Gene Expression Of Metabolizing Enzymes Research Articles

In Vivo Glucose Transporter-2 Regulation of Dorsomedial Versus Ventrolateral VMN Astrocyte Metabolic Sensor and Glycogen Metabolic Enzyme Gene Expression in Female Rat.

Astrocyte glycogenolysis shapes ventromedial hypothalamic nucleus (VMN) regulation of glucostasis in vivo. Glucose transporter-2 (GLUT2), a plasma membrane glucose sensor, controls hypothalamic primary astrocyte culture glycogen metabolism in vitro. In vivo gene silencing tools and single-cell laser-catapult-microdissection/multiplex qPCR techniques were used here to examine whether GLUT2 governs dorsomedial (VMNdm) and/or ventrolateral (VMNvl) VMN astrocyte metabolic sensor and glycogen metabolic enzyme gene profiles. GLUT2 gene knockdown diminished astrocyte GLUT2 mRNA in both VMN divisions. Hypoglycemia caused GLUT2 siRNA-reversible up-regulation of this gene profile in the VMNdm, but down-regulated VMNvl astrocyte GLUT2 transcription. GLUT2 augmented baseline VMNdm and VMNvl astrocyte glucokinase (GCK) gene expression, but increased (VMNdm) or reduced (VMNvl) GCK transcription during hypoglycemia. GLUT2 imposed opposite control, namely stimulation versus inhibition of VMNdm or VMNvl astrocyte 5'-AMP-activated protein kinase-alpha 1 and -alpha 2 gene expression, respectively. GLUT2 stimulated astrocyte glycogen synthase (GS) gene expression in each VMN division. GLUT2 inhibited transcription of the AMP-sensitive glycogen phosphorylase (GP) isoform GP-brain type (GPbb) in each site, yet diminished (VMNdm) or augmented (VMNvl) astrocyte GP-muscle type (GPmm) mRNA. GLUT2 enhanced VMNdm and VMNvl glycogen accumulation during euglycemia, and curbed hypoglycemia-associated VMNdm glycogen depletion. Results show that VMN astrocytes exhibit opposite, division-specific GLUT2 transcriptional responsiveness to hypoglycemia. Data document divergent GLUT2 control of GCK, AMPK catalytic subunit, and GPmm gene profiles in VMNdm versus VMNvl astrocytes. Ongoing studies seek to determine how differential GLUT2 regulation of glucose and energy sensor function and glycogenolysis in each VMN location may affect local neuron responses to hypoglycemia.

A Trp-574-Leu mutation in acetolactate synthase confers imazamox resistance in barnyardgrass (Echinochloa crus-galli) from China

Abstract Barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] is increasingly infesting imidazolinone-tolerant (IMI-T) rice fields in China, imazamox resistance of E. crus-galli has become the major concern for weed management in IMI-T rice fields. In this study, the susceptible population JLGY-3 (S) and the suspected resistant population JHXY-2 (R) collected from IMI-T rice fields were used as research subjects. When treated with imazamox, JHXY-2 (R) population showed a high level of herbicide resistance with a resistance index (RI) of 31.2. JHXY-2 (R) was cross-resistant to all five acetolactate synthase (ALS) inhibitors from different chemical families, but sensitive to herbicides inhibiting acetyl-CoA carboxylase (ACCase). In order to understand the reason why JHXY-2 (R) was resistant to imazamox, we performed experiments to characterize potential TSR and NTSR mechanisms. A trp-574-leu amino acid mutation in ALS and low imazamox ALS sensitivity were the main mechanism underlying imazamox resistance in this JHXY-2 (R) population. There was no significant difference in ALS gene expression and ALS protein abundance between R and S populations. High-performance liquid chromatography-tandem mass spectrometry analysis showed enhanced metabolism of imazamox in JHXY-2 (R), which was in contrast to the results of pretreatment with a metabolic enzyme inhibitor. Treatments with the P450/GST inhibitors did not alter the resistance level of JHXY-2 (R) against imazamox. To further clarify the NTSR mechanism of JHXY-2 (R), transcriptome sequencing showed that there was almost no significant difference in the expression of P450 and GST metabolic enzyme genes between R and S populations, and only GST-U1 showed a significant induction in R population. In conclusion, amino acid mutation and higher enzyme activity of ALS are the main causes of imazamox resistance in JHXY-2 (R). However, given the differences in imazamox residues in the leaves of the E. crus-galli, there may still be undetectable NTSR that are causing imazamox resistance in the R population.

Abstract 7036: Integrative biomarker profiling for predicting immunotherapy outcomes in gynecologic cancers

Abstract Immunotherapy with immune checkpoint inhibitors (ICI) has proven effective for various solid tumors, such as those in the lungs, urinary tract, and skin. However, its efficacy in gynecologic (GYN) cancers has been limited, with response rates of 11-17% in recurrent cases. Only a minority of patients with DNA mismatch repair deficiencies exhibit favorable responses to PD-1 inhibitors. The predictability of PD-L1 expression in GYN cancers remains inconsistent. Our research aimed to identify and prioritize biomarkers and mechanisms linked to ICI resistance in GYN oncology, targeting those who could benefit from these treatments.We gathered clinical and pathological data from 49 patients with ovarian, endometrial, and cervical cancers at various stages, all treated with ICI. We performed comprehensive analyses, including transcriptome-based immune cell abundance estimation, signaling pathway enrichment, and correlation with ICI treatment responses and survival outcomes. Our previous work highlighted TGF-β and CD47's roles in immunotherapy resistance, facilitating an immunosuppressive environment within our GYN cancer cohort. Moreover, we conducted a stoichiometry constraint-based genome-scale metabolic model and computed correlation-grouped metabolic activity scores (cgScore) using metabolic enzyme gene expression as constraints. Among 1533 metabolic reactions, pyruvate transport and folate metabolism stood out as the most upregulated in responders versus those who progressed.Upon identifying several promising biomarkers, we used logistic regression classifiers to decipher the predictive value of each. Our model included clinical-demographic (age, BMI, cancer type, stage) and molecular-genomic features (immune marker gene expression, TopTGF-β score, immune cell abundance from transcriptomic deconvolution, and the top 2 cgScores). Analysis divided into training and testing sets highlighted features such as stage, BMI, eosinophils, CD274, CD47, and the pyruvate transport reaction as significant predictors of ICI response. The model achieved an 80% accuracy rate on the test set, confirmed by permutation importance from scikit-learn. We also used a Regularized Cox model to link predictors with progression-free survival, emphasizing the roles of BMI, plasma cells, CD276, pyruvate transport reaction, and folate metabolism. In conclusion, there are immunological and metabolic markers have the potential to serve as predictive indicators for ICI treatment response and survival in GYN cancers. Understanding these predictors can profoundly influence clinical decision-making in immunotherapy, ultimately leading to enhanced patient outcomes and personalized treatment approaches. Citation Format: Gideon Idumah, Lin Li, Haider Mahdi, Ying Ni. Integrative biomarker profiling for predicting immunotherapy outcomes in gynecologic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7036.

Cadmium Exposure Impairs Development, Detoxification Mechanisms and Gene Expression of Glyphodes pyloalis Walker (Lepidoptera: Pyralidae)

Cadmium (Cd) is the most serious heavy metal pollutant in the agricultural soil of China and can transfer and accumulate through the food chain and affect the growth, development and physiological processes of phytophagous insect. Glyphodes pyloalis Walker (G. pyloalis) is one of the most important pests of mulberry, and there are few studies on the adverse effects of heavy metals on insects, especially mulberry pests. To understand the toxicology of Cd exposure on G. pyloalis, we investigated the effects of three different concentrations of Cd (0, 3.89 and 51.69 mg/kg, labeled as control check (CK), low dose (LD) and high dose (HD)) on the development and detoxification mechanism of G. pyloalis and explored the molecular mechanism of Cd on G. pyloalis larvae using RNA-seq technology. Transcriptome analysis showed that compared with the CK, a total of 63 differentially expressed genes (DEGs) were identified in LD exposure, including 24 upregulated and 39 downregulated candidates. In CK versus HD groups, 395 upregulated DEGs and 436 downregulated DEGs were identified and the expression patterns of 12 genes related to detoxification and metabolism were verified using qPCR. These DEGs were relevant to multiple specific peroxisome and drug metabolism-cytochrome P450 pathways. Gene annotation and quantitative real-time PCR revealed that a high concentration of Cd significantly stimulated the expression of metabolic detoxification enzyme genes. The results revealed that Cd exposure changed the pupal weight, adult emergence rate and the activities of AKP, ACP and GST in G. pyloalis and induced notable adverse effects at the molecular level on detoxification and metabolism such as concentration and time differences. In addition, we silenced CYP12A2 by RNA interference. Bioassays showed that after silencing CYP12A2, the survival rate of G. pyloalis under Cd exposure was lower than that of the control group.

Protein succinylation: regulating metabolism and beyond.

Modifications of protein post-translation are critical modulatory processes, which alters target protein biological activity，function and/or location, even involved in pathogenesis of some diseases. So far, there are at least 16 types of post-translation modifications identified, particularly through recent mass spectrometry analysis. Among them, succinylation (Ksuc) on protein lysine residues causes a variety of biological changes. Succinylation of proteins contributes to many cellular processes such as proliferation, growth, differentiation, metabolism and even tumorigenesis. Mechanically, Succinylation leads to conformation alteration of chromatin or remodeling. As a result, transcription/expression of target genes is changed accordingly. Recent research indicated that succinylation mainly contributes to metabolism modulations, from gene expression of metabolic enzymes to their activity modulation. In this review, we will conclude roles of succinylation in metabolic regulation of glucose, fat, amino acids and related metabolic disease launched by aberrant succinylation. Our goal is to stimulate extra attention to these still not well researched perhaps important succinylation modification on proteins and cell processes.

The Essential Role of H2S-ABA Crosstalk in Maize Thermotolerance through the ROS-Scavenging System.

Hydrogen sulfide (H2S) and abscisic acid (ABA), as a signaling molecule and stress hormone, their crosstalk-induced thermotolerance in maize seedlings and its underlying mechanism were elusive. In this paper, H2S and ABA crosstalk as well as the underlying mechanism of crosstalk-induced thermotolerance in maize seedlings were investigated. The data show that endogenous levels of H2S and ABA in maize seedlings could be mutually induced by regulating their metabolic enzyme activity and gene expression under non-heat stress (non-HS) and HS conditions. Furthermore, H2S and ABA alone or in combination significantly increase thermotolerance in maize seedlings by improving the survival rate (SR) and mitigating biomembrane damage. Similarly, the activity of the reactive oxygen species (ROS)-scavenging system, including enzymatic antioxidants catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POD), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and superoxide dismutase (SOD), as well as the non-enzymatic antioxidants reduced ascorbic acid (AsA), carotenoids (CAR), flavone (FLA), and total phenols (TP), was enhanced by H2S and ABA alone or in combination in maize seedlings. Conversely, the ROS level (mainly hydrogen peroxide and superoxide radical) was weakened by H2S and ABA alone or in combination in maize seedlings under non-HS and HS conditions. These data imply that the ROS-scavenging system played an essential role in H2S-ABA crosstalk-induced thermotolerance in maize seedlings.

Identification of USP29 as a key regulator of nucleotide biosynthesis in neuroblastoma through integrative analysis of multi-omics data

ABSTRACT Cancer cells show enhanced nucleotide biosynthesis, which is essential for their unlimited proliferation, but the underlying mechanisms are not entirely clear. Ubiquitin specific peptidase 29 (USP29) was reported to sustain neuroblastoma progression by promoting glycolysis and glutamine catabolism; however, its potential role in regulating nucleotide biosynthesis in tumor cells remains unknown. In this study, we depleted endogenous USP29 in MYCN-amplified neuroblastoma SK-N-BE2 cells by sgRNAs and conducted metabolomic analysis in cells with or without USP29 depletion, we found that USP29 deficiency caused a disorder of intermediates involved in glycolysis and nucleotide biosynthesis. De novo nucleotide biosynthesis was analyzed using 13C6 glucose as a tracer under normoxia and hypoxia. The results indicated that USP29-depleted cells showed inhibition of nucleotide anabolic intermediates derived from glucose, and this inhibition was more significant under hypoxic conditions. Analysis of RNA sequencing data in SK-N-BE2 cells demonstrated that USP29 promoted the gene expression of metabolic enzymes involved in nucleotide anabolism, probably by regulating MYC and E2F downstream pathways. These findings indicated that USP29 is a key regulator of nucleotide biosynthesis in tumor cells.

Metagenomic characterization of biomethane transformation by lipid-catalyzed anaerobic fermentation of lignite

The present study aims at using lipid in a novel way to improve the efficiency of methane production from lignite anaerobic digestion. The obtained results showed an increase by 3.13 times of the cumulative biomethane content of lignite anaerobic fermentation, when 1.8 g lipid was added. The gene expression of functional metabolic enzymes was also found to be enhanced during the anaerobic fermentation. Moreover, the enzymes related to fatty acid degradation such as long-chain Acyl-CoA synthetase and Acyl-CoA dehydrogenase were increased by 1.72 and 10.48 times, respectively, which consequently, accelerated the conversion of fatty acid. Furthermore, the addition of lipid enhanced the carbon dioxide trophic and acetic acid trophic metabolic pathways. Hence, the addition of lipids was argued to promote the production of methane from lignite anaerobic fermentation, which provided a new insight for the conversion and utilization of lipid waste.

Steroidal glycoalkaloids contribute to anthracnose resistance in Solanum lycopersicum.

Anthracnose is a widespread plant disease caused by various species of the fungal pathogen Colletotrichum. In solanaceous plants such as tomato (Solanum lycopersicum), Colletotrichum infections exhibit a quiescent, asymptomatic state in developing fruit, followed by a transition to necrotrophic infections in ripe fruit. Through analysis of fruit tissue extracts of 95L368, a tomato breeding line that yields fruit with enhanced anthracnose resistance, we identified a role for steroidal glycoalkaloids (SGAs) in anthracnose resistance. The SGA α-tomatine and several of its derivatives accumulated at higher levels, in comparison with fruit of the susceptible tomato cultivar US28, and 95L368 fruit extracts displayed fungistatic activity against Colletotrichum. Correspondingly, ripe and unripe 95L368 fruit displayed enhanced expression of glycoalkaloid metabolic enzyme (GAME) genes, which encode key enzymes in SGA biosynthesis. Metabolomics analysis incorporating recombinant inbred lines generated from 95L368 and US28 yielded strong positive correlations between anthracnose resistance and accumulation of α-tomatine and several derivatives. Lastly, transient silencing of expression of the GAME genes GAME31 and GAME5 in anthracnose-susceptible tomato fruit yielded enhancements to anthracnose resistance. Together, our data support a role for SGAs in anthracnose defense in tomato, with a distinct SGA metabolomic profile conferring resistance to virulent Colletotrichum infections in ripe fruit.

Controlling the confounding effect of metabolic gene expression to identify actual metabolite targets in microsatellite instability cancers

BackgroundThe metabolome is the best representation of cancer phenotypes. Gene expression can be considered a confounding covariate affecting metabolite levels. Data integration across metabolomics and genomics to establish the biological relevance of cancer metabolism is challenging. This study aimed to eliminate the confounding effect of metabolic gene expression to reflect actual metabolite levels in microsatellite instability (MSI) cancers.MethodsIn this study, we propose a new strategy using covariate-adjusted tensor classification in high dimensions (CATCH) models to integrate metabolite and metabolic gene expression data to classify MSI and microsatellite stability (MSS) cancers. We used datasets from the Cancer Cell Line Encyclopedia (CCLE) phase II project and treated metabolomic data as tensor predictors and data on gene expression of metabolic enzymes as confounding covariates.ResultsThe CATCH model performed well, with high accuracy (0.82), sensitivity (0.66), specificity (0.88), precision (0.65), and F1 score (0.65). Seven metabolite features adjusted for metabolic gene expression, namely, 3-phosphoglycerate, 6-phosphogluconate, cholesterol ester, lysophosphatidylethanolamine (LPE), phosphatidylcholine, reduced glutathione, and sarcosine, were found in MSI cancers. Only one metabolite, Hippurate, was present in MSS cancers. The gene expression of phosphofructokinase 1 (PFKP), which is involved in the glycolytic pathway, was related to 3-phosphoglycerate. ALDH4A1 and GPT2 were associated with sarcosine. LPE was associated with the expression of CHPT1, which is involved in lipid metabolism. The glycolysis, nucleotide, glutamate, and lipid metabolic pathways were enriched in MSI cancers.ConclusionsWe propose an effective CATCH model for predicting MSI cancer status. By controlling the confounding effect of metabolic gene expression, we identified cancer metabolic biomarkers and therapeutic targets. In addition, we provided the possible biology and genetics of MSI cancer metabolism.

Comparative study on the gene expression of corticosterone metabolic enzymes in embryonic tissues between Tibetan and broiler chickens

Glucocorticoids (GCs) are an essential mediator hormone that can regulate animal growth, behavior, the phenotype of offspring, and so on, while GCs in poultry are predominantly corticosterones. The biological activity of GCs is mainly regulated by the intracellular metabolic enzymes, including 11β-hydroxysteroid dehydrogenases 1 (11β-HSD1), 11β-hydroxysteroid dehydrogenases 2 (11β-HSD2), and 20-hydroxysteroid dehydrogenase (20-HSD). To investigate the embryonic mechanisms of phenotypic differences between breeds, we compared the expression of corticosterone metabolic enzyme genes in the yolk-sac membrane and chorioallantoic membrane (CAM). We described the tissue distribution and ontogenic patterns of corticosterone metabolic enzymes during embryonic incubation between Tibetan and broiler chickens. Forty fertilized eggs from Tibetan and broiler chickens were incubated under hypoxic and normoxic conditions, respectively. Real-time fluorescence quantitative PCR was used to examine the expression of 11β-HSD1/2, and 20-HSD mRNA in embryonic tissues. The results showed that the expression levels of yolk-sac membrane mRNA of 11β-HSD2 and 20-HSD in Tibetan chickens on E14 (embryonic day of 14) were significantly lower than those of broiler chickens (P < 0.05), and these genes expression of CAM in Tibetan chickens were higher than those of broiler chickens (P < 0.05). In addition, the three genes in the yolk-sac membrane and CAM were followed by a down-regulation on E18 (embryonic day of 18). The 11β-HSD1 and 11β-HSD2 genes followed a similar tissue-specific pattern: the expression level was more abundantly in the liver, kidney, and intestine, with relatively lower abundance in the hypothalamus and muscle, and the expression level of 20-HSD genes in all tissues tested was higher. In the liver, 20-HSD of both Tibetan and broiler chickens showed different ontogeny development patterns, and hepatic mRNA expression of 20-HSD in broiler chickens was significantly higher than that of Tibetan chickens of the same age from E14 to E18 (P < 0.05). This study preliminarily revealed the expression levels of cortisol metabolic genes in different tissues during the development process of Tibetan and broiler chicken embryos. It provided essential information for in-depth research of the internal mechanism of maternal GCs programming on offspring.

Glycine betaine inhibits postharvest softening and quality decline of winter jujube fruit by regulating energy and antioxidant metabolism

Winter jujube fruit easily softens after harvest. To investigate the effects of glycine betaine (N,N,N-trimethylglycine; GB) treatment on the quality of postharvest jujubes, fresh winter jujubes (Zizyphus jujuba Mill. cv. Dongzao) were immersed in 20 mmol·L−1 GB for 20 min. The results showed that GB application can effectively maintain cell wall component content by restraining gene expression and enzyme activities, including PG, CX, PME and β-Glu. Meanwhile, the activities of antioxidant enzymes (APX, CAT, SOD, POD) and the contents of nonenzymatic antioxidants (MDA, H2O2, ASA, GSH) were enhanced in treated jujubes, thereby reducing the content of ROS. In addition, energy metabolism enzyme activities (H+-ATPase, Ca2+-ATPase, SDH and CCO) and gene expression were also significantly increased, thus maintaining higher energy levels (ATP, ADP, AMP and EC). In summary, GB enhances ATP biosynthesis by increasing energy metabolism. It offers essential energy for the antioxidant metabolism, thus retarding the softening of postharvest jujubes.

The ESCRT-III Protein Chmp1 Regulates Lipid Storage in the Drosophila Fat Body.

Defects in how excess nutrients are stored as triglycerides can result in several diseases including obesity, heart disease, and diabetes. Understanding the genes responsible for normal lipid homeostasis will help understand the pathogenesis of these diseases. RNAi screens performed in Drosophila cells identified genes involved in vesicle formation and protein sorting as important for the formation of lipid droplets; however, all of the vesicular trafficking proteins that regulate lipid storage are unknown. Here, we characterize the function of the Drosophila Charged multivesicular protein 1 (Chmp1) gene in regulating fat storage. Chmp1 is a member of the ESCRT-III complex that targets membrane localized signaling receptors to intralumenal vesicles in the multivesicular body of the endosome and then ultimately to the lysosome for degradation. When Chmp1 levels are decreased specifically in the fly fat body, triglyceride accumulates while fat-body-specific Chmp1 overexpression decreases triglycerides. Chmp1 controls triglyceride storage by regulating the number and size of fat body cells produced and not by altering food consumption or lipid metabolic enzyme gene expression. Together, these data uncover a novel function for Chmp1 in controlling lipid storage in Drosophila and supports the role of the endomembrane system in regulating metabolic homeostasis.

Metabolic Pathways, Enzymes, and Metabolites: Opportunities in Cancer Therapy.

Metabolic reprogramming enables cancer cells to proliferate and produce tumor biomass under a nutrient-deficient microenvironment and the stress of metabolic waste. A cancer cell adeptly undergoes a variety of adaptations in metabolic pathways and differential expression of metabolic enzyme genes. Metabolic adaptation is mainly determined by the physiological demands of the cancer cell of origin and the host tissue. Numerous metabolic regulators that assist cancer cell proliferation include uncontrolled anabolism/catabolism of glucose metabolism, fatty acids, amino acids metabolism, nucleotide metabolism, tumor suppressor genes, microRNAs, and many regulatory enzymes and genes. Using this paradigm, we review the current understanding of metabolic reprogramming in tumors and discuss the new strategies of cancer metabolomics that can be tapped into for cancer therapeutics.

Stage specific gene expression of folate mediated one-carbon metabolism enzymes and transporters in buffalo oocytes and pre-implantation embryos

DNA synthesis and methylations are crucial during pre-implantation embryonic development, and are mediated by one-carbon metabolism of folates. Folates, transported into the cells via folate receptors (FOLR1 and FOLR2) and carriers (SLC19A1), are metabolized by various enzymes involved in folate-methionine cycle. However, the variations in temporal expression of folate transporters and folate-methionine cycle enzymes during pre-implantation embryo development is obscure. Thus, the present study aimed to investigate the differential expression of the genes for folate transporters and folate-methionine cycle enzymes. We also examined the expression of folate transport proteins in different pre-implantation development stages. Immature buffalo oocytes were matured in maturation medium followed by in vitro fertilization and culture at standard culture conditions. The temporal pattern of gene expression in buffalo, when compared to previous studies, indicated an inter-specific variation. The transcripts of some enzymes and folate transporters were significantly upregulated after zygotic genome activation. The transcripts as well as proteins for FOLR1, FOLR2 and SLC19A1 were present in oocytes and all the pre-implantation embryo stages. FOLR1 was present in the nuclei of different stages of developing embryos but not in the metaphase (MII) oocytes. As a result, the present study advocates the existence of active folate transport in buffalo oocytes and pre-implantation embryos. The data provided by the analysis of differential gene expression of folate transporters and metabolic enzymes would likely contribute to a better understanding of the role of folates in embryo development as well as advancements in assisted reproductive technologies.

Intestinal Permeability of Drugs in Caco-2 Cells Cultured in Microfluidic Devices.

Microfluidic devices are attracting attention for their ability to provide a biomimetic microenvironment wherein cells are arranged in a particular pattern and provided fluidic and mechanical forces. In this study, we evaluated drug transport across Caco-2 cell layers in microfluidic devices and investigated the effects of fluid flow on drug transport and metabolism. We designed a microfluidic device that comprises two blocks of polydimethylsiloxane and a sandwiched polyethylene terephthalate membrane with pores 3.0 µm in diameter. When cultured in a dynamic fluid environment, Caco-2 cells were multilayered and developed microvilli on the surface as compared with a static environment. Drugs with higher lipophilicity exhibited higher permeability across the Caco-2 layers, as well as in the conventional method using Transwells, and the fluidic conditions had little effect on permeability. In the Caco-2 cell layers cultured in Transwells and microfluidic devices, the basal-to-apical transport of rhodamine 123, a substrate of P-glycoprotein, was greater than the apical-to-basal transport, and the presence of tariquidar, an inhibitor of P-glycoprotein, completely diminished asymmetric transport. Furthermore, fluidic conditions promoted the metabolism of temocapril by carboxylesterases. On the other hand, we showed that fluidic conditions have little effect on gene expression of several transporters and metabolic enzymes. These results provide useful information regarding the application of microfluidic devices in drug transport and metabolism studies.

A Metabolic Gene Signature to Predict Breast Cancer Prognosis.

Background: The existing metabolic gene signatures for predicting breast cancer outcomes only focus on gene expression data without considering clinical characteristics. Therefore, this study aimed to establish a predictive risk model combining metabolic enzyme genes and clinicopathological characteristics to predict the overall survival in patients with breast cancer. Methods: Transcriptomics and corresponding clinical data for patients with breast cancer were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Differentially expressed metabolic genes between tumors and normal tissues were identified in the TCGA dataset (training dataset). A prognostic model was then built using univariate and multifactorial Cox proportional hazards regression analyses in the training dataset. The capability of the predictive model was then assessed using the receiver operating characteristic in both datasets. Pathway enrichment analysis and immune cell infiltration were performed using Kyoto Encyclopedia of Genes and Genomes (KEGG)/Gene Ontology (GO) enrichment and CIBERSORT algorithm, respectively. Results: In breast cancer and normal tissues, 212 metabolic enzyme genes were differentially expressed. The predictive model included four factors: age, stage, and expression of SLC35A2 and PLA2G10. Patients with breast cancer were classified into high- and low-risk groups based on the model; the high-risk group had a significantly poorer overall survival rate than the low-risk group. Furthermore, the two risk groups showed different activation of pathways and alterations in the properties of tumor microenvironment-infiltrating immune cells. Conclusion: We developed a powerful model to predict prognosis in patients with breast cancer by combining the gene expression of metabolic enzymes with clinicopathological characteristics.

Loss of Pkm2 Alters Myocardial Metabolism and Increases Oxidative Stress After Infarction

IntroductionPyruvate kinase (PKM1) directs pyruvate to the Krebs cycle for oxidative metabolism in the healthy heart. Our lab described a hypoxia‐mediated switch to the alternatively spliced isoform PKM2, enhancing pyruvate to lactate conversion. Recently, we have also found that Pkm2 knockout (KO) mice had profound depletion of basal glucose in the heart compared to control mice. Pkm2 has also been shown to reduce oxidative damage and promote cardiomyocyte cell proliferation after myocardial infarction (MI). We hypothesize that upregulation of PKM2 can alter metabolic pathways by promoting glycolysis, and that after injury, this can preserve ATP production, protecting the heart from the stresses of hypoxia and injury.MethodsGlobal Pkm2 KO mice were subjected to permanent ligation of the left anterior descending coronary artery to mimic an MI. RNA‐seq analysis of left ventricles from control (n=8) and Pkm2 KO mice (n=8) before and 3 days after sham or MI surgery was performed. Semi‐quantitative real‐time PCR was used to confirm changes in selected genes of interest.ResultsLoss of Pkm2 moderately altered gene expression at baseline (q&lt;0.05, FDR&lt;0.05). Notably, the mitochondrial gene COX3 was downregulated in Pkm2 KO hearts. 68 genes were differentially expressed in Pkm2 KO hearts after MI, not observed in control MI hearts. MI of Pkm2 KO hearts resulted in considerable reduction of transcripts of enzymes in the insulin signaling pathway, mitochondrial oxidative phosphorylation, mitochondrial uncoupling, fatty acid metabolism, and increase in transcripts encoding enzymes in the pentose phosphate pathway, response to oxidative stress, and apoptotic signaling. Semi‐quantitative PCR of selected genes involved in glucose metabolism confirmed RNA‐seq results.ConclusionsRNA‐seq analysis of Pkm2 KO hearts demonstrated that loss of Pkm2 altered gene expression of metabolic and mitochondrial enzymes. Pkm2 KO hearts also showed increased abundance of pro‐apoptotic markers which may be a result of increased oxidative stress.

Physiological and Molecular Aspects of Two Thymus Species Differently Sensitive to Drought Stress.

Drought is one of the most important threats to plants and agriculture. Here, the effects of four drought levels (90%, 55%, 40%, and 25% field capacity) on the relative water content (RWC), chlorophyll and carotenoids levels, and mRNA gene expression of metabolic enzymes in Thymus vulgaris (as sensitive to drought) and Thymus kotschyanus (as a drought-tolerant species) were evaluated. The physiological results showed that the treatment predominantly affected the RWC, chlorophyll, and carotenoids content. The gene expression analysis demonstrated that moderate and severe drought stress had greater effects on the expression of histone deacetylase-6 (HDA-6) and acetyl-CoA synthetase in both Thymus species. Pyruvate decarboxylase-1 (PDC-1) was upregulated in Thymus vulgaris at high drought levels. Finally, succinyl CoA ligase was not affected by drought stress in either species. Data confirmed water stress is able to alter the gene expression of specific enzymes. Furthermore, our results suggest that PDC-1 expression is independent from HDA-6 and the increased expression of ACS can be due to the activation of new pathways involved in carbohydrate production.

Gene Expression of Antioxidant Metabolic Enzymes in Grape Extracts

Fruit can use its structural characteristics to stabilize the excess electrons of free radicals and prevent cell aging, which is the main source of antioxidants supplementation. In the current study, the expression of antioxidant metabolic genes was evaluated in fruit extract. Seventy mice were divided into one control group and six experimental groups. Six experimental groups were fed 0.1g of grape extract daily for 3, 7, 10, 14, 21, 24 and 30 days, respectively. The antioxidant activity of fruit extract was tested by antioxidant metabolic enzyme gene expression in the liver and kidney of different experimental groups. The results showed that grape extract could effectively enhance SOD activity, LPL relative expression and FAS relative expression in mice liver tissue; grape extract could effectively increase SOD relative expression, TrxR2 relative expression and CAT relative expression in mice kidney tissue; grape extract could effectively reduce MDA activity in mice liver tissue, and increase and decrease the extent of MDA activity and the duration of feeding in mice. The proportion of fruit extracts showed that fruit extracts could effectively improve the antioxidant performance of the body.