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1.
DNA Cell Biol ; 39(3): 389-397, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31905020

RESUMO

Acetate and ß-hydroxybutyrate (BHBA) are the predominant substrates for de novo fatty acid (FA) synthesis in mammary gland of dairy cow. To investigate the nutrigenomic role of acetate and BHBA in bovine mammary epithelial cells during milk fat production, RNA sequencing (RNA-seq) transcriptomic analysis was used to identify differentially expressed genes (DEGs) between acetate- and BHBA-treated cells (high-milk fat cells) and control cells. A total of 625 DEGs (358 upregulated and 267 downregulated) were identified between the high-milk fat cells and control cells. Gene ontology enrichment analysis revealed that the upregulated genes in high-milk fat cells were mainly involved in lipid biosynthetic process, steroid biosynthetic process, oxidation-reduction process, receptor binding, and vesicle and small molecule biosynthetic process. The downregulated genes were mainly associated with immune response, cytokine production, negative regulation of biological process, and peptidyl-threonine modification. Pathway analysis indicated that FA metabolism and steroid biosynthesis were significantly enriched for the upregulated genes in the high-milk fat cells, while apoptosis was enriched for the downregulated genes. This work provides a profile of gene expression changes that occur during acetate- and BHBA-induced milk fat synthesis in bovine mammary epithelial cells, which furthers our understanding of the molecular regulation of lipid metabolism.


Assuntos
Ácido 3-Hidroxibutírico/metabolismo , Acetatos/metabolismo , Bovinos/genética , Células Epiteliais/metabolismo , Glândulas Mamárias Animais/metabolismo , Leite/metabolismo , Transcriptoma , Animais , Células Cultivadas , Ácidos Graxos/metabolismo , Feminino , Perfilação da Expressão Gênica , Glândulas Mamárias Animais/citologia , Nutrigenômica
2.
Prep Biochem Biotechnol ; 50(1): 74-81, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31517565

RESUMO

Ectoine has fostered the development of products for skin care and cosmetics. In this study, we employed the marine bacterial strain Marinococcus sp. MAR2 to increase ectoine production by optimizing medium constituents using Response Surface Methodology (RSM) and a fed-batch strategy. The results from the steepest ascent and central composite design indicated that 54 g/L of yeast extract, 14.0 g/L of ammonium acetate, 74.4 g/L of sodium glutamate, and 6.2 g/L of sodium citrate constituted the optimal medium with maximum ectoine production (3.5 g/L). In addition, we performed fed-batch culture in the bioreactor, combining pH and dissolved oxygen to produce ectoine by Marinococcus sp. MAR2. The ectoine production, content, and productivity of 5.6 g/L, 10%, and 3.9 g/L/day were further reached by a fed-batch culture. Thus, the ectoine production by Marinococcus sp. MAR2 using RSM and fed-batch strategy shows its potential for industrial production.


Assuntos
Diamino Aminoácidos/metabolismo , Bacillaceae/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Microbiologia Industrial/métodos , Acetatos/análise , Acetatos/metabolismo , Bacillaceae/crescimento & desenvolvimento , Técnicas de Cultura Celular por Lotes/instrumentação , Reatores Biológicos , Meios de Cultura/química , Meios de Cultura/metabolismo , Desenho de Equipamento , Fermentação , Microbiologia Industrial/instrumentação , Citrato de Sódio/análise , Citrato de Sódio/metabolismo , Glutamato de Sódio/análise , Glutamato de Sódio/metabolismo
3.
Chemphyschem ; 21(4): 295-306, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-31840917

RESUMO

The Kemp elimination reaction is the most widely used in the de novo design of new enzymes. The effect of two different kinds of electric fields in the reactions of acetate as a base with benzisoxazole and 5-nitrobenzisoxazole as substrates have been theoretically studied. The effect of the solvent reaction field has been calculated using the SMD continuum model for several solvents; we have shown that solvents inhibit both reactions, the decrease of the reaction rate being larger as far as the dielectric constant is increased. The diminution of the reaction rate is especially remarkable between aprotic organic solvents and protic solvents as water, the electrostatic term of the hydrogen bonds being the main factor for the large inhibitory effect of water. The presence of an external electric field oriented in the direction of the charge transfer (z axis) increases it and, so, the reaction rate. In the reaction of the nitro compound, if the electric field is oriented in an orthogonal direction (x axis) the charge transfer to the NO2 group is favored and there is a subsequent increase of the reaction rate. However, this increase is smaller than the one produced by the field in the z axis. It is worthwhile mentioning that one of the main effects of external electric fields of intermediate intensity is the reorientation of the reactants. Finally, the implications of our results in the de novo design of enzymes are discussed.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Hidroliases/metabolismo , Isomerases/metabolismo , Acetatos/química , Acetatos/metabolismo , Biocatálise , Sistema Enzimático do Citocromo P-450/química , Eletricidade , Hidroliases/química , Isomerases/química , Isoxazóis/química , Isoxazóis/metabolismo , Estrutura Molecular
4.
J Photochem Photobiol B ; 199: 111625, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31610430

RESUMO

The cultivated grapevine V. vinifera is a rich source of stilbene compounds such as resveratrol, which are widely believed to provide dietary protection against the development of cardiovascular disease and some forms of cancer. Elicitation is a well-known strategy to increase commercial production of natural products in plant cell suspension culture systems. Callus tissues obtained from berry slices of V. vinifera cv. Shahani grown on an optimized medium were used to develop cell suspension cultures used to study the effects of elicitation on stilbene synthesis. The effect of two light regimes (135.1 µmol. s-1 m-2 radiation, and dark), the concentration of phenylalanine (Phe; 0, 0.1, 0.5 and 1 mM) and of methyl jasmonate elicitor (MeJA; 0 and 25 µM), alone or in combination, were tested. The results showed that cultures grown in darkness resulted in significantly higher levels of the accumulation of total stilbenes (resveratrol + piceid) compared with the high light condition. The combined treatments of dark +1 mM Phe and dark +25 µM MeJA induced the synthesis of high levels of total phenolics, total flavonoids and total stilbenes. Finally, the combined elicitation of dark +1 mM Phe + 25 µM MeJA gave the highest synergistic coefficient (1.24) and proved to be the most effective treatment for the production of total phenolics, total flavonoids, and total stilbenes with mean contents of 384.80 mg GA/g DW, 527.62 mg catechin/g DW and 188.34 µg/g DW, respectively. The results of our study suggest that the combinations of dark together with MeJA and/or Phe can be used as an efficient method for the future scale-up of V. vinifera cell cultures for the production of high value stilbene compounds in a bioreactor system.


Assuntos
Acetatos/metabolismo , Técnicas de Cultura de Células/métodos , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Fenilalanina/metabolismo , Metabolismo Secundário/efeitos dos fármacos , Vitis/citologia , Vias Biossintéticas , Catequina/metabolismo , Linhagem Celular , Condutividade Elétrica , Flavonoides/metabolismo , Glucosídeos/metabolismo , Concentração de Íons de Hidrogênio , Luz , Fenóis/metabolismo , Resveratrol/metabolismo , Estilbenos/metabolismo , Suspensões/metabolismo
5.
Phys Chem Chem Phys ; 21(41): 22849-22856, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31612167

RESUMO

Hyperpolarized metabolites are very attractive contrast agents for in vivo magnetic resonance imaging studies enabling early diagnosis of cancer, for example. Real-time production of concentrated solutions of metabolites is a desired goal that will enable new applications such as the continuous investigation of metabolic changes. To this end, we are introducing two NMR experiments that allow us to deliver high levels of polarization at high concentrations (50 mM) of an acetate precursor (55% 13C polarization) and acetate (17% 13C polarization) utilizing 83% para-state enriched hydrogen within seconds at high magnetic field (7 T). Furthermore, we have translated these experiments to a portable low-field spectrometer with a permanent magnet operating at 1 T. The presented developments pave the way for a rapid and affordable production of hyperpolarized metabolites that can be implemented in e.g. metabolomics labs and for medical diagnosis.


Assuntos
Técnicas de Química Analítica/métodos , Meios de Contraste/síntese química , Campos Magnéticos , Acetatos/química , Acetatos/metabolismo , Meios de Contraste/química , Hidrogênio/química , Imagem por Ressonância Magnética/instrumentação
6.
J Agric Food Chem ; 67(43): 12002-12012, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31595746

RESUMO

In order to achieve rapid xylose utilization in the presence of acetate, improved yeast strains were engineered for higher bioethanol production. First, a six-gene cluster, including XYL1/XYL2/XKS1/TAL1/PYK1/MGT05196, was generated by using an in-depth two-stage (glucose and xylose) transcription reprogramming strategy in an evolutionary adapted strain of CE7, resulting in two improved engineered strains WXY46 and WXY53. Through a combined screening of xylose and glucose stage-specific promoters between tricarboxylic acid (TCA)/HSP and constitutive types, respectively, WXY46 with the constitutive promoters showed a much higher ethanol yield than that of WXY53 with the TCA/HSP promoters. Second, an optimized strain WXY74 was obtained by using more copies of a six-gene cluster, which resulted in a higher ethanol yield of 0.500 g/g total sugars with acetate conditions. At last, simultaneous saccharification and co-fermentation were performed by using the evolved WXY74 strain, which produced 58.4 g/L of ethanol from wheat straw waste and outperformed previous haploid XR-XDH strains.


Assuntos
Acetatos/metabolismo , Etanol/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Xilose/metabolismo , Fermentação , Engenharia Metabólica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Genética
7.
J Anim Sci ; 97(11): 4668-4681, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31603200

RESUMO

Two sets of in vitro rumen fermentation experiments were conducted to determine effects of diets that included wet distiller's grains plus solubles (WDGS) and tannin-rich peanut skin (PS) on the in vitro digestibility, greenhouse gas (GHG) and other gas emissions, fermentation rate, and microbial changes. The objectives were to assess associative effects of various levels of PS or WDGS on the in vitro digestibility, GHG and other gas emissions, fermentation rate, and microbial changes in the rumen. All gases were collected using an ANKOM Gas Production system for methane (CH4), carbon dioxide (CO2), nitrous oxide (N2O), and hydrogen sulfide (H2S) analyses. Cumulative ruminal gas production was determined using 250 mL ANKOM sampling bottles containing 50 mL of ruminal fluid (pH 5.8), 40 mL of artificial saliva (pH 6.8), and 6 g of mixed diets after a maximum of 24 h of incubation. Fermenters were flushed with CO2 gas and held at 39 °C in a shaking incubator for 24 h. Triplicate quantitative real-time polymerase chain reaction (qPCR) analyses were conducted to determine microbial diversity. When WDGS was supplied in the diet, in the absence of PS, cumulative CH4 production increased (P < 0.05) with 40% WDGS. In the presence of PS, production of CH4 was reduced but the reduction was less at 40% WDGS. In the presence of PS, ruminal lactate, succinate, and acetate/propionate (A/P) ratio tended to be less with a WDGS interaction (P < 0.01). In the presence of PS and with 40% WDGS, average populations of Bacteroidetes, total methanogens, Methanobrevibacter sp. AbM4, and total protozoa were less. The population of total methanogens (R2 = 0.57; P < 0.01), Firmicutes (R2 = 0.46: P < 0.05), and Firmicutes/Bacteroidetes (F/B) ratio (R2 = 0.46; P < 0.03) were strongly correlated with ruminal CH4 production. Therefore, there was an associative effect of tannin-rich PS and WDGS, which suppressed methanogenesis both directly and indirectly by modifying populations of ruminal methanogens.


Assuntos
Arachis/química , Bovinos/fisiologia , Suplementos Nutricionais/análise , Metano/metabolismo , Methanobrevibacter/isolamento & purificação , Taninos/metabolismo , Acetatos/metabolismo , Ração Animal/análise , Animais , Dióxido de Carbono/metabolismo , Bovinos/microbiologia , Dieta/veterinária , Digestão/efeitos dos fármacos , Fermentação , Gases/metabolismo , Gases de Efeito Estufa/metabolismo , Masculino , Propionatos/metabolismo , Rúmen/metabolismo , Rúmen/microbiologia
8.
Environ Monit Assess ; 191(10): 628, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31502086

RESUMO

The present study was done to assess the dissipation behavior, decontamination, and half-life time of ready-mix formulation of trifloxystrobin (25% w/w) and tebuconazole (50% w/w) in okra and soil under the crop after foliar spray at fruiting stage. Samples of okra and soil were collected periodically, i.e., zero (2 h after spray), 1, 3, 5, 7, 10, 15, 20, and 25 days after third application at a 7-day interval. Residues of these fungicides were determined by gas liquid chromatography (GLC) equipped with electron capture detector (ECD) and gas chromatography-tandem mass spectrometry (GCMS-triple quadruple). The limits of quantification (LOQ) and detection (LOD) for both the fungicides were 0.01 and 0.003 mg kg-1, respectively. Washing alone with faucet water was found successful in minimizing the residues. Soil was free from residual contamination at fifth day after spraying in case of both the fungicides and at both the doses.


Assuntos
Abelmoschus/metabolismo , Acetatos/metabolismo , Fungicidas Industriais/metabolismo , Iminas/metabolismo , Resíduos de Praguicidas/análise , Solo/química , Estrobilurinas/metabolismo , Triazóis/metabolismo , Abelmoschus/química , Acetatos/análise , Descontaminação , Monitoramento Ambiental , Frutas/química , Frutas/metabolismo , Fungicidas Industriais/análise , Meia-Vida , Iminas/análise , Estrobilurinas/análise , Triazóis/análise
9.
Int J Mol Sci ; 20(17)2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31480584

RESUMO

Powdery mildew is a disease caused by fungal pathogens that harms grape leaves and fruits. The TIFY gene family is a plant-specific super-family involved in the process of plants' development and their biotic and abiotic stress responses. This study aimed to learn the function of the VvTIFY9 gene to investigate molecular mechanisms of grape resistance to powdery mildew. A VvTIFY9 protein encoding a conserved motif (TIF[F/Y]XG) was characterized in grape (Vitis vinifera). Sequence analysis confirmed that VvTIFY9 contained this conserved motif (TIF[F/Y]XG). Quantitative PCR analysis of VvTIFY9 in various grape tissues demonstrated that the expression of VvTIFY9 was higher in grape leaves. VvTIFY9 was induced by salicylic acid (SA) and methyl jasmonate (MeJA) and it also quickly responded to infection with Erysiphe necator in grape. Analysis of the subcellular localization and transcriptional activation activity of VvTIFY9 showed that VvTIFY9 located to the nucleus and had transcriptional activity. Arabidopsis that overexpressed VvTIFY9 were more resistant to Golovinomyces cichoracearum, and quantitative PCR revealed that two defense-related genes, AtPR1 and AtPDF1.2, were up-regulated in the overexpressing lines. These results indicate that VvTIFY9 is intimately involved in SA-mediated resistance to grape powdery mildew. This study provides the basis for exploring the molecular mechanism of grape resistance to disease resistance and candidate genes for transgenic disease resistance breeding of grape plants.


Assuntos
Resistência à Doença , Micoses/metabolismo , Doenças das Plantas , Fatores de Transcrição/metabolismo , Vitis/metabolismo , Acetatos/metabolismo , Sequência de Aminoácidos , Ascomicetos , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Ácido Salicílico/metabolismo , Análise de Sequência de Proteína , Estresse Fisiológico , Fatores de Transcrição/química , Fatores de Transcrição/fisiologia , Vitis/fisiologia
10.
Microb Cell Fact ; 18(1): 151, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31484572

RESUMO

BACKGROUND: Escherichia coli (E. coli) is a bacteria that is widely employed in many industries for the production of high interest bio-products such as recombinant proteins. Nevertheless, the use of E. coli for recombinant protein production may entail some disadvantages such as acetate overflow. Acetate is accumulated under some culture conditions, involves a decrease in biomass and recombinant protein production, and its metabolism is related to protein lysine acetylation. Thereby, the carbon and nitrogen sources employed are relevant factors in cell host metabolism, and the study of the central metabolism of E. coli and its regulation is essential for optimizing the production of biomass and recombinant proteins. In this study, our aim was to find the most favourable conditions for carrying out recombinant protein production in E. coli BL21 using two different approaches, namely, manipulation of the culture media composition and the deletion of genes involved in acetate metabolism and Nε-lysine acetylation. RESULTS: We evaluated protein overexpression in E. coli BL21 wt and five mutant strains involved in acetate metabolism (Δacs, ΔackA and Δpta) and lysine acetylation (ΔpatZ and ΔcobB) grown in minimal medium M9 (inorganic ammonium nitrogen source) and in complex TB7 medium (peptide-based nitrogen source) supplemented with glucose (PTS carbon source) or glycerol (non-PTS carbon source). We observed a dependence of recombinant protein production on acetate metabolism and the carbon and nitrogen source employed. The use of complex medium supplemented with glycerol as a carbon source entails an increase in protein production and an efficient use of resources, since is a sub-product of biodiesel synthesis. Furthermore, the deletion of the ackA gene results in a fivefold increase in protein production with respect to the wt strain and a reduction in acetate accumulation. CONCLUSION: The results showed that the use of diverse carbon and nitrogen sources and acetate metabolism knockout strains can redirect E. coli carbon fluxes to different pathways and affect the final yield of the recombinant protein bioprocess. Thereby, we obtained a fivefold increase in protein production and an efficient use of the resources employing the most suitable strain and culture conditions.


Assuntos
Acetatos/metabolismo , Carbono/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Nitrogênio/metabolismo , Proteínas Recombinantes/biossíntese , Acetatos/química , Acetilação , Carbono/química , Meios de Cultura/química , Proteínas de Escherichia coli/biossíntese , Lisina/metabolismo , Nitrogênio/química , Engenharia de Proteínas , Processamento de Proteína Pós-Traducional
11.
Microb Cell Fact ; 18(1): 130, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387584

RESUMO

BACKGROUND: Acetyl-CoA is an important metabolic intermediate and serves as an acetylation precursor for the biosynthesis of various value-added acetyl-chemicals. Acetyl-CoA can be produced from glucose, acetate, or fatty acids via metabolic pathways in Escherichia coli. Although glucose is an efficient carbon source for acetyl-CoA production, the pathway from acetate to acetyl-CoA is the shortest and fatty acids can produce acetyl-CoA through fatty acid oxidation along with abundant NADH and FADH2. In this study, metabolically engineered E. coli strains for efficiently supplying acetyl-CoA from glucose, acetate, and fatty acid were constructed and applied in one-step biosynthesis of N-acetylglutamate (NAG) from glutamate and acetyl-CoA. RESULTS: A metabolically engineered E. coli strain for NAG production was constructed by overexpressing N-acetylglutamate synthase from Kitasatospora setae in E. coli BW25113 with argB and argA knockout. The strain was further engineered to utilize glucose, acetate, and fatty acid to produce acetyl-CoA. When glucose was used as a carbon source, the combined mutants of ∆ptsG::glk, ∆galR::zglf, ∆poxB::acs, ∆ldhA, and ∆pta were more efficient for supplying acetyl-CoA. The acetyl-CoA synthetase (ACS) pathway and acetate kinase-phosphate acetyltransferase (ACK-PTA) pathway from acetate to acetyl-CoA were investigated, and the ACK-PTA pathway showed to be more efficient for supplying acetyl-CoA. When fatty acid was used as a carbon source, acetyl-CoA supply was improved by deletion of fadR and constitutive expression of fadD under the strong promoter CPA1. Comparison of acetyl-CoA supply from glucose, acetate and palmitic acid revealed that a higher conversion rate of glutamate (98.2%) and productivity (an average of 6.25 mmol/L/h) were obtained when using glucose as a carbon source. The results also demonstrated the great potential of acetate and fatty acid to supply acetyl-CoA, as the molar conversion rate of glutamate was more than 80%. CONCLUSIONS: Metabolically engineered E. coli strains were developed for NAG production. The metabolic pathways of acetyl-CoA from glucose, acetate, or fatty acid were optimized for efficient acetyl-CoA supply to enhance NAG production. The metabolic strategies for efficient acetyl-CoA supply used in this study can be exploited for other chemicals that use acetyl-CoA as a precursor or when acetylation is involved.


Assuntos
Acetilcoenzima A/biossíntese , Carbono/metabolismo , Escherichia coli/metabolismo , Engenharia Metabólica , Acetatos/metabolismo , Vias Biossintéticas , Ácidos Graxos/metabolismo , Glucose/metabolismo
12.
Int J Mol Sci ; 20(15)2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31374948

RESUMO

Autophagy is a well-defined catabolic mechanism whereby cytoplasmic materials are engulfed into a structure termed the autophagosome. Methyl jasmonate (MeJA), a plant hormone, mediates diverse developmental process and defense responses which induce a variety of metabolites. In plants, little is known about autophagy-mediated responses against MeJA. In this study, we used high-throughput comparative proteomics to identify proteins of latex in the laticifers. The isobaric tags for relative and absolute quantification (iTRAQ) MS/MS proteomics were performed, and 298 proteins among MeJA treated groups and the control group of Euphorbia kansui were identified. It is interesting to note that 29 significant differentially expressed proteins were identified and their associations with autophagy and ROS pathway were verified for several selected proteins as follows: α-L-fucosidase, ß-galactosidase, cysteine proteinase, and Cu/Zn superoxide dismutase. Quantitative real-time PCR analysis of the selected genes confirmed the fact that MeJA might enhance the expression of some genes related to autophagy. The western blotting and immunofluorescence results of ATG8 and ATG18a which are two important proteins for the formation of autophagosomes also demonstrated that MeJA could promote autophagy at the protein level. Using the electron microscope, we observed an increase in autophagosomes after MeJA treatment. These results indicated that MeJA might promote autophagy in E. kansui laticifers; and it was speculated that MeJA mediated autophagy through two possible ways: the increase of ROS induces ATG8 accumulation and then aotophagosome formation, and MeJA promotes ATG18 accumulation and then autophagosome formation. Taken together, our results provide several novel insights for understanding the mechanism between autophagy and MeJA treatment. However, the specific mechanism remains to be further studied in the future.


Assuntos
Acetatos/metabolismo , Autofagia , Ciclopentanos/metabolismo , Euphorbia/citologia , Euphorbia/metabolismo , Oxilipinas/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Euphorbia/genética , Euphorbia/ultraestrutura , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteômica , Espécies Reativas de Oxigênio/metabolismo
13.
BMC Plant Biol ; 19(1): 337, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375064

RESUMO

BACKGROUND: Cymbidium goeringii belongs to the Orchidaceae, which is one of the most abundant angiosperm families. Cymbidium goeringii consist with high economic value and characteristics include fragrance and multiple flower colors. Floral scent is one of the important strategies for ensuring fertilization. However, limited genetic data is available in this non-model plant, and little known about the molecular mechanism responsible for floral scent in this orchid. Transcriptome and expression profiling data are needed to identify genes and better understand the biological mechanisms of floral scents in this species. Present transcriptomic data provides basic information on the genes and enzymes related to and pathways involved in flower secondary metabolism in this plant. RESULTS: In this study, RNA sequencing analyses were performed to identify changes in gene expression and biological pathways related scent metabolism. Three cDNA libraries were obtained from three developmental floral stages: closed bud, half flowering stage and full flowering stage. Using Illumina technique 159,616,374 clean reads were obtained and were assembled into 85,868 final unigenes (average length 1194 nt), 33.85% of which were annotated in the NCBI non redundant protein database. Among this unigenes 36,082 were assigned to gene ontology and 23,164 were combined with COG groups. Total 33,417 unigenes were assigned in 127 pathways according to the Kyoto Encyclopedia of Genes and Genomes pathway database. According these transcriptomic data we identified number of candidates genes which differentially expressed in different developmental stages of flower related to fragrance biosynthesis. In q-RT-PCR most of the fragrance related genes highly expressed in half flowering stage. CONCLUSIONS: RNA-seq and DEG data provided comprehensive gene expression information at the transcriptional level that could be facilitate the molecular mechanisms of floral biosynthesis pathways in three developmental phase's flowers in Cymbidium goeringii, moreover providing useful information for further analysis on C. goeringii, and other plants of genus Cymbidium.


Assuntos
Flores/metabolismo , Genes de Plantas/genética , Odorantes , Orchidaceae/genética , Acetatos/metabolismo , Ciclopentanos/metabolismo , Farneseno Álcool/metabolismo , Flores/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/fisiologia , Orchidaceae/metabolismo , Oxilipinas/metabolismo , Filogenia , Análise de Sequência de RNA , Sesquiterpenos/metabolismo , Terpenos/metabolismo
14.
Appl Microbiol Biotechnol ; 103(20): 8631-8645, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31418053

RESUMO

Syntrophic oxidization of acetate and propionate are both critical steps of methanogenesis during thermophilic anaerobic digestion. However, knowledge on syntrophic acetate-oxidizing bacteria (SAOB) and syntrophic propionate-oxidizing bacteria (SPOB) is limited because of the difficulty in pure culture isolation due to symbiotic relationship. In this study, two thermophilic acetate-fed anaerobic chemostats, ATL (dilution rate of 0.025 day-1) and ATH (0.05 day-1) and one thermophilic propionate-fed anaerobic chemostat PTL (0.025 day-1) were constructed, AOB and POB in these chemostats were studied via microbial community analysis and DNA stable-isotope probing (SIP). The results showed that, in addition to Tepidanaerobacter, a known SAOB, species of Thauera, Thermodesulfovibrio, Anaerobaculum, Ruminiclostridium, Comamonas, and uncultured bacteria belonging to Lentimicrobiaceae, o_MBA03, Thermoanaerobacteraceae, Anaerolineaceae, Clostridiales, and Ruminococcaceae were determined to be potential AOB in chemostats. Pelotomaculum was the key SPOB detected in the propionate-fed chemostat. Based on the intense fluorescence of coenzyme F420, majority of Methanosarcina cells in acetate-fed chemostats were involved in hydrogenotrophic methanogenesis, suggesting the existence of highly active SAOB among the detected AOB. In the propionate-fed chemostat, most of the species detected as AOB were similar to those detected in the acetate-fed chemostats, suggesting the contribution of the syntrophic acetate oxidization pathway for methane generation. These results revealed the existence of previously unknown AOB with high diversity in thermophilic chemostats and suggested that methanogenesis from acetate via the syntrophic oxidization pathway is relevant for thermophilic anaerobic digestion.


Assuntos
Acetatos/metabolismo , Bactérias Anaeróbias/classificação , Biota , Microbiologia Ambiental , Metano/metabolismo , Methanosarcina/classificação , Anaerobiose , Bactérias Anaeróbias/genética , Bactérias Anaeróbias/metabolismo , Methanosarcina/genética , Methanosarcina/metabolismo , Oxirredução , Propionatos/metabolismo
15.
Nucl Med Commun ; 40(10): 1081-1085, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31365503

RESUMO

OBJECTIVE: The aim of this study was to evaluate the frequency and relative significance of incidental cardiac uptake on 68Ga-DOTA-peptide PET-CT scans in patients with neuroendocrine tumours/genetic syndromes expressing somatostatin receptors. METHODS: Scans of 1463 patients who underwent 68Ga-DOTA-peptide PET-CT scans in our department between 2013 and 2018 were retrospectively evaluated for the presence of uptake in the heart and/or its appendages. RESULTS: out of 1463 patients (1.3%) demonstrated uptake of radio-peptide in the heart and/or its appendages. In 18 out of these 19 patients, the clinicians were unaware of possible cardiac involvement (one patient was a known case of cardiac paraganglioma). The primary neuroendocrine tumours and genetic syndromes associated with those with cardiac uptake of 68Ga-DOTA-peptide were as follows: unknown primary (n = 7), small bowel (n = 6), colon (n = 2), caecum (n = 1), pancreas (n = 1), cardiac paraganglioma (n = 1), Von Hippel Lindau syndrome (n = 1). Amongst the patients with neuroendocrine tumours, demonstrating cardiac uptake of 68Ga-DOTA-peptide, there was no clear association with carcinoid heart disease. DISCUSSION: Our results are in agreement with established literature. The exact prognostic implication of cardiac involvement in patients with neuroendocrine tumours as well as other genetic syndromes expressing somatostatin receptors is unknown. We hypothesize that early detection and confirmation of cardiac metastasis(es) in these patients and introduction of different treatment regimes (such as Peptide Receptor Radionuclide Therapy), earlier in the course of the disease would reduce the disease burden on the heart and therefore contribute to better patient outcomes. CONCLUSION: Cardiac involvement is a rare and hitherto less well-studied occurrence in neuroendocrine tumours, with an incidence in the range of 1%. To assess the possible prognostic implications, further detailed multicentre studies are required.


Assuntos
Acetatos/metabolismo , Radioisótopos de Gálio/metabolismo , Coração/diagnóstico por imagem , Miocárdio/metabolismo , Peptídeos Cíclicos/metabolismo , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Idoso , Transporte Biológico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos
16.
Plant Mol Biol ; 101(3): 297-313, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31368003

RESUMO

KEY MESSAGE: An enzyme is crucial for the formation of Hedychium coronarium scent and defense responses, which may be responsible for the biosynthesis of allo-ocimene in H. coronarium. Hedychium coronarium can emit a strong scent as its main scent constituents are monoterpenes and their derivatives. Among these derivatives, allo-ocimene is not only a very important volatile substance in flower aroma, but is also crucial to plant defense. However, the molecular mechanism of allo-ocimene biosynthesis has not been characterized in plants. In this study, a new alcohol dehydrogenase gene, HcADH, was cloned. The amino acid sequences encoded by HcADH contained the most conserved motifs of short chain alcohol dehydrogenase/reductases (SDRs), which included NAD+ binding domain, TGxxx[AG]xG and active site YxxxK. Real-time PCR analyses showed that the HcADH was highly expressed in the outer labellum but was almost undetectable in vegetative organs. The change in its expression level in petals was positively correlated with the emission pattern of allo-ocimene during flower development. HcADH expression coincides also the release level of allo-ocimene among different Hedychium species. Although HcADH is not expressed in the leaves, HcADH expression and allo-ocimene release in leaves can be induced by mechanical wounding or methyl jasmonate (MeJA) treatment. In addition, the expression of HcADH induced by mechanical wounding can be prevented by acetylsalicylic acid, a jasmonic acid biosynthesis inhibitor, suggesting that jasmonic acid might participate in the transmission of wounding signals. Using the Barley stripe mosaic virus (BSMV)-VIGS method, it was found that BSMV:HcADH335 inoculation was able to down-regulate HcADH expression, decreasing only the release of allo-ocimene in flowers while the content of other volatile substances did not decrese. In vitro characterization showed that recombinant HcADH can catalyze geraniol into citral, and citral is an intermediate of allo-ocimene biosynthesis. HcADH may be responsible for the biosynthesis of allo-ocimene in H. coronarium, which is crucial for the formation of H. coronarium scent and defense function.


Assuntos
Proteínas de Plantas/metabolismo , Polienos/metabolismo , Redutases-Desidrogenases de Cadeia Curta/metabolismo , Zingiberaceae/enzimologia , Acetatos/metabolismo , Ciclopentanos/metabolismo , Flores/enzimologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Oxilipinas/metabolismo , Proteínas de Plantas/genética , Redutases-Desidrogenases de Cadeia Curta/genética , Transdução de Sinais , Terpenos/metabolismo , Zingiberaceae/genética
17.
Curr Microbiol ; 76(11): 1298-1305, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31428805

RESUMO

Geobacillus thermoglucosidasius NY05 catalyzes calcite single crystal formation at 60 °C by using acetate and calcium. Endospores are embedded at the central part of the calcite single crystal and carbon atoms in the calcite lattice are derived from acetate carbon. Here, we synthesized 21-mer antisense DNA oligonucleotides targeting sporulation transcription factor, acetate-CoA ligase, isocitrate lyase, and malate synthase G mRNAs and evaluated the effect of these oligonucleotides on calcite formation in G. thermoglucosidasius NY05. G. thermoglucosidasius NY05 cells containing antisense DNA oligonucleotides targeting sporulation transcription factor, acetate-CoA ligase, isocitrate lyase, and malate synthase G mRNAs had reduced calcite single crystal formation by 18.7, 50.6, 55.7, and 82.3%, respectively, compared with cells without antisense DNA oligonucleotides. These results support that calcite formation needs endospores as the nucleus to grow, and carbon dioxide generated from acetate, which is metabolized via the glyoxylate pathway and glucogenesis, is supplied to the crystal lattice.


Assuntos
Proteínas de Bactérias/genética , Carbonato de Cálcio/metabolismo , Inativação Gênica , Geobacillus/genética , Acetatos/metabolismo , Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Carbonato de Cálcio/química , Geobacillus/química , Geobacillus/metabolismo , Glioxilatos/metabolismo , Isocitrato Liase/genética , Isocitrato Liase/metabolismo , Malato Sintase/genética , Malato Sintase/metabolismo
18.
Appl Microbiol Biotechnol ; 103(17): 6989-7001, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31267232

RESUMO

The molecular weight of hyaluronic acid (HA) is a critical property which determines its usage in various biomedical applications. This study investigates the correlation between the availability of a critical cofactor, acetyl-CoA, the concentration of a limiting precursor, UDP-N-acetylglucosamine (UDP-GlcNAc), and the molecular weight of HA (MWHA) produced by recombinant Lactococcus lactis MKG6 cultures. This strain expressed three heterologous HA-pathway genes obtained from the has operon of Streptococcus zooepidemicus in an ldh-mutant host strain, L. lactis NZ9020. A flux balance analysis, performed using the L. lactis genome-scale metabolic network, showed a positive correlation of acetyl-CoA flux with the UDP-GlcNAc flux and the experimental data on HA productivity. To increase the intracellular levels of acetyl-CoA, acetate was supplemented as a pulse feed in anaerobic batch cultures. However, acetate is effectively utilized only in the presence of glucose and exhaustion of glucose resulted in decreasing the final MWHA (1.5 MDa). Co-supplementation of acetate resulted in enhancing the acetyl-CoA and UDP-GlcNAc levels as well as the MWHA to 2.5 MDa. This logic was extended to fed-batch cultures, designed with a pH-based feedback control of glucose feeding and pulse acetate supplementation. When the glucose feed concentration was optimally adjusted to prevent glucose exhaustion or accumulation, the acetate utilization was found to be high, resulting in significantly enhanced levels of acetyl-CoA and UDP-GlcNAc as well as a MWHA of 3.4 MDa, which was sustained at this value throughout the process. This study provides the possibility of commercially producing high MWHA using recombinant L. lactis strains.


Assuntos
Acetatos/metabolismo , Acetilcoenzima A/metabolismo , Ácido Hialurônico/biossíntese , Ácido Hialurônico/química , Lactococcus lactis/metabolismo , Acetatos/análise , Proteínas de Bactérias/genética , Técnicas de Cultura Celular por Lotes , Meios de Cultura/química , Meios de Cultura/metabolismo , Glucose/análise , Glucose/metabolismo , Lactococcus lactis/genética , Análise do Fluxo Metabólico , Redes e Vias Metabólicas/genética , Peso Molecular , Proteínas Recombinantes/genética , Streptococcus equi/genética , Uridina Difosfato N-Acetilglicosamina/metabolismo
19.
Nat Commun ; 10(1): 3031, 2019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31292453

RESUMO

Maternal immune dysregulation seems to affect fetal or postnatal immune development. Preeclampsia is a pregnancy-associated disorder with an immune basis and is linked to atopic disorders in offspring. Here we show reduction of fetal thymic size, altered thymic architecture and reduced fetal thymic regulatory T (Treg) cell output in preeclamptic pregnancies, which persists up to 4 years of age in human offspring. In germ-free mice, fetal thymic CD4+ T cell and Treg cell development are compromised, but rescued by maternal supplementation with the intestinal bacterial metabolite short chain fatty acid (SCFA) acetate, which induces upregulation of the autoimmune regulator (AIRE), known to contribute to Treg cell generation. In our human cohorts, low maternal serum acetate is associated with subsequent preeclampsia, and correlates with serum acetate in the fetus. These findings suggest a potential role of acetate in the pathogenesis of preeclampsia and immune development in offspring.


Assuntos
Acetatos/sangue , Feto/imunologia , Pré-Eclâmpsia/imunologia , Efeitos Tardios da Exposição Pré-Natal/imunologia , Linfócitos T Reguladores/imunologia , Acetatos/administração & dosagem , Acetatos/imunologia , Acetatos/metabolismo , Adulto , Animais , Animais Recém-Nascidos , Estudos de Casos e Controles , Desenvolvimento Infantil , Pré-Escolar , Suplementos Nutricionais , Feminino , Feto/citologia , Feto/diagnóstico por imagem , Microbioma Gastrointestinal/imunologia , Vida Livre de Germes/imunologia , Humanos , Tolerância Imunológica/imunologia , Lactente , Recém-Nascido , Estudos Longitudinais , Troca Materno-Fetal/imunologia , Camundongos , Tamanho do Órgão/imunologia , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/diagnóstico , Gravidez , Efeitos Tardios da Exposição Pré-Natal/patologia , Efeitos Tardios da Exposição Pré-Natal/prevenção & controle , Estudos Prospectivos , Timo/citologia , Timo/diagnóstico por imagem , Timo/crescimento & desenvolvimento , Timo/imunologia , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Ultrassonografia Pré-Natal , Adulto Jovem
20.
Food Chem ; 298: 125017, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31260967

RESUMO

The aim of the study was to evaluate an elevated (3.0 °C) and low (1.0 °C) storage temperature combined with dynamic controlled atmosphere monitored by respiratory quotient (DCA-RQ) and chlorophyll fluorescence (DCA-CF) on anaerobic metabolism, physiological storage disorders and overall quality of 'Nicoter' ('Kanzi®') apples after 5.5 and 8.0 months of storage plus 7d shelf-life. Fruit stored under DCA-RQ 2.0 accumulated the highest amounts of anaerobic metabolites (acetaldehyde, ethanol and ethyl acetate), regardless of storage temperature and timing of storage outturn evaluation, but it did not result in higher electrolyte leakage. Flesh breakdown, core breakdown and cavity formation were reduced at 3 °C. Storage at 3 °C combined with DCA maintained higher flesh firmness after 8.0 months storage plus 7d shelf-life. 'Nicoter' apples can be stored at 3 °C using a DCA system, based either on CF or on RQ, to save electrical energy.


Assuntos
Armazenamento de Alimentos/métodos , Frutas/metabolismo , Malus/metabolismo , Acetaldeído/metabolismo , Acetatos/metabolismo , Anaerobiose , Atmosfera , Clorofila/metabolismo , Etanol/metabolismo , Fluorescência , Temperatura Ambiente
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