Citric Production Research Articles

CPT2-mediated Fatty Acid Oxidation Is Dispensable for Humoral Immunity.

B cell activation is accompanied by dynamic metabolic reprogramming, supported by a multitude of nutrients that include glucose, amino acids, and fatty acids. Although several studies have indicated that fatty acid mitochondrial oxidation is critical for immune cell functions, contradictory findings have been reported. Carnitine palmitoyltransferase II (CPT2) is a critical enzyme for long-chain fatty acid oxidation in mitochondria. In this study, we test the requirement of CPT2 for humoral immunity using a mouse model with a lymphocyte-specific deletion of CPT2. Stable [13C] isotope tracing reveals highly reduced fatty acid-derived citrate production in CPT2-deficient B cells. Yet, CPT2 deficiency has no significant impact on B cell development, B cell activation, germinal center formation, and Ab production upon either thymus-dependent or -independent Ag challenges. Together, our findings indicate that CPT2-mediated fatty acid oxidation is dispensable for humoral immunity, highlighting the metabolic flexibility of lymphocytes.

Finite Difference Model of Substrate Channeling in TCA Cycle Enzymes

The Tricarboxylic Acid (TCA) cycle is a highly optimized metabolic pathway. Of particular interest in the TCA cycle is the malate dehydrogenase—citrate synthase (MDH-CS) complex, which accomplishes citrate production from malate with an oxaloacetate intermediate. This complex was studied experimentally by Bulutoglu et al, who showed that mutation of positively-charged residues on the complex surface effected the dynamic response of citrate production as measured by lag time experiments.1 More recently, we have created a Markov State model that was used to predict the transfer efficiency of each complex and determine the reaction pathways on the surface.2 Utilizing the kinetic parameters of both the recombinant and mutant complexes determined experimentally1and Markov state transition matrix produced by previous modeling2, we report a finite difference model to calculate time trajectories of the surface and bulk occupancies by OAA. Solution of a system of differential mass balance equations, based on the transient matrix, provide the occupancy probability at each node in the network. The lag time of MDH-CS was determined computationally to be comparable to experiment for both the recombinant and mutant complex with lag times of 0.44 and 2.1 seconds, respectively.1 Using the model, the dynamics of each of the four possible reaction pathways between the the two source (MDH) active sites and the two sink (CS) sites could be studied independently. This analysis provides a dynamic model for intermediate transport in an electrostatically channeled system, and can be used as a predictive tool to provide mechanistic insight into pathway dominance. We gratefully acknowledge support from the Army Research Office MURI (#W911NF1410263) via The University of Utah. References B. Bulutoglu, K. E. Garcia, F. Wu, S. D. Minteer, and S. Banta, ACS Chemical Biology, 11, 2847–53 (2016). doi:10.1021/acschembio.6b00523Y. Xie, S. D. Minteer, S. Banta, and S. Calabrese Barton, ACS Nanoscience Au, 2, 414-421 (2022). doi:10.1021/acsnanoscienceau.2c00011 Figure 1

Two enzymes contribute to citrate production in the mitochondrion of Toxoplasma gondii

Citrate synthase catalyzes the first and the rate-limiting reaction of the tricarboxylic acid (TCA) cycle, producing citrate from the condensation of oxaloacetate and acetyl-coenzyme A. The parasitic protozoan Toxoplasma gondii has full TCA cycle activity, but its physiological roles remain poorly understood. In this study, we identified three proteins with predicted citrate synthase (CS) activities two of which were localized in the mitochondrion, including the 2-methylcitrate synthase (PrpC) that was thought to be involved in the 2-methylcitrate cycle, an alternative pathway for propionyl-CoA detoxification. Further analyses of the two mitochondrial enzymes showed that both had citrate synthase activity, but the catalytic efficiency of CS1 was much higher than that of PrpC. Consistently, the deletion of CS1 resulted in a significantly reduced flux of glucose-derived carbons into TCA cycle intermediates, leading to decreased parasite growth. In contrast, disruption of PrpC had little effect. On the other hand, simultaneous disruption of both CS1 and PrpC resulted in more severe metabolic changes and growth defects than a single deletion of either gene, suggesting that PrpC does contribute to citrate production under physiological conditions. Interestingly, deleting Δcs1 and Δprpc individually or in combination only mildly or negligibly affected the virulence of parasites in mice, suggesting that both enzymes are dispensable invivo. The dispensability of CS1 and PrpC suggests that either the TCA cycle is not essential for the asexual reproduction of tachyzoites or there are other routes of citrate supply in the parasite mitochondrion.

Targeting Longevity Gene SLC13A5: A Novel Approach to Prevent Age-Related Bone Fragility and Osteoporosis.

Reduced expression of the plasma membrane citrate transporter SLC13A5, also known as INDY, has been linked to increased longevity and mitigated age-related cardiovascular and metabolic diseases. Citrate, a vital component of the tricarboxylic acid cycle, constitutes 1-5% of bone weight, binding to mineral apatite surfaces. Our previous research highlighted osteoblasts' specialized metabolic pathway facilitated by SLC13A5 regulating citrate uptake, production, and deposition within bones. Disrupting this pathway impairs bone mineralization in young mice. New Mendelian randomization analysis using UK Biobank data indicated that SNPs linked to reduced SLC13A5 function lowered osteoporosis risk. Comparative studies of young (10 weeks) and middle-aged (52 weeks) osteocalcin-cre-driven osteoblast-specific Slc13a5 knockout mice (Slc13a5cKO) showed a sexual dimorphism: while middle-aged females exhibited improved elasticity, middle-aged males demonstrated enhanced bone strength due to reduced SLC13A5 function. These findings suggest reduced SLC13A5 function could attenuate age-related bone fragility, advocating for SLC13A5 inhibition as a potential osteoporosis treatment.

Treponema pallidum recombinant protein Tp47 activates NOD-like receptor family protein 3 inflammasomes in macrophages via glycolysis

Glycolysis is a key pathway in cellular glucose metabolism for energy supply and regulates immune cell activation. Whether glycolysis is involved in the activation of NOD-like receptor family protein 3 (NLRP3) inflammasomes during Treponema pallidum (T. pallidum) infection is unclear. In this study, the effect of T. pallidum membrane protein Tp47 on NLRP3 inflammasome activation in rabbit peritoneal macrophages was analysed and the role of glycolysis in NLRP3 inflammasome activation was explored. The results showed that Tp47 promoted NLRP3, caspase-1, and IL-1β mRNA expression in macrophages, enhanced glycolysis and glycolytic capacity of macrophage, and promoted the production of macrophage glycolytic metabolites citrate, phosphoenolpyruvate, and lactate. The M2 pyruvate kinase (PKM2) inhibitor shikonin down-regulated the Tp47-promoted NLRP3, caspase-1, and IL-1β mRNA expression in macrophages, and suppressed the Tp47-enhanced glycolysis and glycolytic capacity. Similarly, si-PKM2 significantly inhibited Tp47-promoted NLRP3, caspase-1, and IL-1β mRNA expression and the Tp47-enhanced glycolysis and glycolytic capacity in macrophages. In conclusion, Tp47 activated NLRP3 inflammasomes via PKM2-dependent glycolysis and provided a new perspective on the effect of T. pallidum infection on host macrophages, which would contribute to the understanding of the infection mechanism and host immune mechanism of T. pallidum.

Malate production, sugar metabolism, and redox homeostasis in the leaf growth zone of Rye (Secale cereale) increase stress tolerance to aluminum stress: A biochemical and genome‐wide transcriptional study

Global soil acidification is increasing, enlarging aluminum (Al) availability in soils, leading to reductions in plant growth. This study investigates the effect of Al stress on the leaf growth zones of Rye (Secale cereale, cv Beira). Kinematic analysis showed that the effect of Al on leaf growth rates was mainly due to a reduced cell production rate in the meristem. Transcriptomic analysis identified 2272 significantly (log2fold > |0.5| FDR < 0.05) differentially expressed genes (DEGs) for Al stress. There was a downregulation in several DEGs associated with photosynthetic processes and an upregulation in genes for heat/light response, and H2O2 production in all leaf zones. DEGs associated with heavy metals and malate transport were increased, particularly, in the meristem. To determine the putative function of these processes in Al tolerance, we performed biochemical analyses comparing the tolerant Beira with an Al sensitive variant RioDeva. Beira showed improved sugar metabolism and redox homeostasis, specifically in the meristem compared to RioDeva. Similarly, a significant increase in malate and citrate production, which are known to aid in Al detoxification in plants, was found in Beira. This suggests that Al tolerance in Rye is linked to its ability for Al exclusion from the leaf meristem.

Transcription factor TEAD4 facilitates glycolysis and proliferation of gastric cancer cells by activating PKMYT1

BackgroundGastric cancer (GC) ranks third for cancer deaths worldwide, and glycolysis is a hallmark of several cancers, including GC. TEAD4 plays a role in establishing an oncogenic cascade in cancers, including GC. Whether TEAD4 can influence the glycolysis of GC cells remains uncovered. Hence, this study attempted to investigate the impact on glycolysis of GC cells by TEAD4. MethodsBy using bioinformatics analysis, differentially expressed mRNAs were screened, and downstream regulatory genes were predicted. Expression levels of TEAD4 and PKMYT1 were assessed by qRT-PCR. The binding sites between TEAD4 and PKMYT1 were predicted by the JASPAR database, meanwhile their modulatory relationship was confirmed through dual-luciferase assay and chromatin Immunoprecipitation (ChIP). Cell viability and proliferation were assayed via CCK-8 and colony formation assays. Glycolysis was measured by assaying extracellular acidification rate, oxygen consumption rate, and production of pyruvic acid, lactate, citrate, and malate. Expression levels of proteins (HK-2 and PKM2) related to glycolysis were assessed by Western blot. ResultsTEAD4 was upregulated in GC tissues and cells. TEAD4 knockdown substantially repressed glycolysis and proliferation of GC cells. PKMYT1, the target gene downstream of TEAD4, was identified via bioinformatics prediction, and its expression was elevated in GC. Dual-luciferase and ChIP assay validated the targeted relationship between the promoter region of PKMYT1 and TEAD4. As revealed by rescue experiments, the knockdown of TEAD4 reversed the stimulative effect on GC cell glycolysis and proliferation by forced expression of PKMYT1. ConclusionTEAD4 activated PKMYT1 to facilitate the proliferation and glycolysis of GC cells. TEAD4 and PKMYT1 may be possible therapeutic targets for GC.

CsTT8 regulates anthocyanin accumulation in blood orange through alternative splicing transcription.

A homologous gene of basic-helix-loop-helix AtTT8 in Arabidopsis thaliana was identified in juice sac cells of pulp tissues from blood orange (Citrus sinensis cv 'Tarocco'), which was designated as CsTT8 in this study. Additionally, the mRNA levels of TT8 with the full-length open reading frame were significantly higher in 'Tarocco' than in mutant fruit lacking pigment in pulp or peel tissues. However, an alternative splicing transcript, Δ15-TT8, with the fourth exon skipped, was also identified from transcripts different in length from that in 'Tarocco'. The mRNA levels of Δ15-TT8 were higher in mutant fruit lacking pigment in pulp or peel tissues than in the wild type. Therefore, the TT8/Δ15-TT8 mRNA level ratio was found to be crucial for sufficient pigment in either pulp or peel tissues. TT8 from blood orange fruit demonstrated the capacity for nucleus localization and binding to other proteins. In contrast, Δ15-TT8, lacking the fourth exon, lost its ability to interact with RUBY1 and to localize at the nucleus. Using a dual luciferase reporter assay and transient overexpression in tobacco, we proved that two regulatory complexes formed by a functional TT8 with different MYB(v-myb avian myeloblastosis viral oncogene homolog)-type partners significantly promoted expression of an anthocyanin biosynthetic gene and a proton pumping gene, leading to anthocyanin and citrate production. Our findings suggest that TT8, rather than dysfunctional Δ15-TT8, is possibly involved in modulating anthocyanin biosynthesis and its transport into vacuoles by proton gradients. However, increased mRNA levels of the dysfunctional alternative splicing transcript may act as a negative feedback to downregulate TT8 expression and limit anthocyanin accumulation in blood oranges.

Efficient esterification over hierarchical Zr-Beta zeolite synthesized via liquid-state ion-exchange strategy

The eco-friendly zeolite-based solid acid catalysts constitute an important alternative to the liquid acid catalyst for the production of plasticizer tributyl citrate (TBC), due to their non-corrosive, high hydrothermal stability and easiness to recycle. Herein, a simple acid treatment to the H-Beta zeolite and subsequent incorporation of ZrIV ions into the vacated Al sites were conducted by liquid-state ion-exchange of ZrO(NO3)2 aqueous solution. The ZrIV ions were incorporated into the framework by the expand of the unit cell size, verified by XRD patterns, the emerging new band at ∼198 nm on UV–vis spectra, the enhanced binding energies of the Zr 3d5/2 and Zr 3d3/2 on XPS spectra, as well as the variations of isolated terminal silanol groups (Si-OH) by FTIR and the change of integrated intensity ratio of Q3/Q4 by 29Si MAS NMR. It was illustrated that the Lewis acid sites was the main active sites for esterification reaction and the produced abundant mesopores in the Zr-Beta-x catalysts would facilitate the transportation of TBC. The maximum conversion of citric acid as high as 80 % was obtained on Zr-Beta-128 (SiO2/ZrO2 molar ratio = 128) catalyst with the highest Lewis acid amount. Furthermore, the Zr-Beta-128 zeolite exhibited high stability without any detectable deactivation after 6 cycles with the 100 % selectivity to TBC. The efficient Zr-Beta catalyst could supply a novel possibility for the development of an environmentally-friendly solid acid catalyst with good performance for the TBC production.

An Industrial Approach to Production of Tofacitinib Citrate (TFC) as an Anti-COVID-19 Agent: A Joint Experimental and Theoretical Study

In this report, we have presented our experience about a facile method for synthesis of tofacitinib citrate (TFC). The developed analytical methods for identification and qualifications are also included. As TFC seems to be effective in treatment of the symptoms of COVID-19 (SARS family), manufacturing of this active pharmaceutical ingredient (API) could be helpful. The API of TFC was prepared from the diamine intermediate in an ambient and solvent-free condition. Elimination of the reaction solvent resulted in decreasing the cost and preventing the rejection of the organic volatile impurity (OVI) test. The final citrate addition step was carried out using water as a solvent (the citrate content was 37.5% by potentiometry). Moreover, the results of the Karl-Fischer (KF) titration analysis was about 0.24%, which showed that the use of water does not increase the water content of the crystal structure.

ROS-activatable biomimetic interface mediates in-situ bioenergetic remodeling of osteogenic cells for osteoporotic bone repair

Bioenergy (ATP) is essentially required for supporting the osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). However, factors such as high ROS levels and decreased glucose metabolism severely limit the bioenergy production in osteoporotic MSCs. We have prepared CaCO3-Quercetin- chromium (CaCO3-Qu-Cr) nanoparticles via ion coordination and packaged them into ROS-responsive gelatin/chitosan coating on titanium surface (Ti/Gel/CaCO3-Qu-Cr), aiming to improve the ATP production and cell mineralization by ameliorating ROS levels via Qu-mediated antioxidative effect and the promotional effect of Qu-Cr combination on glucose metabolism. Characterization results confirmed that Ti/Gel/CaCO3-Qu-Cr could be degraded in an ROS-responsive manner to release CaCO3-Qu-Cr nanoparticles continuously and eliminate excessive ROS in both the MSCs and microenvironment. Meanwhile, Ti/Gel/CaCO3-Qu-Cr significantly increased the glucose uptake and metabolism in osteoporotic MSCs and boosted their ATP and citrate production. This study laid the foundation for the development of functional titanium-based implants for the improvement of osteoporotic osseointegration.

A specialized metabolic pathway partitions citrate in hydroxyapatite to impact mineralization of bones and teeth.

Citrate is a critical metabolic substrate and key regulator of energy metabolism in mammalian cells. It has been known for decades that the skeleton contains most (>85%) of the body's citrate, but the question of why and how this metabolite should be partitioned in bone has received singularly little attention. Here, we show that osteoblasts use a specialized metabolic pathway to regulate uptake, endogenous production, and the deposition of citrate into bone. Osteoblasts express high levels of the membranous Na+-dependent citrate transporter solute carrier family 13 member 5 (Slc13a5) gene. Inhibition or genetic disruption of Slc13a5 reduced osteogenic citrate uptake and disrupted mineral nodule formation. Bones from mice lacking Slc13a5 globally, or selectively in osteoblasts, showed equivalent reductions in cortical thickness, with similarly compromised mechanical strength. Surprisingly, citrate content in mineral from Slc13a5-/- osteoblasts was increased fourfold relative to controls, suggesting the engagement of compensatory mechanisms to augment endogenous citrate production. Indeed, through the coordinated functioning of the apical membrane citrate transporter SLC13A5 and a mitochondrial zinc transporter protein (ZIP1; encoded by Slc39a1), a mediator of citrate efflux from the tricarboxylic acid cycle, SLC13A5 mediates citrate entry from blood and its activity exerts homeostatic control of cytoplasmic citrate. Intriguingly, Slc13a5-deficient mice also exhibited defective tooth enamel and dentin formation, a clinical feature, which we show is recapitulated in primary teeth from children with SLC13A5 mutations. Together, our results reveal the components of an osteoblast metabolic pathway, which affects bone strength by regulating citrate deposition into mineral hydroxyapatite.

The Role of 5' Adenosine Monophosphate-Activated Protein Kinase in the Chemo-Sensitivity and Metabolic Behaviour of Breast Cancer Cells Exposed to Hypoxia and Hyperglycaemia

Background: 5' adenosine monophosphate-activated protein kinase (AMPK) is a key enzyme for maintaining energy homeostasis in the cell and is associated with many downstream targets of metabolic processes such as mTORC1, p53 and fatty acid synthase (FASN) and insulin-like growth factor binding protein-2 (IGFBP-2).&#x0D; Aim: To investigate the interactions between AMPK, FASN and IGFBP-2 and how the activity of AMPK affects the metabolism and response of breast cancer cells to chemotherapy with changes in oxygenation and under different glucose concentrations.&#x0D; Methods: MCF-7 breast cancer cells were exposed to different glucose levels (5mM and 25mM) in the presence or absence of doxorubicin under normoxic and hypoxic conditions with and without AMPK silenced using siRNA. Changes in protein abundance were monitored using Western Immunoblotting. Cell death was measured by the Muse® Cell Analyser using a count and viability assay. Hypoxia was chemically induced using cobalt chloride or with low levels of oxygen (2%). Lactate and citrate levels were measured using commercially available kits.&#x0D; Results: In normoxic conditions, AMPK activity was higher in normal levels of glucose (5mM) compared with high levels of glucose (25mM). Under hypoxic conditions, AMPK phosphorylation remained high in 5mM glucose with levels in 25 mM glucose being equivalent. Upregulation of AMPK in normoxic and hypoxic conditions was associated with a reduction in FASN and IGFBP-2, which resulted in a better response to chemotherapy. Moreover, the cells increased the production of lactate and reduced production of citrate under normoxic conditions in 25mM glucose compared to 5mM glucose. Silencing AMPK under normoxic conditions or inducing hypoxia promoted a more glycogenic phenotype. However, silencing AMPK under hypoxic conditions reduced levels of lactate comparable to normoxic levels. The citrate profile was unaffected by silencing AMPK or altering levels of oxygen.&#x0D; Conclusions: AMPK plays an important role in regulating metabolic signalling and this alters the sensitivity of breast cancer cells to chemotherapy.

Autoantibodies targeting malondialdehyde-modifications in rheumatoid arthritis regulate osteoclasts via inducing glycolysis and lipid biosynthesis

Proteins subjected to post-translational modifications, such as citrullination, carbamylation, acetylation or malondialdehyde (MDA)-modification are targeted by autoantibodies in seropositive rheumatoid arthritis (RA). Epidemiological and experimental studies have both suggested the pathogenicity of such humoral autoimmunity, however, molecular mechanisms triggered by anti-modified protein antibodies have remained to be identified.Here we describe in detail the pathways induced by anti-MDA modified protein antibodies that were obtained from synovial B cells of RA patients and that possessed robust osteoclast stimulatory potential and induced bone erosion in vivo. Anti-MDA antibodies boosted glycolysis in developing osteoclasts via an FcγRI, HIF-1α and MYC-dependent mechanism and subsequently increased oxidative phosphorylation. Osteoclast development required robust phosphoglyceride and triacylglyceride biosynthesis, which was also enhanced by anti-MDA by modulating citrate production and expression of the glycerol-3-phosphate dehydrogenase 1 (GPD1) and glycerol-3-phosphate acyltransferase 2 (GPAT2) genes. In summary, we described novel metabolic pathways instrumental for osteoclast differentiation, which were targeted by anti-MDA antibodies, accelerating bone erosion, a central component of RA pathogenesis.

Sustainable citric acid production from CO2 in an engineered cyanobacterium.

Citric acid is one of the most widely used organic acids in the world, with applications ranging from acidity regulation in food and beverages to metal chelation in hydrometallurgical processes. Most of its production is currently derived from fermentative processes, using plant-derived carbon feedstocks. While these are currently dominant, there is an increasing need to develop closed-loop production systems that reduce process carbon footprint. In this work, we demonstrate for the first time that an engineered marine cyanobacterium Synechococcus sp. PCC 7002 can be used as a sustainable chassis for the photosynthetic conversion of CO2 to citric acid. Decreased citric acid cycle flux, through the use of a theophylline-responsive riboswitch, was combined with improved flux through citrate synthase and enhanced citric acid excretion, resulting in a significant improvement to citric acid production. While allowing citrate production, this strategy induces a growth defect which can be overcome by glutamate supplementation or by fine-tuning aconitase levels, resulting in an increase in production relative to WT of over 100-fold. This work represents a first step toward sustainable production of a commodity organic acid from CO2.

Metabolite Effect on Angiogenesis: Insights from Transcriptome Analysis.

Metabolic status of the cells is important in the expression of the angiogenic phenotype in endothelial cells. Our earlier studies demonstrated the effects of metabolites such as lactate, citrate and lipoxygenase products, on VEGFA-VEGFR2 signaling pathway. Though this link between metabolite status and molecular mechanisms of angiogenesis is becoming evident, it is not clear how it affects genome-level expression in endothelial cells, critical to angiogenesis. In the present study, computational analysis was carried out on the transcriptome data of 4 different datasets where HUVECs were exposed to low and high glucose, both in vitro and in vivo, and the expression of a key enzyme involved in glucose metabolism is altered. The differentially expressed genes belonging to both VEGFA-VEGFR2 signaling pathway, as well as several VEGF signature genes as hub genes were also identified. These findings suggest the metabolite dependence, particularly glucose dependence, of angiogenesis, involving modulation of genome-level expression of angiogenesis- functional genome. This is important in tumor angiogenesis where reprogramming of metabolism is critical.

Metabolic evidence for distinct pyruvate pools inside plant mitochondria.

The majority of the pyruvate inside plant mitochondria is either transported into the matrix from the cytosol via the mitochondria pyruvate carrier (MPC) or synthesized in the matrix by alanine aminotransferase (AlaAT) or NAD-malic enzyme (NAD-ME). Pyruvate from these origins could mix into a single pool in the matrix and contribute indistinguishably to respiration via the pyruvate dehydrogenase complex (PDC), or these molecules could maintain a degree of independence in metabolic regulation. Here we demonstrate that feeding isolated mitochondria with uniformly labelled 13C-pyruvate and unlabelled malate enables the assessment of pyruvate contribution from different sources to intermediate production in the tricarboxylic acid cycle. Imported pyruvate was the preferred source for citrate production even when the synthesis of NAD-ME-derived pyruvate was optimized. Genetic or pharmacological elimination of MPC activity removed this preference and allowed an equivalent amount of citrate to be generated from the pyruvate produced by NAD-ME. Increasing the mitochondrial pyruvate pool size by exogenous addition affected only metabolites from pyruvate transported by MPC, whereas depleting the pyruvate pool size by transamination to alanine affected only metabolic products derived from NAD-ME. PDC was more membrane-associated than AlaAT and NAD-ME, suggesting that the physical organization of metabolic machinery may influence metabolic rates. Together, these data reveal that the respiratory substrate supply in plants involves distinct pyruvate pools inside the matrix that can be flexibly mixed on the basis of the rate of pyruvate transport from the cytosol. These pools are independently regulated and contribute differentially to organic acid export from plant mitochondria.

Multiple metabolic pathways fuel the truncated tricarboxylic acid cycle of the prostate to sustain constant citrate production and secretion

ObjectiveThe prostate is metabolically unique: it produces high levels of citrate for secretion via a truncated tricarboxylic acid (TCA) cycle to maintain male fertility. In prostate cancer (PCa), this phenotype is reprogrammed, making it an interesting therapeutic target. However, how the truncated prostate TCA cycle works is still not completely understood. MethodsWe optimized targeted metabolomics in mouse and human organoid models in ex vivo primary culture. We then used stable isotope tracer analyses to identify the pathways that fuel citrate synthesis. ResultsFirst, mouse and human organoids were shown to recapitulate the unique citrate-secretory program of the prostate, thus representing a novel model that reproduces this unusual metabolic profile. Using stable isotope tracer analysis, several key nutrients were shown to allow the completion of the prostate TCA cycle, revealing a much more complex metabolic profile than originally anticipated. Indeed, along with the known pathway of aspartate replenishing oxaloacetate, glutamine was shown to fuel citrate synthesis through both glutaminolysis and reductive carboxylation in a GLS1-dependent manner. In human organoids, aspartate entered the TCA cycle at the malate entry point, upstream of oxaloacetate. Our results demonstrate that the citrate-secretory phenotype of prostate organoids is supported by the known aspartate–oxaloacetate–citrate pathway, but also by at least three additional pathways: glutaminolysis, reductive carboxylation, and aspartate–malate conversion. ConclusionsOur results add a significant new dimension to the prostate citrate-secretory phenotype, with at least four distinct pathways being involved in citrate synthesis. Better understanding this distinctive citrate metabolic program will have applications in both male fertility as well as in the development of novel targeted anti-metabolic therapies for PCa.

Tributyl citrate production via reactive distillation: Model reconciliation, optimization, scale up and sustainability indicators

• Reconciliation of a reactive distillation (RD) model for tributyl citrate (TBC) production. • A RD Industrial process for TBC production is designed and optimized. • Homogeneous and heterogenous catalysts are assessed. • Comparison with current industrial technology is presented. • Economic and sustainability indicators are presented. This work describes a model reconciliation of a reactive distillation (RD) process for the production of tributyl citrate based upon pilot scale experimental data. Steady state modeling of the process was constructed using previously validated thermodynamics and reaction kinetics that included catalytic and self-catalytic contributions. The set of pilot scale experimental data included input–output flow rates, and composition and temperature profiles of 18 runs under continuous operation. In order to reconcile the RD model, heat losses, catalyst effectiveness, Murphree efficiencies, and reactive and non-reactive liquid holdup were adjusted to fit the measured data. After reconciliation, the obtained model was in good agreement with pilot scale results, with average mass fractions and temperatures deviations < 2%. Once reconciled, the RD model was used to design and optimize the performance of a 5000 ton/yr industrial scale facility. The optimization procedure included decision, design and operating variables of continuous or discrete nature. The best configurations involved a single RD column for the heterogeneous catalyst, or a pre-reactor-RD column scheme for the homogeneously catalyzed process. Reflux ratios ranged from 0.01 to 0.5 and pre-reactor residence times were around 1 h. The optimized configuration for heterogeneous catalyzed process exhibited up to 30% energy savings with respect to the homogeneous case. The unitary TBC production costs were calculated to be 1.66 and 1.69 USD/kg for the heterogeneous and homogeneous case respectively. Finally, the optimized configurations were compared using sustainability indicators.

Effect of lithium citrate on hydration of cement paste

This paper systematically investigated the influence of lithium citrate on the properties of cement. Its effect on hydration heat and internal pore structures was examined by isothermal calorimeter and mercury intrusion porosimetry (MIP) analysis, respectively. The evolution of hydration products and changes of the ion concentration in the pore solution were also investigated by thermal gravimetric (TG), X-ray diffraction (XRD)and Inductively Coupled Plasma (ICP). Thermodynamic model was used to further reveal the relationship between lithium citrate and hydration products. The results showed that lithium citrate first retarded the cement hydration, but improved the strength subsequently functioning as an accelerator as early as 7 curing days. This was because upon the addition in the cement, lithium citrate immediately formed a protective outer layer wrapping around cement clinker particles. As hydration further proceeded, the protective layers were gradually broken down, and the released lithium ions then promoted the hydration of C3A to form a lithium aluminate amorphous gel compound, providing new sites for cement hydration and contributing to a higher content of portlandite and calcite, which in turn augmented the strength.