Blueberry malvidin-3-galactoside modulated gut microbial dysbiosis and microbial TCA cycle KEGG pathway disrupted in a liver cancer model induced by HepG2 cells

HCC(Hepatocellular Carcinoma)is a critical health issue worldwide.Our previous animal experiment has confirmed that blueberry malvidin-3-galactoside(M3G)can regulate the progression of HCC.In this study,feces samples from the same batch of mice were collected to explore the regulatory mechanism of M3G on intestinal microbiota and microbial TCA cycle metabolism KEGG pathway in HCC mice based on 16S rRNA sequencing and metagenomics.Our results showed that blueberry M3G increased the microbial diversity and regulated the structure of intestinal microbiota in mice,such as increasing the abundance of Clostridia(butyric acid-producing bacteria),Oscillospira and Ruminococcus,and reducing the abundance of pathogenic Erysipelotrichi.Compared with the group of liver cancer and 5-fluorouracil,blueberry M3G significantly regulated microbial TCA cycle KEGG pathway via improving the expression of key proteins(porA,DLAT,aceE,PC and OGDH).Additionally,we found which the abundance of Muribaculum intestinale increased by blueberry M3G may be an important factor affecting the microbial TCA cycle KEGG pathway via the pearson correlation(R)analysis of protein and microbiota.Taken together,these results demonstrate that the blueberry M3G has the potential to be an intestinal microbiota regulator and an adjuvant to HCC therapy.

Leptin Causes the Early Inhibition of Glycolysis-and TCA Cycle-Related Genes in the Brain of Ob/Ob Mice to Restore Fertility

Introduction: Polycystic ovarian syndrome (PCOS) is undoubtedly the commonest androgen disorder in woman’s fertile period and certainly one of the most prevalent causes of anovulation. The syndrome has an estimated prevalence of 4% - 10% among women of childbearing age. Previously, our group demonstrated the effect of gonadal white adipose tissue transplantation from wild-type lean and fertile female mice to isogenic obese anovulatory ob/ob mice. These complex metabolic interrelationships between obesity and PCOS have yet to be fully understood. The aim of this study was to evaluate the effect of gonadal white adipose tissue (WAT) transplantation from the wild-type lean and fertile female mice to isogenic obese, anovulatory mice (Lep ob/Lep ob) on the expression of glycolysis- and TCA cycle-related genes and obtain a general view of the glucose metabolism in the brain of these animals. Methods: Fifteen ob/ob mice ranging from 2 to 3 months of age were divided into 3 experimental groups: control normal weight (n = 5), obese control (n = 5) and obese 7 days leptin treated (n = 5). The whole brains of the mice were processed for RNA extraction. The samples from each group were used to perform PCR assays using an array plate containing 84 primers to study the glucose metabolism-related genes. Results: The glycolysis- and TCA cycle-related genes were significantly downregulated. The most significantly affected genes were as follows: for glycolysis (fold regulation with p < 0.05):Pgm1,Bpgm,Aldob, andEno3 (119, 45, 18, and 28 times less, respectively);and for the TCA cycle (fold regulation with p < 0.05):Cs,Idh3b, andMdh2 (84, 27, and 37 times less, respectively).Conclusion: The seven-day leptin treated mice show a decrease in the glucose metabolism. These results confirm the ability of the adipose tissue-derived hormone leptin to regulate early crucial genes that are related to glycolysis mechanisms and to the TCA cycle. This hormone seems to revert early the central physiological conditions that are associated with PCOS;however, the morphological alterations can only be observed within a 45-day treatment.

Two mitochondrial phosphatases,PP2c63 and Sal2,are required for posttranslational regulation of the TCA cycle in Arabidopsis

Protein phosphorylation is a well-established post-translational mechanism that regulates protein functions and metabolic pathways.It is known that several plant mitochondrial proteins are phosphorylated in a reversible manner.However,the identities of the protein kinases/phosphatases involved in this mech-anism and their roles in the regulation of the tricarboxylic acid(TCA)cycle remain unclear.In this study,we isolated and characterized plants lacking two mitochondrially targeted phosphatases(Sal2 and PP2c63)along with pyruvate dehydrogenase kinase(PDK),Protein-protein interaction analysis,quantitative phos-phoproteomics,and enzymatic analyses revealed that PDK specifically regulates pyruvate dehydrogenase complex(PDC),while PP2c63 nonspecifically regulates PDC.When recombinant PP2c63 and Sal2 proteins were added to mitochondria isolated from mutant plants,protein-protein interaction and enzymatic analyses showed that PP2c63 directly phosphorylates and modulates the activity of PDC,while Sal2 only indirectly affects TCA cycle enzymes.Characterization of steady-state metabolite levels and fluxes in the mutant lines further revealed that these phosphatases regulate flux through the TCA cycle,and that altered metabolism in the sa/2 pp2c63 double mutant compromises plant growth.These results are discussed in the context of current models of the control of respiration in plants.

The emerging role and targetability of the TCA cycle in cancer metabolism

The tricarboxylic acid （TCA） cycle is a central route for oxidative phosphorylation in cells, and fulfills their bioenergetic, biosynthetic, and redox balance require- ments. Despite early dogma that cancer cells bypass the TCA cycle and primarily utilize aerobic glycolysis, emerging evidence demonstrates that certain cancer cells, especially those with deregulated oncogene and tumor suppressor expression, rely heavily on the TCA cycle for energy production and macromolecule synthesis. As the field progresses, the importance of aberrant TCA cycle function in tumorigenesis and the potentials of applying small molecule inhibitors to perturb the enhanced cycle function for cancer treatment start to evolve. In this review, we summarize current knowledge about the fuels feeding the cycle, effects of oncogenes and tumor suppressors on fuel and cycle usage, common genetic alterations and deregulation of cycle enzymes, and potential therapeutic opportunities for targeting the TCA cycle in cancer cells. With the application of advanced technology and in vivo model organism studies, it is our hope that studies of this previously overlooked biochemical hub will provide fresh insights into cancer metabolism and tumorigenesis, subsequently revealing vulnerabilities for thera- peutic interventions in various cancer types.

Fatty acid feedstocks enable a highly efficient glyoxylate-TCA cycle for high-yield production of β-alanine

Metabolic engineering to produce tricarboxylic acid(TCA)cycle-derived chemicals is usually associated with problems of low production yield and impaired cellular metabolism.In this work,we found that fatty acid(FA)feedstocks could enable high-yield production of TCA cycle-derived chemicals,while maintaining an efficient and balanced metabolic flux of the glyoxylate-TCA cycle,which is favorable for both product synthesis and cell growth.Here,we designed a novel synthetic pathway for production of β-alanine,an important TCA cycle-derived product,from FAs with a high theortecial yield of 1.391 g/g.By introducing panD,improving aspA,and knocking out iclR,glyoxylate shunt was highly activated in FAs and the yield of β-alanine reached 0.71 g/g from FAs,much higher than from glucose.Blocking the TCA cycle at icd/sucA/fumAC nodes could increase β-alanine yield in a flask cultivation,but severely reduced cell growth and FA utilization during fed-batch processes.Replenishing oxaloacetate by knocking out aspC and recovering fumAC could restore the growth and lead to a titer of 35.57 g/l.After relieving the oxidative stress caused by FA metabolism,β-alanine production could reach 72.05 g/l with a maximum yield of 1.24 g/g,about 86% of the theoretical yield.Our study thus provides a promising strategy for the production of TCA cycle-derived chemicals.

Dihydroartemisinin inhibits plasmid transfer in drug-resistant Escherichia coli via limiting energy supply

Conjugative transfer of antibiotic resistance genes(ARGs)by plasmids is an important route for ARG dissemination.An increasing number of antibiotic and nonantibiotic compounds have been reported to aid the spread of ARGs,highlighting potential challenges for controlling this type of horizontal transfer.Development of conjugation inhibitors that block or delay the transfer of ARG-bearing plasmids is a promising strategy to control the propagation of antibiotic resistance.Although such inhibitors are rare,they typically exhibit relatively high toxicity and low efficacy in vivo and their mechanisms of action are inadequately understood.Here,we studied the effects of dihydroartemisinin(DHA),an artemisinin derivative used to treat malaria,on conjugation.DHA inhibited the conjugation of the IncI2 and IncX4 plasmids carrying the mobile colistin resistance gene(mcr-1)by more than 160-fold in vitro in Escherichia coli,and more than two-fold(IncI2 plasmid)in vivo in a mouse model.It also suppressed the transfer of the IncX3 plasmid carrying the carbapenem resistance gene bla_(NDM-5)by more than twofold in vitro.Detection of intracellular adenosine triphosphate(ATP)and proton motive force(PMF),in combination with transcriptomic and metabolomic analyses,revealed that DHA impaired the function of the electron transport chain(ETC)by inhibiting the tricarboxylic acid(TCA)cycle pathway,thereby disrupting PMF and limiting the availability of intracellular ATP for plasmid conjugative transfer.Furthermore,expression levels of genes related to conjugation and pilus generation were significantly down-regulated during DHA exposure,indicating that the transfer apparatus for conjugation may be inhibited.Our findings provide new insights into the control of antibiotic resistance and the potential use of DHA.

Identification of Potential Therapeutic Targets of Alzheimer's Disease By Weighted Gene Co-Expression Network Analysis

Objective Alzheimer's disease(AD)is the most common cause of dementia.The pathophysiology of the disease mostly remains unearthed,thereby challenging drug development for AD.This study aims to screen high throughput gene expression data using weighted co-expression network analysis(WGCNA)to explore the potential therapeutic targets.Methods The dataset of GSE36980 was obtained from the Gene Expression Omnibus(GEO)database.Normalization,quality control,filtration,and soft-threshold calculation were carried out before clustering the co-expressed genes into different modules.Furthermore,the correlation coefiidents between the modules and clinical traits were computed to identify the key modules.Gene ontology and pathway enrichment analyses were performed on the key module genes.The STRING database was used to construct the protein-protein interaction(PPI)networks,which were further analyzed by Cytoscape app(MCODE).Finally,validation of hub genes was conducted by external GEO datasets of GSE 1297 and GSE 28146.Results Co-expressed genes were clustered into 27 modules,among which 6 modules were identified as the key module relating to AD occurrence.These key modules are primarily involved in chemical synaptic transmission(G0:0007268),the tricarboxylic acid(TCA)cycle and respiratory electron transport(R-HSA-1428517).WDR47,OXCT1,C3orfl4,ATP6V1A,SLC25A14,NAPB were found as the hub genes and their expression were validated by external datasets.Conclusions Through modules co-expression network analyses and PPI network analyses,we identified the hub genes of AD,including WDR47,0XCT1,C3orfl4i ATP6V1A,SLC25A14 and NAPB.Among them,three hub genes(ATP6V1A,SLC25A14,OXCT1)might contribute to AD pathogenesis through pathway of TCA cycle.

The LncRNA FEZF1-AS1 promotes tumor proliferation in colon cancer by regulating the mitochondrial protein PCK2

LncRNAs and metabolism represents two factors involved in cancer initiation and progression.However,the interaction between lncRNAs and metabolism remains to be fully explored.In this study,lncRNA FEZF1-AS1(FEZF1-AS1)was found upregulated in colon cancer after screening all the lncRNAs of colon cancer tissues deposited in TCGA,the result of which was further confirmed by RNAscope staining on a colon tissue chip.The results obtained using FEZF1-AS1 knockout colon cancer cells(SW480 KO and HCT-116 KO)constructed using CRISPR/Cas9 system confirmed the proliferation,invasion,and migration-promoting function of FEZF1-AS1 in vitro.Mechanistically,FEZF1-AS1 associated with the mitochondrial protein phosphoenolpyruvate carboxykinase(PCK2),which plays an essential role in regulating energy metabolism in the mitochondria.Knockdown of FEZF1-AS1 greatly decreased PCK2 protein levels,broke the homeostasis of energy metabolism in the mitochondria,and inhibited proliferation,invasion,and migration of SW480 and HCT-116 cells.PCK2 overexpression in FEZF1-AS1 knockout cells partially rescued the tumor inhibitory effect on colon cancer cells both in vitro and in vivo.Moreover,PCK2 overexpression specifically rescued the abnormal accumulation of Flavin mononucleotide(FMN)and succinate,both of which play an important role in oxidative phosphorylation(OXPHOS).Overall,these results indicate that FEZF1-AS1 is an oncogene through regulating energy metabolism of the cell.This research reveals a new mechanism for lncRNAs to regulate colon cancer and provides a potential target for colon cancer diagnosis and treatment.

The conditional mitochondrial protein complexome in the Arabidopsis thaliana root and shoot

Protein complexes are important for almost all biological processes.Hence,to fully understand how cells work,it is also necessary to characterize protein complexes and their dynamics in response to various cellular cues.Moreover,the dynamics of protein interaction play crucial roles in regulating the(dis)association of protein complexes and,in turn,regulating biological processes such as metabolism.Here,mitochondrial protein complexes were investigated by blue native PAGE and size-exclusion chromatography under conditions of oxidative stress in order to monitor their dynamic(dis)associations.Rearrangements of enzyme interactions and changes in protein complex abundance were observed in response to oxidative stress induced by menadione treatment.These included changes in enzymatic protein complexes involving g-amino butyric acid transaminase(GABA-T),D-ornithine aminotransferase(D-OAT),or proline dehydrogenase 1(POX1)that are expected to affect proline metabolism.Menadione treatment also affected interactions between several enzymes of the tricarboxylic acid(TCA)cycle and the abundance of complexes of the oxidative phosphorylation pathway.In addition,we compared the mitochondrial complexes of roots and shoots.Considerable differences between the two tissues were observed in the mitochondrial import/export apparatus,the formation of super-complexes in the oxidative phosphorylation pathway,and specific interactions between enzymes of the TCA cycle that we postulate may be related to the metabolic/energetic requirements of roots and shoots.

Chromium-containing traditional Chinese medicine, Tianmai Xiaoke Tablet improves blood glucose through activating insulin-signaling pathway and inhibiting PTP1B and PCK2 in diabetic rats

OBJECTIVE： Chromium is an essential mineral that is thought to be necessary for normal glucose homeostasis. Numerous studies give evidence that chromium picolinate can modulate blood glucose and insulin resistance. The main ingredient of-13anmai Xiaoke （TMXK） Tablet is chromium picolinate. In China, TMXK Tablet is used to treat type 2 diabetes. This study investigated the effect of TMXK on glucose metabolism in diabetic rats to explore possible underlying molecular mechanisms for its action. METHODS： Diabetes was induced in rats by feeding a high-fat diet and subcutaneously injection with a single dose of streptozotocin （50 mg/kg, tail vein）. One week after streptozotocin-injection, model rats were divided into diabetic group, low dose of TMXK group and high dose of TMXK group. Eight normal rats were used as normal control. After 8 weeks of treatment, skeletal muscle was obtained and was analyzed using Roche NimbleGen mRNA array and quantitative polymerase chain reaction （qPCR）. Fasting blood glucose, oral glucose tolerance test and homeostasis model assessment of insulin resistance （HOMA-IR） index were also measured. RESULTS： The authors found that the administration of TMXK Tablet can reduce the fasting blood glucose and fasting insulin level and HOMA-IR index. The authors also found that 2 223 genes from skeletal muscle of the high-dose TMXK group had significant changes in expression （1 752 increased, 471 decreased）. Based on Kyoto encyclopedia of genes and genomes pathway analysis, the most three significant pathways were ＂insulin signaling pathway＂, ＂glycolysis/ gluconeogenesis＂ and ＂citrate cycle （-ICA）＂. qPCR showed that relative levels of forkhead box 03 （Fox03）, phosphoenolpyruvate carboxykinase 2 （Pck2）, and protein tyrosine phosphatase 1B （Ptplb） were significantly decreased in the high-dose TMXK group, while v-akt murine thymoma viral oncogene homolog 1 （Aktl） and insulin receptor substrate 2 （Its2） were increased. CONCLUSION： Our data show that TMXK Tablet reduces fasting glucose level and improves insulin resistance in diabetic rats. The mechanism may be linked to the inactivation of PTP1B and PCK enzymes, or through intracellular pathways, such as the insulin signaling pathway.

Unraveling the Acidithiobacillus caldus complete genome and its central metabolisms for carbon assimilation

Acidithiobacillus caldus is one of the dominant sulfur-oxidizing bacteria in bioleaching reactors. It plays the essential role in maintaining the high acidity and oxidation of reduced inorganic sulfur compounds during bioleaching process. In this report, the complete genome sequence of A. caldus SM-1 is presented. The genome is composed of one chromosome （2,932,225 bp） and four plasmids （pLAtcl, pLAtc2, pLAtc3, pLAtcm） and it is rich in repetitive sequences （accounting for 11% of the total genome）, which are often associated with transposable genetic elements. In particular, twelve copies of ISAtfe and thirty-seven copies of ISAtcl have been identified, suggesting that they are active transposons in the genome. A. caldus SM-1 encodes all enzymes for the central metabolism and the assimilation of carbon compounds, among which 29 proteins/enzymes were identifiable with proteomic tools. The SM-1 fixes CO2 via the classical Calvin-Bassham--Benson （CBB） cycle, and can operate complete Embden-Meyerhof pathway （EMP）, pentose phosphate pathway （PPP）, and gluconeogenesis. It has an incomplete tricarboxylic acid cycle （TCA）. Four putative transporters involved in carbohydrate uptake were identified. Taken together, the results suggested that SM-1 was able to assimilate carbohydrates and this was subsequently confirmed experimentally because addition of 1% glucose or sucrose in basic salt medium significantly increased the growth of SM-1. It was concluded that the complete genome of SM-1 provided fundamental data for further investigation of its physiology and genetics, in addition to the carbon metabolism revealed in this study.

Redox Regulation of Arabidopsis Mitochondrial Citrate Synthase

Citrate synthase has a key role in the tricarboxylic （TCA） cycle of mitochondria of all organisms, as it cata- lyzes the first committed step which is the fusion of a carbon-carbon bond between oxaloacetate and acetyl CoA. The regulation of TCA cycle function is especially important in plants, since mitochondrial activities have to be coordinated with photosynthesis. The posttranslational regulation of TCA cycle activity in plants is thus far almost entirely unexplored. Although several TCA cycle enzymes have been identified as thioredoxin targets in vitro, the existence of any thioredoxin-dependent regulation as known for the Calvin cycle, yet remains to be demonstrated. Here we have investigated the redox regulation of the Arabidopsis citrate synthase enzyme by site-directed mutagenesis of its six cysteine residues. Our results indicate that oxidation inhibits the enzyme activity by the formation of mixed disulfides, as the partially oxidized citrate synthase enzyme forms large redox-dependent aggregates. Furthermore, we were able to demonstrate that thioredoxin can cleave diverse intraas well as intermolecular disulfide bridges, which strongly enhances the activity of the enzyme. Activity measurements with the cysteine variants of the enzyme revealed important cysteine residues affecting total enzyme activity as well as the redox sensitivity of the enzyme.

Genome-wide identification and characterization of long non-coding RNAs in Tribolium castaneum

Long non-coding RNAs(lncRNAs)are poorly understood in insects.In this study,we performed genome-wide analysis of lncRNAs in Tribolium castaneum by RNA-seq.In total,4516 lncRNA transcripts corresponding to 3917 genes were identified from late embryos,early larvae,late larvae,early pupae,late pupae and early adults of T.castaneum,including 3152 novel lncRNAs and 1364 known lncRNAs.These lncRNAs have few exons and transcripts,and are short in length.During development,they exhibited nine different expression patterns.Functionally,they can act either by targeting messenger RNAs(1813 lncRNAs)and lncRNAs(45 lncRNAs)or as micro RNA(miRNA)precursors(46 lncRNAs).LncRNAs were observed to target the metabolic enzymes of glycolysis,TCA cycle and amino acids,demonstrating that lncRNAs control metabolism by regulating metabolic enzymes.Moreover,lncRNAs were shown to participate in cell differentiation and development via their targets.As miRNA precursors,lncRNAs could participate in the ecdysone signaling pathway.This study provides comprehensive information for lncRNAs of T.castaneum,and will promote functional analysis and target identification of lncRNAs in the insect.

Cyanobacterial photo-driven mixotrophic metabolism and its advantages for biosynthesis

Cyanobacterium offers a promising chassis for phototrophic production of renewable chemicals. Although engineered cyanobacteria can achieve similar product carbon yields as heterotrophic microbial hosts, their production rate and titer under photoautotrophic conditions are 10 to 100 folds lower than those in fast growing E. colt. Cyanobacterial factories face three indomitable bottlenecks. First, photosynthesis has limited ATP and NADPH generation rates. Second, CO2 fixation by ribulose-l,5-bisphosphate carboxylase/oxygenase （RuBisCO） has poor efficiency. Third, CO2 mass transfer and light supply are deficient within large photobioreac- tors. On the other hand, cyanobacteria may employ organic substrates to promote phototrophic cell growth, Nz fixation, and metabolite synthesis. The photo-fermenta- tions show enhanced photosynthesis, while CO2 loss from organic substrate degradation can be reused by the Calvin cycle. In addition, the plasticity of cyanobacterial path- ways （e.g., oxidative pentose phosphate pathway and the TCA cycle） has been recently revealed to facilitate the catabolism. The use of cyanobacteria as ＂green E. colt＂ could be a promising route to develop robust photobiorefineries.

Thiamine deficiency perturbed energy metabolism enzymes in brain mitochondrial fraction of Swiss mice

BACKGROUND： Thiamine is an essential cofactor associated with several enzymes in energy metabolism and its deficiency may lead to neurological deficits. Current research evaluated the biochemical and molecular changes in TCA cycle enzymes using the mitochondrial fraction of the brain following thiamine deficiency （TD） in mice. METHODS： The investigation was carried out on Swiss mice （6-8 week old） allocated into three groups. First group was control; second and third group were made thiamine deficient for 8 and 10 days. RESULTS： Current study showed that alpha-ketoglutarate dehydrogenase （KGDHC） （thiamine-dependent enzyme） level found to be significantly reduced in experimental groups as compared to control group. In comparison to control group, a significant decrease in the succinate dehydrogenase （SDH） activity was calculated in group II and group III （p 〈 0.0001） mice. Diminished enzymatic activity of fumarase and MDH enzyme in thiamine deficient groups exposed for 8 and 10 days was calculated as compared to control group. The expression analysis of different genes governing TCA cycle enzymes in different experimental groups showed that there was a negotiable change in the expression of fumarase and DLD （dihydrolipoyl dehydrogenase- E3 subunit of KGDHC） whereas a declined in the expression of SDH and two subunits of KGDHC i.e. OGDH （2-oxoglutarate dehydrogenase- E1 subunit of KGDHC） and DLST （dihydrolipoyllysine-residue succinyltransferase- E2 subunit of KGDHC） was observed as compared to control group. CONCLUSIONS： Hence, current findings strongly entail that TD promotes alteration in energy metabolism in brain mitochondria which will decline the neuronal progression which may lead to neurodegenerative diseases such as Alzheimer＇s diseases.