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1.
Sci Rep ; 9(1): 16039, 2019 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-31690751

RESUMO

The CytoFLEX is a novel semiconductor-based flow cytometer that utilizes avalanche photodiodes, wavelength-division multiplexing, enhanced optics, and diode lasers to maximize light capture and minimize optical and electronic noise. Due to an increasing interest in the use of extracellular vesicles (EVs) as disease biomarkers, and the growing desire to use flow cytometry for the analyses of biological nanoparticles, we assessed the light-scatter sensitivity of the CytoFLEX for small-particle detection. We found that the CytoFLEX can fully resolve 70 nm polystyrene and 98.6 nm silica beads by violet side scatter (VSSC). We further analyzed the detection limit for biological nanoparticles, including viruses and EVs, and show that the CytoFLEX can detect viruses down to 81 nm and EVs at least as small as 65 nm. Moreover, we could immunophenotype EV surface antigens, including directly in blood and plasma, demonstrating the double labeling of platelet EVs with CD61 and CD9, as well as triple labeling with CD81 for an EV subpopulation in one donor. In order to assess the refractive indices (RIs) of the viruses and EVs, we devised a new method to inversely calculate the RIs using the intensity vs. size data together with Mie-theory scatter efficiencies scaled to reference-particle measurements. Each of the viruses tested had an equivalent RI, approximately 1.47 at 405 nm, which suggests that flow cytometry can be more broadly used to easily determine virus sizes. We also found that the RIs of EVs increase as the particle diameters decrease below 150 nm, increasing from 1.37 for 200 nm EVs up to 1.61 for 65 nm EVs, expanding the lower range of EVs that can be detected by light scatter. Overall, we demonstrate that the CytoFLEX has an unprecedented level of sensitivity compared to conventional flow cytometers. Accordingly, the CytoFLEX can be of great benefit to virology and EV research, and will help to expand the use of flow cytometry for minimally invasive liquid biopsies by allowing for the direct analysis of antigen expression on biological nanoparticles within patient samples, including blood, plasma, urine and bronchoalveolar lavages.

2.
mSystems ; 4(5)2019 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-31594827

RESUMO

Gut microbiota play important roles in host metabolism, especially in diabetes. However, why different diets lead to similar diabetic states despite being associated with different microbiota is not clear. Mice were fed two high-energy diets (HED) with the same energy density but different fat-to-sugar ratios to determine the associations between the microbiota and early-stage metabolic syndrome. The two diets resulted in different microbiota but similar diabetic states. Interestingly, the microbial gene profiles were not significantly different, and many common metabolites were identified, including l-aspartic acid, cholestan-3-ol (5ß, 3α), and campesterol, which have been associated with lipogenesis and inflammation. Our study suggests that different metabolic-syndrome-inducing diets may result in different microbiota but similar microbiomes and metabolomes. This suggests that the metagenome and metabolome are crucial for the prognosis and pathogenesis of obesity and metabolic syndrome.IMPORTANCE Various types of diet can lead to type 2 diabetes. The gut microbiota in type 2 diabetic patients are also different. So, two questions arise: whether there are any commonalities between gut microbiota induced by different pro-obese diets and whether these commonalities lead to disease. Here we found that high-energy diets with two different fat-to-sugar ratios can both cause obesity and prediabetes but enrich different gut microbiota. Still, these different gut microbiota have similar genetic and metabolite compositions. The microbial metabolites in common between the diets modulate lipid accumulation and macrophage inflammation in vivo and in vitro This work suggests that studies that only use 16S rRNA amplicon sequencing to determine how the microbes respond to diet and associate with diabetic state are missing vital information.

3.
J Agric Food Chem ; 67(39): 10984-10993, 2019 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-31525294

RESUMO

The objective of the present study was to reveal the effects of four types of nitrogen sources (soymeal, yeast extract, KNO3, and ammonium tartrate) on the lipid metabolism of the oleaginous fungus Mortierella alpina using untargeted lipidomics, targeted fatty acid, and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis. Our results showed clear differences in the contents and compositions of lipids between four types of nitrogen sources. Soymeal and ammonium tartrate supplementation favored the accumulation of triglycerides with arachidonic acid (ARA) and C16-18 fatty acids, respectively. These results were further validated by our targeted fatty acid analysis. RT-qPCR analysis of related genes in M. alpina between the four nitrogen source conditions found that soymeal supplementation dramatically increased the expression of GPAT, ELOVL, and Δ12/Δ6 desaturase. Our findings provided new insights into the regulation of lipid biosynthesis in M. alpina and potential avenues for genetic manipulation and highlighted the importance of an optimal nitrogen source for ARA-rich oil production.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Lipídeos/biossíntese , Lipídeos/química , Espectrometria de Massas/métodos , Mortierella/metabolismo , Nitrogênio/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/biossíntese , Ácidos Graxos/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mortierella/química , Mortierella/enzimologia , Mortierella/genética
4.
J Agric Food Chem ; 67(34): 9551-9559, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31379157

RESUMO

In oleaginous micro-organisms, nitrogen limitation activates adenosine monophosphate deaminase (AMPD) and promotes lipogenesis via the inhibition of isocitrate dehydrogenase. We found that the overexpression of homologous AMPD in Mortierella alpina favored lipid synthesis over cell growth. Total fatty acid content in the recombinant strain was 15.0-34.3% higher than that in the control, even though their biomass was similar. During the early fermentation stage, the intracellular AMP level reduced by 40-60%, together with a 1.9-2.7-fold increase in citrate content compared with the control, therefore provided more precursors for fatty acid synthesis. Moreover, the decreased AMP level resulted in metabolic reprogramming, reflected by the blocked TCA cycle and reduction of amino acids, distributing more carbon to lipid synthesis pathways. By coupling the energy balance with lipogenesis, this study provides new insights into cell metabolism under nitrogen-limited conditions and targets the regulation of fatty acid accumulation in oleaginous micro-organisms.


Assuntos
AMP Desaminase/metabolismo , Ácidos Graxos/metabolismo , Proteínas Fúngicas/metabolismo , Mortierella/enzimologia , AMP Desaminase/genética , Monofosfato de Adenosina/metabolismo , Aminoácidos/metabolismo , Proteínas Fúngicas/genética , Metabolismo dos Lipídeos , Mortierella/genética , Mortierella/crescimento & desenvolvimento , Mortierella/metabolismo
5.
Nutr Cancer ; : 1-12, 2019 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-31352802

RESUMO

Kudingcha is implicated in alleviating metabolic disorders in traditional Chinese medicine. However, the role of Kudingcha, one of the Ligustrum robustum species, in metabolic regulations and its antitumor activity in triple-negative breast cancer (TNBC) remains to be determined. Two breast cancer cell lines and immunocompetent and immunodeficient mice were used to evaluate the therapeutic effects of Kudingcha treatment. The production of reactive oxygen species (ROS) and glucose uptake were examined by flow cytometry. Metabolic shift was examined by metabonomics and western blot analysis. In this study, we found that aqueous extract of Kudingcha dose dependently inhibited cell growth and induced apoptosis in vitro and in vivo. Moreover, Kudingcha supplementation significantly reduced cancer metastasis. Kudingcha significantly inhibited glycolysis and glutamine metabolism. In addition, we demonstrated that the antitumor effects of Kudingcha were dependent on ROS production, which was increased by ß-oxidation and oxidative phosphorylation. These findings provide a novel potential benefit of Kudingcha from targeting the cancer metabolism.

6.
Mol Nutr Food Res ; 63(11): e1800987, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30920128

RESUMO

SCOPE: The aim of the study is to explore which properties of selected peptides will positively predict their antidiabetic activity in vitro and in vivo. METHODS AND RESULTS: Streptozotocin-induced diabetic C57BL/6J mice are administered with soybean peptide (SP), mung bean peptide (MP), corn peptide (CP), and wheat peptide (WP) (500 mg kg-1  d-1 ) for 10 weeks. CP and WP improve hyperglycemia homeostasis in streptozotocin-induced diabetic mice. Female nonobese diabetic (NOD) mice are treated with CP, WP, fractions C1 and C2 (isolated from CP), and W1 and W2 (isolated from WP) beginning at 3 weeks of age. CP, C2, and W2 delay the initiation of diabetes and decrease serum IL-6 levels in NOD mice. CP also reduces insulitis and increases the ß-cell area in NOD mice. MIN-6 cells are incubated with the selected peptides. CP, C2, and W2 result in the reduced expression of LPS-induced IL-6 mRNA in MIN-6 cells. CP inhibits signaling pathways related to apoptosis and inflammation. The antioxidative, hydrophobic, and proliferative properties of the selected peptides are analyzed. The hypoglycemic effects of cereal peptides are not associated with their antioxidant activity, hydrophobicity, or proliferative ability. CONCLUSION: Findings suggest that the effect of cereal peptides on the development of T1D is associated with their anti-inflammatory ability.

7.
Metabolomics ; 15(4): 50, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30900034

RESUMO

INTRODUCTION: Metabolomics has been successfully applied to guide the rational engineering of industrial strains and improve the performance of bioprocesses. Mortierella alpina has traditionally been one of the most popular industrial strains for the production of polyunsaturated fatty acids. However, a systematic comparison and optimisation of the metabolomic analysis methods of M. alpina has not yet been reported. OBJECTIVE: We sought to identify potential weaknesses that are important for accurate metabolomic analysis. We also aimed to determine an efficient sample preparation protocol for metabolomics studies in the oleaginous filamentous fungus M. alpina. METHODS: In this study, using GC-MS, we evaluated three sample preparation protocols and five solvent mixtures by assessment of the metabolite profile differences, the sum of peak intensities and the reproducibility of metabolite quantification. RESULTS: The freeze-dried biomass had better reproducibility and recovery than fresh biomass for metabolite extraction and data normalisation that is part of a metabolomics analysis of filamentous fungi M. alpina. Methanol:water (1:1) was superior for the profiling of metabolites in oleaginous fungi M. alpina. The unbiased metabolite profiling difference between the growth phase and lipids synthesis phase revealed that the degradation of amino acids were critical nodes for the efficient synthesis of lipids in M. alpina. CONCLUSION: The use of freeze-dried biomass for metabolite extraction and data normalisation was more efficient at measuring the active state of the intracellular metabolites in M. alpina. We recommend extracting the intracellular metabolites with methanol:water (1:1). An important role of amino acid oxidation in the nitrogen limitation-mediated lipid accumulation was found.

8.
Biochem Biophys Res Commun ; 512(2): 196-201, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30879764

RESUMO

G-protein coupled receptor 120 (GPR120 or FFAR4) functions as a receptor for free fatty acids and plays a critical role in lipid metabolism. Studies have shown a close relationship between GDM and lipid metabolism disorders, whether GPR120 participates in the metabolic regulation of GDM remains unclear. In this study, 29 women with GDM and 33 normal pregnant women were enrolled. Lipid profiles were determined by lipidomics, expression of GPR120 and FGF21 was measured in the white blood cells, and regulation of FGF21 by GPR120 was investigated in THP-1 cells as well as human peripheral blood monocytes. Lipidomics reveal altered lipid metabolism in patients with GDM. The expression of both GPR120 and FGF21 is significantly higher in the GDM than in the control at the 32nd and 37th weeks of pregnancy, but the differences disappear by the 2nd day post-delivery. Generally positive correlations are found between the total amount of lipids and expression levels of GPR120 and FGF21 in GDM patients. FGF21 expression is induced by GPR120 activation in THP-1 cells and WBCs. GPR120 may act as a metabolic regulator, through the induction of FGF21, to control lipid metabolism, and GDM patients may manifest a GPR120 insensitivity.

9.
J Microbiol Methods ; 158: 80-85, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30708087

RESUMO

Screening oleaginous microorganisms capable of accumulating considerable lipids is essential for industrial lipid production. Here we demonstrated forty-seven filamentous fungal isolates were obtained from eight soil samples using a new screening strategy with both triphenyltetrazolium chloride (TTC), a redox indicator used for testing oil presence, and cerulenin, an inhibitor of fatty acid synthase (FAS), supplemented in screening medium. Among these fungal isolates, nineteen have high lipid content (>20% dry biomass weight) and were affiliated with the genus Mortierella by morphology identification and phylogenetic analysis based on ITS gene sequences. Notably, one strain designated as SL-4 reached 32% of its biomass weight as lipid, displaying the highest potential. Two candidates with high lipid content as well as biomass production were selected for exploring the effect of different carbon and nitrogen sources on morphology, biomass and lipid accumulation.

10.
Front Microbiol ; 9: 2193, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30258432

RESUMO

Similar to the gut, the bladder contains urinary microbiota, and its bacterial composition and structure are determined by the individual's health status. Cesarean section is a traumatic event for women and it is correlated with postpartum complications. To better understand the urinary microbiota alterations caused by cesarean section, 16S rDNA sequencing was used to assess urine specimens collected by transurethral catheterization from 30 healthy women undergoing cesarean section pre-delivery (PreD) and post-delivery (PostD). A significant increase in bacterial diversity and more detectable bacteria at the phylum, family, and genus levels was observed in the PostD group compared to the PreD group, indicating that cesarean delivery (a process that includes surgery and delivery) altered the bacterial community. Specifically, the phylum Firmicutes and its affiliated family Lactobacillaceae and genus Lactobacillus dramatically decreased in the PostD group, suggesting that beneficial bacteria decreased after cesarean section, and clinicians should be aware that this might increase the risk of complications. Concurrently, the phylum Proteobacteria and its affiliated bacteria Pseudomonadaceae and Pseudomonas increased in the PostD group compared to the PreD group. This indicates that pathogen growth increases after cesarean section, making it important for clinicians to combat these changes to protect women from infectious diseases. Interestingly, several metabolic pathways, such as metabolism of energy, cofactors and vitamins were strengthened in the PostD group, whereas membrane transport was lessened in this group. This suggests that women's metabolic disorders might be cured by balancing urinary microbiota. In conclusion, the altered urinary microbiota between the PreD and PostD periods appears to provide insight into how to prevent postpartum metabolic disorders.

11.
Appl Microbiol Biotechnol ; 102(22): 9679-9689, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30255230

RESUMO

The ω-3 fatty acid desaturase (ω3Des) is a key enzyme in the biosynthesis of polyunsaturated fatty acids (PUFAs). However, the enzyme exhibits a significant preference towards different fatty acid substrates. To examine the molecular mechanism of its substrate specificity, a series of site-directed mutants were constructed based on the membrane topology model and functionally characterised by heterologous expression in Saccharomyces cerevisiae. Our results revealed that the W106F and V137T mutations markedly decreased the enzyme activity which indicated that these two residues were associated with substrate recognition. In contrast, the A44S, M156I and W291M mutations showed significant increments (30 to 40%) of the conversion rate for AA substrate desaturation, which suggests that these residues play a pivotal role in desaturation of longer chain-length substrates. Through homology modelling of 3-dimensional structures and molecular docking of FADS15, we propose that the critical residues that bind to the CoA groups may affect substrate localisation and govern substrate preference and chain-length specificity. Our work increases the understanding of the structure-function relationships of the microbial membrane-bound desaturases. The growing knowledge of the molecular mechanism will also aid in the efficient production of value-added fatty acids.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ácidos Graxos Dessaturases/química , Ácidos Graxos Dessaturases/genética , Ácidos Graxos/metabolismo , Mortierella/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Análise Mutacional de DNA , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/química , Simulação de Acoplamento Molecular , Mortierella/química , Mortierella/genética , Mutação de Sentido Incorreto , Alinhamento de Sequência , Especificidade por Substrato
12.
Cell Physiol Biochem ; 48(6): 2318-2336, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30114709

RESUMO

BACKGROUND/AIMS: Rapamycin (Rp), the main mammalian target of rapamycin complex inhibitor, is a promising therapeutic agent for breast cancer. However, metabolic disorders and drug resistance reduce its efficacy. Epidemiological, clinical, and experimental studies have demonstrated that omega-3 polyunsaturated fatty acids (ω-3 PUFAs) significantly reduce the incidence and mortality of breast cancer and improve metabolic disorders. METHODS: Three breast cancer cell lines and immunocompetent and immunodeficient mice were used to evaluate the therapeutic effects of Rp plus ω-3 PUFA treatment. The production of reactive oxygen species (ROS) and glucose uptake were examined by flow cytometry. Metabolic shift was examined by metabonomics, seahorse experiments, and western blot analysis. RESULTS: We found that ω-3 PUFAs and Rp synergistically induced cell cycle arrest and apoptosis in vitro and in vivo, accompanied by autophagy blockage. In addition, Rp-induced hypertriglyceridemia and hypercholesterolemia were completely abolished by ω-3 PUFA supplementation. Moreover, the combined treatment of ω-3 PUFA and Rp significantly inhibited glycolysis and glutamine metabolism. The anti-tumor effects of this combination treatment were dependent on ROS production, which was increased by ß-oxidation and oxidative phosphorylation. CONCLUSION: Our study revealed that ω-3 PUFA enhanced the anti-tumor activity of Rp while minimizing its side effects in vitro and in vivo. These results provide novel insights into the mechanisms underlying the potential beneficial effects of Rp combined with ω-3 PUFAs on the prevention of breast cancer.


Assuntos
Apoptose/efeitos dos fármacos , Ácidos Graxos Ômega-3/farmacologia , Sirolimo/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Ácidos Graxos Ômega-3/uso terapêutico , Feminino , Humanos , Ácido Láctico/metabolismo , Células MCF-7 , Malondialdeído/metabolismo , Metabolômica , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Sirolimo/uso terapêutico
13.
J Microbiol Methods ; 152: 179-185, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30096341

RESUMO

The Agrobacterium tumefaciens-mediated transformation (ATMT) method is commonly applied in the oleaginous filamentous fungus Mortierella alpina. During the ATMT process, the spores of M. alpina have traditionally been used as a co-cultivation material, but their long spore-producing cycle and low sporulation rate make the transformation process tedious. This study explores the use of germinating spores, mycelium and single solid colonies of uracil auxotrophic M. alpina CCFM501 as a co-cultivation material with A. tumefaciens AGL1. The results show that A. tumefaciens AGL1 can successfully transform the germinating spores, mycelium and single solid colonies of M. alpina. In addition, the transformation rate of the germinating spores was 50% higher than that of the fresh spores. Due to its concise preparation process, the mycelium was chosen as a co-cultivation material for two plasmids of different lengths and proven to be an efficient co-cultivation material for M. alpina.


Assuntos
Agrobacterium tumefaciens/genética , Técnicas de Cocultura/métodos , Engenharia Genética/métodos , Mortierella/genética , Transformação Genética , Meios de Cultura , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Plasmídeos , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento
14.
Front Microbiol ; 9: 1878, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30154780

RESUMO

Omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) have important therapeutic and nutritional benefits in humans. In the biosynthesis pathways of these LC-PUFAs, omega-3 desaturase plays a critical role. In this study, we report a new omega-3 desaturase (PPD17) from Phytophthora parasitica. This desaturase shares high similarities with the known omega-3 desaturases and was expressed in Saccharomyces cerevisiae for the activity and substrate specificity research. The desaturase has a wide omega-6 fatty acid substrate, containing both 18C and 20C fatty acids, and exhibits a strong activity of delta-17 desaturase but a weak activity of delta-15 desaturase. The new desaturase converted the omega-6 arachidonic acid (AA, C20:4) to EPA (an omega-3 LC-PUFA, C20:5) with a substrate conversion rate of 70%. To obtain a high EPA-producing strain, we transformed PPD17 into Mortierella alpina, an AA-producing filamentous fungus. The EPA content of the total fatty acids in reconstruction strains reached 31.5% and was followed by the fermentation optimization of the EPA yield of up to 1.9 g/L. This research characterized a new omega-3 desaturase and provides a possibility of industrially producing EPA using M. alpina.

15.
J Ind Microbiol Biotechnol ; 45(4): 281-291, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29442208

RESUMO

Thioesterases (TEs) play an essential role in the metabolism of fatty acids (FAs). To explore the role of TEs in mediating intracellular lipid metabolism in the oleaginous fungus Mortierella alpina, the acyl-CoA thioesterase ACOT8I was overexpressed. The contents of total fatty acids (TFAs) were the same in the recombinant strains as in the wild-type M. alpina, whilst the production of free fatty acids (FFAs) was enhanced from about 0.9% (wild-type) to 2.8% (recombinant), a roughly threefold increase. Linoleic acid content in FFA form constituted about 9% of the TFAs in the FFA fraction in the recombinant strains but only about 1.3% in the wild-type M. alpina. The gamma-linolenic acid and arachidonic acid contents in FFA form accounted for about 4 and 25%, respectively, of the TFAs in the FFA fraction in the recombinant strains, whilst neither of them in FFA form were detected in the wild-type M. alpina. Overexpression of the TE ACOT8I in the oleaginous fungus M. alpina reinforced the flux from acyl-CoAs to FFAs, improved the production of FFAs and tailored the FA profiles of the lipid species.


Assuntos
Ácido Araquidônico/química , Ácidos Graxos não Esterificados/química , Metabolismo dos Lipídeos , Mortierella/metabolismo , Palmitoil-CoA Hidrolase/metabolismo , Biomassa , Cromatografia em Camada Delgada , DNA/química , DNA Fúngico/genética , Fermentação , Glucose/química , Concentração de Íons de Hidrogênio , Ácido Linoleico/química , Lipídeos/química , Consumo de Oxigênio , Isoformas de Proteínas , Proteínas Recombinantes/química , Ácido gama-Linolênico/química
16.
Cell Biol Int ; 42(2): 180-186, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28980737

RESUMO

Intake of ω-3 PUFAs reduces the frequency of breast cancer, and GPR120 receptor transduces ω-3 PUFAs signaling to increase insulin sensitivity in mice, but whether GPR120 mediates ω-3 PUFAs signaling to inhibit breast carcinogenesis is currently unknown. In the present study, we found that GPR120 is highly expressed in human breast cancerous tissues but not adjacent normal tissue. Knockdown of GPR120 by siRNA in breast cancer cells significantly reduced cell growth, and dramatically increased ω-3 FFA-induced cell growth inhibition and apoptosis. Thus, these observations indicated that GPR120 promotes breast cancer cell growth, whereas ω-3 PUFA-induce breast cancer cell apoptosis independently of GPR120.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Ácidos Graxos Ômega-3/uso terapêutico , Receptores Acoplados a Proteínas-G/metabolismo , Apoptose , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Receptores Acoplados a Proteínas-G/fisiologia
17.
Appl Microbiol Biotechnol ; 102(1): 211-223, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29082420

RESUMO

Polyunsaturated fatty acids (PUFAs) are essential lipids for cell function, normal growth, and development, serving as key structural components of biological membranes and modulating critical signal transduction events. Omega-3 (n-3) long chain PUFAs (LC-PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to protect against inflammatory diseases and enhance brain development and function. This had led to a marked increase in demand for fish and fish oils in human diets, supplements, and aquaculture and created a need for new, sustainable n-3 LC-PUFA sources. We have studied for the first time homogenous preparations of the membrane-type ω6 and ω3 fatty acid desaturases from the fungus Mortierella alpina, as a model system to produce PUFAs. These desaturases possess a di-iron metal center and are selective for 18:1 n-9 and 18:2 n-6 acyl-CoA substrates, respectively. Sequence alignments and membrane topology predictions support that these enzymes have unique cap regions that may include the rearrangement and repositioning of the active site, especially when compared to the mammalian stearoyl-coenzyme A desaturase-1 (SCD1) and the related sphingolipid α-hydroxylase (Scs7p) that act upon different substrates.


Assuntos
Ácidos Graxos Dessaturases/química , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Insaturados/biossíntese , Mortierella/enzimologia , Sequência de Aminoácidos , Citocromo-B(5) Redutase/genética , Citocromo-B(5) Redutase/isolamento & purificação , Citocromo-B(5) Redutase/metabolismo , Citocromos b/genética , Citocromos b/isolamento & purificação , Citocromos b/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/isolamento & purificação , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-6/metabolismo , Humanos , Ferro/química , Cinética , Membranas/química , Membranas/enzimologia , Mortierella/classificação , Mortierella/genética , Filogenia , Especificidade por Substrato
18.
Anal Biochem ; 557: 62-68, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29224734

RESUMO

This study evaluated the quenching and extraction procedures using different solvents at different temperatures for Lactobacillus plantarum ZS2058. Eight quenching methods that used readily available organic solvents (methanol and ethanol) or cryoprotectants (glycerol and dimethyl sulfoxide) were evaluated quantitatively, and their effects on cell membrane integrity and metabolic inactivation were assessed by propidium iodide and energy charge assays, respectively. The combination of 20% methanol and 0.9% sodium chloride (-4 °C), with the lowest propidium iodide labelling rate of 3.06 ± 0.16% and the highest energy charge value of 0.849 ± 0.003, outperformed most of its counterparts and was adopted for further use. To retrieve most of the cellular metabolites, four extraction solvent systems, including methyl tert-butyl ether/methanol/water, methanol/water, acetonitrile/water and acetonitrile/methnol/water, were evaluated. And acetonitrile/methnol/water was found to be the most efficient one as, among other solvents, it yielded the highest metabolite abundances for most of the metabolites. Furthermore, the protocol was subjected to analytical validation using a series of selected representative metabolites and yielded a linear range of 0.00128-100 µg mL-1 with coefficients of 0.9953-0.9999 and recovery levels of 90%-110%. These results suggest the reliability of the proposed method for L. plantarum ZS2058.


Assuntos
Lactobacillus plantarum/metabolismo , Metabolômica/métodos , Metabolômica/normas
19.
Front Immunol ; 8: 1345, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29097999

RESUMO

Recent evidence indicates that indigenous Clostridium species induce colonic regulatory T cells (Tregs), and gut lymphocytes are able to migrate to pancreatic islets in an inflammatory environment. Thus, we speculate that supplementation with the well-characterized probiotics Clostridium butyricum CGMCC0313.1 (CB0313.1) may induce pancreatic Tregs and consequently inhibit the diabetes incidence in non-obese diabetic (NOD) mice. CB0313.1 was administered daily to female NOD mice from 3 to 45 weeks of age. The control group received an equal volume of sterile water. Fasting glucose was measured twice a week. Pyrosequencing of the gut microbiota and flow cytometry of mesenteric lymph node (MLN), pancreatic lymph node (PLN), pancreatic and splenic immune cells were performed to investigate the effect of CB0313.1 treatment. Early oral administration of CB0313.1 mitigated insulitis, delayed the onset of diabetes, and improved energy metabolic dysfunction. Protection may involve increased Tregs, rebalanced Th1/Th2/Th17 cells and changes to a less proinflammatory immunological milieu in the gut, PLN, and pancreas. An increase of α4ß7+ (the gut homing receptor) Tregs in the PLN suggests that the mechanism may involve increased migration of gut-primed Tregs to the pancreas. Furthermore, 16S rRNA gene sequencing revealed that CB0313.1 enhanced the Firmicutes/Bacteroidetes ratio, enriched Clostridium-subgroups and butyrate-producing bacteria subgroups. Our results provide the basis for future clinical investigations in preventing type 1 diabetes by oral CB0313.1 administration.

20.
Appl Microbiol Biotechnol ; 101(22): 8063-8075, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28965220

RESUMO

Functional genomics of filamentous fungi has gradually uncovered gene information for constructing 'cell factories' and controlling pathogens. Available gene manipulation methods of filamentous fungi include random integration methods, gene targeting technology, gene editing with artificial nucleases and RNA technology. This review describes random gene integration constructed by restriction enzyme-mediated integration (REMI); Agrobacterium-mediated transformation (AMT); transposon-arrayed gene knockout (TAGKO); gene targeting technology, mainly about homologous recombination; and modern gene editing strategies containing transcription activator-like effector nucleases (TALENs) and a clustered regularly interspaced short palindromic repeat/associated protein system (CRISPR/Cas) developed in filamentous fungi and RNA technology including RNA interference (RNAi) and ribozymes. This review describes historical and modern gene manipulation methods in filamentous fungi and presents the molecular tools available to researchers investigating filamentous fungi. The biggest difference of this review from the previous ones is the addition of successful application and details of the promising gene editing tool CRISPR/Cas9 system in filamentous fungi.


Assuntos
Sistemas CRISPR-Cas , Fungos/genética , Técnicas Genéticas , Biologia Molecular/métodos , Elementos de DNA Transponíveis , Edição de Genes/métodos , Técnicas de Inativação de Genes , Marcação de Genes/métodos , Genômica , Recombinação Homóloga , Humanos , Interferência de RNA
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