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1.
Chemistry ; 25(56): 12916-12919, 2019 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-31397017

RESUMO

Inorganic cells bearing calcium silicate membranes were prepared and resembled closed chemical gardens. It was demonstrated that these inorganic cells can successfully be loaded with natural products, proteins and plasmid DNA, and their cargo can be released in a controlled manner. These cells demonstrated the ability of chemical gardens to act as platforms for the sustained delivery of biomolecules and are expected to introduce chemical gardens in the field of biosciences.


Assuntos
Portadores de Fármacos/química , Animais , Compostos de Cálcio/química , Bovinos , Plasmídeos/química , Plasmídeos/metabolismo , Rutina/química , Rutina/metabolismo , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Silicatos/química
2.
DNA Cell Biol ; 38(10): 1048-1055, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31433200

RESUMO

DNA condensed agents can improve the transfection efficiency of the cationic liposome delivery system. However, various condensed agents have distinct transfection efficiency and cellular cytotoxicity. The object of this study was to screen the optimal agents with the high transfection efficiency and low cytotoxicity from four polymer compressive materials, polyethylenimine (PEI), chitosan, poly-l-lysine (PLL), and spermidine. DNA was precompressed with these four agents and then combined to cationic liposomes. Subsequently, the entrapment and transfection efficiency of the obtained complexes were investigated. Finally, the particle sizes, cytotoxicity, and endocytosis fashion of these copolymers (Lipo-PEI, Lipo-chitosan, Lipo-PLL, and Lipo-spermidine) were examined. It was found that these four copolymers had significantly lower cytotoxicity and higher transfection efficiency (45.5%, 42.4%, 36.8%, and 47.4%, respectively) than those in the control groups. The transfection efficiency of Lipo-PEI and Lipo-spermidine copolymers were better than the other two copolymers. In 293T cells, nystatin significantly inhibited the transfection efficiency of Lipo-PEI-DNA and Lipo-spermidine-DNA (51.88% and 46.05%, respectively), which suggest that the endocytosis pathway of Lipo-spermidine and Lipo-PEI copolymers was probably caveolin dependent. Our study indicated that these dual-degradable copolymers especially liposome-spermidine copolymer could be used as the potential biocompatible gene delivery carriers.


Assuntos
Quitosana/química , Lipossomos/química , Polietilenoimina/química , Polilisina/química , Espermidina/química , Transfecção/métodos , Cátions , Caveolina 1/genética , Caveolina 1/metabolismo , Quitosana/metabolismo , Colesterol/química , Colesterol/metabolismo , Endocitose/efeitos dos fármacos , Endocitose/genética , Ácidos Graxos Monoinsaturados/química , Ácidos Graxos Monoinsaturados/metabolismo , Células HEK293 , Humanos , Lipossomos/metabolismo , Nistatina/farmacologia , Tamanho da Partícula , Plasmídeos/química , Plasmídeos/metabolismo , Polietilenoimina/metabolismo , Polilisina/metabolismo , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/metabolismo , Espermidina/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.
Inorg Chem ; 58(13): 8800-8819, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31247881

RESUMO

Very few inorganic antineoplastic drugs have entered the clinic in the last decades, mainly because of toxicity issues. Because copper is an essential trace element of ubiquitous occurrence, decreased side effects could be expected in comparison with the widely used platinum anticancer compounds. In the present work, two novel hydrazonic binucleating ligands and their µ-hydroxo dicopper(II) complexes were prepared and fully characterized. They differ by the nature of the aromatic group present in their aroylhydrazone moieties: while H3L1 and its complex, 1, possess a thiophene ring, H3L2 and 2 contain the more polar furan heterocycle. X-ray diffraction indicates that both coordination compounds are very similar in structural terms and generate dimeric arrangements in the solid state. Positive-ion electrospray ionization mass spectrometry analyses confirmed that the main species present in a 10% dimethyl sulfoxide (DMSO)/water solution should be [Cu2(HL)(OH)]+ and the DMSO-substituted derivative [Cu2(L)(DMSO)]+. Scattering techniques [dynamic light scattering (DLS) and small-angle X-ray scattering] suggest that the complexes and their free ligands interact with bovine serum albumin (BSA) in a reversible manner. The binding constants to BSA were determined for the complexes through fluorescence spectroscopy. Moreover, to gain insight into the mechanism of action of the compounds, calf thymus DNA binding studies by UV-visible and DLS measurements using plasmid pBR322 DNA were also performed. For the complexes, DLS data seem to point to the occurrence of DNA cleavage to Form III (linear). Both ligands and their dicopper(II) complexes display potent antiproliferative activity in a panel of four cancer cell lines, occasionally even in the submicromolar range, with the complexes being more potent than the free ligands. Our data on cellular models correlate quite well with the DNA interaction experiments. The results presented herein show that aroylhydrazone-derived binucleating ligands, as well as their dinuclear µ-hydroxodicopper(II) complexes, may represent a promising structural starting point for the development of a new generation of highly active potential antitumor agents.


Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Hidrazonas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/toxicidade , Bovinos , Linhagem Celular Tumoral , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/toxicidade , Cobre/química , DNA/química , Clivagem do DNA/efeitos dos fármacos , Cães , Humanos , Hidrazonas/síntese química , Hidrazonas/química , Hidrazonas/toxicidade , Isomerismo , Ligantes , Células Madin Darby de Rim Canino , Camundongos , Plasmídeos/química , Multimerização Proteica/efeitos dos fármacos , Soroalbumina Bovina/metabolismo
6.
Nat Biotechnol ; 37(6): 626-631, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110355

RESUMO

Base editing requires that the target sequence satisfy the protospacer adjacent motif requirement of the Cas9 domain and that the target nucleotide be located within the editing window of the base editor. To increase the targeting scope of base editors, we engineered six optimized adenine base editors (ABEmax variants) that use SpCas9 variants compatible with non-NGG protospacer adjacent motifs. To increase the range of target bases that can be modified within the protospacer, we use circularly permuted Cas9 variants to produce four cytosine and four adenine base editors with an editing window expanded from ~4-5 nucleotides to up to ~8-9 nucleotides and reduced byproduct formation. This set of base editors improves the targeting scope of cytosine and adenine base editing.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Adenina/química , Citosina/química , Humanos , Nucleotídeos/química , Nucleotídeos/genética , Plasmídeos/química , Plasmídeos/genética
7.
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
8.
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
9.
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
10.
Mol Vis ; 25: 174-182, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30996586

RESUMO

Purpose: To evaluate the efficacy of using a CRISPR/Cas-mediated strategy to correct a common high-risk allele that is associated with age-related macular degeneration (AMD; rs1061170; NM_000186.3:c.1204T>C; NP_000177.2:p.His402Tyr) in the complement factor H (CFH) gene. Methods: A human embryonic kidney cell line (HEK293A) was engineered to contain the pathogenic risk variant for AMD (HEK293A-CFH). Several different base editor constructs (BE3, SaBE3, SaKKH-BE3, VQR-BE3, and Target-AID) and their respective single-guide RNA (sgRNA) expression cassettes targeting either the pathogenic risk variant allele in the CFH locus or the LacZ gene, as a negative control, were evaluated head-to-head for the incidence of a cytosine-to-thymine nucleotide correction. The base editor construct that showed appreciable editing activity was selected for further assessment in which the base-edited region was subjected to next-generation deep sequencing to quantify on-target and off-target editing efficacy. Results: The tandem use of the Target-AID base editor and its respective sgRNA demonstrated a base editing efficiency of facilitating a cytosine-to-thymine nucleotide correction in 21.5% of the total sequencing reads. Additionally, the incidence of insertions and deletions (indels) was detected in only 0.15% of the sequencing reads with virtually no off-target effects evident across the top 11 predicted off-target sites containing at least one cytosine in the activity window (n = 3, pooled amplicons). Conclusions: CRISPR-mediated base editing can be used to facilitate a permanent and stably inherited cytosine-to-thymine nucleotide correction of the rs1061170 SNP in the CFH gene with minimal off-target effects.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Edição de Genes/métodos , RNA Guia/genética , Sequência de Bases , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Citosina/metabolismo , Expressão Gênica , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Óperon Lac , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Mutação , Plasmídeos/química , Plasmídeos/metabolismo , RNA Guia/metabolismo , Timina/metabolismo
11.
Cell Mol Life Sci ; 76(20): 4155-4164, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31030226

RESUMO

Evolved xCas9(3.7) variant with broad PAM compatibility has been reported in cell lines, while its editing efficiency was site-specific. Here, we show that xCas9(3.7) can recognize a broad PAMs including NGG, NGA, and NGT, in both embryos and Founder (F0) rabbits. Furthermore, the codon-optimized xCas9-derived base editors, exBE4 and exABE, can dramatically improve the base editing efficiencies in rabbit embryos. Our results demonstrated that the optimized xCas9 with expanded PAM compatibility and enhanced base editing efficiency could be used for precise gene modifications in organisms.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Efeito Fundador , Edição de Genes/métodos , Marcação de Genes/métodos , RNA Guia/genética , Animais , Animais Geneticamente Modificados , Proteína 9 Associada à CRISPR/metabolismo , Códon , Distrofina/genética , Distrofina/metabolismo , Embrião de Mamíferos , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Microinjeções , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas de Ligação a Poli(A)/genética , Proteínas de Ligação a Poli(A)/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , RNA Guia/metabolismo , Coelhos , Repetições de Trinucleotídeos , Zigoto
12.
Methods Mol Biol ; 1943: 123-139, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30838613

RESUMO

Lipidic vesicles have been extensively studied for their capacity to condensate and deliver nucleic acids to the cells. Many different amphiphilic lipidic structures have been proposed each of them bringing some advances in nonviral gene transfection. The ionic or neutral nature of the lipids induces tremendous differences in the behavior of the corresponding liposomes, from the complexation of nucleic acid to the delivery to the cell. An efficient delivery in vitro or in vivo also depends closely on the structure of the lipids and very often, efficient liposomes in vitro have been found useless for in vivo administration.We describe in this chapter the chemical synthesis of two different lipids, one cationic and the other essentially neutral, and the formulation to obtain liposomes and DNA-liposome complexes. The different ways and tricks for the formulation of the two different structures are especially highlighted.


Assuntos
Técnicas de Química Sintética/métodos , Lipídeos/química , Transfecção/métodos , Animais , Cátions/química , Linhagem Celular Tumoral , DNA/química , DNA/genética , Lipossomos , Camundongos , Estrutura Molecular , Nanopartículas/química , Plasmídeos/química , Plasmídeos/genética
13.
ACS Appl Mater Interfaces ; 11(12): 11270-11282, 2019 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-30844235

RESUMO

Optogenetics is a recently established neuromodulation technique in which photostimulation is used to manipulate neurons with high temporal and spatial precision. However, sequential genetic and optical insertion with double brain implantation tends to cause excessive tissue damage. In addition, the incorporation of light-sensitive genes requires the utilization of viral vectors, which remains a safety concern. Here, by combining device fabrication design, nanotechnology, and cell targeting technology, we developed a new gene-embedded optoelectrode array for neural implantation to enable spatiotemporal electroporation (EP) for gene delivery/transfection, photomodulation, and synchronous electrical monitoring of neural signals in the brain via one-time implantation. A biotic-abiotic neural interface (called PG) composed of reduced graphene oxide and conductive polyelectrolyte 3,4-ethylenedioxythiophene-modified amphiphilic chitosan was developed to form a nanostructural hydrogel with assembled nanodomains for encapsulating nonviral gene vectors (called PEI-NT-pDNA) formulated by neurotensin (NT) and polyethylenimine (PEI)-coupled plasmid DNA (pDNA). The PG can maintain high charge storage ability to respond to a minimal current of 125 µA for controllable gene delivery. The in vitro analysis of PG-PEI-NT-pDNA on the microelectrode array chip showed that the microelectrodes provided electrically inductive electropermeabilization, which permitted gene transfection into localized rat adrenal pheochromocytoma cells with a strong green fluorescent protein expression that was up to 8-fold higher than that in nontreated cells. Furthermore, the in vivo implantation enabled on-demand spatiotemporal gene transfection to neurons with 10-fold enhancement of targeting ability compared with astrocytes. Finally, using the real optogenetic opsin channelrhodopsin-2, the flexible neural probe incorporated with an optical waveguide fiber displayed photoevoked extracellular spikes in the thalamic ventrobasal region after focal EP for only 7 days, which provided a proof of concept for the use of photomodulation to facilitate neural therapies.


Assuntos
Nanoestruturas/química , Neurônios/fisiologia , Optogenética/métodos , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/química , Sobrevivência Celular/efeitos dos fármacos , Condutividade Elétrica , Eletrodos Implantados , Grafite/química , Hidrogéis/química , Microeletrodos , Microscopia de Fluorescência , Neurotensina/química , Células PC12 , Plasmídeos/química , Plasmídeos/metabolismo , Polietilenoimina/química , Polímeros/química , Ratos , Transfecção
14.
Proc Natl Acad Sci U S A ; 116(13): 6260-6269, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30850542

RESUMO

R-loops are abundant three-stranded nucleic-acid structures that form in cis during transcription. Experimental evidence suggests that R-loop formation is affected by DNA sequence and topology. However, the exact manner by which these factors interact to determine R-loop susceptibility is unclear. To investigate this, we developed a statistical mechanical equilibrium model of R-loop formation in superhelical DNA. In this model, the energy involved in forming an R-loop includes four terms-junctional and base-pairing energies and energies associated with superhelicity and with the torsional winding of the displaced DNA single strand around the RNA:DNA hybrid. This model shows that the significant energy barrier imposed by the formation of junctions can be overcome in two ways. First, base-pairing energy can favor RNA:DNA over DNA:DNA duplexes in favorable sequences. Second, R-loops, by absorbing negative superhelicity, partially or fully relax the rest of the DNA domain, thereby returning it to a lower energy state. In vitro transcription assays confirmed that R-loops cause plasmid relaxation and that negative superhelicity is required for R-loops to form, even in a favorable region. Single-molecule R-loop footprinting following in vitro transcription showed a strong agreement between theoretical predictions and experimental mapping of stable R-loop positions and further revealed the impact of DNA topology on the R-loop distribution landscape. Our results clarify the interplay between base sequence and DNA superhelicity in controlling R-loop stability. They also reveal R-loops as powerful and reversible topology sinks that cells may use to nonenzymatically relieve superhelical stress during transcription.


Assuntos
Sequência de Bases , DNA Super-Helicoidal/química , DNA/química , Conformação de Ácido Nucleico , DNA de Cadeia Simples/química , Modelos Genéticos , Hibridização de Ácido Nucleico , Plasmídeos/química , RNA/química , Transcrição Genética
15.
Bioengineered ; 10(1): 59-70, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30866700

RESUMO

Tryptophan, an aromatic amino acid, has been widely used in food industry because it participates in the regulation of protein synthesis and metabolic network in vivo. In this study, we obtained a strain named TRP03 by enhancing the tryptophan synthesis pathway, which could accumulate tryptophan at approximately 35 g/L in a 5 L bioreactor. We then modified the central metabolic pathway of TRP03, to increase the supply of the precursor phosphoenolpyruvate (PEP), the genes related to PEP were modified. Furthermore, citric acid transport system and TCA were upregulated to effectively increase cell growth. We observed that strain TRP07 that could accumulate tryptophan at approximately 49 g/L with a yield of 0.186 g tryptophan/g glucose in a 5 L bioreactor. By-products such as glutamate and acetic acid were reduced to 0.8 g/L and 2.2 g/L, respectively.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Engenharia Metabólica/métodos , Triptofano/biossíntese , Ácido Acético/metabolismo , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Ácido Cítrico/metabolismo , Ciclo do Ácido Cítrico/genética , Proteínas de Escherichia coli/metabolismo , Fermentação , Ácido Glutâmico/metabolismo , Humanos , Microbiologia Industrial/métodos , Cinética , Fosfoenolpiruvato/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Transformação Bacteriana
16.
Nat Protoc ; 14(4): 1084-1107, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30911173

RESUMO

Bioluminescence resonance energy transfer (BRET) is a transfer of energy between a luminescence donor and a fluorescence acceptor. Because BRET occurs when the distance between the donor and acceptor is <10 nm, and its efficiency is inversely proportional to the sixth power of distance, it has gained popularity as a proximity-based assay to monitor protein-protein interactions and conformational rearrangements in live cells. In such assays, one protein of interest is fused to a bioluminescent energy donor (luciferases from Renilla reniformis or Oplophorus gracilirostris), and the other protein is fused to a fluorescent energy acceptor (such as GFP or YFP). Because the BRET donor does not require an external light source, it does not lead to phototoxicity or autofluorescence. It therefore represents an interesting alternative to fluorescence-based imaging such as FRET. However, the low signal output of BRET energy donors has limited the spatiotemporal resolution of BRET imaging. Here, we describe how recent improvements in detection devices and BRET probes can be used to markedly improve the resolution of BRET imaging, thus widening the field of BRET imaging applications. The protocol described herein involves three main stages. First, cell preparation and transfection require 3 d, including cell culture time. Second, image acquisition takes 10-120 min per sample, after an initial 60 min for microscope setup. Finally, image analysis typically takes 1-2 h. The choices of energy donor, acceptor, luminescent substrates, cameras and microscope setup, as well as acquisition modes to be used for different applications, are also discussed.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Medições Luminescentes/métodos , Imagem Óptica/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzenoacetamidas/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Imidazóis/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Pirazinas/metabolismo , Receptores de Vasopressinas/genética , Receptores de Vasopressinas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Renilla , Transfecção , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismo
17.
Nanoscale ; 11(11): 4970-4986, 2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30839018

RESUMO

Poor success rates and challenges associated with the current therapeutic strategies of inflammatory bowel disease (IBD) have accelerated the emergence of gene therapy as an alternative treatment option with great promise. However, oral delivery of nucleic acids (NAs) to an inflamed colon is challenged by multiple barriers presented by the gastrointestinal, extracellular and intracellular compartments. Therefore, we screened a series of polyaspartic acid-derived amphiphilic cationic polymers with varied hydrophobicity for their ability to deliver NAs into mammalian cells. Using the most effective TAC6 polymer, we then engineered biocompatible and stable nanogels composed of polyplexes (TAC6, NA) and an anionic polymer, sodium polyaspartate, that were able to deliver the NAs across mammalian cells using caveolae-mediated cellular uptake. We then utilized these nanogels for oral delivery of PIAS1 (protein inhibitor of activated STAT1), a SUMO 3 ligase, encoding plasmid DNA since PIAS1 is a key nodal therapeutic target for IBD due to its ability to control NF-κB-mediated inflammatory signaling. We show that plasmid delivery using TAC6-derived nanogels diminished gut inflammation in a murine colitis model. Therefore, our study presents engineering of orally deliverable nanogels that can target SUMOylation machinery to combat gut inflammation with very high efficacy.


Assuntos
Colite/terapia , Técnicas de Transferência de Genes/instrumentação , Terapia Genética/métodos , Polietilenoglicóis/administração & dosagem , Polietilenoimina/administração & dosagem , Sumoilação , Administração Oral , Animais , Cátions/química , Linhagem Celular Tumoral , Colite/patologia , Colite/fisiopatologia , Colo/metabolismo , Colo/patologia , Colo/fisiopatologia , Modelos Animais de Doenças , Endocitose , Expressão Gênica , Terapia Genética/instrumentação , Humanos , Inflamação , Camundongos , Peptídeos/química , Plasmídeos/administração & dosagem , Plasmídeos/química , Plasmídeos/genética , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polietilenoimina/química , Polietilenoimina/metabolismo , Polímeros/química , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Inibidoras de STAT Ativados/metabolismo
18.
Nat Commun ; 10(1): 1133, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850604

RESUMO

Genome editing for therapeutic applications often requires cleavage within a narrow sequence window. Here, to enable such high-precision targeting with zinc-finger nucleases (ZFNs), we have developed an expanded set of architectures that collectively increase the configurational options available for design by a factor of 64. These new architectures feature the functional attachment of the FokI cleavage domain to the amino terminus of one or both zinc-finger proteins (ZFPs) in the ZFN dimer, as well as the option to skip bases between the target triplets of otherwise adjacent fingers in each zinc-finger array. Using our new architectures, we demonstrate targeting of an arbitrarily chosen 28 bp genomic locus at a density that approaches 1.0 (i.e., efficient ZFNs available for targeting almost every base step). We show that these new architectures may be used for targeting three loci of therapeutic significance with a high degree of precision, efficiency, and specificity.


Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/genética , Edição de Genes/métodos , Genoma Humano , Engenharia de Proteínas/métodos , Nucleases de Dedos de Zinco/genética , Pareamento de Bases , Sequência de Bases , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Loci Gênicos , Biblioteca Genômica , Humanos , Mutação INDEL , Células K562 , Biblioteca de Peptídeos , Plasmídeos/química , Plasmídeos/metabolismo , Transformação Genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Nucleases de Dedos de Zinco/metabolismo
19.
RNA ; 25(6): 727-736, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30902835

RESUMO

The human immunodeficiency virus type 1 (HIV-1) genomic RNA (vRNA) has two major fates during viral replication: to serve as the template for the major structural and enzymatic proteins, or to be encapsidated and packaged into assembling virions to serve as the genomic vRNA in budding viruses. The dynamic balance between vRNA translation and encapsidation is mediated by numerous host proteins, including Staufen1. During HIV-1 infection, HIV-1 recruits Staufen1 to assemble a distinct ribonucleoprotein complex promoting vRNA encapsidation and viral assembly. Staufen1 also rescues vRNA translation and gene expression during conditions of cellular stress. In this work, we utilized novel Staufen1-/- gene-edited cells to further characterize the contribution of Staufen1 in HIV-1 replication. We observed a marked deficiency in the ability of HIV-1 to dissociate stress granules (SGs) in Staufen1-deficient cells and remarkably, the vRNA repositioned to SGs. These phenotypes were rescued by Staufen1 expression in trans or in cis, but not by a dsRBD-binding mutant, Staufen1F135A. The mistrafficking of the vRNA in these Staufen1-/- cells was also accompanied by a dramatic decrease in viral production and infectivity. This work provides novel insight into the mechanisms by which HIV-1 uses Staufen1 to ensure optimal vRNA translation and trafficking, supporting an integral role for Staufen1 in the HIV-1 life cycle, positioning it as an attractive target for next-generation antiretroviral agents.


Assuntos
Grânulos Citoplasmáticos/virologia , Proteínas do Citoesqueleto/genética , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Vírion/genética , Transporte Biológico , Grânulos Citoplasmáticos/metabolismo , Proteínas do Citoesqueleto/deficiência , Regulação da Expressão Gênica , Células HCT116 , Humanos , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , RNA Viral/metabolismo , Transdução de Sinais , Transfecção , Vírion/metabolismo , Montagem de Vírus/genética , Replicação Viral/genética
20.
Biomater Sci ; 7(5): 1940-1948, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30785129

RESUMO

Carbon dots have attracted rapidly growing interest in recent years. In this report, we prepared two cationic polymer-derived carbon dots (Taea-CD and Cyclen-CD, collectively called C-dots) via a hydrothermal method. Transmission electron microscopy (TEM) results show that the C-dots were sphere-like and the size distribution was 1.8 ± 0.4 nm for Taea-CD and 5.4 ± 2 nm for Cyclen-CD. The C-dots emitted bright blue fluorescence under UV light (365 nm). Confocal laser scanning microscopy (CLSM) assay indicates that the C-dots-mediated transfection process could be detected in real time, and their tunable fluorescence emission under different wavelengths could satisfy varying requirements. Luciferase assay indicates that the transformation from the polymer to CD is an effective strategy to improve the transfection efficiency (TE) of the materials. Moreover, the C-dots also exhibit higher serum tolerance and cell viability than commercially available polyethyleneimine (PEI). These results demonstrate that the preparation of carbon dots from polymers is a promising method for developing multifunctional gene vectors with high TE and biocompatibility.


Assuntos
Carbono/química , Portadores de Fármacos/química , Imagem Molecular , Nanopartículas/química , Polietilenoimina/química , Transfecção , Células HEK293 , Células HeLa , Humanos , Tamanho da Partícula , Plasmídeos/química , Plasmídeos/genética
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