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1.
Microb Cell Fact ; 18(1): 162, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31581942

RESUMO

BACKGROUND: Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. RESULTS: Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR-Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR-Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR-Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. CONCLUSIONS: This study applied CRISPR-Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR-Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.


Assuntos
Edição de Genes/métodos , Genoma Bacteriano , Zymomonas/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/metabolismo , Francisella/enzimologia , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Zymomonas/metabolismo
2.
Microb Cell Fact ; 18(1): 163, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31581944

RESUMO

BACKGROUND: Sustainable production of microbial fatty acids derivatives has the potential to replace petroleum based equivalents in the chemical, cosmetic and pharmaceutical industry. Most fatty acid sources for production oleochemicals are currently plant derived. However, utilization of these crops are associated with land use change and food competition. Microbial oils could be an alternative source of fatty acids, which circumvents the issue with agricultural competition. RESULTS: In this study, we generated a chimeric microbial production system that features aspects of both prokaryotic and eukaryotic fatty acid biosynthetic pathways targeted towards the generation of long chain fatty acids. We redirected the type-II fatty acid biosynthetic pathway of Escherichia coli BL21 (DE3) strain by incorporating two homologues of the beta-ketoacyl-[acyl carrier protein] synthase I and II from the chloroplastic fatty acid biosynthetic pathway of Arabidopsis thaliana. The microbial clones harboring the heterologous pathway yielded 292 mg/g and 220 mg/g DCW for KAS I and KAS II harboring plasmids respectively. Surprisingly, beta-ketoacyl synthases KASI/II isolated from A. thaliana showed compatibility with the FAB pathway in E. coli. CONCLUSION: The efficiency of the heterologous plant enzymes supersedes the overexpression of the native enzyme in the E. coli production system, which leads to cell death in fabF overexpression and fabB deletion mutants. The utilization of our plasmid based system would allow generation of plant like fatty acids in E. coli and their subsequent chemical or enzymatic conversion to high end oleochemical products.


Assuntos
Arabidopsis/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/biossíntese , Engenharia Metabólica , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/síntese química , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/síntese química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Vias Biossintéticas , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Ácido Graxo Sintases/genética , Ácidos Graxos/química , Isoenzimas/síntese química , Isoenzimas/genética , Isoenzimas/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo
3.
Bioengineered ; 10(1): 335-344, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31322471

RESUMO

Selenium-enriched yeast can transform toxic inorganic selenium into absorbable organic selenium, which is of great significance for human health and pharmaceutical industry. A yeast Rhodotorula glutinis X-20 we obtained before has good selenium-enriched ability, but its selenium content is still low for industrial application. In this study, strategies of process optimization and transport regulation of selenium were thus employed to further improve the cell growth and selenium enrichment. Through engineering phosphate transporters from Saccharomyces cerevisiae into R. glutinis X-20, the selenium content was increased by 21.1%. Through using mixed carbon culture (20 g L-1, glycerol: glucose 3:7), both biomass and selenium content were finally increased to 5.3 g L-1 and 5349.6 µg g-1 (cell dry weight, DWC), which were 1.14 folds and 6.77 folds compared to their original values, respectively. Our results indicate that high selenium-enrichment ability and biomass production can be achieved through combining process optimization and regulation of selenium transport.


Assuntos
Engenharia Metabólica/métodos , Fosfatos/metabolismo , Rhodotorula/genética , Saccharomyces cerevisiae/genética , Selênio/metabolismo , Transgenes , Transporte Biológico , Biomassa , Meios de Cultura/química , Meios de Cultura/farmacologia , Fermentação , Expressão Gênica , Glucose/química , Glucose/metabolismo , Glicerol/química , Glicerol/metabolismo , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Simportadores de Próton-Fosfato/genética , Simportadores de Próton-Fosfato/metabolismo , Rhodotorula/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismo
4.
Chem Commun (Camb) ; 55(57): 8227-8230, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31268107

RESUMO

An original family of multivalent vectors encompassing gemini and facial amphiphilicity, namely cationic Siamese twin surfactants, has been prepared from the disaccharide trehalose; molecular engineering lets us modulate the self-assembling properties and the topology of the nanocomplexes with plasmid DNA for efficient gene delivery in vitro and in vivo.


Assuntos
Nanoestruturas/química , Plasmídeos/química , Tensoativos/química , Transfecção/métodos , Trealose/química , Animais , Linhagem Celular , Cercopithecus aethiops , Humanos , Camundongos , Plasmídeos/metabolismo
5.
Gene ; 712: 143958, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278963

RESUMO

The Wnt signaling pathway has been identified for its function in carcinogenesis and embryonic development. It is known to play a vital role in the initiation and development of colorectal cancer (CRC). Therefore, it is of great importance for CRC research to illuminate the mechanisms which regulate Wnt pathway activity. Here, we intended to examine the effect of hsa-miR-942 (miR-942) on the Wnt signaling activity, cell cycle progression, and its expression in CRC tissues. RT-qPCR results indicated that miR-942 is significantly upregulated in colorectal cancer. Then, overexpression of miR-942 promoted, whereas its inhibition decreased the Wnt signaling activity, detected by RT-qPCR and Top/Fop flash assay. Inhibition of Wnt signaling by using PNU-74654 or IWP-2 small molecules indicated that miR-942 applies its effect to the ß-catenin degradation complex level. Then, RT-qPCR and dual luciferase assay showed that miR-942 upregulated Wnt signaling through direct targeting of APC, which is a tumor suppressor in Wnt signaling pathway. Furthermore, the western blotting analysis indicated that ß.catenin, as a main member of Wnt signaling pathway is upregulated following the overexpression of miR-942. Finally, miR-942 overexpression resulted in cell cycle progression in SW480 cells. Taken together, our findings established an oncogenic role for miR-942 in CRC and indicated that this miRNA might be a crucial target for CRC therapy.


Assuntos
Neoplasias Colorretais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Via de Sinalização Wnt , Regiões 3' não Traduzidas , Carcinogênese/genética , Ciclo Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Biologia Computacional , Ciclina D1/metabolismo , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Luciferases/metabolismo , Masculino , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , RNA Longo não Codificante/genética , Transdução de Sinais , Regulação para Cima , beta Catenina/metabolismo
6.
World J Microbiol Biotechnol ; 35(8): 119, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332541

RESUMO

The microalgal genus of Nannochloropsis is considered one of the most promising organisms for the production of biofuels due to their high lipid content. Transformation systems for marine Nannochloropsis species have been established in the recent decade, however, genetic manipulation of Nannochloropsis limnetica, the only known freshwater species in this genus, is not yet available. Based on established marine Nannochloropsis species electrotransformation protocol, nuclear genetic transformation was established in N. limnetica, meanwhile the appropriate antibiotic selection concentration and electric field strength of electroporation were determined. For the selection of transformants in N. limnetica on plates, 0.07 µg mL-1 of zeocin or 5 µg mL-1 of hygromycin B was proved sufficient, and the transformation efficiency was < 2 × 10-8 with a single pulse ranging from 2200 to 2600 V using 2-mm electroporation cuvettes. Pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundreds of times, and the highest transformation efficiency of 10-11 × 10-6 was obtained with an electric field strength of 12,000 V/cm. Our results help to expand the biotechnological applications of this freshwater species and provide means for successful electrotransformation of other microalgae as well. High-efficiency transformation of freshwater Nannochloropsis pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundreds of times.


Assuntos
Eletroporação , Água Doce/microbiologia , Microalgas/metabolismo , Estramenópilas/metabolismo , Acetatos , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microalgas/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Estramenópilas/genética , Transformação Genética
7.
Nat Commun ; 10(1): 2948, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270316

RESUMO

CRISPR-Cas systems inherently multiplex through CRISPR arrays-whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Biblioteca Gênica , Técnicas Genéticas , RNA/biossíntese , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , DNA/genética , Endonucleases/metabolismo , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Plasmídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo
8.
Int J Nanomedicine ; 14: 4353-4366, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31354265

RESUMO

Purpose: Gene therapy has become a promising remedy to treat disease by modifying the person's genes. The therapeutic potential of related tools such as CRISPR-Cas9 depends on the efficiency of delivery to the targeted cells. Numerous transfection reagents have been designed and lots of efforts have been devoted to develop carriers for this purpose. Therefore, the aim of the present study was to develop novel cholesterol-rich lipid-based nanoparticles to enhance transfection efficiency and serum stability. Materials and methods: We constructed two-, three- and four-component cationic liposomes (CLs) to evaluate the combined effect of cholesterol domain and DOPE (dioleoyl phosphatidylethanolamine), a fusogenic lipid, and the PEG (polyethylene glycol) moiety location inside or outside of the cholesterol domain on transfection efficiency and other properties of the particle. Lipoplex formation and pDNA (plasmid DNA) entrapment were assessed by gel retardation assay at different N/P ratios (3, 5, 7). Physicochemical characteristics, cytotoxicity, serum stability and endosomal escape capability of the lipoplexes were studied and transfection potential was measured by firefly luciferase assay. Next, HEK293 cell line stably expressing GFP was utilized to demonstrate the editing of a reporter through Cas9 and sgRNA plasmids delivery by the selected CL formula, which showed the highest transfection efficiency. Results: Among the designed CLs, the four-component formula [DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane)/DOPE/cholesterol/Chol-PEG (cholesterol-polyethylene glycol)] showed the highest rate of transfection at N/P 3. Finally, transfection of Cas9/sgRNA by this formulation at N/P 3 resulted in 39% gene-editing efficiency to knockout GFP reporter. The results also show that this CL with no cytotoxicity effect can totally protect the plasmids from enzymatic degradation in serum. Conclusion: The novel PEGylated cholesterol domain lipoplex providing serum stability, higher transfection efficiency and endosomal release can be used for in vivo Cas9/sgRNA delivery and other future gene-therapy applications.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Colesterol/química , Edição de Genes , Nanopartículas/química , Transfecção/métodos , Cátions/química , Morte Celular , Colesterol/análogos & derivados , Ensaio de Desvio de Mobilidade Eletroforética , Endossomos/metabolismo , Ácidos Graxos Monoinsaturados/química , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Lipossomos/química , Tamanho da Partícula , Fosfatidiletanolaminas/química , Plasmídeos/metabolismo , Polietilenoglicóis/química , Compostos de Amônio Quaternário/química , RNA Guia/metabolismo , Eletricidade Estática
9.
Int J Nanomedicine ; 14: 4123-4131, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31239671

RESUMO

Purpose: The objective of this work was to formulate a delivery system of pDNA encoded p53 gene-loaded chitosan-sodium deoxycholate (CS-DS) nanoparticles, and to evaluate their influence on in vitro cytotoxicity and transfection efficiency of p53 gene. Methods: The prepared pDNA-loaded CS-DS nanoparticles were evaluated for morphology, particle size, zeta potential, entrapment efficiency %, in vitro release, in vitro cytotoxicity, and transfection efficiency. Results: The mean particle size ranged from from 96.5 ± 11.31 to 405 ± 46.39 nm. All nanoparticles had good positive zeta potential values. Entrapment efficiency % ranged from 38.25 ± 3.25 to 94.89 ± 5.67. The agarose gel electrophoresis confirmed the strong binding between plasmid and CS. The in vitro pDNA release from nanoparticles exhibited an initial burst effect followed by a sustained drug release over a period of 6 days. In vitro cytotoxicity against human Caco-2 cells showed low cell cytotoxicity of plain CS-DS nanoparticles, while pDNA-loaded CS-DS nanoparticles showed a cytotoxic effect with increasing nanoparticles' concentration. Gene transfection, analyzed by PCR and ELISA, showed a direct correlation between gene expression and concentration of pDNA. The highest expression of the gene was achieved with pDNA concentration of 9 µg/mL with 5.7 times increase compared to naked pDNA of the same concentration. Conclusion: The obtained results were very encouraging and offer an alternative approach to enhancing the transfection efficiency of genetic material-loaded chitosan-based delivery systems.


Assuntos
Quitosana/química , DNA/genética , Ácido Desoxicólico/química , Nanopartículas/química , Plasmídeos/metabolismo , Transfecção , Proteína Supressora de Tumor p53/genética , Células CACO-2 , Morte Celular , Liberação Controlada de Fármacos , Humanos , Nanopartículas/ultraestrutura , Tamanho da Partícula , Eletricidade Estática
10.
Emerg Microbes Infect ; 8(1): 857-865, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31169081

RESUMO

Ciprofloxacin resistance in Salmonella has been increasingly reported due to the emergence and dissemination of multiple Plasmid-Mediated Quinolone Resistance (PMQR) determinants, which are mainly located in non-conjugative plasmids or chromosome. In this study, we aimed to depict the molecular mechanisms underlying the rare phenomenon of horizontal transfer of ciprofloxacin resistance phenotype in Salmonella by conjugation experiments, S1-PFGE and complete plasmid sequencing. Two types of non-conjugative plasmids, namely an IncX1 type carrying a qnrS1 gene, and an IncH1 plasmid carrying the oqxAB-qnrS gene, both ciprofloxacin resistance determinants in Salmonella, were recovered from two Salmonella strains. Importantly, these non-conjugative plasmids could be fused with a novel Incl1 type conjugative helper plasmid, which could target insertion sequence (IS) elements located in the non-conjugative, ciprofloxacin-resistance-encoding plasmid through replicative transcription, eventually forming a hybrid conjugative plasmid transmissible among members of Enterobacteriaceae. Since our data showed that such conjugative helper plasmids are commonly detectable among clinical Salmonella strains, particularly S. Typhimurium, fusion events leading to generation and enhanced dissemination of conjugative ciprofloxacin resistance-encoding plasmids in Salmonella are expected to result in a sharp increase in the incidence of resistance to fluoroquinolone, the key choice for treating life-threatening Salmonella infections, thereby posing a serious public health threat.


Assuntos
Antibacterianos/farmacologia , Ciprofloxacino/farmacologia , Conjugação Genética , Farmacorresistência Bacteriana , Plasmídeos/genética , Salmonella/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Plasmídeos/metabolismo , Salmonella/genética , Salmonella/metabolismo , Infecções por Salmonella/microbiologia
11.
Int J Nanomedicine ; 14: 3189-3201, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31118631

RESUMO

Purpose: Early diagnosis is essential for reducing liver cancer mortality, and molecular diagnosis by magnetic resonance imaging (MRI) is an emerging and promising technology. The chief aim of the present work is to use the ferritin gene, modified by the alpha-fetoprotein (AFP) promoter, carried by a highly safe vector, to produce signal contrast on T2-weighted MR imaging as an endogenous contrast agent, and to provide a highly specific target for subsequent therapy. Methods: Polyethyleneimine-ß-cyclodextrin (PEI-ß-CD, PC) was synthesized as a novel vector. The optimal nitrogen/phosphorus ratio (N/P) of the PC/plasmid DNA complex was determined by gel retardation, biophysical properties and transmission electron microscopy morphological analysis. The transfection efficiency was observed under a fluorescence microscope and analyzed by flow cytometry. Cellular iron accumulation caused by ferritin overexpression was verified by Prussian blue staining, and the resulting contrast imaging effect was examined by MRI. Results: The modified cationic polymer PC was much safer than high molecular weight PEI, and could condense plasmid DNA at an N/P ratio of 50 with suitable biophysical properties and a high transfection efficiency. Overexpression of ferritin enriched intracellular iron. The short-term iron imbalance initiated by AFP promoter regulation only occurred in hepatoma cells, resulting in signal contrast on MRI. The specific target TfR was also upregulated during this process. Conclusion: These results illustrate that the regulated ferritin gene carried by PC can be used as an endogenous contrast agent for MRI detection of hepatocellular carcinoma (HCC). This molecular imaging technique may promote safer early diagnosis of HCC, and provide a more highly specific target for future chemotherapy drugs.


Assuntos
Carcinoma Hepatocelular/diagnóstico por imagem , Ferritinas/genética , Neoplasias Hepáticas/diagnóstico por imagem , Imagem por Ressonância Magnética , Imagem Molecular/métodos , Poliaminas/química , Carcinoma Hepatocelular/patologia , Morte Celular , Linhagem Celular Tumoral , DNA/metabolismo , Células HEK293 , Humanos , Ferro/metabolismo , Neoplasias Hepáticas/patologia , Tamanho da Partícula , Plasmídeos/metabolismo , Polietilenoimina/química , Regiões Promotoras Genéticas , Espectroscopia de Prótons por Ressonância Magnética , Receptores da Transferrina/metabolismo , Eletricidade Estática
12.
J Agric Food Chem ; 67(24): 6867-6873, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31134807

RESUMO

Hydroxytyrosol is a high-value-added compound with a variety of biological and pharmacological activities. In this study, a whole-cell catalytic method for the synthesis of hydroxytyrosol was developed: aromatic amino acid aminotransferase (TyrB), l-glutamate dehydrogenase (GDH), α-keto acid decarboxylase (PmKDC), and aldehyde reductase (YahK) were co-expressed in Escherichia coli to catalyze the synthesis of hydroxytyrosol from l-3,4-dihydroxyphenylalanine (l-DOPA). The plasmids with different copy numbers were used to balance the expression of the four enzymes, and the most appropriate strain (pRSF- yahK- tyrB and pCDF- gdh- Pmkdc) was identified. After determination of the optimum temperature (35 °C) and pH (7.5) for whole-cell catalysis, the yield of hydroxytyrosol reached 36.33 mM (5.59 g/L) and the space-time yield reached 0.70 g L-1 h-1.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Levodopa/metabolismo , Engenharia Metabólica , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo
13.
Acta Crystallogr D Struct Biol ; 75(Pt 5): 498-504, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31063152

RESUMO

Bovine meat and milk factors (BMMFs) are circular, single-stranded episomal DNAs that have been detected in bovine meat and milk products. BMMFs are thought to have roles in human malignant and degenerative diseases. BMMFs encode a replication initiator protein (Rep) that is actively transcribed and translated in human cells. In this study, a Rep WH1 domain encoded on a BMMF (MSBI1.176) isolated from a multiple sclerosis human brain sample was determined to 1.53 Šresolution using X-ray crystallography. The overall structure of the MSBI1.176 WH1 domain was remarkably similar to other Rep structures, despite having a low (28%) amino-acid sequence identity. The MSBI1.176 WH1 domain contained elements common to other Reps, including five α-helices, five ß-strands and a hydrophobic pocket. These new findings suggest that the MSBI1.176 Rep might have comparable roles and functions to other known Reps of different origins.


Assuntos
Encéfalo/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Esclerose Múltipla/metabolismo , Plasmídeos/isolamento & purificação , Plasmídeos/metabolismo , Transativadores/química , Transativadores/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Homologia de Sequência
14.
Mol Med Rep ; 19(6): 5097-5104, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059057

RESUMO

Numerous studies have demonstrated the association between senescence and cancer. However, the molecular mechanism regulating senescence in ovarian cancer remains unknown. In the present study, the protein expression level of calbindin 1 (CALB1) in ovarian cancer was examined using western blot and immunohistochemistry. The function of CALB1 in ovarian cancer cells was examined using MTT assay, anchorage­independent growth assay and senescence assay. The molecular mechanisms underlying CALB1 function were investigated using immunoprecipitation and pull­down assays. In the present study, the expression of CALB1 was found to be increased in ovarian cancer. Overexpression of CALB1 promoted the proliferation and colony formation of ovarian cancer cells and inhibited senescence by modulating the expression levels of p21 and p27. Knockdown of CALB1 inhibited the proliferation and colony formation of ovarian cancer cells. Mechanistically, co­immunoprecipitation assays revealed that CALB1 interacts with MDM2 proto­oncogene (MDM2) and promoted the interaction between p53 and MDM2. Collectively, the present study suggested that CALB1 may act as an oncogene in ovarian cancer by inhibiting the p53 pathway.


Assuntos
Calbindina 1/metabolismo , Senescência Celular , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Calbindina 1/antagonistas & inibidores , Calbindina 1/genética , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo
15.
Bioengineered ; 10(1): 87-97, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30957636

RESUMO

Expression of recombinant proteins fused to a novel glycomodule tag, termed hydroxyproline (Hyp)-O-glycosylated peptides (HypGP), was earlier found to boost secreted protein yields up to 500-fold in plant cell culture. Here, this technology was applied to the expression of human protease inhibitor α1-antitrypsin (AAT) in tobacco BY-2 cell culture. A designer HypGP tag composed of a 'Ala-Pro' motif of 20 units, or (AP)20, was engineered either at the N- or C-terminal end of AAT. The (AP)20 tag substantially increased the secreted yields of the recombinant AAT up to 34.7 mg/L. However, the (AP)20-tagged AAT products were frequently subjected to proteolytic processing. The intact AAT-(AP)20 along with some of the truncated AAT domains exhibited desired biological activity in inhibiting elastase. The results from this research demonstrated that the designer (AP)20 module engineered in BY-2 cells could function as a molecular carrier to substantially enhance the secreted yields of the recombinant AAT.


Assuntos
Elastase Pancreática/antagonistas & inibidores , Processamento de Proteína Pós-Traducional , Proteínas Secretadas Inibidoras de Proteinases/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Tabaco/genética , alfa 1-Antitripsina/biossíntese , Sequência de Bases , Técnicas de Cultura de Células , Dipeptídeos/genética , Dipeptídeos/metabolismo , Expressão Gênica , Glicosilação , Humanos , Elastase Pancreática/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Células Vegetais/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Proteínas Secretadas Inibidoras de Proteinases/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Tabaco/citologia , Tabaco/metabolismo , Transformação Genética , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/isolamento & purificação , alfa 1-Antitripsina/farmacologia
16.
ACS Appl Mater Interfaces ; 11(17): 15322-15331, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30986029

RESUMO

Reactive oxygen species (ROS)-induced oxidative stress leads to neuron damage and is involved in the pathogenesis of chronic inflammation in neurodegenerative diseases (NDs), such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis. Researchers, therefore, are looking for antiinflammatory drugs and gene therapy approaches to slow down or even prevent neurological disorders. Combining therapeutics has shown a synergistic effect in the treatment of human diseases. Many nanocarriers could be designed for the simultaneous codelivery of drugs with genes to fight diseases. However, only a few researches have been performed in NDs. In this study, we developed a mesoporous silica nanoparticle (MSN)-based approach for neurodegenerative therapy. This MSN-based platform involved multiple designs in the targeted codelivery of (1) curcumin, a natural antioxidant product, to protect ROS-induced cell damage and (2) plasmid RhoG-DsRed, which is associated with the formation of lamellipodia and filopodia for promoting neurite outgrowth. At the same time, TAT peptide was introduced to the plasmid RhoG-DsRed via electrostatic interaction to elevate the efficiency of nonendocytic pathways and the nuclear plasmid delivery of RhoG-DsRed in cells for enhanced gene expression. Besides, such a plasmid RhoG-DsRed/TAT complex could work as a noncovalent gatekeeper. The release of curcumin inside the channel of the MSN could be triggered when the complex was dissociated from the MSN surface. Taken together, this MSN-based platform combining genetic and pharmacological manipulations of an actin cytoskeleton as well as oxidative stress provides an attractive way for ND therapy.


Assuntos
Curcumina/farmacologia , Portadores de Fármacos/química , Nanopartículas/química , Crescimento Neuronal/efeitos dos fármacos , Plasmídeos/metabolismo , Dióxido de Silício/química , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Curcumina/química , GTP Fosfo-Hidrolases/genética , Camundongos , Estresse Oxidativo , Tamanho da Partícula , Fragmentos de Peptídeos/química , Plasmídeos/química , Porosidade , Espécies Reativas de Oxigênio/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química
17.
Int J Mol Sci ; 20(8)2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31022896

RESUMO

Psychrobacter sp. DAB_AL32B, originating from Spitsbergen island (Arctic), carries the large plasmid pP32BP2 (54,438 bp). Analysis of the pP32BP2 nucleotide sequence revealed the presence of three predicted phenotypic modules that comprise nearly 30% of the plasmid genome. These modules appear to be involved in fimbriae synthesis via the chaperone-usher pathway (FIM module) and the aerobic and anaerobic metabolism of carnitine (CAR and CAI modules, respectively). The FIM module was found to be functional in diverse hosts since it facilitated the attachment of bacterial cells to abiotic surfaces, enhancing biofilm formation. The CAI module did not show measurable activity in any of the tested strains. Interestingly, the CAR module enabled the enzymatic breakdown of carnitine, but this led to the formation of the toxic by-product trimethylamine, which inhibited bacterial growth. Thus, on the one hand, pP32BP2 can enhance biofilm formation, a highly advantageous feature in cold environments, while on the other, it may prevent bacterial growth under certain environmental conditions. The detrimental effect of harboring pP32BP2 (and its CAR module) seems to be conditional, since this replicon may also confer the ability to use carnitine as an alternative carbon source, although a pathway to utilize trimethylamine is most probably necessary to make this beneficial. Therefore, the phenotype determined by this CAR-containing plasmid depends on the metabolic background of the host strain.


Assuntos
Plasmídeos/genética , Psychrobacter/genética , Regiões Árticas , Aderência Bacteriana , Sequência de Bases , Biofilmes/crescimento & desenvolvimento , Carnitina/metabolismo , Elementos de DNA Transponíveis , Fenótipo , Plasmídeos/metabolismo , Psychrobacter/fisiologia
18.
Biomed Res Int ; 2019: 9630793, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30941374

RESUMO

Background: A recombinant BCG strain expressing the genetically detoxified S1 subunit of pertussis toxin 9K/129G (rBCG-S1PT), previously constructed by our research group, demonstrated the ability to develop high protection in mouse models of pertussis challenge which correlated with the induction of a Th1 immune response pattern. The Th1 immune response induced by rBCG-S1PT treatment was also confirmed in the murine orthotopic bladder cancer model, in which the intravesical instillation of rBCG-S1PT resulted in an improved antitumor effect. Based on these observations, we hypothesize that the reengineering of the S1PT expression in BCG could increase the efficiency of the protective Th1 immune response in order to develop a new alternative of immunotherapy in bladder cancer treatment. Objectives: To construct rBCG strains expressing S1PT from extrachromosomal (rBCG-S1PT) and integrative vectors (rBCG-Sli), or their combination, generating the bivalent strain (rBCG-S1+S1i), and to evaluate the respective immunogenicity of rBCG strains in mice. Methods: Mycobacterial plasmids were constructed by cloning the s1pt gene under integrative and extrachromosomal vectors and used to transform BCG, individually or in combination. Antigen expression and localization were confirmed by Western blot. Mice were immunized with wild-type BCG or the rBCG strains, and cytokines quantification and flow cytometry analysis were performed in splenocytes culture stimulated with mycobacterial-specific proteins. Findings: S1PT expression was confirmed in all rBCG strains. The extrachromosomal vector directs S1PT to the cell wall-associated fraction, while the integrative vector directs its expression mainly to the intracellular fraction. Higher levels of IFN-γ were observed in the splenocytes culture from the group immunized with rBCG-S1i in comparison to BCG or rBCG-S1PT. rBCG-S1+S1i showed higher levels of CD4+ IFN-γ + and double-positive CD4+ IFN-γ + TNF-α + T cells. Conclusions: rBCG-S1+S1i was able to express the two forms of S1PT and elicited higher induction of polyfunctional CD4+ T cells, indicating enhanced immunogenicity and suggesting its use as immunotherapy for bladder cancer.


Assuntos
Vacina BCG/imunologia , Linfócitos T CD4-Positivos/imunologia , Imunidade Celular , Mycobacterium bovis/fisiologia , Toxina Pertussis/metabolismo , Subunidades Proteicas/metabolismo , Vacinas Sintéticas/imunologia , Animais , Citocinas/biossíntese , Citocinas/metabolismo , Feminino , Imunização , Mediadores da Inflamação/metabolismo , Camundongos Endogâmicos BALB C , Fenótipo , Plasmídeos/metabolismo , Baço/citologia
19.
BMC Res Notes ; 12(1): 225, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30987672

RESUMO

OBJECTIVE: Delivery of constructs for silencing or over-expressing genes or their modified versions is a crucial step for studying neuronal cell biology. Therefore, efficient transfection is important for the success of these experimental techniques especially in post-mitotic cells like neurons. In this study, we have assessed the transfection rate, using a previously established protocol, in both primary cortical cultures and neuroblastoma cell lines. Transfection efficiencies in these preparations have not been systematically determined before. RESULTS: Transfection efficiencies obtained herein were (10-12%) for neuroblastoma, (5-12%) for primary astrocytes and (1.3-6%) for primary neurons. We also report on cell-type specific transfection efficiency of neurons and astrocytes within primary cortical cultures when applying cell-type selective transfection protocols. Previous estimations described in primary cortical or hippocampal cultures were either based on general observations or on data derived from unspecified number of biological and/or technical replicates. Also to the best of our knowledge, transfection efficiency of pure primary neuronal cultures or astrocytes cultured in the context of pure or mixed (neurons/astrocytes) population cultures have not been previously determined. The transfection strategy used herein represents a convenient, and a straightforward tool for targeted cell transfection that can be utilized in a variety of in vitro applications.


Assuntos
Astrócitos/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Plasmídeos/metabolismo , Transfecção/métodos , Animais , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Técnicas de Cocultura , Expressão Gênica , Genes Reporter , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lipídeos/química , Lipídeos/farmacologia , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Especificidade de Órgãos , Plasmídeos/química , Cultura Primária de Células
20.
Nanoscale ; 11(16): 7931-7943, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30964937

RESUMO

Since mannose receptors (MRs) are expressed on the surfaces of dendritic cells (DCs), the most professional antigen presenting cells in our body, DNA vaccine carriers containing either covalently grafted mannosyl- or mannose-mimicking shikimoyl-ligands are being increasingly used in ex vivo DC-transfection based DNA vaccination. To this end, we have recently demonstrated that ex vivo immunization of mice with liposomes of shikimoylated cationic amphiphiles containing a 6-amino hexanoic acid spacer group in the head-group region in complexation with melanoma antigen (MART1) encoded DNA vaccine (pCMV-MART1) induces long lasting anti-melanoma immune responses (C. Voshavar, et al., J. Med. Chem., 2017, 60, 1605-1610). This finding prompted us to examine, in the present investigation, the efficacies of gold nanoparticles conjugated to the mannose-mimicking shikimoyl ligand (SL) via a 6-amino hexane thiol spacer (AuNPs-SL) for use in ex vivo DC-transfection based genetic immunization. Herein, we report on the design, synthesis, physico-chemical characterization and bioactivities of AuNPs-SL. Dynamic light scattering and transmission electron microscopy studies revealed the hydrodynamic diameters of theAuNPs-SL nanoconjugates to be within the range of 23-44 nm and their surface potentials within the range of 9-28 mV. MTT-assay showed the non-cytotoxic nature of AuNPs-SL and the findings in the electrophoretic gel retardation assays revealed strong DNA binding properties of the AuNPs-SL. Importantly, subcutaneous immunization of C57BL/6J mice with DCs ex vivo transfected with an electrostatic complex of AuNPs-SL & melanoma antigen (MART1) encoded DNA vaccine (p-CMV-MART1) induced a long lasting (100 days) anti-tumor immune response in immunized mice upon subsequent challenge with a lethal dose of melanoma. Notably, mice immunized with either autologous mbmDCs ex vivo pre-transfected with nanoplexes of shikimoylated AuNPs-SL & an irrelevant pCMV-SPORT-ß-gal plasmid (without having encoded melanoma antigen) or untransfected DCs showed no lasting protection against subsequent tumor challenge. The presently described shikimoyl-decorated gold nanoparticles (AuNPs-SL) are expected to find future use in ex vivo DC-transfection based genetic immunization against cancer and other infectious diseases.


Assuntos
Vacinas Anticâncer/imunologia , Ouro/química , Nanopartículas Metálicas/química , Nanoestruturas/química , Safrol/química , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Imunidade Celular , Interferon gama/metabolismo , Ligantes , Antígeno MART-1/genética , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/mortalidade , Camundongos , Camundongos Endogâmicos C57BL , Nanoestruturas/uso terapêutico , Nanoestruturas/toxicidade , Plasmídeos/genética , Plasmídeos/metabolismo , Taxa de Sobrevida , Transplante Homólogo
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