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1.
Front Immunol ; 13: 1005426, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36203584

RESUMO

Mesenchymal stem cell (MSC) is a potential therapeutic material that has self-renewal, multilineage differentiation, and immunomodulation properties. However, the biological function of MSCs may decline due to the influence of donor differences and the in vitro expansion environment, which hinders the advancement of MSC-based clinical therapy. Here, we investigated a method for improving the immunomodulatory function of MSCs with the help of small-molecule compounds, A-83-01, CHIR99021, and Y27632 (ACY). The results showed that small-molecule induced MSCs (SM-MSCs) could enhance their immunosuppressive effects on T cells and macrophages. In vivo studies showed that, in contrast to control MSCs (Ctrl-MSCs), SM-MSCs could inhibit the inflammatory response in mouse models of delayed hypersensitivity and acute peritonitis more effectively. In addition, SM-MSCs showed the stronger ability to inhibit the infiltration of pro-inflammatory T cells and macrophages. Thus, small-molecule compounds ACY could better promote the immunomodulatory effect of MSCs, indicating it could be a potential improving method in MSC culture.


Assuntos
Imunomodulação , Células-Tronco Mesenquimais , Animais , Diferenciação Celular , Imunomodulação/fisiologia , Macrófagos , Camundongos , Linfócitos T
2.
Plant Physiol Biochem ; 192: 320-330, 2022 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-36302334

RESUMO

Melon fruits are popular because of sweet taste and pleasant aroma. Grafting has been extensively used for melons to alleviate abiotic stresses and control soil borne diseases. However, use of grafting for vegetable fruit quality improvement is less studies. In modern age fruit quality particularly sensory quality characteristics have key importance from consumer eye lens. We performed liquid chromatography-mass spectrometry and metabonomic analysis to examine sensory fruit quality of melon grafted onto ten different pumpkin rootstocks. Bases on the result of our study, 478 metabolites were detected and 184 metabolites consisting of lipids, amino acids and organic oxygen compounds were differentially expressed in grafted melon fruits. The results from metabolomic, physiochemical and sensory analysis explain the differences in melon fruit flavor from two contrasting rootstocks. In conclusion the fruits from Tianzhen No. 1 rootstock exhibited better organoleptic characteristics and higher soluble sugars content [glucose (19.87 mg/g), fructose (19.68 mg/g) and sucrose (169.45 mg/g)] compared with other rootstocks used in this study. Moreover, the contents of bitterness causing amino acids such as L-arginine, L-asparagine, Histidinyl-histidine and Acetyl-DL-valine were found lower in Tianzhen No. 1-grafted melon fruits compared with Sizhuang No. 12-grafted melon fruits. These fruit quality characteristics made Tianzhen No. 1 rootstock suitable for commercial cultivation of Yuniang melon.

3.
Front Bioeng Biotechnol ; 10: 819148, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35360405

RESUMO

Mesenchymal stem cells (MSCs) have a variety of unique properties, such as stem cell multipotency and immune regulation, making them attractive for use in cell therapy. Before infusion therapy, MSCs are required to undergo tissue separation, purification, and expansion in vitro for a certain duration. During the process of in vitro expansion of MSCs, the influence of culture time and environment can lead to cell senescence, increased heterogeneity, and function attenuation, which limits their clinical applications. We used a cocktail of three small-molecule compounds, ACY (A-83-01, CHIR99021, and Y-27632), to increase the proliferation activity of MSCs in vitro and reduce cell senescence. ACY inhibited the increase in heterogeneity of MSCs and conserved their differentiation potential. Additionally, ACY maintained the phenotype of MSCs and upregulated the expression of immunomodulatory factors. These results suggest that ACY can effectively improve the quantity and quality of MSCs.

4.
J Exp Bot ; 73(8): 2275-2289, 2022 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-35139196

RESUMO

The flux, distribution, and storage of soluble sugars regulate crop yield in terms of starch, oil, protein, and total carbohydrates, and affect the quality of many horticultural products. Sugar transporters contribute to phloem loading and unloading. The mechanisms of phloem loading have been studied in detail, but the complex and diverse mechanisms of phloem unloading and sugar storage in sink organs are less explored. Unloading and subsequent transport mechanisms for carbohydrates vary in different sink organs. Analyzing the transport and storage mechanisms of carbohydrates in important storage organs, such as cereal seeds, fruits, or stems of sugarcane, will provide information for genetic improvements to increase crop yield and fruit quality. This review discusses current research progress on sugar transporters involved in carbohydrate unloading and storage in sink organs. The roles of sugar transporters in crop yield and the accumulation of sugars are also discussed to highlight their contribution to efficient breeding.


Assuntos
Frutas , Regulação da Expressão Gênica de Plantas , Transporte Biológico/fisiologia , Carboidratos , Frutas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Floema/metabolismo , Melhoramento Vegetal , Sacarose/metabolismo , Açúcares/metabolismo
6.
Eur J Med Chem ; 229: 114067, 2022 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-34973507

RESUMO

Drugs have been largely inspired from natural products, while enzymes underlying their biosynthesis have enabled complex structures and diverse bioactivities. Nevertheless, the high enzyme specificity and limited in vivo precursor types have restricted the natural product reservoir, but Nature has imprinted natural products with active sites, which can be readily modified by chemosynthesis with various functional groups for more favorable druggability. Here in the less exploited fungal natural products, we introduced CtvA, a polyketide synthase for a mycotoxin citreoviridin biosynthesis in Aspergillus, into an endophytic fungus Calcarisporium arbuscula to expand tetrahydrofuran (THF) into a dioxabicyclo-octane (DBO) ring moiety based on versatility and promiscuity of the aurovertin biosynthetic enzyme. Alternative acylations on the hydroxyl groups essential for cell toxicity by chemosynthesis produced compounds with improved anti-tumor activities and pharmacokinetics. Thus, we showed an effective strategic way to optimize the fungal natural product efficiently for more promising drug development.


Assuntos
Antineoplásicos/química , Aurovertinas/química , Produtos Biológicos/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Octanos/química , Policetídeo Sintases/metabolismo , Acilação , Antineoplásicos/farmacocinética , Aspergillus , Produtos Biológicos/farmacocinética , Proliferação de Células , Furanos/química , Humanos , Hypocreales , Micotoxinas/metabolismo
7.
ACS Synth Biol ; 11(2): 547-553, 2022 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-35061355

RESUMO

There is a growing interest in establishing the methylotrophic yeast Pichia pastoris as microbial cell factories for producing fuels, chemicals, and natural products, particularly with methanol as the feedstock. Although CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based genome editing technology has been established for the integration of multigene biosynthetic pathways, long (500-1000 bp) homology arms are generally required, probably due to low homologous recombination (HR) efficiency in P. pastoris. To achieve efficient genome integration of heterologous genes with short homology arms, we aimed to enhance HR efficiency by introducing the recombination machinery from Saccharomyces cerevisiae. First, we overexpressed HR related genes, including RAD52, RAD59, MRE11, and SAE2, and evaluated their effects on genome integration efficiency. Then, we constructed HR efficiency enhanced P. pastoris, which enabled single-, two-, and three-loci integration of heterologous gene expression cassettes with ∼40 bp homology arms with efficiencies as high as 100%, ∼98%, and ∼81%, respectively. Finally, we demonstrated the construction of ß-carotene producing strain and the optimization of betaxanthin producing strain in a single step. The HR efficiency enhanced P. pastoris strains can be used for the construction of robust cell factories, and our machinery engineering strategy can be employed for the modification of other nonconventional yeasts.


Assuntos
Edição de Genes , Pichia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Recombinação Homóloga , Pichia/genética , Pichia/metabolismo , Saccharomycetales
8.
ACS Synth Biol ; 10(4): 698-706, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33720696

RESUMO

Fungal natural products are rich sources of clinical drugs. Particularly, the fungicolous fungi have a large number of biosynthetic gene clusters (BGCs) to produce numerous bioactive natural products, but most BGCs are silent in the laboratory. We have shown that a fungicolous fungus Calcarisporiumarbuscula NRRL 3705 predominantly produces the highly reduced polyketide-type mycotoxins aurovertins. Here after evaluation of the aurovertin-null mutant ΔaurA as an efficient host, we further screened two strong promoters aurBp and A07068p based on RNA-Seq, and successfully activated an endogenous gene cluster from C. arbuscula as well as three additional exogenous BGCs from other fungi to produce polyketide-type natural products. Thus, we showed an efficient expression system from the fungicolous fungus C. arbuscula, which will be highly beneficial and complementary to the conventional Aspergillus and Penicillium fungal cell factories, and provides a useful toolkit for genome-wide mining of bioactive natural products from fungicolous fungi.


Assuntos
Produtos Biológicos/metabolismo , Hypocreales/metabolismo , Aspergillus/genética , Hypocreales/genética , Família Multigênica/genética , Família Multigênica/fisiologia , Penicillium/genética
9.
J Agric Food Chem ; 69(38): 11303-11310, 2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34542281

RESUMO

Liver fibrosis has accounted for liver diseases and overall mortality, but no relevant drug has been developed. Filamentous fungi are important resources of natural products for pharmaceutical development. Calcarisporium arbuscula is a mushroom endophytic fungus, which primarily produces aurovertins. Here, in an aurovertin null-production mutant, one silent gene cluster (mca17) was activated by overexpression of a pathway-specific zinc finger transcriptional regulator, and a tetramic acid-type compound (1, MCA17-1) was identified. Along with detailed structural characterization, its biosynthesis was proposed to be produced from the core PKS-NRPS hybrid enzyme. Moreover, 1 suppressed the activation of LX-2 upon transforming growth factor-ß (TGF-ß) challenge and had stronger bioactivity than the positive control obeticholic acid (OCA) against liver fibrosis. Our work suggested that this engineered fungus could be a producer of 1 for promising pharmaceutical development, and alternatively, it would be developed as a mushroom ingredient in dietary therapy to prevent liver fibrosis.


Assuntos
Agaricales , Hypocreales , Agaricales/genética , Humanos , Hypocreales/genética , Cirrose Hepática/genética , Família Multigênica
10.
BMC Genomics ; 21(1): 424, 2020 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-32580753

RESUMO

BACKGROUND: Secondary metabolites as natural products from endophytic fungi are important sources of pharmaceuticals. However, there is currently little understanding of endophytic fungi at the omics levels about their potential in secondary metabolites. Calcarisporium arbuscula, an endophytic fungus from the fruit bodies of Russulaceae, produces a variety of secondary metabolites with anti-cancer, anti-nematode and antibiotic activities. A comprehensive survey of the genome and transcriptome of this endophytic fungus will help to understand its capacity to biosynthesize secondary metabolites and will lay the foundation for the development of this precious resource. RESULTS: In this study, we reported the high-quality genome sequence of C. arbuscula NRRL 3705 based on Single Molecule Real-Time sequencing technology. The genome of this fungus is over 45 Mb in size, larger than other typical filamentous fungi, and comprises 10,001 predicted genes, encoding at least 762 secretory-proteins, 386 carbohydrate-active enzymes and 177 P450 enzymes. 398 virulence factors and 228 genes related to pathogen-host interactions were also predicted in this fungus. Moreover, 65 secondary metabolite biosynthetic gene clusters were revealed, including the gene cluster for the mycotoxin aurovertins. In addition, several gene clusters were predicted to produce mycotoxins, including aflatoxin, alternariol, destruxin, citrinin and isoflavipucine. Notably, two independent gene clusters were shown that are potentially involved in the biosynthesis of alternariol. Furthermore, RNA-Seq assays showed that only expression of the aurovertin gene cluster is much stronger than expression of the housekeeping genes under laboratory conditions, consistent with the observation that aurovertins are the predominant metabolites. Gene expression of the remaining 64 gene clusters for compound backbone biosynthesis was all lower than expression of the housekeeping genes, which partially explained poor production of other secondary metabolites in this fungus. CONCLUSIONS: Our omics data, along with bioinformatics analysis, indicated that C. arbuscula NRRL 3705 contains a large number of biosynthetic gene clusters and has a huge potential to produce a profound number of secondary metabolites. This work also provides the basis for development of endophytic fungi as a new resource of natural products with promising biological activities.


Assuntos
Proteínas Fúngicas/genética , Perfilação da Expressão Gênica/métodos , Hypocreales/genética , Sequenciamento Completo do Genoma/métodos , Vias Biossintéticas , Regulação Fúngica da Expressão Gênica , Tamanho do Genoma , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Hypocreales/classificação , Hypocreales/metabolismo , Filogenia , Metabolismo Secundário , Análise de Sequência de RNA , Imagem Individual de Molécula
11.
Artigo em Inglês | MEDLINE | ID: mdl-32140461

RESUMO

Precise regulation of gene expression is fundamental for tailor-made gene circuit design in synthetic biology. Current strategies for this type of development are mainly based on directed evolution beginning with a native promoter template. The performances of engineered promoters are usually limited by the growth phase because only one promoter is recognized by one type of sigma factor (σ). Here, we constructed multiple-σ recognizable artificial hybrid promoters (AHPs) composed of tandems of dual and triple natural minimal promoters (NMPs). These NMPs, which use σA, σH and σW, had stable functions in different growth phases. The functions of these NMPs resulted from an effect called transcription compensation, in which AHPs sequentially use one type of σ in the corresponding growth phase. The strength of the AHPs was influenced by the combinatorial order of each NMP and the length of the spacers between the NMPs. More importantly, the output of the precise regulation was achieved by equipping AHPs with synthetic ribosome binding sites and by redesigning them for induced systems. This strategy might offer promising applications to rationally design robust synthetic promoters in diverse chassis to spur the construction of more complex gene circuits, which will further the development of synthetic biology.

12.
Plant Physiol Biochem ; 145: 107-113, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31677541

RESUMO

Hexose transporters play many important roles in plant development. However, the role of hexose transporter in secondary cell wall growth has not been reported before. Here, we report that the hexose transporter gene CsHT3 is mainly expressed in cells with secondary cell walls in cucumber. Spatiotemporal expression analysis revealed that the transcript of CsHT3 mainly accumulates in the stem, petiole, tendril, and peduncle, all of which contain high cellulose levels. Immunolocalization results show that CsHT3 is localized at the sclereids in young peduncles, shifts to the phloem fiber cells during peduncle development, and then shifts again to the companion cells when the development of secondary cell walls is almost completed. Carboxyfluoresce unloading experiment indicated that carbohydrate unloading in the phloem follows an apoplastic pathway. Overexpression of CsHT3 in cucumber plant can improve the cellulose content and cell wall thickness of phloem fiber cells in the peduncle. The expression of cellulose synthase genes were increased in the CsHT3 overexpression plants. These results indicated that CsHT3 may play an important role in cellulose synthesis through promoting the expression of cellulose synthase genes.


Assuntos
Cucumis sativus , Frutas , Regulação da Expressão Gênica de Plantas , Proteínas de Transporte de Monossacarídeos , Proteínas de Plantas , Parede Celular , Celulose/genética , Celulose/metabolismo , Cucumis sativus/genética , Cucumis sativus/metabolismo , Frutas/química , Frutas/genética , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Floema , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
13.
Exp Mol Pathol ; 108: 17-23, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30853612

RESUMO

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. The journal was initially contacted by the corresponding author to report that the article was submitted without the consent of an author. Given the comments of Dr Elisabeth Bik regarding this article "… the Western blot bands in all 400+ papers are all very regularly spaced and have a smooth appearance in the shape of a dumbbell or tadpole, without any of the usual smudges or stains. All bands are placed on similar looking backgrounds, suggesting they were copy/pasted from other sources, or computer generated ", the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request and therefore the Editor-in-Chief decided to retract the article.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ginsenosídeos/farmacologia , MicroRNAs/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Retinoblastoma/genética , Retinoblastoma/metabolismo , Retinoblastoma/patologia , Serina-Treonina Quinases TOR/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacos
14.
Int J Mol Sci ; 19(9)2018 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-30200653

RESUMO

Soil salinity adversely affects the growth and yield of crops, including cucumber, one of the most important vegetables in the world. Grafting with salt-tolerant pumpkin as the rootstock effectively improves the growth of cucumber under different salt conditions by limiting Na⁺ transport from the pumpkin rootstock to the cucumber scion. High-affinity potassium transporters (HKTs) are crucial for the long distance transport of Na⁺ in plants, but the function of pumpkin HKTs in this process of grafted cucumber plants remains unclear. In this work, we have characterized CmHKT1;1 as a member of the HKT gene family in Cucurbita moschata and observed an obvious upregulation of CmHKT1;1 in roots under NaCl stress conditions. Heterologous expression analyses in yeast mutants indicated that CmHKT1;1 is a Na⁺-selective transporter. The transient expression in tobacco epidermal cells and in situ hybridization showed CmHKT1;1 localization at plasma membrane, and preferential expression in root stele. Moreover, ectopic expression of CmHKT1;1 in cucumber decreased the Na⁺ accumulation in the plants shoots. Finally, the CmHKT1;1 transgenic line as the rootstock decreased the Na⁺ content in the wild type shoots. These findings suggest that CmHKT1;1 plays a key role in the salt tolerance of grafted cucumber by limiting Na⁺ transport from the rootstock to the scion and can further be useful for engineering salt tolerance in cucurbit crops.


Assuntos
Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Cucumis sativus/crescimento & desenvolvimento , Cucurbita/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sódio/metabolismo , Simportadores/genética , Simportadores/metabolismo , Membrana Celular/metabolismo , Cucumis sativus/genética , Cucurbita/genética , Cucurbita/metabolismo , Regulação da Expressão Gênica de Plantas , Folhas de Planta/metabolismo , Brotos de Planta/genética , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Tolerância ao Sal , Regulação para Cima
15.
Microb Biotechnol ; 11(5): 930-942, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29984489

RESUMO

Nattokinase (NK) is an important serine-protease with direct fibrinolytic activity involving the prevention of cardiovascular disease as an antithrombotic agent. Dozens of studies have focused on the characterization of intrinsic novel promoters and signal peptides to the secretory production of recombinant proteins in Bacillus subtilis. However, intrinsic genetic elements have several drawbacks, which cannot mediate the production of NK to the desired level. In this study, the genetic elements, which were used to overproduce the recombinant secretory NK, were rationally modified in B. subtilis in a stepwise manner. The first step was to select a suitable signal peptide for the highly efficient secretion of NK. By comparison of the secretory levels mediated by two different signal peptides, which were encoded by the genes of a minor extracellular protease epr (SPepr ) and cell-wall associated protease wapA (SPwapA ), respectively, SPwapA was verified as the superior secretory element. Second, P04, which was a synthetic promoter screened from an array of mutants based on the promoter cloned from the operon of a quorum-sensing associated gene srfA (PsrfA ), was paired to SPwapA. The secretory level of NK was obviously augmented by the combination of these two genetic elements. Third, the cis-acting element CodY-binding sequence positioned at the 5'UTR was deleted (yielding P08), and thus the secretory level was significantly elevated. The activity of NK, which was defined as fibrinolytic units (FU), reached to a level of 270 FU ml-1 . Finally, the superior genetic element composed of P08 and SPwapA was utilized to overproduce NK in the host B. subtilis WB800, which was able to produce the secretory NK at 292 FU ml-1 . The strategy established in this study can not only be used to overproduce NK in B. subtilis but also might be a promising pipeline to modify the genetic element for the synthetic secretory system.


Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Engenharia Metabólica/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Subtilisinas/genética , Subtilisinas/metabolismo , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas/genética , Transporte Proteico
16.
J Microbiol Methods ; 151: 1-6, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29859215

RESUMO

Filamentous fungi are emerging as attractive producers of natural products with novel structures, diverse bioactivities and unprecedented enzymology. But their genetic systems are poorly developed, especially in some non-model endogenic fungi, which have hampered our genetic manipulation of their natural product development. Calcarisporium arbuscula NRRL 3705 is an endophytic filamentous fungus rich in biosynthetic gene clusters and primarily producing mycotoxin aurovertins. Here we optimized Agrobacterium tumefaciens-mediated transformation (ATMT)-based efficient DNA introduction into C. arbuscula. By complementation of the monooxygenase gene aurC in ΔaurC mutant as a model, we showed that a strong but down-regulated promoter aurAp and three strong constitutive promoter gpdAp, tef1p and tubCp could be used for gene overexpression. Meanwhile, red fluorescence protein (RFP) was expressed in this fungus under the control of tubCp, potentially paving the way for enzyme localization determination during natural product biosynthesis. Furthermore, we developed efficient and convenient gene disruption in C. arbuscula based on ATMT, as exemplified by deletion of aurA in ΔaurC mutant. Our efficiency of deletion ran at about 40%. These results suggest that ATMT-based transformation for gene ectopic expression or deletion is an efficient strategy for genetic manipulation of C. arbuscula, and can be readily adapted to other rare filamentous fungi, potentially to promote discovery and development of natural products.


Assuntos
Expressão Ectópica do Gene , Genes Fúngicos/genética , Hypocreales/genética , Família Multigênica , Transformação Genética , Agrobacterium tumefaciens/genética , Regulação para Baixo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Técnicas de Silenciamento de Genes , Oxigenases de Função Mista
17.
J Exp Bot ; 69(3): 511-523, 2018 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-29309616

RESUMO

Fruits are an important part of the human diet and sugar content is a major criterion used to evaluate fruit quality. Melon fruit accumulate high sugar concentrations during their development; however, the mechanism through which these sugars are transported into the vacuoles of fruit cells for storage remains unclear. In this study, three tonoplast sugar transporters (TSTs), CmTST1, CmTST2, and CmTST3, were isolated from melon plants. Analysis of subcellular localization revealed that all these proteins were targeted to the tonoplast, and evaluation of spatial expression and promoter-GUS activity indicated that they had different expression patterns in the plant. RT-PCR and qRT-PCR results indicated that CmTST2 exhibited the highest expression level among the TST isoforms during melon fruit development. Histochemical and immunohistochemistry localization experiments were performed to identify the tissue- and cell-type localization of CmTST2 in the fruit, and the results indicated that both its transcription and translation were in the mesocarp and vascular cells. Overexpressing the CmTST2 gene in strawberry fruit and cucumber plants by transient expression and stable transformation, respectively, both increased sucrose, fructose, and glucose accumulation in the fruit. The results indicate that CmTST2 plays an important role in sugar accumulation in melon fruit.


Assuntos
Cucumis melo/genética , Fragaria/metabolismo , Expressão Gênica , Proteínas de Plantas/genética , Açúcares/metabolismo , Cucumis melo/metabolismo , Fragaria/genética , Frutose/metabolismo , Frutas/metabolismo , Glucose/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Sacarose/metabolismo
18.
Exp Ther Med ; 14(1): 791-796, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28673001

RESUMO

Glaucoma is a major cause of irreversible blindness. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) regulates the expression of numerous antioxidants within cells and is therefore a focus of current ophthalmic research. To determine the roles of Nrf2 in mediating the glaucoma trabecular meshwork (GTM), the present study evaluated the levels of Nrf2 expression in GTM and human trabecular meshwork (HTM) cells by reverse-transcription-quantitative polymerase chain reaction and western blot analysis. It was principally observed that Nrf2 expression was downregulated in GTM cells. In addition, to determine the influence of Nrf2 on the apoptosis and proliferation of GTM and HTM cells, transfection assays and western blotting were performed to evaluate the expression of apoptosis-related proteins. The results of the current study indicated that Nrf2 may promote viability and reduce apoptosis in GTM and HTM cells. Collectively, these data suggest that Nrf2 may be a novel therapeutic target to treat glaucoma.

19.
Appl Microbiol Biotechnol ; 101(5): 2107-2120, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27986992

RESUMO

Tuneable gene expression controlled by synthetic biological elements is of great importance to biotechnology and synthetic biology. The synthetic riboswitch is a pivotal type of elements that can easily control the heterologous gene expression in diverse bacteria. In this study, the theophylline-dependent synthetic riboswitch and the corresponding variants with varied spacings between Shine-Dalgarno (SD) sequence and start codon were employed to comprehensively characterize the induction and regulation properties through combining a strong promoter aprE in Bacillus subtilis. Amongst the sets of newly constructed expression elements, the expression element with 9-bp spacing exhibited the higher expression level, a superior induction fold performance, and a considerably lower leaky expression than those with longer or shorter spacings. The riboswitch expression element with 9-bp spacing showed an approximately linear dose dependence from 0 to 8 mM of theophylline. Modification of the SD sequence through the insertion of a single A base prior to the native sequence enables the increase of the expression level post induction while decreasing the induction fold as a result of the elevated leaky level. The riboswitch elements with the engineered SD and the optimal 9-bp spacing exhibit an altered dose dependency in which the approximately linear range shifts to 0-4 mM, although it has a similar profile to the induction process. These results not only provide comprehensive data for the induced expression by a theophylline riboswitch combined with a strong native promoter from B. subtilis but also provide the two pivotal features of SD essential to the modular design of other synthetic riboswitches.


Assuntos
Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Riboswitch/efeitos dos fármacos , Teofilina/farmacologia , Proteínas de Bactérias/genética , Sequência de Bases/genética , Códon de Iniciação/genética , Elongação Traducional da Cadeia Peptídica/genética , Regiões Promotoras Genéticas/genética , Riboswitch/genética
20.
Sci Rep ; 6: 37566, 2016 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-27917866

RESUMO

Human UC-MSCs are regarded as an attractive alternative to BM-MSCs for clinical applications due to their easy preparation, higher proliferation and lower immunogenicity. However, the mechanisms underlying immune suppression by UC-MSCs are still unclear. We studied the mechanism of inhibition by UC-MSCs during the differentiation of monocytes into DCs and focused on the specific source and the role of the involved cytokines. We found that UC-MSCs suppressed monocyte differentiation into DCs and instructed monocytes towards other cell types, with clear decreases in the expression of co-stimulatory molecules, in the secretion of inflammatory factors and in allostimulatory capacity. IL6, HGF and IL10 might be involved in this process because they were detected at higher levels in a coculture system. UC-MSCs produce IL-6 and HGF, and neutralization of IL-6 and HGF reversed the suppressive effect of UC-MSCs. IL10 was not produced by UC-MSCs but was exclusively produced by monocytes after exposure to UC-MSCs, IL-6 or HGF. In summary, we found that the UC-MSC-mediated inhibitory effect was dependent on IL6 and HGF secreted by UC-MSCs and that this effect induced monocyte-derived cells to produce IL10, which might indirectly strengthen the suppressive effect of UC-MSCs.


Assuntos
Comunicação Celular , Sangue Fetal/metabolismo , Fator de Crescimento de Hepatócito/imunologia , Interleucina-10/imunologia , Interleucina-6/imunologia , Células-Tronco Mesenquimais/metabolismo , Monócitos/metabolismo , Diferenciação Celular , Técnicas de Cocultura , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Sangue Fetal/citologia , Regulação da Expressão Gênica , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Células-Tronco Mesenquimais/citologia , Monócitos/citologia , Fenótipo , Cultura Primária de Células , Transdução de Sinais
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