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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 829 records · Page 46
All sourcesNASA NTRSDOE OSTI
DOE OSTI2016

Concurrent acetylation of FoxO1/3a and p53 due to sirtuins inhibition elicit Bim/PUMA mediated mitochondrial dysfunction and apoptosis in berberine-treated HepG2 cells

Post-translational modifications i.e. phosphorylation and acetylation are pivotal requirements for proper functioning of eukaryotic proteins. The current study aimed to decode the impact of acetylation/deacetylation of non-histone targets i.e. FoxO1/3a and p53 of sirtuins (NAD{sup +} dependent enzymes with lysine deacetylase activity) in berberine treated human hepatoma cells. Berberine (100 μM) inhibited sirtuins significantly (P < 0.05) at transcriptional level as well as at translational level. Combination of nicotinamide (sirtuin inhibitor) with berberine potentiated sirtuins inhibition and increased the expression of FoxO1/3a and phosphorylation of p53 tumor suppressor protein. As sirtuins deacetylate non-histone targets including FoxO1/3a and p53, berberine increased the acetylation load of FoxO1/3a and p53 proteins. Acetylated FoxO and p53 proteins transcriptionally activate BH3-only proteins Bim and PUMA (3.89 and 3.87 fold respectively, P<0.001), which are known as direct activator of pro-apoptotic Bcl-2 family protein Bax that culminated into mitochondria mediated activation of apoptotic cascade. Bim/PUMA knock-down showed no changes in sirtuins' expression while cytotoxicity induced by berberine and nicotinamide was curtailed up to 28.3% (P < 0.001) and it restored pro/anti apoptotic protein ratio in HepG2 cells. Sirtuins inhibition was accompanied by decline in NAD{sup +}/NADH ratio, ATP generation, enhanced ROS production and decreased mitochondrial membrane potential. TEM analysis confirmed mitochondrial deterioration and cell damage. SRT-1720 (1–10 μM), a SIRT-1 activator, when pre-treated with berberine (25 μM), reversed sirtuins expression comparable to control and significantly restored the cell viability (P < 0.05). Thus, our findings suggest that berberine mediated sirtuins inhibition resulting into FoxO1/3a and p53 acetylation followed by BH3-only protein Bim/PUMA activation may in part be responsible for mitochondria-mediated apoptosis. - Highlights: • Berberine inhibits sirtuins at transcriptional as well as at translational level. • Sirtuins inhibition leads to acetylation of non-histone proteins, FoxO1/3a and p53. • Acetylated FoxO and p53 transcriptionally upregulate BH3-only proteins Bim and PUMA respectively. • BH3-only proteins trigger mitochondrial dysfunction culminating into apoptosis. • SRT-1720 (SIRT-1 activator) partially restores Bax/Bcl-2 ratio and reduces berberine induced cytotoxicity in HepG2 cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Distribution, lipid-bilayer affinity and kinetics of the metabolic effects of dinoseb in the liver

Dinoseb is a highly toxic pesticide of the dinitrophenol group. Its use has been restricted, but it can still be found in soils and waters in addition to being a component of related pesticides that, after ingestion by humans or animals, can originate the compound by enzymatic hydrolysis. As most dinitrophenols, dinoseb uncouples oxidative phosphorylation. In this study, distribution, lipid bilayer affinity and kinetics of the metabolic effects of dinoseb were investigated, using mainly the isolated perfused rat liver, but also isolated mitochondria and molecular dynamics simulations. Dinoseb presented high affinity for the hydrophobic region of the lipid bilayers, with a partition coefficient of 3.75 × 10{sup 4} between the hydrophobic and hydrophilic phases. Due to this high affinity for the cellular membranes dinoseb underwent flow-limited distribution in the liver. Transformation was slow but uptake into the liver space was very pronounced. For an extracellular concentration of 10 μM, the equilibrium intracellular concentration was equal to 438.7 μM. In general dinoseb stimulated catabolism and inhibited anabolism. Half-maximal stimulation of oxygen uptake in the whole liver occurred at concentrations (2.8–5.8 μM) at least ten times above those in isolated mitochondria (0.28 μM). Gluconeogenesis and ureagenesis were half-maximally inhibited at concentrations between 3.04 and 5.97 μM. The ATP levels were diminished, but differently in livers from fed and fasted rats. Dinoseb disrupts metabolism in a complex way at concentrations well above its uncoupling action in isolated mitochondria, but still at concentrations that are low enough to be dangerous to animals and humans even at sub-lethal doses. - Highlights: • Dinoseb presents high affinity for the hydrophobic region of the lipid bilayers. • Its cellular concentration may exceed the extracellular one by a factor of 43.9. • One order of magnitude separates the active concentrations in mitochondria and liver. • Hepatic metabolism is disrupted in the concentration range up to 10 μM.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Loss of fructose-1,6-bisphosphatase induces glycolysis and promotes apoptosis resistance of cancer stem-like cells: an important role in hexavalent chromium-induced carcinogenesis

Hexavalent chromium (Cr(VI)) compounds are confirmed human carcinogens for lung cancer. Our previous studies has demonstrated that chronic exposure of human bronchial epithelial BEAS-2B cells to low dose of Cr(VI) causes malignant cell transformation. The acquisition of cancer stem cell-like properties is involved in the initiation of cancers. The present study has observed that a small population of cancer stem-like cells (BEAS-2B-Cr-CSC) exists in the Cr(VI)-transformed cells (BEAS-2B-Cr). Those BEAS-2B-Cr-CSC exhibit extremely reduced capability of generating reactive oxygen species (ROS) and apoptosis resistance. BEAS-2B-Cr-CSC are metabolic inactive as evidenced by reductions in oxygen consumption, glucose uptake, ATP production, and lactate production. Most importantly, BEAS-2B-Cr-CSC are more tumorigenic with high levels of cell self-renewal genes, Notch1 and p21. Further study has found that fructose-1,6-bisphosphatase (FBP1), an rate-limiting enzyme driving glyconeogenesis, was lost in BEAS-2B-Cr-CSC. Forced expression of FBP1 in BEAS-2B-Cr-CSC restored ROS generation, resulting in increased apoptosis, leading to inhibition of tumorigenesis. In summary, the present study suggests that loss of FBP1 is a critical event in tumorigenesis of Cr(VI)-transformed cells. - Highlights: • A small population of cancer-like stem cells exist in Cr(VI)-transformed cells. • Those cancer-like stem cells are low in ROS levels and resistant to apoptosis. • Those cancer-like stem cells are metabolic inactive and loss of FBP1. • Those cancer-like stem cells initiate and sustain tumor growth. • Forced expression of FBP1 inhibits tumorigenecity of those cancer-like stem cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Baculoviruses and nucleosome management

Negatively-supercoiled-ds DNA molecules, including the genomes of baculoviruses, spontaneously wrap around cores of histones to form nucleosomes when present within eukaryotic nuclei. Hence, nucleosome management should be essential for baculovirus genome replication and temporal regulation of transcription, but this has not been documented. Nucleosome mobilization is the dominion of ATP-dependent chromatin-remodeling complexes. SWI/SNF and INO80, two of the best-studied complexes, as well as chromatin modifier TIP60, all contain actin as a subunit. Retrospective analysis of results of AcMNPV time course experiments wherein actin polymerization was blocked by cytochalasin D drug treatment implicate actin-containing chromatin modifying complexes in decatenating baculovirus genomes, shutting down host transcription, and regulating late and very late phases of viral transcription. Moreover, virus-mediated nuclear localization of actin early during infection may contribute to nucleosome management. - Highlights: • Baculoviruses have negatively-supercoiled, circular ds DNA. • Negatively-supercoiled DNA spontaneously forms nucleosomes in the nucleus. • Nucleosomes must be mobilized for replication and transcription to proceed. • Actin-containing chromatin modifiers participate in baculovirus replication.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

ATP/P2X7-NLRP3 axis of dendritic cells participates in the regulation of airway inflammation and hyper-responsiveness in asthma by mediating HMGB1 expression and secretion

Highlights: • ATP/P2X7 axis induces Th2, 17 cell inflammatory response and AHR in allergic asthma. • Inhibiting NLRP3 blocks Th2, 17 cell inflammatory response and AHR in allergic asthma. • Activating ATP/P2X7 axis increases NLRP3 inflammasome complex expression. • Activating ATP/P2X7 axis induces HMGB1 expresssion and release from DCs. • Inhibiting NLRP3 reduces HMGB1 expresssion and release from DCs. The ATP/P2X7 axis of dendritic cells (DCs) mediates the activation of NLRP3 inflammasome and promotes secretion of interleukin (IL)−1β and IL-18 to induce T helper (Th) 2, Th17 differentiation in the pathogenesis of asthma. NLRP3 inflammasome also regulates high mobility protein 1 (HMGB1) release in DCs. Recent studies demonstrated the correlation between HMGB1 expression and airway inflammation and hyper-responsiveness (AHR) in asthma. However, the relationship between the ATP/P2X7-NLRP3 axis and HMGB1 in DCs in asthma is still unclear. ATP, apyrase, Brilliant Blue G, BzATP, glibenclamide, and Z-YVAD-FMK were administered to ovalbumin (OVA)-induced murine asthmatic model. For in vitro studies, bone marrow-derived mononuclear cells (BMDCs) were primed with LPS and stimulated with the same reagents. Activation of the ATP/P2X7 axis aggravated airway inflammation and AHR in the lung and induced Th2, Th17 polarization in asthmatic mice. Inhibition of NLRP3 inflammasome weakened cardinal features of asthma and blocked Th2, Th17 polarization. In vitro and vivo, ATP/P2X7 axis activated NLRP3 inflammasome and induced HMGB1 expression and release from DCs. Inhibition of NLRP3 inflammasome reduced HMGB1 expression and release. The ATP/P2X7-NLRP3 axis of DCs participates in mediating airway inflammation, AHR, and promoting Th2, Th17 inflammatory responses in asthmatic mice by inducing HMGB1 expression and secretion.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

RNA helicase DDX5 participates in oxLDL-induced macrophage scavenger receptor 1 expression by suppressing mRNA degradation

Highlights: • RNA helicase DDX5 positively regulates macrophage lipid uptake. • Macrophages scavenger receptor 1(MSR1) mRNA is stabilized by DDX5. • The m{sup 6}a modification of MSR1 mRNA was regulated by interaction of mettl3 and DDX5. • Degradation of MSR1 mRNA was suppressed by m{sup 6}a modification on mRNA. The DEAD box protein DDX5, an ATP-dependent RNA helicase, plays an important role in transcriptional regulation and is associated with solid tumors and leukemia. However, its role in oxLDL-induced lipid uptake in macrophages remains unclear. In this study, we detected the expression of DDX5 mRNA and protein in oxidized low-density lipoprotein (oxLDL)-treated human primary macrophages that were induced from monocytes isolated from human peripheral blood with or without several chemical inhibitors using quantitative real-time PCR (qRT-PCR) or Western blotting. We found that oxLDL induced DDX5 expression to be independent of both the MAPK and NF-κB pathways. We also found that DDX5 promoted macrophage lipid uptake by evaluating the fluorescence intensity of engulfed dil-oxLDL. Various scavenger receptors that participate in lipid uptake were detected in siR-DDX5 transfected macrophages using qRT-PCR and Western blotting. Macrophage scavenger receptor A (MSR1) was found to be involved the upregulation of DDX5-mediated lipid uptake. Through the use of a dual luciferase reporter assay system and incubation with cycloheximide (CHX) MG132 and actidione (ActD), we found that DDX5 promoted MSR1 protein expression by stabilizing MSR1 mRNA. Moreover, the mechanism involved in DDX5 regulation of MSR1 mRNA was also explored using mass spectrum analysis; Immunoprecipitations (IPs) and RNA- Immunoprecipitations (R-IPs) revealed that mettl3 was involved in DDX5-mediated MSR1 mRNA stabilization. In addition, we also demonstrated that DDX5 inhibited mettl3 to catalyze m{sup 6}a methylation in MSR1 mRNA, which contributed to the maintenance of MSR1 mRNA stability. In conclusion, ox-LDL promotes DDX5 expression in macrophages, which interacts with mettl3 to stabilize MSR1 mRNA by decreasing the m{sup 6}a modification of MSR1 mRNA, ultimately promoting lipid uptake in macrophages.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Myosin Id localizes in dendritic spines through the tail homology 1 domain

Highlights: • Myosin Id is a synapse-related molecule that binds to actin filaments. • Myosin Id localizes in dendritic spines of primary hippocampal neurons. • The tail homology 1 domain is critical for the localization of myosin Id. Dendritic spines, the postsynaptic compartments at excitatory synapses, are capable of changing their shape and size to modulate synaptic transmission. The actin cytoskeleton and a variety of actin-binding proteins play a critical role in the dynamics of dendritic spines. Class I myosins are monomeric motor proteins that move along actin filaments using the energy of ATP hydrolysis. Of these class I myosins, myosin Id, the mammalian homolog of Drosophila Myo31DF, has been reported to be expressed in neurons, whereas its subcellular localization in neurons remained unknown. Here, we investigated the subcellular localization of myosin Id and determined the domain responsible for it. We found that myosin Id is enriched in the F-actin-rich pseudopodia of HEK293T cells and in the dendritic spines of primary hippocampal neurons. Both deletion and substitution of the tail homology 1 (TH1) domain drastically diminishes its colocalization with F-actin. In addition, the mutant form lacking the TH1 domain is less distributed in dendritic spines than is the full-length form. Taken together, our findings reveal that myosin Id localizes in dendritic spines through the TH1 domain.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

SIRT3 aggravates metformin-induced energy stress and apoptosis in ovarian cancer cells

Highlights: • Metformin induces mitochondrial-apoptosis by targeting respiratory chain complex 1. • The expression and activity of sirtuin 3 (SIRT3) are increased by metformin. • SIRT3 aggravates metformin-induced energy stress and apoptosis. • Inhibition of the AMPK / glucose pathway enhances the antitumor effect of metformin. Increasing evidence suggests that mitochondrial respiratory chain complex I participates in carcinogenesis and cancer progression by providing energy and maintaining mitochondrial function. However, the role of complex I in ovarian cancer is largely unknown. In this study we showed that metformin, considered to be an inhibitor of complex I, simultaneously inhibited cell growth and induced mitochondrial-related apoptosis in human ovarian cancer cells. Metformin interrupted cellular energy metabolism mainly by causing damage to complex I that impacted mitochondrial function. Additionally, treatment with metformin increased the activation of sirtuin 3 (SIRT3), a mitochondrial deacetylase. We demonstrated that SIRT3 overexpression aggravated metformin-induced apoptosis, energy stress and mitochondrial dysfunction. Moreover, treatment with metformin or SIRT3 overexpression increased activation of AMP-activated protein kinase (AMPK), a major sensor of cellular energy status. AMPK compensated for energy loss by increasing glycolysis. The impact of this was assessed by reducing glucose levels in the media or by using inhibitors (2-deoxyglucose, Compound C) of glycolysis and AMPK. The combination of these factors with metformin intensified cytotoxicity through further downregulation of ATP. Our study outlines an important role for SIRT3 in the antitumor effect of mitochondrial complex I inhibitors in human ovarian cancer cells. This effect appears to be mediated by induction of energy stress and apoptosis. Strategies that target the mitochondria could be enhanced by modulating glycolysis to further aggravate energy stress that may increase the antitumor effect.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Oocyte-specific deletion of Gsα induces oxidative stress and deteriorates oocyte quality in mice

Highlights: • Oocyte-specific disruption of G{sub s}α leads to female infertility in mice. • Deletion of G{sub s}α in oocytes causes poor oocyte quality and impaired early embryo development. • G{sub s}α deficiency in oocytes leads to oxidative stress. The stimulatory heterotrimeric G{sub s} protein alpha subunit (G{sub s}α) is a ubiquitous guanine nucleotide-binding protein that regulates the intracellular cAMP signaling pathway and consequently participates in a wide range of biological events. In the reproductive system, despite G{sub s}α being associated with oocyte meiotic arrest in vitro, the exact role of G{sub s}α in female fertility in vivo remains largely unknown. Here, we generated oocyte-specific G{sub s}α knockout mice by using the Cre/LoxP system. We observed that the deletion of G{sub s}α caused complete female infertility. Exclusion of post-implantation abnormalities, oogenesis, fertilization, and early embryo development was subsequently monitored; meiosis in G{sub s}α-deficient oocytes precociously resumed in only 43% of antral follicles from mutant mice, indicating that alteration of meiotic pause was not the key factor in infertility. Ovulation process and number were normal, but the rate of morphological abnormal oocytes was apparently increased; spindle organization, fertilization, and early embryo development were impaired. Furthermore, the level of ROS (reactive oxygen species) and the mitochondrial aggregation increased, and antioxidant glutathione (GSH) content, ATP level, mtDNA copy number, and mitochondrial membrane potential decreased in G{sub s}α-deficient oocytes. GV oocytes from mutant mice showed early-stage apoptosis. Meanwhile, the G{sub s}α knockout-induced decline in oocyte quality and low developmental potential was partially rescued by antioxidant supplementation. To sum up, our results are the first to reveal that the profile of G{sub s}α oocyte-specific deletion caused female infertility in vivo, and oxidative stress plays an important role in this event.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

MicroRNA-23a promotes colorectal cancer cell survival by targeting PDK4

Highlights: • miR-23a is overexpressed in CRC cell lines and tissues compared with that of normal cells. • miR-23a repress both the mRNA and protein expressions of PDK4. • PDK4 negatively regulate CRC proliferation via suppressing PDH activity. • miR-23a facilitates PDH activation and oxidative phosphorylation via targeting PDK4. • miR-23a promotes CRC cells proliferation in vitro and in vivo via targeting PDK4. MicroRNA (miR) is important regulators of gene expression, and aberrant miR expression has been linked to oncogenesis; however, little is understood about their contribution to colorectal cancer (CRC). Here, we determined that miR-23a is overexpressed in human colorectal cancer cell lines and tissues compared with that of normal cells. The stable over-expression of miR-23a in CRC cells was sufficient to promote cell proliferation in vitro and in vivo. Further studies showed that miR-23a can directly bind to the 3′untranslated region (3′UTR) of PDK4 mRNA and subsequently repress both the mRNA and protein expressions of PDK4. PDK4 negatively regulate CRC proliferation via suppressing PDH activity. Ectopic expression of PDK4 by transiently transfected with PDK4 vector encoding the entire coding sequence could reverse the effects of miR-23a on CRC proliferation. By this way, miR-23a promotes PDH activation and oxidative phosphorylation to generate sufficient ATP for cell proliferation. Our results illustrated that the up-regulation of miR-23a played an important role in CRC cell proliferation through direct repressing PDK4, suggesting a potential application of miR-23a in prognosis prediction and therapeutic application in CRC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Diacylglycerol kinase γ predicts prognosis and functions as a tumor suppressor by negatively regulating glucose transporter 1 in hepatocellular carcinoma

Highlights: • DGKγ expression was downregulated in hepatocellular carcinoma (HCC). • Lower DGKγ expression in HCC tumor tissues was an independent prognostic factor for poor post-surgical overall survival. • Cell proliferation and migration of HCC cells were suppressed by DGKγ ectopic expression. • DGKγ overexpression led to down regulation of GLUT1 protein level and AMPK activity. Diacylglycerol kinases (DGK) are a family of enzymes catalyzing the transformation of diacylglycerol into phosphatidic acid, which have been recognized as key regulators in cell signaling pathways. The role of DGKγ in human malignancies has seldom been studied. In this study, we investigated the role of DGKγ in hepatocellular carcinoma (HCC). We found that DGKγ was down-regulated in HCC tumor tissues and cell lines as compared to that in non-tumor tissues. The prognostic value of DGKγ expression was evaluated by Cox regression and Kaplan-Meier analyses. Lower DGKγ expression in tumor tissues was an independent prognostic factor for poor post-surgical overall survival. By using HDACs inhibitors treatment and ChIP-PCR, we discovered that histone H3 and H4 deacetylation mainly contributed to the downregulation of DGKγ expression. Functional studies revealed that ectopic expression of DGKγ inhibited cell proliferation and cell migration in HCC cells. Mechanism studies showed that DGKγ overexpression led to down regulation of GLUT1 protein level and AMPK activity, which result in glucose uptake suppression as well as lactate and ATP production declination. The decrease of GLUT1 level could be partially rescued by treatments with either DGK inhibitor and lysosome inhibitor, indicating DGKγ may down-regulate GLUT1 through its kinase activity and lysosome degradation process. Together, this study demonstrated that DGKγ plays a tumor suppressor role in HCC by negatively regulating GLUT1. DGKγ could be a novel prognostic indicator and therapeutic target for HCC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Adaptation of metabolism to multicellular aggregation, hypoxia and obese stromal cell incorporation as potential measure of survival of ovarian metastases

Ovarian metastases exfoliate from the primary tumor and it is thought that aggregation supports their survival in the peritoneal cavity during dissemination but the underlying mechanisms are not clearly identified. We have previously shown that ovarian cancer cells acquire an increasingly glycolytic and metabolic flexible phenotype during progression. In the present study, we investigated how hypoxia, aggregation, and the incorporation of the obese stromal vascular fraction (SVF) affect cellular metabolism and the response to common anti-cancer and anti-diabetic drugs. Our results show a reduction of glucose uptake, lactate secretion, cellular respiration and ATP synthesis in response to hypoxia and aggregation, suggesting that the observed reduced proliferation of cells aggregated into spheroids is the result of a down-regulation of respiration. Recruitment of SVF to spheroids increased the spheroids invasive capacity but reduced respiration only in the most aggressive cells. Further, aggregation and hypoxia reduced the response to the metabolic drugs AICAR and metformin, and the chemotherapeutic agents cisplatin and paclitaxel. Our results suggest that the adaptation of cellular metabolism may contribute to enhanced survival under non-permissive conditions, and that these metabolic alterations may provide targets for future interventions that aim to enhance the survival of women with metastatic ovarian cancer.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

EPS8 supports pancreatic cancer growth by inhibiting BMI1 mediated proteasomal degradation of ALDH7A1

Aldehyde dehydrogenase 7 family member A1 (ALDH7A1) is an enzyme catalyzing lipid peroxidation of fatty aldehydes. It plays a critical role in sustaining high oxygen consumption rate (OCR) and ATP production in pancreatic ductal adenocarcinoma (PADC). However, why PADC cells maintain a relatively high level of ALDH7A1 concentration is still not well understood. In the current study, we explored the interplay between epidermal growth factor receptor kinase substrate 8 (EPS8) and ALDH7A1 in PADC cells. PADC cell lines MIA PaCa-2 and AsPANC-1 were used for in vitro and in vivo studies. The co-IP assay showed mutual interactions between Flag-EPS8 and Myc-ALDH7A1 in both MIA PaCa-2 and AsPANC-1 cells. EPS8 knockdown resulted in decreased ALDH7A1 protein levels and increased poly-ubiquitination. An interaction was observed between ALDH7A1 and BMI1 but not between BMI1 and EPS8. BMI1 knockdown reduced ALDH7A1 poly-ubiquitination and degradation caused by EPS8 knockdown. Dual EPS8 and ALDH7A1 knockdown had a synergistic effect on suppressing PADC cell proliferation in vitro and in vivo. In conclusion, this study revealed that EPS8 supports PADC growth by interacting with ALDH7A1 and inhibiting BMI1 mediated proteasomal degradation of ALDH7A1.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Extracorporeal membrane oxygenation promotes long chain fatty acid oxidation in the immature swine heart in vivo

Extracorporeal membrane oxygenation (ECMO) supports infants and children with severe cardiopulmonary compromise. Nutritional support for these children includes provision of medium- and long-chain fatty acids (FAs). However, ECMO induces a stress response, which could limit the capacity for FA oxidation. Metabolic impairment could induce new or exacerbate existing myocardial dysfunction. Using a clinically relevant piglet model, we tested the hypothesis that ECMO maintains the myocardial capacity for FA oxidation and preserves myocardial energy state. Provision of 13-Carbon labeled medium-chain FA (octanoate), longchain free FAs (LCFAs), and lactate into systemic circulation showed that ECMO promoted relative increases in myocardial LCFA oxidation while inhibiting lactate oxidation. Loading of these labeled substrates at high dose into the left coronary artery demonstrated metabolic flexibility as the heart preferentially oxidized octanoate. ECMO preserved this octanoate metabolic response, but also promoted LCFA oxidation and inhibited lactate utilization. Rapid upregulation of pyruvate dehydrogenase kinase-4 (PDK4) protein appeared to participate in this metabolic shift during ECMO. ECMO also increased relative flux from lactate to alanine further supporting the role for pyruvate dehydrogenase inhibition by PDK4. High dose substrate loading during ECMO also elevated the myocardial energy state indexed by phosphocreatine to ATP ratio. ECMO promotes LCFA oxidation in immature hearts, while maintaining myocardial energy state. These data support the appropriateness of FA provision during ECMO support for the immature heart.

clear magnetic resonance↗
DOE OSTI2016

Comprehensive analysis of glucose and xylose metabolism in Escherichia coli under aerobic and anaerobic conditions by 13 C metabolic flux analysis

Glucose and xylose are the two most abundant sugars derived from the breakdown of lignocellulosic biomass. While aerobic glucose metabolism is relatively well understood in E. coli, until now there have been only a handful of studies focused on anaerobic glucose metabolism and no 13 C-flux studies on xylose metabolism. In the absence of experimentally validated flux maps, constraint-based approaches such as MOMA and RELATCH cannot be used to guide new metabolic engineering designs. In this work, we have addressed this critical gap in current understanding by performing comprehensive characterizations of glucose and xylose metabolism under aerobic and anaerobic conditions, using recent state-of-the-art techniques in 13 C metabolic flux analysis ( 13 C-MFA). Specifically, we quantified precise metabolic fluxes for each condition by performing parallel labeling experiments and analyzing the data through integrated 13C-MFA using the optimal tracers [1,2- 13 C]glucose, [1,6- 13 C]glucose, [1,2- 13 C]xylose and [5- 13 C]xylose. We also quantified changes in biomass composition and confirmed turnover of macromolecules by applying [U- 13 C]glucose and [U-13C]xylose tracers. We demonstrate that under anaerobic growth conditions there is significant turnover of lipids and that a significant portion of CO 2 originates from biomass turnover. Using knockout strains, we also demonstrate that β-oxidation is critical for anaerobic growth on xylose. Quantitative analysis of co-factor balances (NADH/FADH2, NADPH, and ATP) for different growth conditions provided new insights regarding the interplay of energy and redox metabolism and the impact on E. coli cell physiology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Phosphoketolase overexpression increases biomass and lipid yield from methane in an obligate methanotrophic biocatalyst

Microbial conversion of methane to high-value bio-based chemicals and materials offers a path to mitigate GHG emissions and valorize this abundant-yet -underutilized carbon source. In addition to fermentation optimization strategies, rational methanotrophic bacterial strain engineering offers a means to reach industrially relevant titers, carbon yields, and productivities of target products. The phosphoketolase pathway functions in heterofermentative bacteria where carbon flux through two sugar catabolic pathways to mixed acids (lactic acid and acetic acid) increases cellular ATP production. Importantly, this pathway also serves as an alternative route to produce acetyl-CoA that bypasses the CO 2 lost through pyruvate decarboxylation in the Embden-Meyerhof-Parnas pathway. Thus, the phosphoketolase pathway can be leveraged for carbon efficient biocatalysis to acetyl-CoA-derived intermediates and products. Here, we show that the industrially promising methane biocatalyst, Methylomicrobium buryatense , encodes two phosphoketolase isoforms that are expressed in methanol- and methane-grown cells. Overexpression of the PktB isoform led to a 2-fold increase in intracellular acetyl-CoA concentration, and a 2.6-fold yield enhancement from methane to microbial biomass and lipids compared to wild-type, increasing the potential for methanotroph lipid-based fuel production. Off-gas analysis and metabolite profiling indicated that global metabolic rearrangements, including significant increases in post-translational protein acetylation and gene expression of the tetrahydromethanopterin-linked pathway, along with decreases in several excreted products, coincided with the superior biomass and lipid yield observed in the engineered strain. Further, these data suggest that phosphoketolase may play a key regulatory role in methanotrophic bacterial metabolism. As a result, given that acetyl-CoA is a key intermediate in several biosynthetic pathways, phosphoketolase overexpression offers a viable strategy to enhance the economics of an array of biological methane conversion processes.

09 BIOMASS FUELS↗
DOE OSTI2017

High-throughput enzyme screening platform for the IPP-bypass mevalonate pathway for isopentenol production

Isopentenol (or isoprenol, 3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals such as isoprene. Biological production of isopentenol via the mevalonate pathway has been optimized extensively in Escherichia coli , yielding 70% of its theoretical maximum. However, high ATP requirements and isopentenyl diphosphate (IPP) toxicity pose immediate challenges for engineering bacterial strains to overproduce commodities utilizing IPP as an intermediate. To overcome these limitations, we developed an “IPP-bypass” isopentenol pathway using the promiscuous activity of a mevalonate diphosphate decarboxylase (PMD) and demonstrated improved performance under aeration-limited conditions. However, relatively low activity of PMD toward the non-native substrate (mevalonate monophosphate, MVAP) was shown to limit flux through this new pathway. By inhibiting all IPP production from the endogenous non-mevalonate pathway, we developed a high-throughput screening platform that correlated promiscuous PMD activity toward MVAP with cellular growth. Successful identification of mutants that altered PMD activity demonstrated the sensitivity and specificity of the screening platform. Strains with evolved PMD mutants and the novel IPP-bypass pathway increased titers up to 2.4-fold. Further enzymatic characterization of the evolved PMD variants suggested that higher isopentenol titers could be achieved either by altering residues directly interacting with substrate and cofactor or by altering residues on nearby α-helices. These altered residues could facilitate the production of isopentenol by tuning either k cat or K i of PMD for the non-native substrate. The synergistic modification made on PMD for the IPP-bypass mevalonate pathway is expected to significantly facilitate the industrial scale production of isopentenol.

09 BIOMASS FUELS↗
DOE OSTI2017

Advanced water splitting for green hydrogen gas production through complete oxidation of starch by in vitro metabolic engineering

Starch is a natural energy storage compound and is hypothesized to be a high-energy density chemical compound or solar fuel. In contrast to industrial hydrolysis of starch to glucose, an alternative ATP-free phosphorylation of starch was designed to generate cost-effective glucose 6-phosphate by using five thermophilic enzymes (i.e., isoamylase, alpha-glucan phosphorylase, 4-α-glucanotransferase, phosphoglucomutase, and polyphosphate glucokinase). This enzymatic phosphorolysis is energetically advantageous because the energy of α-1,4-glycosidic bonds among anhydroglucose units is conserved in the form of phosphorylated glucose. Additionally, we demonstrated an in vitro 17-thermophilic enzyme pathway that can convert all glucose units of starch, regardless of branched and linear contents, with water to hydrogen at a theoretic yield (i.e., 12 H 2 per glucose), three times of the theoretical yield from dark microbial fermentation. The use of a biomimetic electron transport chain enabled to achieve a maximum volumetric productivity of 90.2 mmol of H 2 /L/h at 20 g/L starch. The complete oxidation of starch to hydrogen by this in vitro synthetic (enzymatic) biosystem suggests that starch as a natural solar fuel becomes a high-density hydrogen storage compound with a gravimetric density of more than 14% H 2 -based mass and an electricity density of more than 3000 W h/kg of starch.

08 HYDROGEN↗