Pentose Phosphate Pathway Pathway Research Articles

Supplemental oxygen alters the pentose phosphate pathway in the developing mouse brain through SIRT signaling

Oxygen support plays a critical role in the management of preterm infants in neonatal intensive care units. On the other hand, the possible effects of oxygen supplementation on cellular functions, specifically glucose metabolism, have been less understood. Purposeof the study is to investigate whether supplemental oxygen alters glucose metabolism and pentose phosphate pathway (PPP) activity in the brain tissue and its relevance with silent information regulator proteins (SIRT) pathway. For this purpose, newborn C57BL/6 pups were exposed to 90% oxygen from birth until postnatal day 7 (PN7) and metabolites of glysolysis and PPP were investigated through metabolomics analysis. SIRT1, glucose-6-phosphate dehydrogenase (G6PD) and transaldolase (TALDO) proteins were examined immunohistochemically and molecularly in the prefrontal and hippocampus regions of the brain. Later on, SIRT1 inhibition was carried out.Our results indicate that supplemental oxygen causes an increase in PPP metabolites as well as activation of G6PD enzyme in the brain tissue, which is reversed by SIRT1 inhibition. Our study underlines a connection between supplemental oxygen, glucose metabolism, PPP pathway and the SIRT signaling. Understanding these intricate relationships not only deepens our knowledge of cellular physiology but also holds promise for therapeutic interventions for creating neuroprotective strategies in preterm brain.

Effects of Foliar Ca and Mg Nutrients on the Respiration of ‘Feizixiao’ Litchi Pulp and Identification of Differential Expression Genes Associated with Respiration

The ‘Feizixiao’ litchi cultivar, predominantly grown in Hainan Province, faces the issue of “sugar receding” during fruit ripening. The application of mixed foliar nutrients containing calcium and magnesium (Ca+Mg) during the fruit pericarp’s full coloring stage was investigated to overcome this issue. Experimental trials unveiled significant alterations in litchi pulp physiochemical properties, including the main nutrient and flavor quality, the total respiration rates of the main respiratory pathways, and the activities of some important enzymes associated with Embden–Meyerhof–Parnas (EMP), the tricarboxylic acid cycle (TCA) and the pentose phosphate pathway (PPP). The Ca+Mg treatment showed higher sugar levels than the control (CK) during ripening. Notably, the application of Ca+Mg in litchi pulp inhibited respiration rates through the EMP, TCA, and PPP pathways, resulting in a strong effect. RNA sequencing analysis revealed the impact of Ca+Mg treatment on respiratory pathways, revealing differentially expressed genes (DEGs) such as pyruvate PK1, PK2 (pyruvate kinase), and PDC (pyruvate dehydrogenase complex), validated through qRT-PCR with a significant correlation to RNA-seq results. In general, Ca+Mg treatment during litchi fruit ripening overcame “sugar receding” by inhibiting the expression of respiration key metabolic pathway genes. These findings provide insights for enhancing cultivation management strategies.

Prolactin receptor potentiates chemotherapy through miRNAs-induced G6PD/TKT inhibition in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with a notoriously dismal prognosis. As a competitive inhibitor of DNA synthesis, gemcitabine is the cornerstone drug for treating PDAC at all stages. The therapeutic effect of gemcitabine, however, is often hindered by drug resistance, and the underlying mechanisms remain largely unknown. It is unclear whether their response to chemotherapeutics is regulated by endocrine regulators, despite the association between PDAC risk and endocrine deregulation. Here, we show that prolactin receptor (PRLR) synergizes with gemcitabine in both invitro and invivo treatment of PDAC. Interestingly, PRLR promotes the expression of miR-4763-3p and miR-3663-5p, two novel miRNAs whose functions are unknown. Furthermore, the analysis of transcriptome sequencing data of tumors from lactating mouse models enriches the PPP pathway, a multifunctional metabolic pathway. In addition to providing energy, the PPP pathway mainly provides a variety of raw materials for anabolism. We demonstrate that two key enzymes of the pentose phosphate pathway (PPP), G6PD and TKT, are directly targeted by miR-4763-3p and miR-3663-5p. Notably, miR-4763-3p and miR-3663-5p diminish the nucleotide synthesis of the PPP pathway, thereby increasing gemcitabine sensitivity. As a result, PRLR harnesses these two miRNAs to suppress PPP and nucleotide synthesis, subsequently elevating the gemcitabine sensitivity of PDAC cells. Also, PDAC tissues and tumors from LSL-KrasG12D/+, LSL-Trp53R172H/+, and PDX1-cre (KPC) mice exhibit downregulation of PRLR. Bisulfite sequencing of PDAC tissues revealed that PRLR downregulation is due to epigenetic methylation. In this study, we show for the first time that the endocrine receptor PRLR improves the effects of gemcitabine by boosting two new miRNAs that block the PPP pathway and nucleotide synthesis by inhibiting two essential enzymes concurrently. The PRLR-miRNAs-PPP axis may serve as a possible therapeutic target to supplement chemotherapy advantages in PDAC.

Gambogic acid exhibits promising anticancer activity by inhibiting the pentose phosphate pathway in lung cancer mouse model

BackgroundThe pentose phosphate pathway (PPP) plays a crucial role in the material and energy metabolism in cancer cells. Targeting 6-phosphogluconate dehydrogenase (6PGD), the rate-limiting enzyme in the PPP metabolic process, to inhibit cellular metabolism is an effective anticancer strategy. In our previous study, we have preliminarily demonstrated that gambogic acid (GA) induced cancer cell death by inhibiting 6PGD and suppressing PPP at the cellular level. However, it is unclear whether GA could suppress cancer cell growth by inhibiting PPP pathway in mouse model. PurposeThis study aimed to confirm that GA as a covalent inhibitor of 6PGD protein and to validate that GA suppresses cancer cell growth by inhibiting the PPP pathway in a mouse model. MethodsCell viability was detected by CCK-8 assays as well as flow cytometry. The protein targets of GA were identified using a chemical probe and activity-based protein profiling (ABPP) technology. The target validation was performed by in-gel fluorescence assay, the Cellular Thermal Shift Assay (CETSA). A lung cancer mouse model was constructed to test the anticancer activity of GA. RNA sequencing was performed to analyze the global effect of GA on gene expression. ResultsThe chemical probe of GA exhibited high biological activity in vitro. 6PGD was identified as one of the binding proteins of GA by ABPP. Our findings revealed a direct interaction between GA and 6PGD. We also found that the anti-cancer activity of GA depended on reactive oxygen species (ROS), as evidenced by experiments on cells with 6PGD knocked down. More importantly, GA could effectively reduce the production of the two major metabolites of the PPP in lung tissue and inhibit cancer cell growth in the mouse model. Finally, RNA sequencing data suggested that GA treatment significantly regulated apoptosis and hypoxia-related physiological processes. ConclusionThese results demonstrated that GA was a covalent inhibitor of 6PGD protein. GA effectively suppressed cancer cell growth by inhibiting the PPP pathway without causing significant side effects in the mouse model. Our study provides in vivo evidence that elucidates the anticancer mechanism of GA, which involves the inhibition of 6PGD and modulation of cellular metabolic processes.

Reactive oxygen species homeostasis and carbohydrate metabolism involved in wound healing of carrot induced by hot water treatment

Although the information on regulation of reactive oxygen species (ROS) homeostasis or carbohydrate metabolism has been published in other wounding-responsive horticultural crops, the involvement of that in carrot remains unknown, especially treatment with hot water. In this study, ROS and carbohydrate metabolism analysis during wound healing was performed to clarify the underlying mechanism for the wounded carrots subjected to hot water treatment. Results showed that 45 ℃ hot water promoted NADPH oxidase (NOX) and superoxide dismutase (SOD) activities in healing carrots, elevated O2·− and H2O2 contents. Also, hot water enhanced catalase (CAT) and peroxidase (POD) activities, the activities of AsA-GSH cycle-related as well as the ascorbic acid and glutathione levels. Additionally, hot water stimulated acid invertase (AI) and neutral invertase (NI) activities with a higher level of sucrose, fructose and glucose, triggered stronger activities of hexokinase (HK), phosphofruc tokinase (PFK), pyruvate kinase (PK) and glucose-6-phosphate dehydrogenase (G-6-PDH) in glycolytic and pentose phosphate pathway (PPP) during healing of carrots. Taken together, hot water can regulate ROS homeostasis and activate sucrose metabolism, glycolysis and PPP pathway to benefit carrot wound healing.

An examination of the carbon metabolic pathways in Acinetobacter sp. TAC-1 in the context of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) utilization

The present study aimed to unravel the carbon metabolism pathway of Acinetobacter sp. TAC-1, a heterotrophic nitrification-aerobic denitrification (HN-AD) strain that utilizes poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) as a carbon source. Sodium acetate was employed as a control to assess the gene expression of carbon metabolic pathways in the TAC-1 strain. The results of genome sequencing demonstrated that the TAC-1 strain possessed various genes encoding carbon metabolic enzymes, such as gltA, icd, sucAB, acs, and pckA. KEGG pathway database analysis further verified the presence of carbon metabolism pathways, including the glycolytic pathway (EMP), pentose phosphate pathway (PPP), glyoxylate cycle (GAC), and tricarboxylic acid (TCA) cycle in the TAC-1 strain. The differential expression of metabolites derived from distinct carbon sources provided further evidence that the carbon metabolism pathway of TAC-1 utilizing PHBV follows the sequential process of PHBV (via the PPP pathway)→gluconate (via the EMP pathway)→acetyl-CoA (entering the TCA cycle)→CO2+H2O (generating electron donors and releasing energy). This study is expected to furnish a theoretical foundation for the advancement and implementation of novel denitrification processes based on HN-AD and solid carbon sources.

Transcriptomics reveal how circadian regulation contributes to starch hyperaccumulation in marine alga Tetraselmis helgolandica

AbstractTetraselmis helgolandica var. Tsingtaoensis is a marine microalga. It can produce a large amount of starch, especially amylose, with addition of carbon source and specific circadian rhythm. The mechanism behind this phenomenon is still unclear. Analysis of this mechanism can help to develop T. helgolandica into a new green bioengineering chassis organism. We explained how circadian rhythm and glucose affect the rate of starch accumulation and starch structure in T. helgolandica based on the transcriptome. The glucose inhibited the photosynthetic system of T. helgolandica, while the circadian rhythm can alleviate the inhibition. Circadian rhythm induced the upregulation of Embden–Meyerhof–Parnas pathway and pentose phosphate pathway (PPP) in T. helgolandica, but had little effect on the tricarboxylic acid cycle. PPP pathway provides Ribulose‐1,5‐bisphosphate, which may be beneficial for dark reactions and nucleotide synthesis. And PPP pathway provides Nicotinamide adenine dinucleotide phosphate, which facilitates energy substance synthesis. This will further upregulate the starch metabolic pathway. The transcript level of the key gene ADP‐Glucose pyrophosphorylase is mainly regulated by glucose. The granule‐bound starch synthase (gbss), a key gene for amylose synthesis, is mainly influenced by circadian rhythm. In general, the increase of starch synthesis and amylose ratio requires both glucose addition and circadian rhythm. We report the first referenced transcriptome of T. helgolandica. Differences between transcripts reveal how circadian rhythm and glucose addition affected the rate of starch synthesis and structural variation. It provides a reference for an in‐depth study of starch synthesis in green algae.

Heterotrophic modification of Phaeodactylum tricornutum Bohlin

Phaeodactylum tricornutum is abundant in eicosapentaenoic acid and fucoxanthin. Heterotrophically modifying P. tricornutum to construct a production system by fermentation has attracted increasing interest. Here, glucose used heterotrophic transformant was obtained by expressing of the human Gult1 gene in P. tricornutum. It was found that glucose metabolism was mainly dependent on the PPP pathway, while the EMP pathway was regularly embolized, and excessive G6P accumulation inhibits the heterotrophic growth of the Gult1 transformant. 6-phosphofructokinase gene (Pfk), glucose-6-phosphate dehydrogenase gene (G6pd), and 6-phosphogluconolactonase gene (Pgl) were expressed in the Gult1 transformant respectively, to enhance the EMP (Embden-Meyerhof-Parnas) and PPP (Pentose Phosphate Pathway) pathways for accelerating G6P metabolic rate. The Pfks expressed transformants have 14–33 % biomass higher than Gult1 transformant under heterotrophy and 4 times higher than autotrophy. The G6pds and Pgls expressed transformants have 40 % biomass higher than Gult1 transformant under heterotrophy and 4.5 times higher than autotrophy. These findings evidenced that heterotrophic modification and further optimizing global glucose metabolism are feasibly promising to construct a cell factory for P. tricornutum industrial fermentation.

Targeted profiling of polar metabolites in cancer metabolic reprogramming by hydrophilic interaction liquid chromatography-tandem mass spectrometry

Metabolic reprogramming of cancer cells is a hallmark of cancer, in which the polar metabolites involving aerobic glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, and glutaminolysis play a crucial role in the occurrence and development of cancer. Therefore, targeted analysis of the polar metabolites in these pathways is of great value for understanding cancers, finding diagnostic biomarkers, and identifying therapeutic targets. However, it is still challenging to directly determine polar metabolites in these pathways without derivatization due to their diverse chemical properties, isomers, and strong polarity. Herein, a highly selective and sensitive HILIC-MS/MS method was developed for direct determination of the polar metabolites in aerobic glycolysis, PPP, TCA cycle, and glutaminolysis pathways. Without derivatization, 19 polar metabolites and their isomers with carbonyl, carboxyl, or phosphoryl groups in human plasma and cell extracts of prostate cancer (PC) were determined with strong retention and high resolution. This method has been widely verified by measuring linearity, precision, sensitivity, repeatability, matrix effect, and accuracy. The analysis of plasma samples by HILIC-MS/MS revealed distinct PC-specific metabolic signatures compared to a healthy control. In addition, this method could also be used to screen the targets of metabolic inhibitors at the cellular level. We conclude that the developed HILIC-MS/MS method provides a valuable means to study the cancer metabolic reprogramming or energy metabolism in living organisms.

Ginseng-derived nanoparticles inhibit lung cancer cell epithelial mesenchymal transition by repressing pentose phosphate pathway activity.

It is unclear whether ginseng-derived nanoparticles (GDNPs) can prevent tumor cell epithelial-mesenchymal transition (EMT). Here, we describe typical characteristics of GDNPs and possible underlying mechanisms for GDNP antitumor activities. First, GDNPs particle sizes and morphology were determined using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), respectively, while cellular uptake of PKH67-labeled GDNPs was also assessed. Next, we evaluated GDNPs antitumor effects by determining whether GDNPs inhibited proliferation and migration of five tumor cell lines derived from different cell types. The results indicated that GDNPs most significantly inhibited proliferation and migration of lung cancer-derived tumor cells (A549, NCI-H1299). Moreover, GDNPs treatment also inhibited cell migration, invasion, clonal formation, and adhesion tube formation ability and reduced expression of EMT-related markers in A549 and NCI-H1299 cells in a dose-dependent manner. Meanwhile, Kaplan-Meier analysis of microarray data revealed that high-level thymidine phosphorylase (TP) production, which is associated with poor lung cancer prognosis, was inhibited by GDNPs treatment, as reflected by decreased secretion of overexpressed TP and downregulation of TP mRNA-level expression. In addition, proteomic analysis results indicated that GDNPs affected pentose phosphate pathway (PPP) activity, with ELISA results confirming that GDNPs significantly reduced levels of PPP metabolic intermediates. Results of this study also demonstrated that GDNPs-induced downregulation of TP expression led to PPP pathway inhibition and repression of lung cancer cell metastasis, warranting further studies of nano-drugs as a new and promising class of anti-cancer drugs.

Thioredoxin 1 regulates the pentose phosphate pathway via ATM phosphorylation after experimental subarachnoid hemorrhage in rats

Subarachnoid hemorrhage (SAH), a type of hemorrhagic stroke, is a neurological emergency with high morbidity and mortality. Early brain injury (EBI) after SAH is the leading cause of poor prognosis in SAH patients. TRX system is a NADPH-dependent antioxidant system which is composed of thioredoxin reductase (TRXR), thioredoxin (TRX). The pentose phosphate pathway (PPP), a pathway through which glucose can be metabolized, is a major source of NADPH. Thioredoxin 1 (TRX1) is a member of thioredoxin system mainly located in cytoplasm. Serine/threonine kinases ataxia telangiectasia mutated (ATM) is an important oxidative stress receptor, and TRX1 can regulate ATM phosphorylation and then affect the activity of PPP key enzyme glucose 6-phosphate dehydrogenase (G6PD). However, whether TRX1 is involved in the regulation of PPP pathway after subarachnoid hemorrhage remains unclear. The results showed that after SAH, the level of TRX1 and phosphor-ATM decreased while the level of TRXR1 increased. G6PD protein level remained unchanged but the activity decreased, and the NADPH contents decreased. Overexpression of TRX1 by lentivirus upregulates the level of phosphor-ATM, G6PD activity and NADPH content. TRX1 overexpression improved short-term and long-term neurobehavioral outcomes and alleviated neuronal impairment in rats. Nissl staining showed that upregulation of TRX1 reduced cortical neuron injury. Our study shows that TRX1 participates in the PPP pathway by regulating phosphorylation ATM, which is accomplished by affecting G6PD activity. TRX1 may be an important target for EBI intervention after SAH.

Combined Inhibition of CXCR4 Signaling and System xc- Transporter Activity Induces Synthetic Lethality in T-ALL Cells By Suppressing Gsh and Inducing Ferroptosis

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that accounts for 10-15% of pediatric and 25% of adult ALL cases. Prior studies have established that most cases pf T-ALL are addicted to CXCR4 signaling. Indeed, strong preclinical data demonstrating therapeutic activity of BL-8040, a potent CXCR4 antagonist, have led to a clinical trial of BL-8040 in combination with nelarabine for patients with relapsed/refractory T-ALL (NCT02763384). However, the molecular mechanisms by which CXCR4 blockade induces T-ALL cell death are unknown. Using a human T-ALL xenotransplantation model, we previously reported that treatment with BL-8040 killed T-ALL cells through a non-apoptotic mechanism. Transcriptome sequencing revealed that BL-8040 induced alterations in genes involved in oxidative phosphorylation and carbohydrate metabolism. Indeed, seahorse experiments show that BL-8040 markedly reduced both oxidative phosphorylation and glycolysis. However, metabolic tracing studies using 13C-labeled glucose show that BL-8040 treatment does not have a major effect on the contribution of glucose to either glycolysis or the citric acid cycle. Instead, the major alteration observed is the reduced entry of glucose into the pentose phosphate pathway (PPP). A major function of the PPP pathway is to generate NADPH, which regulates reactive oxygen species (ROS) by producing reduced glutathione (GSH). Indeed, BL-8040 treatment resulted in a significant decrease in the ratio of reduced glutathione to oxidized glutathione. Together, these data suggest that BL-8040 induces oxidative stress by inhibiting GSH production. One mechanism utilized by cancer cells to regulate GSH levels and oxidative stress is the system xc- amino acid antiporter that mediates the exchange of extracellular L-cystine and intracellular l-glutamate across the plasma membrane, resulting in the production of GSH and oxidative protection. We measured L-cystine levels in the media of T-ALL cells cultured for 24 hours with or without BL-8040. A significant decrease in L-cystine in the media was observed. These data, along with increased expression of the xc- transporter (SLC7A11), suggested that increased system xc- activity was compensating for the loss of GSH induced by BL-8040. To test this possibility, we cultured T-ALL cells in L-cystine deficient media. Loss of L-cystine in the media resulted in a modest decrease in T-ALL cell viability that was markedly increased, in a synergistic fashion, upon treatment with BL-8040. Interestingly, caspase 3 was not activated, suggesting that, similar to in vivo results, BL-8040 induces a non-apoptotic cell death. This observation, coupled with the reduction in GSH, suggested the hypothesis that BL-8040 induces ferroptosis. Consistent with the hypothesis, treatment of T-ALL cells with ACXT-3102, a novel system xc- inhibitor, significantly enhanced BL-8040 killing of T-ALL cells in vitro. Collectively, these data suggest that T-ALL cells are sensitive to perturbations of the glutathione axis. Combined inhibition of CXCR4 signaling and system xc- activity exploits this vulnerability and presents a promising new therapeutic approach for T-ALL. Disclosures Uy: Astellas Pharma: Honoraria; Jazz Pharmaceuticals: Consultancy; Genentech: Consultancy; Agios: Consultancy; Pfizer: Consultancy; Daiichi Sankyo: Consultancy. Sorani:BiolineRx Ltd: Current Employment. Vainstein:BiolineRx Ltd: Current Employment. Davish:BiolineRx Ltd: Current Employment. Hawkins:Accuronix Therapeutics: Membership on an entity's Board of Directors or advisory committees.

The Pentose Phosphate Pathway Dynamics in Cancer and Its Dependency on Intracellular pH.

The Pentose Phosphate Pathway (PPP) is one of the key metabolic pathways occurring in living cells to produce energy and maintain cellular homeostasis. Cancer cells have higher cytoplasmic utilization of glucose (glycolysis), even in the presence of oxygen; this is known as the “Warburg Effect”. However, cytoplasmic glucose utilization can also occur in cancer through the PPP. This pathway contributes to cancer cells by operating in many different ways: (i) as a defense mechanism via the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to prevent apoptosis, (ii) as a provision for the maintenance of energy by intermediate glycolysis, (iii) by increasing genomic material to the cellular pool of nucleic acid bases, (iv) by promoting survival through increasing glycolysis, and so increasing acid production, and (v) by inducing cellular proliferation by the synthesis of nucleic acid, fatty acid, and amino acid. Each step of the PPP can be upregulated in some types of cancer but not in others. An interesting aspect of this metabolic pathway is the shared regulation of the glycolytic and PPP pathways by intracellular pH (pHi). Indeed, as with glycolysis, the optimum activity of the enzymes driving the PPP occurs at an alkaline pHi, which is compatible with the cytoplasmic pH of cancer cells. Here, we outline each step of the PPP and discuss its possible correlation with cancer.

The Role of Non-Glycolytic Glucose Metabolism in Myocardial Recovery Following Mechanical Unloading and Circulatory Support in Chronic Heart Failure

Following left ventricular assist device (LVAD) mechanical unloading significant improvements in myocardial structure and function have been reported in some advanced heart failure (HF) patients, termed as Responders (R). Our prior work demonstrated a post-LVAD mismatch of glycolysis and oxidative phosphorylation characterized by upregulation of glycolysis without subsequent increase in pyruvate oxidation via the Krebs cycle. We hypothesized that the accumulated glycolytic intermediates are channeled into cardioprotective and repair pathways, such as the pentose phosphate pathway (PPP) and one-carbon metabolism (OCM), which may mediate myocardial structural and functional improvement in Responders. We prospectively obtained myocardial tissue from Non-Failing Donor hearts (n=15) and from Responders (n=11) and Non-Responders (n=30) at LVAD implant (Pre-LVAD) and later at transplantation (Post-LVAD). Responders and Non-Responders were defined based on prior published criteria. We conducted protein expression and metabolite profiling and evaluated mitochondrial structure using electron microscopy. Western blot analysis of key PPP and OCM rate-limiting enzymes, glucose-6-phosphate dehydrogenase and phosphoglycerate dehydrogenase were significantly increased in Post-LVAD Responders (Post-R). In agreement with these findings the metabolite levels of these enzyme substrates such as sedoheptulose-6-phosphate (PPP) and serine and glycine (OCM) were decreased in Post-R. Furthermore, Post-R had significantly higher NADPH levels, reduced ROS levels, improved mitochondrial dynamics and enhanced glycosylation of the extracellular matrix (ECM) protein, α-dystroglycan, indicating increased flux of glucose into the PPP and OCM pathways and all together these findings correlated with the observed myocardial structural and functional improvement. The recovering human heart appears to direct glycolytic metabolites into the PPP and OCM pathways which could contribute to cardioprotection and improved myocardial function by generating NADPH to enhance biosynthesis for repair and by reducing oxidative stress.

Placental growth factor regulates the pentose phosphate pathway and antioxidant defense systems in human retinal endothelial cells

The molecular mechanisms whereby placental growth factor (PlGF) mediates its effects in nonproliferative diabetic retinopathy (DR) are unknown. To better understand the role of PlGF in DR, we used tandem mass tags (TMT)-labeled quantitative proteomics to human retinal endothelial cells (HRECs), treated anti-PlGF antibody, and PBS as a control. Functional annotation and pathway enrichments were performed, which suggested that the differentially expressed proteins (DEPs) were involved in key metabolic processes, protein binding, and membrane, pentose phosphate pathway (PPP) and adherens junction. We conducted integrated gene profiles of our previously published transcriptomic data to the TMT-labeled proteomics data. The results showed the sixty genes were found to be changed at the proteome level. The functional annotation conducted for the sixty proteins suggested that 58.3% of proteins were involved in PPP, 25% of proteins were in interleukin-12 singling and 16.7% of proteins were involved in glycolysis and gluconeogenesis pathway. Mass spectrometry results were validated by transendothelial electrical resistance measurement by an electrical cell-impedance sensing (ECIS) and western blot analysis of VE-cadherin, G6PD. These findings suggest that the PPP proteins and antioxidants may act as a downstream target of PlGF and may play a decisive role in HREC biological functions in DR. SignificancePlGF (Placental growth factor) is known to play a pivotal role in pathological angiogenesis and inflammation by stimulating endothelial cell migration and by recruiting pericytes and inflammatory cells such as microglia and macrophages. Despite the well-defined pathophysiological roles of PlGF, the underlying molecular and cellular mechanisms are not completely understood, especially the exact relationships between biochemical events and molecular pathways regulated by PlGF, whose inhibition exhibits a protective role in DR. This study provides new insights into protein expression patterns and enables the identification of many attractive candidates for investigation of PPP pathway role in the activation of the antioxidant defense system in DR. Our findings suggest that the PPP proteins and antioxidants (PRDX6, HMOX1, NQO1 and YES1) may act as downstream targets of PlGF and may play a decisive role in HREC biological functions in DR.

Nanocomposite packaging regulate respiration and energy metabolism in Flammulina velutipes

Energy metabolism is accompanied with postharvest senescence, deterioration and physiological disorder in Flammulina velutipes. In this study, nanocomposite packaging material (Nano-PM) inhibited rapid respiration thus preventing accumulation of reactive oxygen species (ROS). In comparison to the normal packaging material (Normal-PM) and no packaging (No-PM), Nano-PM elevated NADP and NADPH contents by increasing the key enzymes in pentose phosphate pathway (PPP), NADK and G6PDH activities, which was due to ROS removal. Moreover, the package enhanced mitochondrial ATPase activity. Regulation of PPP pathway and elevation of ATPase activity was the main reason for the maintenance of mitochondria membrane and structure integrity. Therefore, Nano-PM maintained sufficient ATP content and energy charge by protecting mitochondria structure and regulating electron transfer of mitochondrial respiratory. Overall, Nano-PM maintained energy metabolism by delaying apoptosis and swelling of mitochondria, thus, it alleviated postharvest senescence of F. velutipes.

PhoU2 but Not PhoU1 as an Important Regulator of Biofilm Formation and Tolerance to Multiple Stresses by Participating in Various Fundamental Metabolic Processes in Staphylococcus epidermidis.

ABSTRACTPhoU, a conserved protein that has been proposed to coordinate phosphate import, is a negative regulator of drug tolerance in most bacteria. In Staphylococcus epidermidis, the role of PhoU in biofilm formation and drug tolerance has not yet been investigated. Two PhoU homologs in the genome of S. epidermidis have been identified by the presence of the conserved motif E(D)XXXD of PhoU. We separately constructed ΔphoU1 and ΔphoU2 mutants of S. epidermidis strain 1457. The ΔphoU2 mutant displayed growth retardation, a weakened biofilm formation capacity, a higher sensitivity to H2O2, and reduced tolerance to multiple antibiotics. However, deletion of phoU1 had no effect on those. We compared the transcriptome profiles of the ΔphoU2 and ΔphoU1 mutants with that of the parent strain. In the ΔphoU2 mutant, expression of genes related to inorganic phosphate uptake was significantly upregulated (pst operon) and the levels of intracellular inorganic polyphosphate (polyP) were increased. In the ΔphoU2 mutant, expression of enzymes in the pentose phosphate pathway (PPP) was downregulated and less NADP (NADPH) was detected, consistent with the high sensitivity to H2O2 and the growth retardation of the ΔphoU2 mutant. The upregulated expression of ATP synthase was consistent with the high intracellular ATP content in the ΔphoU2 mutant, which may have been related to the lower drug tolerance of the ΔphoU2 mutant. This study demonstrates that PhoU2, but not PhoU1, in S. epidermidis regulates bacterial growth, biofilm formation, oxidative stress, and drug tolerance in association with alterations to inorganic phosphate metabolism, the pentose phosphate pathway, galactose metabolism, the tricarboxylic acid (TCA) or citric cycle, glycolysis and gluconeogenesis, and respiratory reactions.IMPORTANCE PhoU is widely conserved throughout the bacterial kingdom and plays an important role in response to stress and metabolic maintenance. In our study, two PhoU homologs were found in S. epidermidis. The function of phoU2, but not phoU1, in S. epidermidis is related to growth, drug tolerance, the oxidative stress response, polyP levels, and ATP accumulation. In addition, phoU2 regulates biofilm formation. Hence, phoU2 is a regulator of both drug tolerance and biofilm formation, which are two bacterial properties that present major challenges to the clinical treatment of infections. Analysis of differential gene expression revealed that phoU2 is involved in fundamental metabolic processes, such as the PPP pathway. These findings indicate that phoU2 is a crucial regulator in S. epidermidis.

Optimal 13C-labeling of glycerol carbon source for precise flux estimation in Escherichia coli

Glycerol is a promising carbon source for bio-production and is particularly attractive because it is produced in excess as a biodiesel byproduct. Elucidating the flux distribution of glycerol catabolism would greatly aid metabolic engineering, but 13C-labeling of glycerol has not yet been optimized for precise flux estimations. In this study, an Escherichia coli wild type strain was aerobically cultured using glycerol as the sole carbon source. [1,3-13C], [2-13C], and [U-13C] glycerols were independently mixed with an equal amount of naturally labeled glycerol; these mixtures were used as 13C-labeled substrates, and flux distributions during exponential growth were estimated based on 13C-enrichment of proteinogenic amino acids. The glycerol catabolism pathway in E.coli has four branches: the oxidative pentose phosphate pathway (PP), Entner-Doudoroff pathway (ED), and malic enzyme (ME) pathways, and the glyoxylate shunt (GX). The 95% confidence intervals of these fluxes were compared across the 13C-labeling experiments. The [2-13C] and [U-13C] glycerols, but not [1,3-13C] glycerol allowed precise characterization of the PP, ED, and ME pathway fluxes. All three types of 13C-labeling aided in successfully determining the GX flux. Based on the above estimated flux distribution, various patterns of 13C-labeling of glycerol were computationally generated. These in silico experiments revealed that the sole use of [2-13C] glycerol or [1,3-13C] glycerol is optimal for precise flux estimation, where simultaneous using glycerols with different types of 13C-labeling failed to improve flux estimation as assessed by confidence intervals.

Multi-omics analyses of red blood cell reveal antioxidation mechanisms associated with hemolytic toxicity of gossypol.

Gossypol is an antiproliferative drug with limited use due to its hemolytic toxicity. In this study, accelerated hemolysis was observed in the cows treated with gossypol. Comparative metabolomics were used to gain responsive pathways in the red blood cell (RBC) to the treatment, which were crossly validated by parallel iTRAQ-based proteomic analysis and enzyme activity assay. We found that gossypol treatment appeared to considerably activate pentose phosphate pathway (PPP) with an increased key product of ribose-5-phosphate and the increased abundance and activity of several key enzymes such as 6-phosphogluconate dehydrogenase, flavin reductase, and ribose-phosphate pyrophesphokinase. Meanwhile, a decreased glycolysis metabolism was observed, as many input metabolites of glycolysis were reduced in the gossypol group, whereas its distal metabolites were unchanged, along with decreased abundance of triosephosphate isomerase and increased abundance of enzymes catalyzing several distal glycolytic steps. Oxidative reduction pathways were also remarkably affected as we found a decreased substrate of flavin reductase, glutathione disulfide, increased glutathione reductase activity, and increased abundance and activity of glutathione S-transferase with the increase of its catalytic product, cysteine. Our results demonstrated that glycolysis, PPP, and oxidative reduction pathways of RBC were all involved in RBC’s response to the hemolytic toxicity of gossypol.

Abstract LB-333: Lymphatic endothelium increases antioxidant capacity of triple-negative breast cancer cells and protects from cell death

Abstract Involvement of lymphatic system with cancer and the extent of lymph node metastases are directly correlated with the poor patient outcome. However, it is not understood whether the presence of lymphatic metastases is only indicative of an aggressive cancer or if the lymphatic vessel microenvironment directly contributes to the metastatic progression. We demonstrate that soluble factors produced by lymphatic endothelial cells (LECs) protect triple negative breast cancer cells from cell death in vitro. Co-culture with LECs or LEC-conditioned medium (LEC-CM) protected cancer cells from death induced by the loss of homotypic cell adhesion, nutrient deprivation, or loss of matrix attachment. High levels of reactive oxygen species (ROS) preceded cell death, and were significantly decreased in tumor cells upon treatment with LEC-CM. Furthermore, LEC-CM protected tumor cells from death induced by exogenous oxidative stress (H2O2), while treatment with the anti-oxidant N-acetyl-cysteine (NAC) recapitulated the cytoprotective effect of LEC-CM. RNA-Seq analysis revealed Nrf2 pathway as the most upregulated stress-pathway induced in tumor cells upon loss of adhesion. Nrf2 and other stress signaling pathways were significantly diminished in the presence of LEC-CM. Pharmacological inhibition of the pentose phosphate pathway (PPP) and the components of thioredoxin and glutathione scavanging systems increased ROS and cell death in LEC-CM indicating that the maintenance of redox homeostasis and cell viability by LEC-CM is dependent on the PPP pathway and in particular thioredoxin system. Furthermore, LEC-CM preserved integrity and function of mitochondria. These results demonstrate that soluble factors produced by lymphatic endothelium promote survival of triple-negative breast cancer cells under stress by regulating tumor cell redox homeostasis and promoting mitochondrial function. Citation Format: Mirela Berisa, Simona Podgrabinska, Jerry Chipuk, Mihaela Skobe. Lymphatic endothelium increases antioxidant capacity of triple-negative breast cancer cells and protects from cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-333. doi:10.1158/1538-7445.AM2017-LB-333