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1.
Microb Cell Fact ; 21(1): 178, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36050762

RESUMO

BACKGROUND: Electro-fermentation (EF) is an emerging tool for bioprocess intensification. Benefits are especially expected for bioprocesses in which the cells are enabled to exchange electrons with electrode surfaces directly. It has also been demonstrated that the use of electrical energy in BES can increase bioprocess performance by indirect secondary effects. In this case, the electricity is used to alter process parameters and indirectly activate desired pathways. In many bioprocesses, oxidation-reduction potential (ORP) is a crucial process parameter. While C. pasteurianum fermentation of glycerol has been shown to be significantly influenced electrochemically, the underlying mechanisms are not clear. To this end, we developed a system for the electrochemical control of ORP in continuous culture to quantitatively study the effects of ORP alteration on C. pasteurianum by metabolic flux analysis (MFA), targeted metabolomics, sensitivity and regulation analysis. RESULTS: In the ORP range of -462 mV to -250 mV, the developed algorithm enabled a stable anodic electrochemical control of ORP at desired set-points and a fixed dilution rate of 0.1 h-1. An overall increase of 57% in the molar yield for 1,3-propanediol was observed by an ORP increase from -462 to -250 mV. MFA suggests that C. pasteurianum possesses and uses cellular energy generation mechanisms in addition to substrate-level phosphorylation. The sensitivity analysis showed that ORP exerted its strongest impact on the reaction of pyruvate-ferredoxin-oxidoreductase. The regulation analysis revealed that this influence is mainly of a direct nature. Hence, the observed metabolic shifts are primarily caused by direct inhibition of the enzyme upon electrochemical production of oxygen. A similar effect was observed for the enzyme pyruvate-formate-lyase at elevated ORP levels. CONCLUSIONS: The results show that electrochemical ORP alteration is a suitable tool to steer the metabolism of C. pasteurianum and increase product yield for 1,3-propanediol in continuous culture. The approach might also be useful for application with further anaerobic or anoxic bioprocesses. However, to maximize the technique's efficiency, it is essential to understand the chemistry behind the ORP change and how the microbial system responds to it by transmitted or direct effects.


Assuntos
Clostridium , Glicerol , Clostridium/metabolismo , Fermentação , Glicerol/metabolismo , Oxirredução , Piruvatos/metabolismo
2.
Elife ; 112022 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-36052997

RESUMO

Acute myeloid leukaemia (AML) cells interact and modulate components of their surrounding microenvironment into their own benefit. Stromal cells have been shown to support AML survival and progression through various mechanisms. Nonetheless, whether AML cells could establish beneficial metabolic interactions with stromal cells is underexplored. By using a combination of human AML cell lines and AML patient samples together with mouse stromal cells and a MLL-AF9 mouse model, here we identify a novel metabolic crosstalk between AML and stromal cells where AML cells prompt stromal cells to secrete acetate for their own consumption to feed the tricarboxylic acid cycle (TCA) and lipid biosynthesis. By performing transcriptome analysis and tracer-based metabolic NMR analysis, we observe that stromal cells present a higher rate of glycolysis when co-cultured with AML cells. We also find that acetate in stromal cells is derived from pyruvate via chemical conversion under the influence of reactive oxygen species (ROS) following ROS transfer from AML to stromal cells via gap junctions. Overall, we present a unique metabolic communication between AML and stromal cells and propose two different molecular targets, ACSS2 and gap junctions, that could potentially be exploited for adjuvant therapy.


Assuntos
Leucemia Mieloide Aguda , Acetatos , Animais , Humanos , Leucemia Mieloide Aguda/metabolismo , Lipídeos , Camundongos , Piruvatos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Células Estromais/metabolismo , Microambiente Tumoral
3.
Cell ; 185(19): 3467-3486.e16, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36113426

RESUMO

Changes in gut microbiota have been associated with several diseases. Here, the International Multiple Sclerosis Microbiome Study (iMSMS) studied the gut microbiome of 576 MS patients (36% untreated) and genetically unrelated household healthy controls (1,152 total subjects). We observed a significantly increased proportion of Akkermansia muciniphila, Ruthenibacterium lactatiformans, Hungatella hathewayi, and Eisenbergiella tayi and decreased Faecalibacterium prausnitzii and Blautia species. The phytate degradation pathway was over-represented in untreated MS, while pyruvate-producing carbohydrate metabolism pathways were significantly reduced. Microbiome composition, function, and derived metabolites also differed in response to disease-modifying treatments. The therapeutic activity of interferon-ß may in part be associated with upregulation of short-chain fatty acid transporters. Distinct microbial networks were observed in untreated MS and healthy controls. These results strongly support specific gut microbiome associations with MS risk, course and progression, and functional changes in response to treatment.


Assuntos
Microbioma Gastrointestinal , Esclerose Múltipla , Ácidos Graxos Voláteis , Humanos , Interferon beta , Ácido Fítico , Piruvatos
4.
Biogerontology ; 23(5): 641-652, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36048311

RESUMO

In the present study, attempts have been made to evaluate the potential role of 3 Bromopyruvate (3-BP) a glycolytic inhibitor and a caloric restriction mimetic (CRM), to exert neuroprotection in rats during aging through modulation of autophagy. Young male rats (4 months), and naturally aged (22 months) male rats were supplemented with 3-BP (30 mg/kg b.w., orally) for 28 days. Our results demonstrate a significant increase in the antioxidant biomarkers (ferric reducing antioxidant potential level, total thiol, superoxide dismutase, and catalase activities) and a decrease in the level of pro-oxidant biomarkers such as protein carbonyl after 3-BP supplementation in brain tissues. A significant increase in reactive oxygen species (ROS) was observed due to the mitohormetic effect of 3-BP supplementation in the treated rats. Furthermore, the 3-BP treatment also enhanced the activities of electron transport chain complexes I and IV in aged brain mitochondria thus proving its antioxidant potential at the level of mitochondria. Gene expression analysis with reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to assess the expression of autophagy, neuroprotective and aging marker genes. RT-PCR data revealed that 3-BP up-regulated the expression of autophagy markers genes (Beclin-1 and LC3 ß), sirtuin-1, and neuronal marker gene (NSE), respectively in the aging brain. The results suggest that 3-BP induces a mitohormetic effect through the elevation of ROS which reinforces defensive mechanism(s) targeted at regulating autophagy. These findings suggest that consistently low-dose 3-BP may be beneficial for neuroprotection during aging and age-related disorders.


Assuntos
Restrição Calórica , Neuroproteção , Envelhecimento/metabolismo , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Autofagia , Biomarcadores/metabolismo , Masculino , Estresse Oxidativo , Piruvatos , Ratos , Espécies Reativas de Oxigênio/metabolismo
5.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 38(3): 247-251, 2022 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-36062794

RESUMO

Objective: To investigate the molecular mechanisms of Gupi Xiaoji decoction on apoptosis of human hepatoma cells HepG2. Methods: HepG2 cells were divided into 4 groups: control group (Control), blank serum group (Blank), Gupi Xiaoji Yin serum group (GPXJY) and cisplatin group (Positive). Eight duplicate holes were set in each group. After treated with Gupi Xiaoji Decoction-containing serum or cisplatin for 24 hours, the cell viability, the number of viable cells, the state of apoptosis, the cell cycle and the mitochondrial membrane potential were detected, and the level of lipid peroxidation (MDA) and glycolysis rate of the cells were detected. The expressions of apoptotic Bax, Bcl-2, and Caspase-3 proteins, and the contents of triacylglycerol (TG), cholesterol (TC), pyruvate and glucose in the cell supernatant were detected. Results: Compared with the control group, in the GPXJY group, the inhibition rate was increased (P<0.05), the number of cells was decreased, the number of apoptosis-positive cells was increased (P<0.01), the number of cells in the G1 phase was increased significantly (P<0.05), and the cell membrane potential was decreased (P<0.05,P<0.01), the glycolytic function was inhibited significantly, the MDA level was increased, the expressions of Bax and Caspase-3 in the GPXJY group were increased, and the expression of Bcl-2 was decreased (P<0.05, P<0.01). In cell supernatant, the TC, TG and glucose contents were decreased significantly, and the pyruvate content was increased significantly (P<0.05,P<0.01). Conclusion: Gupi Xiaoji Decoction can induce apoptosis of HepG2 cells and may play a role in energy metabolism.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Apoptose , Caspase 3/metabolismo , Cisplatino , Medicamentos de Ervas Chinesas , Glucose , Células Hep G2 , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Piruvatos , Proteína X Associada a bcl-2/metabolismo
6.
Int J Mol Sci ; 23(17)2022 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-36077135

RESUMO

Sugarcane (Saccharum spp. hybrid) is an important crop for sugar and biofuels, and often suffers from water shortages during growth. Currently, there is limited knowledge concerning the molecular mechanism involved in sugarcane response to drought stress (DS) and whether chitooligosaccharide could alleviate DS. Here, we carried out a combined transcriptome and metabolome of sugarcane in three different treatment groups: control group (CG), DS group, and DS + chitooligosaccharide group (COS). A total of 12,275 (6404 up-regulated and 5871 down-regulated) differentially expressed genes (DEGs) were identified when comparing the CG and DS transcriptomes (T_CG/DS), and 2525 (1261 up-regulated and 1264 down-regulated) DEGs were identified in comparing the DS and COS transcriptomes (T_DS/COS). GO and KEGG analysis showed that DEGs associated with photosynthesis were significantly enriched and had down-regulated expression. For T_DS/COS, photosynthesis DEGs were also significantly enriched but had up-regulated expression. Together, these results indicate that DS of sugarcane has a significantly negative influence on photosynthesis, and that COS can alleviate these negative effects. In metabolome analysis, lipids, others, amino acids and derivatives and alkaloids were the main significantly different metabolites (SDMs) observed in sugarcane response to DS, and COS treatment reduced the content of these metabolites. KEGG analysis of the metabolome showed that 2-oxocarboxylic acid metabolism, ABC transporters, biosynthesis of amino acids, glucosinolate biosynthesis and valine, leucine and isoleucine biosynthesis were the top-5 KEGG enriched pathways when comparing the CG and DS metabolome (M_CG/DS). Comparing DS with COS (M_DS/COS) showed that purine metabolism and phenylalanine metabolism were enriched. Combined transcriptome and metabolome analysis revealed that pyruvate and phenylalanine metabolism were KEGG-enriched pathways for CG/DS and DS/COS, respectively. For pyruvate metabolism, 87 DEGs (47 up-regulated and 40 down-regulated) and five SDMs (1 up-regulated and 4 down-regulated) were enriched. Pyruvate was closely related with 14 DEGs (|r| > 0.99) after Pearson's correlation analysis, and only 1 DEG (Sspon.02G0043670-1B) was positively correlated. For phenylalanine metabolism, 13 DEGs (7 up-regulated and 6 down-regulated) and 6 SDMs (1 up-regulated and 5 down-regulated) were identified. Five PAL genes were closely related with 6 SDMs through Pearson's correlation analysis, and the novel.31257 gene had significantly up-regulated expression. Collectively, our results showed that DS has significant adverse effects on the physiology, transcriptome, and metabolome of sugarcane, particularly genes involved in photosynthesis. We further show that COS treatment can alleviate these negative effects.


Assuntos
Saccharum , Transcriptoma , Aminoácidos/metabolismo , Quitosana , Secas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Metaboloma , Oligossacarídeos , Fenilalanina/metabolismo , Piruvatos/metabolismo , Saccharum/metabolismo
7.
Int J Mol Sci ; 23(17)2022 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-36077431

RESUMO

Aerobic glycolysis is an emerging hallmark of many human cancers, as cancer cells are defined as a "metabolically abnormal system". Carbohydrates are metabolically reprogrammed by its metabolizing and catabolizing enzymes in such abnormal cancer cells. Normal cells acquire their energy from oxidative phosphorylation, while cancer cells acquire their energy from oxidative glycolysis, known as the "Warburg effect". Energy-metabolic differences are easily found in the growth, invasion, immune escape and anti-tumor drug resistance of cancer cells. The glycolysis pathway is carried out in multiple enzymatic steps and yields two pyruvate molecules from one glucose (Glc) molecule by orchestral reaction of enzymes. Uncontrolled glycolysis or abnormally activated glycolysis is easily observed in the metabolism of cancer cells with enhanced levels of glycolytic proteins and enzymatic activities. In the "Warburg effect", tumor cells utilize energy supplied from lactic acid-based fermentative glycolysis operated by glycolysis-specific enzymes of hexokinase (HK), keto-HK-A, Glc-6-phosphate isomerase, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase, phosphofructokinase (PFK), phosphor-Glc isomerase (PGI), fructose-bisphosphate aldolase, phosphoglycerate (PG) kinase (PGK)1, triose phosphate isomerase, PG mutase (PGAM), glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase isozyme type M2 (PKM2), pyruvate dehydrogenase (PDH), PDH kinase and lactate dehydrogenase. They are related to glycolytic flux. The key enzymes involved in glycolysis are directly linked to oncogenesis and drug resistance. Among the metabolic enzymes, PKM2, PGK1, HK, keto-HK-A and nucleoside diphosphate kinase also have protein kinase activities. Because glycolysis-generated energy is not enough, the cancer cell-favored glycolysis to produce low ATP level seems to be non-efficient for cancer growth and self-protection. Thus, the Warburg effect is still an attractive phenomenon to understand the metabolic glycolysis favored in cancer. If the basic properties of the Warburg effect, including genetic mutations and signaling shifts are considered, anti-cancer therapeutic targets can be raised. Specific therapeutics targeting metabolic enzymes in aerobic glycolysis and hypoxic microenvironments have been developed to kill tumor cells. The present review deals with the tumor-specific Warburg effect with the revisited viewpoint of recent progress.


Assuntos
Glicólise , Neoplasias , Hexoquinase/metabolismo , Humanos , Neoplasias/metabolismo , Fosfofrutoquinase-1/metabolismo , Fosfoglicerato Quinase/metabolismo , Fosfoglicerato Mutase/metabolismo , Piruvatos , Microambiente Tumoral
8.
J Org Chem ; 87(16): 10736-10746, 2022 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-35921209

RESUMO

A palladium-catalyzed Heck reaction between 2-oxyacrylates and aryl bromides was developed, where DavePhos was a unique ligand that efficiently promoted the reaction. The products, 2-oxycinnamates, served as excellent precursors, providing synthetically useful monoaryl pyruvates or ortho ester-protected monoaryl pyruvates depending on the nature of the 2-oxy group. The formation of such ortho esters via alkoxide addition is novel, and computational studies identified a plausible mechanism with an oxyallyl zwitterion as the key intermediate.


Assuntos
Brometos , Ésteres , Catálise , Paládio , Piruvatos
9.
Mol Biol Rep ; 49(9): 8241-8250, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35925486

RESUMO

BACKGROUND: The glycolytic enzyme, α-Enolase (ENO1), catalyzes the production of phosphoenolpyruvate from 2-phosphoglycerate, thereby enhancing glycolysis and contributing to tumor progression. In the present study, we aimed to determine the role of ENO1 in skin cutaneous melanoma (SKCM) and the potential underlying mechanism. METHODS: The Sangerbox database was used to analyze the mRNA expression of ENO1 in SKCM. Western blotting was used to assess the levels of ENO1, c-Myc, ß-catenin, MMP-9, PGAM1, and MMP-13 in SKCM-derived cell lines or tumor tissues from patients with SKCM. The pCMV-SPORT6-ENO1 and pET-28a-ENO1siRNA plasmids were used to overexpress and knockdown ENO1 in SKCM cells, respectively. To determine the function of ENO1 in the malignant behavior of SKCM cells, we performed a wound-healing assay, cell counting kit 8 assay, and transwell chamber analyses. The production of pyruvate and lactic acid in tumor cells was evaluated using their respective kits. RESULTS: Compared with non-tumor tissues, ENO1 was found to be overexpressed in SKCM tissues. In SKCM cells, ENO1 overexpression promoted invasion, migration, and proliferation of tumor cells; increased pyruvate and lactate production; and increased ß-catenin, MMP-9, MMP-13, and c-Myc levels. The opposite effects were observed in SKCM cells silenced for ENO1. CONCLUSIONS: These results indicate that ENO1 is involved in SKCM progression by enhancing the invasion and proliferation of tumor cells. In addition, ENO1 might have an important function in tumor cell glycolysis. Therefore, ENO1 represents a potential therapeutic target for treatment of SKCM.


Assuntos
Melanoma , Neoplasias Cutâneas , Apoptose/genética , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 9 da Matriz , Melanoma/genética , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Piruvatos , Neoplasias Cutâneas/genética , beta Catenina/genética
10.
Tissue Cell ; 78: 101892, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35988475

RESUMO

It is well recognized that the neighbor location between cartilage layer and subchondral bone facilitates the intercellular communication and material exchange. However, the evidence that demonstrates the influence of direct communication between cartilage and subchondral bone on their cell behaviors are still partially unknown. In the current study, we established a co-culture system of chondrocytes and osteoblasts aiming to explore the changes of intracellular metabolism of chondrocytes induced by osteoblasts. By using lactate assay kit, RNA sequencing, qRT-PCR and western blot, we found that osteoblasts enhanced the glycolysis in chondrocytes by characterizing the changes of lactate secretion and cytoplasmic expression, and gene expressions including glucose-6-phosphate isomerase 1 (Gpi1), phosphofructokinase, liver type (Pfkl), lactate dehydrogenase A (Ldha), aldolase, fructose-bisphosphate C (Aldoc), phosphoglycerate kinase 1 (Pgk1), glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and triosephosphate isomerase 1 (Tpi1). The enhanced glycolysis might be due to the activation of HIF-1 signaling and its downstream target, pyruvate dehydrogenase kinase1 (PDK1), by qRT-PCR, western blot and immunofluorescence. We also detected the up-regulation of ERK and p38/MAPK upstream signaling in chondrocytes induced by osteoblasts by western blot and immunofluorescence. The enhanced glycolysis in chondrocytes induced by osteoblasts could help us to better understand the intracellular metabolic mechanism of chondrocytes and cartilage disease occurrence.


Assuntos
Condrócitos , Glucose-6-Fosfato Isomerase , Condrócitos/metabolismo , Técnicas de Cocultura , Frutose-Bifosfato Aldolase/metabolismo , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Glicólise , Lactato Desidrogenase 5 , Lactatos/metabolismo , Osteoblastos/metabolismo , Fosfofrutoquinases/metabolismo , Fosfoglicerato Quinase/genética , Fosfoglicerato Quinase/metabolismo , Piruvatos/metabolismo , Triose-Fosfato Isomerase/metabolismo
11.
Anal Methods ; 14(35): 3397-3404, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-35980164

RESUMO

Heterogeneity in metabolite structure and charge state complicates their analysis in electrospray mass spectrometry (ESI-MS). Complications such as diminished signal response and quantitation can be reduced by sequential dual-stage derivatization and capillary RP LC-ESI-MS analysis. Our sequential dual-stage chemical derivatization reacts analyte primary amine and hydroxyl groups with a linear acyl chloride head containing a tertiary amine moiety. Analyte carboxylate groups are then coupled to a linear amine tag with a tertiary amine moiety. This increase in the number of tags on analytes increases analyte proton affinity and hydrophobicity. We derivatized 250 metabolite standards which on average improved signal to noise by >44-fold, with an average limit of detection of 66 nM and R2 of 0.98. This system detected 107 metabolites from 18 BAECs, 111 metabolites from human urine, and 153 from human serum based on retention time, exact mass, and MS/MS matches from a derivatized standard library. As a proof of concept, aortic endothelial cells were treated with epinephrine and analyzed by the dual-stage derivatization. We observed changes in 32 metabolites with many increases related to energy metabolism, specifically in the TCA cycle. A decrease in lactate levels and corresponding increase in pyruvate levels suggest that epinephrine causes a movement away from glycolytic reliance on energy and a shift towards the more efficient TCA respiration for increasing energy.


Assuntos
Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Aminas/química , Aminas/metabolismo , Aminoácidos/metabolismo , Cloretos , Células Endoteliais/química , Células Endoteliais/metabolismo , Epinefrina , Humanos , Lactatos , Prótons , Piruvatos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos
12.
Biosci Trends ; 16(4): 257-266, 2022 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-35965099

RESUMO

The evidence shows that there is an associated relationship between hepatosteatosis and insulin resistance. While some existing genetic induction animal and patient models challenge this relationship, indicating that hepatosteatosis is dissociated from insulin resistance. However, the molecular mechanisms of this dissociation remain poorly understood due to a lack of available, reliable, and simplistic setup models. Currently, we used primary rat hepatocytes (rHPCs), co-cultured with rat hepatic stellate cells (HSC-T6) or human foreskin fibroblast cells (HFF-1) in stimulation with high insulin and glucose, to develop a model of steatosis charactered as dissociated lipid accumulation from insulin resistance. Oil-Red staining significantly showed intracellular lipid accumulated in the developed model. Gene expression of sterol regulatory element-binding protein 1c (SREBP1c) and elongase of very-long-chain fatty acids 6 (ELOVL6), key genes responsible for lipogenesis, were detected and obviously increased in this model. Inversely, the insulin resistance related genes expression included phosphoenolpyruvate carboxykinase 1 (PCK1), pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4), and glucose-6-phosphatase (G6pase) were decreased, suggesting a dissociation relationship between steatosis and insulin resistance in the developed model. As well, the drug metabolism of this developed model was investigated and showed up-regulation of cytochrome P450 3A (CYP3A) and down-regulation of cytochrome P450 2E1 (CYP2E1) and cytochrome P450 1A2 (CYP1A2). Taken together, those results demonstrate that the in vitro model of dissociated steatosis from insulin resistance was successfully created by our co-cultured cells in high insulin and glucose medium, which will be a potential model for investigating the mechanism of insulin resistance dissociated steatosis, and discovering a novel drug for its treatment.


Assuntos
Fígado Gorduroso , Resistência à Insulina , Insulinas , Animais , Técnicas de Cocultura , Citocromo P-450 CYP1A2 , Citocromo P-450 CYP2E1 , Citocromo P-450 CYP3A , Elongases de Ácidos Graxos , Ácidos Graxos , Glucose , Glucose-6-Fosfatase , Humanos , Resistência à Insulina/fisiologia , Isoenzimas , Lipídeos , Fosfoenolpiruvato , Piruvatos , Ratos , Esteróis
13.
Food Funct ; 13(18): 9610-9621, 2022 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-36004536

RESUMO

Maintenance of intestinal metabolic function is important for optimal growth performance in post-weaning pigs. This study aimed to evaluate the effect of pyrroloquinoline quinone (PQQ) on maintaining intestinal glycolipid metabolism in weaned pigs. Seventy-two Duroc × Landrace × Yorkshire crossbred pigs were divided into two groups: pigs fed a basal diet (CTRL group) and pigs fed a basal diet supplemented with 3.0 mg kg-1 PQQ (PQQ group). On d 14, serum was harvested from six pigs per group and the pigs were slaughtered to sample jejunal tissue. Compared with the CTRL group, pigs in the PQQ group had increased average daily gain (P < 0.05), decreased feed : gain (P < 0.05) and tended to have a reduced diarrhea ratio (P = 0.057). Jejunal villus height and villus height/crypt depth ratio were increased, and the crypt depth was decreased in the PQQ group (P < 0.01). The proteomics results showed that PQQ supplementation acted on three metabolic pathways, type I diabetes mellitus, the pancreatic secretion pathway and immune-related signalling. Compared with the CTRL group, PQQ supplementation increased (P < 0.05) serum insulin and jejunal mucosal pyruvate, triglyceride, total cholesterol and low-density lipoprotein cholesterol in the pigs. Jejunal mucosal lactic dehydrogenase and high-density lipoprotein cholesterol levels in the pigs were decreased by PQQ supplementation (P < 0.05). In addition, PQQ supplementation reduced glucose transporter 5 and phosphorylated-AMP-activated protein kinase expression in the jejunal mucosa of the pigs (P < 0.05). In conclusion, dietary supplementation with PQQ improved the growth performance and jejunal morphology and regulated glycolipid metabolism via inhibiting AMPK phosphorylation in weaned pigs.


Assuntos
Insulinas , Jejuno , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Ração Animal/análise , Animais , Colesterol/metabolismo , Dieta/veterinária , Suplementos Nutricionais , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Glicolipídeos/metabolismo , Insulinas/metabolismo , Jejuno/metabolismo , Lipoproteínas HDL , Lipoproteínas LDL/metabolismo , Oxirredutases/metabolismo , Cofator PQQ , Fosforilação , Piruvatos/metabolismo , Suínos , Triglicerídeos/metabolismo , Desmame
14.
Sci Rep ; 12(1): 13965, 2022 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-35978081

RESUMO

High-temperature ethanol fermentation by thermotolerant yeast is considered a promising technology for ethanol production, especially in tropical and subtropical regions. In this study, optimization conditions for high-temperature ethanol fermentation of pineapple waste hydrolysate (PWH) using a newly isolated thermotolerant yeast, Saccharomyces cerevisiae HG1.1, and the expression of genes during ethanol fermentation at 40 °C were carried out. Three independent variables, including cell concentration, pH, and yeast extract, positively affected ethanol production from PWH at 40 °C. The optimum levels of these significant factors evaluated using response surface methodology (RSM) based on central composite design (CCD) were a cell concentration of 8.0 × 107 cells/mL, a pH of 5.5, and a yeast extract concentration of 4.95 g/L, yielding a maximum ethanol concentration of 36.85 g/L and productivity of 3.07 g/L. Gene expression analysis during high-temperature ethanol fermentation using RT-qPCR revealed that the acquisition of thermotolerance ability and ethanol fermentation efficiency of S. cerevisiae HG1.1 are associated with genes responsible for growth and ethanol stress, oxidative stress, acetic acid stress, DNA repair, the pyruvate-to-tricarboxylic acid (TCA) pathway, and the pyruvate-to-ethanol pathway.


Assuntos
Ananas , Termotolerância , Ananas/genética , Ananas/metabolismo , Etanol/metabolismo , Fermentação , Expressão Gênica , Piruvatos/metabolismo , Saccharomyces cerevisiae/metabolismo , Temperatura , Termotolerância/genética
15.
Food Microbiol ; 107: 104085, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35953179

RESUMO

This study sought to І)investigate the effect of varying concentrations of salt on the survival of Gram-negative bacteria in artificial seawater (ASW; pH 6) stored at 4 °C, ІІ)examine the culturable populations of Vibrio vulnificus persisting in ASW with or without 1000 U/mg catalase or 0.1% sodium pyruvate (pH 6) under aerobic and oxygen-restricted environments at 4 °C, and ІІІ)determine whether double layer agar plate (DLAP) methods can improve the culturability of V. vulnificus and Vibrio parahaemolyticus in ASW (pH 6) stored at 4 °C. Once incubated in ASW containing 10% salt at 4 °C, Escherichia coli O157:H7, Salmonella Typhimurium, and Shigella flexneri survived at levels over at least 3.42-4.65 log CFU/mL on day 28, but V. vulnificus underwent the inability to produce colonies within 7 days, while maintaining its membrane integrity. A positive correlation between sodium pyruvate and maintenance of platable colony-forming capability was observed in V. vulnificus incubated aerobically. Using tryptic soy agar containing 3% salt (TSAS) overlayed by thiosulfate-citrate-bile salts-sucrose agar (TCBS), a higher platable colony count of Vibrio spp. was recovered from ASW after 25 or 30 days of storage at 4 °C, followed by TSAS, TSAS overlayed by marine agar (MA), MA, and TCBS.


Assuntos
Vibrio parahaemolyticus , Vibrio vulnificus , Vibrio , Ágar , Contagem de Colônia Microbiana , Nutrientes , Piruvatos , Refrigeração , Sódio , Temperatura
16.
Int J Mol Sci ; 23(15)2022 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-35955764

RESUMO

BACKGROUND: Oocyte vitrification has been widely used in the treatment of infertility and fertility preservation. However, vitrification-induced mitochondrial damage adversely affects oocyte development. Several studies have reported that mitochondrial calcium uptake protein 1 (MICU1) regulates the uptake of mitochondrial calcium by the mitochondrial calcium uniporter (MCU) and subsequently controls aerobic metabolism and oxidative stress in mitochondria, but research considering oocytes remains unreported. We evaluated whether the addition of MICU1 modulators enhances mitochondrial activity, pyruvate metabolism, and developmental competence after warming of MII oocytes. METHODS: Retrieved MII oocytes of mice were classified as vitrified or control groups. After thawing, oocytes of vitrified group were cultured with or without DS16570511 (MICU1 inhibitor) and MCU-i4 (MICU1 activator) for 2 h. RESULTS: Mitochondrial Ca2+ concentration, pyruvate dephosphorylation level, and MICU1 expression of MII oocytes were significantly increased after vitrification. These phenomena were further exacerbated by the addition of MCU-i4 and reversed by the addition of DS16570511 after warming. However, the mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) in vitrified-warmed MII oocytes drop significantly after vitrification, which was improved after MCU-i4 treatment and decreased significantly after DS16570511 treatment. The vitrification process was able to elicit a development competence reduction. After parthenogenetic activation, incubation of the thawed oocytes with MCU-i4 did not alter the cleavage and blastocyst rates. Moreover, incubation of the thawed oocytes with DS16570511 reduced the cleavage and blastocyst rates. CONCLUSIONS: MICU1-mediated increasing mitochondrial calcium uptake after vitrification of the MII oocytes promoted the pyruvate oxidation, and this process may maintain oocyte development competence by compensating for the consumption of ATP under stress state.


Assuntos
Cálcio , Criopreservação , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Potencial da Membrana Mitocondrial , Metáfase , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Oócitos/metabolismo , Piruvatos/metabolismo
17.
J Am Chem Soc ; 144(34): 15786-15792, 2022 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-35976081

RESUMO

Many biosensing methods rely on signals produced by enzyme-catalyzed reactions and efficient methods to detect and record this activity. Herein, we report a wireless lateral flow device and demonstrate the conversion of oxidase reactions to changes in the resonance of radio frequency identification (RFID) circuits. The detection is triggered by polyoxometalate-catalyzed oxidative doping of polypyrrole (pPy) when exposed to oxidase-generated H2O2. We have integrated this transduction and RFID capability into a lateral flow device to create a low-cost, rapid, and portable method for quantitative biological signal detection. We further report a new method for creating functional coatings from pPy core-shell colloidal particles bioconjugated for streptavidin-biotin recognition with glucose oxidase or pyruvate oxidase. The biofunctionalized pPy particles coalesce on the nitrocellulose membrane to produce a chemiresistive band. Glucose or pyruvate solutions result in formation of H2O2 at the pPy bands, functionalized with the respective oxidase, to produce conductivity enhancements exceeding 7·105%. Placing the pPy band in the RFID circuit converts the resistivity response to a change of RF resonance. The enzymatic response of glucose oxidase is recorded within 30 min with as low as 0.6 mM of glucose using this lateral flow device. Pyruvate is also shown to produce large responses. The oxidase enzymes/pPy transduction establishes a resistivity-based platform for the construction of a new family of lateral flow devices capable of detecting and quantifying biological targets.


Assuntos
Técnicas Biossensoriais , Glucose Oxidase , Ânions , Técnicas Biossensoriais/métodos , Enzimas Imobilizadas , Glucose , Peróxido de Hidrogênio , Polieletrólitos , Polímeros , Pirróis , Piruvatos
18.
Appl Microbiol Biotechnol ; 106(17): 5563-5574, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35932295

RESUMO

Amine transaminases (ATA) convert ketones into optically active amines and are used to prepare active pharmaceutical ingredients and building blocks. Novel ATA can be identified in protein databases due to the extensive knowledge of sequence-function relationships. However, predicting thermo- and operational stability from the amino acid sequence is a persisting challenge and a vital step towards identifying efficient ATA biocatalysts for industrial applications. In this study, we performed a database mining and characterized selected putative enzymes of the ß-alanine:pyruvate transaminase cluster (3N5M) - a subfamily with so far only a few described members, whose tetrameric structure was suggested to positively affect operational stability. Four putative transaminases (TA-1: Bilophilia wadsworthia, TA-5: Halomonas elongata, TA-9: Burkholderia cepacia, and TA-10: Burkholderia multivorans) were obtained in a soluble form as tetramers in E. coli. During comparison of these tetrameric with known dimeric transaminases we found that indeed novel ATA with high operational stabilities can be identified in this protein subfamily, but we also found exceptions to the hypothesized correlation that a tetrameric assembly leads to increased stability. The discovered ATA from Burkholderia multivorans features a broad substrate specificity, including isopropylamine acceptance, is highly active (6 U/mg) in the conversion of 1-phenylethylamine with pyruvate and shows a thermostability of up to 70 °C under both, storage and operating conditions. In addition, 50% (v/v) of isopropanol or DMSO can be employed as co-solvents without a destabilizing effect on the enzyme during an incubation time of 16 h at 30 °C. KEY POINTS: • Database mining identified a thermostable amine transaminase in the ß-alanine:pyruvate transaminase subfamily. • The tetrameric transaminase tolerates 50% DMSO and isopropanol under operating conditions at 30 °C. • A tetrameric structure is not necessarily associated with a higher operational stability.


Assuntos
Aminas , Escherichia coli , 2-Propanol , Burkholderia , Dimetil Sulfóxido , Piruvatos , Especificidade por Substrato , Transaminases , beta-Alanina
19.
Appl Environ Microbiol ; 88(17): e0097622, 2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-35980178

RESUMO

As an important metabolic intermediate, 2-ketoisovalerate has significant potential in the pharmaceutical and biofuel industries. However, a low output through microbial fermentation inhibits its industrial application. The microbial production of 2-ketoisovalerate is representative whereby redox imbalance is generated with two molecules of NADH accumulated and an extra NADPH required to produce one 2-ketoisovalerate from glucose. To achieve efficient 2-ketoisovalerate production, metabolic engineering strategies were evaluated in Escherichia coli. After deleting the competing routes, overexpressing the key enzymes for 2-ketoisovalerate production, tuning the supply of NADPH, and recycling the excess NADH through enhancing aerobic respiration, a 2-ketoisovalerate titer and yield of 46.4 g/L and 0.644 mol/mol glucose, respectively, were achieved. To reduce the main by-product of isobutanol, the activity and expression of acetolactate synthase were modified. Additionally, a protein degradation tag was fused to pyruvate dehydrogenase (PDH) to curtail the conversion of pyruvate precursor into acetyl-CoA and the generation of NADH. The resulting strain, 050TY/pCTSDTQ487S-RBS55, was initially incubated under aerobic conditions to attain sufficient cell mass and then transferred to a microaerobic condition to degrade PDH and inhibit the remaining activity of PDH. Intracellular redox imbalance was relieved with titer, productivity and yield of 2-ketoisovalerate improved to 55.8 g/L, 2.14 g/L h and 0.852 mol/mol glucose. These results revealed metabolic engineering strategies for the production of a redox-imbalanced fermentative metabolite with high titer, productivity, and yield. IMPORTANCE An efficient microbial strain was constructed for 2-ketoisovalerate synthesis. The positive effect of the leuA deletion on 2-ketoisovalerate production was found. An optimal combination of overexpressing the target genes was obtained by adjusting the positions of the multiple enzymes on the plasmid frame and the presence of terminators, which could also be useful for the production of downstream products such as isobutanol and l-valine. Reducing the isobutanol by-product by engineering the acetolactate synthase called for special attention to decreasing the promiscuous activity of the enzymes involved. Redox-balancing strategies such as tuning the expression of the chromosomal pyridine nucleotide transhydrogenase, recycling NADH under aerobic cultivation, switching off PDH by degradation, and inhibiting the expression and activity under microaerobic conditions were proven effective for improving 2-ketoisovalerate production. The degradation of PDH and inhibiting this enzyme's expression would serve as a means to generate a wide range of products from pyruvate.


Assuntos
Acetolactato Sintase , Engenharia Metabólica , Acetolactato Sintase/metabolismo , Butanóis , Escherichia coli/metabolismo , Glucose/metabolismo , Hemiterpenos , Cetoácidos , Engenharia Metabólica/métodos , NAD/metabolismo , NADP/metabolismo , Piruvatos/metabolismo
20.
Physiol Rep ; 10(15): e15415, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35924321

RESUMO

Left ventricular diastolic dysfunction is a structural and functional condition that precedes the development of heart failure with preserved ejection fraction (HFpEF). The etiology of diastolic dysfunction includes alterations in fuel substrate metabolism that negatively impact cardiac bioenergetics, and may precipitate the eventual transition to heart failure. To date, the molecular mechanisms that regulate early changes in fuel metabolism leading to diastolic dysfunction remain unclear. In this report, we use a diet-induced obesity model in aged mice to show that inhibitory lysine acetylation of the pyruvate dehydrogenase (PDH) complex promotes energetic deficits that may contribute to the development of diastolic dysfunction in mouse hearts. Cardiomyocyte-specific deletion of the mitochondrial lysine acetylation regulatory protein GCN5L1 prevented hyperacetylation of the PDH complex subunit PDHA1, allowing aged obese mice to continue using pyruvate as a bioenergetic substrate in the heart. Our findings suggest that changes in mitochondrial protein lysine acetylation represent a key metabolic component of diastolic dysfunction that precedes the development of heart failure.


Assuntos
Cardiomiopatias , Insuficiência Cardíaca , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Dieta Hiperlipídica , Lisina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Oxirredução , Complexo Piruvato Desidrogenase/metabolismo , Piruvatos , Volume Sistólico
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