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1.
J Surg Oncol ; 121(1): 100-108, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31240729

RESUMO

BACKGROUND AND OBJECTIVES: Previously, we have shown that 9-cis retinoic acid (9-cis RA) stimulates lymphangiogenesis and limits postsurgical lymphedema in animal models when administered via daily intraperitoneal injections. In this study, we investigate whether a single-use depot 9-cis RA drug delivery system (DDS) implanted at the site of lymphatic injury can mitigate the development of lymphedema in a clinically relevant mouse limb model. METHODS: Hind limb lymphedema was induced via surgical lymphadenectomy and irradiation. Animals were divided into two treatment groups: (1) 9-cis RA DDS, (2) placebo DDS. Outcomes measured included paw thickness, lymphatic clearance and density, epidermal thickness, and collagen deposition. RESULTS: Compared with control animals, 9-cis RA-treated animals had significantly less paw swelling from postoperative week 3 (P = .04) until the final timepoint at week 6 (P = .0007). Moreover, 9-cis RA-treated animals had significantly faster lymphatic clearance (P < .05), increased lymphatic density (P = .04), reduced lymphatic vessel size (P = .02), reduced epidermal hyperplasia (P = .04), and reduced collagen staining (P = .10). CONCLUSIONS: Animals receiving 9-cis RA sustained-release implants at the time of surgery had improved lymphatic function and structure, indicating reduced lymphedema progression. Thus, we demonstrate that 9-cis RA contained within a single-use depot DDS has favorable properties in limiting pathologic responses to lymphatic injury and may be an effective strategy against secondary lymphedema.


Assuntos
Alitretinoína/administração & dosagem , Excisão de Linfonodo/métodos , Linfedema/prevenção & controle , Animais , Colágeno/metabolismo , Preparações de Ação Retardada , Epiderme/efeitos dos fármacos , Epiderme/patologia , Feminino , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Membro Posterior , Hiperplasia , Excisão de Linfonodo/efeitos adversos , Sistema Linfático/efeitos dos fármacos , Sistema Linfático/metabolismo , Linfedema/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Complicações Pós-Operatórias/prevenção & controle
2.
Microb Cell Fact ; 18(1): 170, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601271

RESUMO

BACKGROUND: Most microorganisms have evolved to maximize growth rate, with rapid consumption of carbon sources from the surroundings. However, fast growing phenotypes usually feature secretion of organic compounds. For example, E. coli mainly produced acetate in fast growing condition such as glucose rich and aerobic condition, which is troublesome for metabolic engineering because acetate causes acidification of surroundings, growth inhibition and decline of production yield. The overflow metabolism can be alleviated by reducing glucose uptake rate. RESULTS: As glucose transporters or their subunits were knocked out in E. coli, the growth and glucose uptake rates decreased and biomass yield was improved. Alteration of intracellular metabolism caused by the mutations was investigated with transcriptome analysis and 13C metabolic flux analysis (13C MFA). Various transcriptional and metabolic perturbations were identified in the sugar transporter mutants. Transcription of genes related to glycolysis, chemotaxis, and flagella synthesis was downregulated, and that of gluconeogenesis, Krebs cycle, alternative transporters, quorum sensing, and stress induced proteins was upregulated in the sugar transporter mutants. The specific production yields of value-added compounds (enhanced green fluorescent protein, γ-aminobutyrate, lycopene) were improved significantly in the sugar transporter mutants. CONCLUSIONS: The elimination of sugar transporter resulted in alteration of global gene expression and redirection of carbon flux distribution, which was purposed to increase energy yield and recycle carbon sources. When the pathways for several valuable compounds were introduced to mutant strains, specific yield of them were highly improved. These results showed that controlling the sugar uptake rate is a good strategy for ameliorating metabolite production.


Assuntos
Carbono/metabolismo , Escherichia coli/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/genética , Glucose/metabolismo , Engenharia Metabólica/métodos , Proteínas Recombinantes/biossíntese , Ciclo do Carbono , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Fluorescência Verde/biossíntese , Licopeno/metabolismo , Análise do Fluxo Metabólico/métodos , Ácido gama-Aminobutírico/biossíntese
3.
ACS Appl Mater Interfaces ; 11(41): 38190-38204, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31550131

RESUMO

Despite broad application of nanotechnology in neuroscience, the nanoneurotoxicity of magnetic nanoparticles in primary hippocampal neurons remains poorly characterized. In particular, understanding how magnetic nanoparticles perturb neuronal calcium homeostasis is critical when considering magnetic nanoparticles as a nonviral vector for effective gene therapy in neuronal diseases. Here, we address the pressing need to systematically investigate the neurotoxicity of magnetic nanoparticles with different surface charges in primary hippocampal neurons. We found that unlike negative and neutral nanoparticles, positively charged magnetic nanoparticles (magnetic poly(lactic-co-glycolic acid) (PLGA)-polyethylenimine (PEI) nanoparticles, MNP-PLGA-PEI NPs) rapidly elevated cytoplasmic calcium levels in primary hippocampal neurons, mainly via extracellular calcium influx regulated by voltage-gated calcium channels. We went on to show that this perturbation of intracellular calcium homeostasis elicited serious cytotoxicity in primary hippocampal neurons. However, our next experiment demonstrated that PEGylation on the surface of MNP-PLGA-PEI NPs shielded the surface charge, thereby preventing the perturbation of intracellular calcium homeostasis. That is, PEGylated MNP-PLGA-PEI NPs reduced nanoneurotoxicity. Importantly, biocompatible PEGylated MNP-PLGA-PEI NPs under an external magnetic field enhanced transfection efficiency (>7%) of plasmid DNA encoding GFP in primary hippocampal neurons compared to NPs without external magnetic field mediation. Moreover, under an external magnetic field, this system achieved gene transfection in the hippocampus of the C57 mouse. Overall, this study is the first to successfully employ biocompatible PEGylated MNP-PLGA-PEI NPs for transfection using a magnetofection strategy in primary hippocampal neurons, thereby providing a nanoplatform as a new perspective for treating neuronal diseases or modulating neuron activities.


Assuntos
Proteínas de Fluorescência Verde , Hipocampo/metabolismo , Nanopartículas/química , Neurônios/metabolismo , Plasmídeos , Transfecção , Animais , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células HEK293 , Hipocampo/citologia , Humanos , Camundongos , Neurônios/citologia , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/farmacologia , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Polietilenoimina/química , Polietilenoimina/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia , Ratos , Ratos Sprague-Dawley
4.
Arch Virol ; 164(10): 2505-2513, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31377888

RESUMO

Infectious hematopoietic necrosis virus (IHNV) was developed as a vector to aid the construction of vaccines against viral diseases such as viral hemorrhagic septicemia virus, spring viremia of carp virus, and influenza virus H1N1. However, the optimal site for foreign gene expression in the IHNV vector has not been determined. In the present study, five recombinant viruses with the green fluorescence protein (GFP) gene inserted into different genomic junction regions of the IHNV genomic sequence were generated using reverse genetics technology. Viral growth was severely delayed when the GFP gene was inserted into the intergenic region between the N and P genes. Real-time fluorescence quantitative PCR assays showed that the closer the GFP gene was inserted towards the 3' end, the higher the GFP mRNA levels. Measurement of the GFP fluorescence intensity, which is the most direct method to determine the GFP protein expression level, showed that the highest GFP protein level was obtained when the gene was inserted into the intergenic region between the P and M genes. The results of this study suggest that the P and M gene junction region is the optimal site within the IHNV vector to express foreign genes, providing valuable information for the future development of live vector vaccines.


Assuntos
Expressão Gênica , Vetores Genéticos , Vírus da Necrose Hematopoética Infecciosa/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Fluorometria , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Reação em Cadeia da Polimerase em Tempo Real , Genética Reversa
5.
Physiol Res ; 68(4): 525-530, 2019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31342754

RESUMO

Green Fluorescent protein (GFP) transgenic animals are accepted tools for studying various physiological processes, including organ development and cell migration. However, several in vivo studies claimed that GFP may impair transgenic animals' health. Glomerulosclerosis was observed in transgenic mice and rabbits with ubiquitous reporter protein expression. Heart-specific GFP expression evoked dilated cardiomyopathy and altered cardiac function in transgenic mouse and zebrafish lines, respectively. Moreover, growth retardation and increased axon swelling were observed in GFP and yellow fluorescent protein (YFP) transgenic mice, respectively. This review will focus on the potential drawbacks of the applications of GFP transgenic animals in biomedical research.


Assuntos
Animais Geneticamente Modificados/genética , Pesquisa Biomédica/tendências , Proteínas de Fluorescência Verde/genética , Animais , Pesquisa Biomédica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/biossíntese , Camundongos , Camundongos Transgênicos , Coelhos , Peixe-Zebra
6.
Biotechnol Appl Biochem ; 66(4): 527-536, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30957320

RESUMO

Green fluorescent protein (GFP) is a globular protein used as biosensor and biomarker in medical and industrial fields. However, due to the expensive production costs of expressing proteins using high-cost inducers like isopropyl-ß-d-1-thiogalactopyranoside (IPTG), the number of GFP applications are still scarce. This work studied the production of enhanced GFP (EGFP) using Escherichia coli BL21 (DE3) [pLysS; pET28(a)], aiming to increase its yield and reduce costs. First, the influence of agitation rate, induction time, and concentration of IPTG in the production of EGFP was evaluated, but only the first two parameters were significant. Subsequently, aiming to reduce costs related to the use of inducer, the IPTG concentration (0.005, 0.010, and 0.025 mM) was decreased and, interestingly, the production levels were maintained or increased. These results show that a proper choice of production conditions, particularly through the decrease of inducer concentration, is effective to reduce the upstream production costs and guarantee high EGFP expression.


Assuntos
Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/economia , Escherichia coli/crescimento & desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/economia , Proteínas Recombinantes/genética
7.
Biotechnol Lett ; 41(6-7): 701-709, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30953310

RESUMO

OBJECTIVES: To investigate the effect of full-length fragment of DNA topoisomerase I gene (TOP1) matrix attachment regions (MARs) originating from the human genome on transgene expression in Chinese hamster ovary (CHO) cells and explore the underlying mechanisms. RESULTS: Results showed that TOP1 MAR cannot only enhance the transient and stable transgenic expression of enhanced green fluorescence protein (EGFP) but also increase long-term stability and ratio of positive colonies in transfected CHO cells with TOP1 MAR at the 5' or 3' ends of the EGFP expression cassette. Interestingly, the CHO cells were transfected with the 5',3' TOP1 MAR-containing vector featured the highest transient and stable expression, whereas those with the 3' TOP1 MAR-containing vector exhibited the most effective stability and ratio of positive colonies. We also observed that transgene copy numbers and mRNA of egfp gene were correlated with the expression levels of EGFP protein in polyclonal CHO cells. However, the heterogeneity of expression in monoclonal CHO cells was unaffected by transgene copy number. CONCLUSIONS: The findings may aid in the potential application of TOP1 MAR in expression enhancement of recombinant proteins in mammalian cells.


Assuntos
Células CHO , Engenharia Celular/métodos , DNA Topoisomerases Tipo I/genética , Expressão Gênica , Proteínas de Fluorescência Verde/biossíntese , Regiões de Interação com a Matriz , Proteínas Recombinantes/biossíntese , Animais , Cricetulus , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas Recombinantes/genética
8.
Biotechnol Lett ; 41(6-7): 691-700, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30941601

RESUMO

OBJECTIVE: To knock-in an EGFP cassette into the γ-globin genes of K562 cells via CRISPR/Cas9, and to assess expression and hydroxyurea (HU)-mediated induction of the targeted EGFP transgene. RESULTS: The EGFP cassettes were specifically knocked into the Gγ gene. EGFP expression was detected in the targeted cell population and isolated clones. Furthermore, EGFP transcript and fluorescence levels were significantly induced following HU-treatment. CONCLUSION: This system is readily utilizable for genome scale studies of cis-acting regulatory elements which are implicated in γ-globin expression or HU-mediated induction.


Assuntos
Engenharia Celular/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Introdução de Genes/métodos , Proteínas de Fluorescência Verde/biossíntese , Hidroxiureia/metabolismo , Proteínas Recombinantes/biossíntese , Ativação Transcricional , Proteína 9 Associada à CRISPR , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas de Fluorescência Verde/genética , Humanos , Células K562 , Proteínas Recombinantes/genética , gama-Globinas/genética
9.
Appl Microbiol Biotechnol ; 103(8): 3501-3510, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30903214

RESUMO

Methods for heterologous protein production in Escherichia coli have revolutionized biotechnology and the bioindustry. It is ultimately important to increase the amount of protein product from bacteria. To this end, a variety of tools, such as effective promoters, have been developed. Here, we present a versatile molecular tool based on a phenomenon termed "translation enhancement by a Dictyostelium gene sequence" ("TED") in E. coli. We found that protein expression was increased when a gene sequence of Dictyostelium discoideum was placed upstream of the Shine-Dalgarno sequence located between the promoter and the initiation codon of a target gene. The most effective sequence among the genes examined was mlcR, which encodes the myosin regulatory light chain, a subunit of myosin II. Serial deletion analysis revealed that at least 10 bases of the 3' end of the mlcR gene enhanced the production of green fluorescent protein in cells. We applied this tool to a T7 expression system and found that the expression level of the proteins tested was increased when compared with the conventional method. Thus, current protein production systems can be improved by combination with TED.


Assuntos
Dictyostelium/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Biossíntese de Proteínas , Sequência de Bases , Proteínas de Escherichia coli/biossíntese , Expressão Gênica , Genes de Protozoários/genética , Proteínas de Fluorescência Verde/biossíntese , Estrutura Secundária de Proteína , RNA Bacteriano/química
10.
Dev Biol ; 450(1): 1-8, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30885528

RESUMO

Lineage analysis plays a central role in exploring the developmental potential of stem and progenitor cell populations. In higher vertebrates, a variety of techniques have been used to label individual cells or cell populations, including interspecies grafting, intracellular microinjection, and Cre-mediated recombination. However, these approaches often suffer from difficulties in progenitor cell targeting, low cellular resolution and/or ectopic labeling. To circumvent these issues, here we utilize replication incompetent avian (RIA) retroviruses to deliver combinations of fluorescent proteins into distinct cellular compartments in chick embryos. In particular, RIA-mediated lineage tracing is optimal for long term mapping of dispersing cell populations like the neural crest. Using this tool, we confirm that trunk neural crest cells are multipotent. Furthermore, our RIA vector is engineered to be fully adaptable for other purposes such as cell fate analysis, gene perturbation studies and time-lapse imaging. Taken together, we present a novel approach of multiplex lineage analysis that can be applied to normal and perturbed development of diverse cell populations in avian embryos.


Assuntos
Linhagem da Célula , Proteínas de Fluorescência Verde/biossíntese , Crista Neural/embriologia , Retroviridae , Coloração e Rotulagem , Animais , Embrião de Galinha , Galinhas , Proteínas de Fluorescência Verde/genética , Histocitoquímica , Crista Neural/citologia
11.
J Vis Exp ; (144)2019 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-30855561

RESUMO

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 µg/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per µg of protein produced or $0.019 per µL of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.


Assuntos
Sistema Livre de Células , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Biossíntese de Proteínas , Escherichia coli/genética
12.
Bioprocess Biosyst Eng ; 42(5): 799-806, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30730009

RESUMO

Stable transfection of mammalian cells using various expression cassettes for exogenous gene expression has been well established. The impact of critical factors in these cassettes, such as promoter and enhancer elements, on recombinant protein production in mammalian cells has been studied extensively to optimize the expression efficiency. However, few studies on the correlation between the strength of selection marker and the expression of gene of interest (GOI) have been reported. Here we investigated the correlation between the strength of a widely used selection marker, glutamine synthetase (GS) gene, and gene of interest in which the expression of GOI is driven by mouse cytomegalovirus (mCMV) major immediate early (MIE) promoter whereas the expression of GS is controlled by SV40E (Simian vacuolating virus 40E) promoter. We used a green fluorescent protein and the adalimumab antibody (heavy and light chain) as two distinct examples for the gene of interest. We then decreased the expression of GS gene by engineering a specific region of its SV40E promoter in these expression cassettes. By comparing the expression of GS and GOI at transcription and translation level before and after the SV40E promoter was weakened, we found that lower GS expression due to weaker SV40E transcription correlated well with the higher expression of recombinant proteins, mainly by increasing the copy number of GS and GOI integration into host cell genome.


Assuntos
Adalimumab , Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde , Regiões Promotoras Genéticas , Transcrição Genética , Adalimumab/biossíntese , Adalimumab/genética , Animais , Células CHO , Cricetulus , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Camundongos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
13.
Stem Cell Res ; 35: 101400, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30735882

RESUMO

Cholangiocytes are the epithelial cells that line bile ducts, and ductal plate malformation is a developmental anomaly of bile ducts that causes severe congenital biliary disorders. However, because of a lack of specific marker genes, methods for the stepwise differentiation and isolation of human induced pluripotent stem cell (hiPSC)-derived cholangiocyte progenitors at ductal plate stages have not been established. We herein generated an AQP1-GFP reporter hiPSC line and developed a combination treatment with transforming growth factor (TGF) ß2 and epidermal growth factor (EGF) to induce hiPSC-derived hepatoblasts into AQP1+ cells in vitro. By confirming that the isolated AQP1+ cells showed similar gene expression patterns to cholangiocyte progenitors at the remodeling ductal plate stage around gestational week (GW) 20, we established a differentiation protocol from hiPSCs through SOX9+CK19+AQP1- ductal plate-like cells into SOX9+CK19+AQP1+ remodeling ductal plate-like cells. We further generated 3D bile duct-like structures from the induced ductal plate-like cells. These results suggest that AQP1 is a useful marker for the generation of remodeling ductal plate cells from hiPSCs. Our methods may contribute to elucidating the differentiation mechanisms of ductal plate cells and the pathogenesis of ductal plate malformation.


Assuntos
Aquaporina 1 , Ductos Biliares , Células Epiteliais , Proteínas de Fluorescência Verde , Células-Tronco Pluripotentes Induzidas , Aquaporina 1/biossíntese , Aquaporina 1/genética , Ductos Biliares/anormalidades , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia
14.
Appl Microbiol Biotechnol ; 103(7): 3085-3097, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30737536

RESUMO

The development of arming yeast strains as whole-cell biocatalysts involves a selection of effective anchoring proteins to display enzymes and proteins on yeast cell surface. To screen for novel anchoring proteins with improved efficiency, a bioinformatics pipeline for the identification of glycosylphosphatidylinositol-anchored cell wall proteins (GPI-CWPs) suitable for attaching passenger proteins to the cell surface of Saccharomyces cerevisiae has been developed. Here, the C-terminal sequences (CTSs) of putative GPI-CWPs were selected based on the criteria that the sequence must contain a serine/threonine-rich (S/T) region of at least 30% S/T content, a total threonine content of at least 10%, a continuous S/T stretch of at least 130 amino acids in length, and a continuous T-rich region of at least 10 amino acids in length. Of the predicted 790 proteins, 37 putative GPI-CWPs were selected from different yeast and fungal species to be evaluated for their performance in displaying yeast-enhanced green fluorescent protein and ß-glucosidase enzyme. This led to the identification of five novel anchoring proteins with higher performance compared to α-agglutinin used as benchmark. In particular, the CTS of uncharacterized protein in Kluyveromyces lactis, namely 6_Kl, is the most efficient anchoring protein of the group. The CTS of 6_Kl protein provided a ß-glucosidase activity of up to 23.5 U/g cell dry weight, which is 2.8 times higher than that of the CTS of α-agglutinin. These identified CTSs could be potential novel anchoring protein candidates for construction of efficient arming yeasts for biotechnology applications in the future.


Assuntos
Parede Celular/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Engenharia de Proteínas , Saccharomyces cerevisiae/metabolismo , beta-Glucosidase/biossíntese , Proteínas de Bactérias/química , Biologia Computacional , Glicosilfosfatidilinositóis/química , Kluyveromyces , Propriedades de Superfície
15.
Neuroscience ; 404: 268-281, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30703506

RESUMO

GIN (GFP-expressing inhibitory interneuron) transgenic mice are believed to express the enhanced GFP (eGFP) in a subset of somatostatin (SST)-expressing interneurons in the neocortex and have been widely used in the study on SST interneurons. Previous studies showed that eGFP+ neurons in the neocortex are distributed in the layer II-IV and upper layer V (cortical eGFP neurons) and contain SST. In this study, we reported a new group of eGFP+ neurons in GIN mice at early postnatal ages, which was located in the deep layer of the lateral neocortex as clusters (cluster eGFP neurons). Cluster eGFP neurons were noticeable at birth but disappeared within two months, in contrast to cortical eGFP neurons that started to appear around postnatal day 3 to 5 and existed through life. Cluster eGFP neurons were not immunoreactive for SST antibodies, contrary to cortical eGFP neurons. They were also not immunolabeled by parvalbumin, a marker for another major type of interneurons, and Ca2+/calmodulin-dependent kinases II, a commonly used marker for excitatory neurons. Firing rate, afterhyperpolarization, and excitatory synaptic activity significantly enhanced in cortical eGFP neurons during postnatal development, but these properties remained mostly unchanged in cluster eGFP neurons. Short-term plasticity of the excitatory synapse showed robust facilitation in cortical eGFP neurons but depression in cluster eGFP neurons. These results implied that eGFP might also be expressed in other types of cortical neurons in addition to SST-containing interneurons in GIN mice at early postnatal ages.


Assuntos
Proteínas de Fluorescência Verde/biossíntese , Interneurônios/metabolismo , Neocórtex/citologia , Neocórtex/metabolismo , Animais , Feminino , Expressão Gênica , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Interneurônios/química , Masculino , Camundongos , Camundongos Transgênicos , Neocórtex/química
16.
Chemphyschem ; 20(5): 727-735, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30672638

RESUMO

Twelve surface-active ionic liquids (SAILs) and surface-active derivatives, based on imidazolium, ammonium, and phosphonium cations and containing one, or more, long alkyl chains in the cation and/or the anion, were synthetized and characterized. The aggregation behavior of these SAILs in water, as well as their adsorption at solution/air interface, were studied by assessing surface tension and conductivity. The CMC values obtained (0.03-6.0 mM) show a high propensity of these compounds to self-aggregate in aqueous media. Their thermal properties were also characterized, namely the melting point and decomposition temperature by using DSC and TGA, respectively. Furthermore, the toxicity of these SAILs was evaluated using the marine bacteria Aliivibrio fischeri (Gram-negative). According to the EC50 values obtained (0.3-2.7 mg L-1 ), the surface-active compounds tested should be considered "toxic" or "highly toxic". Their ability to induce cell disruption of Escherichia coli cells (also Gram-negative), releasing the intracellular green fluorescent protein (GFP) produced, was investigated. The results clearly evidence the capability of these SAILs to act as cell disruption agents.


Assuntos
Escherichia coli/química , Escherichia coli/efeitos dos fármacos , Líquidos Iônicos/síntese química , Líquidos Iônicos/farmacologia , Compostos de Amônio/química , Escherichia coli/citologia , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/química , Imidazóis/química , Líquidos Iônicos/química , Líquidos Iônicos/metabolismo , Compostos Organofosforados/química , Propriedades de Superfície
17.
Biochem Mol Biol Educ ; 47(2): 145-155, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30664332

RESUMO

Undergraduates learn that gene editing in diverse organisms is now possible. How targeted manipulation of genes and genomes is utilized in basic science and biomedicine to address biological questions is challenging for undergraduates to conceptualize. Thus, we developed a lab experience that would allow students to be actively engaged in the full process of design, implementation of a gene editing strategy, and interpretation of results within an 8-week lab period of a Genetics course. The laboratory experience combines two transformative biotechnology tools; the utilization of green fluorescent protein (GFP) as a diagnostic marker of gene expression and the fundamentals and specificity of Clustered Regularly Interspaced Short Palindromic Repeats-cas9 (CRISPR-cas9) gene editing in bacterial cells. The students designed and constructed plasmids that express single guide RNA targeted to GFP, expressed the sgRNA and cas9 in bacteria cells, and successfully deactivated GFP gene expression in the bacterial cells with their designed CRISPR-cas9 tools. Student assessment revealed most students achieved student learning objectives. We conclude this lab experience is an effective and accessible method for engaging students in the scientific practices, knowledge and challenges revolving targeted CRISPR-cas9 gene manipulation. © 2019 International Union of Biochemistry and Molecular Biology, 47(2): 145-155, 2019.


Assuntos
Sistemas CRISPR-Cas/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Fluorescência Verde/deficiência , Proteínas de Fluorescência Verde/genética , Laboratórios , Universidades , Proteínas de Fluorescência Verde/biossíntese , Software , Estudantes
18.
Aging (Albany NY) ; 10(12): 3713-3735, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30513510

RESUMO

Aged liver is usually impaired in response to hepatic injury. Tissue-specific stem cells participate in the repair of tissue injury. However, how oval cells (OCs) respond to injury and how the process is regulated by tissue microenvironment in aged mice have not been fully understood. In this study, taking advantage of well-established murine OC activation model, we demonstrated that OCs were less activated upon injury in aged mice and the impairment was mainly attributed to dysfunction in their niche. Through analyzing global gene expression, we found that the genes differentially expressed in damaged young and aged mouse liver tissues were predominantly those required for the formation and remodeling of extracellular matrix. As one of the most important extracellular matrix components in the OC niche, laminin was shown to promote the proliferation of OCs. Not surprisingly, laminin was downregulated with aging. Consistent with the downregulation of genes encoding DNA-dependent protein kinase (DNA-PK) proteins in aged hepatic stellate cells (HSCs), inhibition of DNA-PK also led to reduced expression of laminin in HSCs. Moreover, impairment in OC activation caused by less supporting from DNA-damaged HSCs could be rescued by laminin. This study reveals a new cellular mechanism underlying impaired OCs functionality during aging.


Assuntos
Comunicação Celular , Proliferação de Células , Células Estreladas do Fígado/metabolismo , Laminina/metabolismo , Hepatopatias/metabolismo , Fígado/metabolismo , Fatores Etários , Animais , Células Cultivadas , Microambiente Celular , Senescência Celular , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células Estreladas do Fígado/patologia , Integrinas/metabolismo , Fígado/patologia , Hepatopatias/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Nucleares/metabolismo , Transdução de Sinais
19.
Pharm Biol ; 56(1): 450-454, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30354840

RESUMO

CONTEXT: Safranal (SAF) is verified to have potential effects in promoting nerve growth. OBJECTIVES: This study verifies the role of SAF in promoting dopaminergic neurons growth in vitro and in vivo. MATERIAL AND METHODS: Rat neural stem cells (NSC) were treated with 1, 20, or 100 ng/mL of SAF, and the expression levels of tyrosine hydroxylase (TH) and dopamine transporter (DAT) were assayed by flow cytometry and real-time PCR and the secretion of dopamine (DA) was assayed by ELISA. Then, 2 × 106 cells of SAF-treated NSC was administrated into PD rat models induced by 6-OHDA. The differentiation and survival of dopaminergic neurons was identified by fluorescence microscope and TH+ cells by immunostaining and DA secretion by ELISA at week 2 and week 4, respectively. RESULTS: After being treated with SAF at 20 and 100 ng/mL for 1 week, TH and DAT positive rates increased 1.4- and 1.7-fold (p < 0.01, respectively). TH and DAT mRNA also increased 8.05- and 4.41-fold, respectively. And the release of DA statistically increased 1.5-fold (p < 0.01). In vivo, the number of rotations decreased to 4.33 ± 0.97 rpm (p < 0.01) and the survival rates increased to 77.66 ± 7.87% (p < 0.05) at week 4 after transplantation of SAF-treated NSC. Moreover, the transplanted cells increased three-fold, TH fluorescence density increased four-fold and DA releases increased 1.4-fold (p < 0.01) at week 4 after transplantation. CONCLUSIONS: SAF promoted the production of functional DA cells and alleviated PD, which may contribute to a new therapy for PD patients.


Assuntos
Cicloexenos/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Células-Tronco Neurais/efeitos dos fármacos , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/patologia , Terpenos/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/biossíntese , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/biossíntese , Células-Tronco Neurais/citologia , Oxidopamina , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/metabolismo , Ratos , Ratos Sprague-Dawley , Transplante de Células-Tronco , Tirosina 3-Mono-Oxigenase/biossíntese , Tirosina 3-Mono-Oxigenase/metabolismo
20.
Sci Rep ; 8(1): 14453, 2018 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-30262904

RESUMO

Site-specific recombinases (SSR) are utilized as important genome engineering tools to precisely modify the genome of mice and other model organisms. Reporter mice that mark cells that at any given time had expressed the enzyme are frequently used for lineage tracing and to characterize newly generated mice expressing a recombinase from a chosen promoter. With increasing sophistication of genome alteration strategies, the demand for novel SSR systems that efficiently and specifically recombine their targets is rising and several SSR-systems are now used in combination to address complex biological questions in vivo. Generation of reporter mice for each one of these recombinases is cumbersome and increases the number of mouse lines that need to be maintained in animal facilities. Here we present a multi-reporter mouse line for loci-of-recombination (X) (MuX) that streamlines the characterization of mice expressing prominent recombinases. MuX mice constitutively express nuclear green fluorescent protein after recombination by either Cre, Flp, Dre or Vika recombinase, rationalizing the number of animal lines that need to be maintained. We also pioneer the use of the Vika/vox system in mice, illustrating its high efficacy and specificity, thereby facilitating future designs of sophisticated recombinase-based in vivo genome engineering strategies.


Assuntos
DNA Nucleotidiltransferases , Proteínas de Escherichia coli , Genes Reporter , Proteínas de Fluorescência Verde , Integrases , Camundongos Transgênicos , Recombinases , Animais , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Transgênicos/genética , Camundongos Transgênicos/metabolismo , Recombinases/genética , Recombinases/metabolismo
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