Functional characterisation of Artemisia annua jasmonic acid carboxyl methyltransferase: a key enzyme enhancing artemisinin biosynthesis.

MAIN CONCLUSION: Overexpression of Artemisia annua jasmonic acid carboxyl methyltransferase (AaJMT) leads to enhanced artemisinin content in Artemisia annua. Artemisinin-based combination therapies remain the sole deterrent against deadly disease malaria and Artemisia annua remains the only natural producer of artemisinin. In this study, the 1101 bp gene S-adenosyl-L-methionine (SAM): Artemisia annua jasmonic acid carboxyl methyltransferase (AaJMT), was characterised from A. annua, which converts jasmonic acid (JA) to methyl jasmonate (MeJA). From phylogenetic analysis, we confirmed that AaJMT shares a common ancestor with Arabidopsis thaliana, Eutrema japonica and has a close homology with JMT of Camellia sinensis. Further, the Clustal Omega depicted that the conserved motif I, motif III and motif SSSS (serine) required to bind SAM and JA, respectively, are present in AaJMT. The relative expression of AaJMT was induced by wounding, MeJA and salicylic acid (SA) treatments. Additionally, we found that the recombinant AaJMT protein catalyses the synthesis of MeJA from JA with a Km value of 37.16 µM. Moreover, site-directed mutagenesis of serine-151 in motif SSSS to tyrosine, asparagine-10 to threonine and glutamine-25 to histidine abolished the enzyme activity of AaJMT, thus indicating their determining role in JA substrate binding. The GC-MS analysis validated that mutant proteins of AaJMT were unable to convert JA into MeJA. Finally, the artemisinin biosynthetic and trichome developmental genes were upregulated in AaJMT overexpression transgenic lines, which in turn increased the artemisinin content.

Insights into the relative contribution of four precursors to 3-sulfanylhexan-1-ol and 3-sulfanylhexylacetate biogenesis during fermentation.

The desirable wine aroma compounds 3-sulfanylhexan-1-ol (3SH) and 3-sulfanylhexyl acetate (3SHA) are released during fermentation from non-volatile precursors present in the grapes. This work explores the relative contribution of four precursors (E-2-hexenal, 3-S-glutathionylhexan-1-ol, 3-S-glutathionylhexanal, and 3-S-cysteinylhexan-1-ol) to 3SH and 3SHA. Through the use of isotopically labelled analogues of these precursors in defined fermentation media, new insights into the role of each precursor have been identified. E-2-Hexenal was shown to contribute negligible amounts of thiols, while 3-S-glutathionylhexan-1-ol was the main precursor of both 3SH and 3SHA. The glutathionylated precursors were both converted to 3SHA more efficiently than 3-S-cysteinylhexan-1-ol. Interestingly, 3-S-glutathionylhexanal generated 3SHA without detectable concentrations of 3SH, suggesting possible differences in the way this precursor is metabolised compared to 3-S-glutathionylhexan-1-ol and 3-S-cysteinylhexan-1-ol. We also provide the first evidence for chemical conversion of 3-S-glutathionylhexan-1-ol to 3-S-(γ-glutamylcysteinyl)-hexan-1-ol in an oenological system.

Multi-modular metabolic engineering and efflux engineering for enhanced lycopene production in recombinant Saccharomyces cerevisiae.

Lycopene has been widely used in the food industry and medical field due to its antioxidant, anti-cancer, and anti-inflammatory properties. However, achieving efficient manufacture of lycopene using chassis cells on an industrial scale remains a major challenge. Herein, we attempted to integrate multiple metabolic engineering strategies to establish an efficient and balanced lycopene biosynthetic system in Saccharomyces cerevisiae. First, the lycopene synthesis pathway was modularized to sequentially enhance the metabolic flux of the mevalonate pathway, the acetyl-CoA supply module, and lycopene exogenous enzymatic module. The modular operation enabled the efficient conversion of acetyl-CoA to downstream pathway of lycopene synthesis, resulting in a 3.1-fold increase of lycopene yield. Second, we introduced acetate as an exogenous carbon source and utilized an acetate-repressible promoter to replace the natural ERG9 promoter. This approach not only enhanced the supply of acetyl-CoA but also concurrently diminished the flux toward the competitive ergosterol pathway. As a result, a further 42.3% increase in lycopene production was observed. Third, we optimized NADPH supply and mitigated cytotoxicity by overexpressing ABC transporters to promote lycopene efflux. The obtained strain YLY-PDR11 showed a 12.7-fold increase in extracellular lycopene level compared to the control strain. Finally, the total lycopene yield reached 343.7 mg/L, which was 4.3 times higher than that of the initial strain YLY-04. Our results demonstrate that combining multi-modular metabolic engineering with efflux engineering is an effective approach to improve the production of lycopene. This strategy can also be applied to the overproduction of other desirable isoprenoid compounds with similar synthesis and storage patterns in S. cerevisiae. ONE-SENTENCE SUMMARY: In this research, lycopene production in yeast was markedly enhanced by integrating a multi-modular approach, acetate signaling-based down-regulation of competitive pathways, and an efflux optimization strategy.

Cancer-associated fibroblast-derived acetate promotes pancreatic cancer development by altering polyamine metabolism via the ACSS2-SP1-SAT1 axis.

The ability of tumour cells to thrive in harsh microenvironments depends on the utilization of nutrients available in the milieu. Here we show that pancreatic cancer-associated fibroblasts (CAFs) regulate tumour cell metabolism through the secretion of acetate, which can be blocked by silencing ATP citrate lyase (ACLY) in CAFs. We further show that acetyl-CoA synthetase short-chain family member 2 (ACSS2) channels the exogenous acetate to regulate the dynamic cancer epigenome and transcriptome, thereby facilitating cancer cell survival in an acidic microenvironment. Comparative H3K27ac ChIP-seq and RNA-seq analyses revealed alterations in polyamine homeostasis through regulation of SAT1 gene expression and enrichment of the SP1-responsive signature. We identified acetate/ACSS2-mediated acetylation of SP1 at the lysine 19 residue that increased SP1 protein stability and transcriptional activity. Genetic or pharmacologic inhibition of the ACSS2-SP1-SAT1 axis diminished the tumour burden in mouse models. These results reveal that the metabolic flexibility imparted by the stroma-derived acetate enabled cancer cell survival under acidosis via the ACSS2-SP1-SAT1 axis.

Gut microbiota regulates hepatic ischemia-reperfusion injury-induced cognitive dysfunction via the HDAC2-ACSS2 axis in mice.

AIMS: Hepatic ischemia-reperfusion injury (HIRI) resulting from hepatic inflow occlusion, which is a common procedure in liver surgery is inevitable. Previous research has confirmed that the cognitive dysfunction induced by HIRI is closely related to dysbiosis of the gut microbiota. This research aims to investigate the mechanisms underlying this complication. METHODS: C57BL/6 mice underwent hepatic ischemia experimentally through the occlusion of the left hepatic artery and portal vein. To assess the HDAC2-ACSS2 axis, gut microbiota transplantation. Enzyme-linked immunosorbent assay and LC/MS short-chain fatty acid detection were utilized. RESULTS: The findings indicated a notable decline in ACSS2 expression in the hippocampus of mice experiencing hepatic ischemia-reperfusion injury, emphasizing the compromised acetate metabolism in this particular area. Furthermore, the cognitive impairment phenotype and the dysregulation of the HDAC2-ACSS2 axis could also be transmitted to germ-free mice via fecal microbial transplantation. Enzyme-linked immunosorbent assay revealed reduced Acetyl-coenzyme A (acetyl-CoA) and Acetylated lysine levels in the hippocampus. CONCLUSION: These findings suggest that acetate metabolism is impaired in the hippocampus of HIRI-induced cognitive impairment mice and related to dysbiosis, leading to compromised histone acetylation.

FFAR2 expressing myeloid-derived suppressor cells drive cancer immunoevasion.

BACKGROUND: Emerging evidences suggest that aberrant metabolites contributes to the immunosuppressive microenvironment that leads to cancer immune evasion. Among tumor immunosuppressive cells, myeloid-derived suppressor cells (MDSCs) are pathologically activated and extremely immunosuppressive, which are closely associated with poor clinical outcomes of cancer patients. However, the correlation between MDSCs mediated immunosuppression and particular cancer metabolism remained elusive. METHODS: Spontaneous lung adenocarcinoma and subcutaneous mouse tumor models, gas chromatography-mass spectrometry (GC-MS) and immunofluorescence assay of patient-derived lung adenocarcinoma tissues, and flow cytometry, RNA sequencing and Western blotting of immune cells, were utilized. RESULTS: Metabolite profiling revealed a significant accumulation of acetic acids in tumor tissues from both patients and mouse model, which contribute to immune suppression and cancer progression significantly through free fatty acid receptor 2 (FFAR2). Furthermore, FFAR2 is highly expressed in the myeloid-derived suppressor cells (MDSCs) from the tumor of lung adenocarcinoma (LUAD) patients which is greatly associated with poor prognosis. Surprisingly, whole or myeloid Ffar2 gene deletion markedly inhibited urethane-induced lung carcinogenesis and syngeneic tumor growth with reduced MDSCs and increased CD8+ T cell infiltration. Mechanistically, FFAR2 deficiency in MDSCs significantly reduced the expression of Arg1 through Gαq/Calcium/PPAR-γ axis, which eliminated T cell dysfunction through relieving L-Arginine consumption in tumor microenvironment. Therefore, replenishment of L-Arginine or inhibition to PPAR-γ restored acetic acids/FFAR2 mediated suppression to T cells significantly. Finally, FFAR2 inhibition overcame resistance to immune checkpoint blockade through enhancing the recruitment and cytotoxicity of tumor-infiltrating T cells. CONCLUSION: Altogether, our results demonstrate that the acetic acids/FFAR2 axis enhances MDSCs mediated immunosuppression through Gαq/calcium/PPAR-γ/Arg1 signaling pathway, thus contributing to cancer progression. Therefore, FFAR2 may serve as a potential new target to eliminate pathologically activated MDSCs and reverse immunosuppressive tumor microenvironment, which has great potential in improving clinical outcomes of cancer immunotherapy.

CD4+ T-Cell Legumain Deficiency Attenuates Hypertensive Damage via Preservation of TRAF6.

BACKGROUND: T cells are central to the immune responses contributing to hypertension. LGMN (legumain) is highly expressed in T cells; however, its role in the pathogenesis of hypertension remains unclear. METHODS: Peripheral blood samples were collected from patients with hypertension, and cluster of differentiation (CD)4+ T cells were sorted for gene expression and Western blotting analysis. TLGMNKO (T cell-specific LGMN-knockout) mice (Lgmnf/f/CD4Cre), regulatory T cell (Treg)-specific LGMN-knockout mice (Lgmnf/f/Foxp3YFP Cre), and RR-11a (LGMN inhibitor)-treated C57BL/6 mice were infused with Ang II (angiotensin II) or deoxycorticosterone acetate/salt to establish hypertensive animal models. Flow cytometry, 4-dimensional label-free proteomics, coimmunoprecipitation, Treg suppression, and in vivo Treg depletion or adoptive transfer were used to delineate the functional importance of T-cell LGMN in hypertension development. RESULTS: LGMN mRNA expression was increased in CD4+ T cells isolated from hypertensive patients and mice, was positively correlated with both systolic and diastolic blood pressure, and was negatively correlated with serum IL (interleukin)-10 levels. TLGMNKO mice exhibited reduced Ang II-induced or deoxycorticosterone acetate/salt-induced hypertension and target organ damage relative to wild-type (WT) mice. Genetic and pharmacological inhibition of LGMN blocked Ang II-induced or deoxycorticosterone acetate/salt-induced immunoinhibitory Treg reduction in the kidneys and blood. Anti-CD25 antibody depletion of Tregs abolished the protective effects against Ang II-induced hypertension in TLGMNKO mice, and LGMN deletion in Tregs prevented Ang II-induced hypertension in mice. Mechanistically, endogenous LGMN impaired Treg differentiation and function by directly interacting with and facilitating the degradation of TRAF6 (tumor necrosis factor receptor-associated factor 6) via chaperone-mediated autophagy, thereby inhibiting NF-κB (nuclear factor kappa B) activation. Adoptive transfer of LGMN-deficient Tregs reversed Ang II-induced hypertension, whereas depletion of TRAF6 in LGMN-deficient Tregs blocked the protective effects. CONCLUSIONS: LGMN deficiency in T cells prevents hypertension and its complications by promoting Treg differentiation and function. Specifically targeting LGMN in Tregs may be an innovative approach for hypertension treatment.

Potential inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced inflammation, hyperproliferation, and hyperplasiogenic responses by celecoxib in mouse skin.

PURPOSE: Skin exposure to noxious agents leads to cutaneous lesion marked by an increase in inflammation, cellular proliferation, and hyperplasiogenic reactions. Studies have demonstrated that these damages breach the skin integrity resulting in the aetiology of various cutaneous disorders like atopic dermatitis, eczema, psoriasis, and development of non-melanoma skin cancer. Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, is an effective treatment for a variety of inflammatory diseases. Its importance in the therapy of skin problems, however, remains under appreciated. METHODS: We tested efficacy of topically applied celecoxib in mitigating skin inflammation, cellular proliferation, and hyperplasia induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in Swiss albino mice. RESULTS: Celecoxib (5 and 10 µmol) markedly reduced TPA (10 nmol) induced prostaglandin E2 (PGE2) production, oedema formation, myeloperoxidase (MPO) activity, and levels of pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and interleukin-6 (IL-6). It also resulted in a considerable decrease in ornithine decarboxylase (ODC) activity and the incorporation of [3H]-thymidine into DNA. In addition, there was a significant reduction in histoarchitectural abnormalities such as epidermal thickness, number of epidermal cell layers, neutrophil infiltration, intercellular oedema, and vasodilation. CONCLUSION: Our results demonstrate that topical celecoxib can reduce the inflammation, hyperproliferation, and hyperplasiogenic events of skin insults suggesting that it may prove to be a valuable management option for cutaneous lesion and associated illnesses such as atopic dermatitis, eczema, and psoriasis, as well as the emergence of non-melanoma cancer.

Dietary Potassium Supplementation Reduces Chronic Kidney Lesions Independent of Blood Pressure in Deoxycorticosterone-Acetate and High Sodium Chloride-Treated Mice.

We have previously shown that an excess of deoxycorticosterone acetate and high sodium chloride intake (DOCA/salt) in one-renin gene mice induces a high urinary Na/K ratio, hypokalemia, and cardiac and renal hypertrophy in the absence of hypertension. Dietary potassium supplementation prevents DOCA/salt-induced pathological processes. In the present study, we further study whether DOCA/salt-treated mice progressively develop chronic inflammation and fibrosis in the kidney and whether dietary potassium supplementation can reduce the DOCA/salt-induced renal pathological process. Results showed that (1) long-term DOCA/salt-treated one-renin gene mice developed severe kidney injuries including tubular/vascular hypertrophy, mesangial/interstitial/perivascular fibrosis, inflammation (lymphocyte's immigration), proteinuria, and high serum creatinine in the absence of hypertension; (2) there were over-expressed mRNAs of plasminogen activator inhibitor-1 (PAI-1), fibronectin, collagen type I and III, interferon-inducible protein-10 (IP-10), monocyte chemotactic protein-1 (MCP1), transforming growth factor-ß (TGF-ß), tumor necrosis factor-alpha (TNF-α), osteopontin, Nuclear factor kappa B (NF-κB)/P65, and intercellular adhesion molecule (ICAM)-1; and (3) dietary potassium supplementation normalized urinary Na/K ratio, hypokalemia, proteinuria, and serum creatinine, reduced renal hypertrophy, inflammations, and fibrosis, and down-regulated mRNA expression of fibronectin, Col-I and III, TGF-ß, TNF-α, osteopontin, and ICAM without changes in the blood pressure. The results provide new evidence that potassium and sodium may modulate proinflammatory and fibrotic genes, leading to chronic renal lesions independent of blood pressure.

Roseomonas populi sp. nov., an acetate-degrading bacteria isolated from the stem of Populus tomentosa.

Strain CN29T, isolated from the stem of 5- to 6-year-old Populus tomentosa in Shandong, China, was characterized using a polyphasic taxonomic approach. Cells of CN29T were Gram-stain negative, aerobic, nonspore-forming, and nonmotile coccoid. Growth occurred at 20-37 °C, pH 4.0-9.0 (optimum, pH 6.0), and with 0-1% NaCl (optimum, 1%). Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain CN29T was closely related to members of the genus Roseomonas and closest to Roseomonas pecuniae N75T (96.6%). This classification was further supported by phylogenetic analysis using additional core genes. The average nucleotide identity and digital DNA‒DNA hybridization values between strain CN29T and Roseomonas populi CN29T were 82.7% and 27.8%, respectively. The genome size of strain CN29T was 5.87 Mb, with a G + C content of 70.9%. The major cellular fatty acids included summed feature 8 (C18:1 ω7c/C18:1 ω6c), C19:0 cyclo ω8c and C16:0. The major respiratory quinone was Q-10. The polar lipids were phosphatidylcholine, aminolipid, phosphatidylglycerol, and diphosphatidylglycerol. Strain CN29T can utilize acetate as a carbon source for growth and metabolism. Additionally, it contains acid phosphatase (2-naphthyl phosphate), which catalyzes the hydrolysis of phosphoric monoesters. The CN29T strain contains several genes, including maeB, gdhB, and cysJ, involved in carbon, nitrogen, and sulfur cycling. These findings suggest that the strain may actively participate in ecosystem cycling, leading to soil improvement and promoting the growth of poplar trees. Based on the phylogenetic, phenotypic, and genotypic characteristics, strain CN29T is concluded to represent a novel species of the genus Roseomonas, for which the name Roseomonas populi sp. nov. is proposed. The type strain is CN29T (= JCM 35579T = GDMCC 1.3267T).

Candidatus Accumulibacter use fermentation products for enhanced biological phosphorus removal.

Previous research suggested that two major groups of polyphosphate-accumulating organisms (PAOs), i.e., Ca. Accumulibacter and Tetrasphaera, play cooperative roles in enhanced biological phosphorus removal (EBPR). The fermentation of complex organic compounds by Tetrasphaera provides carbon sources for Ca. Accumulibacter. However, the viability of the fermentation products (e.g., lactate, succinate, alanine) as carbon sources for Ca. Accumulibacter and their potential effects on the metabolism of Ca. Accumulibacter were largely unknown. This work for the first time investigated the capability and metabolic details of Ca. Accumulibacter cognatus clade IIC strain SCUT-2 (enriched in a lab-scale reactor with a relative abundance of 42.8%) in using these fermentation products for EBPR. The enrichment culture was able to assimilate lactate and succinate with the anaerobic P release to carbon uptake ratios of 0.28 and 0.36 P mol/C mol, respectively. In the co-presence of acetate, the uptake of lactate was strongly inhibited, since two substrates shared the same transporter as suggested by the carbon uptake bioenergetic analysis. When acetate and succinate were fed at the same time, Ca. Accumulibacter assimilated two carbon sources simultaneously. Proton motive force (PMF) was the key driving force (up to 90%) for the uptake of lactate and succinate by Ca. Accumulibacter. Apart from the efflux of proton in symport with phosphate via the inorganic phosphate transport system, translocation of proton via the activity of fumarate reductase contributed to the generation of PMF, which agreed with the fact that PHV was a major component of PHA when lactate and succinate were used as carbon sources, involving the succinate-propionate pathway. Metabolic models for the usage of lactate and succinate by Ca. Accumulibacter for EBPR were built based on the combined physiological, biochemical, metagenomic, and metatranscriptomic analyses. Alanine was shown as an invalid carbon source for Ca. Accumulibacter. Instead, it significantly and adversely affected Ca. Accumulibacter-mediated EBPR. Phosphate release was observed without alanine uptake. Significant inhibitions on the aerobic phosphate uptake was also evident. Overall, this study suggested that there might not be a simply synergic relationship between Ca. Accumulibacter and Tetrasphaera. Their interactions would largely be determined by the kind of fermentation products released by the latter.

Acetate acts as a metabolic immunomodulator by bolstering T-cell effector function and potentiating antitumor immunity in breast cancer.

Acetate metabolism is an important metabolic pathway in many cancers and is controlled by acetyl-CoA synthetase 2 (ACSS2), an enzyme that catalyzes the conversion of acetate to acetyl-CoA. While the metabolic role of ACSS2 in cancer is well described, the consequences of blocking tumor acetate metabolism on the tumor microenvironment and antitumor immunity are unknown. We demonstrate that blocking ACSS2, switches cancer cells from acetate consumers to producers of acetate thereby freeing acetate for tumor-infiltrating lymphocytes to use as a fuel source. We show that acetate supplementation metabolically bolsters T-cell effector functions and proliferation. Targeting ACSS2 with CRISPR-Cas9 guides or a small-molecule inhibitor promotes an antitumor immune response and enhances the efficacy of chemotherapy in preclinical breast cancer models. We propose a paradigm for targeting acetate metabolism in cancer in which inhibition of ACSS2 dually acts to impair tumor cell metabolism and potentiate antitumor immunity.

C-atrial natriuretic peptide (ANP)4-23 attenuates renal fibrosis in deoxycorticosterone-acetate-salt hypertensive mice.

Epithelial-mesenchymal transition (EMT) plays a critical role in hypertension-induced renal fibrosis, a final pathway that leads to end-stage renal failure. C-Atrial natriuretic peptide (ANP)4-23, a specific agonist of natriuretic peptide receptor-C (NPR-C), has been reported to have protective effects against hypertension. However, the role of C-ANP4-23 in hypertension-associated renal fibrosis has not yet been elucidated. In this study, mice were randomly divided into SHAM group, DOCA-salt group and DOCA-salt + C-ANP4-23 group. Renal morphology changes, renal function and fibrosis were detected. Human proximal tubular epithelial cells (HK2) stimulated by aldosterone were used for cell function and mechanism study. The DOCA-salt treated mice exhibited hypertension, kidney fibrosis and renal dysfunction, which were attenuated by C-ANP4-23. Moreover, C-ANP4-23 inhibited DOCA-salt treatment-induced renal EMT as evidenced by decrease of the mesenchymal marker alpha-smooth muscle actin (ACTA2) and vimentin and increase of epithelial cell marker E-cadherin. In HK2 cells, aldosterone induced EMT response, which was also suppressed by C-ANP4-23. The key transcription factors (twist, snail, slug and ZEB1) involved in EMT were increased in the kidney of DOCA-salt-treated mice, which were also suppressed by C-ANP4-23. Mechanistically, C-ANP4-23 inhibited the aldosterone-induced translocation of MR from cytosol to nucleus without change of MR expression. Furthermore, C-ANP4-23 rescued the enhanced expression of NADPH oxidase (NOX) 4 and oxidative stress after aldosterone stimulation. Aldosterone-induced Akt and Erk1/2 activation was also suppressed by C-ANP4-23. Our data suggest that C-ANP4-23 attenuates renal fibrosis, likely through inhibition of MR activation, enhanced oxidative stress and Akt and Erk1/2 signaling pathway.

GPR120 Gene expression and activity in subcutaneous and intramuscular adipose tissues of Angus crossbred steers.

We hypothesized that media long-chain fatty acids (LCFA) would more greatly depress cyclic adenosine monophosphate (cAMP), glycerol, and free fatty acid (FFA) concentrations in subcutaneous (s.c.) adipose tissue than in intramuscular (i.m.) adipose tissue via G protein-coupled receptor 120 (GPR120). The GPR120 receptor binds to LCFA, which reduces cAMP production, thereby causing a depression in lipolysis. Fresh ex vivo explants of s.c. and i.m. adipose tissue from the fifth to eighth longissimus thoracic rib muscle section of 8, 22-mo-old Angus crossbred steers were transferred immediately to 6-well culture plates containing 3 mL of Krebs-Henseleit buffer/Hepes/5 mM glucose. Samples were preincubated with 0.5 mM theophylline plus 10 µM forskolin for 30 min, after which increasing concentrations of acetate or propionate (volatile fatty acids, VFA) (0, 1, 5, and 10 mM) in the absence or presence of 100 µM oleate (18:1n-9) or 100 µM palmitate (16:0) (LCFA) were added to the incubation media and incubated an additional 30 min. Main effects of adipose tissue depot (i.m. vs. s.c) and VFA (acetate vs. propionate) for adipose tissue concentrations of forskolin-stimulated cAMP were P = 0.747 and P = 0.106, respectively. The addition of LCFA to the media depressed adipose tissue concentrations of cAMP (P = 0.006) (LCFA main effects). The Tissue × VFA × LCFA interaction was not significant for any dependent variable (P ≥ 0.872). Therefore, concentrations of cAMP, glycerol, and FFA were analyzed separately for i.m. and s.c. adipose tissue by split-plot analysis. Concentrations of cAMP, glycerol, or FFA in i.m. and s.c. adipose tissue were not affected by increasing concentrations of VFA (P ≥ 0.497). Media LCFA had no effect on i.m. adipose tissue cAMP (P = 0.570) or glycerol (P = 0.470) but depressed i.m. adipose tissue FFA (P < 0.001). In s.c. adipose tissue, LCFA decreased concentrations of cAMP (P = 0.042) and glycerol (P = 0.038), but increased FFA concentration (P = 0.026). Expression of GPR120 (P = 0.804) and stearoyl-CoA desaturase (P = 0.538) was not different between s.c. adipose tissue and i.m. adipose tissue. The binding of VFA to the GPR43 receptor depresses cAMP production, thereby attenuating lipolysis, but GPR43 mRNA was undetectable in those adipose tissue samples. These results provide evidence for functional GPR120 receptors in s.c. adipose tissue but question the role of GPR43 in the accumulation of adipose tissue lipids in growing steers.

We measured the mRNA abundance and activity of the fatty acid receptor, G protein-coupled receptor 120 (GPR120) in bovine subcutaneous and intramuscular (marbling) adipose tissue. The GPR120 receptor binds to long-chain fatty acids, which reduces cyclic adenosine monophosphate (cAMP) production, thereby decreasing lipolysis. The mRNA amount of GPR120 was similar between subcutaneous and intramuscular adipose tissues. In subcutaneous and intramuscular adipose tissue incubated in vitro, the fatty acids oleic acid and palmitic acid (the most abundant fatty acids in bovine adipose tissue) strongly depressed the production of cAMP and glycerol in subcutaneous adipose tissue and decreased the concentration of free fatty acids in intramuscular adipose tissue (all measured with commercial kits). This indicates that elevations in adipose tissue or plasma fatty acids may promote fat accumulation by decreasing the breakdown of stored lipids via GPR120. The volatile fatty acids acetate and propionate, which bind to G protein-coupled receptor 43 (GPR43) had no effect on cAMP, glycerol, or free fatty acids. This questions the role of GPR43 in the accumulation of adipose tissue lipids in growing steers.

H2 generated by fermentation in the human gut microbiome influences metabolism and competitive fitness of gut butyrate producers.

BACKGROUND: Hydrogen gas (H2) is a common product of carbohydrate fermentation in the human gut microbiome and its accumulation can modulate fermentation. Concentrations of colonic H2 vary between individuals, raising the possibility that H2 concentration may be an important factor differentiating individual microbiomes and their metabolites. Butyrate-producing bacteria (butyrogens) in the human gut usually produce some combination of butyrate, lactate, formate, acetate, and H2 in branched fermentation pathways to manage reducing power generated during the oxidation of glucose to acetate and carbon dioxide. We predicted that a high concentration of intestinal H2 would favor the production of butyrate, lactate, and formate by the butyrogens at the expense of acetate, H2, and CO2. Regulation of butyrate production in the human gut is of particular interest due to its role as a mediator of colonic health through anti-inflammatory and anti-carcinogenic properties. RESULTS: For butyrogens that contained a hydrogenase, growth under a high H2 atmosphere or in the presence of the hydrogenase inhibitor CO stimulated production of organic fermentation products that accommodate reducing power generated during glycolysis, specifically butyrate, lactate, and formate. Also as expected, production of fermentation products in cultures of Faecalibacterium prausnitzii strain A2-165, which does not contain a hydrogenase, was unaffected by H2 or CO. In a synthetic gut microbial community, addition of the H2-consuming human gut methanogen Methanobrevibacter smithii decreased butyrate production alongside H2 concentration. Consistent with this observation, M. smithii metabolic activity in a large human cohort was associated with decreased fecal butyrate, but only during consumption of a resistant starch dietary supplement, suggesting the effect may be most prominent when H2 production in the gut is especially high. Addition of M. smithii to the synthetic communities also facilitated the growth of E. rectale, resulting in decreased relative competitive fitness of F. prausnitzii. CONCLUSIONS: H2 is a regulator of fermentation in the human gut microbiome. In particular, high H2 concentration stimulates production of the anti-inflammatory metabolite butyrate. By consuming H2, gut methanogenesis can decrease butyrate production. These shifts in butyrate production may also impact the competitive fitness of butyrate producers in the gut microbiome. Video Abstract.

Combining elicitor treatment of chitosan, methyl jasmonate, and cyclodextrin to induce the generation of immune response bioactive peptides in peanut hairy root culture.

The endogenous peptides from peanut hairy root culture were induced upon elicitor treatment with chitosan (CHT), methyl jasmonate (MeJA), and cyclodextrin (CD): CHT+MeJA+CD. The peptides secreted into the liquid culture medium play an important role in plant signaling and stress responses. By performing gene ontology (GO) analysis, a number of plant proteins involved in biotic and abiotic defense responses were identified, such as endochitinase, defensin, antifungal protein, cationic peroxidase and Bowman-Birk type protease inhibitor A-II. The bioactivity of 14 peptides synthesized from secretome analysis was determined. Peptide BBP1-4, derived from the diverse region of Bowman-Birk type protease inhibitor, displayed high antioxidant activity and mimicked the property of chitinase and ß-1,3-glucanase enzymes. The antimicrobial activity against S. aureus, S. typhimurium, and E. coli was evidenced with different peptide concentrations. Additionally, peptide BBP1-4 has the potential to be a useful candidate for an immune response property, as it was found to increase the expression of some pathogenesis-related (PR) proteins and stilbene biosynthesis genes in peanut hairy root tissues. The findings indicate that secreted peptides may play a role in plant responses to both abiotic and biotic stresses. These peptides, which possess bioactive properties, could be considered as potential candidates for use in the pharmaceutical, agricultural, and food industries.

The adaptive response to alternative carbon sources in the pathogen Candida albicans involves a remodeling of thiol- and glutathione-dependent redox status.

Candida albicans is an opportunist pathogen responsible for a large spectrum of infections, from superficial mycosis to systemic diseases known as candidiasis. During infection in vivo, Candida albicans must adapt to host microenvironments and this adaptive response is crucial for the survival of this organism, as it facilitates the effective assimilation of alternative carbon sources others than glucose. We performed a global proteomic analysis on the global changes in protein abundance in response to changes in micronutrient levels, and, in parallel, explored changes in the intracellular redox and metabolic status of the cells. We show here that each of the carbon sources considered - glucose, acetate and lactate - induces a unique pattern of response in C. albicans cells, and that some conditions trigger an original and specific adaptive response involving the adaptation of metabolic pathways, but also a complete remodeling of thiol-dependent antioxidant defenses. Protein S-thiolation and the overproduction of reduced glutathione are two components of the response to high glucose concentration. In the presence of acetate, glutathione-dependent oxidative stress occurs, reduced thiol groups bind to proteins, and glutathione is exported out of the cells, these changes probably being triggered by an increase in glutathione-S-transferases. Overall, our results suggest that the role of cellular redox status regulation and defenses against oxidative stress, including the thiol- and glutathione-dependent response, in the adaptive response of C. albicans to alternative carbon sources should be reconsidered.

Myocardial External Efficiency in Asymptomatic Severe Primary Mitral Regurgitation Using 11C-Acetate PET.

Subjects with asymptomatic moderate-to-severe or severe primary mitral regurgitation are closely observed for signs of progression or symptoms requiring surgical intervention. The role of myocardial metabolic function in progression of mitral regurgitation is poorly understood. We used 11C-acetate PET to noninvasively measure myocardial mechanical external efficiency (MEE), which is the energetic ratio of external cardiac work and left ventricular (LV) oxygen consumption. Methods: Forty-seven patients in surveillance with mitral regurgitation and no or minimal symptoms prospectively underwent PET, echocardiography, and cardiac MRI on the same day. PET was used to simultaneously measure cardiac output, LV mass, and oxygen consumption to establish MEE. PET findings were compared between patients and healthy volunteers (n = 9). MEE and standard imaging indicators of regurgitation severity, LV volumes, and function were studied as predictors of time to surgical intervention. Patients were followed a median of 3.0 y (interquartile range, 2.0-3.8 y), and the endpoint was reached in 22 subjects (47%). Results: MEE in patients reaching the endpoint (23.8% ± 5.0%) was lower than in censored patients (28.5% ± 4.5%, P = 0.002) or healthy volunteers (30.1% ± 4.9%, P = 0.001). MEE with a cutoff lower than 25.7% was significantly associated with the outcome (hazard ratio, 7.5; 95% CI, 2.7-20.6; P < 0.0001) and retained independent significance when compared with standard imaging parameters. Conclusion: MEE independently predicted time to progression requiring valve surgery in patients with asymptomatic moderate-to-severe or severe primary mitral regurgitation. The study suggests that inefficient myocardial oxidative metabolism precedes clinically observed progression in mitral regurgitation.

Putative metabolism of Ca. Accumulibacter via the utilization of glucose.

Ca. Accumulibacter was the predominant microorganism (relative FISH bio-abundance of 67 ± 5%) in a lab-scale sequential batch reactor that accomplished enhanced biological phosphorus removal (EBPR) while using glucose and acetate as the carbon sources (1:1 COD-based ratio). Both organic compounds were completely anaerobically consumed. The reactor's performance in terms of P/C ratio, phosphorous release and uptake, and overall kinetic and stoichiometric parameters were on the high end of the reported spectrum for EBPR systems (100:9.3 net mg phosphate removal per mg COD consumed when using glucose and acetate in a 1:1 ratio). The batch tests showed that, to the best of our knowledge, this is the first time a reactor enriched with Ca. Accumulibacter can putatively utilize glucose as the sole carbon source to biologically remove phosphate (COD:P (mg/mg) removal ratio of 100:6.3 when using only glucose). Thus, this research proposes that Ca. Accumulibacter directly anaerobically stored the fed glucose primarily as glycogen by utilizing the ATP provided via the hydrolysis of poly-P and secondarily as PHA by balancing its ATP utilization (glycogen generation) and formation (PHA storage). Alternative hypotheses are also discussed. The reported findings could challenge the conventional theories of glucose assimilation by Ca. Accumulibacter, and can be of significance for the biological removal of phosphorus from wastewaters with high contents of fermentable compounds or low VFAs.

Acetate circumvents impaired metabolic switch in skeletal muscle of letrozole-induced PCOS rat model by suppression of PDK4/NLRP3.

OBJECTIVES: Endocrine disorders in women of childbearing age, including polycystic ovarian syndrome (PCOS), have been linked to skeletal muscle insulin resistance with multiple post-receptor intracellular defects, disrupting metabolic flexibility. Short-chain fatty acids, such as acetate have been suggested as a metabolic modulator. However, the effects of acetate on aberrant metabolic switch in skeletal muscle of individuals with PCOS are unknown. This study therefore hypothesized that acetate would circumvent impaired metabolic switch in the skeletal muscle of a letrozole-induced PCOS rat model, probably by suppression of PDK4/NLRP3. METHODS: Eight-wk-old female Wistar rats were assigned into three groups (n = 6), which received vehicle, letrozole (1 mg/kg), and letrozole plus acetate (200 mg/kg), respectively. The administrations were done by oral gavage for 21 d. . RESULTS: Animals with PCOS had insulin resistance, increased testosterone, and leptin, as well as decreased adiponectin level. Additionally, the skeletal muscle was also characterized with increased lipid deposition, malondialdehyde, inflammatory mediators (nuclear factor-κB and tumor necrosis factor-α), lactate dehydrogenase, lactate/pyruvate ratio, HDAC and PDK 4 with corresponding decrease in glycogen synthesis, glutathione and NrF2. Besides, immunohistochemical evaluation showed severe expression of inflammasome and apoptosis in PCOS animals. Nonetheless, supplementation with acetate significantly attenuated these perturbations. CONCLUSIONS: The present results demonstrate aberrant metabolic switch in the skeletal muscle of PCOS animals, which is accompanied by excessive inflammation, oxidative stress and elevated levels of histone deacetylase and PDK4. The results suggested that histone deacetylase inhibitor, acetate circumvents impaired metabolic switch in the skeletal muscle of PCOS rats by suppression of PDK4/NLRP3 inflammasome.