant organics. Additionally, the degradation of organics was limited, exhibiting a “bust” state. This study revealed the metabolic adaption and susceptibility of AOB and AnAOB in response to organics from the leachate, demonstrating the applicability of the two-stage configuration for treating high-strength wastewater containing abundant and diverse organics.

Cuscuta chinensis flavonoids alleviate ovarian damage in offspring female mice induced by BPA exposure during pregnancy by regulating the central carbon metabolism pathway

Pregnancy is a sensitive window period for bisphenol A (BPA) exposure. BPA can pass through the placenta and cause reproductive damage in offspring female mice. Even BPA that is not metabolized during lactation can be passed through milk. Cuscuta chinensis flavonoids (CCFs) can alleviate reproductive damage caused by BPA, but the mechanism of action is unclear. To investigate the potential mitigating impact of CCFs on ovarian damage resulting from BPA exposure during pregnancy, we administered BPA and CCFs to pregnant mice during the gestational period spanning from 0.5 to 17.5 days. Aseptic collection of serum and ovaries from female mice was conducted on postnatal day 21 (PND21). Serum hormone levels and tissue receptor levels were quantified utilizing ELISA and PCR, while ovaries underwent sequencing and analysis through transcriptomics and metabolomics techniques. Additionally, the assessment of ovarian oxidative stress levels was carried out as part of the comprehensive analysis. The results showed that CCFs administration mitigated the adverse effects induced by BPA exposure on ovarian index, hormone levels, receptor expression, and mRNA expression levels in female offspring mice. The joint analysis of transcriptome and metabolome revealed 48 enriched pathways in positive ion mode and 44 enriched pathways in negative ion mode. Among them, the central carbon metabolism pathway is significantly regulated by BPA and CCFs. The screened sequencing results were verified through qPCR and biochemical kits. In this study, CCFs may participate in the central carbon metabolism pathway by reducing the expression of Kit proto-oncogene (Kit), hexokinase 1 gene (Hk1) and pyruvate kinase M (Pkm) mRNA and increasing the expression of h-ras proto-oncogene (Hras), sirtuin 3 (Sirt3), sirtuin 6 (Sirt6) and TP53 induced glycolysis regulatory phosphatase gene (Tigar) mRNA, thereby resisting the effects of BPA on the body. At the same time, the metabolic levels of D-Fructose 1,6-bisphosphate and L-Asparagine tend to be stable. Moreover, CCFs demonstrated a capacity to diminish the BPA-induced escalation in reactive oxygen species (ROS) and malondialdehyde (MDA). Simultaneously, it exhibited the ability to elevate levels of glutathione (GSH) and catalase (CAT), thereby effectively preventing peroxidation. In summary, CCFs alleviate BPA-induced ovarian damage in offspring female mice by regulating the central carbon metabolism pathway. This study will improve the information on BPA reproductive damage antagonist drugs and provide a theoretical basis for protecting animal reproductive health.

Abstract 1790: Pyruvate carboxylase regulates tumor progression through central carbon metabolism and immunosuppression

Abstract The tumor microenvironment (TME) is characterized by metabolic reprogramming and immune evasion, essential features in tumor progression. Tumor metabolic dysregulation, particularly the high production of lactate, plays a pivotal role in suppressing antitumor immunity within the TME. Pyruvate carboxylase (PC), responsible for converting pyruvate to oxaloacetate, is a critical determinant for lung metastasis in triple-negative breast cancer (TNBC). Herein we test the hypothesis that PC-mediated regulation of central carbon metabolism controls TME immunosurrveilance. Wild-type C57BL6/J and NSG mice received orthotopic injections of E0771 or M-Wnt tumor cells with shRNA targeting PC or a scramble control. RNA from tumors underwent transcriptomic profiling using Affymetrix Clariom D microarray (n=3-6/group per experiment). Gene set enrichment analysis identified pathways enriched in each condition, including a PC-associated hypoxia signature. Immunohistochemistry with anti-CD3 antibody and full spectrum flow cytometry assessed T cell infiltration and TME immune cell composition. Untargeted metabolomics, extracellular flux analysis, and high-resolution respirometry determined how loss of PC altered cellular metabolism. We found that suppressing PC expression increased primary tumor growth (~2 fold), potently drove immunosuppression, and remodeled cellular metabolism towards lactate production. Injection of PC-suppressed cells increased regulatory T cell levels in wild type mice. Importantly, PC-suppressed cells injected into NSG mice, which lack T, B, and NK cells, demonstrated slower, rather than accelerated, tumor growth. PC-suppression reduced oxygen consumption rate (OCR), but supply of anaplerotic carbon through exogenous malate or oxaloacetate restored OCR. Conversely, both the rate of and reliance on lactate production was increased by PC suppression. Notably, loss of immunosurveillance following downregulation of PC was reversed by inhibiting lactate transport. Additionally, lower PC expression was associated with increased hypoxia in tumors, supporting in vitro evidence of PC-repression by hypoxia. In summary, hypoxia-induced PC suppression emerges as a crucial mechanism through which primary tumors hinder antitumor immunity, partly via increased lactate production. These findings underscore the significance of PC-directed tumor metabolism as an understudied nexus in the interplay between tumor progression and antitumor immunity. Citation Format: Michael F. Coleman, Eylem Kulkoyluoglu Cotul, Alexander J. Pfeil, Alyssa N. Ho, Emily N. Devericks, Muhammad H. Safdar, Hao Chen, Numair Attaar, Violet A. Kiesel, Dorothy Teegarden, Stephen D. Hursting, Michael K. Wendt. Pyruvate carboxylase regulates tumor progression through central carbon metabolism and immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1790.

Abstract 3170: Epigenetic identification and functional characterization of a novel RING finger protein tumor suppressor in nasopharyngeal carcinoma and esophageal carcinoma

Abstract RING finger proteins play crucial roles in normal physiology and pathophysiology as important regulators of ubiquitin-dependent proteasomal protein degradation. Many RING finger proteins have also been implicated in tumorigenesis either as oncogenes or tumor suppressors. Through cancer CpG methylome studies, we identified RING finger protein 150 (RNF150) as a methylated target gene in nasopharyngeal carcinoma (NPC) and esophageal carcinoma (ESCC). RNF150 was found to be ubiquitously expressed in human normal tissues but frequently downregulated in NPC and ESCC cells due to its promoter methylation. Its downregulation is associated with poor survival in patients with head and neck cancer and esophageal cancer. We also found that RNF150 is a functional tumor suppressor by inhibiting tumor cell growth and inducing cell cycle arrest in NPC and ESCC cells. Furthermore, ectopic expression of RNF150 inhibited NPC and ESCC cell migration/invasion by suppressing epithelial-mesenchymal transition (EMT) through upregulation of epithelial marker E-cadherin and downregulation of mesenchymal markers (vimentin, N-cadherin, Snail, and Slug). RNF150 is located in the cytoplasm and represses several oncogenic signaling pathways including the Wnt/β-catenin and NF-kB signaling pathways. Liquid chromatography-mass spectrometry (LC-MS) was performed to elucidate RNF150 binding proteins. KEGG enrichment analysis showed that glycolysis/gluconeogenesis, pyruvate metabolism, central carbon metabolism in cancer, and AMPK signaling were the top 4 pathways with significant enrichment, suggesting potential targeting of metabolic molecules by RNF150. Thus, our results provide insights into a new ring finger protein member, RNF150, as a functional tumor suppressor for NPC and ESCC. Our study also suggests a potential epigenetic biomarker for the early dection of NPC and ESCC. Citation Format: Xiaoxue Chai, Feifei Luo, Shujie Bao, Liuzhen Meng, Anthony T. Chan, Brigette B. MA, Qian Tao, li LiLi. Epigenetic identification and functional characterization of a novel RING finger protein tumor suppressor in nasopharyngeal carcinoma and esophageal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3170.

Metabolic reprogramming of cancer cells by JMJD6-mediated pre-mRNA splicing associated with therapeutic response to splicing inhibitor.

Dysregulated pre-mRNA splicing and metabolism are two hallmarks of MYC-driven cancers. Pharmacological inhibition of both processes has been extensively investigated as potential therapeutic avenues in preclinical and clinical studies. However, how pre-mRNA splicing and metabolism are orchestrated in response to oncogenic stress and therapies is poorly understood. Here, we demonstrate that jumonji domain containing 6, arginine demethylase, and lysine hydroxylase, JMJD6, acts as a hub connecting splicing and metabolism in MYC-driven human neuroblastoma. JMJD6 cooperates with MYC in cellular transformation of murine neural crest cells by physically interacting with RNA binding proteins involved in pre-mRNA splicing and protein homeostasis. Notably, JMJD6 controls the alternative splicing of two isoforms of glutaminase (GLS), namely kidney-type glutaminase (KGA) and glutaminase C (GAC), which are rate-limiting enzymes of glutaminolysis in the central carbon metabolism in neuroblastoma. Further, we show that JMJD6 is correlated with the anti-cancer activity of indisulam, a 'molecular glue' that degrades splicing factor RBM39, which complexes with JMJD6. The indisulam-mediated cancer cell killing is at least partly dependent on the glutamine-related metabolic pathway mediated by JMJD6. Our findings reveal a cancer-promoting metabolic program is associated with alternative pre-mRNA splicing through JMJD6, providing a rationale to target JMJD6 as a therapeutic avenue for treating MYC-driven cancers.

Metabolic reprogramming of cancer cells by JMJD6-mediated pre-mRNA splicing associated with therapeutic response to splicing inhibitor

Dysregulated pre-mRNA splicing and metabolism are two hallmarks of MYC-driven cancers. Pharmacological inhibition of both processes has been extensively investigated as potential therapeutic avenues in preclinical and clinical studies. However, how pre-mRNA splicing and metabolism are orchestrated in response to oncogenic stress and therapies is poorly understood. Here, we demonstrate that jumonji domain containing 6, arginine demethylase, and lysine hydroxylase, JMJD6, acts as a hub connecting splicing and metabolism in MYC-driven human neuroblastoma. JMJD6 cooperates with MYC in cellular transformation of murine neural crest cells by physically interacting with RNA binding proteins involved in pre-mRNA splicing and protein homeostasis. Notably, JMJD6 controls the alternative splicing of two isoforms of glutaminase (GLS), namely kidney-type glutaminase (KGA) and glutaminase C (GAC), which are rate-limiting enzymes of glutaminolysis in the central carbon metabolism in neuroblastoma. Further, we show that JMJD6 is correlated with the anti-cancer activity of indisulam, a ‘molecular glue’ that degrades splicing factor RBM39, which complexes with JMJD6. The indisulam-mediated cancer cell killing is at least partly dependent on the glutamine-related metabolic pathway mediated by JMJD6. Our findings reveal a cancer-promoting metabolic program is associated with alternative pre-mRNA splicing through JMJD6, providing a rationale to target JMJD6 as a therapeutic avenue for treating MYC-driven cancers.

Succinate utilisation by Salmonella is inhibited by multiple regulatory systems.

Succinate is a potent immune signalling molecule that is present in the mammalian gut and within macrophages. Both of these infection niches are colonised by the pathogenic bacterium Salmonella enterica serovar Typhimurium during infection. Succinate is a C4-dicarboyxlate that can serve as a source of carbon for bacteria. When succinate is provided as the sole carbon source for in vitro cultivation, Salmonella and other enteric bacteria exhibit a slow growth rate and a long lag phase. This growth inhibition phenomenon was known to involve the sigma factor RpoS, but the genetic basis of the repression of bacterial succinate utilisation was poorly understood. Here, we use an experimental evolution approach to isolate fast-growing mutants during growth of S. Typhimurium on succinate containing minimal medium. Our approach reveals novel RpoS-independent systems that inhibit succinate utilisation. The CspC RNA binding protein restricts succinate utilisation, an inhibition that is antagonised by high levels of the small regulatory RNA (sRNA) OxyS. We discovered that the Fe-S cluster regulatory protein IscR inhibits succinate utilisation by repressing the C4-dicarboyxlate transporter DctA. Furthermore, the ribose operon repressor RbsR is required for the complete RpoS-driven repression of succinate utilisation, suggesting a novel mechanism of RpoS regulation. Our discoveries shed light on the redundant regulatory systems that tightly regulate the utilisation of succinate. We speculate that the control of central carbon metabolism by multiple regulatory systems in Salmonella governs the infection niche-specific utilisation of succinate.

Enteric coronavirus PDCoV evokes a non-Warburg effect by hijacking pyruvic acid as a metabolic hub

The Warburg effect, also referred as aerobic glycolysis, is a common metabolic program during viral infection. Through targeted metabolomics combined with biochemical experiments and various cell models, we investigated the central carbon metabolism (CCM) profiles of cells infected with porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus with zoonotic potential. We found that PDCoV infection required glycolysis but decreased glycolytic flux, exhibiting a non-Warburg effect characterized by pyruvic acid accumulation. Mechanistically, PDCoV enhanced pyruvate kinase activity to promote pyruvic acid anabolism, a process that generates pyruvic acid with concomitant ATP production. PDCoV also hijacked pyruvic acid catabolism to increase biosynthesis of non-essential amino acids (NEAAs), suggesting that pyruvic acid is an essential hub for PDCoV to scavenge host energy and metabolites. Furthermore, PDCoV facilitated glutaminolysis to promote the synthesis of NEAA and pyrimidines for optimal proliferation. Our work supports a novel CCM model after viral infection and provides potential anti-PDCoV drug targets.

Network Pharmacology Approach and Partial Experimental Validation of Aidi Injection Solution for the Treatment of Colorectal Cancer

Purpose The objective of this research was to employ network pharmacology to analyze and identify the key active components and target points of action of Aidi injection in relation to colorectal cancer. Additionally, this study aimed to experimentally validate the mechanism of action of hinokinin in treating colorectal cancer. Methods This study employed a network pharmacology methodology to identify the primary components and action targets of Aidi injection in public databases; a similar approach was used to identify effective targets for colorectal cancer treatment. The protein‒protein interaction network, along with gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses, facilitated the prediction of central targets and pathways by which Aidi injection combats colorectal cancer. Molecular docking techniques were harnessed to sift through drugs and their targets for high binding affinity. Experimental validation was carried out to corroborate the findings derived from network pharmacology. Results We identified 37 active constituents of Aidi injection (ADI), 701 potential targets, 768 colorectal cancer (CRC) targets, and 111 overlapping targets. The anti-CRC efficacy of ADI seems to be associated with several pathways: cancer pathways, EGFR tyrosine kinase inhibitor resistance, proteoglycans in cancer, endocrine resistance, central carbon metabolism in cancer, chemo-oncogenic receptor initiation, cancer-specific microRNAs, and signaling pathways such as PI3K-Akt, mitogen-activated protein kinase, prolactin, FoxO, and Ras. Predominantly, ADI exhibited activity against key markers such as EGFR, ERBB2, HSP90AA1, mTOR, HIF1A, CCND1, JUN, AKT1, SRC, and STAT3 to mitigate CRC. Furthermore, Hinokinin has been shown to curtail the proliferation of colorectal cancer cells, amplify apoptosis, and modulate the expression of the mTOR protein. Conclusion Through network pharmacology, we identified 13 shared targets associated with colorectal cancer. Subsequent experimental validation revealed that hinokinin can curtail the proliferation of colorectal cancer cells and enhance their apoptosis, primarily by modulating mTOR expression.