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At least 217 records · Page 12
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Effects of 5-ion 6-beam sequential irradiation in the presence and absence of hindlimb or control hindlimb unloading on behavioral performances and plasma metabolic pathways of Fischer 344 rats

Introduction Effects and interactions between different spaceflight stressors are expected to be experienced by crew on missions when exposed to microgravity and galactic cosmic rays (GCRs). One of the limitations of previous studies on simulated weightlessness using hindlimb unloading (HU) is that a control HU condition was not included. Methods We characterized the behavioral performance of male Fischer rats 2 months after sham or total body irradiation with a simplified 5-ion 6-mixed-beam exposure representative of GCRs in the absence or presence of HU. Six months later, the plasma, hippocampus, and cortex were processed to determine whether the behavioral effects were associated with long-term alterations in the metabolic pathways. Results In the open field without and with objects, interactions were observed for radiation × HU. In the plasma of animals that were not under the HU or control HU condition, the riboflavin metabolic pathway was affected most for sham irradiation vs. 0.75 Gy exposure. Analysis of the effects of control HU on plasma in the sham-irradiated animals showed that the alanine, aspartate, glutamate, riboflavin, and glutamine metabolisms as well as arginine biosynthesis were affected. The effects of control HU on the hippocampus in the sham-irradiated animals showed that the phenylalanine, tyrosine, and tryptophan pathway was affected the most. Analysis of effects of 0.75 Gy irradiation on the cortex of control HU animals showed that the glutamine and glutamate metabolic pathway was affected similar to the hippocampus, while the riboflavin pathway was affected in animals that were not under the control HU condition. The effects of control HU on the cortex in sham-irradiated animals showed that the riboflavin metabolic pathway was affected. Animals receiving 0.75 Gy of irradiation showed impaired glutamine and glutamate metabolic pathway, whereas animals receiving 1.5 Gy of irradiation showed impaired riboflavin metabolic pathways. A total of 21 plasma metabolites were correlated with the behavioral measures, indicating that plasma and brain biomarkers associated with behavioral performance are dependent on the environmental conditions experienced. Discussion Phenylalanine, tyrosine, and tryptophan metabolism as well as phenylalanine and tryptophan as plasma metabolites are biomarkers that can be considered for spaceflight as they were revealed in both Fischer and WAG/Rij rats exposed to simGCRsim and/or HU.

Physiology↗
DOE OSTI2017

Comparative TEA for Indirect Liquefaction Pathways to Distillate-Range Fuels via Oxygenated Intermediates

This paper presents a comparative techno-economic analysis of five conversion pathways from biomass to gasoline-, jet-, and diesel-range hydrocarbons via indirect liquefaction with specific focus on pathways utilizing oxygenated intermediates (derived either via thermochemical or biochemical conversion steps). The four emerging pathways of interest are compared with one conventional pathway (Fischer-Tropsch) for the production of the hydrocarbon blendstocks. The processing steps of the four emerging pathways include: biomass-to-syngas via indirect gasification, gas cleanup, conversion of syngas to alcohols/oxygenates, followed by conversion of alcohols/oxygenates to hydrocarbon blendstocks via dehydration, oligomerization, and hydrogenation. We show that the emerging pathways via oxygenated intermediates have the potential to be cost competitive with the conventional Fischer-Tropsch process. The evaluated pathways and the benchmark process generally exhibit similar fuel yields and carbon conversion efficiencies. The resulting minimum fuel selling prices are comparable to the benchmark at approximately $3.60 per gallon-gasoline equivalent, with potential for two new pathways to be more economically competitive. Additionally, the coproduct values can play an important role in the economics of the processes with oxygenated intermediates derived via syngas fermentation. Major cost drivers for the integrated processes are tied to achievable fuel yields and conversion efficiency of the intermediate steps, i.e., the production of oxygenates/alcohols from syngas and the conversion of oxygenates/alcohols to hydrocarbon fuels.

09 BIOMASS FUELS↗
DOE OSTI2023

Characterizing and Engineering Trehalose Biosynthesis Pathways to Improve Acetate Tolerance in Pseudomonas putida KT2440

Alkaline pretreatment can depolymerize lignin into a diverse mixture of monomers and smaller compounds, enabling bioconversion of these compounds to value-added chemicals in engineered bacterial hosts such as Pseudomonas putida KT2440. Acetate and salts comprise approximately 8% and 32% of the lignin stream, respectively. High concentrations of these components can cause cell death in P. putida, so lignin-derived streams must be fed in lower amounts to decrease stress. Investigation of the mechanisms of bacterial tolerance to chemicals in lignin-rich substrates is therefore required to overcome this obstacle in lignin bioconversion. Previous work has shown that trehalose, an endogenously produced disaccharide, is involved in tolerance to heat, cold, and osmotic stress, as well as implicated in acid stress in other bacteria. As such, trehalose biosynthesis presents a useful target to increase acetate and osmotic tolerance in P. putida. This work investigates the native TreSA, TreSB, and TreY/TreZ trehalose biosynthesis pathways in this bacterial strain as well as the OtsAB pathway of Sphingobium sp. SYK-6. Overexpression of the OtsAB pathway in P. putida increased tolerance to acetate relative to wild-type. Additionally, functional knockouts of treSA, treSB, treY, and treZ in P. putida showed that interruption of any trehalose biosynthesis pathway, and, in the case of TreY/TreZ, interruption of the pathway at any point, significantly hindered growth in acetate as well as on glucose and LB broth. Each trehalose biosynthesis pathway was then engineered for overexpression in P. putida to determine whether overexpression of these genes conferred additional tolerance to acetate. The TreSA, TreSB, OtsA, and OtsB enzymes were also expressed and purified to determine the reaction rates and kinetics of these pathways. The hypothetical glycoside hydrolase family 15 protein, encoded in the gene located in between otsB and otsA in the Sphingobium operon, was purified as well to investigate its potential involvement in the OtsAB pathway. The findings of this work illuminated the mechanisms of trehalose biosynthesis in P. putida and identified genetic targets to improve strain tolerance to common components of alkaline-pretreated lignin streams. These findings could ultimately improve yields of desired products from lignin in engineered strains of P. putida.

alkaline pretreatment↗
DOE OSTI2023

Establishing a versatile toolkit of flux enhanced strains and cell extracts for pathway prototyping

Building and optimizing biosynthetic pathways in engineered cells holds promise to address societal needs in energy, materials, and medicine, but it is often time-consuming. Cell-free synthetic biology has emerged as a powerful tool to accelerate design-build-test-learn cycles for pathway engineering with increased tolerance to toxic compounds. However, most cell-free pathway prototyping to date has been performed in extracts from wildtype cells which often do not have sufficient flux towards the pathways of interest, which can be enhanced by engineering. Here, in this study, to address this gap, we create a set of engineered Escherichia coli and Saccharomyces cerevisiae strains rewired via CRISPR-dCas9 to achieve high-flux toward key metabolic precursors; namely, acetyl-CoA, shikimate, triose-phosphate, oxaloacetate, α-ketoglutarate, and glucose-6-phosphate. Cell-free extracts generated from these strains are used for targeted enzyme screening in vitro. As model systems, we assess in vivo and in vitro production of triacetic acid lactone from acetyl-CoA and muconic acid from the shikimate pathway. The need for these platforms is exemplified by the fact that muconic acid cannot be detected in wildtype extracts provided with the same biosynthetic enzymes. We also perform metabolomic comparison to understand biochemical differences between the cellular and cell-free muconic acid synthesis systems (E. coli and S. cerevisiae cells and cell extracts with and without metabolic rewiring). While any given pathway has different interfaces with metabolism, we anticipate that this set of pre-optimized, flux enhanced cell extracts will enable prototyping efforts for new biosynthetic pathways and the discovery of biochemical functions of enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Single-Amino Acid Modifications Reveal Additional Controls on the Proton Pathway of [FeFe]-Hydrogenase

The proton pathway of [FeFe]-hydrogenase is essential for enzymatic H2 production and oxidation and is composed of four residues and a modeled water molecule. Recently, a computational analysis of this pathway revealed that the solvent-exposed residue of the pathway (Glu282) could form hydrogen bonds to two residues outside of the pathway (Arg286 and Ser320), implicating that these residues could function in regulating proton transfer. Substituting Arg286 with leucine eliminates hydrogen bonding with Glu282 and results in a 2.5-fold enhancement in H2 production activity, suggesting that Arg286 serves an important role in controlling the rate of proton delivery. In contrast, substitution of Ser320 with alanine reduces the rate approximately 5-fold, implying that it either acts as a member of the pathway or influences Glu282 to enable proton transfer. Interestingly, QM/MM and molecular dynamics calculations indicate that Ser320 does not play an electronic or structural role. QM calculations also estimate that including Ser320 in the pathway does not significantly change the barrier to proton movement, providing further support for its role as a member of the proton pathway. While further studies are needed to quantify the role of Ser320, collectively, these data provide evidence that the enzyme scaffold plays a significant role in modulating the activity of the enzyme, demonstrating that the rate of intraprotein proton transfer can be accelerated, particularly in a non-biological context. This work was supported by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science, DE-FC02-07ER64494). In addition, support from the DOE Office of Science Early Career Research Program through the Office of Basic Energy Sciences (WJS, BGP, SR) is gratefully acknowledged. Computational resources were provided at W. R. Wiley Environmental Molecular Science Laboratory (EMSL), a national scientific user facility sponsored by the Department of Energy’s Office of Biological and Environmental Research located at Pacific Northwest National Laboratory, and a portion of the research was performed using PNNL Institutional Computing at Pacific Northwest National Laboratory. Pacific Northwest National Laboratory is operated by Battelle for the U.S. Department of Energy.

catalysis↗
DOE OSTI2018

Parallel Integration and Chromosomal Expansion of Metabolic Pathways

Robust fermentation of biomass-derived sugars into bioproducts demands the reliable microbial expression of metabolic pathways. Plasmid-based expression systems may suffer from instability and result in highly variable titers, rates, and yields. An established mitigation approach, Chemical Induced Chromosomal Expansion (CIChE), expands a singly integrated pathway to plasmid-like copy numbers, while maintaining stability in the absence of antibiotic selection pressure. Here, we report Parallel Integration and Chromosomal Expansion (PIACE), extensions to CIChE that enable independent expansions of pathway components across multiple loci, use suicide vectors to achieve high-efficiency site-specific integration of sequence-validated multi-gene components, and introduce a heat-curable plasmid to obviate recA deletion post pathway expansion. We applied PIACE to stabilize an isopentenol pathway across three loci in E. coli DH1, and to then generate libraries of pathway component copy-number variants to screen for improved titers. Furthermore, polynomial regressor statistical modeling of the production screening data suggests that increasing copy numbers of all isopentenol pathway components would further improve titers.

09 BIOMASS FUELS↗
DOE OSTI2022

Acetyl-CoA Synthesis Through a Bicyclic Carbon-Fixing Pathway in Gas-Fermenting Bacteria

Gas-fermenting acetogens can upgrade one-carbon (C1) compounds (such as CO2 and CO) to the two-carbon (C2) metabolite acetyl coenzyme A (CoA) and convert sugar feedstocks to acetyl-CoA with minimal CO2 emissions. Fulfilling the biosynthetic potential of these microbes requires overcoming challenges in pathway engineering. Here we design a synthetic acetyl-CoA bi-cycle-in addition to the natural carbon-fixing pathways-for C2 metabolite synthesis. This pathway produces an acetyl-CoA by fixation of two CO2 equivalents via three functional modules acting in sequence: carbon fixation, gluconeogenesis and non-oxidative glycolysis. The pathway was examined by in silico thermodynamic and kinetic analyses. The prototypic pathway was implemented in a syngas-fermenting organism, Clostridium ljungdahlii DSM 13528, by expressing a heterologous phosphoketolase that can work with other native enzymes in the host acetogen. The carbon conversion pathway is possible under various growth conditions and is independent of the Wood-Ljungdahl pathway for the valorization of H2 and CO2. This study reports the improvement of carbon conversion using a reductive acetyl-CoA bi-cycle and the potential impact of redox homoeostasis in the acetogenic host for industrial applications of gas fermentation.

Acetyl-CoA↗
DOE OSTI2018

Genomics and biochemistry investigation on the metabolic pathway of milled wood and alkali lignin-derived aromatic metabolites of Comamonas serinivorans SP-35

Background: The efficient depolymerization and utilization of lignin are one of the most important goals for the renewable use of lignocelluloses. The degradation and complete mineralization of lignin by bacteria represent a key step for carbon recycling in land ecosystems as well. However, many aspects of this process remain unclear, for example, the complex network of metabolic pathways involved in the degradation of lignin and the catabolic pathway of intermediate aromatic metabolites. To address these subjects, we characterized the deconstruction and mineralization of lignin with milled wood lignin (MWL, the most representative molecule of lignin in its native state) and alkali lignin (AL), and elucidated metabolic pathways of their intermediate metabolites by a bacterium named Comamonas serinivorans SP-35. Results: The degradation rate of MWL reached 30.9%, and its particle size range was decreased from 6 to 30 µm to 2–4 µm—when cultured with C. serinivorans SP35 over 7 days. FTIR analysis showed that the C–C and C–O–C bonds between the phenyl propane structures of lignin were oxidized and cleaved and the side chain structure was modi- fied. More than twenty intermediate aromatic metabolites were identified in the MWL and AL cultures based on GC–MS analysis. Through genome sequencing and annotation, and from GC–MS analysis, 93 genes encoding 33 enzymes and 5 regulatory factors that may be involved in lignin degradation were identified and more than nine metabolic pathways of lignin and its intermediates were predicted. Of particular note is that the metabolic pathway to form the powerful antioxidant 3,4-dihydroxyphenylglycol is described for the first time in bacteria. Conclusion: Elucidation of the ß-aryl ether cleavage pathway in the strain SP-35 indicates that the ß-aryl ether catabolic system is not only present in the family of Sphingomonadaceae, but also other species of bacteria kingdom. These newly elucidated catabolic pathways of lignin in strain SP-35 and the enzymes responsible for them provide exciting biotechnological opportunities for lignin valorization in future.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

Systems Biology Model of Interactions between Tissue Growth Factors and DNA Damage Pathways: Low Dose Response and Cross-Talk in TGFβ and ATM Signaling

The etiology of radiation carcinogenesis has been described in terms of aberrant changes that span several levels of biological organization. Growth factors regulate many important cellular and tissue functions including apoptosis, differentiation and proliferation. A variety of genetic and epigenetic changes of growth factors have been shown to contribute to cancer initiation and progression. It is known that cellular and tissue damage to ionizing radiation is in part initiated by the production of reactive oxygen species, which can activate cytokine signaling, and the DNA damage response pathways, most notably the ATM signaling pathway. Recently, the transforming growth factor β (TGFβ) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation. The relevance of this interaction with the ATM pathway is not known although p53 becomes phosphorylated and DNA damage responses are involved. However, growth factor interactions with DNA damage responses have not been elucidated particularly at low doses, and further characterization of their relationship to cancer processes is warranted. Our goal will be to use a systems biology approach to mathematically and experimentally describe the low-dose responses and cross-talk between the ATM and TGFβ pathways initiated by low- and high-LET radiation. We will characterize ATM and TGFβ signaling in epithelial and fibroblast cells using 2D models and ultimately extending to 3D organotypic cell culture models to begin to elucidate possible differences that may occur for different cell types and/or inter-cellular communication. We will investigate the roles of the Smad and Activating transcription factor 2 (ATF2) proteins as the potential major contributors to crosstalk between the TGFβ and ATM pathways, and links to cell cycle control and/or the DNA damage response, and potential differences in their responses at low and high doses. We have developed various experimental approaches to apply to these problems using confocal microscopy and flow cytometry to detail changes at low dose/dose-rate in order to understand individual cell responses, and will establish our mathematical models based on the experimental findings resulting from changes in DNA repair, apoptosis and proliferation.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗
DOE OSTI2015

Insulin-like growth factor-1 suppresses the Myostatin signaling pathway during myogenic differentiation

Myogenic differentiation is a complex and well-coordinated process for generating mature skeletal muscle fibers. This event is autocrine/paracrine regulated by growth factors, principally Myostatin (MSTN) and Insulin-like Growth Factor-1 (IGF-1). Myostatin, a member of the transforming growth factor-β superfamily, is a negative regulator of skeletal muscle growth in vertebrates that exerts its inhibitory function by activating Smad transcription factors. In contrast, IGF-1 promotes the differentiation of skeletal myoblasts by activating the PI3K/Akt signaling pathway. This study reports on a novel functional crosstalk between the IGF-1 and MSTN signaling pathways, as mediated through interaction between PI3K/Akt and Smad3. Stimulation of skeletal myoblasts with MSTN resulted in a transient increase in the pSmad3:Smad3 ratio and Smad-dependent transcription. Moreover, MSTN inhibited myod gene expression and myoblast fusion in an Activin receptor-like kinase/Smad3-dependent manner. Preincubation of skeletal myoblasts with IGF-1 blocked MSTN-induced Smad3 activation, promoting myod expression and myoblast differentiation. This inhibitory effect of IGF-1 on the MSTN signaling pathway was dependent on IGF-1 receptor, PI3K, and Akt activities. Finally, immunoprecipitation assay analysis determined that IGF-1 pretreatment increased Akt and Smad3 interaction. These results demonstrate that the IGF-1/PI3K/Akt pathway may inhibit MSTN signaling during myoblast differentiation, providing new insight to existing knowledge on the complex crosstalk between both growth factors. - Highlights: • IGF-1 inhibits Myostatin canonical signaling pathway through IGF-1R/PI3K/Akt pathway. • IGF-1 promotes myoblast differentiation through a direct blocking of Myostatin signaling pathway. • IGF-1 induces the interaction of Akt with Smad3 in skeletal myoblast.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Activation of IRE1α-XBP1 pathway induces cell proliferation and invasion in colorectal carcinoma

Cell proliferation and tumor metastasis are considered as the main reasons for death in colorectal carcinoma (CRC). IRE1α-XBP1 pathway is the most conserved UPR pathways, which are activated during ER stress caused by the accumulation of unfolded or misfolded protein in the lumen of ER. Here, we demonstrated the critical role of IRE1α-XBP1 pathway and underlying molecular mechanism in cell proliferation and tumor metastasis in CRC. By the use of tissue microarray analysis of samples from 119 patients with CRC, IRE1α was determined to be an independent predictor of overall survival as higher expression of IRE1α in CRC patients showed lower survival rates (p = 0.0041). RNA interference and ectopic expression of IRE1α were applied to determine the molecular effects of IRE1α in CRC cells. The silencing of IRE1α inhibited the proliferation and blocked the invasion of CRC cells in vitro, while ectopic expression of IRE1α in turn promoted cell proliferation and invasion. IRE1α-XBP1 pathway regulated the mitosis of CRC cells through the directly binding of XBP1s to Cyclin D1 promoter to activate Cyclin D1 expression. Our results reveal that IRE1α-XBP1 pathway plays an important role in tumor progression and epithelial-to-mesenchymal transition (EMT), and IRE1α could be employed as a novel prognostic marker and a promising therapeutic target for CRC. - Highlights: • IRE1 was determined to be an independent predictor of overall survival in CRC patient. • IRE1-XBP1 pathway promoted CRC cell proliferation through regulating Cyclin D1 expression. • IRE1-XBP1 pathway played important role in EMT of CRC cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Activation of the Wnt/β-catenin pathway in pancreatic beta cells during the compensatory islet hyperplasia in prediabetic mice

The Wnt/β-catenin signaling pathway, also known as the canonical Wnt pathway, plays a role in cell proliferation and differentiation in several tissues/organs. It has been recently described in humans a relationship between type 2 diabetes (T2DM) and mutation in the gene encoding the transcription factor TCF7L2 associated to the Wnt/β-catenin pathway. In the present study, we demonstrated that hyperplastic pancreatic islets from prediabetic mice fed a high-fat diet (HFD) for 60 d displayed nuclear translocation of active β-catenin associated with significant increases in protein content and gene expression of β-catenin as well as of cyclins D1, D2 and c-Myc (target genes of the Wnt pathway) but not of Tcf7l2 (the transcription factor). Meanwhile, these alterations were not observed in pancreatic islets from 30 d HFD-fed mice, that do not display significant beta cell hyperplasia. These data suggest that the Wnt/β-catenin pathway is activated in pancreatic islets during prediabetes and may play a role in the induction of the compensatory beta cell hyperplasia observed at early phase of T2DM. - Highlights: • Exposure to high-fat diet for 60 days induced prediabetes and beta cell mass expansion. • Hyperplastic pancreatic islets displayed nuclear translocation of active β-catenin. • Hyperplastic islets showed increased expression of target genes of the Wnt/β-catenin pathway. • Wnt/β-catenin pathway is activated during compensatory beta cell hyperplasia in mice.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Wnt/β-catenin pathway promotes acute lung injury induced by LPS through driving the Th17 response in mice

T helper cell 17 (Th17), one type of CD4{sup +} T cell, plays an important role in regulating the acute lung injury (ALI) inflammatory response. Recent studies showed that Wnt/β-catenin pathway could modulate the differentiation and the function of CD4{sup +} T cell. However, whether Wnt/β-catenin could regulate the differentiation and function of Th17 in the development and progress of ALI induced by lipopolysaccharide (LPS) is still unknown. To test this, we used dickkopf1 (Dkk-1) to block the Wnt/β-catenin pathway and LiCl to activate the Wnt/β-catenin pathway by instillation to the murine model of ALI. Our results revealed that activation of Wnt/β-catenin pathway significantly aggravated the LPS-induced lung inflammation. Meanwhile, we observed that activation of Wnt/β-catenin pathway promoted Th17 response by analyzing CD4{sup +} T cells and the related cytokines secretions. Enhanced Th17 response was responsible for the further neutrophils infiltration and pro-inflammatory cytokines production. In addition, activation of Wnt/β-catenin pathway resulted in induced expression of retinoic acid related orphan receptor-γt (RORγt) via histone acetyltransferase p300. These data suggested that Wnt/β-catenin pathway might be a potential target to treat the LPS-induced inflammation in ALI.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Brg1 aggravates airway inflammation in asthma via inhibition of the PI3K/Akt/mTOR pathway

Highlights: • Brg1 is highly expressed in HDM-induced asthma. • Brg1 knockout in asthmatic mice increased the activation of the PI3K/Akt/mTOR pathway. • Brg1 aggravates airway inflammation partly via inhibiting the PI3K/Akt/mTOR pathway. • PI3K inhibitor abrogated the effect of Brg1 on airway inflammation in asthmatic mice. The PI3K/Akt/mTOR pathway is thought to be closely associated with airway inflammation and is regulated by various upstream proteins. Brahma-related gene 1 (Brg1) plays an important role in chromatin remodeling and facilitates recruitment of essential transcription factors, leading to regulation of gene expression. Thus, the present study aimed at evaluating the anti-inflammatory role of Brg1 on house dust mite (HDM)-induced asthma through regulating the PI3K/Akt/mTOR pathway. The Brg{sup fl/fl} mice were crossbred with the SFTPC-Cre mice to generate bronchial epithelial cell specific Brg1 knockout mice, and LY294002 was used to inhibit PI3K. Western blot, immunofluorescence, immunoprecipitation, and immunohistochemical staining were used to detect the expression of proteins. An increase in Brg1 and a decrease in the PI3K/Akt/mTOR pathway activity were detected in asthmatic mice, but not in control mice. When Brg1 was knocked out, the asthma severity was ameliorated and the PI3K/Akt/mTOR pathway was activated. However, this protective effect could be suppressed by LY294002. Additionally, we observed that Brg1 was co-localized and co-immunoprecipitated with PI3K, using immunofluorescence and immunoprecipitation assays. Our results suggest that Brg1 might play an essential role in maintaining airway inflammation and affect the PI3K/Akt/mTOR pathway in asthma.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Sanguisorba officinalis L. extracts activate Wnt/β-catenin pathway, and subsequently control adipo-osteogenic differentiation

Highlights: • Identification of herbal extracts as an activator of Wnt/β-catenin pathway. • Activation of Wnt/β-catenin pathway by Sanguisorba officinalis L. extracts (SOE). • Inhibitory effects of SOE on adipogenesis via Wnt/β-catenin pathway activation. • Stimulating effects of SOE on osteogenesis via Wnt/β-catenin pathway activation. • The effectiveness of SOE for accelerating mice calvaroal bone formation ex vivo. Wnt/β-catenin pathway plays an important role in adipogenesis and osteogenesis. To search for novel activators of the Wnt/β-catenin pathway, we screened plant extracts and found that Sangusorba officinalis L. extracts (SOE) could increase β-catenin expression level and nuclear accumulation in 3T3-L1 and MC3T3-E1 cells. It was confirmed that SOE could effectively control adipo-osteogenic differentiation. SOE inhibited adipocyte differentiation of 3T3-L1 cells, markedly decreased intracellular lipid accumulation, and decreased expression levels of key adipogenic transcription factors including PPARγ, C/EBPα, and SREBP-1c. SOE enhanced ALP activity and terminal osteoblast differentiation which was confirmed by Alizarin Red S staining of MC3T3-E1 cells. In ex vivo culture, SOE significantly increased the thickness of calvarial bone in mice. Taken together, our results indicate that SOE could be used as a potential therapeutic agent for dual-treatment of anti-obesity and anti-osteoporosis via activation of Wnt/β-catenin pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Down-regulation of hTERT and Cyclin D1 transcription via PI3K/Akt and TGF-β pathways in MCF-7 Cancer cells with PX-866 and Raloxifene

Human telomerase reverse transcriptase (hTERT) is the catalytic and limiting component of telomerase and also a transcription factor. It is critical to the integrity of the ends of linear chromosomes and to the regulation, extent and rate of cell cycle progression in multicellular eukaryotes. The level of hTERT expression is essential to a wide range of bodily functions and to avoidance of disease conditions, such as cancer, that are mediated in part by aberrant level and regulation of cell cycle proliferation. Value of a gene in regulation depends on its ability to both receive input from multiple sources and transmit signals to multiple effectors. The expression of hTERT and the progression of the cell cycle have been shown to be regulated by an extensive network of gene products and signaling pathways, including the PI3K/Akt and TGF-β pathways. The PI3K inhibitor PX-866 and the competitive estrogen receptor ligand raloxifene have been shown to modify progression of those pathways and, in combination, to decrease proliferation of estrogen receptor positive (ER+) MCF-7 breast cancer cells. We found that combinations of modulators of those pathways decreased not only hTERT transcription but also transcription of additional essential cell cycle regulators such as Cyclin D1. By evaluating known expression profile signatures for TGF-β pathway diversions, we confirmed additional genes such as heparin-binding epidermal growth factor-like growth factor (HB EGF) by which those pathways and their perturbations may also modify cell cycle progression. - Highlights: • PX-866 and raloxifene affect the PI3K/Akt and TGF-β pathways. • PX-866 and raloxifene down-regulate genes up-regulated in cancer. • PX-866 and raloxifene decrease transcription of hTERT and Cyclin D1. • Pathological transcription signatures can identify new defense mechanisms.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

EP300/CBP is crucial for cAMP-PKA pathway to alleviate podocyte dedifferentiation via targeting Notch3 signaling

Podocyte injury is the hallmark of proteinuric glomerular diseases. Notch3 is neo-activated simultaneously in damaged podocytes and podocyte's progenitor cells of FSGS, indicating a unique role of Notch3. We previously showed that activation of cAMP-PKA pathway alleviated podocyte injury possibly via inhibiting Notch3 expression. However, the mechanisms are unknown. In the present study, Notch3 signaling was significantly activated in ADR-induced podocytes in vitro and in PAN nephrosis rats and patients with idiopathic FSGS in vivo, concomitantly with podocyte dedifferentiation. In cultured podocytes, pCPT-cAMP, a selective cAMP-PKA activator, dramatically blocked ADR-induced activation of Notch3 signaling as well as inhibition of cAMP-PKA pathway, thus alleviating the decreased cell viability and podocyte dedifferentiation. Bioinformatics analysis revealed EP300/CBP, a transcriptional co-activator, as a central hub for the crosstalk between these two signaling pathways. Additionally, CREB/KLF15 in cAMP-PKA pathway competed with RBP-J the major transcriptional factor of Notch3 signaling for binding to EP300/CBP. EP300/CBP siRNA significantly inhibited these two signaling transduction pathways and disrupted the interactions between the above major transcriptional factors. These data indicate a crucial role of EP300/CBP in regulating the crosstalk between cAMP-PKA pathway and Notch3 signaling and modulating the phenotypic change of podocytes, and enrich the reno-protective mechanisms of cAMP-PKA pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Comparative Life Cycle Evaluation of the Global Warming Potential (GWP) Impacts of Renewable Natural Gas Production Pathways

Renewable Natural Gas (RNG) sources are being considered in future energy strategy discussions as potential replacements for fossil natural gas (FNG). While today’s supply of RNG resources is insufficient to meet U.S. demands, there is significant interest in its viability to supplement and decarbonize the natural gas supply. However, the resources that compare the life cycle global warming potential (GWP) of various RNG production pathways are lacking and focus mostly on a singular pathway. This effort is an attempt to close this gap and provide a comparison between the life cycle GWP of three major RNG pathways and the FNG pathway. The three RNG pathways evaluated are Anaerobic Digestion (AD), Thermal Gasification (TG), and Power-to-Gas (P2G) using various feedstocks. The functional unit is 1MJ of compressed RNG ready for injection into the natural gas transmission network. The results show that RNG production is not always carbon neutral or negative. Depending on the pathway the GWP impact of RNG production can range from -229 to 27 g CO 2 e/MJ compressed RNG, with AD of animal manure and AD of municipal solid waste being the least and the most impactful pathways, respectively, compared to the 10.1 g CO 2 e/MJ impact for compressed FNG.

54 ENVIRONMENTAL SCIENCES↗